Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF TREATING OCULAR NEOVASCULAR DISEASES USING AAV2 VARIANTS
ENCODING AFLIBERCEPT
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent
Application No. 62/899,070 filed
September 11, 2019, U.S. Provisional Patent Application No. 62/913,648 filed
October 10, 2019,
International Application No. PCT/US2019/062066 filed November 18, 2019, U.S.
Provisional Patent
Application No. 62/959,784 filed January 10, 2020, U.S. Provisional Patent
Application No. 62/971,835
filed February 7, 2020, U.S. Provisional Patent Application No. 63/019,190
filed May 1, 2020, US.
Provisional Patent Application No. 63/030,819 filed May 27, 2020 and U.S.
Provisional Patent
Application No. 63/063,203 filed August 7, 2020, the disclosure of each of
which are hereby incorporated
by reference in its entirety.
FIELD
100021 The present disclosure relates to methods of treating ocular
neovascular disease and disorders in
an individual that comprise administering a single unit dose of a recombinant
adeno associated virus
(rAAV) particles encoding an anti-VEGF agent (e.g., aflibercept) to an eye of
an individual.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0003] The content of the following submission on ASCII text file is
incorporated herein by
reference in its entirety: a computer readable form (CRF) of the Sequence
Listing (file name:
627002001241SEQLIST_TXT, date recorded: September 9, 2020, size: 41 KB).
BACKGROUND
[0004] Age-related macular degeneration (AMD) is a degenerative ocular disease
affecting the macula,
a light sensitive, small area in the center of the retina that is responsible
for reading and fine vision.
Conditions affecting the macula reduce central vision while leaving peripheral
vision intact. In severe
cases, the disease can lead to central blindness. AMD is a notable cause of
vision loss in the US
population among persons 65 years and older, and the estimated prevalence of
any AMD among persons
over 40 years of age is approximately 6.5% (Klein et al,, (2011) Arch
Ophthalmol, 129(1):75-80).
Neovascular or exudative or wet AMD (nAMD, wAMD, or nwAMD) is an advanced form
of AMD. The
hallmark of wAMD is choroidal neovascularization (CNV), which is the
infiltration of abnormal blood
vessels in the retina from the underlying choroid layer, resulting in retinal
cell damage and central
blindness. This abnormal angiogenic process is modulated by growth factors, in
particular, vascular
endothelial growth factor (VEGF). The standard of care of wAMD is a class of
molecules that bind to and
sequester VEGF, such as ranibizumab (Lucentis) and aflibercept (Eylea).
[0005] Diabetic retinopathy (DR) is a major complication of diabetes mellitus,
and is a leading cause of
visual loss in the working age population. DR may be non-proliferative (NPDR),
with no new blood
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vessel growth, or proliferative DR (PDR), with new abnomial blood vessel
growth within the retina or
choroid. Diabetic macular edema (DME) is a complication of DR, and is another
example of an ocular
disease affecting the macula. DME affects up to 10% of people with diabetes
and is caused by fluid
accumulation in the macula. DME is the most frequent cause of sight loss in
people with DR. Available
therapies for treating DME include laser and anti-vascular endothelial growth
factor (anti-VEGF) drugs
such as aflibercept.
100061 Aflibercept is a recombinant fusion protein that acts as a decoy
receptor for vascular endothelial
growth factor subtypes A and B (VEGF-A and VEGF-B) and placental growth factor
(PGF). By binding
to these ligands, aflibercept is able to prevent them from binding to vascular
endothelial growth factor
receptors (VEGFR), VEGFR-1 and VEGFR-2, to suppress neovascularization and
decrease vascular
permeability. Aflibercept consists of domain 2 of VEGFR-1 and domain 3 of
VEGFR-2 fused with the Fc
fragment of IgGl.
100071 Current standard of care anti-VEGF agents such as aflibercept need to
be re-administered via
intravitreal (WI) injection every 4 to 8 weeks to achieve optimal therapeutic
outcomes and maintain
visual acuity. Compliance with such a regimen is burdensome to patients, their
caregivers, and the
healthcare system, and most patients fall out of compliance with the optimal
regimen over time, which is
correlated with vision loss (Khanani AM, et al.). In addition, there are
complications including
endophthalmitis, retinal detachments, traumatic cataract, and elevated
intraocular pressure (lOP); the risks
of these complications are likely to increase with repeated IVT injections
(Falavarjani et al., (2013) Eye
(Lond), 27(7).787-794).
100081 Therefore, there is a need in the art for therapies for ocular
neovascular diseases such as wAMD,
DR, or DME that are effective, reduce the risk of adverse effects, and are
amenable to high long-term
patient compliance.
SUMMARY OF THE DISCLOSURE
100091 In one aspect, provided herein is a method for treating an ocular
neovascular disease in an
individual, the method comprising administering a unit dose of about 6 x 10"
vector genomes (vg) or less
of recombinant adeno-associated virus (rAAV) particles to one eye of the
individual, wherein the
individual is a human, and wherein the rAAV particles comprise: (a) a nucleic
acid encoding a
polypeptide comprising an amino acid sequence with at least about 95% identity
to the amino acid
sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats
(ITRs), and (b) an AAV2
capsid protein comprising, or consisting of, an amino acid sequence LGETTRP
(SEQ ID NO: 14) inserted
between positions 587 and 588 of the capsid protein, wherein the amino acid
residue numbering
corresponds to an AAV2 VP1 capsid protein. In some embodiments, the method
comprises reducing
retinal fluid in an eye of the individual.
100101 In another aspect, provided herein is a method for reducing retinal
fluid in an eye of an
individual with an ocular neovascular disease, the method comprising
administering a unit dose of rAAV
particles to one eye of the individual, wherein the individual is a human, and
wherein the rAAV particles
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comprise: (a) a nucleic acid encoding a polypeptide comprising an amino acid
sequence with at least
about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by
AAV2 inverted terminal
repeats (ITRs), and (b) an AAV2 capsid protein comprising, or consisting of,
an amino acid sequence
LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid
protein, wherein the
amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In
some embodiments, the
individual has received at least one treatment of an anti-VEGF agent in about
last 12 weeks prior to
administration of the unit dose of rAAV particles. In some embodiments, the
amount or presence of
retinal fluid in the one eye of the individual is refractory to prior
treatment with an anti-VEGF agent. In
some embodiments, the anti-VEGF agent is aflibercept. In some embodiments, the
retinal fluid in the one
eye is reduced by at least about 60%. In some embodiments, the retinal fluid
in the one eye is reduced by
about 80% compared to the level of retinal fluid in the one eye of the
individual prior to administration of
the rAAV to the individual. In some embodiments, the retinal fluid is
subretinal fluid (SRF) or intraretinal
fluid (WY). In some embodiments, the unit dose of rAAV particles is about 6 x
1011 vector genomes per
eye (vg/eye) or less.
100111 In another aspect, provided herein is a method for treating an ocular
neovascular disease in an
individual, the method comprising: (a) administering an anti-VEGF agent to one
eye of the individual; and
(b) administering a unit dose of about 6 x 10"- vector genomes (vg) or less of
recombinant adeno-
associated virus (rAAV) particles to the one eye of the individual after
administration of the anti-VEGF
agent, wherein the individual is a human, and wherein the rAAV particles
comprise: (i) a nucleic acid
encoding a polypeptide comprising an amino acid sequence with at least about
95% identity to the amino
acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats
(ITRs), and (ii) an
AAV2 capsid protein comprising an amino acid sequence LGETTRP (SEQ ID NO: 14)
inserted between
positions 587 and 588 of the capsid protein, wherein the amino acid residue
numbering corresponds to an
AAV2 VP1 capsid protein. In some embodiments, the method comprises
administering the unit dose of
rAAV particles to the one eye of the individual about 1 week or about 7 days
after administration of the
anti-VEGF agent. In some embodiments, the method comprises administering the
unit dose of rAAV
particles to the one eye of the individual about 1 week to about 2 weeks after
administration of the anti-
VEGF agent. In some embodiments, the method comprises administering the unit
dose of rAAV particles
to the one eye of the individual about 1 day, about 2 days, about 3 days,
about 4 days, about 5 days, about
6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11
days, about 12 days, about 13
days, about 14 days, or about 15 days after administration of the anti-VEGF
agent. In some embodiments,
the method comprises administering the anti-VEGF agent to the one eye of the
individual on Day 1, and
administering the unit dose of rAAV particles to the one eye of the individual
on Day 8. In some
embodiments, the anti-VEGF agent comprises aflibercept. In some embodiments,
the aflibercept is
administered at a dose of about 2 mg by intravitreal injection. In some
embodiments, the method further
comprises administering a topical steroid treatment. In some embodiments, the
topical steroid treatment is
a difluprednate treatment. In some embodiments, the topical steroid treatment
comprises about four
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administrations of topical steroid per day for about four weeks, followed by
about three administrations of
topical steroid per day for about one week, followed by about two
administrations of topical steroid per
day for about one week, and followed by about one administration of topical
steroid per day for about one
week; timing starting with and following administration of the anti-VEGF
agent. In some embodiments,
the topical steroid treatment comprises about four administrations of topical
steroid per day for about one
month, followed by about three administrations of topical steroid per day for
about one month, followed
by about two administrations of topical steroid per day for about one month,
and followed by about one
administration of topical steroid per day for about one month; timing starting
with and following
administration of the anti-VEGF agent. In some embodiments, the topical
steroid treatment comprises
about four administrations of topical steroid per day for about one month,
followed by about three
administrations of topical steroid per day for about one month, followed by
about two administrations of
topical steroid per day for about one month, and followed by about one
administration of topical steroid
per day for about one month; timing starting with and following administration
of the rAAV particles_ In
some embodiments, the topical steroid comprises difluprednate 0.05% at a dose
of about litg to about 3
rig. In some embodiments, the topical steroid comprises diflupreclnate 0.05%
at a dose of about 15pg.
[0012] In some embodiments that may be combined with any of the preceding
embodiments, the unit
dose of rAAV particles is about 6 x 1010 to about 2 x 1011 vector genomes per
eye (vg/eye). In some
embodiments, the unit dose of rAAV particles is about 2 x 10" or about 6 x 101
vector genomes per eye
(vg/eye). In some embodiments that may be combined with any of the preceding
embodiments, the unit
dose of rAAV particles is between about 6 x 1010 to about 6 x 101' vector
genomes per eye (vg/eye). In
some embodiments that may be combined with any of the preceding embodiments,
the unit dose of rAAV
particles is between about 6 x 1010 to about 2 x 10" vector genomes per eye
(vg/eye). In some
embodiments that may be combined with any of the preceding embodiments, the
unit dose of rAAV
particles is between about 2 x 1011 to about 6 x 10" vector genomes per eye
(vg/eye). In some
embodiments, the unit dose of rAAV particles is about 2 x 1011 or about 6 x
1010 vector genomes per eye
(vg/eye). In some embodiments, the unit dose of rAAV particles is about 2 x
101' vg/eye. In some
embodiments, the unit dose of rAAV particles is about 6 x 1011 vg/eye.
[0013] In some embodiments that may be combined with any of the preceding
embodiments, the
individual has one or more symptoms of an ocular neovascular disease in the
contralateral eye.
[0014] In some embodiments that may be combined with any of the preceding
embodiments, the
methods provided herein further comprise administering a unit dose of rAAV
particles to the contralateral
eye of the individual. In some embodiments, the administering the unit dose of
rAAV particles to the
contralateral eye is up to about 2 weeks after administering the unit dose of
rAAV particles to the one eye.
In some embodiments, the administering the unit dose of rAAV particles to the
contralateral eye is on the
same day as the administering the unit dose of rAAV particles to the one eye;
or the administering the unit
dose of rAAV particles to the contralateral eye is between about 1 day to
about 14 days after
administering the unit dose of rAAV particles to the one eye. In some
embodiments, the unit dose of
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rAAV particles administered to the contralateral eye of the individual
comprises the same or less vector
genomes per eye (vg/eye) than the unit dose of rAAV particles administered to
the one eye of the
individual. In some embodiments, the administering the unit dose of rAAV
particles to the contralateral
eye is at least about 2 weeks after administering the unit dose of rAAV
particles to the one eye. In some
embodiments, the unit dose of rAAV particles administered to the contralateral
eye of the individual
comprises more vector genomes per eye (vg/eye) than the unit dose of rAAV
particles administered to the
one eye of the individual.
100151 In some embodiments that may be combined with any of the preceding
embodiments, the nucleic
acid comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having
at least 85% identity
thereto.
100161 In some embodiments that may be combined with any of the preceding
embodiments, the
polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some
embodiments that may be
combined with any of the preceding embodiments, the polypeptide comprises the
amino acid sequence of
SEQ ID NO: 41. In some embodiments, the polypeptide is aflibercept.
100171 In some embodiments that may be combined with any of the preceding
embodiments, the nucleic
acid further comprises a first enhancer region, a promoter region, a 5'UTR
region, a second enhancer
region, and a polyadenylation site. In some embodiments, the nucleic acid
comprises, in the 5' to 3' order:
(a) a first enhancer region; (b) a promoter region; (c) a 5'UTR region; (d) a
nucleic acid encoding a
polypeptide comprising an amino acid sequence with at least about 95% identity
to the amino acid
sequence of SEQ ID NO: 35; (e) a second enhancer region; and (f) a
polyadenylation site; and flanked by
AAV2 inverted terminal repeats (11R.$). In some embodiments, the first
enhancer region comprises a
CMV sequence comprising the sequence of SEQ ID NO: 22 or a sequence having at
least 85% identity
thereto. In some embodiments, the promoter region comprises a CMV sequence
comprising the sequence
of SEQ ID NO: 23 or a sequence having at least 85% identity thereto. In some
embodiments, the nucleic
acid encoding a polypeptide comprises the nucleic acid sequence of SEQ ID NO:
40 or a sequence having
at least 85% identity thereto. In some embodiments, the polypeptide comprises
the amino acid sequence
of SEQ ID NO: 35 or a sequence having at least 85% identity thereto. In some
embodiments, the
polypeptide comprises the amino acid sequence of SEQ ID NO: 41 or a sequence
having at least 85%
identity thereto. In some embodiments, the polypeptide is aflibercept. In some
embodiments, the 5'UTR
region comprises, in 5' to 3' order, a TPL sequence comprising the sequence of
SEQ ID NO: 24 or a
sequence having at least 85% identity thereto, and an eMLP sequence comprising
the sequence of SEQ ID
NO: 25 or a sequence having at least 85% identity thereto. In some
embodiments, the second enhancer
region comprises a full EES sequence comprising the sequence of SEQ NO: 26 or
a sequence having at
least 85% identity thereto. In some embodiments, the polyadenylation site
comprises a HGH
polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence
having at least 85%
identity thereto. In some embodiments, the nucleic acid further comprises (a)
a first enhancer region
comprising a CMV sequence comprising the sequence of SEQ ID NO: 22 or a
sequence having at least
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85% identity thereto; (b) a promoter region, comprising a CMV sequence
comprising the sequence of
SEQ ID NO: 23 or a sequence having at least 85% identity thereto; (c) a 5'UTR
region comprising, in 5' to
3' order, a TPL sequence comprising the sequence of SEQ ID NO: 24 or a
sequence haying at least 85%
identity thereto, and an eMLP sequence comprising the sequence of SEQ ID NO:
25 or a sequence haying
at least 85% identity thereto; (d) a second enhancer region comprising a full
EES sequence comprising the
sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto;
and (e) a HGH
polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence
having at least 85%
identity thereto. In some embodiments, the nucleic acid comprises AAV figs
flanking the elements.
100181 In some embodiments that may be combined with any of the preceding
embodiments, the nucleic
acid comprises the sequence of SEQ ID NO: 39 or a sequence haying at least 85%
identity thereto.
100191 In some embodiments that may be combined with any of the preceding
embodiments, the rAAV
particles comprise an AAV2 VP1 capsid protein comprising a GH loop that
comprises the amino acid
sequence of SEQ ID NO: 38 or an amino acid sequence having at least 90%
sequence identity to SEQ ID
NO: 38. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid
protein comprising a
OH loop that comprises an amino acid sequence having any of at least 90%, at
least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100%
sequence identity to SEQ ID NO: 38.
100201 In some embodiments that may be combined with any of the preceding
embodiments, the rAAV
particles comprise an AAV2 VP1 capsid protein comprising the amino acid
sequence of SEQ ID NO: 37
or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:
37. In some
embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising
an amino acid
sequence having any of at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity to SEQ ID NO: 37.
100211 In some embodiments that may be combined with any of the preceding
embodiments, the AAV2
capsid protein comprises, or consists of, the amino acid sequence LGETTRP (SEQ
ID NO: 14) inserted
between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ
ID NO: 13. In some
embodiments, the AAV2 capsid protein comprises the amino acid sequence
LALGETTRPA (SEQ ID
NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein
the amino acid residue
numbering corresponds to an AAV2 VP! capsid protein. In some embodiments, the
AAV2 capsid protein
comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between
positions 587 and
588 of the AAV2 VP1 comprising the sequence of SEQ ID NO: 13. In some
embodiments, the AAV2
capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1)
inserted between
positions 587 and 588 of the capsid protein, wherein the amino acid residue
numbering corresponds to an
AAV2 VP1 capsid protein. In some embodiments, the AAV2 capsid protein
comprises the amino acid
sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of
the AAV2 VP1
comprising the sequence of SEQ ID NO: 13.
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[0022] In some embodiments that may be combined with any of the preceding
embodiments, the
administration of the unit dose of rAAV particles to the one eye and/or the
contralateral eye is by
intravitreal administration.
100231 In some embodiments that may be combined with any of the preceding
embodiments, the unit
dose of rAAV particles is in a pharmaceutical formulation. In some
embodiments, the pharmaceutical
formulation comprises the rAAV particles, sodium chloride, sodium phosphate
and a surfactant. In some
embodiments, the pharmaceutical formulation comprises about 150 to about 200
mM sodium chloride,
about 1 to about 10 mM monobasic sodium phosphate, about 1 to about 10 mM
dibasic sodium
phosphate, about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188, and about
6 x 1013 to about 6 x
10' vector genomes (vg) per mL (vg/mL) of the rAAV particles, wherein the
pharmaceutical formulation
has a pH of about 7.0 to about 7.5. In some embodiments, the pharmaceutical
formulation comprises
about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5
mM dibasic sodium
phosphate, about 6 x 10'2vg/mL of the rAAV particles, and about 0.001% (w/v)
poloxamer 188, wherein
the pharmaceutical formulation has a pH of about 7.3. In some embodiments, the
pharmaceutical
formulation comprises about 180 mM sodium chloride, about 5 mM monobasic
sodium phosphate, about
mM dibasic sodium phosphate, about 2 x1012vg/mL of the rAAV particles, and
about 0.001% (w/v)
poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3.
In some embodiments, the
pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM
monobasic sodium
phosphate, about 5 mM dibasic sodium phosphate, about 6 x10" vg,/mL of the
rAAV particles, and about
0.001% (w/v) poloxamer 188, wherein the pharmaceutical formulation has a pH of
about 7.3.
100241 In some embodiments that may be combined with any of the preceding
embodiments, the unit
dose of rAAV particles comprises a volume of about 25 L to about 250 L. In
some embodiments, the
unit dose of rAAV particles comprises a volume of about 1001.,L. In some
embodiments, the unit dose of
rAAV particles comprises a volume of about 30 L. In some embodiments that may
be combined with any
of the preceding embodiments, the unit dose of rAAV particles administered to
the one eye and/or to the
contralateral eye comprises a volume of about 25 jiL to about 250 L. In some
embodiments, the unit
dose of rAAV particles administered to the one eye and/or to the contralateral
eye comprises a volume of
about 100 L. In some embodiments, the unit dose of rAAV particles administered
to the one eye and/or to
the contralateral eye comprises a volume of about 30pL.
100251 In some embodiments that may be combined with any of the preceding
embodiments, the
individual received prior treatment for the ocular neovascular disease with an
anti-VEGF agent. In some
embodiments, the individual has received 1 or 2 injections of an anti-VEGF
agent in the one eye and/or in
the contralateral eye prior to administration of the rAAV particles in the one
eye and/or in the
contralateral eye. In some embodiments, the individual has not received prior
treatment for the ocular
neovascular disease with an anti-VEGF agent. In some embodiments, the anti-
VEGF agent is aflibercept.
[0026] In some embodiments that may be combined with any of the preceding
embodiments, the ocular
neovascular disease is wet age-related macular degeneration (AMD), retinal
neovascularization, choroidal
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neovascularization diabetic retinopathy, proliferative diabetic retinopathy,
retinal vein occlusion, central
retinal vein occlusion, branched retinal vein occlusion, diabetic macular
edema, diabetic retinal ischemia,
ischemic retinopathy, diabetic retinal edema, or any combination thereof.
100271 In some embodiments that may be combined with any of the preceding
embodiments, the unit
dose of rAAV particles is administered in combination with steroid treatment.
In some embodiments, the
steroid treatment is a corticosteroid treatment In some embodiments, the
steroid treatment is a systemic
steroid treatment. In some embodiments, the steroid treatment is an oral
steroid treatment. In some
embodiments, the steroid treatment is a prednisone treatment. In some
embodiments, the oral prednisone
treatment comprises administering prednisone at a dose of about 60 mg per day
for a total of 6 days
starting at 3 days before administration of the unit dose of rAAV particles to
the one eye and/or to the
contralateral eye, followed by administering prednisone at a dose of about 40
mg per day for a total of 3
days, followed by administering prednisone at a dose of about 20 mg per day
for a total of 2 days, and
followed by administering prednisone at a dose of about 10 mg per day for a
total of 2 days. In some
embodiments, the steroid treatment is a topical steroid treatment. In some
embodiments, the steroid
treatment is a difluprednate treatment. In some embodiments, the steroid is
administered before, during
and/or after administration of the unit dose of rAAV particles. In some
embodiments, the steroid is
administered before, during and/or after administration of the unit dose of
rAAV particles to the one eye
and/or to the contralateral eye.
100281 In some embodiments that may be combined with any of the preceding
embodiments, the steroid
treatment is a topical steroid treatment and the topical steroid treatment is
a daily steroid treatment for up
to about 4 weeks, up to about 6 weeks, or up to about 8 weeks from
administering the unit dose of rAAV
particles. In some embodiments, the topical steroid treatment comprises about
four administrations of
topical steroid on about week 1, about three administrations of topical
steroid on about week 2, about two
administrations of topical steroid on about week 3, and about one
administration of topical steroid on
about week 4; timing starting with and following administration of the unit
dose of rAAV particles. In
some embodiments, the topical steroid treatment comprises about four
administrations of topical steroid
per day for about 3 weeks after administration of the unit dose of rAAV
particles, followed by about 3
administrations of topical steroid per day for about 1 week, followed by about
2 administrations of topical
steroid per day for about 1 week, and followed by about 1 administration of
topical steroid per day for
about 1 week. In some embodiments, the topical steroid treatment comprises
about four administrations of
topical steroid per day for about four weeks, followed by about three
administrations of topical steroid per
day for about one week, followed by about two administrations of topical
steroid per day for about one
week, and followed by about one administration of topical steroid per day for
about one week; timing
starting at about one week prior to administration of the unit dose of rAAV
particles. In some
embodiments, the topical steroid treatment comprises about four
administrations of topical steroid per day
for about one month, followed by about three administrations of topical
steroid per day for about one
month, followed by about two administrations of topical steroid per day for
about one month, and
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followed by about one adminisnation of topical steroid per day for about one
month; timing starting at
about the administration of the unit dose of rAAV particles. In some
embodiments, the topical steroid
treatment comprises about four administrations of topical steroid per day for
about one month, followed
by about three administrations of topical steroid per day for about one month,
followed by about two
administrations of topical steroid per day for about one month, and followed
by about one administration
of topical steroid per day for about one month; timing starting at about one
week prior to administration of
the unit dose of rAAV particles. In some embodiments, the topical steroid
comprises difluprednate 0.05%
at a dose of about 1 pg to about 3 pg. In some embodiments, the topical
steroid comprises difluprednate
0.05% at a dose of about 2.5pg.
[0029] In some embodiments, the ocular neovascular disease is wet age-related
macular degeneration
(wAMD).
[0030] In some embodiments that may be combined with any of the preceding
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in maintenance or a decrease of retinal thickness compared
to the retinal thickness prior
to administration of the unit dose of rAAV particles. In some embodiments, the
maintenance or the
decrease of retinal thickness compared to the retinal thickness prior to
administration of the unit dose of
rAAV particles is present at about 30 weeks, about 34 weeks, about 44 weeks,
about 6 months, about 1
year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10
years, or more after
administration of the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual. In some embodiments, the administering the unit dose of rAAV
particles to the one eye and/or
to the contralateral eye of the individual results in a decrease in retinal
thickness compared to the retinal
thickness prior to administration of the unit dose of rAAV particles. In some
embodiments, the decrease in
retinal thickness is at least about 10% compared to the retinal thickness
prior to administration of the unit
dose of rAAV particles. In some embodiments, retinal thickness is central
subfield thickness (CST) or
central retinal thickness (CRT).
[0031] In some embodiments that may be combined with any of the preceding
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in maintenance or a decrease in macular volume compared to
the macular volume prior
to administration of the unit dose of rAAV particles. In some embodiments, the
maintenance or the
decrease in macular volume compared to the macular volume prior to
administration of the unit dose of
rAAV particles is present at about 30 weeks, about 34 weeks, about 44 weeks,
about 6 months, about 1
year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10
years, or more after
administration of the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual. In some embodiments, the administering the unit dose of rAAV
particles to the one eye and/or
to the contralateral eye of the individual results in a decrease in macular
volume compared to the macular
volume prior to administration of the unit dose of rAAV particles. In some
embodiments, the decrease in
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macular volume is at least about 10% compared to the macular volume prior to
administration of the unit
dose of rAAV particles.
100321 In some embodiments that may be combined with any of the preceding
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in maintenance or an improvement of visual acuity compared
to the visual acuity prior
to administration of the unit dose of rAAV particles. In some embodiments, the
maintenance or the
improvement of visual acuity compared to the visual acuity prior to
administration of the unit dose of
rAAV particles is present at about 30 weeks, about 34 weeks, about 44 weeks,
about 6 months, about I
year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10
years, or more after
administration of the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual. In some embodiments, the administering the unit dose of rAAV
particles to the one eye and/or
to the contralateral eye of the individual results in an improvement of visual
acuity compared to the visual
acuity prior to administration of the unit dose of rAAV particles. In some
embodiments, visual acuity is
best corrected visual acuity (BCVA).
100331 In some embodiments that may be combined with any of the preceding
embodiments,
administration of a single unit dose of rAAV particles to the one eye and/or
to the contralateral eye of the
individual provides a therapeutic benefit (e.g., treatment of an ocular
neovascular disease, reduction of
retinal fluid, maintenance or a decrease of retinal thickness, maintenance or
a decrease in macular volume,
and/or maintenance or an improvement of visual acuity). In some embodiments,
administration of a single
unit dose of rAAV particles to the one eye and/or to the contralateral eye of
the individual provides a
therapeutic benefit that is present at about 30 weeks or more, about 34 weeks
or more, about 44 weeks or
more, about 6 months or more, about 1 year or more, about 1.5 years or more,
or about 2 years, about 3
years, about 5 years, about 10 years, or more after administration of the unit
dose of rAAV particles to the
one eye and/or to the contralateral eye of the individual.
100341 In some embodiments that may be combined with any of the preceding
embodiments,
administration of the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of a
plurality of individuals results in at least about 50% of the individuals in
the plurality not requiring an
anti-VEGF rescue treatment. In some embodiments, at least about 50% of the
individuals in the plurality
do not require an anti-VEGF rescue treatment for at least about 20 weeks, at
least about 36 weeks, at least
about 52 weeks, at least about 56 weeks, or more after administration of the
unit dose of rAAV particles.
100351 In some embodiments that may be combined with any of the preceding
embodiments,
administration of a single unit dose of rAAV particles to the one eye and/or
to the contralateral eye of a
plurality of individuals results in at least about 67% of the individuals in
the plurality not requiring an
anti-VEGF rescue treatment. In some embodiments, at least about 67% of the
individuals in the plurality
do not require an anti-VEGF rescue treatment for at least about 20 weeks, at
least about 36 weeks, at least
about 52 weeks, at least about 60 weeks, at least about 64 weeks, or at least
about 66 weeks after
administration of the unit dose of rAAV particles.
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[0036] In some embodiments that may be combined with any of the preceding
embodiments,
administration of the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of a
plurality of individuals results in at least about 78% of the individuals in
the plurality not requiring an
anti-VEGF rescue treatment. In some embodiments, at least about 78% of the
individuals in the plurality
do not require an anti-VEGF rescue treatment for at least about 20 weeks, at
least about 36 weeks, or
more after administration of the unit dose of rAAV particles.
[0037] In some embodiments that may be combined with any of the preceding
embodiments,
administration of the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of a
plurality of individuals results in 100% of the individuals in the plurality
not requiring an anti-VEGF
rescue treatment. In some embodiments, 100% of the individuals in the
plurality do not require an anti-
VEGF rescue treatment for at least about 64 weeks, at least about 68 weeks, at
least about 72 weeks, at
least about 76 weeks, at least about 80 weeks, at least about 84 weeks, or
more after administration of the
unit dose of rAAV particles.
[0038] In some embodiments that may be combined with any of the preceding
embodiments,
administration of the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of a
plurality of individuals results in a reduction in the annualized anti-VEGF
injection rate of at least about
80%, at least about 85%, at least about 87%, at least about 90%, at least
about 95%, at least about 99%, or
100% compared to the annualized anti-VEGF injection rate prior to
administration of the unit dose of
rAAV particles.
[0039] In some embodiments, the ocular neovascular disease is diabetic macular
edema (DME). In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a 2-step or in a 3-step improvement in
Diabetic Retinopathy Severity Scale
(DRSS). In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in a 2-step improvement in
Diabetic Retinopathy Severity
Scale (DRSS). In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in a 3-step
improvement in Diabetic Retinopathy
Severity Scale (DRSS).
INCORPORATION BY REFERENCE
[0040] All references cited herein, including patent applications and
publications, are incorporated by
reference in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The novel features of the invention are set forth with particularity in
the appended claims. A
better understanding of the features and advantages of the present invention
will be obtained by reference
to the following detailed description that sets forth illustrative
embodiments, in which the principles of the
invention are utilized, and the accompanying drawings of which:
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[0042] FIGS. 1A-1B provide schematics of the investigational medicinal product
and the phase I study
described in Examples 1 and 2. FIG. 1A is a schematic of AAV2.7m8-aflibercept.
AAV2.7m8-aflibercept
is a recombinant, replication-deficient adeno-associated viral (rAAV) vector
containing the AAV2.7m8
protein capsid and a vector genome containing an expression cassette of a
oodon-optimized version of the
aflibercept cDNA under the control of a ubiquitous chimeric promoter (C11).
The AAV2.7m8-aflibercept
vector genome also contains two AAV2 inverted terminal repeat sequences (ITR)
flanking the aflibercept
cDNA expression cassette. FIG. 1B is a diagram summarizing the study design
for the phase I study
described in Examples 1 and 2.
100431 FIG. 1C provides a diagram of the AAV2.7m8-aflibercept vector genome
(SEQ ID NO: 39).
The vector genome comprises two inverted terminal repeats (flits) of AAV
serotype 2 (at positions 1-145
and 3772-3916 of SEQ ID NO: 39), an expression cassette comprised of the CMV
promoter (at positions
180-693 of SEQ ID NO: 39), a 5' Untranslated Region (UTR) comprised of
Adenovirus Tripartite Leader
Sequence and Synthetic hitron (at positions 694-1314 of SEQ ID NO: 39), a
Kozak sequence (at positions
1329-1340 of SEQ ID NO: 39), a codon-optimized aflibercept cDNA (at positions
1338-2714 of SEQ ID
NO: 39), a 3' UTR comprised of human scaffold attachment region (at positions
2717-3527 of SEQ ID
NO: 39), and human growth hormone polyadenylation/transcription stop signal
(at positions 3546-3748 of
SEQ ID NO: 39). AAV = adeno-associated virus; CMV = cytomegalovirus; GH =
growth hormone; ITR
= inverted terminal repeat; polyA = polyadenylation; SAR = scaffold-attached
region; TPL = tripartite
leader sequence.
[0044] FIGS. 2A-2L show optical coherence tomography (OCT) images and retinal
thickness maps
derived from OCT images taken from subjects in Cohort 1 of the study described
in Example 1. The OCT
images were taken at the indicated times before and after administration of
AAV2.7m8-aflibercept (Day
1). The anti-VEGF WT treatment interval is indicated for all subjects. FIG. 2A
provides OCT images
and retinal thickness maps derived from OCT images taken from Subject 1 at
five office visits at the times
indicated prior to the Screening aflibercept injection. OCT images were taken
immediately prior to
treatment with aflibercept standard of care. Subject 1 required aflibercept
!VT every 5-7 weeks and
exhibited refractory subretinal fluid and a pigment epithelial detachment
(PED) despite aflibercept
standard of care treatment. FIG. 2B provides OCT images and retinal thickness
maps derived from OCT
images taken from Subject 1 at the Screening aflibercept injection (Day -7),
at the AAV2.7m8-aflibercept
injection (Day 1), and at follow-up visits at the times indicated. Subject 1
did not require any rescue
injections after the AAV2.7m8-aflibercept injection. Subject 1 exhibited
resolution of subretinal fluid
beginning at week 4, and remained free of subretinal and intraretinal fluid
(remained dry). FIG. 2C
provides OCT images and retinal thickness maps derived from OCT images taken
from Subject 2 at five
office visits at the times indicated prior to the Screening aflibercept
injection. OCT images were taken
immediately prior to treatment with aflibercept standard of care. Subject 2
required six aflibercept IVT
treatments in the 8 months prior to AAV2.7m8-aflibercept treatment to maintain
retinal anatomy. FIG.
2D provides OCT images and retinal thickness maps derived from OCT images
taken from Subject 2 at
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the Screening aflibercept injection (Day -7), at the AAV2.7m8-aflibercept
injection (Day 1), and at
follow-up visits at the times indicated. Subject 2 did not require any rescue
injections after the
AAV2.7m8-aflibercept injection. Subject 2 exhibited stable retinal anatomy
with no subretinal or
intraretinal fluid through week 24. FIG. 2E provides OCT images and retinal
thickness maps derived
from OCT images taken from Subject 3 at four office visits at the times
indicated prior to the Screening
aflibercept injection. OCT images taken on Week -27 are not shown. OCT images
were taken
immediately prior to treatment with aflibercept standard of care. Subject 3
exhibited subretinal fluid,
which increased when the interval between aflibercept IVT was increased from 5
to 7 weeks. FIG. 2F
provides OCT images and retinal thickness maps derived from OCT images taken
from Subject 3 at the
Screening aflibercept injection (Day -7), the AAV2.7m8-aflibercept injection
(Day 1), and at follow-up
visits at the times indicated. Subject 3 did not require any rescue injections
after the AAV2.7m8-
aflibercept injection. Subject 3 exhibited resolution of refractory subretinal
fluid by week 8, and stable
retinal anatomy through week 24. FIG. 2G provides OCT images and retinal
thickness maps derived from
OCT images taken from Subject 4 at five office visits at the times indicated
prior to the Screening
aflibercept injection. OCT images were taken immediately prior to treatment
with ranibizumab 0.5 mg
IVT standard of care. Subject 4 exhibited subretinal fluid that was refractory
to ranibizurnab IVT
injections. FIG. 2H provides OCT images and retinal thickness maps derived
from OCT images taken
from Subject 4 at the Screening aflibercept injection (Day -14), the AAV2.7m8-
aflibercept injection (Day
1), and at follow-up visits at the times indicated. Subject 4 did not require
any rescue injections after the
AAV2.7m8-aflibercept injection. Subject 4 exhibited resolution of refractory
subretinal fluid by week 8,
and stable retinal anatomy through week 24. FIG. 21 provides OCT images and
retinal thickness maps
derived from OCT images taken from Subject 5 at five office visits at the
times indicated prior to the
Screening aflibercept injection. OCT images were taken immediately prior to
treatment with aflibercept
standard of care. FIG. 2J provides OCT images and retinal thickness maps
derived from OCT images
taken from Subject 5 at the Screening aflibercept injection (Day -14), the
AAV2.7m8-aflibercept injection
(Day 1), and at follow-up visits at the times indicated. Subject 5 did not
require any rescue injections after
the AAV2.7m8-aflibercept injection. Subretinal fluid and PEDs present at the
time of AAV2.7m8-
aflibercept treatment resolved overtime, and retinal anatomy remained stable
and free of subretinal or
intraretinal fluid through week 24. FIG. 2K provides OCT images and retinal
thickness maps derived
from OCT images taken from Subject 6 at five office visits at the times
indicated prior to the Screening
aflibercept injection. OCT images were taken immediately prior to treatment
with either bevacizumab 1.5
mg IVT standard of care or ranibizumab 0.5 mg IVT standard of care, as
indicated. The appearance of
Subject 6's retina was consistent with polypoidal choroidal vasculopathy
(PCV). FIG. 2L provides OCT
images and retinal thickness maps derived from OCT images taken from Subject 6
at the Screening
aflibercept injection (Day -10), the AAV2.7m8-aflibercept injection (Day 1),
and at follow-up visits at the
times indicated. Subject 6 did not require any rescue injections after the
AAV2.7m8-aflibercept injection.
Subject 6 did not exhibit any increase in subretinal fluid and achieved some
anatomical improvement
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through week 24. The contralateral eye of Subject 6 received standard of care
aflibercept injections every
4 weeks over the course of the trial, and exhibited similar retinal morphology
to the AAV2.7m8-
aflibercept-treated eye.
100451 FIG. 3 shows the change in mean central retinal thickness (CST) for
subjects in Cohort 1 of the
study described in Example 1 at the indicated time points. Error bars indicate
the 90% confidence
interval, calculated using the T-distribution. Baseline (BL) indicates the
measurement taken prior to the
Screening aflibercept injection 7 to 15 days (e.g., 7-14 days) prior to
AAV2.7m8-aflibercept treatment on
day 1. At 24 weeks post AAV2.7m8-aflibercept treatment, subjects on average
exhibited a change in
CRT of-52.7 pm (90% CI 46.5, -18.8). BL = baseline; D = day; W = week. The day
1 visit occurred 7-
14 days after the baseline visit.
100461 FIG. 4 shows the mean best corrected visual acuity (BCVA) measurements
based on Early
Treatment Diabetic Retinopathy Study (ETDRS) letters assessments for subjects
in Cohort 1 of the study
described in Example 1 at the indicated time points. Error bars indicate the
90% confidence interval,
calculated using the T-distribution. Baseline (BL) indicates the measurement
taken prior to the Screening
aflibercept injection 7 to 15 days (e.g., 7-14 days) prior to AAV2.7m8-
aflibercept treatment on clay 1. At
24 weeks post AAV2.7m8-aflibercept treatment, subjects on average exhibited a
change in BCVA of -2
letters (90% CI -9,1, 5.1). BL = baseline; D = day; W = week. The day 1 visit
occurred 7-14 days after the
baseline visit.
100471 FIG. 5 provides the nucleic acid sequence of aflibercept (SEQ ID NO:
36).
100481 FIG. 6 shows plots of anterior chamber cell and vitreous cell counts
following treatment with
AAV2.7m8-aflibercept for subjects 1-6 of the study described in Example 1. The
steroid treatment
administered to each patient is indicated below each plot. Aqueous cell count
categories were based on the
Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et at, J
Ophthalmol. 2005;140:509-
516). Vitreous cell count categories were based on National Institutes of
Health (NIH) guidelines. For
aqueous cells, a cell count value of 0.5+ indicates 1-5 cells; a cell count
value of 1+ indicates 6-15 cells; a
cell count value of 2+ indicates 16-25 cells; a cell count value of 3+
indicates 26-50 cells; and a cell
count value of 4+ indicates >50 cells. For vitreous cells, a cell count value
of 0.5+ indicates 1-10 cells; a
cell count value of 1+ indicates 11-20 cells; a cell count value of 2+
indicates 21-30 cells; a cell count
value of 3+ indicates 31-100 cells; and a cell count value of 4+ indicates
>100 cells. Rare cells were
captured as 0.5+ for the analysis shown in this figure.
100491 FIGS. 7A-7B show optical coherence tomography (OCT) images and retinal
thickness maps
derived from OCT images taken from subjects 1-6 in Cohort 1 of the study
described in Example 1 at a
median follow up time of 34 weeks. In addition, the change in BCVA from
Baseline, the number of anti-
VEGF IVT injections in the 8 months prior to administration of AAV2.7m8-
aflibercept, and the number
of administered rescue anti-VEGF IVT injections during the study are also
provided for each of subjects
1-6. The actual week during which the OCT images and retinal thickness maps
were obtained for each
subject are indicated (Subject 1 = Week 44; Subject 2 = Week 40; Subject 3 =
Week 36; Subject 4=
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Week 32; Subject 5 = Week 28; and Subject 6= Week 28). No subjects required
rescue anti-VEGF IVT
injections during the study and no retreatment criteria were met at any point
during the follow-up period
of up to 44 weeks. No subjects exhibited signs of disease re-activation on OCT
imaging.
100501 FIG. 8 shows plots of aqueous and vitreous cell counts following
treatment with AAV2.7m8-
aflibercept for subjects 1-6 in Cohort 1 of the study described in Example 1
up to a median follow-up time
of 44 weeks (40-52 weeks). The steroid treatment administered to each subject
is indicated below each
plot. Aqueous cell grade categories were based on the Standardization of
Uveifis Nomenclature (SUN)
criteria (Jabs, DA et al. J Ophthalmol. 2005; 140:509-516). Vitreous cell
grade categories were based on
National Institutes of Health (NH-I) guidelines. For aqueous cells, a cell
grade of 05+ indicates 1-5 cells; a
cell grade of 1+ indicates 6-15 cells; a cell grade of 2+ indicates 16-25
cells; a cell grade of 3+ indicates
26-50 cells; and a cell grade of 4+ indicates >50 cells. For vitreous cells, a
cell grade of 0.5+ indicates 1-
cells; a cell grade of 1+ indicates 11-20 cells; a cell grade of 2+ indicates
21-30 cells; a cell grade of 3+
indicates 31-100 cells; and a cell grade of 4+ indicates >100 cells. Rare
cells were captured as a cell
grade of 0.5+.
100511 FIGS. 9A-9B show optical coherence tomography (OCT) images and retinal
thickness maps
derived from OCT images taken from subjects 1-6 in Cohort 1 of the study
described in Example 1 at a
median follow-up time of 44 weeks. The change in BCVA ETDRS letters and CST
from Baseline are also
provided. The actual weeks during which the OCT images and retinal thickness
maps were obtained for
each subject are indicated. The asterisk indicates that Subject 4 underwent
retinal detachment repair with
gas bubble injection in order to repair a spontaneous pseudophakic inferior
macula off rhegmatogenous
retinal detachment (RRD) (FIG. 98). Subject 4 remains under follow-up; OCT
images and BCVA values
for Subject 4 correspond to the last observations prior to retinal detachment.
100521 FIG. 10 shows the mean best corrected visual acuity (BCVA) measurements
based on Early
Treatment Diabetic Retinopathy Study (ETDRS) letters assessments for subjects
in Cohort 2 of the study
described in Examples 2 and 3 at the indicated time points. Error bars
indicate the 90% confidence
interval of the mean absolute BCVA calculated using the T-distribution.
Baseline (BL) indicates the
measurement taken prior to the Screening aflibercept injection 7 to 15 days
(e.g., 7-14 days) prior to
AAV2.7m8-aflibercept treatment on day 1. At 24 weeks post AAV2.7m8-aflibercept
treatment, subjects
exhibited a mean change in BCVA of ¨4.8 letters. BL = baseline; D = day; W =
week. The day 1 visit
occurred 7-15 days after the baseline visit.
100531 FIG. 11 shows the mean central retinal thickness (CST) for subjects in
Cohort 2 of the study
described in Examples 2 and 3 at the indicated time points. Error bars
indicate the 90% confidence
interval of the mean absolute CST calculated using the T-distribution.
Baseline (BL) indicates the
measurement taken prior to the Screening aflibercept injection 7 to 15 days
(e.g., 7-14 days) prior to
AAV2.7m8-aflibercept treatment on day 1. At 24 weeks post AAV2.7m8-aflibercept
treatment, subjects
exhibited a mean change in CST of ¨27.8 pin. BL = baseline; D = day; W = week.
The day I visit
occurred 7-15 days after the baseline visit.
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[0054] FIG. 12 shows plots of aqueous cell and vitreous cell counts following
treatment with
AAV2.7m8-aflibercept for subjects in Cohort 2 of the study described in
Examples 2 and 3 up to a follow-
up time of 24 weeks. The steroid treatment administered to each subject is
indicated below each plot; the
frequency of topical steroid (difluprednate) eye drop administration is
indicated (e.g., lx = once per day;
2x = twice per day; 3x = 3 times per day; 4x =4 times per day). Aqueous cell
grade categories were based
on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al.
J Ophthalmol. 2005;
140:509-516). Vitreous cell grade categories were based on National Institutes
of Health (Nil-1)
guidelines. For aqueous cells, a cell grade of 0.5+ indicates 1-5 cells; a
cell grade of 1+ indicates 6-15
cells; a cell grade of 2+ indicates 16-25 cells; a cell grade of 3+ indicates
26-50 cells; and a cell grade of
4+ indicates >50 cells. For vitreous cells, a cell grade of 0.5+ indicates 1-
10 cells; a cell grade of 1+
indicates 11-20 cells; a cell grade of 2+ indicates 21-30 cells; a cell grade
of 3+ indicates 31-100 cells;
and a cell grade of 4+ indicates >100 cells. Rare cells were captured as a
cell grade of 0.5+ for this
analysis.
[0055] FIGS. 13A-13B show optical coherence tomography (OCT) images and
retinal thickness maps
derived from OCT images taken from subjects 1-6 in Cohort 2 of the study
described in Examples 2 and 3
up to a follow-up time of 24 weeks. The change in BCVA from Baseline, the
change in CST from
baseline, and the number of rescue anti-VEGF injections administered during
the 24-week follow up
period are also provided. In FIG. 13A, the asterisks indicate that Subject 3
received 3 rescue anti-VEGF
injections, including at week 24, all due to loss of >10 letters in BCVA from
baseline. This was attributed
to intraretinal or subretinal fluid. In FIG. 13B, the asterisks indicate that
Subject 5 received 3 rescue anti-
VEGF injections, the last of which occurred on week 20, due to an increase in
central subfield thickness
of >75 m from baseline.
[0056] FIG. 14 is a Swimmer's Lane Plot showing the number of anti-VEGF
injections for subjects in
Cohorts 1 and 2 of the study described in Examples 1-3. The x-axis represents
time in weeks relative to
the time AAV2.7m8-aflibercept was administered. The y-axis shows each
individual subject in Cohorts 1
and 2. The circular shapes represent anti-VEGF NT injections administered
before and after treatment
with AAV2.7m8-aflibercept. The asterisk indicates that Subject 6 of Cohort 2
was diagnosed with nAMD
6.4 months prior to administration of AAV2.7m8-aflibercept. The vertical line
bisecting the plot indicates
day 1, when AAV2.7m8-aflibercept was administered. To the right of the
bisecting vertical line are each
of the subsequent 61 study visits.
[0057] FIGS. 15A-15C show plots of aqueous cell and vitreous cell counts as
measured by slit lamp
examination following treatment with AAV2.7m8-aflibercept for subjects in
Cohorts 1, 2 and 3 of the
study described in Examples 1-5, FIG. 15A shows plots of aqueous cell and
vitreous cell counts for
subjects in Cohort 1 up to a follow up time of 64 weeks (median = 60 weeks;
range = 52-64 weeks). FIG.
15B shows plots of aqueous cell and vitreous cell counts for subjects in
Cohort 2 up to a follow up time of
40 weeks (median = 36 weeks; range = 3240 weeks). FIG. 15C shows plots of
aqueous cell and vitreous
cell counts for subjects in Cohort 3 up to a follow up time of 20 weeks. In
FIGS. 15A-15C, the steroid
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treatment administered to each subject is indicated below each plot; aqueous
cell grade categories were
based on the Standardization of Uveitis Nomenclature (SUN) criteria Jabs DA,
et al. J Ophthalmol
2005;140:509-516; vitreous cell grade categories were based on National
Institutes of Health (Nil-I)
guidelines; aqueous cells: 0.5+ = 1-5 cells; 1+ = 6-15 cells; 2+ = 16-25
cells; 3+ = 26-50 cells; 4+ = >50
cells; vitreous cells: 0.5+ = 1-10 cells; 1+ = 11-20 cells; 2+ = 21-30 cells;
3+ = 31-100 cells; 4+ = >100
cells; rare cells were captured as 0.5+ for this analysis. QM = four times per
day; TID = three time per
day; BID = twice per day; QD = once per day; QOD = once every other day; "QD
>>" indicates that the
subject continues to receive topical steroids once per day; "BID >>" indicates
that the subject continues to
receive topical steroids twice per day; and "QID >>" indicates that the
subject continues to receive topical
steroids four times per day.
100581 FIGS. 16A-168 provide the mean BCVA and mean CST for subjects in Cohort
1 of the study
described in Examples 1-5 (n=6) from baseline up to Week 52. FIG. 16A shows
the mean BCVA
(ETDRS letters) from baseline up to Week 52. The asterisk indicates that one
subject had low BCVA
scores at Weeks 44 and 48 due to retinal detachment. FIG. 16B shows the mean
CST (pm) from baseline
up to Week 52. The asterisk indicates that one subject had no CST data at
Weeks 44 and 48 due to retinal
detachment. In FIGS. 16A-16B, the error bars indicate the 90% confidence
intervals of the mean absolute
BCVA and CST values using the T-distribution; BL = baseline; D = day; W =
week.
100591 FIGS. 17A-17B provide the mean BCVA and mean CST for subjects in Cohort
2 of the study
described in Examples 1-5 (n=6) from baseline up to Week 36. FIG. 17A shows
the mean BCVA
(ETDRS letters) from baseline up to Week 36. FIG. 17B shows the mean CST (pm)
from baseline up to
Week 36. In FIGS. 17A-17B, the error bars indicate the 90% confidence
intervals of the mean absolute
BCVA and CST values using the T-distribution; BL = baseline; D = day; W =
week; one subject missed
the Week 36 visit.
100601 FIGS. 18A-18B provide the mean BCVA and mean CST for five subjects in
Cohort 3 of the
study described in Examples 1-5 from baseline up to Week 20. FIG. 18A shows
the mean BCVA
(ETDRS letters) from baseline up to Week 20. FIG. 18B shows the mean CST (pin)
from baseline up to
Week 20. In FIGS. 18A-18B, the error bars indicate the 90% confidence
intervals of the mean absolute
BCVA and CST values using the T-distribution; BL = baseline; D = day; W =
week; one subject missed
the Week 36 visit.
100611 FIG. 19 is a Swimmer's Lane Plot showing the number of anti-VEGF
injections administered to
subjects in Cohorts 1, 2 and 3 of the study described in Examples 1-5. The x-
axis represents time in weeks
relative to the time AAV2.7m8-aflibercept was administered. The y-axis shows
each individual subject in
Cohorts 1-3. The circular shapes represent anti-VEGF IVT injections
administered before and after
treatment with AAV2.7m8-aflibercept. The vertical line bisecting the plot
indicates Day 1, when
AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical
line are each of the
subsequent study visits.
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[0062] FIGS. 20A-20B show optical coherence tomography (OCT) images and
retinal thickness maps
derived from OCT images taken from Subject 4 in Cohort 1 of the study
described in Examples 1-5 at the
indicated times before and after administration of AAV2.7m8-aflibercept. FIG.
20A shows OCT images
and retinal thickness maps for Subject 4 in Cohort 1 at the indicated times
(weeks) prior to administration
of AAV2.7m8-aflibercept, during which the subject was administered anti-VEGF
IVT injections
(ranibiziunab). FIG. 20B shows OCT images and retinal thickness maps for
Subject 4 in Cohort 1 at the
indicated times (weeks) before and after administration of AAV2.7m8-
aflibercept. The times of
administration of the screening anti-VEGF IVT injection ("Screening anti-VEGF
IVT") and of
AAV2.7m8-aflibercept ("AAV2.7m8-aflibercept") are indicated. The BCVA (ETDRS
letters) and CST
(pm) at each indicated time before and after administration of AAV2.7m8-
aflibercept are provided. The
asterisk indicates that the subject had a retinal detachment event unrelated
to AAV2.7m8-aflibercept.
100631 FIGS. 21A-218 show optical coherence tomography (OCT) images and
retinal thickness maps
derived from OCT images taken from Subject 5 in Cohort 3 of the study
described in Examples 1-5 at the
indicated times before and after administration of AAV2.7m8-aflibercept. FIG.
21A shows OCT images
and retinal thickness maps for Subject 5 in Cohort 3 at the indicated times
(weeks) prior to administration
of AAV2.7m8-aflibercept, during which the subject was administered anti-VEGF
IVT injections
(aflibercept). FIG. MB shows OCT images and retinal thickness maps for Subject
5 in Cohort 3 at the
indicated times (weeks) before and after administration of AAV2.7m8-
aflibercept. The times of
administration of the screening anti-VEGF IVT injection ("Screening anti-VEGF
IVT") and of
AAV2.7m8-aflibercept (4'AAV2.7m8-aflibercept") are indicated. The BCVA (ETDRS
letters) and CST
(pm) at each indicated time before and after administration of AAV2.7m8-
aflibercept are provided.
[0064] FIGS. 22A-22D show plots of aqueous cell and vitreous cell counts as
measured by slit lamp
examination following treatment with AAV2.7m8-aflibercept for subjects in
Cohorts 1, 2, 3, and 4 of the
study described in Examples 1-5 and 7. FIG. 22A shows plots of aqueous cell
and vitreous cell counts for
subjects in Cohort 1 up to a follow up time of 80 weeks. FIG. 22B shows plots
of aqueous cell and
vitreous cell counts for subjects in Cohort 2 up to a follow up time of 56
weeks. FIG. 22C shows plots of
aqueous cell and vitreous cell counts for subjects in Cohort 3 up to a follow
up time of 36 weeks. FIG.
22D shows plots of aqueous cell and vitreous cell counts for subjects in
Cohort 4 up to a follow up time of
8 weeks. In FIGS. 22A-22D, the steroid treatment administered to each subject
is indicated below each
plot; aqueous cell grade categories were based on the Standardization of
Uveitis Nomenclature (SUN)
criteria: Jabs DA, et al. J Ophthalmol 2005;140:509-516; vitreous cell grade
categories were based on
National Institutes of Health (NIH) guidelines; aqueous cells: 0.5+ = 1-5
cells; 1+ = 6-15 cells; 2+ = 16-
25 cells; 3+ = 26-50 cells; 4+ = >50 cells; vitreous cells: 0.5+ = 1-10 cells;
1+ = 11-20 cells; 2+ = 21-30
cells; 3+ = 31-100 cells; 4+ = >100 cells; rare cells were captured as 0.5+
for this analysis. QID = four
times per day; TID = three time per day; BID = twice per day; QD = once per
day; QOD = once every
other day; "QD >>" indicates that the subject continues to receive topical
steroids once per day; "BID >>"
indicates that the subject continues to receive topical steroids twice per
day; "TID >>" indicates that the
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subject continues to receive topical steroids three times per day; and "QID
>>" indicates that the subject
continues to receive topical steroids four times per day.
[0065] FIGS. 23A-238 provide the mean BCVA and mean CST for subjects in Cohort
1 of the study
described in Examples 1-5 and 7 (n=6) from baseline up to Week 72. FIG. 23A
shows the mean BCVA
(ETDRS letters) from baseline up to Week 72, One subject had low BCVA scores
at Weeks 44 and 48 due
to retinal detachment. *n = 5 from Week 56 to Week 72. FIG. 23B shows the mean
CST (gm) from
baseline up to Week 72. One subject had no CST data at Weeks 44 and 48 due to
retinal detachment. *n =
from Week 56 to Week 72. In FIGS. 23A-23B, the error bars indicate the 90%
confidence intervals of
the mean absolute BCVA and CST values using the T-distribution; BL = baseline;
D = day; W = week.
[0066] FIGS. 24A-248 provide the mean BCVA and mean CST for subjects in Cohort
2 of the study
described in Examples 1-5 and 7 (n=6) from baseline up to Week 52. FIG. 24A
shows the mean BCVA
(ETDRS letters) from baseline up to Week 52. *n =5 for Week 36 and Week 40.
FIG. 24B shows the
mean CST (gm) from baseline up to Week 52. *n = 5 for Week 36 and Week 40. In
FIGS. 24A-24B, the
error bars indicate the 90% confidence intervals of the mean absolute BCVA and
CST values using the T-
distribution; BL = baseline; D = day; W = week.
[0067] FIGS. 25A-258 provide the mean BCVA and mean CST for subjects in Cohort
3 of the study
described in Examples 1-5 and 7 (n=9) from baseline up to Week 20. FIG. 25A
shows the mean BCVA
(ETDRS letters) from baseline up to Week 20. *n = 8 for Weeks 4, 16, and 20.
FIG. 25B shows the mean
CST (gm) from baseline up to Week 20. *n = 8 for Weeks 4, 16, and 20. In FIGS.
25A-258, the error
bars indicate the 90% confidence intervals of the mean absolute BCVA and CST
values using the T-
distribution; BL = baseline; D = day; W = week,
[0068] FIG. 26 is a Swimmer's Lane Plot showing the number of anti-VEGF
injections administered to
subjects in Cohorts 1, 2, 3, and 4 of the study described in Examples 1-5 and
7. The x-axis represents time
in weeks relative to the time AAV2.7m8-aflibercept was administered. The y-
axis shows each individual
subject in Cohorts 1-4. The circular shapes represent anti-VEGF IVT injections
administered before and
after treatment with AAV2.7m8-aflibercept. The vertical line bisecting the
plot indicates Day 1, when
AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical
line are each of the
subsequent study visits. Five subjects were diagnosed <1 year prior to
AAV2.7m8-aflibercept injection (1
subject in each of Cohorts 2 and 3, and 3 subjects in Cohort 4). In Cohort 4,
data before Day 1 are
incomplete for Subject 2 due to relocation, and Subject 5 because the subject
participated in a clinical trial
with unknown medication(s) after diagnosis.
[0069] FIGS. 27A-27B show the mean annualized anti-VEGF injection rate for
subjects in Cohorts 1-3
of the study described in Examples 1-5 and 7. FIG. 27A provides a comparison
of the mean annualized
anti-VEGF injection rates for subjects in Cohort 1 (administered the "High
Dose" of 6 x 1011vg/eye; n =
6) and for subjects in Cohorts 2 and 3 (administered the "Low Dose" of 2 x 10"
vg/eye; n = 15). FIG.
27B provides a comparison of the mean annualized anti-VEGF injection rates for
subjects in each of
Cohorts 1 (n = 6), 2 (n = 6), and 3 (n = 9). In FIGS. 27A-27B, Annualized rate
(Prior) = (number of anti-
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VEGF IVT injections in the 12 months prior to AAV2.7m8-aflibercept) / (days
from the first anti-VEGF
IVT injection in the past 12 months to the AAV2.7m8-aflibercept injection /
365.25); and Annualized rate
(Post) = (number of anti-VEGF IVT injections since AAV2.7m8-aflibercept) /
(days from AAV2.7m8-
aflibercept to the last study follow-up 1365.25).
[0070] FIGS. 28A-28B show optical coherence tomography (OCT) images and
retinal thickness maps
derived from OCT images taken from Subject 5 in Cohort 3 of the study
described in Examples 1-5 and 7
at the indicated times before and after administration of AAV2.7m8-
aflibercept. FIG. 28A shows OCT
images and retinal thickness maps for Subject 5 in Cohort 3 at the indicated
times (weeks) prior to
administration of AAV2.7m8-aflibercept, during which the subject was
administered anti-VEGF IVT
injections (aflibercept). FIG. 28B shows OCT images and retinal thickness maps
for Subject 5 in Cohort 3
at the indicated times (weeks) before and after administration of AAV2.7m8-
aflibercept. The times of
administration of the screening anti-VEGF IVT injection ("Aflibercept IVT")
and of AAV2.7m8-
aflibercept are indicated. The BCVA (ETDRS letters) and CST (pm) at each
indicated time before and
after administration of AAV2.7m8-aflibercept are provided.
[0071] FIG. 29 shows a diagram of the design of the study described in Example
6, evaluating the
durability of a single intravitreal (IVT) injection of AAV2.7m8-aflibercept in
subjects with diabetic
macular edema (DME). DRSS = Diabetic Retinopathy Severity Score; OCT= Optical
Coherence
Tomography; CST = Central Subfield Thickness. *All subjects receive a 7-week
course of difiuprednate
eye drops, starting at QID and tapering to QD. **PE Primary Endpoint
assessment. ***EOS= End of
Study assessment.
DETAILED DESCRIPTION
[0072] Several aspects are described below with reference to example
applications for illustration. It
should be understood that numerous specific details, relationships, and
methods are set forth to provide a
full understanding of the features described herein. One having ordinary skill
in the relevant art, however,
will readily recognize that the features described herein can be practiced
without one or more of the
specific details or with other methods. The features described herein are not
limited by the illustrated
ordering of acts or events, as some acts can occur in different orders and/or
concurrently with other acts or
events. Furthermore, not all illustrated acts or events are required to
implement a methodology in
accordance with the features described herein.
Definitions
[0073] Unless otherwise defined, all technical terms used herein have the same
meaning as conunonly
understood by one of ordinary skill in the art.
[0074] The terminology used herein is for the purpose of describing particular
examples only and is not
intended to be limiting. As used herein, the singular forms "a", "an" and
"the" are intended to include the
plural forms as well, unless the context clearly indicates otherwise.
Furthermore, to the extent that the
terms "including", "includes", "having", "has", "with", or variants thereof
are used in either the detailed
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description and/or the claims, such terms are intended to be inclusive in a
manner similar to the term
"comprising". The term "comprising" as used herein is synonymous with
"including" or "containing",
and is inclusive or open-ended.
100751 Any reference to "or" herein is intended to encompass "and/or" unless
otherwise stated. As used
herein, the term "about" a number refers to that number plus or minus 10% of
that number. The term
"about" a range refers to that range minus 10% of its lowest value and plus
10% of its greatest value.
Reference to "about" a value or parameter herein includes (and describes)
embodiments that are directed
to that value or parameter per se.
100761 The term "subject", "patient", or "individual" refers to primates, such
as humans and non-human
primates, e.g., African green monkeys and rhesus monkeys. In some embodiments,
the subject is a human.
100771 The terms "treat," "treating", "treatment," "ameliorate" or
"ameliorating" and other grammatical
equivalents as used herein, refer to alleviating, abating or ameliorating an
ocular neovascular disease or
disorder, or symptoms of the ocular neovascular disease or disorder,
preventing additional symptoms of
the ocular neovascular disease or disorder, ameliorating or preventing the
underlying metabolic causes of
symptoms, inhibiting the ocular neovascular disease or disorder, e.g.,
arresting the development of the
ocular neovascular disease or disorder, relieving the ocular neovascular
disease or disorder, causing
regression of the ocular neovascular disease or disorder, or stopping the
symptoms of the ocular
neovascular disease or disorder, and are intended to include prophylaxis. The
terms further include
achieving a therapeutic benefit and/or a prophylactic benefit. The term
"therapeutic benefit" refers to
eradication or amelioration of the ocular neovascular disease or disorder
being treated. Also, a therapeutic
benefit is achieved with the eradication or amelioration of one or more of the
physiological symptoms
associated with the ocular neovascular disease or disorder such that an
improvement is observed in the
subject, notwithstanding that, in some embodiments, the subject is still
afflicted with the ocular
neovascular disease or disorder. For prophylactic benefit, the pharmaceutical
compositions are
administered to a subject at risk of developing the ocular neovascular disease
or disorder, or to a subject
reporting one or more of the physiological symptoms of the ocular neovascular
disease or disorder, even if
a diagnosis of the disease or disorder has not been made.
100781 The terms "administer," "administering", "administration," and the
like, as used herein, can refer
to the methods that are used to enable delivery of therapeutics or
pharmaceutical compositions to the
desired site of biological action. These methods include intravitreal or
subretinal injection to an eye.
100791 The terms "effective amount", "therapeutically effective amount" or
"pharmaceutically effective
amount" as used herein, can refer to a sufficient amount of at least one
pharmaceutical composition or
compound being administered which will relieve to some extent one or more of
the symptoms of the
ocular disease or disorder being treated. An "effective amount",
'Therapeutically effective amount" or
"pharmaceutically effective amount" of a pharmaceutical composition may be
administered to a subject in
need thereof as a unit dose (as described in further detail elsewhere herein).
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[0080] The term "pharmaceutically acceptable" as used herein, can refer to a
material, such as a carrier
or diluent, which does not abrogate the biological activity or properties of a
compound disclosed herein,
and is relatively nontoxic (i.e., when the material is administered to an
individual it does not cause
undesirable biological effects nor does it interact in a deleterious manner
with any of the components of
the composition in which it is contained).
[0081] The term "pharmaceutical composition," or simply "composition" as used
herein, can refer to a
biologically active compound, optionally mixed with at least one
pharmaceutically acceptable chemical
component, such as, though not limited to carriers, stabilizers, diluents,
dispersing agents, suspending
agents, thickening agents, excipients and the like.
[0082] An "AAV vector" or "rAAV vector" as used herein refers to an adeno-
associated virus (AAV)
vector or a recombinant AAV (rAAV) vector comprising a polynucleotide sequence
not of AAV origin
(e.g., a polynucleotide heterologous to AAV such as a nucleic acid sequence
that encodes a therapeutic
transgene, e.g., aflibercept) for transduction into a target cell or to a
target tissue. In general, the
heterologous polynucleotide is flanked by at least one, and generally by two,
AAV inverted terminal
repeat sequences (ITRs). The tenn rAAV vector encompasses both rAAV vector
particles and rAAV
vector plasmids. A rAAV vector may be either single-stranded (ssAAV) or self-
complementary (scAAV).
[0083] An "AAV virus" or "AAV viral particle" or "rAAV vector particle" or
"rAAV particle" refers to
a viral particle comprising at least one AAV capsid protein and a
polynucleotide rAAV vector. In some
cases, the at least one AAV capsid protein is from a wild type AAV or is a
variant AAV capsid protein
(e.g., an AAV capsid protein with an insertion, e.g., an insertion of the 7m8
amino sequence as set forth
below). If the particle comprises a heterologous polynucleotide (e.g., a
polynucleotide other than a wild-
type AAV genome such as a transgene to be delivered to a target cell or target
tissue), it is referred to as a
"rAAV particle", "rAAV vector particle" or a "rAAV vector". Thus, production
of rAAV particles
necessarily includes production of a rAAV vector, as such a vector contained
within a rAAV particle.
[0084] The term "packaging" as used herein can refer to a series of
intracellular events that can result in
the assembly and encapsidation of a rAAV particle.
[0085] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding
replication and
encapsidation proteins of adeno-associated virus. AAV rep and cap are referred
to herein as AAV
packaging genes."
[0086] The term "polypeptide" can encompass both naturally occurring and non-
naturally occurring
proteins (e.g., a fusion protein), peptides, fragments, mutants, derivatives
and analogs thereof A
polypeptide may be monomeric, dimeric, trimeric, or polymeric. Further, a
polypeptide may comprise a
number of different domains, each of which has one or more distinct
activities. For the avoidance of
doubt, a "polypeptide" may be any length greater two amino acids.
[0087] As used herein, "polypeptide variant" or simply "variant" refers to a
polypeptide whose sequence
contains an amino acid modification. In some embodiments, the modification is
an insertion, duplication,
deletion, rearrangement or substitution of one or more amino acids compared to
the amino acid sequence
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of a reference protein or polypeptide, such as a native or wild type protein.
A variant may have one or
more amino acid point substitutions, in which a single amino acid at a
position has been changed to
another amino acid, one or more insertions and/or deletions, in which one or
more amino acids are
inserted or deleted, respectively, in the sequence of the reference protein,
and/or truncations of the amino
acid sequence at either or both the amino or carboxy termini. A variant can
have the same or a different
biological activity compared to the reference protein, or the unmodified
protein.
[0088] In some embodiments, a variant can have, for example, at least about
80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence homology to its
counterpart reference
protein. In some embodiments, a variant can have at least about 90% overall
sequence homology to the
wild-type protein. In some embodiments, a variant exhibits at least about 95%,
at least about 98%, at least
about 99%, at least about 99_5%, or at least about 99.9% overall sequence
identity.
[0089] As used herein, "recombinant" can refer to a biomolecule, e.g., a gene
or protein, that (1) has
been removed from its naturally occurring environment, (2) is not associated
with all or a portion of a
polynucleotide in which the gene is found in nature, (3) is operatively linked
to a polynucleotide which it
is not linked to in nature, or (4) does not occur in nature. The term
"recombinant" can be used in reference
to cloned DNA isolates, chemically synthesized polynucleotide analogs, or
polynucleotide analogs that
are biologically synthesized by heterologous systems, as well as proteins
and/or mRNAs encoded by such
nucleic acids. Thus, for example, a protein synthesized by a microorganism is
recombinant, for example,
if it is synthesized from an mRNA synthesized from a recombinant gene present
in the cell.
[0090] The term "anti-VEGF agent" includes any therapeutic agent, including
proteins, polypeptides,
peptides, fusion protein, multimeric proteins, gene products, antibody, human
monoclonal antibody,
antibody fragment, aptamer, small molecule, kinase inhibitor, receptor or
receptor fragment, or nucleic
acid molecule, that can reduce, interfere with, disrupt, block and/or inhibit
the activity or function of an
endogenous VEGF and/or an endogenous VEGF receptor (VEGFR), or the VEGF-VEGFR
interaction or
pathway in vivo. An anti-VEGF agent can be any one of the known therapeutic
agents that can reduce new
blood vessel growth or formation and/or oedem, or swelling, when delivered
into a cell, tissue, or a
subject in vivo, e.g., ranibizurnab, brolucizumab, or bevacizumab. hi some
embodiments, an anti-VEGF
agent can be naturally occurring, non-naturally occurring, or synthetic. In
some embodiments, an anti-
VEGF agent can be derived from a naturally occurring molecule that was
subsequently modified or
mutated to confer an anti- VEGF activity. In some embodiments, an anti-VEGF
agent is a fusion or
chimeric protein. In such proteins, functional domains or polypeptides are
artificially fused to a moiety or
a polypeptide to make a fusion or chimeric protein that can sequester VEGF in
vivo or function as a
VEGFR decoy. In some embodiments, an anti-VEGF agent is a fusion or chimeric
protein that blocks
endogenous VEGFR from interacting with its ligands.
100911 As used herein, "VEGF" can refer to any isoform of VEGF, unless
required otherwise,
including, but not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F,
or any
combination, or any fitnctional fragment or variant thereof Unless required
otherwise, "VEGF" can refer
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to any member of the VEGF family, including members: VEGF-A, placenta growth
factor (PGF), VEGF-
B, VEGF-C, and VEGF-D, or any combination, functional fragment, or variant
thereof As used herein,
"VEGF receptor" or "VEGFR" or "VEGF-R" can be used to refer to any one of the
receptors of VEGF,
including, but not limited to, VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1/KDR), and
VEGFR-3 (or Flt-4).
VEGFR can be a membrane bound or soluble form, or a functional fragment or
truncation of a receptor.
Examples of anti-VEGF agents include, but are not limited to, ranibizumab,
bevacizumab, broluciziunab,
or any combination, variant, or functional fragment thereof
[0092] "Operatively linked" or "operably linked" or "coupled" can refer to a
juxtaposition of genetic
elements, wherein the elements are in a relationship permitting them to
operate in an expected manner.
For instance, a promoter can be operatively linked to a coding region if the
promoter helps initiate
transcription of the coding sequence. There may be intervening residues
between the promoter and coding
region so long as this functional relationship is maintained.
[0093] The term "expression vector" or "expression construct" or "cassette" or
"plasmid" or simply
"vector" can include any type of genetic construct, including AAV or rAAV
vectors, containing a nucleic
acid Of polynucleotide coding for a gene product in which part or all of the
nucleic acid encoding
sequence is capable of being transcribed and is adapted for gene therapy. The
transcript can be translated
into a protein. In some embodiments, the transcript is partially translated or
not translated. In certain
aspects, expression includes both transcription of a gene and translation of
mRNA into a gene product. In
other aspects, expression only includes transcription of the nucleic acid
encoding genes of interest. An
expression vector can also comprise control elements operatively linked to the
encoding region to
facilitate expression of the protein in target cells. The combination of
control elements and a gene or
genes to which they are operably linked for expression can sometimes be
referred to as an "expression
cassette," a large number of which are known and available in the art or can
be readily constructed from
components that are available in the art.
[0094] The term "heterologous" can refer to an entity that is genotypically
distinct from that of the rest
of the entity to which it is being compared. For example, a polynucleotide
introduced by genetic
engineering techniques into a plasmid or vector derived from a different
species can be a heterologous
polynucleotide. A promoter removed from its native coding sequence and
operatively linked to a coding
sequence with which it is not naturally found linked can be a heterologous
promoter.
[0095] As used herein, "7m8" refers to the amino acid sequence LALGETTRPA (SEQ
ID NO: 1).
[0096] "7m8 variant" refers to a rAAV, which can be of any serotype, with the
amino acid sequence
LALGETTRPA (SEQ ID NO: 1) inserted in the solvent exposed GH loop of the
capsid protein.
[0097] When 7m8 is inserted in a rAAV2 (also referred to as AAV2.7m8), the
amino acid sequence
LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 570-
611 of the AAV2
capsid protein, e.g., between positions 587 and 588 of the AAV2 capsid
protein, VP!. In some cases,
when 71118 is inserted in a rAAV2 (also referred to as AAV2.7m8), the amino
acid sequence
LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop of the AAV2 capsid
protein, e.g., between
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positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13.
When 7m8 is inserted
in a rAAV I (also referred to as AAV1.7m8), the amino acid sequence LALGETTRPA
(SEQ ID NO: 1) is
inserted into the GH loop within amino acids 571-612 of the AAV1 capsid
protein, e.g., between amino
acids 590 and 591 of the AAV1 capsid protein. When 7m8 is inserted in a rAAV5
(also referred to as
AAV5.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into
the Gil loop
within amino acids 560-601 of the AAV5 capsid protein, e.g., between amino
acids 575 and 576 of the
AAV5 capsid protein. When 7m8 is inserted in a rAAV6 (also referred to as
AAV6.7m8), the amino acid
sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino
acids 571 to 612 of
the AAV6 capsid protein, e.g., between amino acids 590 and 591 of the AAV6
capsid protein. When 7m8
is inserted in a rAAV7 (also referred to as AAV7.7m8), the amino acid sequence
LALGETTRPA (SEQ
ID NO: 1) is inserted into the GH loop within amino acids 572 to 613 of the
AAV7 capsid protein, e.g.,
between amino acids 589 and 590 of the AAV7 capsid protein. When 7m8 is
inserted in a rAAV8 (also
referred to as AAV8.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is
inserted into the
GH loop within amino acids 573 to 614 of the AAV8 capsid protein, e.g.,
between amino acids 590 and
591 of the AAV8 capsid protein. When 7m8 is inserted in a rAAV9 (also referred
to as AAV9.7m8), the
amino acid sequence LALGETTRPA (SEQ NO: 1) is inserted into the (WI loop of
the AAV9 capsid
protein, e.g., between amino acids 588 and 589 of the AAV9 capsid protein.
When 7m8 is inserted in a
rAAV10 (also referred to as AAV10.7m8), the amino acid sequence LALGETTRPA
(SEQ II) NO: 1) is
inserted into the GH loop within amino acids 573 to 614 of the AAV10 capsid
protein, e.g., between
amino acids 589 and 590 of the AAV10 capsid protein.
Overview
[0098] Current therapies (e.g., aflibercept recombinant protein, ranibiztunab
recombinant protein) for
ocular neovascular diseases such as wAMD require lifelong PIT administration
approximately every 4-8
weeks. This can increase the risk of inflammation, infection, and other
adverse effects in some patients.
Further, current therapies create compliance challenges due to repeated and/or
frequent trips to medical
offices for administration of the therapy, especially in elderly patients, who
are most affected with
wAMD. Reduction in frequency of administration is associated with vision loss
and deterioration of the
eye disease or condition. The ability of AAV vectors to efficiently transduce
target retinal cells following
IVT injection has been exploited to successfully transfer therapeutic genes
into photoreceptors, retinal
pigment epithelium, and the inner retina to treat a variety of retinal
diseases. Thus, administration of
rAAV particles encoding an anti-VEGF agent (e.g., aflibercept) can provide
prolonged and/or sustained
release of the anti-VEGF agent in vivo.
[0099] Surprisingly, administration of a single low unit dose of 6 x 10"
vector genomes (vg) per eye of
rAAV particles encoding aflibercept to the eyes of individuals with an ocular
neovascular disease led to
stabilization of the disease and a robust anatomical response in all treated
individuals (See Example 1). In
addition, visual acuity was stabilized in all treated individuals and none of
the individuals required rescue
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anti-VEGF treatment (e.g., aflibercept IVT injections) after administration of
the single low unit dose of 6
x loll vg/eye of rAAV particles encoding aflibercept. Moreover, administration
of the single unit dose of
rAAV particles encoding aflibercept to the eyes of individuals with an ocular
neovascular disease
unexpectedly caused a reduction (e.g., resolution) of symptoms, including
intraretinal and subretinal fluid
that were refractory to prior anti-VEGF treatments (e.g., chronic WT
injections of aflibercept,
ranibizumab, or bevaciziunab).
[0100] Accordingly, the present disclosure provides methods of treating an
ocular neovascular disease
in an individual by administering a single unit dose of 6 x 1011 vg/eye or
less of rAAV particles encoding
an anti-VEGF agent (e.g., aflibercept). In addition, the present disclosure
provides methods for reducing
retinal fluid in the eye of an individual with an ocular neovascular disease
by administering a single unit
dose of rAAV particles encoding an anti-VEGF agent (e.g., aflibercept). The
methods disclosed herein
reduce or eliminate the need for repeated IVT injections while providing long-
term efficacy, thereby
addressing the non-compliance and non-adherence problem. In addition, the
methods provided herein
reduce the adverse effects associated with multiple IVT injections.
Methods of Treatment
[0101] Provided herein is a method for treating an ocular neovascular disease
in an individual, the
method comprising administering a unit dose of recombinant adeno-associated
virus (rAAV) particles to
an eye of the individual.
[0102] Also provided herein is a method for reducing retinal fluid in the eye
of an individual with an
ocular neovascular disease, the method comprising administering a unit dose of
rAAV particles to an eye
of the individual.
[0103] Also provided herein is a method for treating an ocular neovascular
disease in an individual, the
method comprising administering an anti-VEGF agent (e.g., aflibercept) to an
eye of the individual, and
administering a unit dose of recombinant adeno-associated virus (rAAV)
particles to the eye of the
individual after administration of the anti-VEGF agent.
[0104] In some embodiments, the ocular neovascular disease is wet age-related
macular degeneration
(wAMD), retinal neovascularization, choroidal neovascularization diabetic
retinopathy, proliferative
diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion,
branched retinal vein occlusion,
diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy,
diabetic retinal edema, or any
combination thereof
[0105] In some embodiments, the term ocular neovascular disease also
encompasses VEGF-driven pre-
neovascular diseases which, if left untreated, progress to a neovascular form.
In some embodiments, the
ocular neovascular disease is the pre-neovascular disease, non-proliferative
diabetic retinopathy.
[0106] In some embodiments, the individual is a human. In some embodiments,
the individual received
at least one prior treatment (e.g., at least one, at least two, at least
three, at least four, at least 5 or more
treatments) for the ocular neovascular disease with an anti-VEGF agent (e.g.,
bevacizmnab,
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brolucizumab, ranibizutnab, faricimab, abicipar pegol, conbercept, OPT-302,
KSI-301, injectable
sunitinib maleate (GB-102), PAN-90806 (PanOptica), and/or aflibercept) in
about the last 8 weeks, about
the last 9 weeks, about the last 10 weeks, about the last 11 weeks, about the
last 12 weeks, about the last
13 weeks, about the last 14 weeks, about the last 15 weeks, or about the last
16 weeks prior to
administration of the unit dose of MAY particles. In some embodiments, the
individual demonstrated a
meaningful response to a prior treatment with anti-VEGF agent (e.g.,
bevacizwnab, broluciztunab,
ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301,
injectable sunitinib maleate (GB-
102), PAN-90806 (PanOptica), and/or aflibercept). In some embodiments, the
anti-VEGF agent is
aflibercept, a functional variant thereof, or a functional fragment thereof.
In some embodiments, the anti-
VEGF agent comprises a polypeptide comprising an amino acid sequence with at
least about 95% identity
to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the retinal
fluid in the eye of an
individual is intraretinal fluid (IRF) and/or subretinal fluid (SRF). In some
embodiments, the amount or
presence of retinal fluid in the eye of the individual is refractory to prior
treatment with an anti-VEGF
agent (e.g., bevacizumab, brolucizturtab, ranibiztunab, faricimab, abicipar
pegol, conbercept, OPT-302,
KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and/or
aflibercept). In some
embodiments, the anti -VEGF agent is aflibercept, a ftmctional variant
thereof, or a functional fragment
thereof In some embodiments, the anti-VEGF agent comprises a polypeptide
comprising an amino acid
sequence with at least about 95% identity to the amino acid sequence of SEQ ID
NO: 35.
101071 In some embodiments, the ocular neovascular disease is diabetic macular
edema (DME). In some
embodiments, the individual is a human. In some embodiments, the individual
has type 1 or type 2
diabetes mellitus. In some embodiments, the individual has vision impairment
that is due to center
involving diabetic macular edema. In some embodiments, the individual has
visual acuity (BCVA) of
between about 78 to 50 ETDRS letters (e.g., any of 50, 51, 52, 53 54, 55, 56,
57, 58, 59, 60, 61,62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78 ETDRS letters)
in the eye administered the
rAAV particles prior to administration of the unit dose of rAAV particles. In
some embodiments, the
individual has visual acuity (Snellen equivalent) of between about 20/32 to
about 20/100 in the eye
administered the rAAV particles prior to administration of the unit dose of
rAAV particles. In some
embodiments, the individual has a central subfield thickness (CST) of? 325um
using Heidelberg
Spectralis with center-involving IRF (center 1 mm) in the eye administered
the rAAV particles prior to
administration of the unit dose of MAY particles. In some embodiments, the
individual has a decrease in
vision in the eye administered the rAAV particles prior to administration of
the unit dose of rAAV
particles that is primarily due to diabetic macular edema. In some
embodiments, the individual was
diagnosed with diabetic macular edema in the eye administered the rAAV
particles about 6 months or less
prior to administration of the unit dose of rAAV particles, e.g., any of about
6 months, about 5 months,
about 4 months, about 3 months, about 2 months, about 1 month, or less, prior
to administration of the
unit dose of rAAV particles. In some embodiments, the individual received 0,
1, or 2 prior treatments for
DME in the eye administered the rAAV particles, e.g., 0, 1, or 2 intravitreal
injections with an anti-VEGF
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agent, e.g., aflibercept, prior to administration of the unit dose of rAAV
particles. In some embodiments,
the individual received the prior treatments with an anti-VEGF agent in the
eye administered the rAAV
particles at least about 60 days (i.e., about 2 months) prior to
administration of the unit dose of rAAV
particles. In some embodiments, the individual exhibited a meaningful response
in central subfield
thickness to the prior treatments with an anti-VEGF agent in the eye
administered the rAAV particles
prior to administration of the unit dose of rAAV particles, for example, at
least a 10% reduction in central
subfield thickness. In some embodiments, the individual did not experience an
adverse reaction to the
prior treatments with an anti-VEGF agent prior to administration of the unit
dose of rAAV particles. In
some embodiments, the individual does not have neutralizing antibodies to
AAV2.7m8 prior to
administration of the unit dose of rAAV particles. In some embodiments, the
individual does not have an
anti-AAV2.7m8 neutralizing antibody titer of greater than 1:125 prior to
administration of the unit dose of
rAAV particles, e.g., within about 6 months prior to administration of the
unit dose of rAAV particles. In
some embodiments, the individual does not have a history of allergy to
aflibercept, corticosteroids, or
fluorescein dye or sodium fluorescein (e.g., used in angiography) prior to
administration of the unit dose
of rAAV particles. In some embodiments, the individual has a history of mild
allergy to aflibercept,
corticosteroid, or fluorescein dye or sodium fluorescein (e.g., used in
angiography) prior to administiation
of the unit dose of rAAV particles, wherein the allergy is amenable to
treatment. In some embodiments,
the individual does not have uncontrolled diabetes, e.g., HbAlC of greater
than 10%, prior to
administration of the unit dose of rAAV particles. In some embodiments, the
individual does not have a
history of diabetic ketoacidosis within about 3 months prior to administration
of the unit dose of rAAV
particles. In some embodiments, the individual has not initiated intensive
insulin treatment, e.g., with an
insulin pump or multiple daily insulin injections, prior to administration of
the unit dose of rAAV
particles. In some embodiments, the individual does not plan to initiate
intensive insulin treatment, e.g.,
with an insulin pump or multiple daily insulin injections, within about 3
months after administration of the
unit dose of rAAV particles. In some embodiments, the individual does not have
a history of systemic
autoimmune disease that requires treatment with systemic steroids or
immunosuppressive treatments, e.g.,
methotrexate or adalimumab, prior to administration of the unit dose of rAAV
particles. In some
embodiments, the individual is not being administered a systemic drug known to
cause macular edema,
such as fingolimod, tamoxifen, chloroquine, or hydroxychloroquine, prior to
administration of the unit
dose of rAAV particles. In some embodiments, the individual is not being
administered a systemic anti-
VEGF treatment prior to administration of the unit dose of rAAV particles. In
some embodiments, the
individual does not have high-risk proliferative diabetic retinopathy (PDR) in
the eye administered the
rAAV particles prior to administration of the unit dose of rAAV particles. In
some embodiments, PDR is
defined as any vitreous or preretinal hemorrhage, neovascularization elsewhere
>1/2-disc area within an
area equivalent to standard ETDRS 7-field on clinical examination, or
neovascularization of disc > 1/3-
disc area on clinical examination. In some embodiments, the individual does
not have focal or grid laser
photocoagulation in the eye administered the rAAV particles prior to
administration of the unit dose of
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rAAV particles. In some embodiments, the individual does not have any prior
pan retinal
photocoagulation (PRP) in the eye administered the rAAV particles prior to
administration of the unit
dose of rAAV particles. In some embodiments, the individual has not received
an anti-VEGF therapy
(e.g., aflibercept IVT injections) in the eye administered the rAAV particles
prior to administration of the
unit dose of rAAV particles. In some embodiments, the individual has not
received an anti-VEGF therapy
(e.g., aflibercept IVT injections) in the eye administered the rAAV particles
for at least 60 days prior to
administration of the unit dose of rAAV particles. In some embodiments, the
individual has not received
more than two anti-VEGF treatments (e.g., aflibercept IVT injections) in the
eye administered the rAAV
particles prior to administration of the unit dose of rAAV particles. In some
embodiments, the individual
does not have a history of any of anterior segment neovascularization (e.g.,
neovascularization of the iris
[NVI] or neovascular glaucoma [WC-ft significant vitreous hemorrhage,
fibrovascular proliferation, or
tractional retinal detachment in the eye administered the rAAV particles prior
to administration of the unit
dose of rAAV particles. In some embodiments, the individual does not have
structural abnormalities at the
fovea (e.g., any of dense hard exudates, pigment abnormalities, foveal
atrophy, vitreomacular traction or
epiretinal membrane) in the eye administered the rAAV particles that
contribute to macular edema or
visual impairment prior to administration of the unit dose of rAAV particles.
In some embodiments,
structural abnormalities at the fovea are assessed on clinical examination or
OCT. In some embodiments,
the individual does not have a history of retinal disease other than diabetic
retinopathy (e.g., age-related
macular degeneration (in either eye), retinal vein occlusion, retinal arterial
occlusion, or pathologic
myopia) in the eye administered the rAAV particles prior to administration of
the unit dose of rAAV
particles. In some embodiments, the individual does not have history of ocular
disease other than diabetic
macular edema in the eye administered the rAAV particles, es., a significant
cataract or macular traction,
or evidence of posterior subcapsular cataract, prior to administration of the
unit dose of rAAV particles.
In some embodiments, the individual does not have history of cataract
extraction or Yttrium Aluminum
Garnet (YAG) capsulotomy in the eye administered the rAAV particles within at
least about 3 months
prior to administration of the unit dose of rAAV particles. In some
embodiments, the individual does not
have a history of retinal detachment (with or without repair) in the eye
administered the rAAV particles
prior to administration of the unit dose of rAAV particles. In some
embodiments, the individual does not
have a history of any of trabeculectomy, glaucoma shunt, or minimally invasive
glaucoma surgery
(MIGS) in the eye administered the rAAV particles prior to administration of
the unit dose of rAAV
particles. In some embodiments, the individual does not have a history of
vitrectomy or other filtration
surgery in the eye administered the rAAV particles prior to administration of
the unit dose of rAAV
particles. In some embodiments, the individual does not have aphalcia or
presence of an anterior chamber
intraocular lens in the eye administered the rAAV particles prior to
administration of the unit dose of
rAAV particles. In some embodiments, the individual does not have uncontrolled
ocular hypertension or
glaucoma in the eye administered the rAAV particles, e.g., IOP >22 mmHg
despite treatment with anti-
glaucoma medication or current use of >2 IOP lowering medications, prior to
administration of the unit
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dose of rAAV particles. In some embodiments, the individual does not have a
history of intraocular or
periocular steroid treatment for any ocular condition (e.g., IVT Triesence,
Iluvien or Ozurdex) in the eye
administered the rAAV particles prior to administration of the unit dose of
rAAV particles. In some
embodiments, the individual has not had refractive surgery in the eye
administered the rAAV particles
within at least about 90 days prior to administration of the unit dose of rAAV
particles. In some
embodiments, the individual does not have previous penetrating keratoplasty,
endothelial keratoplasty, or
ocular radiation in the eye administered the rAAV particles prior to
administration of the unit dose of
rAAV particles. In some embodiments, the individual has not had any prior
vitreoretinal surgery in the
eye administered the rAAV particles prior to administration of the unit dose
of rAAV particles. In some
embodiments, the individual does not have a history of uveitis or intraocular
inflammation, e.g., grade
trace or above except mild anticipated post-operative inflammation that
resolved, prior to administration
of the unit dose of rAAV particles. In some embodiments, the individual does
not have a history of IOP
elevation that is related to topical steroid administration prior to
administration of the unit dose of rAAV
particles. In some embodiments, the individual does not have a history of
ocular Herpes Simplex Virus
(HSV), Varicella-zoster virus (VZV), or Cytomegalovims (CMV), including viral
uveitis, retinitis or
keratitis prior to administration of the unit dose of rAAV particles. In some
embodiments, the individual
does not have evidence of any of external ocular infection, including
conjunctivitis, chalazion, or
significant blepharitis prior to administration of the unit dose of rAAV
particles. In some embodiments,
the individual does not have history of ocular toxoplasmosis prior to
administration of the unit dose of
rAAV particles.
[0108] In some embodiments, the unit dose is expressed as the number of vector
genomes (vg). In some
embodiments, the unit dose is about 6 x 10" vector genomes (vg) or less of the
rAAV particles. In some
embodiments, the unit dose is expressed as the number of vector genomes (vg)
per eye (vg/eye). In some
embodiments, the unit dose is about 6 x 10" vg/eye or less of the rAAV
particles. In some embodiments,
the unit dose of rAAV particles is about 6 x 1010 to about 2 x loll vg/eye. In
some embodiments, the unit
dose of rAAV particles is about 2 x 10" or about 6 x 1010 vg/eye.
[0109] In some embodiments, the unit dose of rAAV particles is administered to
one eye of the
individual. In some embodiments, the one eye of the individual is the right
eye or the left eye. In some
embodiments, the one eye of the individual is the right eye. In some
embodiments, the one eye of the
individual is the left eye. In some embodiments, the methods provided herein
further comprise
administering a unit dose of rAAV particles to the contralateral eye of the
individual. In some
embodiments, the one eye of the individual is the right eye and the
contralateral eye is the left eye_ In
some embodiments, the one eye of the individual is the left eye and the
contralateral eye is the right eye.
[0110] In some embodiments, the administering the unit dose of rAAV particles
to the contralateral eye
of the individual is up to about 2 weeks (e.g., about 0 days, I day, 2 days, 3
days, 4 days, 5 days, 6 days, 7
days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days) after
administering the unit dose of
rAAV particles to the one eye. In some embodiments, the unit dose of rAAV
particles administered to the
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contralateral eye of the individual is about the same as (e.g., less than 1%
higher or lower, less than 5%
higher or lower, less than 10% higher or lower, or less than 20% higher or
lower) or lower (e.g., about
5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about
70%, about 80%, or
about 90% lower) than the unit dose of rAAV particles administered to the one
eye of the individual.
[0111] In some embodiments, the administering the unit dose of rAAV particles
to the contralateral eye
is at least about 2 weeks (e.g., at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at
least about 1 month, at least about 2 months, at least about 3 months, at
least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9
months, at least about 10 months, at least about 11 months, at least about 12
months, at least about 1 year,
at least about 2 years, at least about 3 years, at least about 4 years, at
least about 5 years, or more) after
administering the unit dose of rAAV particles to the one eye. In some
embodiments, the unit dose of
rAAV particles administered to the contralateral eye of the individual is
higher (e.g., any of about 5%,
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,
about 80%, about
90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%,
about 250%, about
275%, about 300%, or more, higher) than the unit dose of rAAV particles
administered to the one eye of
the individual.
[0112] In some embodiments, the rAAV particles comprise a) a nucleic acid
encoding a polypeptide
comprising an amino acid sequence with at least about 95%, at least about 96%,
at least about 97%, at
least about 98%, at least about 99%, at least about 99.99%, or 100% identity
to the amino acid sequence
of SEQ 113 NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and b)
an AAV2 capsid
protein comprising an amino acid sequence LGETTRP (SEQ ID NO: 14) inserted
between positions 587
and 588 of the capsid protein, wherein the amino acid residue numbering
corresponds to an AAV2 VP1
capsid protein. The sequence of SEQ ID NO: 35 is provided below:
SDTGRPFVEMY SE IPEI I HMT EGRELVI PCRVTSPNITVTLKKFPLDTL I P DGKR I I WDSRKGF I
I SNATY
KEIGLLTCEATVNGHLY KTNY LT HRQTNT I I DVVLSPSHGIELSVGEKLVLNCTARTELNVGI DFNWEYPS
SKHQHKKLVNRDLKTQSGSEMKKFLSTLT I DGVTRSDQGLYTCAASSGLMTKKNST FVRVHEKDKTHTCPP
C PAPELLGGPSVFLFPPKPKDTLMI SRT PEVTCVVVDVS HE DPEVKFNWYVDGVEVHNAKTKPREEQYNST
Y RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
G FY PSDIAVEWE SNGQPENNY KTTPPVLDSDGS FFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPG (SEQ ID NO: 35)
[0113] In some embodiments, the rAAV particles comprise a) a nucleic acid
encoding a polypeptide
comprising an amino acid sequence with at least about 95% identity to the
amino acid sequence of SEQ
ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and b) an AAV2
capsid protein
comprising an amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between
positions 587 and 588
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of the capsid protein, wherein the amino acid residue numbering corresponds to
an AAV2 VP1 capsid
protein.
[0114] In some embodiments, the rAAV particles comprise a nucleic acid
encoding a polypeptide
comprising an amino acid sequence with at least about 80%, at least about 85%,
at least about 90%, at
least about 95%, at least about 96%, at least about 97%, at least about 98%,
at least about 99%, at least
about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35 and
flanked by AAV2
inverted terminal repeats (ITRs). In some embodiments, the rAAV particles
comprise a nucleic acid
encoding a polypeptide comprising an amino acid sequence with at least about
95% identity to the amino
acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats
(ITRs). In some
embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:
35. In some
embodiments, the polypeptide is aflibercept or a functional variant thereof or
functional fragment thereof
[0115] In some embodiments, the rAAV particles comprise a nucleic acid
comprising a codon-
optimized sequence encoding an amino acid sequence with at least about 80%, at
least about 85%, at least
about 90%, at least about 95%, at least about 96%, at least about 97%, at
least about 98%, at least about
99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ
ID NO: 35 and flanked
by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV
particles comprise a nucleic
acid comprising a codon-optimized sequence encoding an amino acid sequence
with at least about 95%
identity to the amino acid sequence of SEQ 113 NO: 35 and flanked by AAV2
inverted terminal repeats
(ITRs). In some embodiments, the rAAV particles comprise a nucleic acid
comprising a codon-optimized
sequence encoding an amino acid sequence with 100% identity to the amino acid
sequence of SEQ ID
NO: 35 and flanked by AAV2 inverted temlinal repeats (11Rs).
[0116] In some embodiments, the rAAV particles comprise a nucleic acid
comprising the cDNA
sequence of aflibercept or a functional variant thereof or functional fragment
thereof and flanked by
AAV2 inverted terminal repeats (Hits). In some embodiments, the rAAV particles
comprise a nucleic
acid comprising a codon-optimized cDNA sequence of aflibercept or a functional
variant thereof or
functional fragment thereof and flanked by AAV2 inverted terminal repeats
(ITRs). In some
embodiments, the rAAV particles comprise a nucleic acid comprising the nucleic
acid sequence of SEQ
ID NO: 36.
[0117] In some embodiments, the nucleic acid further comprises (a) a first
enhancer region comprising a
CMV sequence; (b) a promoter region comprising a CMV sequence; (c) a 5'UTR
region comprising, in
the 5' to 3' order, a TPL sequence and an eMLP sequence; (d) a second enhancer
region comprising a full
EES sequence; and (e) a HGH polyadenylation site. In some embodiments, the
enhancer region
comprising a CMV sequence comprises the sequence of SEQ ID NO: 22. In some
embodiments, the
promoter region comprising a CMV sequence comprises the sequence of SEQ ID NO:
23. In some
embodiments, the TPL sequence comprises the sequence of SEQ ID NO: 24. In some
embodiments, the
eMLP sequence comprises the sequence of SEQ ID NO: 25. In some embodiments,
the second enhancer
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region comprising a full EES sequence comprises the sequence of SEQ ID NO: 26.
In some embodiments,
the HGH polyadenylation site comprises the sequence of SEQ ID NO: 27.
101181 In some embodiments, the rAAV particles comprise an AAV2 capsid protein
comprising the
amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and
588 of the AAV2
VP1 comprising the sequence of SEQ ID NO: 13. The sequence of SEQ ID NO: 13 is
provided below:
MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKRAERHKDDSRGLVLPGYKYLGPFNGLD
KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ
AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD
SVPDPQPLGOPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHODSTWMGDRVI
TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI
NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSARQG
CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFFSQMLRTGNNFTFSYTFEDVPF
HSSYABSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG
PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL
IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGV
LPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTT
FSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVY
SEPRPIGTRYLTRNL (SEQ ID NO: 13)
101191 In some embodiments, the rAAV particles comprise an AAV2 capsid protein
comprising the
amino acid sequence LALGETTRPA (SEQ ID NO: I) inserted between positions 587
and 588 of the
capsid protein, wherein the amino acid residue numbering corresponds to an
AAV2 VP1 capsid protein.
In some embodiments, the rAAV particles comprise an AAV2 capsid protein
comprising the amino acid
sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of
the AAV2 VP1
comprising the sequence of SEQ ID NO: 13.
101201 In some embodiments, the rAAV particles comprise an AAV2 capsid protein
comprising any of
the following amino acid sequences inserted between positions 587 and 588 of
the capsid protein, wherein
the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein:
LALGETTRPA (SEQ ID
NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ
1D
NO: 4), KDTDITR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO:
7),
STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10),
LAAVDTTICFA (SEQ ED NO: 11), LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO:
14),
NETITRP (SEQ ID NO: 15), ICAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17),
KDTDTTR
(SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN
(SEQ
ID NO: 21). In some embodiments, the rAAV particles comprise an AAV2 capsid
protein comprising any
of the following amino acid sequences inserted between positions 587 and 588
of the AAV2 VP1
comprising the sequence of SEQ ID NO: 13: LALGETTRPA (SEQ ID NO: 1);
LANETITRPA (SEQ ID
NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAICDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID
NO:
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5), FtAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8),
LAICDTDITRA (SEQ ID NO: 9), LARAGGSVGA (SEQ 1D NO: 10), LAAVDTTKFA (SEQ ID NO:
11), LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO:
15),
ICAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18),
RAGGSVG (SEQ ID NO: 19), AVDTT1CF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO:
21).
101211 In some embodiments, the administration of the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual is by intravitreal (WT)
injection, intraocular
administration, or intraretina1 injection. In some embodiments, the
administration of the unit dose of
rAAV particles to the one eye and/or to the contralateral eye of the
individual is by intravitreal (IVT)
injection.
101221 In some embodiments, the unit dose of rAAV particles is in a
pharmaceutical fonnulation. In
some embodiments, the pharmaceutical formulation comprises the rAAV particles,
one or more osmotic
or ionic strength agents, one or more buffering agents, one or more
surfactants, and one or mom solvents.
In some embodiments, the osmotic or ionic strength agent is sodium chloride.
In some embodiments, the
one or more buffering agents are sodium phosphate monobasic and/or sodium
phosphate dibasic. In some
embodiments, the surfactant is Poloxamer 188. In some embodiments, the solvent
is water. In some
embodiments, the pharmaceutical formulation comprises the rAAV particles,
sodium chloride, sodium
phosphate and a surfactant. In some embodiments, the pharmaceutical
formulation comprises about
lx 101" vg/mL to about lx 10" vg/mL of rAAV particles. In some embodiments,
the pharmaceutical
formulation comprises about 6x 10" vg/mL to about 6x 1012 vg/mL of rAAV
particles. In some
embodiments, the pharmaceutical formulation comprises about 150 mM to about
200 mM sodium
chloride (e.g., any of about 150 mM, about 160 mM, about 170 mM, about 180 mM,
about 190 mM, or
about 200 mM). In some embodiments, the pharmaceutical formulation comprises
about 1 mM to about
mM monobasic sodium phosphate (e.g., about 1mM, about 2 mM, about 3 mM, about
4 mM, about 5
mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM). In some
embodiments, the
pharmaceutical formulation comprises about 1 mM to about 10 mM dibasic sodium
phosphate (e.g., about
1mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM,
about 8 mM, about
9 mM, or about 10 mM). ht some embodiments, the pharmaceutical formulation
comprises about
0.0005% (w/v) to about 0.005% (w/v) poloxamer 188 (e.g., any of about 0.0005%
(w/v), 0.0006% (w/v) ,
0.0007% (w/v) , 0.0008% (w/v) , 0.0009% (w/v), 0.001% (w/v) 0.002% (w/v) ,
0.003% (w/v) , 0.004%
(w/v), or about 0.005% (w/v)). In some embodiments, the pharmaceutical
formulation has a pH of about
7.0 to about 7.5 (e.g., any of about 71), about 7,1, about 7.2, about 7.3,
about 7.4, or about 7.5). In some
embodiments, the pharmaceutical formulation comprises about 6x 1012 vg/mL of
rAAV particles, about
180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM
dibasic sodium
phosphate, and about 0.001% (w/v) poloxamer 188, wherein the pharmaceutical
formulation has a pH of
about 7.3. In some embodiments, the pharmaceutical formulation comprises about
6x10" vg/mL of rAAV
particles, about 180 mM sodium chloride, about 5 mM monobasic sodium
phosphate, about 5 mM dibasic
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sodium phosphate, and about 0.001% (w/v) poloxamer 188, wherein the
pharmaceutical formulation has a
pH of about 7.3.
[0123] In some embodiments, the unit dose of rAAV particles comprises a volume
of between about 25
pL to about 250 pL (e.g., any of about 25 pL, about 30 jiL, about 40 gL, about
50 pL, about 60 pL, about
70 jilt about 80 pL, about 90 ILL, about 100 gL, about 110 pL, about 120 pL,
about 130 gL, about 140
gL, about 150 pL, about 160 L, about 170 L, about 180 gL, about 190 pL,
about 200 pL, about 210 pL,
about 220 itL, about 230 pL, about 240 pL, or about 250 itL). In some
embodiments, the concentration of
rAAV particles in the pharmaceutical formulation is adjusted such that the
volume of the unit dose of
rAAV particles administered to an eye of the individual is between about 25 gL
to about 250 L. In some
embodiments, the unit dose of rAAV particles comprises a volume of about 100
pL. In some
embodiments, the unit dose of rAAV particles comprises a volume of about 30
L.
[0124] In some embodiments, the unit dose of rAAV particles is administered in
combination with
steroid treatment. In some embodiments, the steroid treatment is a
corticosteroid treatment. In some
embodiments, the steroid treatment is a systemic steroid treatment. In some
embodiments, the steroid
treatment is an oral steroid treatment. In some embodiments, the steroid
treatment is a prednisone
treatment. In some embodiments, the steroid treatment is an ophthalmic steroid
treatment. In some
embodiments, the ophthalmic steroid treatment is a topical steroid treatment
(e.g., a drop), a periocular
steroid treatment (e.g., subtenons, subconjunctival), an intravitreal steroid
treatment, or a superchoroidal
steroid treatment. In some embodiments, the topical steroid treatment is a
difluprednate treatment, a
medrysone treatment, a loteprednol treatment, a prednisolone treatment, a
fluocinolone treatment, a
triamcinolone treatment, a rimexolone treatment, a dexamethasone treatment, a
fluorometholone
treatment, a fluocinolone treatment, a rimexolone treatment, or a prednisone
treatment. In some
embodiments, the topical steroid treatment is a difluprednate treatment. In
some embodiments, the steroid
treatment is administered before, during, and/or after administration of the
unit dose of rAAV particles. In
some embodiments, the steroid treatment is administered before administration
of the unit dose of rAAV
particles. In some embodiments, the steroid treatment is administered during
administration of the unit
dose of rAAV particles. In some embodiments, the steroid treatment is
administered after administration
of the unit dose of rAAV particles. In some embodiments, the steroid treatment
is administered before and
during administration of the unit dose of rAAV particles. In some embodiments,
the steroid treatment is
administered before and after administration of the unit dose of rAAV
particles. In some embodiments,
the steroid treatment is administered during, and after administration of the
unit dose of rAAV particles.
In some embodiments, the steroid treatment is administered before, during, and
after administration of the
unit dose of rAAV particles.
[0125] In some embodiments, the steroid treatment is an ophthalmic steroid
treatment (e.g.,
difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g.,
difluprednate) is a daily
steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks
from administering the unit
dose of rAAV particles. In some embodiments, the ophthalmic steroid treatment
comprises about four
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administrations of ophthalmic steroid on about week 1, about three
administrations of ophthalmic steroid
on about week 2, about two administrations of ophthalmic steroid on about week
3, and about one
administration of ophthalmic steroid on about week 4; timing starting with and
following administration
of the unit dose of rAAV particles. In some embodiments, the ophthalmic
steroid is about 0.005% to about
0.5% difluprednate. In some embodiments, the ophthalmic steroid is any of
about 0.005%, about 0.006%,
about 0.007%, about 01)08%, about 0.009%, about 0.01%, about 0.02%, about
0.03%, about 0.4%, about
0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1%
difluprednate. In some
embodiments, the ophthalmic steroid is difluprednate 0.05%. In some
embodiments, a dose of
difluprednate 0.05% is one drop of ophthalmic solution. In some embodiments,
one drop is about 50 pl
(e.g., about 25 gl to about 50 p.tl, about 50 gl to about 100 pi). In some
embodiments, a dose of
difluprednate comprises about 1 pg to about 5 pg, or about 2 pg to about 3 pg,
or about 2.5 pg
difluprednate. In some embodiments, a dose of difluprednate comprises about
2.5 pg difluprednate.
[0126] In some embodiments, the steroid treatment is an ophthalmic steroid
treatment (e.g.,
difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g.,
difluprednate) is a daily
topical steroid treatment for up to about 4 weeks, about 6 weeks, or about 8
weeks from administering the
unit dose of rAAV particles. In some embodiments, the topical steroid
treatment comprises about four
administrations of topical steroid on about week 1, about three
administrations of topical steroid on about
week 2, about two administrations of topical steroid on about week 3, and
about one administration of
topical steroid on about week 4; timing starting with and following
administration of the unit dose of
rAAV particles. In some embodiments, the topical steroid treatment comprises
about four administrations
of topical steroid (/. a, QID) per day for about 3 weeks after administration
of the unit dose of rAAV
particles, followed by about 3 administrations of topical steroid per day
(i.e., T1D) for about 1 week,
followed by about 2 administrations of topical steroid per day (i.e., BID) for
about 1 week, and followed
by about 1 administration of topical steroid per day (i.e., QD) for about 1
week. In some embodiments, the
topical steroid comprises difluprednate 0.05% at a dose of about 1pg to about
3 pg. In some
embodiments, the topical steroid comprises difluprednate 0.05% at a dose of
about 2.5pg. In some
embodiments, the topical steroid is about 0.005% to about 05% difluprednate.
In some embodiments, the
topical steroid is any of about 0.005%, about 0.006%, about 0.007%, about
0.008%, about 0.009%, about
0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about
0.07%, about 0.08%,
about 0.09%, or about 0.1% difluprednate. In some embodiments, the topical
steroid is difluprednate
0.05%. In some embodiments, a dose of difluprednate 0.05% is one drop of
ophthalmic solution. In some
embodiments, one drop is about 50 pl (e.g., about 25 pl to about 50 pl. about
50 pl to about 100 ply In
some embodiments, a dose of difluprednate comprises about 1 pg to about 5 pg,
or about 2 pig to about 3
pg, or about 2.5 pg difluprednate. In some embodiments, a dose of
difluprednate comprises about 2.5 pg
difluprednate.
[0127] In some embodiments, the retinal fluid in the eye of the individual
(e.g., SRF and/or IRE) is
reduced by more than any of about 5%, about 10%, about 15%, about 20%, about
30%, about 40%, about
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50%, about 60%, about 70%, about 80%, about 90%, or about 100% after
administration of the unit dose
of rAAV particles to the one eye and/or to the contralateral of the
individual. In some embodiments, the
retinal fluid in the eye of the individual (e.g., SRF and/or IRF) is reduced
by more than any of about 5%,
about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about
80%, about 90%, or about 100% after administration of the unit dose of rAAV
particles to the one eye
and/or to the contralateral of the individual compared to the level of retinal
fluid in the eye of the
individual prior to administration of the unit dose or rAAV particles. In some
embodiments, the retinal
fluid in the eye of the individual (e.g., SRF and/or IRF) is reduced by about
100% after administration of
the unit dose of rAAV particles to the one eye and/or to the contralateral of
the individual compared to the
level of retinal fluid in the eye of the individual prior to administration of
the unit dose or rAAV particles.
101281 In some embodiments, the methods provided herein further comprise
monitoring the level of
retinal fluid (e.g., SRF and/or IRF) in the one eye and/or the contralateral
eye of the individual after
administration of the unit dose of rAAV particles. In some embodiments, the
reduction of retinal fluid
(e.g., SRF and/or IRF) in the eye is first observed any of about 1 day, about
3 days, about 8 days, about 2
weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16
weeks, about 20 weeks,
about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40
weeks, about 44 weeks, about
48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks,
about 68 weeks, about 72
weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about
92 weeks, about 96
weeks, about 100 weeks, about 104 weeks, or more after administration of the
unit dose of rAAV
particles. In some embodiments, the reduction of retinal fluid (e.g., SRF
and/or IRF) in the eye continues
or is maintained for at least 1 week, at least 2 weeks, at least 4 weeks, at
least 6 weeks, at least 8 weeks, at
least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at
least 28 weeks, at least 32 weeks,
at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at
least 52 weeks, at least 56
weeks, at least 60 weeks, at least 64 weeks, at least 68 weeks, at least 72
weeks, at least 76 weeks, at least
80 weeks, at least 84 weeks, at least 88 weeks, at least 92 weeks, at least 96
weeks, at least 100 weeks, at
least 104 weeks, or more after administration of the unit dose of rAAV
particles.
101291 In some embodiments, the reduction of retinal fluid (e.g., SRF and/or
IRF) in the eye is
determined by any method known in the art. In some embodiments, the reduction
of retinal fluid (e.g.,
SRF and/or IRF) in the eye is determined by optical coherence tomography
(OCT), spectral domain OCT
(SD-OCT), OCT angiography, fluorescein angiography, or by direct retinal
observation. In some
embodiments, the reduction of retinal fluid (e.g., SRF and/or IRF) in the eye
is determined by optical
coherence tomography (OCT). In some embodiments, the reduction of retinal
fluid (ea., SRF and/or IRF)
in the eye is determined by spectral domain OCT (SD-OCT). In some embodiments,
the reduction of
retinal fluid (e.g., SRF and/or 1RF) in the eye is determined by OCT
angiography. In some embodiments,
the reduction of retinal fluid (e.g., SRF and/or IRF) in the eye is determined
by fluorescein angiography.
In some embodiments, the reduction of retinal fluid (e.g., SRF and/or IRF) in
the eye is determined by
direct retinal observation,
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101301 In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of rAAV particles to the one eye and/or the
contralateral eye is assessed
based on the level of retinal fluid (e.g., intraretinal fluid (IRF) and/or
subretinal fluid (SRF)) compared the
level of retinal fluid (e.g., SRF and/or HU) prior to administration of the
unit dose of rAAV particles to
the one eye and/or the contralateral eye (e.g., as described above). In some
embodiments, the retinal fluid
is subretinal fluid (SRF) or intraretinal fluid (1RF). In some embodiments,
the retinal fluid is subretinal
fluid (SRF). In some embodiments, the retinal fluid is intraretinal fluid
(IRF). In some embodiments,
treatment of an ocular neovascular disease in an individual after
administration of the unit dose of rAAV
particles to the one eye and/or the contralateral eye is determined if a
reduction in retinal fluid (e.g., IRF
and/or SRF) is observed after administration of the unit dose of rAAV
particles to the one eye and/or the
contralateral eye compared to the levels of retinal fluid (e.g., IRF and/or
SRF) prior to administration of
the unit dose of rAAV particles in the one eye and/or the contralateral eye
(e.g., as described above). In
some embodiments, the ocular neovascular disease is wAMD.
101311 In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in maintenance or a decrease
of retinal thickness compared
to the retinal thickness prior to administration of the unit dose of rAAV
particles. In some embodiments,
the administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a decrease of retinal thickness compared to the retinal
thickness prior to
administration of the unit dose of rAAV particles. In some embodiments,
retinal thickness is central
subfield thickness (CST) or central retinal thickness (CRT). In some
embodiments, the administering the
unit dose of rAAV particles to the one eye and/or to the contralateral eye of
the individual results in a
decrease of retinal thickness of more than any of about 5%, about 10%, about
15%, about 20%, about
25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about
60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%,
or about 100%
compared to the retinal thickness prior to administration of the unit dose of
rAAV particles. In some
embodiments, the retinal thickness (e.g., CST or CRT) is determined by OCT or
SD-OCT.
101321 In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in a decrease of central
retinal thickness (CRT) or central
subfield thickness (CST) of about 10 pm to about 100 pm (e.g., more than any
of about 10 p.m, about 15
p.m, about 20 pm, about 25 pm, about 30 pun, about 35 pm, about 40 pm, about
45 pm, about 50 pm,
about 55 tun, about 60 pm, about 65 gm, about 70 p.m, about 75 pm, about 80
pm, about 85 pm, about 90
tun, about 95 pm, about 100 pin, or more). In some embodiments, the
administering the unit dose of
rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a decrease of
central retinal thickness (CRT) or central subfield thickness (CST) of about 5
pm to about 50 pm (e.g.,
any of about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about
30 pm, about 35 pm,
about 40 gm, about 45 pm, or about 50 pm). In some embodiments, the
administering the unit dose of
rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a decrease of
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central retinal thickness (CRT) or central subfield thickness (CST) of about 5
p.m to about 40 pm (e.g.,
any of about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 p.m, about
30 pm, about 35 pm, or
about 40 pm). In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in a decrease of
central retinal thickness (CRT) or
central subfield thickness (CST) of about 5 pm to about 30 pm (e.g., any of
about 5 pm, about 6 pm,
about 7 pm, about 8 pm, about 9 pin, about 10 pm, about 11 pm, about 12 pm,
about 13 pm, about 14 pm,
about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm,
about 21 pm, about 22
pm, about 23 pm, about 24 pm, about 25 gm, about 26 pm, about 27 pm, about 28
pm, about 29 pm, or
about 30 pm). In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in a decrease of
central retinal thickness (C(T) or
central subfield thickness (CST) of about 5 pm to about 25 pm (e.g., any of
about 5 pm, about 6 pm,
about 7 pm, about 8 pm, about 9 pm, about 10 gm, about 11 pm, about 12 pm,
about 13 pm, about 14 inn,
about 15 pm, about 16 pm, about 17 gm, about 18 pm, about 19 pm, about 20 pm,
about 21 pm, about 22
pm, about 23 pm, about 24 pm, or about 25 gm). In some embodiments, the
administering the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a decrease of
central retinal thickness (CRT) or central subfield thickness (CST) of about 5
pm to about 20 pm (e.g.,
any of about 5 gm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10
pm, about 11 pm, about 12
pm, about 13 pm, about 14 pm, about 15 pun, about 16 pm, about 17 pm, about 18
pm, about 19 pm, or
about 20 pm). In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in a decrease of
central retinal thickness (CRT) or
central subfield thickness (CST) of between about 18 pm to about 75 pm. In
some embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a decrease of central retinal thickness (CRT) or central
subfield thickness (CST) of
about 18.5 pm. In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in a decrease of
central retinal thickness (CRT) or
central subfield thickness (CST) of about 21.0 pm. In some embodiments, the
administering the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a decrease of
central retinal thickness (CRT) or central subfield thickness (CST) of about
8.3 pm. In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a decrease of central retinal thickness (CR1)
or central subfield thickness
(CST) of about 25.5 pm. In some embodiments, the administering the unit dose
of rAAV particles to the
one eye and/or to the contralateral eye of the individual results in a
decrease of central retinal thickness
(CRT) or central subfield thickness (CST) of about 24.8 gm. In some
embodiments, the administering the
unit dose of rAAV particles to the one eye and/or to the contralateral eye of
the individual results in a
decrease of central retinal thickness (CRT) or central subfield thickness
(CST) of about 75 pm. In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a decrease of central retinal thickness (CRT)
or central subfield thickness
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(CST) of about 100 pm or more (e.g., any of about 100 pm or more, about 110 pm
or more, about 120 pm
or more, about 130 pm or more, about 140 pm or more, about 150 pm or more,
about 160 pm or more,
about 170 pm or more, about 180 pm or more, about 190 pm or more, or about 200
pm or more). In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a decrease of central retinal thickness (CRT)
or central subfield thickness
(CST) of between about 10 pm to about 200 pm (e.g., any of about 10 pin, about
20 pm, about 30 pm,
about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 gm, about 90 pm,
about 100 pm, about
110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm,
about 170 pm, about
180 pm, about 190 pm, or about 200 pm). In some embodiments, the administering
the unit dose of rAAV
particles to the one eye and/or to the contralateral eye of the individual
results in a decrease of central
retinal thickness (CRT) or central subfield thickness (CST) of between about
50 pm to about 200 pm
(e.g., any of about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 gm,
about 100 pm, about
110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm,
about 170 pm, about
180 pm, about 190 pm, or about 200 pin). In some embodiments, the
administering the unit dose of rAAV
particles to the one eye and/or to the contralateral eye of the individual
results in a decrease of central
retinal thickness (CRT) or central subfield thickness (CST) of between about
60 pm to about 200 pm
(e.g., any of about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100
pm, about 110 pm, about
120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm,
about 180 p.m, about
190 pm, or about 200 pm). In some embodiments, the administering the unit dose
of rAAV particles to the
one eye and/or to the contralateral eye of the individual results in a
decrease of central retinal thickness
(CRT) or central subfield thickness (CST) of between about 70 pm to about 200
pm (e.g., any of about 70
pm, about 80 pm, about 90 pm, about 100 p.m, about 110 pm, about 120 pm, about
130 pm, about 140
pm, about 150 pm, about 160 pm, about 170 gm, about 180 pm, about 190 pm, or
about 200 pm). In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a decrease of central retinal thickness (CRT)
or central subfield thickness
(CST) of between about 80 pm to about 200 pm (e.g., any of about 80 pm, about
90 pm, about 100 pm,
about 110 pm, about 120 pm, about 130 p.m, about 140 pm, about 150 pm, about
160 pm, about 170 pm,
about 180 pm, about 190 pm, or about 200 pm). In some embodiments, the
administering the unit dose of
rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a decrease of
central retinal thickness (CRT) or central subfield thickness (CST) of between
about 90 pm to about 200
pm (e.g., any of about 90 pm, about 100 gm, about 110 pm, about 120 pm, about
130 pin, about 140 pm,
about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, or about
200 pin). In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a decrease of central retinal thickness (CR1)
or central subfield thickness
(CST) of between about 100 pm to about 200 pm (e.g., any of about 100 pm,
about 110 pm, about 120
pm, about 130 pm, about 140 pin, about 150 pm, about 160 pin, about 170 pm,
about 180 gm, about 190
pm, or about 200 pm). In some embodiments, the administering the unit dose of
rAAV particles to the one
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eye and/or to the contralateral eye of the individual results in a decrease of
central retinal thickness (CRT)
or central subfield thickness (CST) of between about 110 pm to about 200 pm
(e.g., any of about 110 pm,
about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about
170 m, about 180 pm,
about 190 pm, or about 200 pm). In some embodiments, the administering the
unit dose of rAAV particles
to the one eye and/or to the contralateral eye of the individual results in a
decrease of central retinal
thickness (CRT) or central subfield thickness (CST) of between about 115 pm to
about 200 pm (e.g., any
of about 115 m, about 120 iun, about 125 p.m, about 130 pm, about 135 pm,
about 140 pm, about 145
pm, about 150 pm, about 155 pm, about 160 pm, about 165 pm, about 170 pm,
about 175 pm, about 180
pm, about 185 pm, about 190 pm, about 195 gm, or about 200 pm). In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a decrease of central retinal thickness (CRT) or central
subfield thickness (CST) of
between about 120 pm to about 200 pm (e.g., any of about 120 pin, about 125
turn, about 130 pm, about
135 pm, about 140 pm, about 145 pin, about 150 gm, about 155 pm, about 160 pm,
about 165 pm, about
170 tun, about 175 pm, about 180 pin, about 185 pm, about 190 pin, about 195
pm, or about 200 pm). In
some embodiments, the administering the unit dose of rAAV particles to the one
eye and/or to the
contralateral eye of the individual results in a decrease of central retinal
thickness (CRT) or central
subfield thickness (CST) of between about 125 pm to about 200 pm (e.g., any of
about 125 pm, about 130
pm, about 135 pin, about 140 pm, about 145 pm, about 150 pm, about 155 pm,
about 160 pm, about 165
pm, about 170 pm, about 175 pm, about 180 pm, about 185 inn, about 190 m,
about 195 gm, or about
200 pm). In some embodiments, the administering the unit dose of rAAV
particles to the one eye and/or
to the contralateral eye of the individual results in a decrease of central
retinal thickness (CRT) or central
subfield thickness (CST) of between about 130 pm to about 200 pm (e.g., any of
about 130 pin, about 135
pm, about 140 pm, about 145 pm, about 150 pm, about 155 iun, about 160 pm,
about 165 gm, about 170
gm, about 175 pm, about 180 pin, about 185 pin, about 190 pm, about 195 pm, or
about 200 pm). In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a decrease of central retinal thickness (CRT)
or central subfield thickness
(CST) of between about 135 pm to about 200 pm (e.g., any of about 135 pm,
about 140 pin, about 145
pm, about 150 pm, about 155 pm, about 160 pm, about 165 rn, about 170 m,
about 175 gm, about 180
gm, about 185 fam, about 190 pm, about 195 pm, or about 200 pm). In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a decrease of central retinal thickness (CRT) or central
subfield thickness (CST) of
between about 140 pm to about 200 pm (e.g., any of about 140 pm, about 145 pm,
about 150 pm, about
155 pm, about 160 pm, about 165 pm, about 170 pm, about 175 pm, about 180 gm,
about 185 pm, about
190 pin, about 195 pm, or about 200 pin). In some embodiments, the
administering the unit dose of rAAV
particles to the one eye and/or to the contralateral eye of the individual
results in a decrease of central
retinal thickness (CRT) or central subfield thickness (CST) of between about
145 pm to about 200 pm
(e.g., any of about 145 pm, about 150 pm, about 155 pm, about 160 pm, about
165 pm, about 170 pm,
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about 175 pm, about 180 pm, about 185 pm, about 190 pm, about 195 pm, or about
200 pm). In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a decrease of central retinal thickness (CRT)
or central subfield thickness
(CST) of between about 150 gm to about 200 pm (e.g., any of about 150 gm,
about 155 pm, about 160
pm, about 165 pm, about 170 pm, about 175 p.m, about 180 pm, about 185 tun,
about 190 pm, about 195
pm, or about 200 pm). In some embodiments, the administering the unit dose of
rAAV particles to the one
eye and/or to the contralateral eye of the individual results in a decrease of
central retinal thickness (CRT)
or central subfield thickness (CST) of between about 155 gm to about 200 pm
(e.g., any of about 155 pm,
about 160 pm, about 165 tun, about 170 pm, about 175 pm, about 180 pm, about
185 pm, about 190 pm,
about 195 pm, or about 200 pm). In some embodiments, the administering the
unit dose of rAAV particles
to the one eye and/or to the contralateral eye of the individual results in a
decrease of central retinal
thickness (CRT) or central subfield thickness (CST) of between about 160 pm to
about 200 pm (e.g., any
of about 160 pm, about 165 pm, about 170 pm, about 175 gm, about 180 pm, about
185 pm, about 190
pm, about 195 pm, or about 200 gm). In some embodiments, the administering the
unit dose of rAAV
particles to the one eye and/or to the contralateral eye of the individual
results in a decrease of central
retinal thickness (CRT) or central subfield thickness (CST) of between about
165 pm to about 200 pm
(e.g., any of about 165 pm, about 170 turn, about 175 pm, about 180 pm, about
185 pm, about 190 pm,
about 195 pm, or about 200 pm). In some embodiments, the administering the
unit dose of rAAV particles
to the one eye and/or to the contralateral eye of the individual results in a
decrease of central retinal
thickness (CRT) or central subfield thickness (CST) of between about 170 pm to
about 200 pin (e.g., any
of about 170 pm, about 175 tun, about 180 gm, about 185 pm, about 190 pm,
about 195 pm, or about 200
gm). In some embodiments, the administering the unit dose of rAAV particles to
the one eye and/or to the
contralateral eye of the individual results in a decrease of central retinal
thickness (CRT) or central
subfield thickness (CST) of between about 175 pm to about 200 pm (e.g., any of
about 175 pm, about 180
gm, about 185 pm, about 190 pm, about 195 pm, or about 200 pm). In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a decrease of central retinal thickness (CRT) or central
subfield thickness (CST) of
between about 180 pinto about 200 pm (e.g., any of about 180 pm, about 185 pm,
about 190 pin, about
195 pm, or about 200 pm). In some embodiments, the administering the unit dose
of rAAV particles to the
one eye and/or to the contralateral eye of the individual results in a
decrease of central retinal thickness
(CRT) or central subfield thickness (CST) of about 21.0 pm compared to the
central retinal thickness
(CRT) or central subfield thickness (CST) prior to administration of the unit
dose of rAAV particles. In
some embodiments, the administering the unit dose of rAAV particles to the one
eye and/or to the
contralateral eye of the individual results in a decrease of central retinal
thickness (CRT) or central
subfield thickness (CST) of about 8.3 pm compared to the central retinal
thickness (CRT) or central
subfield thickness (CST) prior to administration of the unit dose of rAAV
particles. In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
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eye of the individual results in a decrease of central retinal thickness (CR1)
or central subfield thickness
(CST) of about 26.2 p.m compared to the central retinal thickness (CRT) or
central subfield thickness
(CST) prior to administration of the unit dose of rAAV particles. In some
embodiments, the administering
the unit dose of rAAV particles to the one eye and/or to the contralateral eye
of the individual results in a
decrease of central retinal thickness (CRT) or central subfield thickness
(CST) of about 24.8 pm
compared to the central retinal thickness (CRT) or central subfield thickness
(CST) prior to administration
of the unit dose of rAAV particles. In some embodiments, the administering the
unit dose of rAAV
particles to the one eye and/or to the contralateral eye of the individual
results in a decrease of central
retinal thickness (CRT) or central subfield thickness (CST) of about 40.8 pm
compared to the central
retinal thickness (CRT) or central subfield thickness (CST) prior to
administration of the unit dose of
rAAV particles. In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in a decrease of
central retinal thickness (CRT) or
central subfield thickness (CST) of about 30_0 pm compared to the central
retinal thickness (CRT) or
central subfield thickness (CST) prior to administration of the unit dose of
rAAV particles. In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a decrease of central retinal thickness (CRT)
or central subfield thickness
(CST) of about 118.6 pm compared to the central retinal thickness (CRT) or
central subfield thickness
(CST) prior to administration of the unit dose of rAAV particles. In some
embodiments, the administering
the unit dose of rAAV particles to the one eye and/or to the contralateral eye
of the individual results in a
decrease of central retinal thickness (CRT) or central subfield thickness
(CST) of about 119.0 pm
compared to the central retinal thickness (CRT) or central subfield thickness
(CST) prior to administration
of the unit dose of rAAV particles. In some embodiments, the administering the
unit dose of rAAV
particles to the one eye and/or to the contralateral eye of the individual
results in a decrease of central
retinal thickness (CRT) or central subfield thickness (CST) of about 137.8 p.m
compared to the central
retinal thickness (CRT) or central subfield thickness (CST) prior to
administration of the unit dose of
rAAV particles. In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in a decrease of
central retinal thickness (CR1) or
central subfield thickness (CST) of about 152.7 gm compared to the central
retinal thickness (CRT) or
central subfield thickness (CST) prior to administration of the unit dose of
rAAV particles. In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a decrease of central retinal thickness (CRT)
or central subfield thickness
(CST) of about 153.3 pm compared to the central retinal thickness (CRT) or
central subfield thickness
(CST) prior to administration of the unit dose of rAAV particles. In some
embodiments, the administering
the unit dose of rAAV particles to the one eye and/or to the contralateral eye
of the individual results in a
decrease of central retinal thickness (CRT) or central subfield thickness
(CST) of about 149.8 pm
compared to the central retinal thickness (CRT) or central subfield thickness
(CST) prior to administration
of the unit dose of rAAV particles.
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101331 In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in a change in the central
retinal thickness (CRT) or central
subfield thickness (CST) of between about -200 [um to about +40 tun (e.g., any
about -200 jun, about -180
pm, about -160 gm, about -140 i.un, about -120 pm, about -100 pm, about -80
pm, about -60 pm, about -
40 pm, about -20 pm, about 0 pm, about +5 pm, about +10 pm, about +15 pm,
about +20 pm, about +25
pm, about +30 pm, about +35 pm, or about +40 pm) compared to the retinal
thickness prior to
administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a decrease in
the central retinal thickness (CRT) or central subfield thickness (CST) of any
of about 8 gm, about 11 pm,
about 16 pm, about 29 pm, about 33 pm, about 38 pm, about 55 um, about 61 pm,
or about 117 pm. In
some embodiments, the administering the unit dose of rAAV particles to the one
eye and/or to the
contralateral eye of the individual results in a decrease in the central
retinal thickness (CRT) or central
subfield thickness (CST) of any of about 27.8 pm or about 30.8 pm. In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in an increase in the central retinal thickness (CRT) or
central subfield thickness (CST)
of any of about 4 pm, about 12 pm, or about 32 pm. In some embodiments, the
administering the unit
dose of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a change
in the central retinal thickness (CRT) or central subfield thickness (CST) of
about -21.0 pm compared to
the central retinal thickness (CRT) or central subfield thickness (CST) prior
to administration of the unit
dose of rAAV particles. In some embodiments, the administering the unit dose
of rAAV particles to the
one eye and/or to the contralateral eye of the individual results in a change
in the central retinal thickness
(CRT) or central subfield thickness (CST) of about -8.3 pm compared to the
central retinal thickness
(CRT) or central subfield thickness (CST) prior to administration of the unit
dose of rAAV particles. In
some embodiments, the administering the unit dose of rAAV particles to the one
eye and/or to the
contralateral eye of the individual results in a change in the central retinal
thickness (CRT) or central
subfield thickness (CST) of about -26.2 pm compared to the central retinal
thickness (CRT) or central
subfield thickness (CST) prior to administration of the unit dose of rAAV
particles. In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a change in the central retinal thickness
(CRT) or central subfield thickness
(CST) of about -24.8 pm compared to the central retinal thickness (CRT) or
central subfield thickness
(CST) prior to administration of the unit dose of rAAV particles. In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a change in the central retinal thickness (CRT) or
central subfield thickness (CST) of
about -40.8 pm compared to the central retinal thickness (CRT) or central
subfield thickness (CST) prior
to administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit
dose of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a change
in the central retinal thickness (CRT) or central subfield thickness (CST) of
about -30.0 pm compared to
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the central retinal thickness (CRT) or central subfield thickness (CST) prior
to administration of the unit
dose of rAAV particles. In some embodiments, the administering the unit dose
of rAAV particles to the
one eye and/or to the contralateral eye of the individual results in a change
in the central retinal thickness
(CRT) or central subfield thickness (CST) of about -118.6 pm compared to the
central retinal thickness
(CRT) or central subfield thickness (CST) prior to administration of the unit
dose of rAAV particles. In
some embodiments, the administering the unit dose of rAAV particles to the one
eye and/or to the
contralateral eye of the individual results in a change in the central retinal
thickness (CRT) or central
subfield thickness (CST) of about -119.0 gm compared to the central retinal
thickness (CRT) or central
subfield thickness (CST) prior to administration of the unit dose of rAAV
particles. In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a change in the central retinal thickness
(CRT) or central subfield thickness
(CST) of about -137.8 pm compared to the central retinal thickness (CRT) or
central subfield thickness
(CST) prior to administration of the unit dose of rAAV particles. In some
embodiments, the administering
the unit dose of rAAV particles to the one eye and/or to the contralateral eye
of the individual results in a
change in the central retinal thickness (CRT) or central subfield thickness
(CST) of about -152.7 pin
compared to the central retinal thickness (CRT) or central subfield thickness
(CST) prior to administration
of the unit dose of rAAV particles. In some embodiments, the administering the
unit dose of rAAV
particles to the one eye and/or to the contralateral eye of the individual
results in a change in the central
retinal thickness (CRT) or central subfield thickness (CST) of about -153.3 pm
compared to the central
retinal thickness (CRT) or central subfield thickness (CST) prior to
administration of the unit dose of
rAAV particles. In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in a change in the
central retinal thickness (CRT) or
central subfield thickness (CST) of about -149.8 gm compared to the central
retinal thickness (CRT) or
central subfield thickness (CST) prior to administration of the unit dose of
rAAV particles.
101341 In some embodiments, the change (e.g., the decrease) in the central
retinal thickness (CRT) or
central subfield thickness (CST) compared to the central retinal thickness
(CRT) or central subfield
thickness (CST) prior to administration of the unit dose of rAAV particles is
present at any of about 1 day,
about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 16 weeks,
about 24 weeks, about 30
weeks, about 32 weeks, about 34 weeks, about 40 weeks, about 44 weeks, about
48 weeks, about 52
weeks, or more after administration of the unit dose of rAAV particles to the
one eye and/or to the
contralateral eye of the individual. In some embodiments, the change (e.g.,
the decrease) in the central
retinal thickness (CRT) or central subfield thickness (CST) compared to the
central retinal thickness
(CRT) or central subfield thickness (CST) prior to administration of the unit
dose of rAAV particles is
present at any of about 1 day, about 1 week, about 2 weeks, about 4 weeks,
about 8 weeks, about 12
weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about
32 weeks, about 36
weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about
56 weeks, about 60
weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about
80 weeks, about 84
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weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about
104 weeks, about 108
weeks, or more after administration of the unit dose of rAAV particles to the
one eye and/or to the
contralateral eye of the individual.
[0135] In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in a decrease of central
retinal thickness (CRT) or central
subfield thickness (CST) of more than any of about 5%, about 10%, about 15%,
about 20%, about 25%,
about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or
about 100% compared to
the retinal thickness prior to administration of the unit dose of rAAV
particles. In some embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a decrease of central retinal thickness (CRT) or central
subfield thickness (CST) of at
least about 10% compared to the retinal thickness prior to administration of
the unit dose of rAAV
particles. In some embodiments, the administering the unit dose of rAAV
particles to the one eye and/or
to the contralateral eye of the individual results in a decrease of central
retinal thickness (CRT) or central
subfield thickness (CST) of about 15% or more compared to the retinal
thickness prior to administration
of the unit dose of rAAV particles.
[0136] In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in an increase of central
retinal thickness (CRT) or central
subfield thickness (CST) of less than about 40 gm (e.g., any of less than
about 40 pm, less than about 35
pm, less than about 30 pm, less than about 25 pin, less than about 20 gm, less
than about 15 gin, less than
about 10 gm, less than about 5 gm, less than about 1 prn, or less) compared to
the retinal thickness prior to
administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in an increase of
central retinal thickness (CRT) or central subfield thickness (CST) of about
32 gm or less compared to the
retinal thickness prior to administration of the unit dose of rAAV particles.
[0137] In some embodiments, the maintenance, the decrease, or the increase of
retinal thickness
compared to the retinal thickness prior to administration of the unit dose of
rAAV particles is present at
about 30 weeks or more after administration of the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual. In some embodiments, the maintenance,
the decrease, or the
increase of retinal thickness compared to the retinal thickness prior to
administration of the unit dose of
rAAV particles is present at any of about 30 weeks, about 34 weeks, about 44
weeks, about 6 months,
about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years,
about 10 years, or more, after
administration of the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual.
[0138] In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in maintenance or a decrease
in macular volume compared to
the macular volume prior to administration of the unit dose of rAAV particles.
In some embodiments, the
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administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a decrease in macular volume compared to the macular
volume prior to
administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a decrease in
macular volume of more than any of about 5%, about 10%, about 15%, about 20%,
about 25%, about
30%, about 35%, about 40%, about 45%, or about 50% compared to the macular
volume prior to
administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a decrease in
macular volume of at least about 10% compared to the macular volume prior to
administration of the unit
dose of rAAV particles. In some embodiments, the macular volume is determined
by OCT or SD-OCT. In
some embodiments, the administering the unit dose of rAAV particles to the one
eye and/or to the
contralateral eye of the individual results in a decrease in macular volume of
at least about 10% compared
to the macular volume prior to administration of the unit dose of rAAV
particles. In some embodiments,
the administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a decrease in macular volume of about 15% or more
compared to the macular volume
prior to administration of the unit dose of rAAV particles. In some
embodiments, the macular volume is
determined by OCT or SD-OCT.
[0139] In some embodiments, the maintenance or the decrease in macular volume
compared to the
macular volume prior to administration of the unit dose of rAAV particles is
present at about 30 weeks or
more after administration of the unit dose of rAAV particles to the one eye
and/or to the contralateral eye
of the individual. In some embodiments, the maintenance or the decrease in
macular volume compared to
the macular volume prior to administration of the unit dose of rAAV particles
is present at any of about 30
weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5
years, about 2 years,
about 3 years, about 5 years, about 10y, or more after administration of the
unit dose of rAAV
particles to the one eye and/or to the contralateral eye of the individual.
[0140] In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in maintenance or an
improvement of visual acuity compared
to the visual acuity prior to administration of the unit dose of rAAV
particles. In some embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in an improvement of visual acuity compared to the visual
acuity prior to administration
of the unit dose of rAAV particles. In some embodiments, the administering the
unit dose of rAAV
particles to the one eye and/or to the contralateral eye of the individual
results in an improvement of visual
acuity of more than any of about 5%, about 10%, about 20%, about 30%, about
40%, about 50%, about
60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%,
about 175%, about
200%, about 225%, about 250%, about 275%, about 300%, or more, compared to the
visual acuity prior
to administration of the unit dose of rAAV particles. In some embodiments,
visual acuity is best corrected
visual acuity (BCVA). In some embodiments, the administering the unit dose of
rAAV particles to the one
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eye and/or to the contralateral eye of the individual results in an
improvement of BCVA compared to the
BCVA prior to administration of the unit dose of rAAV particles. In some
embodiments, BCVA is
expressed as an ETDRS score, which corresponds to the number of letters
correctly read (Vitale et al.,
(2016) JAMA Opthalmol 134(9):1041:1047).
[0141] In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in an improvement of BCVA of
at least 15 ETDRS letters
(Vitale etal., (2016) JAMA Opthalmol 134(9): 1041:1047) (e.g., at least about
15, at least about 20, at
least about 30, at least about 40, at least about 50, at least about 60, or
about 70 letters) compared to the
BCVA prior to administration of the unit dose of rAAV particles. In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in an improvement of BCVA of between about 1 to about 15
(e.g., any of about 1, about
2, about 3, about 4, about 5, about 6, about?, about 8, about 9, about 10,
about 11, about 12, about 13,
about 14, or about 15) ETDRS letters compared to the BCVA prior to
administration of the unit dose of
rAAV particles. In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in an improvement of
BCVA of about 5 ETDRS
letters compared to the BCVA prior to administration of the unit dose of rAAV
particles. In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in an improvement of BCVA of any of about 1,
about 2, about 3, about 4,
about 5, about 6, or about? ETDRS letters compared to the BCVA prior to
administration of the unit dose
of rAAV particles. In some embodiments, the administering the unit dose of
rAAV particles to the one
eye and/or to the contralateral eye of the individual results in an
improvement of BCVA of about 3
ETDRS letters or more compared to the BCVA prior to administration of the unit
dose of rAAV particles.
In some embodiments, the administering the unit dose of rAAV particles to the
one eye and/or to the
contralateral eye of the individual results in an improvement of BCVA of about
4 ETDRS letters or more
compared to the BCVA prior to administration of the unit dose of rAAV
particles. In some embodiments,
the administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in an improvement of BCVA of about 5.1 ETDRS letters or
more compared to the
BCVA prior to administration of the unit dose of rAAV particles. In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in an improvement of BCVA of about 6.4 ETDRS letters or
more compared to the
BCVA prior to administration of the unit dose of rAAV particles. In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in an improvement of BCVA of about 6.8 ETDRS letters
compared to the BCVA prior
to administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit
dose of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in an
improvement of BCVA of about 8.8 ETDRS letters compared to the BCVA prior to
administration of the
unit dose of rAAV particles. In some embodiments, the administering the unit
dose of rAAV particles to
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the one eye and/or to the contralateral eye of the individual results in an
improvement of BCVA of about
2.3 ETDRS letters compared to the BCVA prior to administration of the unit
dose of rAAV particles_
101421 In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in maintenance of BCVA,
wherein the individual loses fewer
than 15 ETDRS letters (Vitale etal., (2016) JAMA Opthalmol 134(9):1041:1047)
(e.g., any of 15 or less,
14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or
less, 7 or less, 6 or less, 5 or less, 4 or
less, 3 or less, 2 or less, 1, or 0 letters) compared to the BCVA prior to
administration of the unit dose of
rAAV particles. In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in maintenance of
BCVA, wherein the individual
loses about 2 letters compared to the BCVA prior to administration of the unit
dose of rAAV particles. In
some embodiments, the administering the unit dose of rAAV particles to the one
eye and/or to the
contralateral eye of the individual results in maintenance of BCVA, wherein
the individual loses any of
about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or
about 9 ETDRS letters compared
to the BCVA prior to administration of the unit dose of rAAV particles. In
some embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in maintenance of BCVA, wherein the individual loses 0
letters compared to the BCVA
prior to administration of the unit dose of rAAV particles. In some
embodiments, the administering the
unit dose of rAAV particles to the one eye and/or to the contralateral eye of
the individual results in
maintenance of BCVA, wherein the individual loses about 1 letter compared to
the BCVA prior to
administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in maintenance of
BCVA, wherein the individual loses about 2.7 letters compared to the BCVA
prior to administration of
the unit dose of rAAV particles. In some embodiments, the administering the
unit dose of rAAV particles
to the one eye and/or to the contralateral eye of the individual results in
maintenance of BCVA, wherein
the individual loses about 2.8 letters compared to the BCVA prior to
administration of the unit dose of
rAAV particles. In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in maintenance of
BCVA, wherein the individual
loses about 2 letters or less compared to the BCVA prior to administration of
the unit dose of rAAV
particles. In some embodiments, the administering the unit dose of rAAV
particles to the one eye and/or
to the contralateral eye of the individual results in maintenance of BCVA,
wherein the individual loses
about 3.2 letters or less compared to the BCVA prior to administration of the
unit dose of rAAV particles.
In some embodiments, the administering the unit dose of rAAV particles to the
one eye and/or to the
contralateral eye of the individual results in maintenance of BCVA, wherein
the individual loses between
about 15 to about 0 letters (e.g., any of about 15, about 14, about 13, about
12, about 11, about 10, about
9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, or
0 letters) compared to the
BCVA prior to administration oldie unit dose of rAAV particles. In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
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individual results in maintenance of BCVA, wherein the individual loses
between about 10 to about 0
letters (e.g., any of about 10, about 9, about 8, about 7, about 6, about 5,
about 4, about 3, about 2, about
1, or 0 letters) compared to the BCVA prior to administration of the unit dose
of rAAV particles. In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in maintenance of BCVA, wherein the individual
loses between about 5 to
about 0 letters (e.g., any of about 5, about 4, about 3, about 2, about 1, or
0 letters) compared to the BCVA
prior to administration of the unit dose of rAAV particles. In some
embodiments, the administering the
unit dose of rAAV particles to the one eye and/or to the contralateral eye of
the individual results in
maintenance of BCVA, wherein the individual loses between about 4 to about 0
letters (e.g., any of about
4, about 3, about 2, about 1, or 0 letters) compared to the BCVA prior to
administration of the unit dose of
rAAV particles. In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in maintenance of
BCVA, wherein the individual
loses between about 3 to about 0 letters (e.g., any of about 3, about 2, about
1, or 0 letters) compared to
the BCVA prior to administration of the unit dose of rAAV particles. In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in maintenance of BCVA, wherein the individual loses
between about 2 to about 0
letters (e.g., any of about 2, about 1, or 0 letters) compared to the BCVA
prior to administration of the unit
dose of rAAV particles. In some embodiments, the administering the unit dose
of rAAV particles to the
one eye and/or to the contralateral eye of the individual results in
maintenance of BCVA, wherein the
individual loses between about 1 to about 0 letters compared to the BCVA prior
to administration of the
unit dose of rAAV particles.
[0143] In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in a change in BCVA of between
about -20 to +7 or more
(e.g., any of -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9,4, -7,
-6, -5,4, -3, -2, -1,0, +1, +2,
+3, +4, +5, +6, +7, +8, -F9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19,
+20, or more) ETDRS
letters, compared to the BCVA prior to administration of the unit dose of rAAV
particles. In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in an increase in BCVA of about any of 16, 7 or
5 ETDRS letters compared to
the BCVA prior to administration of the unit dose of rAAV particles. In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a decrease in BCVA of about any of 19, 14, 7, 6, 5, 4,
3, 2, or 1 ETDRS letters
compared to the BCVA prior to administration of the unit dose of rAAV
particles. In some embodiments,
the administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a decrease in BCVA of about 4.8 or about 0.8 ETDRS
letters compared to the BCVA
prior to administration of the unit dose of rAAV particles. In some
embodiments, the administering the
unit dose of rAAV particles to the one eye and/or to the contralateral eye of
the individual results in a
decrease in BCVA of about 2 ETDRS letters or less compared to the BCVA prior
to administration of the
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unit dose of rAAV particles. In some embodiments, the administering the unit
dose of rAAV particles to
the one eye and/or to the contralateral eye of the individual results in a
decrease in BCVA of about 3.2
ETDRS letters or less compared to the BCVA prior to administration of the unit
dose of rAAV particles.
101441 In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in a change in BCVA of between
about -15 to +7 or more
(e.g., any of -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -
1, 0, +1, +2, +3, +4, +5, +6, +7, +8,
+9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS
letters, compared to the
BCVA prior to administration of the unit dose of rAAV particles. In some
embodiments, the
administering the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual results in a change in BCVA of between about -10 to +7 or more
(e.g., any of -10, -9, -8, -7,-
6, -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, +5, -1-6, +7, +8, -F9, +10, +11,
+12, +13, +14, +15, +16, +17, +18,
+19, +20, or more) ETDRS letters, compared to the BCVA prior to administration
of the unit dose of
rAAV particles. In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in a change in BCVA
of between about -5 to +7 or
more (e.g., any of -5, 4, -3, -2, -1,0, +1, +2, +3, +4, +5, +6, +7, +8, +9,
+10, +11, +12, +13, +14, +15,
+16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to
administration of the
unit dose of rAAV particles. In some embodiments, the administering the unit
dose of rAAV particles to
the one eye and/or to the contralateral eye of the individual results in a
change in BCVA of between about
4 to +7 or more (e.g., any of 4, -3, -2, -1, 0, +1, +2, +3, +4, +5, +6, +7,
+8, +9, +10, +11, +12, +13, +14,
+15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA
prior to a,c1ministration of
the unit dose of rAAV particles. In some embodiments, the administering the
unit dose of rAAV particles
to the one eye and/or to the contralateral eye of the individual results in a
change in BCVA of between
about -3 to +7 or more (e.g., any of -3, -2, -1,0, +1, +2, +3, +4, +5, +6, +7,
+8, +9, +10, +11, +12, +13,
+14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the
BCVA prior to
administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a change in
BCVA of between about -2 to +7 or more (e.g., any of -2, -1, 0, +1, +2, +3,
+4, +5, +6, +7, +8, +9, +10,
+11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters,
compared to the BCVA prior
to administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit
dose of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a change
in BCVA of between about -1 to +7 or more (e.g., any of-1, 0, +1, +2, +3, +4,
+5, +6, +7, +8, +9, +10,
+11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters,
compared to the BCVA prior
to administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit
dose of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a change
in BCVA of between about 0 to +7 or more (e.g., any of 0, +1, +2, +3, +4, +5,
+6, +7, +8, +9, +10, +11,
+12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared
to the BCVA prior to
administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit dose
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of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a change in
BCVA of between about +1 to +7 or more (es., any of +1, +2, +3, +4, +5, +6,
+7, +8, +9, +10, +11, +12,
+13, +14, +15, +16, +17, -i-18, +19, +20, or more) ETDRS letters, compared to
the BCVA prior to
administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a change in
BCVA of between about +2 to +7 or more (es., any of +2, +3, +4, +5, +6, +7,
+8, +9, +10, +11, +12,
+13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to
the BCVA prior to
administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a change in
BCVA of between about +310 +7 or more (e.g., any of +3, +4, +5, +6, +7, +8,
+9, +10, +11, +12, +13,
+14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the
BCVA prior to
administration of the unit dose of rAAV particles. In some embodiments, the
administering the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in a change in
BCVA of between about +410 +7 or more (e.g., any of +4, +5, +6, +7, +8, +9,
+10, +11, +12, +13, +14,
+15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA
prior to administration of
the unit dose of rAAV particles. In some embodiments, the administering the
unit dose of rAAV particles
to the one eye and/or to the contralateral eye of the individual results in a
change in BCVA of between
about +5 to +7 or more (e.g., any of +5, +6, +7, +8,+9, +10, +11, +12, +13,
+14, +15, +16, +17, +18,
+19, +20, or more) ETDRS letters, compared to the BCVA prior to administration
of the unit dose of
rAAV particles. In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in a change in BCVA
of about +6 or about +7
ETDRS letters, compared to the BCVA prior to administration of the unit dose
of rAAV particles.
[0145] In some embodiments, administration of the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in transient inflammation
(e.g., inflammation driven by
aqueous cells and/or vitreous cells, aqueous flare, posterior synchiae, poor
pupil dilation). In some
embodiments, administration of the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in inflammation (e.g., inflammation driven by
aqueous cells and/or vitreous
cells, aqueous flare, posterior synchiae, poor pupil dilation) that is
improved after administration of oral
and/or topical steroid treatment and/or mydryatics. In some embodiments,
administration of the unit dose
of rAAV particles to the one eye and/or to the contralateral eye of the
individual results in inflammation
(e.g., inflammation driven by aqueous cells and/or vitreous cells) that
resolves after administration of oral
and/or topical steroid treatment. Inflammation (e.g., inflammation driven by
aqueous cells and/or vitreous
cells, aqueous flare, posterior synchiae, poor pupil dilation) may be measured
using any method known in
the art, such as the slit lamp exam.
[0146] In some embodiments, the maintenance or the improvement of visual
acuity (e.g., BCVA)
compared to the visual acuity prior to administration of the unit dose of rAAV
particles is present at any
of about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks,
about 12 weeks, about 16
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weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about
36 weeks, about 40
weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about
60 weeks, about 64
weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about
84 weeks, about 88
weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 104 weeks, about
108 weeks, or more,
after administration of the unit dose of rAAV particles to the one eye and/or
to the contralateral eye of the
individual. In some embodiments, the maintenance or the improvement of visual
acuity (e.g., BCVA)
compared to the visual acuity prior to administration of the unit dose of rAAV
particles is present at
about 30 weeks or more after administration of the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual. In some embodiments, the maintenance
or the improvement of
visual acuity (e.g., BCVA) compared to the visual acuity prior to
administration of the unit dose of rAAV
particles is present at any of about 30 weeks, about 34 weeks, about 44 weeks,
about 6 months, about 1
year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10
years, or more, after
administration of the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual.
[0147] In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye is assessed
based on best corrected visual acuity (BCVA) in the one eye and/or the
contralateral eye. In some
embodiments, BCVA is expressed as an ETDRS score, which corresponds to the
number of letters
correctly read (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047). In
some embodiments, an
individual is determined to have maintenance of vision and/or visual acuity if
the individual loses fewer
than 15 letters in an ETDRS score (e.g., any of 15 or less, 14 or less, 13 or
less, 12 or less, 11 or less, 10
or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3
or less, 2 or less, 1, or 0 letters)
compared to prior to administration of the unit dose of rAAV particles in the
one eye and/or the
contralateral eye. In some embodiments, an individual is determined to have an
improvement of vision
and/or visual acuity if the individual gains at least 15 letters (e.g., any of
at least about 15, at least about
20, at least about 30, at least about 40, at least about 50, at least about
60, or about 70 letters) comparted
to prior to administration of the unit dose of rAAV particles in the one eye
and/or the contralateral eye.
[0148] In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye is assessed
based on central subfield thickness (CST) or central retinal thickness (CRT)
in the one eye and/or the
contralateral eye. In some embodiments, CST or CRT is determined by SD-OCT. In
some embodiments,
treatment of an ocular neovascular disease in an individual after
administration of the unit dose of rAAV
particles in the one eye and/or the contralateral eye is determined if the CST
or CRT assessed by SD-OCT
is decreased after administration of the unit dose of rAAV particles in the
one eye and/or the contralateral
eye compared to prior to administration of the unit dose of rAAV particles in
the one eye and/or the
contralateral eye. In some embodiments, treatment of an ocular neovascular
disease in an individual after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye is determined
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if the CST or CRT assessed by SD-OCT is maintained after administration of the
unit dose of rAAV
particles in the one eye and/or the contralateral eye compared to prior to
administration of the unit dose of
rAAV particles in the one eye and/or the contralateral eye.
101491 In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of MAY particles in the one eye and/or the
contralateral eye is assessed
based on macular volume in the one eye and/or the contralateral eye. In some
embodiments, macular
volume is determined by SD-OCT. In some embodiments, treatment of an ocular
neovascular disease in
an individual after administration of the unit dose of MAY particles in the
one eye and/or the contralateral
eye is determined if the macular volume assessed by SD-OCT is decreased after
administration of the unit
dose of rAAV particles in the one eye and/or the contralateral eye compared to
prior to administration of
the unit dose of rAAV particles in the one eye and/or the contralateral eye.
In some embodiments,
treatment of an ocular neovascular disease in an individual after
administration of the unit dose of rAAV
particles in the one eye and/or the contralateral eye is determined if the
macular volume assessed by SD-
OCT is maintained after administration of the unit dose of rAAV particles in
the one eye and/or the
contralateral eye compared to prior to administration of the unit dose of rAAV
particles in the one eye
and/or the contralateral eye.
101501 In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye is assessed
based on retinal thickness (e.g., central retinal thickness (CRT) or central
subfield thickness (CST)) and
macular volume in the one eye and/or the contralateral eye. In some
embodiments, CST and macular
volume are determined by SD-OCT. In some embodiments, treatment of an ocular
neovascular disease in
an individual after administration of the unit dose of MAY particles in the
one eye and/or the contralateral
eye is determined if the CST and macular volume assessed by SD-OCT are
decreased after administration
of the unit dose of rAAV particles in the one eye and/or the contralateral eye
compared to prior to
administration of the unit dose of MAY particles in the one eye and/or the
contralateral eye. In some
embodiments, treatment of an ocular neovascular disease in an individual after
administration of the unit
dose of rAAV particles in the one eye and/or the contralateral eye is
determined if the CST and macular
volume assessed by SD-OCT are maintained after administration of the unit dose
of rAAV particles in the
one eye and/or the contralateral eye compared to prior to administration of
the unit dose of rAAV particles
in the one eye and/or the contralateral eye.
101511 In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of IAA'S,. particles in the one eye and/or the
contralateral eye is assessed
based on the number of rescue therapy treatments (e.g., aflibercept
injections) required by the individual
after administration of the unit dose of rAAV particles in the one eye and/or
the contralateral eye. In some
embodiments, treatment of an ocular neovascular disease in an individual after
administration of the unit
dose of rAAV particles in the one eye and/or the contralateral eye is
determined if an individual requires
less than one rescue therapy treatment (e.g., aflibercept injection) any of
every 4 weeks, every 5 weeks,
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every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, or
more, after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye.
101521 In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye is determined
if an individual does not require any rescue therapy treatment (e.g.,
aflibercept injection) for any of at
least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5
weeks, at least 6 weeks, at least
7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 15
weeks, at least 20 weeks, at least
30 weeks, at least 40 weeks, at least 50 weeks, at least 60 weeks, at least 70
weeks, at least 80 weeks, at
least 90 weeks, at least 100 weeks, at least 110 weeks, or more, after
administration of the unit dose of
rAAV particles in the one eye and/or the contralateral eye.
101531 In some embodiments, the individual does not require any rescue therapy
treatment (e.g.,
aflibercept injection) for any of at least about 24 months, at least about 23
months, at least about 22
months, at least about 21 months, at least about 20 months, at least about 19
months, at least about 18
months, at least about 17 months, at least about 16 months, at least about 15
months, at least about 14
months, at least about 13 months, at least about 12 months, at least about 11
months, at least about 10
months, at least about 9 months, at least about 8 months, at least about 7
months, at least about 6 months,
at least about 5 months, at least about 4 months, at least about 3 months, at
least about 2 months, at least
about 1 month, at least about 3 weeks, at least about 2 weeks, or at least
about 1 week after administration
of the unit dose of MAY particles in the one eye and/or the contralateral eye.
In some embodiments, the
individual does not require any rescue therapy treatment (e.g., aflibercept
injection) for at least about 12
months after administration of the unit dose of MAY particles in the one eye
and/or the contralateral eye.
In some embodiments, the individual does not require any rescue therapy
treatment (e.g., aflibercept
injection) for at least about 10 months after administration of the unit dose
of MAY particles in the one
eye and/or the contralateral eye. In some embodiments, the individual does not
require any rescue therapy
treatment (e.g., aflibercept injection) for at least about 7 months after
administration of the unit dose of
rAAV particles in the one eye and/or the contralateral eye. In some
embodiments, the individual does not
require any rescue therapy treatment (e.g., aflibercept injection) for at
least about 6 months after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye. In some
embodiments, the individual does not require any rescue therapy treatment
(e.g., aflibercept injection) for
at least about 2 months after administration of the unit dose of rAAV
particles in the one eye and/or the
contralateral eye. In some embodiments, the individual does not require any
rescue therapy treatment
(e.g., aflibercept injection) for at least about 1 month after administration
of the unit dose of rAAV
particles in the one eye and/or the contralateral eye.
[0154] In some embodiments, administration of a single unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of a plurality of individuals results in at
least about 50% (e.g., any of at
least about 50%, at least about 55%, at least about 60%, at least about 65%,
at least about 70%, at least
about 75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, at least about
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99%, or 100%) of the individuals in the plurality not requiring an anti-VEGF
rescue treatment (e.g.,
aflibercept injection). In some embodiments, administration of a single unit
dose of rAAV particles to the
one eye and/or to the contralateral eye of a plurality of individuals results
in at least about 67% (e.g., any
of at least about 67%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at
least about 90%, at least about 95%, at least about 99%, or 100%) of the
individuals in the plurality not
requiring an anti-VEGF rescue treatment (e.g., aflibercept injection). In some
embodiments,
administration of a single unit dose of rAAV particles to the one eye and/or
to the contralateral eye of a
plurality of individuals results in at least about 50% of the individuals in
the plurality not requiring an
anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments,
administration of a single
unit dose of rAAV particles to the one eye and/or to the contralateral eye of
a plurality of individuals
results in at least about 78% of the individuals in the plurality not
requiring an anti-VEGF rescue
treatment (e.g., aflibercept injection). In some embodiments, administration
of a single unit dose of rAAV
particles to the one eye and/or to the contralateral eye of a plurality of
individuals results in at least about
80% of the individuals in the plurality not requiring an anti-VEGF rescue
treatment (e.g., aflibercept
injection). In some embodiments, administration of a single unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of a plurality of individuals results in at
least about 82% of the individuals
in the plurality not requiring an anti-VEGF rescue treatment (e.g.,
aflibercept injection). In some
embodiments, administration of a single unit dose of rAAV particles to the one
eye and/or to the
contralateral eye of a plurality of individuals results in 100% of the
individuals in the plurality not
requiring an anti-VEGF rescue treatment (e.g., aflibercept injection).
101551 In some embodiments, administration of a single unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of a plurality of individuals results in at
least about 50% (e.g., any of at
least about 50%, at least about 55%, at least about 60%, at least about 65%,
at least about 70%, at least
about 75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, at least about
99%, or 100%) of the individuals in the plurality not requiring an anti-VEGF
rescue treatment (e.g.,
aflibercept injection) for at least about 4 weeks after administration of the
rAAV particles, e.g., any of at
least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least
about 16 weeks, at least about
20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32
weeks, at least about 36
weeks, at least about 40 weeks, at least about 44 weeks, at least about 48
weeks, at least about 52 weeks,
at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at
least about 68 weeks, at least
about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least
about 84 weeks, at least about 88
weeks, at least about 92 weeks, at least about 96 weeks, at least about 100
weeks, at least about 104
weeks, at least about 108 weeks, or more after administration of the rAAV
particles. In some
embodiments, administration of a single unit dose of rAAV particles to the one
eye and/or to the
contralateral eye of a plurality of individuals results in at least about 50%
of the individuals in the
plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept
injection) for about 52 weeks or
more, or about 56 weeks or more, after administration of the rAAV particles.
In some embodiments,
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administration of a single unit dose of rAAV particles to the one eye and/or
to the contralateral eye of a
plurality of individuals results in at least about 67% (e.g., any of at least
about 67%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least about 90%,
at least about 95%, at least
about 99%, or 100%) of the individuals in the plurality not requiring an anti-
VEGF rescue treatment (e.g.,
aflibercept injection) for at least about 20 weeks after administration of the
rAAV particles, e.g., any of at
least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at
least about 32 weeks, at least
about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least
about 48 weeks, at least about 52
weeks, at least about 56 weeks, at least about 60 weeks, at least about 64
weeks, at least about 66 weeks,
or more after administration of the rAAV particles. In some embodiments,
administration of a single unit
dose of rAAV particles to the one eye and/or to the contralateral eye of a
plurality of individuals results in
at least about 78% of the individuals in the plurality not requiring an anti-
VEGF rescue treatment (e.g.,
aflibercept injection) for at least about 4 weeks after administration of the
rAAV particles, e.g., any of at
least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least
about 16 weeks, at least about
20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32
weeks, at least about 36
weeks, at least about 40 weeks, at least about 44 weeks, at least about 48
weeks, at least about 52 weeks,
at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at
least about 68 weeks, at least
about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least
about 84 weeks, at least about 88
weeks, at least about 92 weeks, at least about 96 weeks, at least about 100
weeks, at least about 104
weeks, at least about 108 weeks, or more, after administration of the rAAV
particles. In some
embodiments, administration of a single unit dose of rAAV particles to the one
eye and/or to the
contralateral eye of a plurality of individuals results in at least about 78%
of the individuals in the
plurality not requiring an anti-VEGF rescue treatment (e,g., aflibercept
injection) for about 20 weeks or
more, or about 36 weeks or more, after administration of the rAAV particles.
In some embodiments,
administration of a single unit dose of rAAV particles to the one eye and/or
to the contralateral eye of a
plurality of individuals results in at least about 80% of the individuals in
the plurality not requiring an
anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 20
weeks after administration of
the rAAV particles, e.g., any of at least about 20 weeks, at least about 24
weeks, at least about 28 weeks,
at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at
least about 44 weeks, at least
about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least
about 60 weeks, at least about 64
weeks, at least about 66 weeks, or more after administration of the rAAV
particles. In some embodiments,
administration of a single unit dose of rAAV particles to the one eye and/or
to the contralateral eye of a
plurality of individuals results in at least about 82% of the individuals in
the plurality not requiring an
anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 20
weeks after administration of
the rAAV particles, e.g., any of at least about 20 weeks, at least about 24
weeks, at least about 28 weeks,
at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at
least about 44 weeks, at least
about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least
about 60 weeks, at least about 64
weeks, at least about 66 weeks, or more, after administration of the rAAV
particles. In some
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embodiments, administration of a single unit dose of rAAV particles to the one
eye and/or to the
contralateral eye of a plurality of individuals results in 100% of the
individuals in the plurality not
requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at
least about 20 weeks after
administration of the rAAV particles, e.g., any of at least about 20 weeks, at
least about 24 weeks, at least
about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least
about 40 weeks, at least about 44
weeks, at least about 48 weeks, at least about 52 weeks, at least about 56
weeks, at least about 60 weeks,
at least about 64 weeks, at least about 66 weeks, or more, after
administration of the rAAV particles. In
some embodiments, administration of a single unit dose of rAAV particles to
the one eye and/or to the
contralateral eye of a plurality of individuals results in 100% of the
individuals in the plurality not
requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at
least about 4 weeks after
administration of the rAAV particles, e.g., any of any of at least about 4
weeks, at least about 8 weeks, at
least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at
least about 24 weeks, at least
about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least
about 40 weeks, at least about 44
weeks, at least about 48 weeks, at least about 52 weeks, at least about 56
weeks, at least about 60 weeks,
at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at
least about 76 weeks, at least
about 80 weeks, at least about 84 weeks, at least about 88 weeks, at least
about 92 weeks, at least about 96
weeks, at least about 100 weeks, at least about 104 weeks, at least about 108
weeks, or more after
administration of the rAAV particles. In some embodiments, administration of a
single unit dose of
rAAV particles to the one eye and/or to the contralateral eye of a plurality
of individuals results in 100%
of the individuals in the plurality not requiring an anti-VEGF rescue
treatment (e.g., aflibercept injection)
for any of about 64 weeks or more, 72 weeks or more, or 84 weeks or more,
after administration of the
rAAV particles.
[0156] In some embodiments, administration of a unit dose of rAAV particles in
the one eye and/or the
contralateral eye of a plurality of individuals results in about 78% or less
(e.g., any of about 78% or less,
about 75% or less, about 70% or less, about 65% or less, about 60% or less,
about 55% or less, about 50%
or less, about 45% or less, about 40% or less, about 35% or less, about 30% or
less, about 25% or less,
about 20% or less, about 15% or less, about 10% or less, about 5% or less,
about 2.5% or less, about 1%
or less, or about 0.5% or less) of the individuals in the plurality requiring
any rescue treatment (e.g.,
aflibercept injection) in the one eye and/or the contralateral eye. In some
embodiments, administration of
a unit dose of rAAV particles in the one eye and/or the contralateral eye of a
plurality of individuals
results in about 50% or less (e.g., any of about 50% or less, about 45% or
less, about 40% or less, about
35% or less, about 30% or less, about 25% or less, about 20% or less, about
15% or less, about 10% or
less, about 5% or less, about 2.5% or less, about 1% or less, or about 0.5% or
less) of the individuals in
the plurality requiring any rescue treatment (e.g., aflibercept injection) in
the one eye and/or the
contralateral eye. In some embodiments, administration of a unit dose of rAAV
particles in the one eye
and/or the contralateral eye of a plurality of individuals results in less
than about 30% (e.g., less than any
of about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about
2.5%, about 1%, or about
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0.5%) of the individuals in the plurality requiring any rescue treatment
(e.g., aflibercept injection) in the
one eye and/or the contralateral eye. In some embodiments, administration of a
unit dose of rAAV
particles in the one eye and/or the contralateral eye of a plurality of
individuals results in less than about
30% of the individuals in the plurality requiring any rescue treatment (e.g.,
aflibercept injection) in the
one eye and/or the contralateral eye. In some embodiments, administration of a
unit dose of rAAV
particles in the one eye and/or the contralateral eye of a plurality of
individuals results in less than about
20% of the individuals in the plurality requiring any rescue treatment (e.g.,
aflibercept injection) in the
one eye and/or the contralateral eye. In some embodiments, administration of a
unit dose of rAAV
particles in the one eye and/or the contralateral eye of a plurality of
individuals results in 0% of the
individuals in the plurality requiring any rescue treatment (e.g., aflibercept
injection) in the one eye and/or
the contralateral eye.
[0157] In some embodiments, administration of a unit dose of rAAV particles in
the one eye and/or the
contralateral eye of a plurality of individuals results in about 78% or less
(e.g., any of about 78% or less,
about 75% or less, about 70% or less, about 65% or less, about 60% or less,
about 55% or less, about 50%
or less, about 45% or less, about 40% or less, about 35% or less, about 30% or
less, about 25% or less,
about 20% or less, about 15% or less, about 10% or less, about 5% or less,
about 2.5% or less, about 1%
or less, or about 0.5% or less) of the individuals in the plurality requiring
any rescue treatment (e.g.,
aflibercept injection) for at least about 4 weeks after administration of the
rAAV particles, e.g., any of at
least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least
about 16 weeks, at least about
20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32
weeks, at least about 36
weeks, at least about 40 weeks, at least about 44 weeks, at least about 48
weeks, at least about 52 weeks,
at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at
least about 68 weeks, at least
about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least
about 84 weeks, at least about 88
weeks, at least about 92 weeks, at least about 96 weeks, at least about 100
weeks, at least about 104
weeks, at least about 108 weeks, or more, after administration of the rAAV
particles. In some
embodiments, administration of a unit dose of rAAV particles in the one eye
and/or the contralateral eye
of a plurality of individuals results in less than about 30% (e.g., less than
any of about 30%, about 25%,
about 20%, about 15%, about 10%, about 5%, about 2.5%, about 1%, or about
0.5%) of the individuals in
the plurality requiring any rescue treatment (e.g., aflibercept injection) for
at least about 20 weeks after
administration of the rAAV particles, e.g., any of at least about 20 weeks, at
least about 24 weeks, at least
about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least
about 40 weeks, at least about 44
weeks, at least about 48 weeks, at least about 52 weeks, at least about 56
weeks, at least about 60 weeks,
at least about 64 weeks, at least about 66 weeks, or more, after
administration of the rAAV particles. In
some embodiments, administration of a unit dose of rAAV particles in the one
eye and/or the contralateral
eye of a plurality of individuals results in less than about 30% of the
individuals in the plurality requiring
any rescue treatment (e.g., aflibercept injection) in the one eye and/or the
contralateral eye for at least
about 20 weeks after administration of the rAAV particles, e.g., any of at
least about 20 weeks, at least
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about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least
about 36 weeks, at least about 40
weeks, at least about 44 weeks, at least about 48 weeks, at least about 52
weeks, at least about 56 weeks,
at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or
more, after administration of
the rAAV particles. In some embodiments, administration of a unit dose of rAAV
particles in the one eye
and/or the contralateral eye of a plurality of individuals results in less
than about 20% of the individuals in
the plurality requiring any rescue treatment (e.g., aflibercept injection) in
the one eye and/or the
contralateral eye for at least about 20 weeks after administration of the rAAV
particles, e.g., any of at least
about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least
about 32 weeks, at least about 36
weeks, at least about 40 weeks, at least about 44 weeks, at least about 48
weeks, at least about 52 weeks,
at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at
least about 66 weeks, or more
after administration of the rAAV particles. In some embodiments,
administration of a unit dose of rAAV
particles in the one eye and/or the contralateral eye of a plurality of
individuals results in 0% of the
individuals in the plurality requiring any rescue treatment (e.g., aflibercept
injection) in the one eye and/or
the contralateral eye for at least about 20 weeks after administration of the
rAAV particles, e.g., any of at
least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at
least about 32 weeks, at least
about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least
about 48 weeks, at least about 52
weeks, at least about 56 weeks, at least about 60 weeks, at least about 64
weeks, at least about 66 weeks,
or more after administration of the rAAV particles. In some embodiments,
administration of a unit dose of
rAAV particles in the one eye and/or the contralateral eye of a plurality of
individuals results in 0% of the
individuals in the plurality requiring any rescue treatment (e.g., aflibercept
injection) in the one eye and/or
the contralateral eye for at least about 4 weeks after administration of the
rAAV particles, e.g., any of at
least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least
about 16 weeks, at least about
20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32
weeks, at least about 36
weeks, at least about 40 weeks, at least about 44 weeks, at least about 48
weeks, at least about 52 weeks,
at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at
least about 68 weeks, at least
about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least
about 84 weeks, at least about 88
weeks, at least about 92 weeks, at least about 96 weeks, at least about 100
weeks, at least about 104
weeks, at least about 108 weeks, or more, after administration of the rAAV
particles.
101581 In some embodiments, administration of the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of a plurality of individuals results in a reduction in
the mean annualized anti-VEGF
injection rate of any of at least about 80%, at least about 85%, at least
about 87%, at least about 90%, at
least about 95%, at least about 99%, or 100%, compared to the mean annualized
anti-VEGF injection rate
prior to administration of the unit dose of rAAV particles. In some
embodiments, administration of the
unit dose of rAAV particles to the one eye and/or to the contralateral eye of
a plurality of individuals
results in a reduction in the mean annualized anti-VEGF injection rate of
about 87% or more compared to
the mean annualized anti-VEGF injection rate prior to administration of the
unit dose of rAAV particles.
In some embodiments, administration of the unit dose of rAAV particles to the
one eye and/or to the
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contralateral eye of a plurality of individuals results in a reduction in the
mean annualized anti-VEGF
injection rate of 100% compared to the mean annualized anti-VEGF injection
rate prior to administration
of the unit dose of rAAV particles.
101591 In some embodiments, the mean annualized anti-VEGF injection rate prior
to administration of
the unit dose of rAAV particles is calculated according to the formula:
Annualized rate prior to administration of the unit dose of rAAV particles =
(number of anti-VEGF
injections in 12 months prior to administration of the unit dose of rAAV
particles) / (days from the first
anti-VEGF injection in the past 12 months prior to administration of the unit
dose of rAAV particles to the
administration of the unit dose of rAAV particles / 365.25)
101601 In some embodiments, the mean annualized anti-VEGF injection rate after
administration of the
unit dose of rAAV particles is calculated according to the formula:
Annualized rate after administration of the unit dose of rAAV particles =
(number of anti-VEGF
injections since administration of the unit dose of rAAV particles) / (days
from administration of the unit
dose of rAAV particles / 365.25).
101611 In some embodiments, an individual is determined to require a rescue
treatment (e.g., anti-VEGF
intravitreal injection, such as aflibercept injection) after administration of
the rAAV particles if the
individual exhibits loss of 10 or more letters in BCVA (e.g., using the ETDRS
protocol) in the one eye
and/or the contralateral eye administered the rAAV particles that is
attributed to intraretinal or subretinal
fluid (e.g., as determined by SD-OCT) compared to the BCVA in the one eye
and/or the contralateral eye
administered the rAAV particles prior to administration of the rAAV particles.
In some embodiments, an
individual is determined to require a rescue treatment (e.g., anti-VEGF
intravitreal injection, such as
aflibercept injection) after administration of the rAAV particles if the
individual exhibits an increase in
central subfield thickness (e.g., CST or CRT) greater than 75 gm in the one
eye and/or the contralateral
eye administered the rAAV particles compared to the central subfield thickness
in the one eye and/or the
contralateral eye administered the rAAV particles prior to administration of
the rAAV particles, e.g., as
determined by SD-OCT. In some embodiments, an individual is determined to
require a rescue treatment
(e.g., anti-VEGF intravitreal injection, such as aflibercept injection) after
administration of the rAAV
particles if the individual exhibits vision-threatening hemorrhage due to AMD
in the one eye and/or the
contralateral eye administered the rAAV particles.
101621 In some embodiments, a rescue treatment comprises administration of a
standard of care anti-
VEGF therapy. Such standard of care anti-VEGF therapy comprises one or more
anti-VEGF treatments
(e.g., anti-VEGF intravitreal injections). In some embodiments, a rescue
treatment comprises one or more
aflibercept NT injections. In some embodiments, a rescue treatment comprises
one or more aflibercept
NT injections comprising about 2 mg of aflibercept.
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101631 In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye is assessed
based on the level of retinal fluid compared the level of retinal fluid prior
to administration of the unit
dose of rAAV particles in the one eye and/or the contralateral eye. In some
embodiments, treatment of an
ocular neovascular disease in an individual after administration of the unit
dose of rAAV particles in the
one eye and/or the contralateral eye is determined if a reduction in retinal
fluid is observed after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye compared to
the level of retinal fluid prior to administration of the unit dose of rAAV
particles in the one eye and/or
the contralateral eye. In some embodiments, the ocular neovascular disease is
wAMD.
101641 In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye is assessed
based on the resolution of pigment epithelial detachment (FED) compared to PED
prior to administration
of the unit dose of rAAV particles in the one eye and/or the contralateral
eye. In some embodiments,
treatment of an ocular neovascular disease in an individual after
administration of the unit dose of rAAV
particles in the one eye and/or the contralateral eye is determined if
resolution of PIED after administration
of the unit dose of rAAV particles in the one eye and/or the contralateral eye
is observed, compared to
FED prior to administration of the unit dose of rAAV particles in the one eye
and/or the contralateral eye.
In some embodiments, the ocular neovascular disease is wAMD.
101651 In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye is assessed
based on choroidal neovascularization (CNV) lesion growth as determined by
fluorescein angiography. In
some embodiments, treatment of an ocular neovascular disease in an individual
after administration of the
unit dose of rAAV particles in the one eye and/or the contralateral eye is
determined if CNV lesions
shrink (e.g., by more than any of about 5%, about 10%, about 20%, about 30%,
about 40%, about 50%,
about 60%, about 70%, about 80%, or 100%) after administration of the unit
dose of rAAV particles in
the one eye and/or the contralateral eye compared to CNV lesions present prior
to administration of the
unit dose of rAAV particles in the one eye and/or the contralateral eye. In
some embodiments, treatment
of an ocular neovascular disease in an individual after administration of the
unit dose of rAAV particles in
the one eye and/or the contralateral eye is determined if CNV lesions do not
grow (e.g., grow less than
any of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,
about 8%, about 9%,
about 10%, about 15%, or about 20%) after administration of the unit dose of
rAAV particles in the one
eye and/or the contralateral eye compared to CNV lesions present prior to
administration of the unit dose
of rAAV particles in the one eye and/or the contralateral eye. In some
embodiments, the ocular
neovascular disease is wAMD.
101661 In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye is assessed
based on the anatomical features of the one eye and/or the contralateral eye
based on any methods known
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in the art (e.g., SD-OCT, OCT, fluorescein angiography, digital color fundus
photography, etc.). In some
embodiments, treatment of an ocular neovascular disease in an individual after
administration of the unit
dose of rAAV particles in the one eye and/or the contralateral eye is
determined if an improvement in
anatomical features of the one eye and/or the contralateral eye is observed
after administration of the unit
dose of rAAV particles in the one eye and/or the contralateral eye. In some
embodiments, the ocular
neovascular disease is wAMD.
[0167] In some embodiments, treatment of an ocular neovascular disease in an
individual after
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye is assessed
based on ophthalmologic examination, intraocular pressure (e.g., using a
(loldman/1 applanation
tonometer or Tono-pen), indirect ophthalmoscopy, examination of the one eye
and/or the contralateral eye
and adnexa, eyelid and/or pupil responsiveness, belpharoptosis, abnormal pupil
shape, unequal pupils,
abnormal reaction to light, afferent pupillary defects, slit-lamp examination
(including of the eyelids,
conjunctiva, cornea, lens, iris, and anterior chamber), posterior segment
abnormalities of the vitreous,
optic nerve, peripheral retina, and retinal vasculature, SD-OCT, fluorescein
angiography, digital color
fundus photography (including images of the retina, optic disc, and/or
macula), aqueous humor sampling,
vitreous humor sampling, OCT-angiography (OCT-A), refraction and/or visual
acuity (BCVA). In some
embodiments, SD-OCT is performed to evaluate retinal thickness (e.g., central
retinal thickness or central
subfield thickness), macular volume, and/or the presence of fluid (e.g.,
subretinal fluid or intraretinal
fluid). In some embodiments, the ocular neovascular disease is wAMD.
[0168] The unit dose of rAAV particles may be administered to the one eye
and/or to the contralateral
eye of the individual by any method known in the art. For example, the unit
dose of rAAV particles may
be administered to the one eye and/or to the contralateral eye of the
individual intraocularly, or by
intravitreal injection. In some embodiments, the administration of the unit
dose of rAAV particles to the
one eye and/or to the contralateral eye of the individual is intraocular. In
some embodiments, the
administration of the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual is by intravitreal injection (D/T) or subretinal injection. In some
embodiments, the
administration of the unit dose of rAAV particles to the one eye and/or to the
contralateral eye of the
individual is by WT injection. In some embodiments, aseptic technique is
employed to administer a unit
dose of rAAV particles by intravitreal injection. In some embodiments, aseptic
technique with providone-
iodine is employed to administer a unit dose of rAAV particles by intravitreal
injection.
[0169] In some embodiments, the individual has not received a prior treatment
for an ocular neovascular
disease. In some embodiments, the individual has not received a prior
treatment in the one eye and/or the
contralateral eye for an ocular neovascular disease. In some embodiments, the
individual has not received
a prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab,
ranibizurnab, faricimab,
abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-
102), PAN-90806
(PanOptica), and/or allibercept). In some embodiments, the individual has not
received a prior treatment
with an anti-VEGF agent (e.g., bevacizumab, brolucizurnab, ranibizumab,
faricimab, abicipar pegol,
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conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806
(PanOptica), and/or
aflibercept) in the one eye and/or the contralateral eye. In some embodiments,
the individual has not
received a prior aflibercept treatment. In some embodiments, the individual
has not received a prior
aflibercept treatment in the one eye and/or the contralateral eye.
Steroid Treatments
[0170] In some embodiments, the unit dose of rAAV particles is administered in
combination with
steroid treatment. In some embodiments, the steroid treatment is a
corticosteroid treatment. Exemplary
corticosteroids include, without limitation, aclometasone, amcinomide,
beclometasone, betamethasone,
budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol,
cortivazol, deflazacort,
deoxycorticosterone, desonide desoximetasone, dexamethasone, diflorasone,
diflucortolone,
difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumetasone,
flunisolide, fluocinolone
acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone,
fluperolone, fluticasone,
faprednidene, formocortal, halcinonide, halometasone, hydrocortisone
aceponate, hydrocortisone
buteprate, hydrocortisone butyrate, loteprednol, medlysone, meprednisone,
methylprednisolone,
methylprednisolone aceponate, mometasone furoate, paramethasone,
prednicarbate, prednisone,
prednisolone, prednylidene, remexolone, tixocortol, niamcinolone, and
ulobetasol. In some embodiments,
the steroid treatment is a systemic steroid treatment. In some embodiments,
the steroid treatment is an oral
steroid treatment. In some embodiments, the steroid treatment is an ophthalmic
steroid treatment. In some
embodiments, the ophthalmic steroid treatment is a topical steroid treatment
(e.g. a drop), a periocular
steroid treatment (e.g., subtenons, subconjunctival), an intravitreal steroid
treatment, or a superchoroidal
steroid treatment. In some embodiments, the topical steroid treatment is a
difluprednate treatment, a
medrysone treatment, a loteprednol treatment, a prednisolone treatment, a
fluocinolone treatment, a
triamcinolone treatment, a rimexolone treatment, a dexamethasone treatment, a
fluorometholone
treatment, a fluocinolone treatment, a rimexolone treatment, or a prednisone
treatment. In some
embodiments, the ophthalmic steroid treatment is a difluprednate treatment. In
some embodiments, the
steroid treatment is a prednisone treatment In some embodiments, the steroid
treatment is a difluprednate
treatment.
[0171] In some embodiments, the steroid treatment comprises a systemic steroid
treatment and a topical
steroid treatment. In some embodiments, the systemic steroid treatment is an
oral steroid treatment. In
some embodiments, the systemic steroid treatment is a prednisone treatment. In
some embodiments, the
topical steroid treatment is a difluprednate treatment. In some embodiments,
the systemic steroid
treatment and the topical steroid treatment are administered simultaneously
(e.g., on the same day). k
some embodiments, the systemic steroid treatment and the topical steroid
treatment are administered
separately (e.g., on different days).
[0172] In some embodiments, the steroid is administered before, during, and/or
after administration of
the unit dose of rAAV particles. In some embodiments, the steroid is
administered before, during, and
after administration of the unit dose of rAAV particles. In some embodiments,
the steroid is administered
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during, and after administration of the unit dose of rAAV particles. In some
embodiments, the steroid is
administered before administration of the unit dose of rAAV particles. In some
embodiments, the steroid
is administered during administration of the unit dose of rAAV particles. In
some embodiments, the
steroid is administered before and during administration of the unit dose of
rAAV particles. In some
embodiments, the steroid is administered after administration of the unit dose
of rAAV particles. In some
embodiments, the steroid is administered during and after administration of
the unit dose of rAAV
particles. In some embodiments, the steroid is administered before and/or
after administration of the unit
dose of rAAV particles. In some embodiments, the steroid is administered
before and after administration
of the unit dose of rAAV particles.
[0173] In some embodiments, the steroid treatment is a systemic steroid
treatment. In some
embodiments, the systemic steroid treatment is an oral steroid treatment.
[0174] In some embodiments, the steroid treatment is an oral prednisone
treatment. In some
embodiments, the oral prednisone treatment is initiated prior to
administration of the unit dose of rAAV
particles. In some embodiments, an initial oral prednisone treatment is
administered at a dose of any of
about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg,
or about 70 mg of
prednisone per day any of about 7 days, about 6 days, about 5 days, about 4
days, about 3 days, about 2
days, about 1 day, or 0 days before administration of the unit dose of rAAV
particles, and is continued for
any of about 3 days, about 4 days, about 5 days, about 6 days, about 7 days,
about 8 days, about 9 days, or
about 10 days, or more. In some embodiments, an initial oral prednisone
treatment is administered at a
dose of about 60 mg of prednisone per day about 3 days before administration
of the unit dose of rAAV,
and is continued for about 3 days.
[0175] In some embodiments, the initial oral prednisone treatment is followed
by an oral prednisone
treatment dose taper. In some embodiments, the oral prednisone treatment dose
taper is administered at a
dose of any of about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40
mg, about 45 mg, or about
50 mg of prednisone per day for a total of any of about 1 day, about 2 days,
about 3 days, about 4 days,
about 5 days, about 6 days, or about 7 days, followed by a dose of about 10
mg, about 15 mg, about 20 mg
or about 25 mg of prednisone per day for any of about 1 day, about 2 days,
about 3 days, or about 4 days,
followed by a dose of about 5 mg, about 10 mg, or about 15 mg of prednisone
per day for about I day,
about 2 days, about 3 days, or about 4 days. In some embodiments, the
prednisone dose taper is
administered at a dose of any of about 40 mg of prednisone per day for 3 days,
followed by a dose of
about 20 mg of prednisone per day for 2 days, followed by a dose of about 10
mg of prednisone per day
for 2 days.
[0176] In some embodiments, an initial oral prednisone treatment is initiated
3 days before
administration of the unit dose of rAAV particles at a dose of 60 mg of
prednisone per day for a total of 6
days, followed by a dose of 40 mg of prednisone per day for a total of 3 days,
followed by a dose of 20
mg of prednisone per day for 2 days, followed by a dose of 10 mg of prednisone
per day for 2 days.
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101771 In some embodiments, the steroid treatment is an ophthalmic steroid
treatment. In some
embodiments, the ophthalmic steroid treatment is a difluprednate treatment. In
some embodiments, the
steroid treatment is administered before, during, and/or after administration
of the unit dose of rAAV
particles. In some embodiments, the steroid treatment is administered before
administration of the unit
dose of rAAV particles. In some embodiments, the steroid treatment is
administered during administration
of the unit dose of rAAV particles. In some embodiments, the steroid treatment
is administered after
administration of the unit dose of rAAV particles. In some embodiments, the
steroid treatment is
administered before and during administration of the unit dose of rAAV
particles. In some embodiments,
the steroid treatment is administered before and after administration of the
unit dose of rAAV particles. In
some embodiments, the steroid treatment is administered during, and after
administration of the unit dose
of rAAV particles. In some embodiments, the steroid treatment is administered
before, during, and after
administration of the unit dose of rAAV particles.
101781 In some embodiments, the steroid treatment is an ophthalmic steroid
treatment, e.g., a topical
steroid treatment. In some embodiments, the ophthalmic steroid treatment,
e.g., a topical steroid treatment,
is a daily steroid treatment for up to 4 weeks, up to 6 weeks, up to 8 weeks,
up to 3 months, up to 4
months, up to 5 months, or up to 6 months after administration of the unit
dose of rAAV particles. In
some embodiments, the topical steroid treatment comprises about four
administrations of topical steroid
on about week 1, about three administrations of topical steroid on about week
2, about two
administrations of topical steroid on about week 3, and about one
administration of topical steroid on
about week 4; timing starting with and following administration of the unit
dose of rAAV particles. In
some embodiments, the topical steroid treatment comprises about four
administrations of topical steroid
per day (i.e., QID) for about 3 weeks after administration of the unit dose of
rAAV particles, followed by
about 3 administrations of topical steroid per day (te., TID) for about 1
week, followed by about 2
administrations of topical steroid per day (i.e., BID) for about 1 week, and
followed by about 1
administration of topical steroid per day (i.e., QD) for about 1 week. In some
embodiments, the
ophthalmic steroid treatment is extended at the discretion of the treating
physician.
101791 In some embodiments, the ophthalmic steroid is about 0.005% to about
0.5% difluprednate. In
some embodiments, the ophthalmic steroid is any of about 0.005%, about 0.006%,
about 0.007%, about
0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.4%, about
0.05%, about 0.06%,
about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some
embodiments, the
ophthalmic steroid is difluprednate 0.05%. In some embodiments, a dose of
difluprednate 0.05% is one
drop of ophthalmic solution. In some embodiments, one drop is about 50 pi
(e.g., about 25 pi to about 50
or about 50 gl to about 100 pl). In some embodiments, a dose of difluprednate
comprises about 1 pig
to about 5 pg, or about 2 pg to about 3 pg, or about 2.5 pg difluprednate. In
some embodiments, a dose of
difluprednate comprises about 2.5 pig difluprednate.
101801 In some embodiments, the topical steroid treatment comprises a 7-week
topical steroid treatment,
e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment
comprises about four
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administrations of topical steroid per day (i.e., QID) for about four weeks,
followed by about three
administrations of topical steroid per day (i.e., `HD) for about one week,
followed by about two
administrations of topical steroid per day (i.e., BID) for about one week, and
followed by about one
administration of topical steroid per day (i.e., QD) for about one week;
timing starting at about one week
prior to administration of the unit dose of rAAV particles. In some
embodiments, the topical steroid
treatment comprises about four administrations of topical steroid per day
(i.e., QID) for about 28 days,
followed by about three administrations of topical steroid per day (i.e., TID)
for about 7 days, followed by
about two administrations of topical steroid per day (i.e., BID) for about 7
days, and followed by about
one administration of topical steroid per day (i.e., QD) for about 7 days;
timing starting at about 7 days
prior to administration of the unit dose of rAAV particles. In some
embodiments, the topical steroid
treatment comprises about four administrations of topical steroid per day
QID) on Day 1 to about
Day 28, followed by about three administrations of topical steroid per day
(i.e., TID) on about Day 29 to
about Day 35, followed by about two administrations of topical steroid per day
(i.e., BID) on about Day
36 to about Day 42, and followed by about one administration of topical
steroid per day (i.e., QD) on
about Day 43 to about Day 49; timing starting at Day 1. In some embodiments,
the topical steroid
treatment is continued if inflammation is present.
101811 In some embodiments, the methods of treatment provided herein comprise
administering an anti-
VEGF agent (e.g., an aflibercept WT injection) to one eye of the individual
prior to administration of the
unit dose of rAAV particles to the one eye of the individual. In some
embodiments, the anti-VEGF agent
is administered about 7 days or about 1 week prior to administration of the
unit dose of rAAV particles. In
some embodiments, the anti-VEGF agent is administered on about Day 1 and the
unit dose of rAAV
particles is administered on about Day 8. In some embodiments, the topical
steroid treatment comprises a
7-week topical steroid treatment, e.g., 0.05% difluprednate. In some
embodiments, the topical steroid
treatment comprises about four administrations of topical steroid per day
(i.e., QM) for about four weeks,
followed by about three administrations of topical steroid per day (i.e., TID)
for about one week, followed
by about two administrations of topical steroid per day (i.e., BID) for about
one week, and followed by
about one administration of topical steroid per day (i.e., QD) for about one
week; timing starting with and
following administration of the anti-VEGF agent. In some embodiments, the
topical steroid treatment
comprises about four administrations of topical steroid per day (i.e., QID)
for about 28 days, followed by
about three administrations of topical steroid per day (i.e., TB)) for about 7
days, followed by about two
administrations of topical steroid per day (i.e., BID) for about 7 days, and
followed by about one
administration of topical steroid per day (i.e., QD) for about 7 days; timing
starting with and following
administration of the anti-VEGF agent. In some embodiments, the topical
steroid treatment comprises
about four administrations of topical steroid per day (i.e., QID) on Day 1 to
about Day 28, followed by
about three administrations of topical steroid per day (i.e., TID) on about
Day 29 to about Day 35,
followed by about two administrations of topical steroid per day (i.e., BID)
on about Day 36 to about Day
42, and followed by about one administration of topical steroid per day (i.e..
QD) on about Day 43 to
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about Day 49; timing starting at Day 1. In some embodiments, the topical
steroid treatment is continued if
inflammation is present
101821 In some embodiments, the topical steroid treatment comprises a 4-month
topical steroid
treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid
treatment comprises about
four administrations of topical steroid per day (i.e., QID) for about one
month, followed by about three
administrations of topical steroid per day (i.e., TID) for about one month,
followed by about two
administrations of topical steroid per day (Le., BID) for about one month, and
followed by about one
administration of topical steroid per day (i.e., QD) for about one month;
timing starting at about one week
prior to administration of the unit dose of rAAV particles. In some
embodiments, the topical steroid
treatment comprises about four administrations of topical steroid per day
(i.e., QID) for about 30 days,
followed by about three administrations of topical steroid per day (Le., TIT))
for about 30 days, followed
by about two administrations of topical steroid per day (i.e., BID) for about
30 days, and followed by
about one administration of topical steroid per day (i.e., QD) for about 30
days; timing starting at about 7
days prior to administration of the unit dose of rAAV particles. In some
embodiments, the topical steroid
treatment comprises about four administrations of topical steroid per day
(i.e., QID) on Day 1 to about
Day 30, followed by about three administrations of topical steroid per day
(i.e., TID) on about Day 31 to
about Day 60, followed by about two administrations of topical steroid per day
(La, BID) on about Day
61 to about Day 90, and followed by about one administration of topical
steroid per day (i.e., QD) on
about Day 91 to about Day 120; timing starting at Day 1. In some embodiments,
the topical steroid
treatment is continued if inflammation is present.
101831 In some embodiments, the methods of treatment provided herein comprise
administering an anti-
VEGF agent (e.g., an aflibercept IVT injection) to one eye of the individual
prior to administration of the
unit dose of MAY particles to the one eye of the individual. In some
embodiments, the anti-VEGF agent
is administered about 7 days or about 1 week prior to administration of the
unit dose of rAAV particles. In
some embodiments, the anti-VEGF agent is administered on about Day 1 and the
unit dose of rAAV
particles is administered on about Day 8. In some embodiments, the topical
steroid treatment comprises a
4-month topical steroid treatment, e.g., 0.05% difluprednate. In some
embodiments, the topical steroid
treatment comprises about four administrations of topical steroid per day
(Le., QID) for about one month,
followed by about three administrations of topical steroid per day (i.e., TID)
for about one month,
followed by about two administrations of topical steroid per day (i.e., BID)
for about one month, and
followed by about one administration of topical steroid per day (i.e., QD) for
about one month; timing
starting with and following administration of the anti-VEGF agent. In some
embodiments, the topical
steroid treatment comprises about four administrations of topical steroid per
day (i.e., QID) for about 30
days, followed by about three administrations of topical steroid per day
(i.e., TI)) for about 30 days,
followed by about two administrations of topical steroid per day (i.e., BID)
for about 30 days, and
followed by about one administration of topical steroid per day (i.e., QD) for
about 30 days; timing
starting with and following administration of the anti-VEGF agent. In some
embodiments, the topical
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steroid treatment comprises about four administrations of topical steroid per
day (i.e., QED) on Day 1 to
about Day 30, followed by about three administrations of topical steroid per
day (i.e., TID) on about Day
31 to about Day 60, followed by about two administrations of topical steroid
per day (i.e.. BID) on about
Day 61 to about Day 90, and followed by about one administration of topical
steroid per day (i.e., QD) on
about Day 91 to about Day 120; timing starting at Day 1. In some embodiments,
the topical steroid
treatment is continued if inflammation is present.
Vectors for Delivering Trans genes to Target Cells
[0184] In some embodiments, the recombinant adeno-associated virus (rAAV)
particles comprise a
recombinant viral vector derived from adeno-associated virus (AAV) that has
been altered so that it is
replication-defective in the subject (e.g., a human or a non-human primate).
In some embodiments, the
adeno-associated virus (AAV) is a recombinant AAV (rAAV).
[0185] AAV or rAAV are small non-enveloped single-stranded DNA viruses. rAAVs
are non-
pathogenic human parvoviruses and can be made to be dependent on helper
viruses, including adenovirus,
herpes simplex virus, vaccinia virus and CMV, for replication.
[0186] Exposure to wild type (wt) AAV is not associated or known to cause any
human pathologies and
is conunon in the general population, making AAV or rAAV a suitable delivery
system for gene therapy.
AAV and rAAV used for gene therapy for delivery of an anti-VEGF agent, e.g.,
aflibercept, can be of any
serotype. In some embodiments, the methods of the disclosure provide for use
of any suitable AAV
serotype, including AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAV9,
AAVIO, AAV11, AAV12, rh10, AAV-DJ, and any hybrid or chimeric AAV thereof. In
some
embodiments, the serotype used is based on tropism of the virus, or
infectivity of a target cell of interest.
In some embodiments, several AAV vectors may be generated to allow selection
of the most optimal
serotype for use with an anti-VEGF agent transgene (e.g., aflibercept
transgene).
[0187] In some embodiments, the methods of the present disclosure provide for
the use of pseudotyped
AAV. Pseudotyped AAV particles comprise AAV genome inverted terminal repeats
(flits) of one AAV
serotype encatpsidated by an AAV capsid of another AAV serotype. Typically,
pseudotyped AAV is
designated as "AAV#/#", where the first "4" indicates the AAV ITR serotype and
the second "-14"
indicates capsid serotype. For example, an AAV particle comprising AAV2 ITRs
and an AAV1 capsid
would be designated "AAV2/1".
[0188] In some embodiments, the rAAV particles comprise a nucleic acid, e.g.,
a heterologous nucleic
acid. In some embodiments, the nucleic acid encodes a transgene, e.g., an anti-
VEGF agent (e.g.,
aflibercept). In some embodiments, the encoded transgene, e.g., anti-VEGF
agent, is under the
transcriptional control of a promoter that initiates transcription of the
nucleic acid. In some embodiments,
the promoter is a "ubiquitous" promoter. In some embodiments, the promoter is
a "strong" or
constitutively active promoter, e.g., a cytomegalovirus (CMV) promoter, an
elongation factor 1 alpha
(EF1a) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter,
or a connexin36 (or
"Cx36") promoter. In some embodiments, the promoter is a tissue-specific
promoter that is activated in
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specific tissues or cells, such as retinal cells, to reduce potential toxicity
or undesirable effects to non-
targeted cells. In some aspects, several AAV vectors may be generated to allow
selection of the most
optimal serotype and promoter for use with the anti-VEGF agent transgene
(e.g., aflibercept transgene). In
some embodiments, the nucleic acid is flanked by AAV inverted terminal repeats
(ITRs). In some
embodiments, the nucleic acid is flanked by AAV2 ITRs.
[0189] In some embodiments, the AAV vector comprises a polynucleotide cassette
for enhanced
expression of a transgene (e.g., an anti-VEGF agent such as aflibercept) in a
target cell (e.g., a retinal
cell). In some embodiments, the polynucleotide cassette comprises in 5' to 3'
order (a) a first enhancer
region comprising a CMV sequence (SEQ ID NO: 22); (b) a promoter region,
comprising a CMV
sequence (SEQ ID NO: 23); (c) a 51UTR region comprising, in 5' to 3' order,
TPL and eMLP sequences
(SEQ ID NO: 24 and SEQ ID NO: 25, respectively); (d) a coding sequence
encoding a peptide or
polypeptide (e.g., an anti-VEGF agent such as aflibercept); (e) a second
enhancer region comprising a full
EES sequence (SEQ ID NO: 26); and (0 a HGH polyadenylation site (SEQ ID NO:
27), In certain of
these embodiments, the polynucleotide cassette comprises one or more sequences
selected from SEQ
NOs: 28-32, or a sequence with at least 85% identity thereto. In certain of
these embodiments, the 5 arm
of the polynucleotide cassette comprises or consists of SEQ ID NO: 33 or a
sequence with at least 85%
identity thereto. In certain of these embodiments, the 3' arm of the
polynucleotide cassette comprises or
consists of SEQ NO: 34 or a sequence with at least 85% identity thereto. The
nucleic acid sequences of
SEQ ID NOs: 22-34 are provided below:
ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT TGACGTCAAT
AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC AATGGGTGGA
GTATTTACGG TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC CAAGTCCGCC
CCCTATTGAC GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT
ACGGGACTTT CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCA (SEQ ID NO:
22)
TGCTGATGCG GTTTTGGCAG TACACCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT
TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG
ACTTTCCAAA ATGTCGTAAT AACCCCGCCC CGTTGACGCA AATGGGCGGT AGGCGTGTAC
GGTGGGAGGT CTATATAAGC AGAGCTCGTT TAGTGAACCG (SEQ ID NO: 23)
CTCACTCTCT TCCGCATCGC TGTCTGCGAG GGCCAGCTGT TGGGCTCGCG GTTGAGGACA
AACTCTTCGC GGTCTTTCCA GTACTCTTGG ATCGGAAACC CGTCGGCCTC CGAACGGTAC
TCCGCCACCG AGGGACCTGA GCGAGTCCGC ATCGACCGGA TCGGAAAACC TCTCGAGAAA
GGCGTCTAAC CAGTCACAGT CGCAAGGTAG GCTGAGCACC GTGGCGGGCG GCAGCGGGTG
GCGGTCGGGG TTGTTTCTGG CGGAGGTGCT GCTGATGATG TAATTAAAGT AGGCGGTCTT
GAGACGGCGG ATGGTCGA (SEQ ID NO: 24)
CCAGCTGTTG GGGTGAGTAC TCCCTCTCAA AAGCGGGCAT TACTTCTGCG CTAAGATTGT
CAGTTTCCAA AAACGAGGAG GATTTGATAT TCACCTGGCC CG (SEQ ID NO: 25)
CTGTTCTCAT CACATCATAT CAAGGTTATA TACCATCAAT ATTGCCACAG ATGTTACTTA
GCCTTTTAAT ATTTCTCTAA TTTAGTGTAT ATGCAATGAT AGTTCTCTGA TTTCTGAGAT
TGAGTTTCTC ATGTGTAATG ATTATTTAGA GTTTCTCTTT CATCTGTTCA AATTTTTGTC
TAGTTTTATT TTTTACTGAT TTGTAAGACT TCTTTTTATA ATCTGCATAT TACAATTCTC
TTTACTGGGG TGTTGCAAAT ATTTTCTGTC ATTCTATGGC CTGACTTTTC TTAATGGTTT
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TTTAATTTTA AAAATAAGTC TTAATATTCA TGCAATCTAA TTAACAATCT TTTCTTTGTG
GTTAGGACTT TGAGTCATAA GAAATTTTTC TCTACACTGA AGTCATGATG GCATGCTTCT
ATATTATTTT CTAAAAGATT TAAAGTTTTG CCTTCTCCAT TTAGACTTAT AATTCACTGG
AATTTTTTTG TGTGTATGGT ATGACATATG GGTTCCCTTT TATTTTTTAC ATATAAATAT
ATTTCCCTGT TTTTCTAAAA AAGAAAAAGA TCATCATTTT CCCATTGTAA AATGCCATAT
TTTTTTCATA GGTCACTTAC ATATATCAAT GGGTCTGTTT CTGAGCTCTA CTCTATTTTA
TCAGCCTCAC TGTCTATCCC CACACATCTC ATGCTTTGCT CTAAATCTTG ATATTTAGTG
GAACATTCTT TCCCATTTTG TTCTACAAGA ATATTTTTGT TATTGTCTTT GGGCTTTCTA
TATACATTTT GAAATGAGGT TGACAAGTTA (SEQ ID NO: 26)
CTGCCCGGGT GGCATCCCTG TGACCCCTCC CCAGTGCCTC TCCTGGCCCT GGAAGTTGCC
ACTCCAGTGC CCACCAGCCT TGTOCTAATA AAATTAAGTT GCATCATTTT GTCTGACTAG
GTGTCCTTCT ATAATATTAT GGGGTGGAGG GGGGTGGTAT GGAGCAAGGG GCCCAAGTTG
GGAAGAAACC TGTAGGGCCT GC (SEQ ID NO: 27)
AGGCGGTCTT GAGACGGCGG ATGGTCGAGG TGAGGTGTGG CAGGCTTGAG ATCCAGCTGT
TGGGGTGA (SEQ ID NO: 28)
CGCTGTTTTG ACCTCCATAG TGGACACCGG GACCGATCCA GCCTCCGCGT CTCAGGGGAG
ATCTCGTTTA GTGAACCGTC AGATCCTCAC TCTCTTCCGC ATCGCTGTCT GCGAGGGCCA
GCTGTTGGG (SEQ ID NO: 29)
TTGATATTCA CCTGGCCCGA TCTGGCCATA CACTTG (SEQ OD NO: 30)
CCCAGGTCCA AGTTTAAACG CC (SEQ ID NO: 31)
TCTTTGGGCT TTCTATATAC ATTTTGAAAT GAGGTTGACA AGTTACCTAG GAAAACTGTC
TTCCTGCCCG GGTGGCA (SEQ ID NO: 32)
CTCTGGAGAC GACTTACGGT AAATGGCCCG CCTGGCTGAC CGCCCAACGA CCCCCGCCCA
TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA TAGGGACTTT CCATTGACGT
CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG
CCAAGTCCGC CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG
TACATGACCT TACGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT CATCGCTATT
ACCATGCTGA TGCGGTTTTG GCAGTACACC AATGGGCGTG GATAGOGGTT TGACTCACGG
GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA
CGGGACTTTC CAAAATGTCG TAATAACCCC GCCCCGTTGA CGCAAATGGG CGGTAGGCGT
GTACGGTGGG AGGTCTATAT AAGCAGAGCT CGTTTAGTGA ACCGTCAGAT CGCCTGGAGA
GGCCATCCAC GCTGTTTTGA CCTCCATAGT GGACACCGGG ACCGATCCAG CCTCCGCGTC
TCAGGGGAGA TCTCGTTTAG TGAACCGTCA GATCCTCACT CTCTTCCGCA TCGCTGTCTG
CGAGGGCCAG CTGTTGGGCT CGCGGTTGAG GACAAACTCT TCGCGGTCTT TCCAGTACTC
TTGGATCGGA AACCCGTCGG CCTCCGAACG GTACTCCGCC ACCGAGGGAC CTGAGCGAGT
CCGCATCGAC CGGATCGGAA AACCTCTCGA GAAAGGCGTC TAACCAGTCA CAGTCGCAAG
GTAGGCTGAG CACCGTGGCG GGCGGCAGCG GGTGGCGGTC GGGGTTGTTT CTGGCGGAGG
TGCTGCTGAT GATGTAATTA AAGTAGGCGG TCTTGAGACG GCGGATGGTC GAGGTGAGGT
GTGGCAGGCT TGAGATCCAG CTGTTGGGGT GAGTACTCCC TCTCAAAAGC GGGCATTACT
TCTGCGCTAA GATTGTCAGT TTCCAAAAAC GAGGAGGATT TGATATTCAC CTGGCCCGAT
CTGGCCATAC ACTTGAGTGA CAATGACATC CACTTTGCCT TTCTCTCCAC AGGTGTCCAC
TCCCAGGTCC AAGTTTAAAC GCCGCCACCA TG (SEQ ID NO: 33)
ACTGTTCTOA TCACATCATA TCAAGGTTAT ATACCATCAA TATTGCCACA GATGTTACTT
AGCCTTTTAA TATTTCTCTA ATTTAGTGTA TATGCAATGA TAGTTCTCTG ATTTCTGAGA
TTGAGTTTCT CATGTGTAAT GATTATTTAG AGTTTCTCTT TCATCTGTTC AAATTTTTGT
CTAGTTTTAT TTTTTACTGA TTTGTAAGAC TTCTTTTTAT AATCTGCATA TTACAATTCT
CTTTACTGGG GTGTTGCAAA TATTTTCTGT CATTCTATGG CCTGACTTTT CTTAATGGTT
TTTTAATTTT AAAAATAAGT CTTAATATTC ATGCAATCTA ATTAACAATC TTTTCTTTGT
GGTTAGGACT TTGAGTCATA AGAAATTTTT CTCTACACTG AAGTCATGAT GGCATGCTTC
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TATATTATTT TCTAAAAGAT TTAAAGTTTT GCCTTCTCCA TTTAGACTTA TAATTCACTG
GAATTTTTTT GTGTGTATGG TATGACATAT GGGTTCCCTT TTATTTTTTA CATATAAATA
TATTTCCCTG TTTTTCTAAA AAAGAAAAAG ATCATCATTT TCCCATTGTA AAATGCCATA
TTTTTTTCAT AGGTCACTTA CATATATCAA TGGGTCTGTT TCTGAGCTCT ACTCTATTTT
ATCAGCCTCA CTGTCTATCC CCACACATCT CATGCTTTGC TCTAAATCTT GATATTTAGT
GGAACATTCT TTCCCATTTT GTTCTACAAG AATATTTTTG TTATTGTCTT TGGGCTTTCT
ATATACATTT TGAAATGAGG TTGACAAGTT ACCTAGGAAA ACTGTCTTCC TGCCCGGGTG
GCATCCCTGT GACCCCTCCC CAGTGCCTCT CCTGGCCCTG GAAGTTGCCA CTCCAGTGCC
CACCAGCCTT GTCCTAATAA AATTAAGTTG CATCATTTTG TCTGACTAGG TGTCCTTCTA
TAATATTATG GGGTGGAGGG GGGTGGTATG GAGCAAGGGG CCCAAGTTGG GAAGAAACCT
GTAGGGCCTG CGAAGACAGT CAG (SEQ ID NO: 34)
101901 In some embodiments, the polynucleotide cassette comprises or consists
of SEQ ID NO: 39 or a
sequence with at least 85% identity thereto.
gcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcct
cagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgatt
aacccgccatgetacttatctacgtactctggagacgacttacggtaaatggcccgcctggctgaccgccc
aacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattg
acgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtc
cgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttacgggac
tttcctacttggcagtacatctacgtattagtcatcgctattaccatgctgatgcggttttggcagtacac
caatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtt
tgttttggcaccaaaatcaacgggactttccaaaatgtcgtaataaccccgccccgttgacgcaaatgggc
ggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagagg
ccatccacgctgttttgacctccatagtggacaccgggaccgatccagcctccgcgtctcaggggagatct
cgtttagtgaaccgtcagatcctcactctcttccgcatcgctgtctgcgagggccagctgttgggctcgcg
gttgaggacaaactcttcgcggtotttccagtactcttggatcggaaacccgtcggcctccgaacggtact
ccgccaccgagggacctgagcgagtccgcatcgaccggatcggaaaacctctcgagaaaggcgtctaacca
gtcacagtcgcaaggtaggctgagcaccgtggcgggcggcagcgggtggcggtcggggttgtttctggcgg
aggtgctgctgatgatgtaattaaagtaggcggtcttgagacggcggatggtcgaggtgaggtgtggcagg
cttgagatccagctgttggggtgagtactccctctcaaaagcgggcattacttctgcgctaagattgtcag
tttccaaaaacgaggaggatttgatattcacctggcccgatctggccatacacttgagtgacaatgacatc
cactttgcctttctctccacaggtgtccactcccaggtccaagtttaaacgccgccaccatggtgtcatac
tgggatactggagtcttgctttgtgccctgctgtcctgcctcctcctgactggctccagctcgggctcaga
taccggtcgccccttcgtggagatgtactccgagatcccggaaattatccacatgactgaggggcgcgaac
ttgtgatcccctgccgggtcaccagcccgaacattactgtgactttgaagaagttccccctggacaccctg
attccggatgggaagagaattatctgggattcacggaagggattcatcatcagcaacgcgacctacaagga
aattggcctcctcacttgcgaagccactgtgaacggacacttgtacaagaccaactacctgacccaccgcc
agaccaacaccatcatcgacgtcgtcctgtccccttcgcacgggatcgagctctcggtgggagagaagttg
gtgcttaactgcaccgcccggacggaactgaatgtgggaatcgacttcaactgggaatacccgtccagcaa
gcatcagcataagaagctggtgaaccgggacctcaagactcagtccggcagcgaaatgaagaagttcctgt
cgaccctcactattgacggagtgaccagatccgaccagggcctctacacttgcgccgcttccagcggactc
atgaccaagaagaacagcactttcgtgagggtgcatgagaaggacaagacccacacgtgtccgccgtgccc
agccccagagctgctgggaggccottccgtgttcctgtttccgcccaagccaaaggataccctgatgatct
caaggacccctgaggtcacatgcgtcgtggtggatgtgtcgcacgaggaccctgaagtcaaattcaattgg
tatgtggacggagtggaagtccacaacgcgaaaaccaagccgagagaagaacagtacaattccacctaccg
ggtggtgtcggtgctgactgtgctgcaccaggactggctcaacggaaaggagtacaagtgcaaggtgtcca
acaaggctctgcccgcacctattgaaaagaccatctccaaggccaagggtcaacctcgcgagcctcaggtg
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tacactctgcctccaagccgggacgaactgactaagaa.ccaagtctctctgacctgtttggtgaagggctt
ctacccgtcagacatcgcagtggagtgggagtcaaacggtcagccggagaacaactacaaaacaacccocc
ccgtgctggactccgacggctccttcttcctgtactccaagcttaccgtggataagagccgctggcaacag
ggcaacgtgttttcctgctccgtcatgcacgaagccctgcacaaccattatacccagaagtccctgtcgct
gtcccccgggaaatagtgactgttctcatcacatcatatcaaggttatataccatcaatattgccacagat
gttacttagccttttaatatttctctaatttagtgtatatgcaatgatagttctctgatttctgagattga
gtttctcatgtgtaatgattatttagagtttctctttcatctgttcaaatttttgtctagttttatttttt
actgatttgtaagacttctttttataatctgcatattacaattctctttactggggtgttgcaaatatttt
ctgtcattctatggcctgacttttcttaatggttttttaattttaaaaataagtettaatattcatgcaat
ctaattaacaatcttttctttgtggttaggactttgagtcataagaaatttttctctacactgaagtcatg
atggcatgcttctatattattttctaaaagatttaaagttttgccttctccatttagacttataattcact
ggaatttttttgtgtgtatggtatgacatatgggttcccttttattttttacatataaatatatttccctg
tttttctaaaaaagaaaaagatcatcattttcccattgtaaaatgccatatttttttcataggtcacttac
atatatcaatgggtctgtttctgagctctactctattttatcagcctcactgtctatccccacacatctca
tgctttgctctaaatcttgatatttagtggaacattctttcccattttgttctacaagaatatttttgtta
ttgtctttgggctttctatatacattttgaaatgaggttgacaagttacctaggaaaactgtcttcctgcc
cgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccacca
gccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtgg
aggggggtggtatggagcaaggggcccaagttgggaagaaacctgtagggcctgcgtacgtagataagtag
catggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcg
ctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcg
agcgagcgcgc (SEQ ID NO: 39)
[0191] SEQ ID NO: 39 shown above comprises, in the 5' to 3' direction, an
inverted terminal repeat
(ITR) of AAV serotype 2 comprising nucleotides 1-145 of SEQ ID NO: 39; a CMV
promoter comprising
nucleotides 180-693 of SEQ ID NO: 39; a 5' Untranslated Region (UTR),
including an Adenovirus
Tripartite Leader Sequence and Synthetic Intron, and comprising nucleotides
694-1314 of SEQ 1.13 NO:
39; a Kozak sequence comprising nucleotides 1329-1340 of SEQ ID NO: 39; a
codon-optimized
aflibercept cDNA sequence comprising nucleotides 1338-2714 of SEQ ID NO: 39; a
3' UTR including a
human scaffold attachment region and comprising nucleotides 2717-3527 of SEQ
ID NO: 39; a human
growth hormone polyadenylation/transcription stop signal comprising
nucleotides 3546-3748 of SEQ ID
NO: 39; and an inverted terminal repeat (ITR) of AAV serotype 2 comprising
nucleotides 3772-3916 of
SEQ ID NO: 39. See, e.g., FIG. 1C.
[0192] Additional polynucleotide cassettes for enhanced expression of a
transgene (e.g., a transgene
encoding an anti-VEGF agent such as aflibercept) in a target cell (such as a
retinal cell) are disclosed in
W02018/170473, the contents of which related to polynucleotide cassettes for
enhanced expression of a
transgene in a target cell are incorporated herein by reference.
[0193] In some embodiments, the rAAV particles comprise a variant capsid
protein having increased
infectivity of target cells, e.g. retinal cells, are used to increase
transduction of retinal cells or to increase
targeting of gene delivery to retinal cells in an individual. In some
embodiments, the rAAV particle
comprises an amino acid modification in a capsid protein GH loop/loop IV of
the AAV capsid protein. In
some embodiments, the site of modification is a solvent-accessible portion of
the GH loop/loop IV of the
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AAV capsid protein. Fora description of the GH loop/loop IV of AAV capsid,
see, e.g., van Vliet et al.
(2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et
al. (2007) Mol. Ther.
15:1955. Several AAV capsid variants are known, including the 7m8 variant. In
some embodiments, a
rAAV particle comprises a variant AM/ capsid protein that comprises an
insertion of from 5 amino acids
to 11 amino acids, e.g., 7 amino acid sequence, in the GH loop of a capsid
protein relative to a
corresponding parental AAV capsid protein, and wherein the variant capsid
protein confers increased
infectivity of a retinal cell compared to the infectivity of the retinal cell
by an AAV particle comprising
the corresponding parental or unmodified AAV capsid protein. In some
embodiments, any one of the
following amino acid sequences can be inserted in the GH loop of a capsid
protein: LALGETTRPA (SEQ
ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA
(SEQ
ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTT1CF (SEQ ID
NO: 7),
STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10),
LAAVDTTKFA (SEQ ID NO: 11), and LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID
NO: 14),
NETITRP (SEQ ID NO: 15), ICAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17),
KDTDTTR
(SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN
(SEQ
ID NO: 21). In some embodiments, any one of the amino acid sequences set forth
in SEQ ID NOs: 1-12
and 14-21 is inserted in the solvent-exposed GH loop of VP1 capsid protein in
a rAAV. Additional details
regarding amino acid sequences that can be inserted into the GH loop of a
capsid protein, e.g., to facilitate
transduction of a nucleic acid of interest to a retinal cell following IVT
injection, are provided in
W02012145601, US9587282, US10202657, and US10214785, the contents of which
related to amino
acid sequences that can be inserted into the GH loop of a capsid protein are
incorporated herein by
reference.
[0194] In some embodiments, the rAAV particles comprise an AAV capsid protein,
e.g., an AAV2
capsid protein, that includes any one of the following amino acid sequences:
LALGETTRPA (SEQ ID
NO: 1); LANETTTRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ
ID
NO: 4), ICDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO:
7),
STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10),
LAAVDTTKFA (SEQ ID NO: 11), and LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID
NO: 14),
NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17),
KDTDTTR
(SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN
(SEQ
ID NO: 21) inserted at the following positions: between positions 587 and 588
of the AAV2 capsid
protein; between amino acids 590 and 591 of the AAV1 capsid protein; between
amino acids 575 and 576
of the AAV5 capsid protein; between amino acids 590 and 591 of the AAV6 capsid
protein; between
amino acids 589 and 590 of the AAV7 capsid protein; between amino acids 590
and 591 of the AAV8
capsid protein; between amino acids 588 and 589 of the AAV9 capsid protein; or
between amino acids
589 and 590 of the AAVIO capsid protein. In some embodiments, the rAAV
particles comprise AAV2
capsid proteins comprising an amino acid sequence LALGETTRPA (SEQ ID NO: 1)
inserted between
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positions 587 and 588 of the capsid protein, wherein the amino acid residue
numbering corresponds to an
AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise AAV2
capsid proteins
comprising the amino acid sequence LALGETTRPA (SEQ ID NO: I) inserted between
positions 587 and
588 of the AAV2 VP! comprising the sequence of SEQ ID NO: 13.
101951 In some embodiments, rAAV particles comprise the 7m8 variant capsid
protein from AAV2
comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted in the
OH loop of the
AAV2 VP1 protein between positions 587 and 588 of the AAV2 VP!. In some
embodiments, the rAAV
particles comprise an AAV2 VP1 capsid protein comprising a OH loop that
comprises the amino acid
sequence of SEQ ID NO: 38 or an amino acid sequence having at least 90%
sequence identity to SEQ ID
NO: 38. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid
protein comprising a
GI-I loop that comprises an amino acid sequence having any of at least 90%, at
least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100%
sequence identity to SEQ ID NO: 38_
FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSCITTQSFtLQFSQAGASDI
RDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATICYHLNGRDSLVNPGPANIASHKDDE
EKFFPQSGVLIFGKQGSEKTNVD1EKVMTTDEEEIRTTNPVATEQYGSVSTNLQRGNLAL
GETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK
FIPPPQILIKN (SEQ ID NO: 38)
101961 In some embodiments, rAAV particles comprise the 7m8 variant capsid
protein from AAV2
comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between
positions 587 and
588 of the AAV2 VP!. The sequence of the 7m8 variant capsid protein from AAV2
comprising the amino
acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted between positions 587 and
588 of the AAV2
VP1 is provided below:
MAADGYLPDWLEDTLSEGIRQWWKLICPGPPPPICPAERHKDDSRGLVLPGYKYLGPFNGLDKGE
PVNEADAAALEFIDICAYDRQLDSGDNPYLKYNHADAEFQERLICEDTSFGGNLGRAVFQAKKRV
LEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGICAGQQPARKRLNFGQTGDADSVPDPQPLG
QPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWNIGDRVITTSTRTWALPT
YNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFECHFSPRDWQRLINNNWGFRPKELNFKL
FNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTL
NNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYL
SRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATICYH
LNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATE
QYGSVSTNLQRGNLALGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDG
FIFHPSPLMGGFGLKHPPPQ1LIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR
WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 37)
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[0197] In some embodiments, the rAAV particles comprise a capsid protein VP1
comprising the amino
acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588
of the capsid protein,
wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid
protein. In some
embodiments, the rAAV particles comprise a capsid protein VP2 comprising the
amino acid sequence
LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid
protein, wherein the
amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In
some embodiments, the
rAAV particles comprise a capsid protein VP3 comprising the amino acid
sequence LGETTRP (SEQ 11)
NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein
the amino acid residue
numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the
rAAV particles
comprise capsid proteins VP1, VP2, and VP3, wherein each of VP!, VP2, and VP3
comprise the amino
acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588
of the capsid protein,
wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid
protein.
[0198] In some embodiments, the rAAV particles comprise a capsid protein VP1
comprising the amino
acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588
of the capsid
protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1
capsid protein. In some
embodiments, the rAAV particles comprise a capsid protein VP2 comprising the
amino acid sequence
LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid
protein, wherein
the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In
some embodiments,
the rAAV particles comprise a capsid protein VP3 comprising the amino acid
sequence LALGETTRPA
(SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein,
wherein the amino acid
residue numbering corresponds to an AAV2 VP1 capsid protein. In some
embodiments, the rAAV
particles comprise capsid proteins VP1, VP2, and VP3, wherein each of VP!,
VP2, and VP3 comprise the
amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587
and 588 of the
capsid protein, wherein the amino acid residue numbering corresponds to an
AAV2 VP1 capsid protein.
[0199] In some embodiments, a recombinant virus and/or plasmid used to
generate a rAAV virus
comprises other transcriptional or regulatory elements, such as a poly A
(polyadenylation) sequence,
untranslated regions (UTRs), 3' UTRs, or termination sequences. In some
embodiments, more than one
gene is expressed from the vector or plasmid using internal ribosome entry
site (IRES) or similar element
that allows co-expression of two or more proteins or create multigene, or
polycistronic mRNA.
[0200] In some embodiments, the rAAV and/or plasmid used to generate the rAAV
comprises one or
more of the following nucleic acid elements: a first ITR sequence; a promoter
sequence; an intron
sequence; a first UTR sequence; a heterologous nucleic acid encoding an anti-
VEGF agent (e.g.,
aflibercept); a second UTR sequence; a polyA sequence; and a second ITR
sequence. In some
embodiments, linker sequence(s) are inserted between two or more of the
nucleic acid elements. In some
embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide,
es., encodes aflibercept
(or a functional fragment or functional variant thereof).
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[0201] In some embodiments, the vector is a targeted vector, especially a
targeted rAAV (e.g.,
AAV2.7m8) that shows higher infectivity of a specific cell, such as a retinal
cell (e.g., a photoreceptor, a
retinal ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a
horizontal cell, or a retinal pigmented
epithelium cell). Viral vectors for use in the disclosure can include those
that exhibit low toxicity and/or
low immunogenicity in an individual and expresses therapeutically effective
quantities of the anti-VEGF
agent (e.g., aflibercept) in an individual, e.g., a human. Any suitable method
known in the art can be used
in the biochemical purification of recombinant viruses (e.g., rAAV), e.g., for
the preparation of
pharmaceutical compositions described elsewhere herein. Recombinant AAV
viruses can be harvested
directly from cells, or from the culture media comprising cells. Virus can be
purified using various
biochemical means, such as gel filtration, filtration, chromatography,
affinity purification, gradient
uhracentrifugation, or size exclusion methods. In some embodiments, the virus
is lyophilized.
[0202] In some embodiments, the rAAV particles comprise a 7m8 variant capsid
protein, e.g.,
rAAV2.7m8, and a nucleic acid sequence that encodes an anti-VEGF agent (e.g.,
aflibercept, or a
fimctional fragment or functional variant thereof). In some embodiments, the
rAAV particles (e.g., the
7m8 variant) have an increase in retinal cell infectivity of any of at least
5%, at least 10%, at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, or at least
100% as compared to an AAV particle comprising the corresponding parental or
unmodified AAV capsid
protein. In some embodiments, the increase in infectivity of retinal cells is
an increase of any of between
5% to 100%, between 5% to 95%, between 5% to 90%, between 5% to 85%, between
5% to 80%,
between 5% to 75%, between 5% to 70%, between 5% to 65%, between 5% to 60%,
between 5% to 55%,
between 5% to 50%, between 5% to 45%, between 5% to 40%, between 5% to 35%,
between 5% to 30%,
between 5% to 25%, between 5% to 20%, between 5% to 15%, or between 5% to 10%,
as compared to an
AAV particle comprising the corresponding parental or unmodified AAV capsid
protein.
[0203] In some embodiments, the increase in retinal cell infectivity of a rAAV
variant, e.g.,
rAAV2.7m8, is any of at least 1-fold, at least 1.1-fold, at least 1.2-fold, at
least 1.3-fold, at least 1.4-fold,
at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at
least 1.9-fold, or at least 2-fold
compared to an AAV particle comprising the corresponding parental or
unmodified AAV capsid protein.
In some embodiments, the increase in infectivity is any of at least 2-fold, at
least 3-fold, at least 4-fold, at
least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-
fold, or at least 10-fold, as compared to
an AAV particle comprising the corresponding parental AAV capsid protein. In
some embodiments, the
increase in infectivity is any of at least 15-fold, at least 20-fold, at least
25-fold, at least 30-fold, at least
35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-
fold, at least 60-fold, at least 65-fold,
at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at
least 90-fold, or at least 100-fold
compared to an AAV particle comprising the corresponding parental or
unmodified AAV capsid protein.
[0204] In some embodiments, the increase in retinal cell infectivity of a rAAV
variant, e.g.,
rAAV2.7m8, is between 10-fold to 100-fold, between 10-fold to 95-fold, between
10-fold to 90-fold,
between 10-fold to 85-fold, between 10-fold to 80-fold, between 10-fold to 75-
fold, between 10-fold to
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70-fold, between 10-fold to 65-fold, between 10-fold to 60-fold, between 10-
fold to 55-fold, between 10-
fold to 50-fold, between 10-fold to 45-fold, between 10-fold to 40-fold,
between 10-fold to 35-fold,
between 10-fold to 30-fold, between 10-fold to 25-fold, between 10-fold to 20-
fold, or between 10-fold to
15-fold, as compared to an AAV particle comprising the corresponding parental
or unmodified AAV
capsid protein.
102051 In some embodiments, the increase in retinal cell infectivity is
between 2-fold to 20-fold,
between 2-fold to 19-fold, between 2-fold to 18-fold, between 2-fold to 17-
fold, between 2-fold to 16-
fold, between 2-fold to 15-fold, between 2-fold to 14-fold, between 2-fold to
13-fold, between 2-fold to
12-fold, between 2-fold to 11-fold, between 2-fold to 10-fold, between 2-fold
to 9-fold, between 2-fold to
8-fold, between 2-fold to 7-fold, between 2-fold to 6-fold, between 2-fold to
5-fold, between 2-fold to 4-
fold, or between 2-fold to 3-fold, as compared to an AAV particle comprising
the corresponding parental
or unmodified AAV capsid protein.
[0206] In some embodiments, an amino acid modification of a capsid protein
described herein can
confer an increase in an ability to cross an internal limiting membrane (ILM)
in an eye of an individual,
e.g., a human, as compared to the ability of an AAV particle comprising the
corresponding parental or
unmodified AAV capsid protein to cross the ILM in the eye of the subject. In
some embodiments, the
increase in the ability to cross the ILM of a rAAV variant, e.g., rAAV2.7m8,
is an increase of any of at
least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at
least 80%, at least 90%, or at least 100% as compared to an AAV particle
comprising the corresponding
parental or unmodified AAV capsid protein. In some embodiments, the increase
in the ability to cross the
ILM is an increase of between 5% to 100%, between 5% to 95%, between 5% to
90%, between 5% to
85%, between 5% to 80%, between 5% to 75%, between 5% to 70%, between 5% to
65%, between 5% to
60%, between 5% to 55%, between 5% to 50%, between 5% to 45%, between 5% to
40%, between 5% to
35%, between 5% to 30%, between 5% to 25%, between 5% to 20%, between 5% to
15%, or between 5%
to 10%, as compared to the parental or unmodified AAV capsid protein.
[0207] In some embodiments, the increase in the ability to cross the ILM of a
rAAV variant, e.g.,
rAAV2.7m8, is any of at least 1-fold, at least 1.1-fold, at least 1.2-fold, at
least 1.3-fold, at least 1.4-fold,
at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at
least 1.9-fold, or at least 2-fold
compared to an AAV particle comprising the corresponding parental AAV capsid
protein. In some
embodiments, the increase in the ability to cross the ILM is any of at least 2-
fold, at least 3-fold, at least 4-
fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at
least 9-fold, or at least 10-fold, as
compared to an AAV particle comprising the corresponding parental AAV capsid
protein. In some
embodiments, the increase in the ability to cross the ILM is any of at least
15-fold, at least 20-fold, at least
25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-
fold, at least 50-fold, at least 55-fold,
at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at
least 80-fold, at least 85-fold, at least
90-fold, or at least 100-fold compared to an AAV particle comprising the
corresponding parental or
unmodified AAV capsid protein.
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[0208] In some embodiments, the increase in the ability to cross the ILM of a
rAAV variant, e.g.,
rAAV2.7m8, is between 10-fold to 100-fold, between 10-fold to 95-fold, between
10-fold to 90-fold,
between 10-fold to 85-fold, between 10-fold to 80-fold, between 10-fold to 75-
fold, between 10-fold to
70-fold, between 10-fold to 65-fold, between 10-fold to 60-fold, between 10-
fold to 55-fold, between 10-
fold to 50-fold, between 10-fold to 45-fold, between 10-fold to 40-fold,
between 10-fold to 35-fold,
between 10-fold to 30-fold, between 10-fold to 25-fold, between 10-fold to 20-
fold, or between 10-fold to
15-fold as compared to an AAV particle comprising the corresponding parental
or unmodified AAV
capsid protein.
[0209] In some embodiments, the increase in the ability to cross the ILM of a
rAAV variant, e.g.,
rAAV2.7m8, is between 2-fold to 20-fold, between 2-fold to 19-fold, between 2-
fold to 18-fold, between
2-fold to 17-fold, between 2-fold to 16-fold, between 2-fold to 15-fold,
between 2-fold to 14-fold,
between 2-fold to 13-fold, between 2-fold to 12-fold, between 2-fold to 11-
fold, between 2-fold to 10-
fold, between 2-fold to 9-fold, between 2-fold to 8-fold, between 2-fold to 7-
fold, between 2-fold to 6-
fold, between 2-fold to 5-fold, between 2-fold to 4-fold, or between 2-fold to
3-fold, as compared to an
AAV particle comprising the corresponding parental or unmodified AAV capsid
protein.
[0210] In some embodiments, rAAV.7m8 comprising nucleic acid encoding
aflibercept is used for
gene therapy. In some embodiments, AAV2 or rAAV2 is used to deliver a nucleic
acid sequence
encoding an anti-VEGF agent (e.g., aflibercept) into an eye or retinal cells
of a subject via intravitreal or
subretinal injection. In some embodiments, AAV2 or rAAV2 is used to deliver a
nucleic acid sequence
encoding an anti-VEGF agent (e.g., aflibercept) into an eye or retinal cells
of a subject via intravitreal
injection. In some embodiments, rAAV2.7m8 is used to deliver the nucleic acid
sequence of the anti-
VEGF agent (e.g., aflibercept) into the retinal cells of a subject. In some
embodiments, the heterologous
nucleic acid (e.g., a nucleic acid that encodes an anti-VEGF agent such as
aflibercept) integrates into the
target cell genome (e.g., retinal cell genome), resulting in long-term
expression of, e.g., the anti-VEGF
agent (such as aflibercept), in the target cell. In some embodiments, the
viral vector delivers a plasmid or
other extrachromosomal genetic element that comprises the heterologous nucleic
acid (e.g., a nucleic acid
that encodes an anti-VEGF agent such as aflibercept) to the target cell (e.g.,
retinal cell).
[0211] In some embodiments, the rAAV particles comprise a nucleic acid
encoding a polypeptide
comprising an amino acid sequence with any of at least about 95%, at least
about 96%, at least about 97%,
at least about 98%, at least about 99%, or at least about 100% identity to the
amino acid sequence of SEQ
ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some
embodiments, the rAAV
particles comprise a nucleic acid encoding a polypeptide comprising an amino
acid sequence with at least
about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by
AAV2 inverted terminal
repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic
acid encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2
inverted terminal repeats
(ITRs). In some embodiments, the rAAV particles comprise a nucleic acid
encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the
rAAV particles
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comprise a nucleic acid encoding aflibercept and flanked by AAV2 inverted
terminal repeats (ITRs). The
sequence of SEQ ID NO: 35 is provided below:
S DTGRP FVEMY SE IPEII HMT EGRELVI PCRVT S PNITVTLKKFPLDTL I P DGKRI IWDSRKG F
I I SNATY
KEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPS
SKHQHKKLVNRDLETQSGSEMKKELSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPP
CPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLITVLHQDWLINGKEYKOKVSNKALIPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPG (SEQ ID NO: 35)
102121 In some embodiments, the rAAV particles comprise a nucleic acid with
any of at least about
75%, at least about 80%, at least about 81%, at least about 82%, at least
about 83%, at least about 84%, at
least about 85%, at least about 86%, at least about 87%, at least about 88%,
at least about 89%, at least
about 90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about
95%, at least about 96%, at least about 97%, at least about 98%, at least
about 99%, at least about 993%,
or at least about 100% sequence homology to the nucleic acid sequence of SEQ
ID NO: 36, and wherein
the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs). The
sequence of SEQ ID NO: 36 is
provided in FIG. 5. In some embodiments, the rAAV particles comprise a nucleic
acid with any of at
least about 75%, at least about 80%, at least about 81%, at least about 82%,
at least about 83%, at least
about 84%, at least about 85%, at least about 86%, at least about 87%, at
least about 88%, at least about
89%, at least about 90%, at least about 91%, at least about 92%, at least
about 93%, at least about 94%, at
least about 95%, at least about 96%, at least about 97%, at least about 98%,
at least about 99%, at least
about 99.9%, or at least about 100% sequence homology to the nucleic acid
sequence of aflibercept (e.g.,
SEQ ID NO: 36), and wherein the nucleic acid is flanked by AAV2 inverted
terminal repeats (ITRs). In
some embodiments, the nucleic acid sequence of aflibercept is derived from its
amino acid sequence. In
some embodiments, the nucleic acid sequence of aflibercept is codon optimized
to improve its expression
in a subject. In some embodiments, the rAAV particles comprise a nucleic acid
with any of at least about
75%, at least about 80%, at least about 81%, at least about 82%, at least
about 83%, at least about 84%, at
least about 85%, at least about 86%, at least about 87%, at least about 88%,
at least about 89%, at least
about 90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about
95%, at least about 96%, at least about 97%, at least about 98%, at least
about 99%, at least about 993%,
or 100% sequence homology to the nucleic acid sequence of SEQ ID NO: 40, and
wherein the nucleic
acid is flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments,
the rAAV particles
comprise a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 40.
In some embodiments,
the rAAV particles comprise a nucleic acid comprising the nucleic acid
sequence of SEQ ID NO: 40, and
wherein the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs).
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atggtgtcatactgggatactggagtcttgctttgtgccctgctgtcctgcctcctcctgactggctccag
ctcgggctcagataccggtcgccocttcgtggagatgtactccgagatcccggaaattatccacatgactg
aggggcgcgaacttgtgatcccctgccgggtcaccagcccgaacattactgtgactttgaagaagttcccc
ctggacaccctgattccggatgggaagagaattatctgggattcacggaagggattcatcatcagcaacgc
gacctacaaggaaattggcctcctcacttgcgaagccactgtgaacggacacttgtacaagaccaactacc
tgacccaccgccagaccaacaccatcatcgacgtcgtcctgtccccttcgcacgggatcgagctctcggtg
ggagagaagttggtgcttaactgcaccgcccggacggaactgaatgtgggaatcgacttcaactgggaata
cccgtccagcaagcatcagcataagaagctggtgaaccgggacctcaagactcagtccggcagegaaatga
agaagttcctgtcgaccctcactattgacggagtgaccagatccgaccagggcctctacacttgcgccgct
tccagcggactcatgaccaagaagaacagcactttcgtgagggtgcatgagaaggacaagacccacacgtg
tccgccgtgcccagccccagagctgctgggaggcccttccgtgttcctgtttccgcccaagccaaaggat
accctgatgatctcaaggacccctgaggtcacatgcgtcgtggtggatgtgtcgcacgaggaccctgaagt
caaattcaattggtatgtggacggagtggaagtccacaacgcgaaaaccaagccgagagaagaacagtaca
attccacctaccgggtggtgtcggtgctgactgtgctgcaccaggactggctcaacggaaaggagtacaag
tgcaaggtgtccaacaaggctctgcccgcacctattgaaaagaccatctccaaggccaagggtcaacctcg
cgagcctcaggtgtacactctgcctccaagccgggacgaactgactaagaaccaagtctctotgacctgtt
tggtgaagggcttctacccgtcagacatcgcagtggagtgggagtcaaacggtcagccggagaacaactac
aaaacaaccccccccgtgctggactccgacggctccttcttcctgtactccaagcttaccgtggataagag
ccgctggcaacagggcaacgtgttttcctgctccgtcatgcacgaagccctgcacaaccattatacccaga
agtccctgtcgctgtcccccgggaaatag (SEQ ID NO: 40)
102131 In some embodiments, the rAAV particles comprise a nucleic acid
encoding a polypeptide
comprising an amino acid sequence with any of at least about 95%, at least
about 96%, at least about 97%,
at least about 98%, at least about 99%, or 100% identity to the amino acid
sequence of SEQ ID NO: 41
and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the
rAAV particles
comprise a nucleic acid encoding a polypeptide comprising an amino acid
sequence with at least about
95% identity to the amino acid sequence of SEQ ID NO: 41 and flanked by AAV2
inverted terminal
repeats (ITRs). In some embodiments, The rAAV particles comprise a nucleic
acid encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO: 41 and flanked by AAV2
inverted terminal repeats
(ITRs). In some embodiments, the rAAV particles comprise a nucleic acid
encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO: 41.
MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFP
LDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSV
GEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAA
SSGLMTKKNSTEVRVHEKDKTHTCPPCRAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
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EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWOQGNVFSCSVMHEALHNHYTOKSLSLSPCK (SEQ ID NO: 41)
102141 In some embodiments, the nucleic acid sequence of aflibercept is codon-
optimized for
expression in a primate or a human subject. Construction of a synthetic gene
corresponding to the
aflibercept amino acid sequence has been described in literature, e.g., ICanda
A, Noda K, Saito W, Ishida
S. Aflibercept Traps Galectin-1, an Angiogenic Factor Associated with Diabetic
Retinopathy. Scientific
Reports 5:17946 (2015) (describing "VEGF-Traptuitz (corresponding to
aflibercept) cDNA was generated
as a synthetic gene by IDT (Coralville, IA)"). Given the available amino acid
sequence of aflibercept, any
method known in the art can be used to generate the cDNA of aflibercept for
use in a gene therapy or a
rAAV described herein.
[0215] Codon optimization can be achieved with any method known in the art.
Codon optimization
refers to a process of modifying a nucleic acid sequence for enhanced
expression of a gene in target or
host cells of interest, e.g., human retinal cells, by replacing at least one
codon (e.g., about or more than 1,
2, 3, 4, 5, 10, 15, 20, 25, 50, 100 or more codons) of a native sequence with
codons that are used more
frequently or are most frequently used in the host cell while maintaining the
native amino acid sequence.
Codon usage tables are readily available, including for examples, GenScript
Codon Usage Frequency
Table Tool at www(dot)genscript(dot)comitools/codon-frequency-table; Codon
Usage Database at
www(doOkazusa(dot)or(dot)jp/codont; and Nakamura, Y., et at. "Codon triage
tabulated from the
international DNA sequence databases: status for the year 2000" Nucl. Acids
Res. 28:292 (2000).
[0216] Homology refers to the percent conservation of residues of an alignment
between two sequences,
including, but not limited to functional fragments, sequences comprising
insertions, deletions,
substitutions, pseudofrag;ments, pseudogenes, splice variants or artificially
optimized sequences.
[0217] In some embodiments, the rAAV particles comprise a nucleic acid
encoding aflibercept. In some
embodiments, the polypeptide is aflibercept.
[0218] As used herein, "aflibercept" refers to a polypeptide or protein
sequence, or a functional
fragment or variant or mutant thereof, with any of at least 75%, 80%, 81%,
82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or
100% homology to
the aflibercept amino acid sequence identified above (SEQ NO: 35). Homology
refers to the percent
conservation of residues of an alignment between two sequences, including, but
not limited to functional
fragments, sequences comprising insertions, deletions, substitutions,
pseudofragments, pseudogenes,
splice variants or artificially optimized sequences.
[0219] In some embodiments, the amino acid sequence of aflibercept is any of
at least 75%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%,
99.9%, or 100% homologous to the aflibercept amino acid sequence of SEQ NO:
35. In some
embodiments, the nucleic acid sequence encoding aflibercept disclosed herein
is compared to the
corresponding cDNA sequence of the aflibercept amino acid sequence identified
above, and shows any of
at least 75%, 80%, 810/u, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%,
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96%, 97%, 98%, 99%, 99.9%, or 100 4 sequence homology between the nucleic acid
sequences of
aflibercept (e.g., SEQ ID NO: 36). In some embodiments, aflibercept is any of
at least 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% spatially
homologous to aflibercept
(e.g., in terms of its secondary, tertiary, and quaternary structure or
conformation). In some embodiments,
aflibercept is any of at most 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, 99.9%,
or 100% spatially homologous to the aflibercept used in the standard of care
(e.g., secondary, tertiary, and
quaternary structure or conformation).
102201 In some embodiments, the aflibercept gene product, or aflibercept
transgene, as included in a
gene therapy based on a rAAV, comprises a capsid variant as disclosed herein
(e.g., the 7m8 variant),
encodes a protein, fusion protein, or polypeptide that has any of at least
75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least
97%, at least 98%, at least 99%, or at least 100% homology to the above amino
acid sequence of SEQ 113
NO: 35, or between the corresponding cDNA sequences of aflibercept (e.g., cDNA
of aflibercept
sequence used in a gene therapy compared to SEQ ID NO: 36). In some
embodiments, the methods
compositions disclosed herein comprise a functional fragment of aflibercept,
or a variant or mutant
thereof In some embodiments, the nucleic acid sequence of aflibercept is
modified or codon-optimized to
enhance its activity, expression, stability, and/or solubility in vivo.
102211 Aflibercept is a 115 kDa fusion protein, which can be glycosylated.
Aflibercept comprises an
IgG backbone fused to extracelltdar VEGF receptor sequences of the human VEGFR-
1 and VEGFR-2,
and functions like a soluble decoy receptor by binding VEGF-A with a greater
affinity than its natural or
endogenous receptors. See, for example, Stewart MW. Aflibercept (VEGF Trap-
eye): the newest anti-
VEGF drug. Br. J. Ophthalrnol. 2012 Sep;96(9):1157-8. Aflibercept's high
affinity for VEGF interferes or
disrupts subsequent binding and activation of native or endogenous VEGF
receptors. Reduced VEGF
activity can lead to decreased angiogenesis and vascular permeability.
Inhibition of placental growth
factor PIGF and VEGF-B by aflibercept may also contribute to the treatment of
ocular diseases or
disorders characterized by abnormal (e.g., excessive) angiogenesis and/or
neovascularization. PIGF has
been associated with angiogenesis and certain ocular diseases or disorders,
such as wet AMD, may be
associated with elevated levels of PIGF. VEGF-B overexpression can be
associated with breakdown of
the blood-retinal barrier and retinal angiogenesis. Thus, inhibition of VEGF-
A, VEGF-B, and PIGF may
all contribute to the efficacy of aflibercept.
Methods for Preparation of Vectors for Delivering Trans genes to Target Cells
102221 In some embodiments, the rAAV particles are manufactured using any
method known in the art.
In some embodiments, the rAAV particles are manufactured using a baculovirus
expression vector system
in Sf9 cells. Sf9 cells are an insect cell culture cell line commonly used for
recombinant protein
production using baculovirus. In some embodiments, the rAAV particles are
manufactured using two
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baculoviruses in Sf9 cells. In some embodiments, the rAAV particles are
manufactured using two
baculoviruses in Sf9 cells, wherein a first baculovirus encodes the genes for
AAV2 Rep and AAV2.7m8
Cap proteins and a second baculovirus encodes an anti-VEGF agent. In some
embodiments, the rAAV
particles are manufactured using two baculoviruses in Sf9 cells, wherein a
first baculovirus encodes the
genes for AAV2 Rep and AAV2.7m8 Cap proteins and a second baculovirus encodes
an aflibercept (e.g.,
human aflibercept) cDNA expression cassette. In some embodiments, the rAAV
particles are
manufactured using two baculoviruses in Sf9 cells, wherein a first baculovirus
encodes the genes for
AAV2 Rep and AAV2.7m8 Cap proteins and a second baculovirus comprises a
nucleic acid encoding a
polypeptide comprising an amino acid sequence with at least about 95% identity
to the amino acid
sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats
(1TRs). In some
embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:
35. In some
embodiments, the polypeptide is aflibercept.
Doses
102231 In some embodiments, the unit dose of rAAV particles is administered to
one eye of the
individual. In some embodiments, the one eye of the individual is the right
eye or the left eye. hi some
embodiments, the one eye of the individual is the right eye. In some
embodiments, the one eye of the
individual is the left eye. In some embodiments, the methods provided herein
further comprise
administering a unit dose of rAAV particles to the contralateral eye of the
individual. In some
embodiments, the one eye of the individual is the right eye and the
contralateral eye is the left eye. In
some embodiments, the one eye of the individual is the left eye and the
contralateral eye is the right eye.
102241 In some embodiments, the administering the unit dose of rAAV particles
to the contralateral eye
is at least about 2 weeks (e.g., at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at
least about 1 month, at least about 2 months, at least about 3 months, at
least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9
months, at least about 10 months, at least about 11 months, at least about 12
months, at least about 1 year,
at least about 2 years, at least about 3 years, at least about 4 years, at
least about 5 years, or more) after
administering the unit dose of rAAV particles to the one eye. In some
embodiments, the administering the
unit dose of rAAV particles to the contralateral eye is at least about 2 weeks
after administering the unit
dose of rAAV particles to the one eye and the unit dose of rAAV particles
administered to the
contralateral eye of the individual is higher (e.g., any of about 5%, about
10%, about 20%, about 30%,
about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%,
about 125%, about
150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%,
or more, higher)
than the unit dose of rAAV particles administered to the one eye of the
individual.
102251 In some embodiments, the administering the unit dose of rAAV particles
to the contralateral eye
of the individual is up to about 1 week, up to about 2 weeks, up to about 3
weeks, or up to about 4 weeks
after administering the unit dose of rAAV particles to the one eye. In some
embodiments, the
administering the unit dose of MAY particles to the contralateral eye of the
individual is up to about 2
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weeks (e.g., about 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7
days, 8 days, 9 days, 10 days, 11
days, 12 days, 13 days, or 14 days) after administering the unit dose of rAAV
particles to the one eye. In
some embodiments, the administering the unit dose of rAAV particles to the
contralateral eye of the
individual is up to about 2 weeks (e.g., about 0 days, 1 day, 2 days, 3 days,
4 days, 5 days, 6 days, 7 days,
8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days) after
administering the unit dose of rAAV
particles to the one eye and the unit dose of rAAV particles administered to
the contralateral eye of the
individual is about the same as (e.g., less than 1% higher or lower, less than
5% higher or lower, less than
10% higher or lower, or less than 20% higher or lower), or lower (e.g., about
5%, about 10%, about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%
lower) than the unit
dose of rAAV particles administered to the one eye of the individual. In some
embodiments, the
administering the unit dose of rAAV particles to the contralateral eye of the
individual is up to about 2
weeks after administering the unit dose of rAAV particles to the one eye and
the unit dose of rAAV
particles administered to the contralateral eye of the individual is about the
same (e.g., less than 1% higher
or lower, less than 5% higher or lower, less than 10% higher or lower, or less
than 20% higher or lower)
as the unit dose of rAAV particles administered to the one eye of the
individual. In some embodiments,
the administering the unit dose of rAAV particles to the contralateral eye of
the individual is up to about
2 weeks after administering the unit dose of rAAV particles to the one eye and
the unit dose of rAAV
particles administered to the contralateral eye of the individual is lower
(e.g., about 5%, about 10%, about
20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or
about 90% lower) than
the unit dose of rAAV particles administered to the one eye of the individual.
102261 In some embodiments, the unit dose of rAAV particles is administered to
one eye and/or to the
contralateral eye of the individual. In some embodiments, the unit dose of
rAAV particles is expressed as
the number of vector genomes (vg). In some embodiments, the unit dose is about
6 x 10" vector genomes
(vg) or less of the rAAV particles. In some embodiments, the unit dose is
about lx101 to about 2x 1010
,
between about 2x 1010 to about 3x10' , between about 3 x101 to about 4 x101 ,
between about 4 x101 to
about 5x 10' , between about 5 x10 w to about 6 x10' , between about 6x101 to
about 7x101 , between
about 7x101 to about 8x10' , between about 8x10' to about 9x10' , between
about 9x101 to about
x101 , between about lx 10" to about 2 x10", between about 2x 10" to about 3x
10", between about
3x 10" to about 4x10", between about 4x 10" to about 5x10", or between about
5x 10" to about 6x 10"
vg of the rAAV particles, including any value within these ranges, of the rAAV
particles. In some
embodiments, the unit dose is about 6x low vector genomes (vg) to about 2x10"
vg of the rAAV
particles. In some embodiments, the unit dose is about 6x 1010 vg to about 2x
10" vg, about 7x 101 vg to
about 2x 1011vg, about 8x 101 vg to about 2x10" vg, about 9x101 vg to about
2x10" vg, about 10x101
vg to about 2x 10" vg, or about lx loll vg to about 2x 1011 vg of the rAAV
particles. In some
embodiments, the unit dose is about 6x101 vg to about 2x10" vg of the rAAV
particles. In some
embodiments, the unit dose is about 6x101 vg to about 7x101 vg, about 7x lOw
vg to about 8x 1010 vg,
about 8x 1010 vg to about 9x 1010 vg, about 9x 1010 vg to about 10x 1010 vg,
about 10x10w vg to about
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lx 101' vg, or about lx 1011 vg to about 2x 101' vg of the rAAV particles. In
some embodiments, the unit
dose is about 6 x 10" vg, about 7 x101 vg, about 8 x 1010 vg, about 9x 10'
vg, about 10x 10' vg, about
lx 10" vg, or about 2 x1011 vg of the rAAV particles. In some embodiments, the
unit dose is about 6 x101
vg or about 2 x10" vg of the rAAV particles. In some embodiments, the unit
dose is about 6x 1010 vg of
the rAAV particles. In some embodiments, the unit dose is about 6 x 1010 vg,
about 2 x 10" vg, or about
6x 10" vg. In some embodiments, the unit dose is about 6 x 1010 vg. In some
embodiments, The unit dose
is about 2 x 10" vg. In some embodiments, the unit dose is about 6x 10" vg.
102271 In some embodiments, the unit dose of rAAV particles is administered to
one eye and/or to the
contralateml eye of the individual. In some embodiments, the unit dose is
expressed as the number of
vector genomes (vg) per eye (vg/eye). In some embodiments, the unit dose is
about 6 x 10" vg/eye or less
of the rAAV particles. In some embodiments, the unit dose is about lx 10' to
about 2x 1010, between
about 2x 10' to about 3x 1010, between about 3 x10" to about 4x 1010, between
about 4x 10" to about
5x 1010, between about 5 x101 to about 6x 1010, between about 6x 1010 to
about 7 X 101 , between about
7x 1010 to about 8x10' , between about 8X 1010
to about 9x10' , between about 9x10' to about 10x 1010,
between about lx 10" to about 2 x 10", between about 2 x10" to about 3 x1011,
between about 3x 10" to
about 4x 10", between about 4x1011 to about 5 x10", or between about 5x 1011
to about 6x 10" vg/eye of
the rAAV particles, including any value within these ranges, of the rAAV
particles. In some
embodiments, the unit dose is about 6x101 vg/eye to about 2x 1011 vg/eye of
the rAAV particles. In some
embodiments, the unit dose is about 6x101 vg/eye to about 2x 10" vg/eye,
about 7x 101 vg/eye to about
2x 10" vg/eye, about 3x low vg/eye to about 2x 10 "vg/eye, about 9x low vg/eye
to about 2x 101' vg/eye,
about 10x 10" vg/eye to about 2x 1011 vg/eye, or about lx loll vg/eye to about
2 x10" vg/eye of the rAAV
particles. In some embodiments, the unit dose is about 6x 1010 vg/eye to about
2 x10" vg/eye of the rAAV
particles. In some embodiments, the unit dose is about 6x 1010 vg/eye to about
7x 101 vg/eye, about 7x101
vg/eye to about 8 x101 vg/eye, about 8x 101 vg/eye to about 9x 10" vg/eye,
about 9x HP vg/eye to about
lox 101 vg/eye, about 10x 10" vg/eye to about lx 10 vg/eye, or about lx 10"
vg/eye to about 2x10"
vg/eye of the rAAV particles. In some embodiments, the unit dose is about 6x
10' vg/eye, about 7x 1010
vgjeye, about 8x 1010 vg/eye, about 9x 1010 vg/eye, about 10x10' vg/eye, about
lx 10" vg/eye, or about
2 x10" vg/eye of the rAAV particles. In some embodiments, the unit dose is
about 6x 10" vg/eye or about
2 x10" vg/eye of the rAAV particles. In some embodiments, the unit dose is
about 6x 1010 vg/eye of the
rAAV particles. In some embodiments, the unit dose is about 6 x 10" vg/eye,
about 2 x 10" vg/eye, or
about 6x 10" vg/eye. In some embodiments, the unit dose is about 6 x 10"
vgkye. In some embodiments,
the unit dose is about 2 x 10" vg/eye. In some embodiments, the unit dose is
about 6x 10" vg/eye.
[0228] In some embodiments, the unit dose of rAAV particles is administered to
one eye and/or to the
contralateral eye of the individual. In some embodiments, E is a shorthand for
base 10 for exponentiation,
and xEy refers to x multiplied by base 10 to the y power/exponent. In some
embodiments, the unit dose is
expressed as the number of vector genomes (vg). In some embodiments, the unit
dose is about 6E" vector
genomes (vg) or less of the rAAV particles. In some embodiments, the unit dose
is about lErn to about
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2E", between about 2E1 to about 3E", between about 3E" to about 4E", between
about 4E" to about
5E", between about 5E1 to about 6E1 , between about 6E" to about 7E", between
about 7E1 to about
8E", between about 8E" to about 9E', between about 9E" to about 10E", between
about 1E" to about
2E", between about 2E" to about 3E", between about 3E" to about 4E", between
about 4E" to about
5E", or between about 5E" to about 6E" vg of the rAAV particles, including any
value within these
ranges, of the rAAV particles. In some embodiments, the unit dose is about 6E1
vector genomes (vg) to
about 2E" vg of the rAAV particles. In some embodiments, the unit dose is
about 6E" vg to about 2E"
vg, about 7E1 vg to about 2E" vg, about 8E" vg to about 2E" vg, about 9E1 vg
to about 2E" vg, about
10E" vg to about 2E" vg, or about 1E" vg to about 2E" vg of the rAAV
particles. In some embodiments,
the unit dose is about 6E' vg to about 2E" vg of the rAAV particles. In some
embodiments, the unit dose
is about 6E" vg to about 7E" vg, about 7E" vg to about 8E1 vg, about 8E" vg
to about 9E" vg, about
9Eio vg to about 10E" vg, about 10E" vg to about 1E" vg, or about 1E" vg to
about 2E" vg of the rAAV
particles. In some embodiments, the unit dose is about 6E1 vg, about 7E" vg,
about 8E" vg, about 9E1
vg, about 10E1 vg, about 1E" vg, or about 2E" vg of the rAAV particles. In
some embodiments, the unit
dose is about 6E1 vg or about 2E" vg of the rAAV particles. In some
embodiments, the unit dose is about
6E1 vg of the rAAV particles. In some embodiments, the unit dose is about 6E"
vg, about 2E" vg, or
about 6E11 vg. In some embodiments, the unit dose is about 6E1 vg. In some
embodiments, the unit dose
is about 2E" vg. In some embodiments, the unit dose is about 6E" vg.
102291 In some embodiments, the unit dose of rAAV particles is administered to
one eye and/or to the
contralateral eye of the individual. In some embodiments, the unit dose is
expressed as the number of
vector genomes (vg) per eye (vg/eye). In some embodiments, the unit dose is
about 6E" vg/eye or less of
the rAAV particles. In some embodiments, the unit dose is about 1E" to about
2E', between about 2E"
to about 3E', between about 3E' to about 4E', between about 4E" to about 5E1
, between about 5E" to
about 6E1 , between about 6E" to about 7E", between about 7E' to about 8E",
between about SE ' to
about 9E1 , between about 9E1 to about 10E", between about 1E" to about 2E",
between about 2E" to
about 3E", between about 3E" to about 4E", between about 4E" to about 5E", or
between about 5E" to
about 6E" vg/eye of the rAAV particles, including any value within these
ranges, of the rAAV particles.
In some embodiments, the unit dose is about 6E1 vg/eye to about 2E" vg/eye of
the rAAV particles. In
some embodiments, the unit dose is about 6E1 vg/eye to about 2E" vg/eye,
about 7E" vg/eye to about
2E" vg/eye, about 8E" vg/eye to about 2E" vg/eye, about 9E" vg/eye to about
2E" vg/eye, about 10E"
vg/eye to about 2E" vg/eye, or about 1E" vg/eye to about 2E" vg/eye of the
rAAV particles. In some
embodiments, the unit dose is about 6E" vg/eye to about 2E" vg/eye of the rAAV
particles. In some
embodiments, the unit dose is about 6E" vg/eye to about 7E" vg/eye, about 7E1
vg/eye to about 8E"
vg/eye, about 8E1 vg/eye to about 9E" vg/eye, about 9E1 vg/eye to about 10E1
vg/eye, about 10E10
vg/eye to about 1E" vg/eye, or about lE" vg/eye to about 2E" vg/eye of the
rAAV particles. In some
embodiments, the unit dose is about 6E1 vg/eye, about 7E" vg/eye, about 8E"
vg/eye, about 9E" vg/eye,
about 10E" vg/eye, about 1E" vg/eye, or about 2E" vg/eye of the rAAV
particles. In some embodiments,
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the unit dose is about 6E'' vg/eye or about 2E" vg/eye of the rAAV particles.
In some embodiments, the
unit dose is about 6W vg/eye of the rAAV particles. In some embodiments, the
unit dose is about 6E'
vgkye, about 2E" vg/eye, or about 6E" vg/eye. In some embodiments, the unit
dose is about 6E' vg/eye..
In some embodiments, the unit dose is about 2E" vg/eye. In some embodiments,
the unit dose is about
6E" vg/eye.
[0230] In some embodiments, the unit dose of rAAV particles is administered to
one eye and/or to the
contralateral eye of the individual. In some embodiments, the unit dose of
rAAV particles is a unit dose
sufficient to cause expression of the therapeutic protein (e.g., an anti-VEGF
agent such as aflibercept) in
the vitreous fluid. In some embodiments, the unit dose of rAAV particles is a
unit dose sufficient to
achieve a concentration of the therapeutic protein (e.g., an anti-VEGF agent
such as aflibercept) at about
any one of 3, 3.5, 4, 4.5, 5, 5.5, 6, 63,7, 7.5, 8, 8.5, 9, 9.5, 10 pg/ml, or
more, including any range in
between these values, in the vitreous fluid. In some embodiments, the unit
dose of rAAV particles is a unit
dose sufficient to cause expression of aflibercept in the vitreous fluid. In
some embodiments, the unit dose
of rAAV particles is a unit dose sufficient to achieve a concentration of
aflibercept at about any one of 3,
3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10 tg/ml, or more,
including any range in between these
values, in the vitreous fluid.
[0231] In some embodiments, the unit dose of rAAV particles administered to
the one eye and/or to the
contralateral eye of the individual is a unit dose sufficient to cause
expression of the therapeutic protein
(e.g., an anti-VEGF agent such as aflibercept) in the aqueous fluid. In some
embodiments, the unit dose of
rAAV particles is a unit dose sufficient to achieve a concentration of the
therapeutic protein (e.g., an anti-
VEGF agent such as aflibercept) of at least about 0.5, 0.55, 0.6, 0.65, 0.7,
0.75, 0.8, 0.85, 0.9, 0.95, 1.0
pg,/ml, or more, including any range in between these values, in the aqueous
fluid. In some embodiments,
the unit dose of rAAV particles is a unit dose sufficient to cause expression
of aflibercept in the aqueous
fluid. In some embodiments, the unit dose of rAAV particles is a unit dose
sufficient to achieve a
concentration of aflibercept of at least about 0.5, 0.55, 0.6, 0.65, 0.7,
0.75, 0.8, 0.85, 0.9, 0.95, 1.0 Wm!,
or more, including any range in between these values, in the aqueous fluid.
[0232] In some embodiments, the unit dose of rAAV particles is administered to
one eye and/or to the
contralateral eye of the individual. In some embodiments, the unit dose of
rAAV particles is a unit dose
sufficient to cause expression of the therapeutic protein (e.g., an anti-VEGF
agent such as aflibercept) in
the retina. In some embodiments, the unit dose of rAAV particles is a unit
dose sufficient to achieve a
concentration of the therapeutic protein (e.g., an anti-VEGF agent such as
aflibercept) of at least about 3,
33, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10 Reg, or more, including
any range in between these
values, in the retina. In some embodiments, the unit dose of rAAV particles is
a unit dose sufficient to
cause expression of aflibercept in the retina. In some embodiments, the unit
dose of rAAV particles is a
unit dose sufficient to achieve a concentration of aflibercept of at least
about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7,
7.5, 8, 8.5, 9, 9.5, 10 pg/g, or more, including any range in between these
values, in the retina.
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102331 In some embodiments, the unit dose of rAAV particles is administered to
one eye and/or to the
contralateral eye of the individual. In some embodiments, the unit dose of
rAAV particles is a unit dose
sufficient to cause expression of the therapeutic protein (e.g., an anti-VEGF
agent such as aflibercept) in
the choroid. In some embodiments, the unit dose of rAAV particles is a unit
dose sufficient to achieve a
concentration of the therapeutic protein (e.g., an anti-VEGF agent such as
aflibercept) at about any one of
3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10 tigig, or more,
including any range in between these
values, in the choroid. ht some embodiments, the unit dose of rAAV particles
is a unit dose sufficient to
cause expression of aflibercept in the choroid. In some embodiments, the unit
dose of rAAV particles is a
unit dose sufficient to achieve a concentration of aflibercept at about any
one of 3, 3.5, 4, 4.5, 5, 5.5, 6,
6.5,7, 7.5, 8, 8.5, 9, 9.5, 10 nig, or more, including any range in between
these values, in the choroid.
102341 In some embodiments, the unit dose of rAAV particles is administered to
one eye and/or to the
contralateral eye of the individual. In some embodiments, the unit dose of
rAAV particles is a
therapeutically effective dose.
102351 hi some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if the
unit dose is sufficient to cause maintenance or a decrease of retinal
thickness compared to the retinal
thickness prior to administration of the unit dose of rAAV particles. In some
embodiments, the unit dose
of rAAV particles is a therapeutically effective dose if the unit dose is
sufficient to cause a decrease of
retinal thickness compared to the retinal thickness prior to administration of
the unit dose of rAAV
particles. In some embodiments, retinal thickness is central subfield
thickness (CST) or central retinal
thickness (CRT). In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose
if the unit dose is sufficient to cause a decrease of retinal thickness of
more than any of about 5%, about
10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%,
about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,
about 90%, about
95%, about 99%, or about 100% compared to the retinal thickness prior to
administration of the unit dose
of rAAV particles. In some embodiments, the retinal thickness (e.g., CST or
CRT) is determined by OCT
or SD-OCT.
102361 In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if the
unit dose is sufficient to cause maintenance or a decrease in macular volume
compared to the macular
volume prior to administration of the unit dose of rAAV particles. In some
embodiments, the unit dose of
rAAV particles is a therapeutically effective dose if the unit dose is
sufficient to cause a decrease in
macular volume compared to the macular volume prior to administration of the
unit dose of rAAV
particles. In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if the
unit dose is sufficient to cause a decrease in macular volume of more than any
of about 5%, about 10%,
about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,
about 50%, about
55%, about 60%, or about 65% compared to the macular volume prior to
administration of the unit dose
of rAAV particles. In some embodiments, the macular volume is determined by
OCT or SD-OCT.
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102371 In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if the
unit dose is sufficient to cause maintenance or an improvement of visual
acuity compared to the visual
acuity prior to administration of the unit dose of rAAV particles. In some
embodiments, the unit dose of
rAAV particles is a therapeutically effective dose if the unit dose is
sufficient to cause an improvement of
visual acuity compared to the visual acuity prior to administration of the
unit dose of rAAV particles. In
some embodiments, the unit dose of rAAV particles is a therapeutically
effective dose if the unit dose is
sufficient to cause an improvement of visual acuity of more than any of about
5%, about 10%, about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,
about 100%, about
125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%,
about 300%, or
more compared to the visual acuity prior to administration of the unit dose of
rAAV particles. In some
embodiments, visual acuity is best corrected visual acuity (BCVA). In some
embodiments, the unit dose
of rAAV particles is a therapeutically effective dose if the unit dose is
sufficient to cause an improvement
of BCVA compared to the BCVA prior to administration of the unit dose of rAAV
particles. In some
embodiments, BCVA is expressed as an ETDRS score, which corresponds to the
number of letters
correctly read (Vitale etal., (2016) JAMA Opthalmol 134(9).1041:1047). In some
embodiments, the unit
dose of rAAV particles is a therapeutically effective dose if the unit dose is
sufficient to cause an
improvement of BCVA of at least 15 ETDRS letters (Vitale et al., (2016) JAMA
Opthalmol
134(9):1041:1047) (e.g., at least about 15, at least about 20, at least about
30, at least about 40, at least
about 50, at least about 60, or about 70 letters) compared to the BCVA prior
to administration of the unit
dose of rAAV particles. In some embodiments, the unit dose of rAAV particles
is a therapeutically
effective dose if the unit dose is sufficient to cause maintenance of BCVA,
wherein the individual loses
fewer than 15 ETDRS letters (Vitale etal., (2016) JAMA Opthalmol
134(9):1041:1047) (es., 15 or less,
14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or
less, 7 or less, 6 or less, 5 or less, 4 or
less, 3 or less, 2 or less, 1, or 0 letters) compared to the BCVA prior to
administration of the unit dose of
rAAV particles.
102381 In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if, after
administration of the unit dose of rAAV particles, the individual is
determined to have maintenance of
vision. In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if, after
administration of the unit dose of rAAV particles, the individual is
determined have an improvement of
vision. In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if, after
administration of the unit dose of rAAV particles, the CST or CRT assessed by
SD-OCT is decreased
compared to prior to administration of the unit dose of rAAV particles. In
some embodiments, the unit
dose of rAAV particles is a therapeutically effective dose if, after
administration of the unit dose of rAAV
particles, the CST or CRT assessed by SD-OCT is maintained compared to prior
to administration of the
unit dose of rAAV particles.
102391 In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if, after
administration of the unit dose of rAAV particles, the macular volume is
decreased compared to prior to
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administration of the unit dose of rAAV particles. In some embodiments, the
unit dose of rAAV particles
is a therapeutically effective dose if, after administration of the unit dose
of rAAV particles, the macular
volume is maintained compared to prior to administration of the unit dose of
rAAV particles.
[0240] In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if, after
administration of the unit dose of MAY particles, the retinal thickness (e.g.,
central retinal thickness
(CRT) or central subfield thickness (CST)) and macular volume are decreased
compared to prior to
administration of the unit dose of rAAV particles. In some embodiments, the
unit dose of rAAV particles
is a therapeutically effective dose if, after administration of the unit dose
of rAAV particles, the retinal
thickness (e.g., central retinal thickness (CRT) or central subfield thickness
(CST)) and macular volume
are maintained compared to prior to administration of the unit dose of rAAV
particles.
[0241] In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if, after
administration of the unit dose of rAAV particles, the individual requires
less than one rescue therapy
treatment (e.g., aflibercept injection) about any of every 4 weeks, every 5
weeks, every 6 weeks, every 7
weeks, every 8 weeks, every 9 weeks, every 10 weeks, or more after
administration of the unit dose of
rAAV particles in the one eye and/or the contralateral eye. In some
embodiments, the unit dose of rAAV
particles is a therapeutically effective dose if, after administration of the
unit dose of rAAV particles, the
individual does not require any rescue therapy treatment (e.g., aflibercept
injection) for at least about any
of 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5
weeks, at least 6 weeks, at least 7
weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 15
weeks, at least 20 weeks, at least 30
weeks, at least 40 weeks, at least 50 weeks, at least 60 weeks, at least 70
weeks, at least 80 weeks, at least
90 weeks, at least 100 weeks, at least 110 weeks, or more.
[0242] In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if, after
administration of the unit dose of rAAV particles, the individual is
determined to have a reduction in
retinal fluid compared to the level of retinal fluid prior to administration
of the unit dose of rAAV
particles in the one eye and/or the contralateral eye. In some embodiments,
the unit dose of rAAV
particles is a therapeutically effective dose if, after administration of the
unit dose of rAAV particles, the
individual is determined to have maintenance in retinal fluid compared to the
level of retinal fluid prior to
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye. In some
embodiments, the unit dose of rAAV particles is a therapeutically effective
dose if, after administration of
the unit dose of rAAV particles, the individual is determined to have a
reduction in IRF and/or SRF in the
one eye and/or the contralateral eye compared to the levels of IRF and/or SRF
prior to administration of
the unit dose of rAAV particles in the one eye and/or the contralateral eye.
[0243] In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if, after
administration of the unit dose of MAY particles, the individual is determined
to have a resolution of
pigment epithelial detachment (PH)) compared to PED prior to administration of
the unit dose of rAAV
particles in the one eye and/or the contralateral eye.
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[0244] In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if, after
administration of the unit dose of rAAV particles, CNV lesions shrink compared
to CNV lesions present
prior to administration of the unit dose of rAAV particles in the one eye
and/or the contralateral eye. In
some embodiments, the unit dose of rAAV particles is a therapeutically
effective dose if, after
administration of the unit dose of MAY particles, CNV lesions shrink by more
than any of about 5%,
10%, 20%, 30%, 40%, 5004, 60%, 70%, 80%, 90%, or 100% compared to CNV lesions
present prior to
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye. In some
embodiments, the unit dose of rAAV particles is a therapeutically effective
dose if, after administration of
the unit dose of rAAV particles, CNV lesions do not grow compared to CNV
lesions present prior to
administration of the unit dose of rAAV particles in the one eye and/or the
contralateral eye. In some
embodiments, the unit dose of rAAV particles is a therapeutically effective
dose if, after administration of
the unit dose of rAAV particles, CNV lesions do not grow by more than about
any of about 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% compared to CNV lesions present prior
to administration of
the unit dose of MAY particles in the one eye and/or the contralateral eye.
[0245] In some embodiments, the unit dose of rAAV particles is a
therapeutically effective dose if, after
administration of the unit dose of rAAV particles, the individual is
determined to have an improvement in
anatomical features of the one eye and/or the contralateral eye compared to
the anatomical features prior
to administration of the unit dose of rAAV particles. In some embodiments, the
unit dose of rAAV
particles is a therapeutically effective dose if, after administration of the
unit dose of rAAV particles, the
individual is determined to have a stabilization and/or maintenance of
anatomical features of the one eye
and/or the contralateral eye compared to the anatomical features prior to
administration of the unit dose of
rAAV particles.
[0246] In some embodiments, the unit dose of rAAV particles is therapeutically
effective if
administration of the dose to the one eye and/or the contralateral eye of the
individual reduces, stops, or
prevents at least one symptom of the ocular neovascular disease or disorder.
In the cases of ocular
neovascular diseases or disorders characterized by abnormal (e.g., excessive)
angiogenesis, such
symptoms include, but are not limited to, e.g., visual distortions (such as
impaired color vision, blurred
vision, deterioration of central vision) and vision loss. In some embodiments,
the unit dose of rAAV
particles administered to the one eye and/or to the contralateral eye of the
individual is a therapeutically
effective dose if administration of the unit dose to the one eye and/or to the
contralateral eye of the
individual results in the maintenance, partial resolution, or complete
resolution of one or more clinical
features of the ocular neovascular disease. For example, the unit dose of rAAV
particles administered to
the one eye and/or to the contralateral eye of the individual is
therapeutically effective if administration of
the dose to the one eye and/or to the contralateral eye of the individual
results in complete resolution,
partial resolution or maintenance of the ocular neovascular disease as
measured by any method known in
the art. In some embodiments, the unit dose of rAAV particles administered to
the one eye and/or to the
contralateral eye of the individual is therapeutically effective if
administration of the dose to the one eye
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and/or to the contralateral eye of the individual results in complete
resolution, partial resolution or
maintenance of the ocular neovascular disease as assessed by best corrected
visual acuity (BCVA) (e.g.,
based on an ETDRS score; Vitale el al., (2016) JAMA Opthalmol
134(9):1041:1047), central retinal
thickness as determined by SD-OCT, the number of rescue therapy treatments
(e.g., aflibercept injections)
required by the individual after administration of the unit dose of rAAV
particles in the one eye and/or the
contralateral eye, the presence of intraretinal fluid (LRF) and/or subretinal
fluid (SRF), the resolution of
pigment epithelial detachment (PED), choroidal neovascularization (CNV) lesion
growth, anatomical
features based on any methods known in the art (e.g., SD-OCT, OCT, fluorescein
angiography, digital
color fundus photography, etc.). In some embodiments, the unit dose of rAAV
particles administered to
the one eye and/or to the contralateral eye of the individual is
therapeutically effective if administration of
the dose to the one eye and/or to the contralateral eye of the individual
results in complete resolution,
partial resolution or maintenance of the ocular neovascular disease as
assessed by ophthalmologic
examination, intraocular pressure (e.g., using a Goldinann applanation
tonometer or Tono-pen), indirect
ophthalmoscopy, examination of the one eye and/or the contralateral eye and
adnexa, eyelid and/or pupil
responsiveness, belpharoptosis, abnormal pupil shape, unequal pupils,
abnornial reaction to light, afferent
pupillary defects, slit-lamp examination (including of the eyelids,
conjunctiva, cornea, lens, iris, and
anterior chamber), posterior segment abnormalities of the vitreous, optic
nerve, peripheral retina, and
retinal vasculature, SD-OCT, fluorescein angiography, digital color fundus
photography (including images
of the retina, optic disc, and/or macula), aqueous humor sampling, vitreous
humor sampling, OCT-
angiography (OCT-A), refraction, and visual acuity (BCVA).
102471 In some embodiments, the unit dose of rAAV particles administered to
the one eye of the
individual is the same as the unit dose of rAAV particles administered to the
contralateral eye of the
individual. In some embodiments, the unit dose of rAAV particles administered
to the one eye of the
individual is different from the unit dose of rAAV particles administered to
the contralateral eye of the
individual. In some embodiments, the unit dose of rAAV particles administered
to the one eye of the
individual is higher, e.g., more than any of about 10%, about 20%, about 30%,
about 40%, about 50%,
about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about
150%, about 175%, about
200%, about 225%, about 250%, about 275%, about 300% or more, than the unit
dose of rAAV particles
administered to the contralateral eye of the individual. In some embodiments,
the unit dose of rAAV
particles administered to the contralateral eye of the individual is higher,
e.g., more than any of about
10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about
80%, about 90%,
about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about
250%, about 275%,
about 300% or more, than the unit dose of rAAV particles administered to the
one eye of the individual.
In some embodiments, the unit dose of rAAV particles is expressed as the
number of vector genomes (vg)
per eye (vg/eye). In some embodiments, The unit dose of rAAV particles is
about 6 x 10" vg/eye or less of
the rAAV particles. In some embodiments, the unit dose of rAAV particles is
about lx 10' to about
2 x101 , between about 2x 10' to about 3 x10", between about 3x 1010 to about
4 x 10m, between about
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4x1010 to about 5x10' between about 5 x 10" to about 6x101 , between about
6x10' to about 7x 1010
,
between about 7x 1010 to about 8x101 , between about 8x 1010 to about 9x 1010,
between about 9 x101 to
about 10x 10", between about 1x10" to about 2x 1011, between about 2x 10" to
about 3x 10", between
about 3x 10" to about 4x 1011, between about 4x10'1 to about 5x10", or between
about 5x10" to about
6x 10" vg/eye of the rAAV particles, including any value within these ranges,
of the rAAV particles. In
some embodiments, the unit dose of rAAV particles is about 6x 1010 vg/eye to
about 2x 10" vg,/eye of the
rAAV particles. In some embodiments, the unit dose of rAAV particles is about
6x 1010 vg/eye to about
2x 10" vg/eye, about 7x 101 vg/eye to about 2x 101' vg/eye, about 8 x 101
vg/eye to about 2x 1011 vg/eye,
about 9x 1010 vg/eye to about 2x 10" vg/cyc, about 10x 1010 vg/eye to about 2x
1011 vg/eye, or about lx loll
vgjeye to about 2x 10" vg/eye of the rAAV particles. In some embodiments, the
unit dose of rAAV
particles is about 6 x10" vg/eye to about 2 10" vg/eye of the rAAV particles.
In some embodiments, the
unit dose of rAAV particles is about 6x 1010 vg/eye to about 7x 1010 vg/eye,
about 7x 1010 vg/eye to about
8x 10,0 vg/eye, about 8)< 1010 vg/eye to about 9x 101 vg/eye, about 9x 1010
vg/eye to about 10 x 1010 vg/eye,
about 10x 1019 vg/eye to about lx 10H vgjeye, or about lx 1011 vg/eye to about
2x 10" vg/eye of the rAAV
particles. In some embodiments, the unit dose of rAAV particles is about 6x
10' vgkye, about 7x 1010
vg/eye, about 8x 1010 vg/eye, about 9x 1010 vgkye, about 10x10" vg/eye, about
lx 10" vg/eye, or about
2x 10" vg/eye of the rAAV particles. In some embodiments, the unit dose of
rAAV particles is about
6x 1010 vg/eye or about 2x10" vWeye of the rAAV particles. In some
embodiments, the unit dose of
rAAV particles is about 6x101 vg/eye of the rAAV particles. In some
embodiments, the unit dose of
rAAV particles is about 6 x 1010 vg/eye, about 2 x 10" vg/eye, or about 6x
1011 vg/eye. In some
embodiments, the unit dose of rAAV particles is about 6 x 10" vg/eye. In some
embodiments, the unit
dose of rAAV particles is about 2 x 1011 vg/eye. In some embodiments, the unit
dose of rAAV particles is
about 6x 10'1 vg/eye.
102481 In some embodiments, the unit dose of rAAV particles administered to
the one eye of the
individual and the unit dose of rAAV particles administered to the
contralateral eye of the individual are
administered at the same time. In some embodiments, the unit dose of rAAV
particles administered to the
one eye of the individual and the unit dose of rAAV particles administered to
the contralateral eye of the
individual are administered at different times. In some embodiments, the unit
dose administered to the
contralateral eye is administered any of at least about 1 hour, at least about
2 hours, at least about 4 hours,
at least about 8 hours, at least about 12 hours, at least about 24 hours, at
least about 1 day, at least about 2
days, at least about 3 days, at least about 4 days, at least about 5 days, at
least about 6 days, at least about
7 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks,
at least about 4 weeks, or
more after administering of the unit dose to the one eye. In some embodiments,
the unit dose administered
to the contralateral eye is administered at least about 2 weeks after
administering of the unit dose to the
one eye.
102491 In some embodiments, a single unit dose of rAAV particles is
administered to the one eye and/or
the contralateral eye of the individual. In some embodiments, the single unit
dose of rAAV particles
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administered to the one eye and/or to the contralateral eye is a
therapeutically effective dose. In some
embodiments, more than one dose of rAAV particles (e.g., more than any of
about 2, 3, 4, 5, or more unit
doses) are administered to the one eye and/or the contralateral eye of the
individual. In some
embodiments, the more than one doses of rAAV particles administered to the one
eye and/or to the
contralateral are therapeutically effective doses.
[0250] In some embodiments, an anti-VEGF treatment, e.g., an IVT injection
with an anti-VEGF agent
such as aflibercept, is administered to the one eye and/or to the
contralateral eye administered the rAAV
particles at least about one week, e.g. at least about 7 days, prior to
administration of the unit dose of
rAAV particles (e.g., a unit dose of between about 2 x 1011 vg/eye to about 6
x 10" vg/eye of rAAV
particles). In some embodiments, an anti-VEGF treatment, e.g., an PIT
injection with an anti-VEGF agent
such as aflibercept, is administered to an eye on about Day 1 and the unit
dose of rAAV particles, e.g., a
unit dose of between about 2 x 10" vg/eye to about 6 x 1011vg/eye of rAAV
particles, is administered to
the eye on about Day 8. In some embodiments, the unit dose of rAAV particles
is about 2 x loll vg/eye or
about 6 x 10" vg/eye of rAAV particles. In some embodiments, the ocular
neovascular disease is diabetic
macular edema.
Pharmaceutical Formulations
[0251] In some embodiments, the unit dose of rAAV particles is in a
pharmaceutical formulation. In
some embodiments, the pharmaceutical formulation comprises the rAAV particles,
one or more osmotic
or ionic strength agents, one or more buffering agents, one or more
surfactants, and one or more solvents.
In some embodiments, the osmotic or ionic strength agent is sodium chloride.
In some embodiments, the
one or more buffering agents are sodium phosphate monobasic and/or sodium
phosphate dibasic. In some
embodiments, the surfactant is Poloxamer 188. In some embodiments, the solvent
is water. In some
embodiments, the pharmaceutical formulation comprises the rAAV particles,
sodium chloride, sodium
phosphate monobasic, sodium phosphate dibasic, and a surfactant.
[0252] In some embodiments, the pharmaceutical formulation
comprises about lx101 vg/mL to
about lx10's vg/mL of rAAV particles, about 150 mM to about 200 mM sodium
chloride, about 1 mM to
about 10 mM monobasic sodium phosphate, about 1 mM to about 10 mM dibasic
sodium phosphate, and
about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188, wherein the
pharmaceutical formulation has
a pH of about 7.0 to about 7.5. In some embodiments, the pharmaceutical
formulation comprises about
6 x10" vg/mL to about 6x 1012 vg/mL of rAAV particles, about 150 mM to about
200 mM sodium
chloride, about 1 mM to about 10 mM monobasic sodium phosphate, about 1 mM to
about 10 mM dibasic
sodium phosphate, and about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188,
wherein the
pharmaceutical formulation has a pH of about 7.0 to about 7.5. In some
embodiments, the pharmaceutical
formulation comprises about 6x10" vg/mL of rAAV particles, about 150 mM to
about 200 mM sodium
chloride, about 1 mM to about 10 mM monobasic sodium phosphate, about 1 mM to
about 10 mM dibasic
sodium phosphate, and about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188,
wherein the
phannaceutical formulation has a pH of about 7.0 to about 7.5. In some
embodiments, the pharmaceutical
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formulation comprises about 6 x1012 vg/mL of rAAV particles, about 150 mM to
about 200 mM sodium
chloride, about 1 mM to about 10 mM monobasic sodium phosphate, about 1 mM to
about 10 mM dibasic
sodium phosphate, and about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188,
wherein the
pharmaceutical formulation has a pH of about 7.0 to about 7.5.
[0253] In some embodiments, the rAAV particles in the pharmaceutical
formulation are present at a
concentration of about 1 x 10" vgkril to about lx 10" vg/ml. In some
embodiments, the rAAV particles in
the pharmaceutical formulation are present at a concentration of about lx 10"
vg/m1 to about 6 x10"
vg/ml. In certain embodiments, the rAAV particles in the pharmaceutical
formulation are present at a
concentration of about 1 x 1009 vg/ml to about 2 x10" vg/ml, about 2 x 1009
vg/ml to about 3 x10", about
3x 1009 vg/ml to about 4 x10", about 4x 10" vg/ml to about 5 x10", about 5x
10" vg/ml to about 6x10",
about 6x 10 91/FAA to about 7x 10 9, about 7x 10 9vg/ml to about 8 x 10 9,
about 8 x10" vg/m1 to about
9x j 09, about 9x 10 9vg/m1 to about 10x10 , about 10 x 10 9 vg/ml to about lx
1010, about lx10"vg/m1 to
about 2x 1010, about 2 x10" vg/ml to about 3x 1010, about 3x 10' vg/ml to
about 4 x101 , about 4x 101 vg/ml
to about 5 x10", about 5x 1 01 vg/ml to about 6x 10', about 6x 1010 vg/ml to
about 7x 1010, about 7 x 10'
vg/ml to about 8x 1010, about 8 x101 vg/ml to about 9x 1010, about 9x le
vg/ml to about 10x 1010, about
lox 1010 vg/ml to about lx 1011, about 1 x 1011vg/m1 to about 2x 10", about
2x10" vg/ml to about 3x 10",
about 3x 10" vg/ml to about 4 x10", about 4x10" vg/ml to about 5x10", about 5
x10" vg/m1 to about
6 x1011, about 6x loll vg/ml to about 7x 1011, about 7x 10" vg/ml to about 8x
1011, about 8x 1011vg/m1 to
about 9x 10", about 9 x10" vg/ml to about 10x 10", about 1 x 10" vg/ml to
about 2x 1012, about 2 x1012
n
vg/ml to about 3x1012, about 3 x1012vg/m1 to about 4x1012, about 4x in12 vg/ml
to about 5x1012, about
x1012 vg/m1 to about 6 x1012, about 6x 1012 vg/ml to about 7 x1012, about 7x
10" vg/ml to about 8x10`2,
about 3x 10" vg/ml to about 9x10'2, about 9x 1 On vg/ml to about 10x 1012,
about 1 x1013 vg/ml to about
2x1013, about 2x10" vg/ml to about 3x10', about 3 x10" vg/ml to about 4x 10",
about 4x 10" vg/ml to
about 5 x 1013, about 5 x 10" vg/ml to about 6x 1013, about 6x 10" vg/m1 to
about 7 x1013, about 7x 10" vg/m1
to about 8 x 10'3, about x 10"vg/m1 to about 9x 101s, about 9x 1013 vg/ml to
about 10 x10'3, about lx 10'4
vg/ml to about 2x1014, about 2 x 1014 vg,/m1 to about 3x10'4, about 3 x10"
vg/ml to about 4 X 10'4, about
4 x1014 vg/ml to about 5 x10", or about 5 x1014vg/m1 to about 6x 10" vg/mL. In
some embodiments, the
ri12
pharmaceutical formulation comprises about 6x 10" vg/mL to about 6x ivvg/mL of
rAAV particles. In
some embodiments, the pharmaceutical formulation comprises about 6x 1012 vg/mL
of rAAV particles. In
some embodiments, the pharmaceutical formulation comprises about 6x 10" vg/mL
of rAAV particles.
102541 In some embodiments, the sodium chloride in the pharmaceutical
formulation is present at a
concentration of about 150 mM to about 200 mM. In certain embodiments, the
sodium chloride in the
pharmaceutical formulation is present at a concentration of about 150 mM,
about 160 mM, about 170
mM, about 180 mM, about 190 mM, or about 200 mM. In certain embodiments, the
sodium chloride in
the pharmaceutical formulation is present at a concentration of about 180 mM.
102551 In some embodiments, the sodium phosphate monobasic is present in the
pharmaceutical
formulation at a concentration of about 1 mM to about 10 mM. In some
embodiments, the sodium
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phosphate monobasic is present in the pharmaceutical formulation at a
concentration of any of about 1
mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM,
about 8 mM, about
9 mM, or about 10 mM. In certain embodiments, the sodium phosphate monobasic
is present in the
pharmaceutical formulation at a concentration of about 5 mM.
[0256] In some embodiments, the sodium phosphate dibasic is present in the
pharmaceutical
formulation at a concentration of about 1 mM to about 10 mM. In some
embodiments, the sodium
phosphate dibasic is present in the pharmaceutical formulation at a
concentration of any of about 1 mM,
about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about
8 mM, about 9
mM, or about 10 mM. In certain embodiments, the sodium phosphate dibasic is
present in the
pharmaceutical formulation at a concentration of about 5 mM.
[0257] In some embodiments, the Poloxamer 188 is present in the pharmaceutical
formulation at a
concentration of about 0.0005% (w/v) to about 0.005% (w/v). In some
embodiments, the Poloxamer 188
is present in the pharmaceutical formulation at a concentration of any of
about 0.0005% (w/v), about
0.0006% (w/v), about 0.0007% (w/v), about 0.0008% (w/v), about 0.0009% (w/v),
about 0.001% (w/v),
about 0.002% (w/v), about 0.003% (w/v), about 0.004% (w/v), or about 0.005%
(w/v). In certain
embodiments, the Poloxamer 188 is present in the phamiaceutical formulation at
a concentration of about
0.001% (w/v).
[0258] In some embodiments, the pharmaceutical formulation has a pH of about
7.0 to about 7.5. In
some embodiments, the pharmaceutical formulation has a pH of about 7.0, about
7.1, about 7.2, about 7.3,
about 7.4, or about 7.5. In certain embodiments, the pharmaceutical
formulation has a pH of about 7.3. In
some embodiments, hydrochloric acid and sodium hydroxide are used to adjust
the pH of the
pharmaceutical formulation.
[0259] In some embodiments, the pharmaceutical formulation comprises about 6
x10" vg/mL of rAAV
particles, about 180 mM sodium chloride, about 5 mM monobasic sodium
phosphate, about 5 mM dibasic
sodium phosphate, and about 0.001% (w/v) poloxamer 188, wherein the
pharmaceutical formulation has a
pH of about 7.3. In some embodiments, the pharmaceutical formulation comprises
about 6x101' vg/mL of
rAAV particles, about 180 mM sodium chloride, about 5 mM monobasic sodium
phosphate, about 5 mM
dibasic sodium phosphate, and about 0.001% (w/v) poloxamer 188, wherein the
pharmaceutical
formulation has a pH of about 7.3.
102601 In some embodiments, the pharmaceutical formulations
are suitable for administration to the
one eye and/or the contralateral eye of the individual, e.g., a human patient,
via intravitreal (IVT) injection
to achieve a desired therapeutic or prophylactic effect. In some embodiments,
the pharmaceutical
formulation is supplied as a reconstituted homogenous solution. In some
embodiments, the solution is a
suspension. In some embodiments, the pharmaceutical formulation is supplied as
a frozen suspension, and
is thawed prior to administration to the one eye and/or the contralateral eye
of the individual. In some
embodiments, the solution is isotonic.
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102611 In other embodiments, the pharmaceutical composition comprising e.g.,
an AAV2.7m8 vector
that comprises a nucleic acid sequence encoding the anti-VEGF agent (e.g.,
aflibercept or a functional
fragment or variant thereof), is supplied in a lyophilized form, and is
reconstituted prior to administration
to the one eye and/or the contralateral eye of the individual. In some
embodiments, the methods provided
herein further comprise the steps of reconstituting, dissolving, or
solubilizing a lyophilized pharmaceutical
composition comprising rAAV (e.g., AAV2.7m8) and encoding the anti-VEGF agent
(e.g., aflibercept or
a functional fragment or variant thereof) in a buffer prior to administration
to the subject. In some
embodiments, such lyophilized pharmaceutical composition comprises one or more
of the following: a
cryoprotectant, a surfactant, a salt, a stabilizer, or any combination
thereof.
102621 In some embodiments, the pharmaceutical formulation is a homogenous
solution. In some
embodiments, the homogenous solution is supplied in a pre-filled syringe. In
some embodiments, the
pharmaceutical formulation is supplied as a suspension. In some embodiments, a
suspension is a solution.
In some embodiments, the suspension is refrigerated. In some embodiments, the
suspension is frozen. In
some embodiments, methods provided herein further comprise the step of warming
the refrigerated
suspension to room temperature and/or agitating the suspension to ensure that
the active ingredient(s) are
dissolved and/or evenly distributed in solution prior to administering to the
one eye and/or the
contralateral eye of the individual (e.g., via IVT injection). In some
embodiments, methods provided
herein thither comprise the step of thawing the frozen suspension and warming
to mom temperature
and/or agitating the suspension to ensure that the active ingredient(s) are
dissolved and/or evenly
distributed in solution prior to administering to the one eye and/or the
contralateral eye of the individual
(e.g., via IVT injection). In some embodiments, the suspension is diluted
prior to administration to the
subject (e.g., via IVT injection). In some embodiments, the suspension is
supplied as a pre-filled syringe.
[0263] In some embodiments, the pharmaceutical formulation is provided as a
frozen suspension. In
some embodiments, the suspension comprises a pharmaceutically acceptable
excipient, e.g., surfactant,
glycerol, non-ionic surfactant, buffer, glycol, salt, and any combination
thereof.
[0264] In some embodiments, the suspension is a solution. In some embodiments,
the suspension
comprises micelles.
[0265] In some embodiments, for storage stability and convenience of handling,
a pharmaceutical
formulation, comprising rAAV (e.g., AAV2.7m8) and a nucleic acid sequence that
encodes the anti-
VEGF agent (e.g., aflibercept or a functional fragment or variant thereof), is
formulated as a lyophilized,
freeze dried, or vacuum dried powder that is reconstituted with saline,
buffer, or water prior to
administration to the one eye and/or the contralateral eye of the individual.
Alternately, the
pharmaceutical formulation is formulated as an aqueous solution, such as a
suspension or a homogeneous
solution. A pharmaceutical formulation can contain rAAV particles comprising a
nucleic acid sequence
that encodes aflibercept. Various excipients, such as phosphate, PBS, or Tris
buffer, glycol, glycerol,
saline, surfactant (e.g., pluronic or polysorbate), or any combination
thereof, can be used to stabilize a
pharmaceutical formulation. Additionally, cryoprotectants, such as alcohols
can be used as a stabilizer
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under freezing or drying conditions. In some embodiments, the gene therapy is
provided as a suspension,
a refrigerated suspension, or a frozen suspension.
102661 In some embodiments, a suspension of the pharmaceutical formulation as
disclosed herein has a
volume of any of about 20 ;AL, 30 L, 40 L, 50 pL, 60 pL, 70 L, 80 gL, 90
pL, 100 gL, 200 pL, 300
ML, 400 gL, 500 gL, 600 I_õ 700 L, 800 L, 900 pL, or 1000 L. In some
embodiments, a suspension
of the pharmaceutical formulation as disclosed herein has a volume of about
250 pL. In some
embodiments, the suspension of the pharmaceutical formulation as disclosed
herein has a volume of
between 0.1 to 0.5 mL, between 0.1 to 0.2 mL, between 0.3 to 0.5 mL, between
0.5-1.0 mL, between 0.5-
0.7 mL, between 0.6 to 0.8 mL, between 0.8 to 1 mL, between 0.9 to 1.1 mL,
between 1.0 to 1.2 mL, or
between 1.0 to 1.5 mL. In other embodiments, the volume is no more than 0.1
mL, 0.2 mL, 0.3 mL, 0.4
mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL,
1.4 mL, or 1.5 mL. In
some embodiments, the suspension of the pharmaceutical formulation as
disclosed herein has a volume of
about 0.25 mL.
102671 In some embodiments, a suspension of the pharmaceutical formulation as
disclosed herein is
provided as a sterile-filtered, frozen suspension in a sterile, ready-to-use
vial (e.g., a 0.5 mL vial; e.g., a
Crystal Zenith vial) with a ready-to-use stopper (e.g., a stopper made of
chlorobutyl), and sealed (e.g.,
with a sterile aluminum tear-off seal). In some embodiments, a suspension of
the pharmaceutical
formulation as disclosed herein is provided as a sterile-filtered, frozen
suspension in a sterile, ready-to-use
vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith vial) with, a ready-to-use
stopper (e.g., a stopper made of
chlorobutyl), and sealed (e.g., with a sterile aluminum tear-off seal),
wherein the vial contains a volume of
between 0.1 to 0.5 mL, between 0.1 to 0.2 mL, between 0.2 to 0.3 mL, between
0.3 to 0.4 mL, or between
0.4 mL to 0.5 mL of the suspension of the pharmaceutical formulation. In some
embodiments, a
suspension of the pharmaceutical formulation as disclosed herein is provided
as a sterile-filtered, frozen
suspension in a sterile, ready-to-use vial (e.g., a 0.5 mL vial; e.g., a
Crystal Zenith vial) with a ready-to-
use stopper (e.g., a stopper made of chlorobutyl), and sealed (e.g., with a
sterile aluminum tear-off seal),
wherein the vial contains a volume of about 0.25 mL of the suspension of the
pharmaceutical formulation.
102681 In some embodiments, pharmaceutical formulations disclosed herein are
designed, engineered,
or adapted for administration to a primate (e.g., non-human primate and human
subjects) via intravitreal
or subretinal injection. In some embodiments, a pharmaceutical formulation
comprising rAAV particles
comprising a nucleic acid sequence that encodes the anti-VEGF agent (e.g.,
aflibercept) is formulated for
intravitreal injection into an eye of an individual. In some embodiments, the
pharmaceutical composition
is formulated to or reconstituted to a concentration that allows intravitreal
injection of a volume not more
than about or not more than any of 25 L, 30 L, 35 pL, 40 pL, 45 L, 50 p.L,
55 pL, 60 L, 65 L, 70
pL, 75 pL, 80 pL, 85 L, 90 pL, 95 pL, 100 pL, 110 pL, 120 pL, 130 pL, 140 pL,
150 pL, 160 L, 170
ML, 180 p.L, 190 gL, 200 L, 210 L, 220 L, 230 pL, 240 L, or 250 L. In
some embodiments, a unit
dose of the pharmaceutical formulation comprises a volume not more than about
or not more than any of
25 L, 30 ;IL, 35 pL, 40 !IL, 45 L, 50 L, 55 pL, 60 pL, 65 L, 70 gL, 75 pL,
80 pL, 85 gL, 90 gL, 95
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1AL, 100 pL, 110 pL, 120 L, 130 AL, 140 L, 150 pL, 160 L, 170 pL, 180 L,
190 L, 200 L, 210
pL, 220 pL, 230 pL, 240 L, 01 250 L. In some embodiments, methods disclosed
herein comprise
intravitreal injection of a volume of any of about 25 pit, 30 L, 35 L, 40
pL, 45 lit, 50 AL, 55 pL, 60
IA, 65 pL, 70 pL, 75 pL, 80 L, 85 pL, 90 pL, 95 pL, 100 pL, 110 pL, 120 pit
130 L, 140 L, 150 pL,
160 L, 170 pL, 180 L, 190 pL, 200 AL, 210 L, 220 pL, 230 p.L, 240 pL, or
250 AL of a solution or
suspension of a pharmaceutical formulation comprising a rAAV (e.g., AAV2.7m8)
and a nucleic acid
sequence that encodes the anti-VEGF agent (e.g., aflibercept). In some
embodiments, methods disclosed
herein comprise intravitreal injection of a volume of about 30 p.L or about
100 L of a solution or
suspension of a pharmaceutical formulation comprising a rAAV (e.g., AAV2.7m8)
and a nucleic acid
sequence that encodes the anti-VEGF agent (e.g., aflibercept). In some
embodiments, methods disclosed
herein comprise intravitreal injection of a volume of about 30 FiL of a
solution or suspension of a
pharmaceutical formulation comprising a rAAV (e.g., AAV2.7m8) and a nucleic
acid sequence that
encodes the anti-VEGF agent (e.g., aflibercept). In some embodiments, methods
disclosed herein
comprise intravitreal injection of a volume of about 100 L of a solution or
suspension of a
pharmaceutical formulation comprising a rAAV (e.g., AAV2.7m8) and a nucleic
acid sequence that
encodes the anti-VEGF agent (e.g., aflibercept).
[0269] In some embodiments, an AAV2.7m8 particle comprising a nucleic acid
sequence of the anti-
VEGF agent (e.g., aflibercept) transgene described herein is a component of a
gene therapy
pharmaceutical formulation. In some embodiments, a rAAV particle of any
serotype comprising the 7m8
variant capsid protein as described herein is used to make a frozen suspension
or a freeze-dried or
lyophilized formulation composition. In some embodiments, the gene therapy is
formulated as a
refrigerated or frozen suspension. In some embodiments, the rAAV particle is
rAAV2. In some
embodiments, the lyophilized or suspension of the pharmaceutical formulation
comprises rAAV2
comprising the 7m8 variant capsid protein and a DNA sequence that encodes the
anti-VEGF agent (e.g.,
aflibercept). In some embodiments, the suspension is refrigerated or frozen.
[0270] In some embodiments, the administration of the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual is by intravitreal (IVT)
injection. For IVT injection, the
rAAV particles can be delivered in the form of a suspension of a
pharmaceutical formulation (e.g., as
described herein). Initially, topical anesthetic is applied to the surface of
the eye followed by an
ophthalmic antiseptic solution. The eye is held open, with or without
instrumentation, and the rAAV
particles are injected through the sclera with a short, narrow needle, e.g., a
30-gauge needle, into the
vitreous cavity of the one eye and/or the contralateral eye of the individual
under direct observation.
Typically, a volume of between about 25 pL to about 250 jIL (e.g., any of
about 25 p.L, about 30 pL,
about 40 L about 50 L, about 60 L, about 70 L, about 80 pL, about 90 pL,
about 100 L, about 110
pL, about 120 AL, about 130 L, about 140 L, about 150 jiL, about 160 L,
about 170 pL, about 180 pL,
about 190 pL, about 200 pL, about 210 pL, about 220 pie, about 230 L, about
240 AL, or about 250 AL)
of an rAAV particle suspension may be delivered to the eye by IVT injection.
In some embodiments, the
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unit dose of rAAV particles comprises a volume of about 100 L. In some
embodiments, the unit dose of
rAAV particles comprises a volume of about 30 L. In some embodiments, the IVT
injection is
performed in combination with removal of vitreous fluid. In some embodiments,
a vitrectomy may be
performed, and the entire volume of vitreous gel is replaced by an infusion of
the rAAV particle
suspension (e.g., about 4 mL of the rAAV particle suspension). A vitrectomy is
performed using a cannula
of appropriate bore size (e.g., 20 gauge to 27 gauge), wherein the volume of
vitreous gel that is removed
is replaced by infusion of fluid, e.g., saline, an isotonic solution, a rAAV
particle suspension, from the
infusion cannula. IVT administration is generally well tolerated. At the
conclusion of the procedure, there
is sometimes mild redness at the injection site. There is occasional
tenderness, but most patients do not
report any pain. No eye patch or eye shield is necessary after this procedure,
and activities are not
restricted. Sometimes, an antibiotic eye drop is prescribed for several days
to help prevent infection_
[0271] In some embodiments, the pharmaceutical formulation is a unit dose
(e.g., a therapeutically
effective dose) to be administered to the one eye and/or the contralateral eye
of an individual (e.g., a
human or non-human primate) via IVT injection for the treatment of an ocular
disease or disorder
characterized by abnormal (e.g., excessive) angiogenesis or neovascularizatioa
In some embodiments,
the pharmaceutical formulation comprises a unit dose (e.g., a therapeutically
effective dose) as described
in further detail elsewhere herein. In some embodiments, the volume of the
unit dose (e.g., a
therapeutically effective dose) of a viral vector (e.g., an rAAV vector
disclosed herein) administered to the
subject is no more than any one of about 25 L, 30 L, 35 pL, 40 pL, 45 L, 50
L, 55 pL, 60 L, 65 L,
70 pL, 75 pL, 80 pL, 85 pL, 90 pL, 95 pL, 100 pL, 110 pL, 120 pL, 130 pL, 140
pL, 150 pL, 160 pL,
170 L, 180 pL, 190 IA, 200 L, 210 L, 220 L, 230 pL, 240 pt, or 250 AL,
including any range in
between these values. Minimizing the volume of the unit dose to be
administered to the subject may
obviate or mitigate changes in ocular pressure and other adverse effects
associated with WT injection
(e.g., elevated intraocular pressure, inflammation, irritation, or pain).
[0272] Pharmaceutical formulations suitable for ocular use include sterile
aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile
injectable solutions,
suspension, or dispersion. For intravitreal administration, suitable carriers
include physiological saline,
bacteriostatic water, phosphate buffered saline (PBS), and/or an isotonic
agent, e.g., glycerol. In certain
embodiments, the pharmaceutical formulation is sterile and fluid to the extent
that easy syringability or
injectability exists. In certain embodiments, the pharmaceutical formulation
is stable under the conditions
of manufacture and storage and is preserved against the contaminating action
of microorganisms such as
bacteria and fungi. In some embodiments, the pharmaceutical composition can
include an isotonic agent,
such as a salt or glycerol. In some embodiments, a surfactant or a stabilizer
is added to the pharmaceutical
composition to prevent aggregation.
[0273] In some embodiments, the pharmaceutical formulation contains an
excipient or a carrier. A
carrier is a solvent or dispersion medium containing, for example, water,
saline, ethanol, a polyol (for
example, glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and any combination
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thereof The proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by
the maintenance of the required particle size in the case of dispersion and by
the use of surfactants such as
polysorbates (e.g., TweenTm, polysorbate 20, polysorbate 80), sodium dodecyl
sulfate (sodium lauryl
sulfate), lauryl dimethyl amine oxide, cetyltrimethylammonium bromide (CTAB),
polyethoxylated
alcohols, polyoxyethylene sorbitan, octoxynol (Triton X100"), N,N-
dimethyldodecylamine-N-oxide,
hexadecyltrimethylammonium bromide (HTAB), polyoxyl 10 lauryl ether, Brij
721Tm, bile salts (sodium
deoxycholate, sodium cholate), pluronic acids (F-68, F-127), polyoxyl castor
oil (CremophorTM)
nonylphenol ethoxylate (TergitolTm), cyclodextrins, and ethylbenzethonium
chloride (HyamineTm).
Prevention of the action of microorganisms can be achieved by various
antibacterial and antifungal
agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, cresol,
thimerosal, and the like. In
many embodiments, isotonic agents are included in the pharmaceutical
formulation, for example, sugars,
polyalcohols such as mannitol, sorbitol, and/or sodium chloride. Prolonged
absorption of the internal
compositions can be brought about by including in the composition an agent
that delays absorption, for
example, aluminum monostearate and gelatin. In some embodiments, the
pharmaceutical carrier includes
sodium phosphate, sodium chloride, polysorbate, and sucrose. In some
embodiments, a pharmaceutical
formulation comprises a surfactant, e.g., non-ionic surfactant such as
polysorbate, poloxamer, or pluronic.
In some embodiments, the addition of a non-ionic surfactant reduces
aggregation in the pharmaceutical
composition.
102741 Also provided herein are kits comprising at least one pharmaceutical
formulation described
herein. In some embodiments, the kit comprises a frozen suspension of a
pharmaceutical formulation
(e.g., one unit dose in a vial). In some embodiments, the kit comprises a
lyophilized or freeze-dried
pharmaceutical formulation (e.g_, one unit dose in a vial) disclosed herein
and a solution for dissolving,
diluting, and/or reconstituting the lyophilized pharmaceutical composition. In
some embodiments, the
solution for reconstituting or dilution is supplied as a pre-filled syringe.
In some embodiments, a kit
comprises a freeze-dried or lyophilized pharmaceutical composition comprising
rAAV (e.g., AAV2.7m8)
and a solution for reconstituting the pharmaceutical composition to a desired
concentration or volume. In
some embodiments, the kit includes a buffer that helps to prevent aggregation
upon reconstituting the
pharmaceutical composition disclosed herein. In some embodiments, the
pharmaceutical composition is
provided in a pre-filled syringe. In some embodiments, a kit comprises a dual-
chamber syringe or
container wherein one of the chambers contains a buffer for dissolving or
diluting the pharmaceutical
composition. In some embodiments, the kit comprises a syringe for injection.
In some embodiments, the
reconstituted solution is filtered before administration. In some embodiments,
the kit comprises a filter or
a filter syringe for filtering the reconstituted pharmaceutical composition
before administration to a
patient. In some embodiments, the kit comprises a suspension of the
pharmaceutical formulation
comprising the rAAV particles as disclosed herein provided as a sterile-
filtered, frozen suspension in a
sterile, ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith vial)
with a ready-to-use stopper
(e.g., a stopper made of chlorobutyl), and sealed (e.g., with a sterile
aluminum tear-off seal). In some
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embodiments, the kit comprises a suspension of the pharmaceutical formulation
comprising the rAAV
particles as disclosed herein provided as a sterile-filtered, frozen
suspension in a sterile, ready-to-use vial
(e.g., a 0.5 mL vial; e.g., a Crystal Zenith vial) with a ready-to-use
stopper (e.g., a stopper made of
chlorobutyl), and sealed (e.g., with a sterile aluminum tear-off seal),
wherein the vial contains a volume of
between 0.1 to 0.5 ml-e, between 0.1 to 0.2 mL, between 0.2 to 0.3 mL, between
0.3 to 0.4 mL, or between
0.4 mL to 0.5 mL of the suspension of the pharmaceutical formulation. In some
embodiments, the kit
comprises a suspension of the pharmaceutical formulation comprising the rAAV
particles as disclosed
herein provided as a sterile-filtered, frozen suspension in a sterile, ready-
to-use vial (e.g., a 0.5 mL vial;
e.g., a Crystal Zenith vial) with a ready-to-use stopper (e.g., a stopper
made of chlorobutyl), and sealed
(e.g., with a sterile aluminum tear-off seal), wherein the vial contains a
volume of about 0.25 mL of the
suspension of the pharmaceutical formulation. In some embodiments, the kit
further comprises
instructions for use, e.g., instructions for treating an ocular neovascular
disease with the rAAV particles
disclosed herein.
Ocular Neovascular Diseases
[0275] In one aspect, the present disclosure provides methods for treating an
ocular neovascular disease
in an individual. In another aspect, the present disclosure provides methods
for reducing retinal fluid in
the eye of an individual with an ocular neovascular disease.
[0276] In some embodiments, the ocular neovascular disease is age-related
macular degeneration
(AMD), wet-AMD, retinal neovascularization, choroidal neovascularization
diabetic retinopathy,
proliferative diabetic retinopathy, retinal vein occlusion, central retinal
vein occlusion, branched retinal
vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic
retinopathy, diabetic retinal
edema, or any combination thereof In some embodiments, the ocular neovascular
disease is active
choroidal neovascularization (CNV) secondary to age-related macular
degeneration (AMD).
[0277] In some embodiments, the ocular neovascular disease is recurrent and/or
persistent wAMD. In
some embodiments, the ocular neovascular disease is active subfoveal CNV
secondary to AMU. In some
embodiments, the active subfoveal CNV secondary to AMD occupies 50% of the
total lesion size. In
some embodiments, the active subfoveal CNV secondary to AMD occupies > 50% of
the total lesion size
with evidence of leakage on fluorescein angiogram (FA), fluid on spectral
domain optical coherence
tomography (SD-OCT), and/or subretinal hemorrhage on color fundus photography.
In some
embodiments, the active subfoveal CNV secondary to AMD occupies > 50% of the
total lesion size with
evidence of leakage on fluorescein angiogram (FA), fluid on spectral domain
optical coherence
tomography (SD-OCT), and/or subretinal hemorrhage on color fundus photography,
and the entire
dimension of the lesion does not exceed 12 macular photocoagulation study disc
areas. In some
embodiments, the one eye and/or the contralateral eye of the individual
exhibited best corrected visual
acuity (BCVA) based on an ETDRS letters assessment of 78-25 (e.g., less than
any of about 78, about 75,
about 70, about 65, about 60, about 55, about 50, about 45, about 40, about
35, about 30, or about 25)
prior to administration of the unit dose of rAAV particles of the present
disclosure. In some
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embodiments, the one eye and/or the contralateral eye of the individual
exhibited best corrected visual
acuity (BCVA) based on an ETDRS letters assessment of more than any of about
5, about 10, about 15,
about 20, about 25, about 30, about 35, about 40, about 45, about 50, about
55, about 60, about 65, about
70, about 75, about 80, about 85, about 90, about 95, or about 100 prior to
administration of the unit dose
of rAAV particles of the present disclosure.
[0278] In some embodiments, the individual had polypoidal choroidal
vasculopathy (PCV) in the one
eye and/or the contralateral eye prior to administration of the unit dose of
rAAV particles.
[0279] In some embodiments, ETDRS letters assessment is done at about 0.5
meters, about 1 meter,
about 2 meters, about 3 meters, or about 4 meters. In some embodiments, ETDRS
letters assessment is
done at about 4 meters.
[0280] In some embodiments, the individual received at least one prior
treatment (e.g., at least one, at
least two, at least three, at least four, at least 5 or more treatments) with
an anti-VEGF agent (e.g.,
bevaciziunab, brolucizumab, ranibizumab, faricimab, abicipar pegol,
conbercept, OPT-302, KSI-301,
injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and/or
aflibercept) in about the last 12
weeks (e.g., about 3 or about 4 months) prior to administration of the unit
dose of rAAV particles. In
some embodiments, the individual received 2 or 3 prior treatments with an anti-
VEGF agent (e.g.,
bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept,
OPT-302, KSI-301,
injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and/or
aflibercept) in the one eye and/or
in the contralateral eye during about the last 12 weeks (e.g., about 3 or
about 4 months) prior to
administration of the unit dose of rAAV particles to the one eye and/or the
contralateral eye. In some
embodiments, the individual received at least about 1, at least about 5, at
least about 10, at least about 20,
at least about 30, at least about 40, at least about 50, at least about 60, at
least about 70, at least about 80,
at least about 90, at least about 100, at least about 110, at least about 120,
or more prior treatments with an
anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab,
abicipar pegol, conbercept,
OPT-302, KSI-301, injectable sunitinib maleate ((18-102), PAN-90806
(PanOptica), and/or aflibercept) in
the one eye and/or the contralateral eye. In some embodiments, the individual
had a calculated anti-VEGF
agent (e.g., bevacizumab, broluciziunab, ranibizumab, and/or aflibercept)
injection interval in the one eye
and/or the contralateral eye of about 2 weeks, about 3 weeks, 4 weeks, about 5
weeks, about 6 weeks,
about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks,
about 12 weeks, or more.
In some embodiments, the individual had a calculated anti-VEGF (e.g.,
bevacizumab, brolucizumab,
ranibizumab, and/or aflibercept) injection interval in the one eye and/or the
contralateral eye of about 5-7
weeks, about 4-10 weeks, about 4-7 weeks, or about 4-6 weeks. In some
embodiments, the individual
received a prior treatment with an anti-VEGF agent (e.g., bevacizumab,
brolucizumab, ranibizumab,
faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib
maleate (GB-102), PAN-
90806 (PanOptica), and/or aflibercept) in the one eye and/or in the
contralateral eye any of at least about 5
days, at least about 6 days, at least about 7 days, at least about 8 days, at
least about 9 days, at least about
days, at least about 11 days, at least about 12 days, at least about 13 days,
at least about 14 days, at
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least about 15 days, at least about 16 days, at least about 17 days, at least
about 18 days, at least about 19
days, or at least about 20 days prior to administration of the unit dose of
rAAV particles to the one eye
and/or the contralateral eye. In some embodiments, the individual received a
prior treatment with an anti-
VEGF agent (e.g., bevacizuntab, brolucizumab, ranibizumab, faricimab, abicipar
pegol, conbercept, OPT-
302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica),
and/or aflibercept) in the
one eye and/or the contralateral eye about 7 days, about 10 days, or about 14
days prior to administration
of the unit dose of rAAV particles to the one eye and/or the contralateral
eye. In some embodiments, the
prior treatment comprises an intraocular, subretinal or intravitreal injection
with an anti-VEGF agent. In
some embodiments, the anti-VEGF agent is bevaciztunab, brolucizumab,
ranibizumab, faricimab, abicipar
pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102),
PAN-90806 (PanOptica),
and/or aflibercept. In some embodiments, the anti-VEGF agent is aflibercept.
In some embodiments, the
individual received between 1 and 20 (e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 01 20) prior treatments with an anti-VEGF agent (es., bevacizumab,
brolucizumab, ranibizumab,
faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib
maleate (GB-102), PAN-
90806 (PanOptica), and/or aflibercept) in the one eye and/or in the
contralateral eye during the last about
12 months prior to administration of the unit dose of rAAV particles to the
one eye and/or the
contralateral eye. In some embodiments, the individual received about 9 or
about 10 prior treatments with
an anti-VEGF agent (e.g., bevacizurnab, brolucizumab, ranibizumab, faricimab,
abicipar pegol,
conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806
(PanOptica), and/or
aflibercept) in the one eye and/or in the contralateral eye during the last
about 12 months prior to
administration of the unit dose of rAAV particles to the one eye and/or the
contralateral eye.
[0281] In some embodiments, the individual demonstrated a meaningful response
to a prior treatment
with anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept,
a functional variant
thereof, or a functional fragment thereof In some embodiments, the anti-VEGF
agent comprises a
polypeptide comprising an amino acid sequence with at least about 95% identity
to the amino acid
sequence of SEQ ID NO: 35. In some embodiments, the individual demonstrated a
meaningful response
to a prior anti-VEGF treatment (e.g., aflibercept, a functional variant
thereof, or a functional fragment
thereof) for the ocular neovascular disease in the one eye and/or in the
contralateral eye prior to
administration of the unit dose of rAAV particles to one eye and/or the
contralateral eye. In some
embodiments, the individual is determined to have a meaningful response to a
prior anti-VEGF treatment
(e.g., aflibercept, a functional variant thereof, or a functional fragment
thereof) for the ocular neovascular
disease if a reduction of 30% (e.g., any of at least 30%, at least 40%, at
least 50%, at least 60%, at least
70%, at least 80%, at least 90%, Or 100%) of central retinal thickness (CRT)
or central subfield thickness
(CST) is observed any of at least about 5 days, at least about 6 days, at
least about 7 days, at least about 8
days, at least about 9 days, at least about 10 days, at least about 11 days,
at least about 12 days, at least
about 13 days, at least about 14 days, at least about 15 days, at least about
16 days, at least about 17 days,
or more, after the anti-VEGF treatment, compared to the central retinal
thickness (CRT) or central
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subfield thickness (CST) at the initial diagnosis in the one eye and/or the
contralateral eye. In some
embodiments, the individual is determined to have a meaningful response to a
prior anti-VEGF treatment
(e.g., aflibercept, a functional variant thereof, or a functional fragment
thereof) for the ocular neovascular
disease if a reduction of 30% (e.g., any of at least 30%, at least 40%, at
least 50%, at least 60%, at least
70%, at least 80%, at least 90%, or 100%) in central retinal thickness (CRT)
or central subfield thickness
(CST) is observed more than any of about 7 days, about 10 days, or about 14
days after the anti-VEGF
treatment, compared to the central retinal thickness (CRT) or central subfield
thickness (CST) prior to
administration of the prior anti-VEGF treatment in the one eye and/or the
contralateral eye.
[0282] In some embodiments, the central subfield thickness and/or central
retinal thickness is
determined by SD-OCT in the one eye and/or the contralateral eye. Central
subfield thickness is the mean
thickness of the retina across the central subfield of an ETDRS grid, a 1 nun
diameter circle centered on
the fovea.
[0283] In some embodiments, the individual is determined to have a meaningful
response to a prior
treatment with anti-VEGF agent (e.g., aflibercept, a functional variant
thereof, or a functional fragment
thereof) for the ocular neovascular disease if a reduction of 220% (es., any
of at least 20%, at least 30%,
at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, or 100%) in central
subfield thickness and/or central retinal thickness is observed compared to
the central subfield thickness
and/or central retinal thickness prior to administration of the prior
treatment with the anti-VEGF agent. In
some embodiments, the individual is determined to have a meaningful response
to a prior treatment with
anti-VEGF agent (e.g., aflibercept, a functional variant thereof, or a
functional fragment thereof) for the
ocular neovascular disease if a reduction of 20% (e.g., any of at least 20%,
at least 30%, at least 40%, at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) in
central subfield thickness
and/or central retinal thickness is observed any of at least about 5 days, at
least about 6 days, at least about
7 days, at least about 8 days, at least about 9 days, at least about 10 days,
at least about 11 days, at least
about 12 days, at least about 13 days, at least about 14 days, at least about
15 days, at least about 16 days,
at least about 17 days, or more, after administration of the prior treatment
with an anti-VEGF agent,
compared to the central subfield thickness and/or central retinal thickness
prior to administration of the
prior treatment with an anti-VEGF agent. In some embodiments, the individual
is determined to have a
meaningful response to a prior treatment with an anti-VEGF agent (e.g.,
aflibercept, a functional variant
thereof, or a functional fragment thereof) for the ocular neovasc War disease
if a reduction of >20% (e.g.,
any of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at
least 90%, or 100%) in central subfield thickness and/or central retinal
thickness is observed about 7
days, about 10 days, or about 14 days after administration of the prior
treatment with the anti-VEGF
agent, compared to the central subfield thickness and/or central retinal
thickness prior to administration of
the prior treatment with an anti-VEGF agent. In some embodiments, the central
subfield thickness and/or
central retinal thickness is determined by SD-OCT in the one eye and/or the
contralateral eye. In some
embodiments, the individual is determined to have a meaningful response to a
prior treatment with an
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anti-VEGF agent (e.g., aflibercept, a functional variant thereof, or a
functional fragment thereof) for the
ocular neovascular disease if normalization of CST is observed with no
observable vascular exudation
after the anti-VEGF treatment in the one eye and/or the contralateral eye.
Normalization refers to a CST
value that is normal for that class of patient (e.g., based on age, sex, etc.)
[0284] In some embodiments, the individual is determined to have a meaningful
response to a prior anti-
VEGF treatment (e.g., aflibercept, a functional variant thereof, or a
functional fragment thereof) for the
ocular neovascular disease if a reduction of >20% (e.g., any of at least 20%,
at least 30%, at least 40%, at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) in
central retinal thickness
(CRT) or central subfield thickness (CST) is observed any of at least about 5
days, at least about 6 days, at
least about 7 days, at least about 8 days, at least about 9 days, at least
about 10 days, at least about 11
days, at least about 12 days, at least about 13 days, at least about 14 days,
at least about 15 days, at least
about 16 days, at least about 17 days, or more, after the anti-VEGF treatment,
compared to the central
retinal thickness (CRT) or central subfield thickness (CST) prior to
administration of the anti-VEGF
treatment (e.g., aflibercept), as determined by SD-OCT in the one eye and/or
the contralateral eye. In
some embodiments, the individual is determined to have a meaningful response
to a prior anti-VEGF
treatment (e.g., aflibercept, a functional variant thereof, or a functional
fragment thereof) for the ocular
neovascular disease if a reduction of ?20% (e.g., any of at least 20%, at
least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) in
central retinal thickness (CRT) or
central subfield thickness (CST) is observed about 7 days, about 10 days, or
about 14 days after the anti-
VEGF treatment, compared to the central retinal thickness (CRT) or central
subfield thickness (CST) prior
to administration of the anti-VEGF treatment, as determined by SD-OCT in the
one eye and/or the
contralateral eye.
[0285] In some embodiments, the individual has not received a prior treatment
for an ocular neovascular
disease. In some embodiments, the individual has not received a prior
treatment in the one eye and/or the
contralateral eye for an ocular neovascular disease. In some embodiments, the
individual has not received
a prior anti-VEGF treatment. In some embodiments, the individual has not
received a prior anti -VEGF
treatment in the one eye and/or the contralateral eye. In some embodiments,
the individual has not
received a prior aflibercept treatment. In some embodiments, the individual
has not received a prior
aflibercept treatment in the one eye and/or the contralateral eye.
102861 In some embodiments, the ocular disease or disorder treated according
to the methods described
herein is diabetic macular edema. Diabetic macular edema (DME) is a swelling
of the retina in diabetes
mellitus due to leaking of fluid from blood vessels within the macula. The
macula is the central portion of
the retina, a small area rich in cones, the specialized nerve endings that
detect color and upon which
daytime vision depends. As macular edema develops, blurring occurs in the
middle or just to the side of
the central visual field. Visual loss from diabetic macular edema can progress
over a period of months and
make it impossible to focus clearly. Common symptoms of DME are blurry vision,
floaters, double vision,
and eventually blindness if it goes untreated. In some embodiments, methods
and pharmaceutical
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compositions as disclosed herein are used to treat DME. In some embodiments,
treatment of DME is
assessed by measuring refraction or visual acuity (e.g., BCVA using ETDRS
letters). In some
embodiments, visual acuity is measured starting at a distance of about 4
meters prior to dilation of the one
eye and/or the contralateral eye. In some embodiments, visual acuity is
considered to be maintained if the
individual loses fewer than 15 letters in the ETDRS score after administration
of the unit dose of rAAV
particles compared to prior to administration of the unit dose of rAAV
particles. In some embodiments,
treatment of DME is assessed by SD-OCT and/or OCT-A. In some embodiments,
treatment of DME is
assessed by measuring central subfield thickness and/or macular volume using
SD-OCT. In some
embodiments, treatment of DME is assessed by the number of treatments with an
anti-VEGF agent (e.g.,
aflibercept) administered after administration of the unit dose of rAAV
particles (e.g., aflibercept rescue
treatments). In some embodiments, one or more aflibercept rescue treatments
are administered after
administration of the unit dose of rAAV particles if an increase in central
subfield thickness (CST) of > 50
p.m occurs, assessed by SD-OCT, compared to the lower of the two CST
measurements recorded prior to
administration of the unit dose of rAAV particles (e.g., on Day 1) and about 3
weeks after administration
of the unit dose of rAAV particles (e.g., on Week 4). In some embodiments, one
or more aflibercept
rescue treatments are administered after administration of the unit dose of
rAAV particles if loss of > 5
letters in BCVA occurs due to worsening DME disease activity compared to the
higher of the two BCVA
measurements recorded prior to administration of the unit dose of rAAV
particles (e.g., on Day 1) and
about 3 weeks after administration of the unit dose of rAAV particles (e.g.,
on Week 4). In some
embodiments, treatment of DME is assessed using the Diabetic Retinopathy
Severity Scale (DRSS), e.g.,
using ultra-wide field color fundus photography, compared to DRSS prior to
administration of the unit
dose of rAAV particles. In some embodiments, treatment of DME is assessed by
the occurrence of vision
threatening complications (e.g., anterior segment neovascularization, diabetic
macular edema, high-risk
PDR development, vitreous hemorrhage, or tractional retinal detachment). In
some embodiments, vision
threatening complications are assessed by ultra-wide field imaging and
clinical examination. In some
embodiments, the administering the unit dose of rAAV particles to the one eye
and/or to the contralateral
eye of the individual results in a 2-step or in a 3-step improvement in
Diabetic Retinopathy Severity Scale
(DRSS). In some embodiments, the administering the unit dose of rAAV particles
to the one eye and/or to
the contralateral eye of the individual results in a 2-step improvement in
Diabetic Retinopathy Severity
Scale (DRSS). In some embodiments, the administering the unit dose of rAAV
particles to the one eye
and/or to the contralateral eye of the individual results in a 3-step
improvement in Diabetic Retinopathy
Severity Scale (DRSS).
[0287] In some embodiments, the ocular disease or disorder treated according
to the methods described
herein is a retinal vein occlusion. Retinal vein occlusion is a blockage of
the small veins that carry blood
away from the retina. The retina is the layer of tissue at the back of the
inner eye that converts light
images to nerve signals and sends them to the brain. Retinal vein occlusion is
most often caused by
hardening of the arteries (atherosclerosis) and the formation of a blood clot.
Blockage of smaller veins
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(branch veins or BRVO) in the retina often occurs in places where retinal
arteries that have been
thickened or hardened by atherosclerosis cross over and place pressure on a
retinal vein. Symptoms of
retinal vein occlusion can include a sudden blurring or vision loss in all or
part of one eye.
[0288] In some embodiments, the ocular disease or disorder treated according
to the methods described
herein is choroidal neovascularization (CNV), also known as wet age-related
macular degeneration
(wAMD). Choroidal neovascularization can involve the growth of new blood
vessels that originate from
the choroid through a break in the Bruch membrane into the sub¨retinal pigment
epithelium (sub-RPE) or
subretinal space, which can be a major cause of visual loss. CNV can create a
sudden deterioration of
central vision, noticeable within a few weeks. Other symptoms can include
color disturbances, and
metamorphopsia (distortions in which straight lines appears wavy).
Hemorrhaging of the new blood
vessels can accelerate the onset of symptoms of CNV. CNV may also include
feeling of pressure behind
the eye.
[0289] The advanced "wet" form (neovascular or exudative) of AMD may
frequently cause a rapid and
often substantial loss of central vision in patients. In the wet form of AMD,
choroidal neovascularization
forms and develops into a network of vessels that may grow under and through
the retinal pigment
epithelium. As this is accompanied by leakage of plasma and/or hemorrhage into
the subretinal space,
there could be severe sudden loss of central vision if this occurs in the
macula. The present disclosure
contemplates treatment or prevention of AMD, wet AMD. In some embodiments,
methods and
pharmaceutical compositions as disclosed herein are used to treat AMD.
[0290] In some embodiments, methods described herein are used to prevent or
treat an ocular disease or
disorder in a subject who has received prior treatment with bevaciztunab,
brolucizumab, ranibizumab,
faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable surtitinib
ma1eate (GB-102), PAN-
90806 (PanOptica), and/or aflibercept. In some embodiments, methods described
herein are used to
prevent or treat an ocular disease or disorder that is responsive to treatment
with bevacizumab,
brolucizumab, ranibizumab, and/or aflibercept.
[0291] In some embodiments, the individual was diagnosed with the ocular
neovascular disease at least
I day, at least 1 week, at least 1 month, at least 2 months, at least 4
months, at least 6 months, at least 12
months, at least 18 months, at least 24 months, at least 30 months, at least
36 months, at least 42 months,
at least 48 months, at least 54 months, at least 60 months, at least 66
months, at least 72 months, at least
78 months, at least 84 months, at least 90 months, 96 months, at least 102
months, at least 108 months, at
least 114 months, at least 120 months, at least 126 months, at least 132
months, or more, prior to
administration of the unit dose of rAAV particles to the one eye and/or the
contralateral eye.
[0292] The following description is presented to enable a person of ordinary
skill in the art to make and
use the various embodiments. Descriptions of specific devices, techniques, and
applications are provided
only as examples. Various modifications to the examples described herein will
be readily apparent to
those of ordinary skill in the art, and the general principles defined herein
may be applied to other
examples and applications without departing from the spirit and scope of the
various embodiments. Thus,
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the various embodiments are not intended to be limited to the examples
described herein and shown, but
are to be accorded the scope consistent with the claims.
EXAMPLES
Example 1: An open label Phase 1 study of AAV2.7m8-aflibercept in neovascular
(wet) age-related
macular degeneration at a dose of 6 x 1011vg/eye.
[0293] This example describes an open label Phase 1 study
of AAV2.7m8-aflibercept, a rAAV vector
containing the VEGF inhibitor aflibercept and the AAV2.7m8 protein capsid, for
the treatment of age-
related macular degeneration (AMD) with choroidal neovascularization (wet AMD;
wAMD).
I. Study Objectives
A. Primary Objective
[0294] The primary objective of this study was to assess the safety and
tolerability of a single
inuavitreal (IVT) injection of AAV2,7m8-aflibercept in subjects with wAMD.
Primcny Endpoints
[0295] The primary endpoints of this study were the type, severity, and
incidence of ocular and systemic
adverse events (AEs).
B. Secondary Objectives
[0296] The secondary objectives of this study were:
= To evaluate the effect of AAV2.7m8-aflibercept on Best Corrected Visual
Acuity (BCVA).
= To evaluate the effect of AAV2.7m8-aflibercept on central subfield
thickness (CST), also
known as central retinal Thickness (CRT).
= To assess the need for rescue therapy from Week 4 to Week 104.
= To evaluate the effect of AAV2.7m8-aflibercept on the presence of
intraretinal fluid (1RF)
and subretinal fluid (SRF).
= To evaluate the effect of AAV2.7m8-allibercept on pigment epithelial
detachment (PED)
resolution among patients with a FED at baseline.
Secondary Endpoints
[0297] The secondary endpoints of this study were:
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= The mean change in BCVA from baseline over time, as assessed by ETDRS
letters over time
from Day 8 to Week 104, compared to baseline.
= The percentage of subjects with a BCVA gain of >15 ETDRS letters from
baseline overtime,
as assessed from Day 8 to Week 104, compared to baseline.
= The percentage of subjects with a BCVA decline of <15 ETDRS letters from
baseline over
time, as assessed from Day 8 to Week 104, compared to baseline.
= The mean change in CST and macular volume from Baseline over time, as
assessed from Day
8 to Week 104, compared to baseline.
= The mean number of aflibercept injections over time, as assessed from
Weeks 4-104.
= The percentage of subjects requiring aflibercept injections overtime, as
assessed from
Weeks 4-104.
= The percentage of subjects without IRF over time, as assessed from Day 8
to Week 104.
= The percentage of subjects without SRF over time, as assessed from Day 8
to Week 104.
= The percentage of subjects without a PED over time, as assessed from Day
8 to Week 104
among subjects with a PED at baseline.
IL Study Subjects
[0298] Subjects in this study were diagnosed with active choroidal
neovascularization (CNV)
secondary to age-related macular degeneration (AMD), had a history of recent
responsiveness to anti-
VEGF treatment, and required frequent injections of anti-VEGF therapy.
[0299] Only one eye was selected as the study eye for the duration of the
study. If both eyes met all of
the inclusion and exclusion criteria, the eye with the worst BCVA assessed at
Screening was selected as
the study eye. If both eyes met all of the inclusion and exclusion criteria
and BCVA values were identical
for both eyes, the subject chose to select his/her non-dominant eye for
treatment, or by default the right
eye was selected as the study eye.
A. Inclusion Criteria
[0300] Subjects that met the following inclusion criteria were enrolled in
this study:
= Male or female subjects, age 50 years of age.
= The study eye had prior or current evidence of active subfoveal CNV
secondary to AMD
occupying 50% of total lesion size with:
o Leakage on fluorescein angiogram (FA),
fluid on Spectral Domain Optical Coherence
Tomography (SD-OCT), or subretinal hemorrhage on color fundus photo; and
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o The entire dimension of the lesion did not exceed 12 Macular
Photocoagulation Study
disc areas.
= Subjects were under active anti-VEGF treatment for wAMD with a minimum of
2 injections
within 4 months prior to Screening.
= Vision of study eye at Screening visit (prior to aflibercept injection):
o BCVA ETDRS of 78-25.
= Vision of non-study eye:
o BCVA ETDRS of >35.
= Demonstrated a meaningful anti-VEGF response as confirmed by the
Investigator and defined
as:
o Reduction from initial diagnosis in central subfield thickness by > 30%
as assessed
using SD-OCT; or
o Reduction from screening in central subfield thickness by > 20% as
assessed using
SD-OCT; or
o Normalization of CST with no observable vascular exudation.
103011 VEGF responsiveness was confirmed by the Investigator for purposes of
confirmation of anti-
VEGF response at Day 1 visit prior to dosing with AAV2.7m8-aflibercept.
Subjects determined not to
have a meaningful anti-VEGF response failed screening and were not enrolled in
this study.
B. Exclusion Criteria
103021 Subjects that met the following exclusion criteria were not enrolled in
this study:
Neutralizing Antibodies
= Although them is not an established correlation between levels of
neutralizing antibodies
(NAbs) to AAVs in serum and vitreous fluid (Lukason et aL, (2011) Mol Titer
19(2):260-
265), subjects were screened for NAbs to the AAV2.7m8 vector as a precaution.
Subjects
with documented anti-AAV2.7m8 neutralizing antibody titer levels > 1:125
within 6 months
prior to dosing with AAV2.7m8-aflibercept were excluded from this study.
CNV Lesion
= Known history or evidence of the following CNV lesion characteristics:
o Fibrosis or atrophy, retinal epithelial tear in the center of the fovea
in the study eye,
or any condition preventing visual acuity improvement.
o Scarring or fibrosis making up > 50% of total lesion area.
o Lesion size > 12 Macular Photocoagulation Study disc areas (30.5 min2),
including
blood, scars, and neovascularization as assessed by Fluorescein Angiography
(FA).
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o Subretinal hemorrhage that was > 50% of
the total lesion area, or the presence of
blood under the fovea that was > 1 disc area in size in the study eye (if
blood was
under the fovea, then the fovea was surrounded 270 degrees by visible CNV).
Ocular Conditions (Retina/Posterior Eye)
= Significant epiretina1 membrane or vitreomacular traction (VMT) syndrome
in the study eye
at time of dosing with AAV2.7m8-aflibereept or history of a full thickness
macular hole
((kiss Stage 2 and above) in the study eye.
= History of retinal disease in the study eye other than wAMD, including
diabetic retinopathy (in
either eye), retinal vein occlusion, uveitis, suspected retinal angiomatous
proliferation,
polypoidal choroidopathy, or CNV due to other causes (e.g., ocular
histoplasmosis, trauma, or
pathologic myopia), or any other vascular disease in the eye (benign
conditions of the vitreous
or peripheral retina were non-exclusionary).
= History of retinal detachment (with or without repair) in the study eye.
Other Conditions (Non-Retinal)
= Known history or evidence of significant non-retinal disease or media
opacity in the study eye
that could have compromised vision during the course of the study, required
surgery, and/or
precluded proper visualization or imaging of the retina (e.g., central corneal
scarring,
significant cataract, corneal dystrophy, scleromalacia).
= Uncontrolled ocular hypertension or glaucoma in the study eye at time of
dosing with
AAV2.7m8-aflibereept (defined as intraocular pressure [TOP] > 22 mrnHg despite
treatment
with anti-glaucoma medication) or use of > 2 IOP lowering medications at the
lime of
screening.
= Active or history of ocular or periocular infection in either eye within
4 weeks prior to dosing
with AAV2.7m8-aflibercept.
Ocular Surgeries/Procedures
= Any previous intraocular or periocular surgery on the study eye within 6
months of dosing
with AAV2.7m8-aflibercept, or any planned major surgical procedure within 6
months of
dosing with AAV2.7m8-aflibercept. Lid surgery > 1 month of dosing with
AAV2.7m8-
aflibercept was not an exclusion criterion.
= History of vitrectomy, trabeculectomy, or other filtration surgery in the
study eye.
= Yttrium aluminum garnet (Y AG) posterior capsulotomy within 3 months
prior to dosing with
AAV2.7m8-aflibercept.
= Any prior treatment with photodynamic therapy or retinal laser for the
treatment of wAMD
and any previous therapeutic radiation in the region of the study eye.
General/Systemic Conditions
= History or evidence of any of the following cardiovascular diseases
within 6 months of dosing
unless specified:
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o Severe cardiac disease (e.g., New York Heart Association [NYHA]
Functional Class
III or IV) history or clinical evidence of unstable angina
o Acute coronary syndrome, myocardial infarction or coronary artery
revascularization.
o Ventricular tachyarrhythmias requiring ongoing treatment, or uncontrolled
arrhythmia.
o Uncontrolled hypertension defmed as average systolic blood pressure (SBP)
160
mmHg or an average diastolic blood pressure (DBP) >100 mmHg, despite using BP-
lowering medication within the screening period prior to dosing.
o History of cerebrovascular accident or transient ischemic attack.
= Any history of ongoing bleeding disorders or international normalized
ratio (1NR) >3Ø The
use of aspirin or other anticoagulants (e.g, Factor Xa inhibitors) was not an
exclusion
criterion. INR was repeated during the Screening period to confirm eligibility
criteria were
met.
= Evidence of uncontrolled diabetes with an HbA lc >7.0% within the
screening period prior to
dosing with AAV2.7m8-aflibercept.
= History of malignancy within the last 5 years except for the following,
if adequately treated:
o Local basal cell or squarnous cell carcinoma of the skin.
o Carcinoma in situ of the cervix or breast.
o Papillary, noninvasive bladder cancer.
o Prostate cancer Stage 1 and 2 for which observation was clinically
indicated with
stable prostate-specific antigen (PSA) for 6 months.
o Any other cancer that was in complete remission for at least 2 years or
considered
surgically cured.
= Positive I-11V, Hepatitis B, or Hepatitis C (unless treated with a
documented cure).
= Within 36 hours prior to dosing with AAV2.7m8-aflibercept, evidence or
suspicion of
systemic active infection of any type deemed clinically significant by the
Investigator based
on clinical exam and/or temperature >38.5 C.
= Known serious allergies to:
o Fluorescein dye or sodium fluorescein used in angiography (mild allergy
amenable to
treatment was allowable); or
o Aflibercept.
= Women who were pregnant, breastfeeding, or intend to become pregnant
during the study.
= Other significant laboratory abnormalities or medical conditions that
compromised the
subject's safety in the view of the Investigator.
Medications
= Use of systemic anti-inflammatory steroids or inununosuppressant
medications (other than
protocol-specified prednisone) within 5 half-lives prior to dosing with
AAV2.7m8-
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aflibercept. Inhaled or topical steroids and nonsteroidal anti-inflammatory
drugs (NSA1Ds)
were permitted.
= Received any of the following:
o Investigational medicinal product within 30 days or 5 half-lives prior to
dosing with
AAV2.7m8-aflibercept, whichever was longer.
o Prior gene therapy.
III. Investigational Medicinal Product
103031 The investigational medicinal product (IMP), AAV2.7m8-aflibercept, was
a recombinant,
replication-deficient adeno-associated viral (rAAV) vector containing the
AAV2.7m8 protein capsid
derived by in vivo directed evolution on the AAV2 capsid (Dalkara et at ,
(2013) Sci Transl Med
5(189):189ra76; US2014/0364338). AAV2.7m8-aflibercept carried an expression
cassette of a codon-
optimized version of the aflibercept cDNA under the control of a ubiquitous
chimeric promoter (FIG. 1A)
(See W02018170473A1). AAV2.7m8-aflibercept was manufactured using a
baculovirus expression
vector system in Sf9 cells where two different baculoviruses were used, one
encoding the genes for AAV2
Rep and AAV2.7m8 Cap proteins, and the other encoding the human aflibercept
cDNA expression
cassette.
103041 AAV2.7m8-aflibercept was supplied as a sterile-filtered, frozen
suspension in a sterile, ready-to-
use 0.5 mL Crystal Zenith vial which contained 0.25 mL of IMP, formulated as
shown in Table 1.
Table 1: Formulation of the AAV27m8-aflibercept investigational medicinal
product for Colson I.
Formulation (pH 7.3)
Components Concentration Function
AAV2.7m8-aflibercept 6 x 1012 vg/mL
Active ingredient
Sodium Chloride 180 mM
Osmotic/ionic strength agent
Sodium Phosphate Monobasic 5 mM
Buffering agent
Sodium Phosphate Dibasic 5 mM
Buffering agent
Poloxamer 188 0.001% (w/v) Surfactant
Water for Injection Quantum saris (Q.S.)
Solvent
Injection Volume 100 uL
IV. Study Design
A. Dose and Method of Administration
Cohort I
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[0305] Subjects in Cohort I were administered a single NT injection of
AAV2.7m8-aflibercept at Dose
1 of 6 x 1011vg/eye in an injection volume of 100 p.L.
[0306] AAV2.7m8-aflibercept vials were removed from the frozen storage at < -
60 C and thawed at
room temperature. AAV2.7m8-aflibercept was administered via NT injection.
Aseptic technique with
povidone-iodine was employed, along with topical or subconjunctival
anesthesia. Post-injection care and
medication regimen were given based on institutional standard of care.
B. Study Visits
Screening (Days -15 to -7)
[0307] As shown in FIG. 1B, subjects received a single NT injection of
aflibercept 2 mg during
Screening on Days -15 to -7 (e.g., Days -14- to -7), consistent with standard
of care. Subjects received
routine institutional standard post-injection care.
Study Day 1
[0308] On Study Day 1 (between 7 and 15 days, e.g., between 7 and 14 days,
after the NT injection of
aflibercept), study subjects underwent SD-OCT studies to confirm that they
were responsive to anti-
VEGF therapy, prior to dosing with AAV2.7m8-aflibercept. Anti-VEGF
responsiveness was confirmed
by the Investigator. Only subjects confirmed to have a meaningful anti-VEGF
response, as described
above (See Inclusion Criteria), were eligible to enroll in this study.
[0309] Subjects responsive to anti-VEGF therapy were sequentially enrolled
into the study cohort and
received a single AAV2.7m8-aflibercept NT injection in the study eye. As
described above, only one eye
was selected as the study eye for the duration of the study.
Post-AAV2.7m8-ajlibercept Administration
[0310] Subjects returned on Days 3 and 8, during Weeks 2, 4, 6, and 8, and
every 4 weeks thereafter
(i.e., weeks 12, 16, 20, and 24) for clinical evaluation and treatment (if
necessary). A safety and efficacy
analysis of Cohort I was performed at week 24.
[0311] Starting on Week 4, subjects were eligible to receive rescue injections
of aflibercept 2 mg NT if
there was evidence of increased disease activity according to the retreatment
criteria (see below). The
presence of any one of the following warranted resumption of standard anti-
VEGF treatment with 2 mg
NT aflibercept:
= Loss of? 10 letters in BCVA (using the ETDRS protocol) from Baseline and
intraretinal or
subretinal fluid observed by SD-OCT and judged by the Investigator to be the
cause of the BCVA
loss.
= An increase in central subfield thickness > 75 jun from Baseline as
assessed by SD-OCT.
= Presence of vision-threatening hemorrhage due to macular degeneration.
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[0312] Subjects are evaluated again on week 52 following administration of
AAV2.7m8-aflibercept for
safety and efficacy. The follow up period continues until week 104 following
administration of
AAV2.7m8-aflibercept.
C Cortieofflteroid Regimen
103131 To reduce the possible risk of post-injection ocular inflammation,
subjects were administered a
prophylactic corticosteroid regimen (e.g., prednisone) and monitored closely
for ocular and systemic
tolerability of the vector.
103141 Subjects in Cohort I were administered a prophylactic 13-day oral
corticosteroid regimen,
starting with 60 mg/day of prednisone from 3 days before (day -3) to 3 days
after AAV2.7m8-aflibercept
treatment for a total of 6 days. This was followed by a 7-day prednisone
taper. A summary of the oral
prednisone regimen is provided in Table 2.
Table 2: Oral prednisone regimen.
Study Days Total Number of
Days Prednisone Dose per Day
Days -3 to +3 6
60 mg
Days 4-6 3
40 mg
Days 7-8 2
20 mg
Days 9-10 2
10 mg
Day 11 0
STOP
[0315] Initiating immunosuppression (i.e., prednisone) prior to AAV2.7m8-
aflibercept iv-r injection
was designed to limit the immune response upon exposure to capsid antigens.
Subjects self-administered
prednisone for the 13-day regimen.
103161 Subjects received topical or oral corticosteroids (prednisone) as
needed during weeks 2-24 of the
study.
D. Prohibited Medications and Treatments
[0317] The following medications were prohibited during the study:
= Any systemic anti-VEGF agent including bevacizmnab.
= Any anti-VEGF agent in the study eye other than the study drug or
aflibercept injection 2 mg,
according to the rescue anti-VEGF injection criteria in this study.
= IVT steroids in the study eye.
= Immune suppression drugs. Systemic, inhaled or topical steroids and
NSAIDs were allowed.
= Use of and participation in any other investigational studies.
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= Cataract Surgery in the study eye could be performed if clinically
indicated and was scheduled >
90 days after IVT administration and/or >7 days after the last injection of
aflibercept.
= Subjects who developed AMD in the non-study eye could receive standard of
care therapy in The
non-study eye.
E. Summary of Study Design for Cohort 1
[0318] Six subjects were enrolled into Cohort 1.
[0319] Subjects in Cohort 1 were administered a single NT injection of
AAV2.7m8-aflibercept at Dose
1 of 6 x 1011vg/eye. The first (sentinel) subject enrolled in Cohort I
received an IVT injection of
AAV2.7m8-aflibercept and was evaluated for 29 days prior to dosing the
subsequent 5 subjects (Subjects
2-6) within the cohort.
[0320] A summary of the study design for Cohort 1 is provided in Table 3.
Table 3: Summary of study design for Cohort L
Cohort AAV2.7m8-aflibercept Dose Corticosteroid
Regimen
Cohort 1 6 x 10" vg/eye Prophylactic oral
prednisone
regimen.
F. Study Duration
[0321] Duration of subject participation in the study is approximately 108
weeks for each subject. This
includes a screening period of 4 weeks and an additional 104-week study
period.
[0322] Upon completion or discontinuation of the study, if appropriate,
subjects are offered the
opportunity to enroll in a long-term follow-up study to further assess the
safety of this gene therapy.
V. Study Assessments
A. General Physical Examination and Vital Signs
[0323] Each subject's relevant medical and ophthalmic history was collected
and recorded. A general
physical examination consisted of height (at Screening only), body weight, and
vital signs.
[0324] Vital signs consisted of blood pressure, pulse rate measurements, body
temperature, and
respiratory rate. A 12-lead electrocardiogram (ECG) was taken for each
subject. The following clinical
laboratory and antibody tests were performed: chemistry, complete blood count
(CBC), coagulation
studies, urinalysis, serological evidence of HIV or Hepatitis, and pregnancy
testing.
[0325] At the Week 104, end of study (EOS), and/or Early Termination visit, a
physical examination
assesses if any changes in the subject's physical condition occurred since the
Screening examination.
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B. Immune Response and aibercept Expression
[0326] Total anti-AAV2.7m8 antibodies were measured. Neutralizing anti-
AAV2.7m8 antibodies in
subject's serum were determined using a reporter gene-based transduction
interference assay assessed by
cutpoint.
[0327] The humoral immune response against anti-aflibercept antibodies was
measured in serum using
an ELISA-based cutpoint antibody assay.
[0328] Serum was collected to determine the presence of aflibereept protein.
[0329] Cellular immunity against AAV2.7m8 capsid proteins and aflibercept
protein were measured
using an ELISPOT assay.
C Full Ophthalmic Examination and Other Assessment Methods
[0330] Study assessments included an ophthalmologic exam, intraocular pressure
(lOP), and indirect
ophthalmoscopy.
[0331] The ophthalmic examination consisted of an external examination of the
eye and adnexa, routine
screening for eyelid/pupil responsiveness (including but not limited to
blepharoptosis, abnormal pupil
shape, unequal pupils, abnormal reaction to light, and afferent pupillary
defect), and slit-lamp examination
(eyelids, conjunctiva, cornea, lens, iris, anterior chamber). The slit-lamp
examination examined the
anterior ocular structures and was used for grading any findings. If any
finding was noted during the slit-
lamp examination, at any visit, the severity was graded by the Investigator
and the finding was described
as clinically significant or not clinically significant.
[0332] The IOP measurements were performed using a Goldmann applanation
tonometer or Tono-
penTm. IOP measurements were performed prior to any 1VT injection and prior to
dilating eyes. Day I
visit required pre-injection and post-injection (30 minutes after injection)
IOP measurements.
[0333] The dilated indirect ophthalmoscopy examination included an evaluation
of posterior segment
abnormalities of the vitreous, optic nerve, peripheral retina, and retinal
vasculature. If any finding was
noted during the ophthalmoscopy, at any visit, the severity was graded by the
Investigator and the finding
was described as clinically significant or not clinically significant. Day I
visit required pre-injection and
post-injection indirect ophthalmoscopy assessments.
Spectral Domain Optical Coherence Tomography (SD-OCT)
[0334] SD-0C1' was used to obtain depth-resolved tissue structure information
encoded in the
magnitude and delay of the back-scattered light by spectral analysis of the
interference fringe pattern.
Fluorescein Angiography
[0335] Fluorescein angiography images were used to confirm patient eligibility
for study enrollment, to
assess the efficacy of CNV lesion growth, and to evaluate leakage compared to
Baseline.
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Digital Color Fundus Photography
[0336] Color fundus images of the retina, optic disc, and macula were taken.
Optical Coherence Tomography Angiography (OCT-A)
[0337] OCT-A imaging (swept-source or spectral-domain) was used to obtain
volumetric, three-
dimensional maps of the retina and choroid as well as information on blood
flow.
Refraction and Visual Acuity
[0338] Refraction and BCVA were measured by a trained and certified visual
acuity examiner at the
study sites. Visual acuity was measured at a starting distance of 4 meters,
prior to dilating eyes.
D. Safety Assessments
[0339] To mitigate the risks associated with the NT administration of AAV2.7m8-
aflibercept, subjects
were closely monitored on the day of the NT AAV2.7m8-aflibercept
administration and thereafter post-
treatment.
[0340] The safety of AAV2.7m8-aflibercept was assessed through the collection
of AEs, vital signs,
physical and eye examinations, ECG, pregnancy testing, and laboratory
evaluations.
[0341] Intense monitoring of subjects was done in the first 8 weeks of the
study, followed by regular
safety assessments for safety and efficacy thereafter. All subjects had their
visual acuity tested by Early
Treatment Diabetic ltetinopathy Study (ETDRS) letters assessments at each
study visit, and standard of
care affibercept NT injections were used as a rescue treatment.
[0342] The seventy, or intensity, of an AE was rated on the following scale:
= Mild: the AE was noticeable but did not significantly impair the
subject's daily activities.
= Moderate: the AE reduced or impaired normal daily activity but was not
incapacitating.
= Severe: the AE was incapacitating and resulted in an inability to perform
normal daily
activity.
[0343] Safety is assessed over 104 weeks post-administration of study
treatment. Upon completion of
the End of Study (EOS) Visit, subjects are offered enrollment in a long-term
extension study to further
assess the safety and durability of transgene expression.
E. Efficacy Assessments
[0344] The efficacy of AAV2.7m8-aflibercept in the treatment of wAMD was
assessed by the
following measures. A key assessment time point was at 24 weeks. The baseline
values of BCVA and SD-
OCT refer to the values taken during the screening visit on Days -15 to -7
(e.g., Days -14 to -7) prior to
aflibercept injection. The baseline values were used to compare for analysis.
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[0345] Vision was assessed primarily through the BCVA expressed as an ETDRS
score (number of
letters correctly read) (Vitale etal., (2016) JAMA Ophtalmol 134(9):1041-
1047). Maintenance of vision
was classified if the subject lost fewer than 15 letters in the ETDRS score
compared to Baseline.
Calculated endpoints included the mean change from Baseline, the percent
gaining at least 15 letters
compared to Baseline, and the percent losing 15 or more letters compared to
Baseline.
[0346] FA was performed to assess CNV lesions using a standard technique to
evaluate leakage
compared to Baseline.
[0347] SD-OCT was performed using approved equipment and standard techniques
to evaluate retinal
thickness (e.g., central retinal thickness or central subfield thickness),
macular volume, and the presence
of fluid (e.g., subretinal fluid and intraretinal fluid) compared to baseline
values.
[0348] The number of aflibercept injections given post AAV2.7m8-aflibercept
treatment per subject,
overtime, from Week 4 to Week 104 were determined. In addition, the time to
the first aflibercept
injection post treatment with AAV2.7m8-aflibercept and the proportion of
subjects who did not require an
aflibercept rescue treatment were determined.
[0349] The proportion of subjects without IRE over time from week 4 to week
104 was determined.
[0350] The proportion of subjects without SRF over time from week 4 to week
104 was determined.
[0351] The proportion of subjects without a PED over time from week 4 to week
104 among subjects
with a PED at baseline was determined.
F. Statistics
[0352] The Safety population included all subjects who received AAV2.7m8-
aflibercept and were
analyzed according to the dose received.
[0353] All other safety parameters were summarized by cohort. AEs were coded
using the Medical
Dictionary for Regulatory Activities (MedDRA, version 21) classification to
give a preferred term (PT)
and system organ class (SOC) for each event. SAEs and AEs leading to study
withdrawal were listed
separately.
[0354] Efficacy analyses included all subjects. Efficacy analysis endpoints
were evaluated, and
descriptive statistics will be calculated by cohort. The key assessment time
point was at 24 weeks.
Efficacy was assessed according to dose received and in aggregate.
VI. Results
A. Subject Characteristics
[0355] All six subjects enrolled in Cohort 1 were diagnosed wAMD. At the time
of enrollment, subjects
had a high requirement for anti-VEGF treatment (e.g., required frequent anti-
VEGF treatment), ftmctional
vision around 20/50, some excess central subfield thickness on OCT, and were
undergoing regular IVT
injections of anti-VEGF treatment and responding to therapy. Disease
characteristics and treatment
histories for all subjects are provided in Tables 4-5.
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103561 As shown in Table 4, subjects in Cohort I were diagnosed with wAMD
between approximately
one year (Subject 5) to approximately a decade or more (Subject 4) prior to
being administered
AAV2.7m8-aflibercept. Subjects had received a wide range of prior anti-VEGF
IVT injections in the
study eye, from 7 (Subject 5) to 109 (Subject 4) prior injections. The
calculated average anti-VEGF PIT
injection interval prior to this study ranged from every 4 weeks to every 10
weeks. All subjects received 2
or 3 anti-VEGF injections in the four months prior to screening for enrollment
in this study. Subjects were
administered AAV2.7m8-aflibercept 7 days (Subjects 1-3) or 14 days (Subjects 4-
6) after the Screening
anti-VEGF injection prior to the start of the study.
Table 4: Baseline characteristics of subjects in Cohort 1.
Characteristic
Value
Mean age, years
79.0
Mean time since nAMD diagnosis, years 3.3
Mean number anti-VEGF injections since initial
35.3 (7-109)
diagnosis (range)
Mean number of anti-VEGF injections in 4 2A
months prior to screening
Mean number anti-VEGF injections in 8 months 6.2
prior to screening
Mean number anti-VEGF injections in 12 months 9.2
prior to screening
Average annualized injection frequency 9.3
Raceline BCVA in study eye (ETDRS letters),
65.8
mean
Approximate Snellen equivalent
20/50
Baseline mean CST study eye, Fun
369.2
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Table 5: Disease characteristics and treatment histories of study subjects in
Cohort 1.
Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6
Age 76 years 83 years 87
years 62 years 88 years 77 years
Gender Male Male
Male Male Male Female
Time between
neovascular AMD
diagnosis and 2.3 years 1.1 years 1.2
years 11.0 years 1.0 years 7.0 years
AAV2.7m8-aflibercept
IVT injection:
Number of prior anti-
VEGF injections in 18 9
29 109 7 8
study eye:
Calculated anti-VEGF
injection interval 5-7 weeks 4-10 weeks 4-7 weeks 4-6
weeks 4-7 weeks 4-6 weeks
average pre-study:
Number of anti-VEGF
injections received in 4
3 2
3 3 2 4
months prior to
screening:
Number of anti-VEGF
injections received in 8
6 6 6 6 8
months prior to
screening:
Average annualized
7.5 9
9 9 9 12
injection frequency
Days between
Screening aflibercept
IVT injection and 7 days 7 days 7
days 14 days 14 days 10 days
AAV2.7m8-aflibercept
IVT injection:
B. Safety
[0357] During the 24 weeks following administration of AAV2.7m8-aflibercept,
no SAEs occurred, nor
were there any AEs that met the criteria for dose limiting toxicity (DLT). No
drug-related non-ocular AEs
were observed. Ocular inflammation was observed in all subjects, and was
manageable with topical
steroids. In addition, no vasculitis, retinitis, or choroiditis were observed.
Nineteen ocular AEs were
observed that were deemed potentially related to AAV2.7m8-aflibercept; 14 were
mild, and 5 were
moderate (2 AEs were intermediate uveitis, 1 AE was vitreous cells, 2 AEs were
anterior chamber cells).
One patient had two anterior chamber cell events (one mild event and one
moderate event). Mild-to-
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moderate intraocular inflammation, responsive to topical or oral
corticosteroids, was frequently observed
during early follow-up. OCT images showed resolution of persistent fluid in
most subjects, with no signs
of worsening. Visual acuity was generally stable. A summary of all safety
events that were related to
AAV2.7m8-aflibercept through 24 weeks is provided in Table 6.
Table 6: All safety events related to AAV2.7m8-aflibercept.
AAV2.7m8-
No. of
aflibercept
Patients, Number of Events, n
-related
n
ocular AEs
Patient Patient Patient Patient Patient Patient
1 2 3
4 5 6 Total
Anterior
3 0 1 0 1* 0 21
4
Chamber Cells
AC Flare 3 1 0 2
0 1 0 4
Vitreous Cells 3 0 1* 1 0 0
1 3
Intermediate
2 0 1* 0 0 0 1*
2
Uveitis
Keratic
1 0 0 2 0 0 0
2
Precipitates
Poor Pupil
1 0 0 0 0 1 0
1
Dilation
Ocular Floaters 1 0 1 0 0 0
0 1
Vitreous
1 0 0 0 0 0 1
1
Debris
Vitreous Haze 1 0 0 1 0 0
0 1
* Moderate Event; All events were either mild (14) or moderate (5) in
intensity. 1 Patient had two anterior
chamber cell events (one mild event and one moderate event).
103581 All subjects received 60 mg of oral prednisone for 6 days starting at
day -3, followed by a 7-day
tapering course of prednisone. Clinical assessment of ocular cellular
inflammation revealed that no
clinically significant inflammation occurred early post-AAV2.7m8-aflibercept.
In addition, as shown in
FIG. 6, no worsening or new inflammation was observed when subjects were
receiving steroid eye drops.
By week 24, anterior chamber cellular inflammation was resolved or improving.
Observed cellular
inflammation was generally mild. The aqueous cell count categories shown in
FIG. 6 were based on the
Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al., J
Ophthalmol. 2005;140:509-
516), whereas vitreous cell count categories were based on National Institutes
of Health (Nil-I) guidelines.
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[0359] Overall, safety assessments showed that AAV2.7m8-aflibercept was well
tolerated, with no
DLTs or SAEs reported. All safety events were mild to moderate, and most were
inflammation-related.
Continued follow-up of AEs showed that mean visual acuity remained stable and
no anti-VEGF rescue
injections were required. Thus, the results indicate that AAV2.7m8-aflibercept
administered as a single
IVT injection at a dose of 6x10" vg/eye in wAMD patients previously requiring
frequent anti-VEGF
injections has an acceptable safety profile.
C Efficacy
[0360] Following AAV2.7m8-aflibercept administration, all six subjects showed
stabilization of disease
activity based on OCT assessments. As shown in FIGS. 2A-2L, OCT images taken
prior to and after
treatment with AAV2.7m8-aflibercept indicated that a robust anatomical
response was evident in all six
subjects in Cohort 1. For example, subretinal fluid persisted with standard of
care anti-VEGF treatment,
but resolved and remained resolved following administicition of AAV2.7m8-
aflibercept in Subject 1 (e.g.,
compare FIGS. 2A and 2B), Subject 2 (e.g., compare FIGS. 2C and 2D), Subject 3
(e.g., compare FIGS.
2E and 2F), Subject 4 (e.g., compare FIGS. 2G and 2H), and Subject 5 (e.g.,
compare FIGS. 21 and 2J).
Subject 6 exhibited retinal morphology consistent with polypoidal choroidal
vasculopathy (PCV) (FIG.
2K), and while some fluid persisted after treatment with AAV2.7m8-aflibercept,
there was no evidence of
disease progression (FIG. 2L). In addition, as shown in FIG. 3, none of the
subjects exhibited an
increase in central subfield thickness (CST) and a mean decrease of -52.7 tun
was observed (90% CI -
86.5, -18.8). Increased CST is indicative of disease progression in wAMD.
Importantly, none of the
subjects were administered or required any standard of care allibercept rescue
injections ("rescue
injections").
[0361] Best corrected visual acuity (BCVA) was measured based on ETDRS letters
assessments in all
subjects throughout the study. As shown in FIG. 4, BCVA was stable across
subjects in Cohort 1 with a
mean decrease of -2 letters (90% CI -9.1, 5.1).
[0362] At a median follow up time of 34 weeks after administration of AAV2.7m8-
aflibercept, no
subjects exhibited signs of disease reactivation on OCT imaging (FIGS. 7A-7B).
In addition, no subjects
required any rescue anti-VEGF NT injections and no subjects met retreatment
criteria at any point during
the follow-up period of up to 44 weeks. Finally, during the additional follow-
up period beyond 24 weeks,
BCVA was maintained (i.e., no patient lost or gained greater than 10 ETDRS
letters), the anatomic
improvements (i.e., resolution of subretinal and intraretinal fluid, and
reductions in CST) observed at
week 24 were maintained, and no safety concerns arose.
[0363] A summary of safety and efficacy results evaluated at the median follow
up time of 34 weeks is
provided in Table 7. A summary of efficacy results evaluated at the median
follow up time of 44 weeks
is provided in Table 8. One patient experienced a macula-off retinal
detachment unrelated to study
treatment. For that patient, the last observations prior to detachment were
used to calculate CST and
BCVA mean values and ranges shown in Table 8.
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Table 7: Summary of safety and efficacy results at median follow up time of 34
weeks.
Value
Median follow-up, weeks
34.0
Follow-up (min, max), weeks
28, 44
Grade 3 adverse events, n
0
Serious adverse events, n
0
Dose-limiting toxicities, n
0
Mean BCVA change from baseline, ETDRS letters
-1.5
BCVA change from baseline (min, max), ETDRS letters -9, +5
Total number of rescue injections, n
0
Table 8: Summary of efficacy results at median follow up time of 44 weeks.
Value
Mean BCVA change from baseline, ETDRS letters
-LS
BCVA change from baseline (min, max), ETDRS letters
-7, -1-7
Mean CST change from baseline, gm
-18.5
CST change from baseline (min, max), gm
-75, +32
Total number of rescue injections, n
0
103641 A summary of an additional analysis of efficacy results at the median
follow up time of 44 weeks
(range of 40-52 weeks) after administration of AAV2.7m8-aflibercept is
provided in Table 9.
Table 9: Summary of efficacy results at the follow up time of 40-52 weeks
(median 44
weeks).
Value
Mean BCVA change from baseline, ETDRS letters
-1.0
BCVA change from baseline (min, max), ETDRS letters
-7, +7
Mean CST change from baseline, pm
-25.5
CST change from baseline (min, max), gin
-117, +32
Total number of rescue injections, n
0
103651 Cellular inflammatory events and steroid treatments given to each
subject were analyzed up to
the follow-up time of 52 weeks. As shown in FIG. 8, Subjects 1 and 5 had no
cellular reactions beyond
day 3. Both subjects started topical steroids in the second 6 months of the
study for aqueous cell (AC)
flare and posterior synechiae. Both subjects received topical steroids and
mydriatic eye drops, and
Subject 1 has stopped receiving topical steroid eye drops. Subjects 2 and 6
exhibited early onset
inflammation by the first month after administration of AAV2.7m8-aflibercept,
with an AC response and
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some spillover to vitreous cells. The inflammation in both subjects resolved
following treatment with
topical steroids. Both subjects continued twice daily eye drops (BID).
Subjects 3 and 4 experienced more
delayed cellular inflammation onset, after topical steroids were discontinued
(by months 2-3 after
administration of AAV2.7m8-aflibercept). The inflammation in these subjects
was responsive to topical
steroids. Subject 3 has stopped receiving topical steroid eye drops. Subject 4
experienced an unrelated
retinal detachment and is receiving BID eye drops.
[0366] Overall, inflammation was low grade, with no early clinically
significant inflammation reported.
All inflammation was responsive to topical steroids, with a trend towards
resolution over time.
Inflammation was primarily driven an aqueous cell process.
[0367] FIGS. 9A-9B show OCT imaging of Subjects 1-6 through follow-up weeks 40
to 52. No
subjects exhibited signs of disease reactivation on OCT imaging, and BCVA and
anatomic improvements
were maintained. In addition, no subjects required any rescue anti-VEGF PIT
injections or met
retreatment criteria
11 Conclusions
103681 The current standard of care therapy for wAMD is anti-VEGF PIT
injections that are typically
required long term, about every 4-8 weeks. Compliance with this regimen can be
difficult for patients,
caregivers, and healthcare systems, leading to suboptimal dosing and loss of
vision due to undertreatment.
[0369] The results presented in this example show that AAV2.7m8-aflibercept
provided a clear benefit
for improving retinal anatomy and stabilizing vision, while exhibiting an
acceptable safety profile.
Specifically, patients maintained vision during the study, and AAV2.7m8-
aflibercept was shown to be
safe and well tolerated, with observed inflammation generally being mild and
responsive to steroid eye
drops.
[0370] It was noted that subjects in Cohort 1 of this study previously
required frequent anti-VEGF
injections (Table 4) in order to slow or prevent progression of their disease,
however no anti-VEGF
rescue injections were required during this study following administration of
AAV2.7m8-aflibercept.
[0371] It was noted that subretinal fluid was present on OCT scans of subjects
undergoing standard of
care anti-VEGF treatment for more than 20 weeks, and continued 1-2 weeks after
the Screening
aflibercept PIT injection. This fluid was present even though subjects had
been treated repeatedly and
was thus refractory to standard of care PIT bolus of anti-VEGF protein.
Unexpectedly, this refractory
SRF resolved after treatment with AAV2.7m8-aflibercept, a result which would
not have been predicted
from preclinical studies.
[0372] The observed safety and efficacy of AAV2.7m8-aflibercept administered
at a dose of 6x10"
vg/eye led to the continuation of this Phase 1 study to assess the safety and
efficacy of AAV2.7m8-
aflibercept administered at lower doses and with topical corticosteroids, as
described in Example 2.
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Example 2: An open label Phase I study of AAV2.7m8-aflibercept in neovascular
(wet) age-related
macular degeneration at doses lower than 6 x 101-1vg/eye and with topical
corticosteroids.
[0373] The following example describes a continuation of the Phase 1 study
described in Example 1 to
assess the safety and efficacy of AAV2.7m8-aflibercept administered at doses
lower than 6x10" vg/eye
and with topical corticosteroids in subjects with wAMD.
I. Study Objectives and Endpoints
103741 The primary objective, secondary objectives, and primary and secondary
endpoints are as
described in Section I of Example 1.
H. Study Subjects
[0375] The study subjects in this study are as described in Section II of
Example 1.
III. Investigational Medicinal Product
[0376] The investigational medicinal product is AAV2.7m8-aflibercept, as
described in detail in Section
III of Example 1, and as depicted in FIG. 1A, with the exception that the
concentration of AAV2.7m8-
aflibercept was varied in order to maintain a suitable injection volume as
described in Table 10, below.
Table 10: Formulation of the AAV2.7m8-aflibercept investigational medicinal
product for cohorts 2-4.
AAV2.7m8-aflibercept
Injection Volume
Concentration
Cohort 2 6 x 1012
vg/mL 30 iaL
Cohort 3 6 x 1012
vg/mL 30 it
Cohort 4a 6 x 1012
vg/mL 100 id,
Cohort 4b 6 x 1011
vg/mL 100 tiL
IV. Study Design
A. Dose and Method of Administration
103771 AAV2.7m8-aflibercept at the doses described below was administered as
described in Section IV
of Example 1.
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Study Cohorts 2-4
[0378] Cohort 2: Six subjects who are diagnosed with wAMD are enrolled into
Cohort 2. Subjects in
Cohort 2 are administered a single NT injection of AAV2.7m8-aflibercept at
Dose 2 of 2 x 10Hvg/eye
with a prophylactic oral prednisone regimen.
[0379] Cohort 3: Nine subjects who are diagnosed with wAMD are enrolled into
Cohort 3. Subjects in
Cohort 3 are administered a single NT injection of AAV2.7m8-aflibercept at
Dose 2 of 2 x 1011vg/eye
with a prophylactic topical corticosteroid regimen.
[0380] Cohort 4: Nine subjects who are diagnosed with wAMD are enrolled into
Cohort 4. If signs of
choroidal neovascularization exudation requiring rescue therapy are observed
in a majority of subjects in
Cohorts 2 and 3, the subjects in Cohort 4 are administered a single NT
injection of AAV2.7m8-
aflibercept at Dose 3 of 6x101 vg/eye with a topical corticosteroid regimen
(Cohort 4b). If signs of
choroidal neovascularization exudation requiring rescue therapy are not
observed in a majority of subjects
in Cohorts 2 and 3, the subjects in Cohort 4 are administered a single NT
injection of AAV2.7m8-
aflibercept at Dose 1 of 6x10'' vg/eye with a topical corticosteroid regimen
(Cohort 4a).
R. Study Visits
[0381] The study visits are as described in Section IV of Example 1 and FIG.
1B.
C Corticorteroid Regimen
[0382] Subjects in Cohort 2 are administered a prophylactic 13-day oral
corticosteroid regimen as
described in detail for Cohort in Example 1:
[0383] Cohort 2: Subjects in Cohort 2 are administered a single NT injection
of AAV2.7m8-
aflibercept at Dose 2 of 2 x 1011vgjeye with a prophylactic 13-day oral
corticosteroid regimen, starting
with 60 mg of prednisone 3 days before and 3 days after AAV2.7m8-aflibercept
treatment for a total of 6
days. This is followed by a 7-day prednisone taper. A summary of the oral
prednisone regimen for Cohort
2 is provided in Table 2 of Example 1.
[0384] Subjects in Cohorts 3 and 4 are administered a prophylactic tapering
regimen of topical
corticosteroid (difluprednate 0.05% drops) as described below:
[0385] Cohort 3: Subjects in Cohort 3 are administered a single NT injection
of AAV2.7m8-
aflibercept at Dose 2 of 2 x 1011vg/eye with a prophylactic topical 4-week
tapering corticosteroid
regimen.
[0386] Cohort 4: Subjects in Cohort 4b are administered a single NT injection
of AAV2.7m8-
aflibercept at Dose 3 of 6x101 vg/eye with a prophylactic topical 4-week
tapering corticosteroid regimen
that can be extended at the discretion of the treating physician. Subjects in
Cohort 4a are administered a
single NT injection of AAV2.7m8-aflibercept at Dose 3 of 6x1011 vg/eye with a
prophylactic topical 4-
week tapering difluprednate regimen that can be extended at the discretion of
the treating physician.
[0387] A summary of the topical difluprednate regimen is provided in Table HA,
below.
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Table 11A: Summary of topical difluprednate regimen.
Study Weeks Difluprednate administrations per day
Week 1
4 times
Week 2
3 times
Week 3
2 times
Week 4
1 time
The difluprednate regimen can be extended at the discretion of the treating
physician.
103881 Subjects in Cohorts 3 and 4 may also be administered a 6-week
prophylactic corticosteroid
regimen that can be extended at the discretion of the treating physician. A
summary of the 6-week
prophylactic corticosteroid regimen is provided in Table 1113.
Table .1.11i Summary of the 6-week topical dithrprednate regimen.
Study Days Total
Difluprednate administrations
Number of
per day
Days
Days 1 to 21 21
4 times
Days 22 to 28 7
3 times
Days 29 to 35 7
2 times
Days 36 t,o 42 7
1 time
Day 43 and 0 The
difluprednate regimen can be extended at the
beyond
discretion of the treating physician.
103891 Initiating immunosuppression immediately after exposure to AAV2.7m8-
aflibercept IVT
injection is designed to limit the immune response upon exposure to capsid
antigens. Subjects self-
administer one drop of difluprednate 0_05% solution (2.5 mg difluprednate) to
the conjunctival sac of the
treated eye 4 times per day for the first week beginning on the day of
injection of AAV2.7m8-aflibercept
(Day 1, post-injection). This is followed by 3 times per day for second week,
2 times per day for the third
week, and 1 time per day for the fourth week. The tapering corticosteroid
regimen may be extended at the
discretion of the treating physician.
D. Prohibited Medications and Treatments
103901 The prohibited medications and treatments for this study are as
described in Section IV of
Example 1.
E Summary of Study Design for Cohorts 2-4
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[0391] A summary of the study design for Cohorts 24 is provided in Table 12.
Table 12: Summary of study design for Cohorts 2-4.
Cohort AAV2.7m8-aflibercept
Dose Corticosteroid Regimen
Cohort 2 2 x 101'
vg/eye Prophylactic oral
prednisone
regimen.
Cohort 3 2 x 101'
vg/eye Prophylactic topical
corticosteroid.
Cohort 4a 6 x 101'
vg/eye Prophylactic topical
corticosteroid.
Cohort 4b 6 x 1010
vg/eye Prophylactic topical
corticosteroid.
F. Study Duration
[0392] The duration of subject participation in this study is as described in
Section IV of Example 1.
V. Study Assessments
[0393] Assessments performed in this study are as described in Section V of
Example 1. In addition,
samples are taken of aqueous and vitreous humors.
Aqueous Humor Sampling
[0394] Aqueous humor samples are obtained at screening (prior to aflibercept
injection) and on Day 1
(prior to AAV2.7m8-aflibercept injection), Weeks 12, 24, 36, 52, 76, 88, 104,
and at any visit requiring
the first aflibercept (Eylea) rescue treatment, or early termination. Aqueous
humor samples are analyzed
for aflibercept, VEGF-A, and NAb concentrations.
Vitreous Humor Sampling
[0395] Vitreous hurnor samples are obtained at any point during the study when
a vitrectomy is
performed. Vitreous humor samples are analyzed primarily for aflibercept
concentrations. Remaining
samples are analyzed for VEGF-A concentrations.
VI. Results
A. Subject Characteristics
[0396] Disease characteristics and treatment histories for all subjects are
recorded. In particular, the date
or year of neovascular AMD diagnosis, the number of prior anti-VEGF injections
in the study eye, the
calculated anti-VEGF injection interval average pre-study, the number of anti-
VEGF injections received
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in the 4 months prior to screening, the date of Screening aflibercept IVT
dose, and the date of
AAV2.7m8-aflibercept injection are recorded.
B. Safety
[0397] Subjects are closely monitored on the day of the IVT AAV2.7m8-
aflibercept administration and
thereafter post-treatment.
[0398] The safety of AAV2.7m8-aflibercept is assessed through the collection
of vital signs, physical
and eye examinations, ECG, pregnancy testing, and laboratory evaluations.
[0399] The incidence, severity, and relationship to treatment of AEs, SAEs,
and DLTs are assessed in
all subjects.
[0400] The occurrence, severity, and relationship to treatment of intraocular
inflammation is assessed in
all subjects.
[0401] Visual acuity is assessed in all subjects through BCVA expressed as an
ETDRS score (number of
letters correctly read) (Vitale et al., (2016) JAMA Ophtalmol 134(9):1041-
1047).
[0402] The requirement for anti-VEGE rescue injections is monitored in all
subjects.
[0403] The existence of a correlation between preexisting anti-AAV NAb-levels
in subject's sera and
AEs, SAEs, and DLTs is determined.
[0404] The effect of steroid prophylaxis (topical or oral)
on the occurrence and resolution of AEs,
SAEs, DLTs, and intraocular inflammation is assessed.
104051 Standard of care aflibercept IVT injections are used as a rescue
treatment.
C. Efficacy Assessments
[0406] Disease stabilization is assessed in all subjects. Retinal thickness,
central subfield thickness, fluid
and other anatomical features are measured (e.g., by OCT imaging) prior to and
after administration of
study treatment.
[0407] The efficacy of AAV2.7m8-aflibercept in the treatment of wAMD is
assessed by the following
measures. A key assessment time point is at 24 weeks. The baseline values of
BCVA and SD-OCT refer
to the values taken during the screening visit on Days -15 to -7 (e.g., Days -
14 to -7) prior to aflibercept
injection. The baseline values are used to compare for analysis.
[0408] Vision is assessed primarily through BCVA expressed as an ETDRS score
(number of letters
correctly read) (Vitale et al., (2016) JAMA Ophtalmol 134(9):1041-1047).
Maintenance of vision is
classified if the subject loses fewer than 15 letters in the ETDRS score
compared to Baseline. Calculated
endpoints include the mean change from Baseline, the percent gaining at least
15 letters compared to
Baseline, and the percent losing 15 or more letters compared to Baseline.
[0409] FA is performed to assess CNV lesions using a standard technique to
evaluate leakage compared
to Baseline.
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[0410] SD-OCT is performed using approved equipment and standard techniques to
evaluate retinal
thickness (e.g., central retinal thickness or central subfield thickness),
macular volume, and the presence
of fluid (e.g., subretinal fluid and intraretinal fluid) and fluid compared to
Baseline values.
[0411] The number of aflibercept injections given post AAV2.7m8-aflibercept
treatment per subject,
overtime, from Week 4 to Week 104 are determined. In addition, the time to the
first aflibercept injection
post treatment with AAV2.7m8-aflibercept and the proportion of subjects who
did not require an
aflibercept re-treatment are determined.
[0412] The proportion of subjects without IRF over time from week 4 to week
104 is determined.
[0413] The proportion of subjects without SRF over time from week 4 to week
104 is determined.
[0414] The proportion of subjects without a PED over time from week 4 to week
104 among subjects
with a PED at baseline is determined.
Example 3: Results of an open label Phase 1 study of AAV2.7m8-aflibercept in
neovascular (wet) age-
related macular degeneration at doses lower than 6 x 1011vgfeye and with
topical corticosteroids.
[0415] This Example describes the results of the Phase 1 study described in
Example 2 that assessed the
safety and efficacy of AAV2.7m8-aflibercept administered at doses lower than
6x10" vg/eye and with
topical corticosteroids in subjects with wAMD.
Results
Cohort 2
[0416] The baseline characteristics of subjects in Cohort 2 are provided in
Table 13.
Table 13. Baseline characteristics of subjects in Cohort 2.
Characteristic
Value
Mean age, years
79.8
Mean time since nAMD diagnosis, years 4.0
Mean number anti-VEGF injections since initial
34.0 (4-69)
diagnosis (range)
Mean number anti-VEGF injections in 12 months 9.2
prior to screening
Baseline BCVA in study eye (ETDRS letters),
64.7
mean
Approximate Snellen equivalent
20/50
Baseline mean CST study eye, vim
307/
[0417] As described in Example 2, subjects in Cohort 2 were administered a
single PIT injection of
AAV2.7m8-aflibercept at Dose 2 of 2 x 10"vg/eye with a prophylactic 13-day
oral corticosteroid
regimen, starting with 60 mg of prednisone 3 days before and 3 days after
AAV2.7m8-aflibercept
treatment for a total of 6 days. This was followed by a 7-day prednisone
taper. A summary of the oral
prednisone regimen for Cohort 2 is provided in Table 2 of Example 1.
[0418] As shown in FIG. 10, 24 weeks after administration of AAV2.7m8-
aflibercept, BCVA was
stable across subjects in Cohort 2, with a mean decrease of -4.8 ETDRS
letters. In addition, as shown in
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FIG. 11, subjects exhibited improved central retinal thickness on OCT, with an
average decrease in CST
of 27.8 prn. Four out of six subjects (66%) had not received a rescue anti-
VEGF injection at the follow-up
time of 24 weeks.
[0419] Cellular inflammatory events and steroid treatments
given for each subject were analyzed up to
a follow-up time of 24 weeks. As shown in FIG. 12, inflammation was responsive
to topical steroids.
Subjects 4 and 5 had no clinically relevant cellular inflammation (Subject 5
had <5 AC cells at week 20)
and required no steroid treatments. Subjects 2 and 3 showed 1/2 + aqueous and
vitreous cell grades, or
between 1 and 5 cells observed in the aqueous and between 1 and 10 cells
observed in the vitreous that
resolved following a short course of topical steroids. Subject 1 had an early
inflammatory response
following cessation of oral steroids that responded quickly to steroid eye
drops; the subject remained on
steroid eye drops four times per day (QID). Subject 6 had an AC cellular
response that worsened on BID
drops and resolved on QID drops. Aqueous cell grade categories were based on
the Standardization of
Uveitis Nomenclature (SUN) criteria (Jabs, DA et al. J Ophthalmol.
2005;140:509-516). Vitreous cell
grade categories were based on National Institutes of Health (NMI) guidelines.
[0420] Overall, inflammation was generally mild. Oral steroids did not provide
long enough coverage in
certain subjects. However, all inflammation events were responsive to topical
steroids.
[0421] OCT imaging at 24 weeks after administration of AAV2.7m8-aflibercept
showed anatomic
improvements and maintenance of visual acuity (BCVA). As shown in FIGS. 13A-
13B, Subjects 1,2, 4,
and 6 exhibited resolution of fluid. Subject 3 received 3 rescue aflibercept
injections, all for a loss of 10
BCVA letters attributable to retinal fluid. Subject 5 received 3 rescue
aflibercept injections, all for a CST
increase of 75 p.m.
[0422] Through week 24 after administration of AAV2.7m8-aflibercept, four out
of six subjects had not
received any anti-VEGF rescue injections. As shown in the Swimmer's Lane Plot
in FIG. 14, Subjects 3
and 5 received three anti-VEGF IVT injections each during the 24-week follow-
up period.
[0423] Overall, the results for Cohort 2 at 24 weeks after administration of
AAV2.7m8-aflibercept show
that AAV2.7m8-aflibercept has robust efficacy. In particular, mean BCVA was
maintained and retinal
anatomy and CST were improved in subjects not requiring rescue anti-VEGF
injections. In addition,
AAV2.7m8-aflibercept was safe and well tolerated, with low-grade inflammation
being responsive to
steroid eye drops.
Example 4: Overview of results of an open label Phase 1 study of AAV2.7m8-
aflibercept in neovascular
(wet) age-related macular degeneration.
[0424] This Example provides an overview of the results of the open label
Phase 1 study of AAV2.7m8-
aflibercept for the treatment of age-related macular degeneration (AM])) with
choroidal
neovascularization described in Examples 1-3.
Safely
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[0425] Results available for Cohort 1 up to the median follow up time of 44
weeks and for Cohort 2 up
to the follow up time of 24 weeks show that AAV2.7m8-aflibercept was well
tolerated. Specifically, no
AAV2.7m8-aflibercept or procedure-related serious adverse events have been
reported. In addition, no
AAV2.7m8-aflibercept-related systemic adverse events or adverse events meeting
the criteria for dose-
limiting toxicities have been reported. AAV2.7m8-aflibercept-related adverse
events were mild (71%) or
moderate (29%). Low-grade inflammation was commonly reported and was
responsive to steroid eye
drops. No vasculitis, retinitis, or choroiditis were reported. One serious
adverse event of spontaneous,
pseudophakic retinal detachment unrelated to AAV2.7m8-aflibercept was
reported. The retinal
detachment was surgically repaired and the subject remains under follow-up.
Efficacy
[0426] Results available for Cohort 1 up to The median follow up time of 44
weeks and for Cohort 2 up
to the follow up time of 24 weeks show that AAV2.7m8-aflibercept had robust
efficacy. In Cohort 1, no
subjects were administered an anti-VEGF rescue injection. In Cohort 2, four
out of six subjects were not
administered anti-VEGF rescue injections. Overall, in the 10 out of 12
subjects (83%) in Cohorts 1 and 2
that were not administered anti-VEGF rescue injections, the mean BCVA was
maintained and mean CST
was improved.
[0427] A summary of the available safety and efficacy results for Cohorts 1
and 2 is provided in Table
14.
Table 14. Summary of safety and efficacy results for subjects in Cohorts 1 and
2.
Results Following a Single AAV2.7m8-
Cohort 1 Cohort 2
aflibercept Dose:
Subjects 6
6
Dose of AAV2.7m8-aflibercept Higher Dose
Lower Dose
6x 10" vg/eye
2x 10" vg/eye
Rescue Injections:
Follow-up 50 weeks
(median) 24 weeks (median)
Number of subjects requiring anti-VEGF 0/6 subjects
2/6 subjects
rescue injections
Total anti-VEGF rescue injections 0 injections
6 injections
Safety:
Follow-up 50 weeks
(median) 24 weeks (median)
Systemic adverse events 0
0
Dose-limiting toxicities (DLTs) 0
0
Serious adverse events (SAEs)i 1
0
Drug/procedure related SAEs 0
0
Change in BCVAz:
Full cohort Rescue-free
subjects
Follow-up 44 weeks
(median) 24 weeks 24 weeks
Mean (ETDRS letters)3 -1.0
-4.8 -0.8
Range (ETDRS letters) -71 7
-191 -14 / +16
+16
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Change in CRV:
Follow-up 44 weeks
(median) 24 weeks 24 weeks
Mean (itm)4 -25.5
-27,8 -30,8
Range ( mt -1171+32
-611-8 -61 / -8
This event was deemed unrelated to AAV2.7m8-aflibercept or any study
procedure.
2 Best corrected visual acuity (BCVA) as measured by Early Treatment Diabetic
Retinopathy Study
(E'TDRS) (it., sight charts).
3 Central retinal thickness (CRT), also referred to as central subfield
thickness (CST) assessed using
Optical Coherence Tomography (OCT) imaging.
BCVA and CST values for subject with retinal detachment (unrelated to study
treatment) used last
observations prior to detachment.
Example 5: Additional results of an open label Phase .1 study of AAV27m8-
ajlibercept in neovascular
(wet) age-related macular degeneration.
104281 This Example describes results for Cohorts 1, 2, and 3 of the Phase 1
study described in
Examples 1-4 that assessed the safety and efficacy of a single intravitreal
injection of AAV2.7mS-
allibercept in subjects with wAMD. Safety and efficacy results are provided
for Cohort 1 at a median
follow up time of 60 weeks (range of 52-64 weeks), for Cohort 2 at a median
follow up time of 36 weeks
(range of 32-40 weeks), and for Cohort 3 at a follow up time of up to 20
weeks.
Results
104291 Subjects in Cohort I were administered a single IVT injection of
AAV2.7m8-aflibercept at a
dose of 6 x 10"vg/eye. Subjects in Cohort 2 were administered a single IVT
injection of AAV2.7m8-
aflibercept at a dose of 2 x 10"vg/eye. Subjects in Cohorts 1 and 2 were also
administered a prophylactic
oral prednisone regimen (see Table 2).
104301 Subjects in Cohort 3 were administered a single IVT injection of
AAV2.7m8-aflibercept at a
dose of 2 x 10"vg/eye with a 6-week prophylactic topical corticosteroid
regimen (see Table JAB). The
topical corticosteroid regimen consisted of corticosteroid eye drops (0.05%
difluprednate [2.5ng1)
administered four times per day for 3 weeks (QID for 3 weeks), starting on the
same day as the
administration of AAV2.7m8-aflibercept (administered after the IVT injection
of AAV2.7m8-aflibercept),
followed by a 3-week taper which consisted of corticosteroid eye drops
administered three times per day
for one week (TID for 1 week), followed by corticosteroid eye drops
administered two times per day for
one week (BID for 1 week), followed by corticosteroid eye drops administered
once per day for one week
(QD for 1 week).
Subject Characteristics
104311 The baseline subject characteristics for subjects in Cohorts 1, 2, and
3 are provided in Table 15.
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Table 15. Baseline characteristics of subjects in Cohorts 1-3.
Baseline Characteristics Cohort 1 (N=6)
Cohort 2 (N=6) Cohort 3 (N=9)
Mean age, years 79.0
79.8 77.4
Mean years since nAMD
3.5 4.1 3.3
diagnosis
Mean (range) number anti-
VEGF injections since initial 35.3 (7-109)
34.0 (4-69) 24.8 (9-70)
diagnosis
Mean number anti-VEGF
injections in 12 months prior to 9.2
9.2 9.1
AAV2.7m8-aflibercept
Mean BCVA study eye, ETDRS
65.8 64.7 65_9
letters
20/50
20/50 20/50
Approximate Snellen equivalent
Mean CST study eye, jun 369.2
307.7 472.3
104321 As shown in Table 15, subjects in Cohorts 1-3 previously required
frequent anti-VEGF
injections to maintain vision. The baseline characteristics for subjects in
Cohorts 1-3 were well balanced,
with similar ages, similar mean prior anti-VEGF injections, and a similar need
for frequent injections
(e.g., over 9 injections in the year prior to administration of AAV2.7m8-
aflibercept) to maintain a
relatively high baseline vision of about 65 ETDRS letters across cohorts.
Subjects in Cohort 3 were, on
average, younger and had fewer prior anti-VEGF injections since initial
diagnosis and a thicker average
baseline CST.
Safety Results
104331 As shown in Table 16, across Cohorts 1-3, no AAV2.7m8-aflibercept- or
procedure-related
serious adverse events (SAEs) occurred. In addition, no AAV2.7m8-aflibercept-
related systemic adverse
events (AEs) were observed. An SAE of retinal detachment that was unrelated to
study treatment occurred
and was surgically repaired and resolved (a previous cataract extraction and
artificial lens implantation
were key risk factors). Two subjects had adverse events of intraocular
pressure (lOP) elevation that
resolved. One subject had two mild IOP elevation events (highest TOP was
24mmHg) that were both
treated with Combigan eye drops. The second subject was taking Combigan0 for
ocular hypertension,
and the IOP AE spontaneously resolved within a month with no change to
treatment.
104341 Subjects in Cohort 3 (administered steroid eye drop prophylaxis) had a
low number of ocular
adverse events.
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Table 16. Adverse events in Cohorts 1, 2, and 3.
Cohort 1 (n=6)
Cohort 2 (n=6) Cohort 3 (n=9)
Category
Subjects Events Subjects Events Subjects Events
Ocular AE (Total) 6 49
5 32 7 16
Serious AE 1 1*
0 0 0 0
AAV2.7m8-aflibercept-
related** 6 29
5 21 4 8
Non-ocular AE (Total)t 5 17
5 5 4 6
Serious AE: 1 1
0 0 2 2
* Retinal detachment (unrelated to AAV2.7m8-aflibercept); **AAV2.7m8-
aflibercept-related ocular
events were mild (69%) or moderate (31%); I-None of the non-ocular AEs were
AAV2.7m8-
aflibercept-related; tSerious non-ocular AEs included degenerative
intervertebral disc disease (1) in
Cohort 1, and COPD exacerbation (1) and stable angina pectoris (1) in Cohort
3.
104351 As shown in FIGS. 15A-15C, mild and low-grade inflammation, primarily
affecting the anterior
segment, was common across Cohorts 1-3 and was responsive to topical steroids.
No clinical or
fluorescein (fluorescein angiography of posterior pole) evidence of
vasculitis, retinitis, or choroiditis were
observed.
104361 Three subjects in Cohort 1 continue to be managed with topical
steroids, but only 1 subject has
any evidence of inflanunatory cells. Subject 2 is on topical steroids once per
day; Subject 4 is on topical
steroids twice per day; and Subject 6 is on topical steroids once per day.
Overall, none of the subjects in
Cohort 1 ever had inflammatory cell counts above 2+. In addition, subjects in
Cohort 1 only had
inflammatory cell counts of 2+ for one time point.
104371 Two subjects in Cohort 2 continue to be managed with topical steroids
and have evidence of
mild inflammatory cells. Subject 1 is on topical steroids once per day and
Subject 6 is on topical steroids
once per day. Subjects 1 and 6 had some breakthrough inflammation of up to 3+
inflammatory cell counts,
but both subjects rapidly resolved with topical steroid treatment.
104381 Subjects in Cohort 3 were administered a 6-week prophylactic steroid
eye drop regimen. This
regimen minimized early ocular inflammation (FIG. 15C). Certain subjects in
Cohort 3 also continue to
be managed with topical steroids. Subject 3 in Cohort 3 is receiving topical
steroids four times per day.
Subject 5 is no longer receiving topical steroids but was administered topical
steroids as follows: four
times per day for 3 weeks following administration of AAV2.7m8-aflibercept,
then twice per day for one
week, then four times per day for a week starting at Week 5, and then was
decreased from three times per
day for a week, to twice per day for a week, to once per day for a week.
Subjects 7-9 of Cohort 3 continue
on the 6-week prophylactic steroid eye drop regimen. ht addition to the
prophylactic steroid regimen
described, Subject 6 was hospitalized for chronic obstructive pulmonary
disease (COPD) following
treatment with AAV2.7m8-aflibercept and was prescribed oral steroids. The COPD
was found not to be
related to AAV2.7m8-aflibercept.
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Efficacy
104391 FIG, 16A shows the mean BCVA (ETDRS letters) from baseline up to Week
52 for subjects in
Cohort 1. At a median follow up time of 60 weeks (range of 52-64 weeks),
subjects in Cohort 1 had a
mean BCVA change from baseline of -2.7 letters and 100% of subjects (6/6) had
not received a rescue
anti-VEGF injection. The apparent decrease in mean BCVA at Weeks 44 and 48 was
due to one subject
that experienced an SAE of retinal detachment. FIG. 16B shows the mean CST
(tun) from baseline up to
Week 52 for subjects in Cohort 1. At a median follow up time of 60 weeks
(range of 52-64 weeks),
subjects in Cohort 1 had a mean CST change from baseline of -26.2 pm.
104401 FIG. 17A shows the mean BCVA (ETDRS letters) from baseline up to Week
36 for subjects in
Cohort 2. At a median follow up time of 36 weeks (range of 32-40 weeks),
subjects in Cohort 2 had a
mean BCVA change from baseline of -2.8 letters and 67% of subjects (4/6) had
not received a rescue anti-
VEGF injection. The subjects that had not received a rescue anti-VEGF
injection had a mean BCVA
change from baseline of +2.3 letters. FIG. 17B shows the mean CST (pm) from
baseline up to Week 36
for subjects in Cohort 2. At a median follow up time of 36 weeks (range of
3240 weeks), subjects in
Cohort 2 had a mean CST change from baseline of -40.8 pm. In addition, the
subjects that had not
received a rescue anti-VEGF injection had a mean CST change from baseline of -
30.0 pm. Overall, in
Cohort 2, CST improvements were maintained out to Week 36.
104411 FIG. 18A shows the mean BCVA (ETDRS letters) from baseline up to Week
20 for five
subjects in Cohort 3. At a follow up time of 20 weeks, the five subjects from
Cohort 3 had a mean BCVA
change from baseline of +6.8 letters and 80% (4/5) of the five subjects from
Cohort 3 had not received a
rescue anti-VEGF injection. The subjects that had not received a rescue anti-
VEGF injection had a mean
BCVA change from baseline of +8.8 letters. Overall, in the five subjects in
Cohort 3, BCVA improved
through Week 20 (i.e., a 6.8 letter mean improvement for the five subjects in
Cohort 3). FIG. 18B shows
the mean CST from baseline up to Week 20 for five subjects in Cohort 3. At a
follow up time of 20
weeks, the five subjects from Cohort 3 had a mean CST change from baseline of -
137.8 pm. In addition,
the subjects that had not received a rescue anti-VEGF injection had a mean CST
change from baseline of -
149.8 pm. Overall, in the five subjects in Cohort 3, the mean CST was improved
through Week 20, with a
substantial decrease in CST (i.e., a mean decrease of 137.8 pm for the five
subjects in Cohort 3).
104421 As shown in the Swimmer's Lane Plot in FIG. 19, no subjects in Cohort 1
received a rescue
anti-VEGF injection at a median follow up time of 60 weeks (range of 52-64
weeks). Subjects 3 and 5 in
Cohort 2 each received 4 anti-VEGF rescue injections (aflibercept). Subject 3
in Cohort 3 received 2 anti-
VEGF rescue injections (aflibercept).
Subject Case Studies
104431 At baseline, Subject 4 in Cohort 1 (62 year old male) had received 109
prior anti-VEGF
injections (9 in the last 12 months prior to AAV2.7m8-aflibercept
administration) and had persistent
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subretinal fluid even with frequent anti-VEGF injections (FIG. 20A). As shown
in FIG. 20B, some
reduction in subretinal fluid after the screening 1VT aflibercept injection
was observed in Subject 4. After
administration of AAV2.7m8-aflibercept, the subretinal fluid continued to
resolve, and the retina was dry
by Week 12. This effect continued with no signs of fluid recurrence until the
subject experienced an
unrelated retinal detachment involving the macula that was diagnosed at Week
44. Due to the macular
involvement, the vision was reduced and the subject underwent a successful
retinal detaclunent repair
surgery with vitrectomy and gas bubble. However, due to the gas bubble, OCT
imaging was not possible
at Week 48 and the subject was unable to read any letters on the sight chart.
As the gas bubble dissolved,
the vision improved, and there have been no signs of wet AMD disease activity
or recurrence of fluid. The
fundus topography has changed somewhat due to the retinal detachment and the
foveal center has been
manually located to match pre-operative reference scans in the retinal
thickness maps presented in FIG.
20B at weeks 44, 52, and 60. At Week 60, the retina remained dry, vision was
within 10 letters of
baseline (BCVA), and the subject remained rescue injection-free.
[0444] At baseline, Subject 5 in Cohort 3 (82 year old male) had received 19
prior anti-VEGF injections
(9 in the last 12 months prior to AAV2.7m8-aflibercept administration) and had
persistent subretinal fluid
even with frequent anti-VEGF injections (FIG. 21A). As shown in FIG. 21B,
Subject 5 exhibited
responsiveness to the screening anti-VEGF injection, however, the subretinal
fluid returned within 2
weeks. Thus, Subject 5 is an example of a subject that was responsive to anti-
VEGF treatment but
required frequent injections to keep the fluid away. Based on the history of
Subject 5, the subject would
have required bolus anti-VEGF injections on a monthly basis (e.g., about once
every 4 weeks). However,
one week after AAV2.7m8-aflibercept administration, the subretinal fluid had
not increased, and no fluid
was observed at Weeks 12, 16, and 20 after AAV2.7m8-aflibercept
administration. These results
demonstrate that the course of the disease in Subject 5 was altered by a
single administration of
AAV2.7m8-aflibercept.
Discussion
[0445] The results described in this Example showed that AAV2.7m8-aflibercept
was well tolerated and
showed robust efficacy across Cohorts 1, 2, and 3. In particular, these
results showed that a single dose of
AAV2.7m8-aflibercept reduced the anti-VEGF injection burden in subjects in
Cohorts 1, 2, and 3.
[0446] The results for subjects in Cohort 1, which were administered AAV17m8-
aflibereept at a dose
of 6x10nvg/eye, showed that zero subjects required anti-VEGF rescue injections
at 1 year and beyond
after administration of AAV2.7m8-aflibercept, thus demonstrating the long-term
durability of the efficacy
of AAV2.7m8-aflibercept. In addition, the majority of subjects in Cohorts 2
and 3, which were
administered AAV2.7m8-aflibercept at a dose of 2x10nvg/eye (3-fold lower dose
than Cohort 1), also
remained anti-VEGF rescue injection-free.
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[0447] Overall, out of 17 subjects administered AAV2.7m8-aflibercept in
Cohorts 1-3 (including 5
subjects from Cohort 3 that were followed for 20 weeks), 14 (82%) remained
anti-VEGF rescue injection-
free, maintained mean BCVA, and had improvements in mean CST.
[0448] The results from Cohorts 1-3 also showed all adverse events related to
AAV2.7m8-aflibercept
were mild to moderate, and that ocular inflammation observed following
administration of AAV2.7m8-
aflibercept was low grade and responsive to steroid eye drops. In addition,
subjects in Cohort 3, which
were administered a 6-week prophylactic steroid eye drop regimen, exhibited
minimal inflammation, thus
demonstrating that the 6-week prophylactic steroid eye drop regimen was
effective at minimizing early
ocular inflammation. No events of vasculitis, retinitis, or choroiditis were
observed.
Example 6: A Phase 2, Multi-Center, Randomized, Double-Masked, Active
Controlled Study of
AAV2.7m8-aflibercept in Subjects with Diabetic Macular Edemas
[0449] This Example describes a Phase 2, multi-center, randomized, double-
masked, active controlled
study that evaluated the durability of a single intravitreal (IVT) injection
of AAV2.7m8-aflibercept in
subjects with diabetic macular edema.
I. Study Objectives and Endpoints
A. Primary Objective
[0450] The primary objective of this study is to assess the durability of a
single IVT injection of
AAV2.7m8-aflibercept.
B. Secondary Objectives
[0451] Secondary objectives of this study include:
= Assessment of the safety and tolerability of AAV2.7m8-aflibercept.
= Evaluation of the effect of AAV2.7m8-aflibercept on macular edema.
= Evaluation of the effect of AAV2.7m8-aflibercept on Best Corrected Visual
Acuity (BCVA).
= Evaluation of the effect of AAV2.7m8-aflibercept on Diabetic Retinopathy
Severity Scale
(DRSS) score.
= Assessment of the need for rescue aflibercept (2 mg IVT).
= Assessment of the effect of a preceding dose of aflibercept (2 mg IVT)
prior to AAV2.7m8-
aflibercept administration.
= Evaluation of the effect of AAV2.7m8-aflibercept on development of vision
threatening
complications (anterior segment neovascularization, vitreous hemorrhage, or
tractional retinal
detachment).
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C. Primary Endpoints
104521 The primary endpoint of this study is the time to worsening of diabetic
macular edema (DME)
disease activity in the study eye, as defined by the occurrence of either of.
= An increase in central subfield thickness (CST) >50 gin as assessed by SD-
OCT compared to
the lower of the two CST measurements recorded at Day 1 or Week 4.
= A loss of > 5 letters in BCVA due to worsening DME disease activity
compared to the higher
of the two BCVA measurements recorded at Day 1 or Week 4.
IA Secondary Endpoints
104531 The secondary endpoints for this study are based on outcome measures
for the study eye (unless
otherwise specified) and include:
= Incidence and severity of ocular and non-ocular adverse events (AEs).
= Change from Baseline in CST and macular volume over time through Week 48.
= Change from Baseline in BCVA over time through Week 48.
= Frequency of rescue aflibercept (2 mg IVT) in the study eye over time
during the study.
= Incidence of 2-step and 3-step improvement in DRSS score over time
through Week 48.
= Incidence of 2-step and 3-step worsening in DRSS score over time through
Week 48_
= Occurrence of vision threatening complication (anterior segment
neovasculanzation, vitreous
hemorrhage, or any other high-risk proliferative diabetic retinopathy (DR), or
tractional
retinal detachment) over time through Week 48.
= Incidence of CST <300 gm over time through Week 48.
= Incidence of clinically significant findings via physical examinations,
ocular examinations,
imaging, and laboratory evaluation over time through Week 48.
II. Study Population
A. Inclusion Criteria
104541 Subjects with newly diagnosed DME DME
diagnosis within 6 months of screening) that
have received up to 2 prior injections of anti-VEGF therapy in the study eye
are included in this study if
they meet the following inclusion criteria:
Age?= 18 years of age.
= Type 1 or 2 diabetes mellitus.
= Vision impairment due to center involving diabetic macular edema.
= Vision at Screening:
o Study Eye: BCVA 78 to 50 ETDRS letters,
inclusive (approximate Snellen equivalent
20/32 to 20/100).
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o Non-study eye: BCVA 35 ETDRS letters or more (approximate Snellen
equivalent of
20/200 or better).
= CST of study eye at Screening visit of? 325Eun using Heidelberg
Spectralis with center-
involving IRF (center I ram).
= A decrease in vision in the study eye determined to be primarily due to
DME.
= Initial DME diagnosis within 6 months from screening.
= Up to 2 prior injections (0, 1, or 2) of anti-VEGF in the study eye.
o If a prior anti-VEGF has been administered to the study eye, there must
have been a
meaningful CST response (e.g., ? 10% reduction) and no adverse reaction to
anti-
VEGF (e.g., inflammation).
= A minimum 60-day interval between the last anti-VEGF injection in the
study eye and
randomization on Day 1.
B. Exclusion Criteria
104551 Subjects meeting any of the following criteria are excluded from this
study:
= Documented anti-AAV2.7m8 neutralizing antibody titer > 1:125 within 6
months prior to
randomization.
= Prior ocular gene therapy.
= History of allergy to aflibercept, corticosteroid, or fluorescein dye or
sodium fluorescein used
in angiography (mild allergy amenable to treatment is allowable).
= History or evidence of any of the following cardiovascular disease within
6 months of dosing:
o Severe cardiac disease (e.g., New York Heart Association [NYHA]
Functional Class
III or IV) or clinical evidence of unstable angina.
o Acute coronary syndrome, myocardial infarction or coronary artery
revascularization,
cerebrovascular accident (CVA), transient ischemic attack OW
o Ventricular tachyarrhythmias requiring ongoing treatment, or uncontrolled
arrhythmia.
o Uncontrolled hypertension defmed as systolic blood pressure (SBP) >160
mmHg or a
diastolic blood pressure (DBP) >100 mmHg, despite using BP¨lowering medication
within the screening period. If BP-lowering medications are required, subject
should
be on a stable dose of the same medication continuously for 30 days prior to
randomization.
= Any history of ongoing bleeding disorders. The use of aspirin or other
anticoagulants (e.g.,
Factor Xa inhibitors) is not an exclusion criterion.
= Uncontrolled diabetes defined as HbAlC >10%; or history of diabetic
ketoacidosis within 3
months prior to randomization; or subjects who, within the last 3 months,
initiated intensive
insulin treatment (a pump or multiple daily injection) or plan to do so in the
next 3 months.
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= History of systemic autoimmune disease requiring treatment with systemic
steroids or
immtmosuppressive treatments (e.g. methotrexate, adalimumab).
= Systemic drugs known to cause macular edema (e.g., fingolomod, tamoxifen,
chloroquine/hydroxychloroquine) or any prior systemic anti-VEGF therapy.
= Known to be positive for I-11V or active viral hepatitis (unless
documented cure after
treatment for Hepatitis C); known history of syphilis
= Known severe renal impairment, as indicated by estimated CrC1 <30 mL/min
(by Cockcroft-
Gault calculation); need or anticipated need for hemodialysis during the study
period.
= Any febrile illness within 1 week prior to randomization.
104561 In addition, subjects meeting any of the following ocular exclusion
criteria in the study eye are
excluded from this study:
= High-risk proliferative diabetic retinopathy (PDR) at time of screening,
defined as: any
vitreous or preretinal hemorrhage, neovascularization elsewhere >1/2-disc area
within an area
equivalent to standard ETDRS 7-field on clinical examination, or
neovascularization of disc >
1/3-disc area on clinical examination.
= Any prior focal or grid laser photocoagulation or any prior pan retinal
photocoagulation
(PRY) in the study eye.
= Any anti-VEGF therapy in the preceding 60 days prior to randomization (up
to 2 prior anti-
VEGF injections are allowed but they cannot have occurred in the prior 60
days).
= History of anterior segment neovascularization (e.g., neovascularization
of the iris [NV!' or
neovascular glaucoma [NVG1), significant vitreous hemorrhage, fibrovascular
proliferation or
tractional retinal detachment.
= Examination evidence of structural abnormalities at the fovea (e.g.,
dense hard exudates,
pigment abnormalities, fovea' atrophy, vitreomacular traction or epiretinal
membrane), either
on clinical examination or OCT, thought to be contributing to macular edema or
visual
impairment.
= History of retinal disease in the study eye other than diabetic
retinopathy including age-
related macular degeneration (in either eye), retinal vein occlusion, retinal
arterial occlusion,
pathologic myopia, etc.
= Any current or history of ocular disease other than DME that could reduce
the potential for
visual improvement, confound assessment of the macula or require medical or
surgical
intervention during the study (e.g. significant cataract, macular traction) or
any evidence of
posterior subeapsular cataract.
= History of cataract extraction or Yttrium Aluminum Garnet (YAG)
capsulotomy within 3
months before Day 1.
= History of retinal detachment (with or without repair) in the study eye.
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= History of trabeculectomy or glaucoma shunt or minimally invasive
glaucoma surgery
(MIGS).
= History of vitrectomy or other filtration surgery.
= Aphakia or presence of an anterior chamber intraocular lens.
= Uncontrolled ocular hypertension or glaucoma in the study eye at time of
randomization
(defined as kW >22 imnHg despite treatment with anti-glaucoma medication) or
current use
of >2 IOP lowering medications.
= Any history of intraocular or periocular steroid treatment for any ocular
condition (e.g., IVT
Triesence, fluvien or Ozurdex),
= Refractive surgery within the 90 day period prior to Screening.
= Previous penetrating keratoplasty, endothelial keratoplasty, or ocular
radiation.
= Any prior vitreoretinal surgery.
104571 In addition, subjects meeting any of the following ocular exclusion
criteria in the study eye or in
the non-study eye (i.e., the "fellow eye') are excluded from this study:
= Any history of uveitis or intraocular inflammation (grade trace or above)
except mild
anticipated post-operative inflammation that resolved.
= History of IOP elevation related to topical steroid administration.
= Known history of ocular Herpes Simplex Virus (HSV), Varicella-zoster
virus (VZV), or
Cytomegalovirus (CMV) including viral uveitis, retinitis or keratitis.
= Evidence of external ocular infection, including conjunctivitis,
chalazion or significant
blepharitis.
= History of ocular toxoplasmosis.
IlL Study DesiRn
A. Study Treatment
104581 This is a multi-center, randomized, double-masked, controlled, parallel-
group study to evaluate
the efficacy, safety and tolerability of a single 0.10 inL IVT injection of
AAV2.7m8-afliberc.ept. Two
doses of AAV2.7m8-aflibercept are investigated.
104591 Subjects with initial diagnosis of DME within 6 months of screening and
that have received up
to 2 prior injections of anti-VEGF therapy are eligible for enrollment.
104601 Approximately 33 eligible subjects are randomized to receive one of two
doses of AAV2.7m8-
aflibercept (6 x 10" vg/eye or 2 x 10" vg/eye), or to a control arm to receive
a sham ocular injection with
a preceding affibercept injection. Subjects who are assigned to receive
AAV2.7m8-aflibercept are further
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randomized to receive a preceding aflibercept or sham ocular injection. The
study arms are summarized
below:
= Arm 1 (n=6): Subjects receive AAV2.7m8-aflibercept at a dose of 6 x 10"
vg/eye with a
preceding aflibercept dose.
= Arm 2 (n=6): Subjects receive AAV2.7m8-aflibercept at a dose of 6 x 10"
vg/eye without a
preceding aflibercept dose.
= Arm 3 (n=6): Subjects receive AAV2.7m8-aflibercept at a dose of 2 x 10"
vg/eye with a
preceding aflibercept dose.
= Arm 4 (n=6): Subjects receive AAV2.7m8-aflibercept at a dose of 2 x 10"
vg/eye without a
preceding aflibercept dose.
= Arm 5 (n=9): Subjects receive aflibercept only (active control).
104611 To maintain masking of the treatment assignment, subjects assigned to
the arms with no
preceding aflibercept on Day 1 or to the arm with no AAV2.7m8-aflibercept on
Day 8 receive a sham
ocular injection on the corresponding visit. Only one eye per subject is
selected as the study eye. If both
eyes are eligible, the eye with the worse BCVA is selected as the study eye.
104621 Both AAV2.7m8-aflibercept and aflibercept are administered via IVT
injection. IVT injections
are not performed if active inflammation is present. Aseptic technique with
povidone-iodine is used with
topical or subconjunctival anesthesia. The sham ocular injection procedure is
done under the same
conditions but with an empty syringe without a needle (using the blunt end)
pressed against the eye to
mimic an injection.
104631 A summary of the arms in this study is provided in Table 17.
Table 17. Study Arms.
Day!
Day 8
Ann N
IVT Aflibercept or AAV2.7m8-aflibercept
Sham
Dose (vg/eye)
1 6
Aflibercept 6 x 10"
2 6
Sham 6 x 10"
3 6
Aflibercept 2 x 10"
4 6
Sham 2 x 10"
9 Aflibercept Sham
[0464] After the assigned IVT injections on Day 1 and Day 8, all subjects are
followed up on Week 2,
Week 4, and then every 4 weeks up to Week 48 after Day 1 (e.g., Week 8, Week
12, Week 16, etc.). To
maintain masking, both subjects and personnel conducting assessments are
masked to the treatment
assignments throughout the study.
[0465] All subjects are followed for 48 weeks after randomization.
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[0466] An overview of the study design is provided in FIG. 29.
B. Prophylactic Topical Steroid Regimen
[0467] All subjects are administered a prophylactic 7-week topical
corticosteroid regimen of
difluprednate (0.05%; e.g., Durezol) starting on Day 1. Subjects are
instructed to self-administer
difluprednate four times per day (QID) for 4 weeks (i.e., from Day 1 to Day
28), followed by three times
per day (TID) for 1 week (La, for 7 days), followed by two times per day (BID)
for 1 week (La, 7 days),
and finally once per day (QD) for 1 week (La, 7 days). Tapering is not
commenced in the presence of
active inflammation. This regimen is prolonged should signs of inflammation
occur. A summary of the
difluprednate regimen is provided in Table 18.
Table 18. Difluprednate Regimen.
Study Days Total Number of
Days Difluprednate
Administrations per
Day
Days I to 28 28
4 times
Days 29 to 35 7
3 times
Days 36 to 42 7
2 times
Days 43 to 49 7
1 time
Day 50 0
STOP ¨ if no signs of
inflammation
C. Rescue Treatment
[0468] Starting at Week 8, subjects receive rescue aflibercept (2 mg 1VT) if
they meet any of the
following:
= Increase in CST > 50 pm as assessed by SD-OCT compared to the lower of
the two CST
measurements recorded at Day 1 or Week 4.
= Loss of > 5 letters in BCVA due to worsening DME disease activity
compared to the higher
of the two BCVA measurements recorded at Day 1 or Week 4.
104691 Aflibercept is not injected in eyes with active inflammation. A minimum
of 21 days is required
between rescue aflibercept injections.
IA Medications and Treatments
[0470] The following medications are prohibited during the study:
= Any systemic anti-VEGF agent, including bevacizumab.
= Systemic drugs known to cause macular edema (e.g., fingolomod, tamoxifen,
chloroquinc/hydroxychloroquine).
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= Any anti-VEGF agent in the study eye other than the study drug or
aflibercept IVT 2 mg.
= IVT steroids in the study eye (e.g., Ozurdex or Illuvien Triesence).
= Systemic immunosuppressive drugs (e.g., intravenous steroids,
methotrexate, azathioprine,
ciclosporin, adalimumab, infliximab, etanercept). Inhaled or topical steroids
and NSAIDs are
allowed.
[0471] Subjects who develop high-risk PDR in the study eye receive panretinal
photocoagulation (PRP)
after receiving rescue aflibercept.
[0472] Subjects with visually significant cataract are not enrolled in the
study, but if a cataract develops
during the study, cataract surgery in the study eye may be performed if
clinically indicated and is
scheduled > 90 days after AAV2.7m8-aflibercept administration and/or > 7 days
after the last injection of
aflibercept
[0473] Subjects who develop DME in the fellow (non-study) eye may receive
standard of care therapy.
IV. Study Assessments
A. General Physical Examination and Vital Signs
[0474] A general physical examination (PE) is conducted at the screening and
End of Study (EOS) or
Early Termination visit. It consists of body system examination for general
appearance, neurologic,
HEENT (head, eyes, ears, nose, and throat), neck, cardiovascular, respiratory,
abdomen, extremities, skin,
weight, and height. At the EOS or Early Termination visit, the physical
examination assesses if any
changes in the subject's physical condition have occurred since the Screening
examination. A targeted
physical examination is conducted as needed for the evaluation of AEs.
[0475] Vital signs consist of blood pressure, pulse rate, body temperature,
and respiratory rate. A 12-
lead Electrocardiogram (ECG) is taken for each subject at Screening and EOS or
Early Termination Visit
R Laboratory Tests, Vector Expression and Immune Response
104761 The following Clinical Laboratory Tests are conducted for the study:
chemistry, complete blood
count, HbAlC, urinalysis, and HLA-B27 genotyping.
104771 Subjects' samples (both blood and/or aqueous humor) are collected to
measure the following:
= Total antibodies to AAV2.7m8: serum for total anti-AAV2.7m8 antibodies
are measured in
an ELISA assay.
= Neutralizing antibodies to AAV2.7m8: serum for neutralizing anti-AAV2.7m8
antibodies are
measured in a cell-based assay.
= Anti-aflibercept antibodies: serum for the htunoral immune response
against aflibercept is
measured in an ELISA assay.
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= Aflibercept protein expression: Serum and aqueous humor samples are
collected for the
presence of aflibercept protein to be measured in a MesoScale Discovery assay.
= Cell-mediated immune response: cellular immunity against AAV2.7m8 capsid
and aflibereept
protein are measured in an ELISPOT assay.
C. Full Ophthalmk Examination
[0478] Study assessments include an ophthalmologic exam, Intraocular Pressure
(lOP), and indirect
ophthalmoscopy.
[0479] The ophthalmic examination consists of an external examination of the
eye and adnexa, routine
screening for eyelid/pupil responsiveness (including but not limited to
blepharoptosis, abnormal pupil
shape, unequal pupils, abnormal reaction to light, and afferent pupillary
defect), and slit-lamp examination
(eyelids, conjunctiva, cornea, lens, iris, anterior chamber). The slit-lamp
examination examines the
anterior ocular structures and is used for grading any findings. If any
finding is noted during the slit-lamp
examination, at any visit, the severity is graded and the finding is described
as clinically significant or not
clinically significant.
[0480] IOP measurements are performed using a Goldrnann applanation tonorneter
or Tono-penTm. The
same method of IOP measurement is used throughout the study for each
individual subject. IOP
measurements are performed prior to any IVT injection and prior to dilating
eyes, using the same method
throughout the study. Day 1 and Day 8 visits require pre-injection and post-
injection (30 minutes after
injection) IOP measurements.
[0481] The dilated indirect ophthalmoscopy examination includes an evaluation
of posterior segment
abnormalities of the vitreous, optic nerve, peripheral retina, and retinal
vasculattu-e. If any finding is noted
during the ophthalmoscopy, at any visit, the severity is graded and the
finding is described as clinically
significant or not clinically significant. Day 1 and Day 8 visits require pre-
injection and post-injection
indirect ophthalmoscopy assessments.
D. Refraction and Visual Acuity
[0482] Refraction and BCVA are measured. Visual acuity measurements are
measured at a starting
distance of 4 meters, prior to dilating eyes.
E Imaging
[0483] Spectral Domain Optical Coherence Tomography (SD-OCT) is an
interferometric technique that
provides depth-resolved tissue structure information encoded in the magnitude
and delay of the back-
scattered light by spectral analysis of the interference fringe pattern. If a
subject received anti-VEGF
injections at visits prior to study randomization, OCTs from those visits are
collected and delivered to the
central reading center
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[0484] Optical Coherence Tomography Angiography (OCT-A) is an imaging
technology that provides
volumetric, three-dimensional maps of the retina and choroid as well as
information on blood flow. There
are two types of OCT-A, swept-source and spectral-domain. Swept-source imaging
is used where
available. If a swept-source instrument is not available and a spectral-domain
instrument is available, then
the spectral domain instrument is used.
[0485] Standardized procedures for the collection of ultra-wide field fundus
digital photographic
images of the retina, optic disc, and macula are followed. In addition,
photographs of the iris are taken
prior to dilation.
[0486] A standardized procedure for examining the retinal circulation and
vessel permeability using a
dye-tracing method is followed. This involves injection of sodium fluorescein
into the systemic
circulation, after which an angiogram is obtained by digitally photographing
the fluorescence emitted after
illumination of the retina with blue light at a wavelength of 490 nm.
F. Laboratory, Biornarker and Other Biological Specimens
[0487] Aqueous humor samples are collected and analyzed for levels of
aflibercept, VEGF-A,
neutralizing antibodies (NAbs), and additional biomarkers. Vitreous humor
samples are obtained and
analyzed for aflibercept concentrations and other biomarkers.
a Safety
[0488] After study treatment administration, all clinically significant
adverse events (AEs) are reported.
Each subject is followed until a) the end of the AE reporting period at 30
days after the last study visit or
b) for any ongoing study treatment related AEs and/or serious AEs (SAEs) until
resolved or stable. Any
clinically significant safety assessment that is associated with DME is not
reported as an AE or SAE,
unless judged to be more severe than expected for the subject's condition.
Progression of the disease
under study is captured as an efficacy outcome.
[0489] Adverse events of special interest for this study include:
= Sight-threatening adverse events: an adverse event is considered to be
sight-threatening if it
meets one or more of the following criteria:
o Causes a decrease of > 30 letters in BCVA compared with the prior visit.
o Requires surgical or medical intervention (i.e., conventional surgery,
vitrectomy) to
prevent permanent loss of sight.
o Causes severe intraocular inflanunation (i.e., endophthalmitis, 4+
anterior chamber
cell/flare, or 4+ vitreous cells).
[0490] All of the above-listed sight-threatening adverse events are reported
as serious adverse events,
listing the underlying cause (if known) of the event as the primary event
term.
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H. Efficacy
104911 The efficacy of AAV2.7m8-aflibercept in the treatment of DME is
assessed by the following
measures. Baseline values for BCVA and SD-OCT endpoints refer to pre-treatment
measurements taken
on the Day I visit when aflibercept 1VT or sham ocular injection are
administered.
= BCVA: Vision is assessed primarily through BCVA expressed as an ETDRS
score (number
of letters correctly read). Maintenance of vision is classified if the subject
has lost fewer than
15 letters in the ETDRS score compared to Baseline. Calculated endpoints
include the mean
change from Baseline, the percent gaining at least 15 letters compared to
Baseline, and the
percent losing 15 or more letters compared to Baseline.
= Central subfield thickness: SD-OCT is performed using approved equipment
and standard
techniques to evaluate thickness and fluid compared to Baseline values.
Endpoints include
CST and macular volume.
= Aflibercept re-treatments: The incidence and timing of aflibercept
injections given post-
AAV2.7m8-aflibercept treatment over time.
= For each timepoint, Diabetic Retinopathy Severity Scale (DRSS) is
determined using ultra-
wide field color fundus photography and compared to Day 1.
= Vision threatening complications (anterior segment neovascularization,
diabetic macular
edema, high-risk PDR development, vitreous hemorrhage, or tractional retinal
detachment),
as determined by ultra-wide field imaging and clinical examination.
L Statistical Analyses
104921 The main analysis population includes all randomized subjects who
receive the study treatment
on Day 8 (AAV2.7m8-aflibercept IVT or Sham ocular injection). All safety and
efficacy variables are
summarized descriptively by treatment arm. Mean, standard deviation (SD),
median and range are
provided for continuous variables; and frequency counts and percentages are
provided for categorical
variables. Confidence intervals of the means and percentages are provided at
both 90% and 95% levels.
Kaplan-Meier survival analysis is utilized to derive median time to the first
occurrence of DME disease
worsening_ All rescue aflibercept (2 mg IVT) received by each subject during
the study are summarized
using statistical models for recurrent events. Mean cumulative function (MCF)
curve over time is plotted
for the mean cumulative number of injections. Mixed-effect models for repeated
measures (MMRM) are
employed to explore the treatment effect on the change over time in BCVA and
CST. The treatment effect
on DRSS changes over time is explored using generalized mixed models for
categorical outcomes. An
interim analysis (IA) occurs after all subjects have been followed for 24
weeks.
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Example 7: Additional results of an open label Phase I study of AAV2.7m8-
aflibercept in neovascular
(wet) age-related macular degeneration.
[0493] This Example describes results for Cohorts 1, 2, 3, and 4 of the Phase
1 study described in
Examples 1-5 that assessed the safety and efficacy of a single intravitreal
injection of AAV2.7m8-
aflibercept in subjects with wAMD. Safety and efficacy results are provided
for Cohort 1 at median
follow up time of 72 weeks (range of 64-84 weeks), for Cohort 2 at a median
follow up time of 52 weeks
(range of 52-56 weeks), for Cohort 3 at a median follow up time of 36 weeks
(range 20-40 weeks), and for
Cohort 4 at a median follow up time of 4 weeks (range 2-8 weeks).
Results
[0494] Subjects in Cohort 1 were administered a single IVT injection of
AAV2,7m8-aflibercept at a
dose of 6 x 10"vgjeye. Subjects in Cohort 2 were administered a single IVT
injection of AAV2.7m8-
aflibercept at a dose of 2 x 10"vg/eye. Subjects in Cohorts 1 and 2 were also
administered a prophylactic
oral prednisone regimen (see Table 2).
[0495] Subjects in Cohort 3 were administered a single IVT injection of
AAV2.7m8-aflibercept at a
dose of 2 x 10"vg/eye. Subjects in Cohort 4 were administered a single IVT
injection of AAV2.7m8-
aflibercept at a dose of 6 x 10"vg/eye. Subjects in Cohorts 3 and 4 were
administered a 6-week
prophylactic topical corticosteroid regimen (see Example 5 and Table 11B).
Subject Characteristics
[0496] The baseline characteristics for subjects in Cohorts 14 were well
balanced, with similar ages and
mean prior anti-VEGF injections, and a similar need for frequent injections to
maintain baseline vision of
about 65 ETDRS letters across cohorts. Some subjects in Cohort 4 were
diagnosed more recently than in
other cohorts, e.g., less than 12 months prior to administration of AAV2.7m8-
aflibercept, therefore the
mean number of prior anti-VEGF injections is lower. Overall, subjects in
Cohorts 1-4 had good vision,
and were difficult to treat, with some fluid with frequent treatment with anti-
VEGF injections. The
baseline subject characteristics for subjects in Cohorts 1-4 are provided in
Table 19.
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Table 19. Baseline characteristics of subjects in Cohorts 1-4.
Cohort 1
Cohort 2 Cohort 3 Cohort 4
Baseline Characteristics (N)
(N=6) (N=9) (N=9)
79.0 79.8 77.4 79.9
Mean (range) age, years
(62-88)
(74-90) (65-90) (68-88)
Mean (range) years since nAMD 3.5
4.1 3.3 3.2
diagnosis (0,9-10.6)
(0,5-6.8) (0.7-8.0) (0.2-8.0)
Mean (range) number anti-VEGF 32.2
34.0 24.8 28.5
injections since initial diagnosis* (7-109)
(4-69) (9-70) (2-58)**
Mean (range) number anti-VEGF
9.2 9.2 9.1 6.8
injections in 12 months prior to
(8-11)
(5-11) (7-10) (3-12)**
AAV2.7m8-aflibercept
Mean (range) BCVA, ETDRS 65.8
64.7 65.9 65.0
letters (57-77)
(53-72) (53-75) (54-77)
Approximate Snellen equivalent 20/50
20/50 20/50 20/50
369.2
307.7 473.4 398.6
Mean (range) CST, gm
(293-561)
(235-339) (301-857) (255-538)
*Not including the mandated aflibercept at Screening; **Excluding Subject 2
with incomplete prior
anti-VEGF data due to relocation.
BCVA, best corrected visual acuity; CST, central subfield thickness; ETDRS,
Early Treatment
Diabetic Retinopathy Study; nAMD, neovascular age-related macular
degeneration; VEGF, vascular
endothelial growth factor.
Safety
[0497] No deaths, treatment discontinuations, or AAV2.7m8-aflibercept-related
non-ocular adverse
events have occurred.
[0498] When observed, inflammation was responsive to and managed by topical
steroids. No clinical or
fluorescein (fluorescein angiography of posterior pole) evidence of posterior
inflammation has been
observed. In addition, no vasculitis, retinitis, choroiditis, vascular
occlusions, or endophthalmitis have
occurred.
[0499] All AAV2.7m8-afliberc,ept-related ocular adverse events were mild (78%)
or moderate (22%).
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105001 One adverse event of special interest (AESI) of recurrent moderate
uveitis occurred and was
deemed related to AAV2.7m8-aflibercept (Cohort 1). The recurrent moderate
uveitis AESI was
responsive to steroid eye drops. hi addition, a treatment-unrelated ocular
serious adverse event (SAE) of
retinal detachment occurred and was surgically repaired and resolved (Cohort
1).
[0501] Two subjects had mild adverse events of mild intraocular pressure (lOP)
elevation that resolved.
One subject from Cohort 1 had two mild IOP elevations (highest of 24nunHg)
that were both treated with
Combigan eye drops. The second subject was taking Combigan for ocular
hypertension, and the IOP
adverse event spontaneously resolved within a month with no change to
treatment.
105021 Overall, AAV2.7m8-aflibercept has been well tolerated and there have
been no systemic
adverse events attributed to AAV2.7m8-aflibercept.
105031 A suunmary of safety results in Cohorts 1-4 of this study is provided
in Table 20.
Table 20. Adverse events in Cohorts 1-4.
Cohort 1 Cohort 2
Cohort 3 Cohort 4
(N) (NS)
(N=9) (N=9)
Adverse
6x1011vg/eye 2 x1011 vg/eye
2 x10" vg/eye 6 x10" vg/eye
events
Oral steroids Oral steroids
Steroid eye drops Steroid eye drops
13-day prophylaxis 13-day prophylaxis
6-week prophylaxis 6-week prophylaxis
Ocular Subjects Events Subjects Events Subjects Events Subjects Events
Serious 2 2* 0 0
0 0 0 0
Related
6 30 5 21
5 14 5 12
**
Total
6 51 5 32
8 26 7 15
ocular
Non-
Subjects Events Subjects Events Subjects Events Subjects Events
oculart
Serious 1 1 0 0
2 2 0 0
Total
non- 5 18 6 7
4 9 1 1
ocular/.
*Retinal detachment (unrelated to AAV2.7m8-aflibercept) and recurrent moderate
uveitis (likely related to
AAV2.7m8-aflibercept).
**AAV2.7m8-aflibercept-related ocular events were mild (78%) or moderate
(22%).
t None of the non-ocular AEs were AAV2.7m8-aflibercept-related.
Serious non-ocular AEs included degenerative intervertebral disc disease (1)
in Cohort 1; and COPD
exacerbation (1), and stable angina pectoris (1) in Cohort 3.
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[0504] AAV2.7m8-aflibercept-related adverse events that occurred in Cohorts 1-
4, reported by
MedDRA preferred term, are provided in Table 21.
Table 21. AAV2.7m8-aflibereept-related AEs by preferred term in Cohorts 1-4.
Cohort 1 (N=6) Cohort 2 (N=6) Cohort 3 (N=9) Cohort 4 (N=9)
MedDRA Preferred Term Events Subjects Events Subjects Events Subjects Events
Subjects
Any Events 30 6 21
5 14 5 12 5
Anterior chamber cell 4 3 7
4 2 2 4 4
Anterior chamber flare 5 3 3
2 0 0 2 2
Vitreal cells 4 3 2
2 1 1 2 2
Keratic precipitates 2 1 3
2 2 2 1 1
Anterior chamber 3 1 0
0 2 1 0 0
inflammation
Iris hyperpigmentation 2 2 1
1 1 1 0 0
Anterior chamber 0 0 1
1 2 1 0 0
pigmentation
Uveitis 3 2 0
0 0 0 0 0
Vitreous floaters 2 2 0
0 0 0 1 1
Iris adhesions 0 0 0
0 2 2 0 0
his transillumination defect 0 0 0
0 1 1 1 1
Vitreous haze 1 1 1
1 0 0 0 0
Iridocyclitis 0 0 1
1 0 0 0 0
Iris bombe 1 1 0
0 0 0 0 0
!this 0 0 0
0 1 1 0 0
Lenticular pigmentation 0 0 1
1 0 0 0 0
Miosis 1 1 0
0 0 0 0 0
Pupil fixed 1 1 0
0 0 0 0 0
Retinal pigmentation 1 1 0
0 0 0 0 0
Visual acuity reduced 0 0 1
1 0 0 0 0
Vitritis 0 0 0
0 0 0 1 1
105051 Ocular adverse events that occurred with a frequency of 2 events,
reported by MedDRA
preferred term, are provided in Table 22.
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Table 22. Ocular AEs by preferred term (in events).
MedDRA Preferred Cohort 1 (N) Cohort 2 (N=6)
Cohort 3 (N=9) Cohort 4 (N=9)
Term Events Subjects Events Subjects
Events Subjects Events Subjects
Any Events* 51 6 32
5 26 8 15 7
Anterior chamber cell 5 4 7
4 2 2 4 4
Anterior chamber flare 5 3 3
2 0 0 2 2
Conjunctival haemorrhage 3 3 2
2 1 1 3 3
Vitreal cells 4 3 2
2 1 1 2 2
Keratic precipitates 2 1 3
2 2 2 1 1
Vitreous floaters 4 3 0
0 2 2 1 1
Anterior chamber 3 1 0
0 2 1 0 0
inflammation
Anterior chamber 0 0 1
1 3 2 0 0
pigmentation
Iris hyperpigmentation 2 2 1
1 1 1 0 0
Posterior capsule 0 0 2
2 2 2 0 0
opacification
Intraocular pressure 2 1 0
0 1 1 0 0
increased
Iris adhesions 1 1 0
0 2 2 0 0
Macular oedema 0 0 3
1 0 0 0 0
Retinal haemorrhage 2 1 0
0 1 1 0 0
Uveitis 3 2 0
0 0 0 0 0
Dry eye 2 2 0
0 0 0 0 0
Iris transillumination 0 0 0
0 1 1 1 1
defect
Lenticular pigmentation 1 1 1
1 0 0 0 0
Retinal pigmentation 1 1 0
0 1 1 0 0
Vitreous haze 1 1 1
1 0 0 0 0
*Includes terms reported in only one event.
10506] Non-ocular adverse events that occurred with a frequency of a2 events,
reported by MedDRA
preferred term, are provided in Table 23.
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Table 23. Non-ocular AL's by preferred term (in events).
Cohort 1 (N) Cohort 2 (N) Cohort 3 (N=9) Cohort 4 (N=9)
MedDRA Preferred Term Events Subjects Events Subjects Events Subjects Events
Subjects
Any Events* 18 5 7
6 9 4 1 1
Arthritis 1 1 1
1 0 0 0 0
Atrial fibrillation 1 1 1
1 0 0 0 0
Bronchitis 0 0 0
0 2 1 0 0
Chronic obstructive 0 0 0 0 2
1 0 0
pulmonary disease
Hypertension 0 0 2
2 0 0 0 0
Nasopharyngitis 2 1 0
0 0 0 0 0
Post-traumatic pain 0 0 0 0 2
1 0 0
*Includes terms reported in only one event.
105071 A summary of ocular and non-ocular serious, mild, and moderate adverse
events observed in
Cohorts 1-4 of this study is provided in Table 24,
Table 24. Adverse events in Cohorts 1-4.
Cohort 1 Cohort 2
Cohort 3 Cohort 4
(b15) (N)
(14=9) (14=9)
6x10n vg/eye 2x10n vg/eye 2x10n vg/eye 6x10n vgleye
Adverse Oral steroids
Oral steroids Steroid eye drops Steroid eye drops
events 13-day prophylaxis
13-day prophylaxis 6-week prophylaxis 6-week prophylaxis
Ocular Subjects Events Subjects Events Subjects Events Subjects Events
Serious 2 2* 0 0
0 0 0 0
Related" 6 30 5 21
5 14 5 12
Mild 6 22 4 14
5 12 5 12
Moderate 3 8 2 7
2 2 0 0
Total
6 51 5 32 8 26 7
15
ocular
Non-
ocularf Subjects Events Subjects Events Subjects Events Subjects Events
Serious t 1 1 0 0
2 2 0 0
Total
non- 5 18 6 7
4 9 1 1
000lart
*Retinal detachment (unrelated to study Iteattnent) and recurrent moderate
uveitis (likely related to study
treatment).
**Study treatment-related ocular events were mild (78%) or moderate (22%).
tNone of the non-ocular AEs were related to study treatment.
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:Serious non-ocular AEs included degenerative intervertebral disc disease (1)
in Cohort 1; and COPE)
exacerbation (1), and stable angina pectoris (1) in Cohort 3.
[0508] As shown in FIGS. 22A-22D, when observed, inflammation was primarily in
the anterior
segment and responsive to and managed by topical steroids. No evidence of
posterior inflammation has
been observed, and there have been no cases of vasculitis.
[0509] As shown in FIG. 22A, two subjects (Subjects 2 and 6) in Cohort 1 who
were previously on QD
steroid eye drops with no active inflammation subsequently discontinued drops.
Subject 2 had recurrent
moderate uveitis following discontinuation and restarted steroid eye drops.
Inflammation improved and
the subject resumed tapering down steroid eye drops. At the time of data cut,
Subject 4 was receiving
steroid eye drops BID and had trace inflammation after retinal detachment
surgery. Subject 6 was not
receiving any steroid eye drops and had no inflammation. Overall, inflammation
in subjects in Cohort 1
was resolving, with only 2 out of 6 subjects continuing on steroid eye drop
treatment at the time of data
cut. The results of Cohort 1 have shown that all subjects were responsive to
topical steroids and trending
towards tapering of the steroids at the time of data cut.
[0510] In Cohort 2,4 out of 6 subjects were inflammation-free and 2 out of 6
subjects continued to
receive steroid eye drops at the time of data cut (FIG. 22B). Subject 3
received topical steroids around
Week 46 after cataract surgery. At the time of data cut, Subject 1 was
receiving steroid eye drops QD, and
Subject 6 had low grade inflammation and continued on steroid eye drops.
Overall, inflammation in
subjects in Cohort 2 was resolving with all subjects past the 52-week (1 year)
milestone at the time of data
cut.
[0511] In Cohort 3, 6 out of 9 subjects were inflammation-free at the time of
data cut (FIG. 22C), and
all were beyond the 20-week milestone. At the time of data cut, Subjects 2, 3
and 4 were receiving steroid
eye drops. Subject 4 started durezol treatment BID at Week 24 due to poor
pupil dilation and posterior
synechiae likely related to subclinical inflammation. 3 out of 9 subjects
continued to receive steroid eye
drops at the time of data cut.
[0512] As shown in FIG. 22D, subjects in Cohort 4 have shown minimal to zero
early inflammation
with steroid eye drops, consistent with the results observed for Cohort 3.
Subjects 1, 2 and 3 had anterior
inflammation and continued to receive steroid eye drops at the time of data
cut.
[0513] The 6-week prophylactic topical steroid regimen administered to Cohorts
3 and 4 showed
evidence of less active inflammation (FIGS. 22C-22D) compared to the oral
steroid regimen administered
to Cohorts 1 and 2 (FIGS. 22A-22B).
Efficacy
[0514] FIG. 23A shows the mean BCVA (ETDRS letters) from baseline up to Week
72 for subjects in
Cohort 1. At a median follow up time of 72 weeks (range of 64-84 weeks),
subjects in Cohort I had a
mean BCVA change from baseline of -3.2 letters and 100% of subjects (6/6) had
not received a rescue
anti-VEGF injection. The apparent decrease in mean BCVA at Weeks 44 and 48 was
due to one subject
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that experienced an SAE of retinal detachment. Overall, BCVA scores in
subjects in Cohort I remained
stable out to 72 weeks. FIG. 23B shows the mean CST (pm) from baseline up to
Week 72 for subjects in
Cohort 1. At a median follow up time of 72 weeks (range of 64-84 weeks),
subjects in Cohort 1 had a
mean CST change from baseline of-21.0 pm. Overall, CST measurements in Cohort
1 showed that
anatomy was maintained out to week 72 for all subjects. The observed drop and
change in the error bars at
Weeks 68 and 72 in FIG. 23B was due to exclusion of 1 subject that has always
had a very thick retina
[0515] FIG, 24A shows the mean BCVA (ETDRS letters) from baseline up to Week
52 for subjects in
Cohort 2. At a median follow up time of 52 weeks (range of 52-56 weeks),
subjects in Cohort 2 had a
mean BCVA change from baseline of -2.0 letters and 50% of subjects (3/6) had
not received a rescue anti-
VEGF injection. The subjects that had not received a rescue anti-VEGF
injection had a mean BCVA
change from baseline of +0 letters. Overall, BCVA scores in subjects in Cohort
2 remained stable beyond
52 weeks. FIG. 24B shows the mean CST (pm) from baseline up to Week 52 for
subjects in Cohort 2. At
a median follow up time of 52 weeks (range of 52-56 weeks), subjects in Cohort
2 had a mean CST
change from baseline of -24.8 gm. In addition, the subjects that had not
received a rescue anti-VEGF
injection had a mean CST change from baseline of -8.3 Fun. Overall, CST
measurements in Cohort 2
showed that anatomy was maintained beyond week 52.
[0516] FIG. 25A shows the mean BCVA (ETDRS letters) from baseline up to Week
20 for subjects in
Cohort 3. At a median follow up time of 36 weeks (range 20-40 weeks), subjects
in Cohort 3 had a mean
BCVA change from baseline of +4.0 letters and 78% (7/9) of subjects from
Cohort 3 had not received a
rescue anti-VEGF injection. The subjects that had not received a rescue anti-
VEGF injection had a mean
BCVA change from baseline of +6.4 letters. Overall, BCVA scores in subjects in
Cohort 3 improved.
FIG. 25B shows the mean CST (pm) from baseline up to Week 20 for subjects in
Cohort 3. At a median
follow up time of 36 weeks (range 20-40 weeks), subjects in Cohort 3 had a
mean CST change from
baseline of -118.6 pm. In addition, the subjects that had not received a
rescue anti-VEGF injection had a
mean CST change from baseline of -152.7 gm. Overall, the mean CST continued to
improve in all 9
subjects in Cohort 3.
[0517] As shown in the Swimmer's Lane Plot in FIG.26, no subjects in Cohort 1
received a rescue anti-
VEGF injection at a median follow up time of 72 weeks (range of 64-84 weeks).
In Cohort 2 (median
follow up time of 52 weeks; range of 52-56 weeks), Subject 2 received one anti-
VEGF rescue injection
(aflibercept), and Subjects 3 and 5 each received six anti-VEGF rescue
injections (aflibercept). In Cohort
3 (median follow up time of 36 weeks; range 2040 weeks), Subject 2 received
one anti-VEGF rescue
injection (aflibercept), and Subject 3 received four anti-VEGF rescue
injections (aflibercept). Overall, 10
out of 15 subjects in Cohorts 2-3 remained rescue injection-free. Overall,
even with rescue injections in
certain cohorts, there was a dramatic reduction in the anti-VEGF injection
burden.
105181 A substantial reduction in the annualized anti-VEGF injection rate was
observed in subjects in
Cohorts 1-3 (FIGS. 27A-27B). Specifically, no subjects in Cohort I have
received any rescue anti-VEGF
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injections (100% reduction), and subjects in Cohorts 2 and 3 combined had an
87% reduction in the mean
annual rate of anti-VEGF injections (FIG. 27A).
Case Studies
105191 At baseline, Subject 5 in Cohort 3 (82 year old male with wAMD in the
right eye) had received
19 prior anti-VEGF injections (9 in the last 12 months prior to AAV2.7m8-
aflibercept administration).
Subject 5 had persistent subretinal fluid even with frequent anti-VEGF
injections every 5 weeks (FIG.
28A). The white area in the center of the thickness maps in FIG. 28A reflects
a very thick macula.
105201 As shown in FIG. 28B, Subject 5 received a screening aflibercept
injection 2 weeks prior to the
planned AAV2.7m8-aflibercept injection. Subject 5 showed a good response to
the screening aflibercept
injection, with resolution of IRF and improvement of SRF. Thus, Subject 5 was
responsive to anti-VEGF
treatment but required very frequent injections to keep the fluid away. One
week after the AAV2.7m8-
aflibercept injection, fluid improved. If this subject was only on bolus
aflibercept injections, the fluid
would have started returning within 4-5 weeks and the OCT images would look
like the baseline OCT
image, with significant fluid. However, as shown in FIG. 28B, at 12 weeks, 16
weeks, 20 weeks, 28
weeks, and 36 weeks following AAV2.7m8-aflibercept injection, there was no
fluid and vision improved.
Thus, in Subject 5, AAV2.7m8-aflibercept injection addressed the treatment
burden by eliminating the
need for injections, improved vision, and controlled disease activity. These
results demonstrate that the
course of the disease in Subject 5 was altered by a single administration of
AAV2.7m8-aflibercept.
Discussion
105211 The results described in -this Example showed that AAV2.7m8-aflibercept
continues to be well
tolerated and has a favorable safety profile in all cohorts (n = 30). All
AAV2.7m8-aflibercept-related
ocular adverse events were mild (78%) or moderate (22%), and ocular
inflammation, when observed, was
responsive to steroid eye drops.
105221 In addition, the results described in this Example showed that AAV2.7m8-
aflibercept has robust
and sustained efficacy.
105231 A clear dose response was observed, with all subjects administered
AAV2.7m8-aflibercept at a
dose of 6 x 10"vg/eye remaining rescue anti-VEGF injection-free, and 10 out of
15 subjects administered
AAV2.7m8-aflibercept at a dose of 2 x 1011vg/eye remaining rescue anti-VEGF
injection-free. All
subjects in Cohort 1 (dose of 6 x 10"vg/eye) remain rescue injection-free
beyond 15 months after
administration of AAV2.7m8-aflibercept.
105241 The results described in this Example also showed that a single NT
injection of AAV2.7m8-
aflibercept resulted in a substantial reduction in the annualized anti-VEGF
injection rate. Mean BCVA
was generally maintained to improved and the mean CST generally improved.
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[0525] Results from Cohort 4 showed that the 6-week prophylactic regimen of
steroid eye drops was
effective at minimizing early ocular inflammation, consistent with results
observed in Cohort 3.
[0526] Although the present disclosure has been described in some detail by
way of illustration and
example for purposes of clarity of understanding, the descriptions and
examples should not be construed
as limiting the scope of the present disclosure. The disclosures of all patent
and scientific literature cited
herein are expressly incorporated in the entirety by reference.
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