Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS TO TREAT VIRAL INFECTIONS
Cross-Reference to Related Application
This application claims the benefit and priority of both U.S. Provisional
Application No.
62/168,259, filed May 29, 2015, and U.S. Provisional Application No.
62/168,262, filed May 29,
2015, the contents of which are incorporated by reference.
Technical Field
The invention generally relates to treatment of viral infections and cleaving
foreign
nucleic acids in cells.
Background
Viral infections present significant medical problems. For example, herpes
simplex virus
(HSV) may cause oral herpes, which manifest as blisters around face or mouth,
or genital herpes
that manifest as blisters that can break open and result in small ulcers.
Tingling or shooting pains
may occur before the blisters appear. Additionally, HSV has the ability to lie
dormant within a
cell indefinitely in a latent infection and not be fully eradicated even after
treatment. Because
latent infections can evade immune surveillance and reactivate the lytic cycle
at any time, there
is a persistent risk to an infected individual of outbreak and the pain and
suffering associated
with it.
Worldwide, about 90% of people are infected with one or both of HSV-1 and HSV-
2
with HSV-1 infection much more prevalent than HSV-2 infection. About 65% of
persons in the
United States have antibodies to HSV-1 and about 16% of Americans between the
ages of 14 and
49 are infected with HSV-2.
Cytomegalovirus (CMV) is an example of another problematic virus. Some people
get
infected when they receive an organ transplant. The CMV infection disseminates
to the lungs,
liver, pancreas, kidneys, stomach, intestine, brain, and parathyroid glands,
causing organ failure
and even death. When disseminated to the eye, the virus can cause retinal
detachment and
blindness. Additionally, a fetus may be infected with cytomegalovirus (CMV) in
utero,
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ultimately causing hearing loss, vision impairment, mental retardation,
coordination problems,
and death. Even when born healthy, a baby infected with CMV must be monitored
throughout
the developmental years for hearing or vision loss, or signs of mental
impairment.
Summary
The invention provides methods and compositions for treating viral infections.
A
nuclease is used to cleave viral nucleic acid in an infected cell. The
nuclease cleaves viral nucleic
acid in a sequence-specific manner and thus does not cleave genes or other
important genomic
features from a genome of the infected host. In a preferred embodiment, the
nuclease is a
CRISPR-associated protein such as Cas9 and is delivered to the infected cells
as a
ribonucleoprotein that includes the Cas9 and a guide RNA designed to target
HSV or CMV
nucleic acid. Additionally or alternatively, the nuclease can be delivered
encoded on a plasmid or
as mRNA to be expressed within the target cells. Methods and compositions may
be used to treat
a patient or may be used to treat tissues, cells, or organs ex vivo.
Embodiments of the invention relate to HSV.
The invention provides compositions and methods for selectively treating HSV
infection
using a guided nuclease system that targets a specific region of the HSV
genome such as oriS,
UL9, RL2, or LAT. Methods of the invention may be used to remove even latent
HSV genetic
material from a host organism, without interfering with the integrity of the
host's genetic
material. A nuclease may be targeted to HSV nucleic acid where it can then
disrupt the nucleic
acid, thereby interfering with viral replication or transcription or even
excising the viral genetic
material from the host genome. Through use of a sequence-specific targeting
moiety directed to
HSV, the nuclease may be specifically targeted to remove only the HSV nucleic
acid without
acting on host material whether the viral nucleic acid exists as a particle
within the cell or is
integrated into the host genome. A sequence-specific moiety can include a
guide RNA that
targets viral genomic material for destruction by the nuclease and does not
target the host cell
genome. In some embodiments, a CRISPR/Cas9 nuclease and guide RNA (gRNA) that
together
target and selectively edit or destroy viral genomic material is used. CRISPR
(clustered regularly
interspaced short palindromic repeats) is a naturally-occurring element of the
bacterial immune
system that protects bacteria from phage infection. The guide RNA localizes
the CRISPR/Cas9
complex to a HSV target sequence, such as oriS, UL9, RL2, or LAT. Binding of
the complex
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localizes the Cas9 endonuclease to the HSV genomic target sequence causing
breaks in the viral
genome.
The guided nuclease system may be introduced into an infected host
transdermally
through application of, for instance, a topical solution. Topical solutions
may be applied directly
to infected tissue.
The sequence specific moiety can target other nuclease systems to HSV nucleic
acid
including, for example, zinc finger nucleases, transcription activator-like
effector nucleases
(TALENs), meganucleases, or any other system that can be used to degrade or
interfere with
HSV nucleic acid without interfering with the regular function of the host's
genetic material.
Aspects of the invention include a composition for treatment of a herpes
simplex virus
(HSV) infection. The composition comprises a vector encoding a nuclease and a
sequence-
specific targeting moiety complementary to HSV nucleic acid and capable of
directing the
nuclease to the HSV nucleic acid.
In certain embodiments, the HSV nucleic acid may comprise one or more of the
following regions: origin of replication S (oriS), long unique region 9 (UL9),
or long repeat
region 2 (RL2). The composition may be configured to be administered
transdermally, for
example, through a topical solution. The nuclease may be a zinc-finger
nuclease, a transcription
activator-like effector nuclease, and a meganuclease.
In various embodiments, the nuclease may a Cas9 endonuclease and the sequence-
specific binding module may comprise a guide RNA that specifically targets a
portion of a viral
genome. Compositions of the invention may be packaged for delivery to a human
patient.
In certain embodiments, the targeting sequence may be a guide RNA that has no
match >
60% within a human genome.
The vector may include a retrovirus, a lentivirus, an adenovirus, a
herpesvirus, a
poxvirus, an alphavirus, a vaccinia virus, an adeno-associated viruses, a
plasmid, a nanoparticle,
a cationic lipid, a cationic polymer, metallic nanoparticle, a nanorod, a
liposome, microbubbles,
a cell-penetrating peptide, or a lipo sphere.
In certain aspects, the invention includes a method for treating an HSV
infection
including the step of introducing into a cell of a host, a vector encoding a
nuclease and a
sequence-specific targeting moiety complementary to HSV nucleic acid.
Additional steps include
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causing the sequence-specific targeting moiety to target the nuclease to the
HSV nucleic acid and
to cleave the HSV nucleic acid.
The HSV nucleic acid can include a portion of or all of one or more genomic
regions
including an origin of replication S (oriS), long unique region 9 (UL9) or
long repeat region 2
(RL2). Methods of the invention may include transdermally administering the
vector to the host
and transdermal administration may include applying a topical solution
comprising the vector.
The nuclease may be a zinc-finger nuclease, a transcription activator-like
effector
nuclease, or a meganuclease. In certain embodiments, the nuclease may include
a Cas9
endonuclease and the sequence-specific binding module may include a guide RNA
that
specifically targets a portion of a viral genome.
In various methods of the invention, the host may be a living human subject
and the steps
may be performed in vivo. The targeting sequence may be a guide RNA and have
no match >
60% within a human genome.
In certain methods, the vector may include a retrovirus, a lentivirus, an
adenovirus, a
herpesvirus, a poxvirus, an alphavirus, a vaccinia virus, an adeno-associated
viruses, a plasmid, a
nanoparticle, a cationic lipid, a cationic polymer, metallic nanoparticle, a
nanorod, a liposome,
microbubbles, a cell-penetrating peptide, or a liposphere.
The presented methods allow for HSV genome editing or destruction, which
results in the
inability of the HSV virus to proliferate without adversely affecting a host's
uninfected cells.
Compositions and methods of the invention can accordingly be used to treat an
HSV infection
through targeted disruption of HSV genomic function or by digestion of viral
nucleic acid via
one or multiple breaks caused by targeting sites such as oriS, UL9, RL2, or
LAT for
endonuclease action in the HSV genome.
In preferred aspects, the invention provides a composition for treatment of a
herpes
simplex virus (HSV) infection. The composition includes a ribonucleoprotein
that includes: a
nuclease; and a sequence-specific targeting moiety complementary to HSV
nucleic acid and
capable of directing the nuclease to the HSV nucleic acid. The HSV nucleic
acid may include an
origin of replication S (oriS) region, a long unique region 9 (UL9), a long
repeat region 2 (RL2),
or a combination thereof. Preferably, the nuclease comprises a Cas9
endonuclease and the
sequence-specific binding module comprises a guide RNA that specifically
targets a portion of a
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viral genome. The targeting sequence is a guide RNA and has no match > 60%
within a human
genome.
The composition may be configured to be administered transdermally. In some
embodiments, the composition includes a pharmaceutically acceptable carrier
for topical
application to infected tissue.
Embodiments of the invention relate to CMV.
The invention provides methods for treating cytomegalovirus (CMV) infections
in
tissues, so that infected individuals will not suffer the effects of CMV
infections, such as
hearing/vision loss, mental impairment, multi-organ failure, and death. CMV
infection is treated
by using a nuclease that targets CMV nucleic acid. The nuclease cleaves and
thus interferes with
the function of the CMV nucleic acid, which prevents CMV from infecting the
tissue or patient.
The nuclease may be introduced into a patient with a CMV infection, or into an
organ prior to
transplant. For example, a heart needed for a life-saving transplant may be
exposed to the CMV
targeted nuclease to incapacitate the CMV genome prior to transplantation.
Thus, the heart
transplant recipient does not need to choose between accepting the infected
heart and passing,
only to wait and hope that the next heart will be CMV-free.
In certain aspects, the invention provides compositions for treatment of a
cytomegalovirus (CMV) infection The composition comprise a nuclease; and a
sequence-specific
targeting moiety complementary to CMV nucleic acid and capable of directing
the nuclease to
the CMV nucleic acid.
In certain aspects, the invention provides methods for treating a
cytomegalovirus (CMV)
infection in an organ. The methods comprise introducing a nuclease that
cleaves the CMV
nucleic acid into an organ and then causing the nuclease to target and cleave
CMV nucleic acid.
The organ can be from a transplant donor, or the organ can be grown or created
from cells. In
some aspects, the organ is a heart, liver, kidney, eye, lung, pancreas,
intestine, thymus, or any
other biological tissue to be transplanted into a transplant recipient. It
should be appreciated that
preparing the nuclease for delivery can involve associating the nuclease with
a viral or non-viral
vector. For example, the nuclease could be prepared for delivery into an organ
by encasing or
binding the nuclease within a liposome.
In some embodiments of the invention, the tissue (or organ, used
interchangeably herein)
may be a tissue that is to be used in transplantation. For example, the tissue
may be an organ
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supplied by a donor, to be transplanted into a recipient. In some embodiments,
the tissues of the
organ are treated with the nuclease to cleave and incapacitate the CMV nucleic
acid.
In some embodiments, the transplant donor is treated with or exposed to the
nuclease.
According to methods of the invention, the donor receives the nuclease as a
therapeutic agent to
eliminate CMV from tissues within the patient. It should be appreciated that
any means of
delivery into the patient may be used. For example, the nuclease may be
delivered by oral,
parenteral, inhalation, topical, rectal, or vaginal means.
In some embodiments, the transplant recipient is treated with or exposed to
the nuclease
following transplantation. According to methods of the invention, the
transplant recipient is
treated with the nuclease to prevent CMV from spreading within and throughout
the patient. It
should be appreciated that the nuclease may be delivered by any known means in
the art.
In some embodiments, the tissue is located within a patient. According to
methods of the
invention, a patient may be treated with the CMV targeting nuclease to prevent
infection. For
example, the patient may be a pregnant woman. The nuclease targets the CMV
genome and
destroys or disrupts the function of the virus. Thus, the pregnant woman does
not pass the virus
to the fetus in utero.
In some aspects, the invention provides methods of treating a cytomegalovirus
(CMV)
infection in a fetus. The methods comprise providing a nuclease that cleaves
CMV nucleic acid
and preparing the nuclease for delivery into a pregnant woman. It should be
appreciated that the
nuclease may be delivered into the pregnant woman, the uterus of the pregnant
women, or the
fetus in utero, by techniques known in the art.
It should also be appreciated that any type of nuclease may be used to cleave
CMV
nucleic acid. The nuclease may be a zinc finger nuclease, a transcription
activator-like effector
nuclease, or a meganuclease. The nuclease may be a structure specific nuclease
or a sequence
specific nuclease. In some embodiments, the nuclease is a Cas9 endonuclease.
The methods of
the invention may further comprise cleaving the CMV nucleic acid using the
nuclease.
In some embodiments, the methods comprise delivering to the cell or tissue a
guide RNA
that targets the nuclease to a portion of the CMV nucleic acid. In some
embodiments, for
example, a guide RNA targets the Cas9 endonuclease to a portion of the CMV
nucleic acid. In
certain embodiments, the guide RNA is designed to have no perfect match in a
human genome.
The guide RNAs may target the nuclease to a regulatory element in the genome
of CMV.
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The nuclease may be delivered to the tissue by a viral vector. The viral
vector may be
retrovirus, lentivirus, adenovirus, herpesvirus, poxvirus, alphavirus,
vaccinia virus or adeno-
associated viruses. In some embodiments, the nuclease may be delivered by a
plasmid, a
nanoparticle, a cationic lipid, a cationic polymer, metallic nanoparticle, a
nanorod, a liposome, a
cell-penetrating peptide, a liposphere, and polyethyleneglycol (PEG).
In some embodiments, the nuclease is a CRISPR/Cas9 endonuclease. A guide RNA
that
specifically targets one or more portions of a genome of CMV within a cell or
tissue of the
transplant may be used. The CRISPR/Cas9 complex binds to and alters the CMV
genome. In
other embodiments, the invention may make use of a CRISPR/Cas9 nuclease and
guide RNA
(gRNA) that together target and selectively edit or destroy CMV genomic
material. The CRISPR
(clustered regularly interspaced short palindromic repeats) is an element of
the bacterial immune
system that protects bacteria from phage infection. The guide RNA localizes
the CRISPR/Cas9
complex to a CMV target sequence. Binding of the complex localizes the Cas9
endonuclease to
the CMV genomic target sequence causing breaks in the CMV genome. In a
preferred
embodiment, the guide RNA is designed to target multiple sites on the CMV
genome in order to
disrupt the CMV nucleic acid and reduce the chance that it will functionally
recombine.
The presented methods allow for CMV genome destruction, which results in the
inability
of the virus to proliferate, with no observed cytotoxicity to the cells.
Aspects of the invention
provide for designing a CRISPR/gRNA/Cas9 complex to selectively target CMV
genomic
material (DNA or RNA), delivering the CRISPR/gRNA/Cas9 complex to a cell or
tissue
containing the CMV genome, and cutting the CMV genome in order to incapacitate
the virus.
The presented methods allows for targeted disruption of CMV genomic function
or, in a
preferred embodiment, digestion of CMV nucleic acid via multiple breaks caused
by targeting
multiple sites for nuclease action in the CMV genome. Aspects of the invention
provide for
transfection of a CRISPR/gRNA/Cas9 complex cocktail to completely suppressed
CMV
proliferation. Additional aspects and advantages of the invention will be
apparent upon
consideration of the following detailed description thereof.
Brief Description of the Drawings
FIG. 1 diagrams a method of targeting an HSV infection.
FIG. 2 is a map of an HSV genome.
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FIG. 3 shows amplicons of HSV genomic regions before and after digestion with
cas9.
FIG. 3 is a gel with lanes showing genomic DNA size bands for cells treated
with the
RL2, LATi, LATp, UL9, OriS, and US12 guide sequences with and without Cas9.
FIG. 4 shows the results of quantitative PCR assays showing different levels
of
decreasing of HSV DNA in the CRISPR treated samples.
FIG. 5 shows the EGFP marker fused after the Cas9 protein, allowing selection
of Cas9-
positive cells.
FIG. 6 shows that including an oni-P in the plasmid promoted active plasmid
replication
inside the cells, which increased the transfection efficiency to >60%.
FIG. 7 is a diagram of an EBV genome, with structure-, transformation-, and
latency-
related targets called out.
FIG. 8 shows the genome context around guide RNA sgEBV2 and PCR primer
locations.
FIG. 9 shows the large deletion induced by sgEBV2 (lanes 1-3 are before, 5
days after,
and 7 days after sgEBV2 treatment, respectively).
FIG. 10 shows the genome context around guide RNA sgEBV3/4/5 and PCR primer
locations.
FIG. 11 shows the large deletions induced by sgEBV3/5 and sgEBV4/5. Lane 1 and
2 are
3F/5R PCR amplicons before and 8 days after sgEBV3/5 treatment. Lane 3 and 4
are 4F/5R PCR
amplicons before and 8 days after sgEBV4/5 treatment.
FIG. 12 shows that Sanger sequencing confirmed genome cleavage and repair
ligation 8
days after sgEBV3/5.
FIG. 13 shows that Sanger sequencing confirmed genome cleavage and repair
ligation 8
days after sgEBV4/5.
FIG. 14 shows relative cell proliferation after targeting various combinations
of regions
in an EBV genome with guide RNAs.
FIG. 15 gives flow cytometry scattering signals from before sgEBV1-7
treatments.
FIG. 16 gives flow cytometry scattering signals from 5 days after sgEBV1-7
treatments
FIG. 17 gives flow cytometry scattering signals from 8 days after sgEBV1-7
treatments.
FIG. 18 shows Annexin V A1exa647 and DAPI staining results before sgEBV1-7
treatments.
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FIG. 19 shows Annexin V A1exa647 and DAPI staining results 5 days after sgEBV1-
7
treatments.
FIG. 20 shows Annexin V A1exa647 and DAPI staining results 8 days after sgEBV1-
7
treatments.
FIGS. 21 and 22 show microscopy revealed apoptotic cell morphology after
sgEBV1-7
treatment.
FIG. 23 shows nuclear morphology before sgEBV1-7 treatment.
FIGS. 24-26 show nuclear morphology after sgEBV1-7 treatment.
FIG. 27 shows EBV load after different CRISPR treatments by digital PCR. Cas9
and
Cas9-oriP had two replicates, and sgEBV1-7 had 5 replicates.
FIGS. 28 shows a single Raji cell as captured on a microfluidic chip.
FIG. 29 shows a single sgEBV1-7 treated cell as captured on the chip.
FIG. 30 is a histogram of EBV quantitative PCR Ct values from single cells
before
treatment.
FIG. 31 is a histogram of EBV quantitative PCR Ct values from single live
cells 7 days
after sgEBV1-7 treatment.
FIG. 32 represents SURVEYOR assay of EBV CRISPR (lanes numbered from left to
right: Lane 1: NEB 100bp ladder; Lane 2: sgEBV1 control; Lane 3: sgEBV1; Lane
4: sgEBV5
control; Lane 5: sgEBV5; Lane 6: 5gEBV7 control; Lane 7: 5gEBV7; Lane 8:
5gEBV4).
FIG. 33 shows that the CRISPR treatments were not cytotoxic to the EBV-
negative
Burkitt's lymphoma cell line DG-75
FIG. 34 shows that the CRISPR treatments were not cytotoxic to primary human
lung
fibroblasts IMR90.
FIG. 35 shows ZFN being used to cut viral nucleic acid.
FIG. 36 shows a composition for treating a viral infection.
FIG. 37 depicts a flow chart of embodiments of the invention for targeting
CMV.
FIG. 38 is a map of a CMV genome.
FIG. 39 depicts results from an in vitro CRISPR endonuclease assay, showing
PCR
amplicons from CMV genome and the corresponding products from in vitro CRISPR
digestion
(shown in pairs).
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FIG. 40 depicts results from an in vitro CRISPR endonuclease assay, showing
PCR
amplicons from CMV genome and the corresponding products from in vitro CRISPR
digestion
(shown in pairs).
FIG. 41 depicts results from an in vitro CRISPR endonuclease assay, showing
PCR
amplicons from CMV genome and the corresponding products from in vitro CRISPR
digestion
(shown in pairs).
FIG. 42 shows a method 3201 for treating a cell 3237 to remove foreign nucleic
acid.
Detailed Description
Embodiments of the invention relate to compositions and methods for treating
HSV
infections including HSV-1 and HSV-2 infections using guided nuclease systems
targeted to
specific regions of the HSV genome such as RL2, LAT, UL9, or OriS.
Compositions of the
invention disrupt HSV viral nucleic acid in host cells through nuclease
activity without affecting
the host's genome. Through disruption of the HSV genome in infected host
cells, methods and
compositions of the invention may eradicate even latent HSV infections.
Additionally or alternatively, embodiments of the invention related to methods
for
treating cytomegalovirus (CMV). Methods of the invention are used to
incapacitate or disrupt
CMV within a cell, a tissue, or a patient by systematically causing large or
repeated insertions or
deletions in the CMV genome, reducing the probability of reconstructing the
full genome. The
insertions or deletions in the genome incapacitates or destroys the virus,
thus treating CMV. In
some embodiments, the nuclease may be a CRISPR/Cas9 complex. In some
embodiments, the
nuclease is guided by a sequence, such as a guided RNA. In some embodiments,
tissues, such as
organs are treated with the nuclease prior to transplantation. In some
embodiments, a transplant
donor or recipient are treated before and after a transplantation surgery. In
some embodiments, a
patient infected with CMV is treated with the nuclease.
FIG. 1 diagrams a method of targeting an HSV infection. The method includes
obtaining
a targetable nuclease (e.g., as a protein or a gene for a nuclease). Any
suitable nuclease can be
used such as ZFN, TALENs, or meganucleases. In a preferred embodiment, the
nuclease is Cas9.
A sequence is provided that targets the nuclease to specific targets on the
HSV genome such as
oriS, UL9, RL2, or LAT. The nuclease gene and encoded gRNAs may be provided in
a DNA
vector, such as a plasmid or an adenovirus based vector, and the vector may
further optionally
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include a promoter. That composition is then introduced into the HSV-infected
cells. Any
suitable transfection or delivery method may be used. Once in the cell, the
genes are expressed
and the Cas9 enzyme uses the gRNA to target, and cleave, the HSV genome. Since
the gRNA is
specific to the HSV genome with no match to the human genome according to
methods and
criteria described herein, the method leaves the host genome intact and does
not interfere with
normal human genetic function.
Discussion of HSV as well as regions for targeting can be found in Summers, et
al., 2002,
Herpes Simplex Virus Type 1 Origins of DNA Replication Play No Role in the
Regulation of
Flanking Promoters, J Virol., 76(14); Eom, et al., 2003, Replication-initiator
protein (UL9) of the
herpes simplex virus 1 binds NFB42 and is degraded via the
ubiquitin¨proteasome pathway,
Proc Natl Acad Sci U S A, 100(17): 9803-9807; McGeoch, et al., 1991,
Comparative sequence
analysis of the long repeat regions and adjoining parts of the long unique
regions in the genomes
of herpes simplex viruses types 1 and 2, J Gen Virol., 72 ( Pt 12):3057-75;
the contents of each
of which are incorporated by reference in their entirety for all purposes.
FIG. 2 shows the HSV genome and the HSV oriS palindrome dimer (oriS), RL2-ICP0
(RL2), LAT Intron (LATi), LAT promoter (LATp), UL9, and US12 genes which may
be
targeted by CRISPR guide RNAs.
FIG. 3 shows amplicons of HSV genomic regions before and after digestion with
cas9
and corresponding guide RNAs. T7 in vitro transcription was used to produce
the complete guide
RNA with scaffold for RL2, LATi, LATp, UL9, oriS, and US12. Flanking regions
of the genome
targets were PCR amplified from HSV2 strain G genomic DNA (from ATCC). Cas9
protein
(from PNA Bio), guide RNA and target DNA were mixed and incubated for in vitro
endonuclease assay. High endonuclease activities were revealed by DNA gel
electrophoresis of
the digested DNA as shown in FIG. 3 which shows PCR amplicons of RL2, LATi,
LATp, UL9,
oriS, and US12 before and after digestion with cas9 and the guide RNA
targeting each respective
genomic region.
FIG. 37 depicts a flow chart of embodiments of the invention for targeting
CMV. In
general, the method 100 comprises introducing a nuclease 105 that targets the
CMV genome.
The nuclease can be introduced into an organ or a pregnant woman. The organ or
the fetus can
be suspected of having a CMV infection. The method further comprises causing
the nuclease to
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target and cleave 110 a CMV nucleic acid. The nuclease is able to bind to and
alter the CMV
genome.
i. Treating infected cell
Cells may be treated with a nuclease encoded in a nucleic acid (e.g.,
delivered as mRNA
or a plasmid) or delivered in active form. Where the nuclease is a CRISPR-
associated protein
such as Cas9, an active ribonucleoprotein (RNP) may be delivered to the cells.
FIG. 42 shows a method 3201 for treating a cell 3237 to remove foreign nucleic
acid such
as a viral nucleic acid 3251 from a herpes simplex virus. The method 3201 may
be used to treat
an infection, or the method 3201 may be used in vitro in research and
development to remove
foreign nucleic acid from subject cells such as cells from a human.
The method 3201 includes the steps of: forming 3225 a ribonucleoprotein (RNP)
3231
that includes a nuclease 3205 and an RNA 3213; obtaining a cell 3237 from a
donor; delivering
3245 (preferably in vitro) the RNP 3231 to the cell 3237; and cleaving viral
nucleic acid 3251
within the cell 3237 with the RNP 3231. The method 3201 may include providing
the cell 3237
for transplantation into a patient.
The delivering 3245 may include electroporation, or the RNP may be packaged in
a
liposome for the delivering 3245. In some embodiments, the viral nucleic acid
3251 will exist as
an episomal viral genome, i.e., an episome or episomal vector, of a virus. The
RNA 3213 has a
portion that is substantially complementary to a target within a viral nucleic
acid 3251 and
preferably not substantially complementary to any location on a human genome.
In the preferred
embodiments, the virus is a herpes family virus such as one selected from the
group consisting of
HSV-1, HSV-2, Varicella zoster virus, Epstein-Barr virus, and Cytomegalovirus.
The virus may
be in a latent stage in the cell.
In a preferred embodiment, the nuclease 3205 is a Crisper-associated protein
such as,
preferably, Cas9. The RNA 3213 may be a single guide RNA (sgRNA) (providing
the
functionality of crRNA and tracrRNA). In the preferred embodiment, the
nuclease 3205 and the
RNA 3213 are delivered to the cell as the RNP 3231.
In preferred embodiments, the cell 3237 has the viral nucleic acid 3251
therein, and the
method further comprises cleaving the viral nucleic acid using the nuclease.
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In some embodiments, it may be found that RNP is preferable (e.g., to plasmid
DNA) for
clinical applications, particularly for parenteral delivery. RNP is the active
pre-formed drug
which offers advantages to DNA (AAV) or mRNA. No need to transcribe,
translate, or assemble
drug components within cell. Delivery of RNP 3231 may offer improved drug
properties, e.g.
earlier onset activity and controlled clearance (toxicity).
FIG. 36 shows a composition for treating a viral infection according to
certain
embodiments. The composition preferably includes a vector (which may be a
plasmid, linear
DNA, or a viral vector) that codes for a nuclease and a targeting moiety
(e.g., a gRNA) that
targets the nuclease to HSV and may be complimentary to a genomic region of
HSV such as
RL2, LAT, UL9, or OriS. The composition may optionally include one or more of
a promoter,
replication origin, other elements, or combinations thereof as described
further herein.
ii. Nuclease
Methods of the invention include using a programmable or targetable nuclease
to
specifically target viral nucleic acid for destruction. Any suitable targeting
nuclease can be used
including, for example, zinc-finger nucleases (ZFNs), transcription activator-
like effector
nucleases (TALENs), clustered regularly interspaced short palindromic repeat
(CRISPR)
nucleases, meganucleases, other endo- or exo-nucleases, or combinations
thereof. See Schiffer,
2012, Targeted DNA mutagenesis for the cure of chronic viral infections, J
Virol 88(17):8920-
8936, incorporated by reference.
CRISPR methodologies employ a nuclease, CRISPR-associated (Cas9), that
complexes
with small RNAs as guides (gRNAs) to cleave DNA in a sequence-specific manner
upstream of
the protospacer adjacent motif (PAM) in any genomic location. CRISPR may use
separate guide
RNAs known as the crRNA and tracrRNA. These two separate RNAs have been
combined into a
single RNA to enable site-specific mammalian genome cutting through the design
of a short
guide RNA. Cas9 and guide RNA (gRNA) may be synthesized by known methods.
Cas9/guide-
RNA (gRNA) uses a non-specific DNA cleavage protein Cas9, and an RNA oligo to
hybridize to
target and recruit the Cas9/gRNA complex. See Chang et al., 2013, Genome
editing with RNA-
guided Cas9 nuclease in zebrafish embryos, Cell Res 23:465-472; Hwang et al.,
2013, Efficient
genome editing in zebrafish using a CRISPR-Cas system, Nat. Biotechnol 31:227-
229; Xiao et
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al., 2013, Chromosomal deletions and inversions mediated by TALENS and
CRISPR/Cas in
zebrafish, Nucl Acids Res 1-11.
CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats) is found in
bacteria
and is believed to protect the bacteria from phage infection. It has recently
been used as a means
to alter gene expression in eukaryotic DNA, but has not been proposed as an
anti-viral therapy or
more broadly as a way to disrupt genomic material. Rather, it has been used to
introduce
insertions or deletions as a way of increasing or decreasing transcription in
the DNA of a
targeted cell or population of cells. See for example, Horvath et al., Science
(2010) 327:167-170;
Terns et al., Current Opinion in Microbiology (2011) 14:321-327; Bhaya et al.
Annu Rev Genet
(2011) 45:273-297; Wiedenheft et al. Nature (2012) 482:331-338); Jinek Metal.
Science
(2012) 337:816-821; Cong Let al. Science (2013) 339:819-823; Jinek M et al.
(2013) eLife
2:e00471; Mali Pet al. (2013) Science 339:823-826; Qi LS et al. (2013) Cell
152:1173-1183;
Gilbert LA et al. (2013) Cell 154:442-451; Yang H et al. (2013) Cell 154:1370-
1379; and Wang
H et al. (2013) Cell 153:910-918); the contents of each of which are
incorporated by reference in
their entirety for all purposes.
In an aspect of the invention, the Cas9 endonuclease causes a double strand
break in at
least two locations in the genome. These two double strand breaks cause a
fragment of the
genome to be deleted. Even if viral repair pathways anneal the two ends, there
will still be a
deletion in the genome. One or more deletions using the mechanism will
incapacitate the viral
genome. The result is that the host cell will be free of viral infection.
In embodiments of the invention, nucleases cleave the genome of the target
virus. A
nuclease is an enzyme capable of cleaving the phosphodiester bonds between the
nucleotide
subunits of nucleic acids. Endonucleases are enzymes that cleave the
phosphodiester bond within
a polynucleotide chain. Some, such as Deoxyribonuclease I, cut DNA relatively
nonspecifically
(without regard to sequence), while many, typically called restriction
endonucleases or
restriction enzymes, cleave only at very specific nucleotide sequences. In a
preferred
embodiment of the invention, the Cas9 nuclease is incorporated into the
compositions and
methods of the invention, however, it should be appreciated that any nuclease
may be utilized.
In preferred embodiments of the invention, the Cas9 nuclease is used to cleave
the
genome. The Cas9 nuclease is capable of creating a double strand break in the
genome. The Cas9
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nuclease has two functional domains: RuvC and HNH, each cutting a different
strand. When
both of these domains are active, the Cas9 causes double strand breaks in the
genome.
In some embodiments of the invention, insertions into the genome can be
designed to
cause incapacitation, or altered genomic expression. Additionally,
insertions/deletions are also
used to introduce a premature stop codon either by creating one at the double
strand break or by
shifting the reading frame to create one downstream of the double strand
break. Any of these
outcomes of the NHEJ repair pathway can be leveraged to disrupt the target
gene. The changes
introduced by the use of the CRISPR/gRNA/Cas9 system are permanent to the
genome.
In some embodiments of the invention, at least one insertion is caused by the
CRISPR/gRNA/Cas9 complex. In a preferred embodiment, numerous insertions are
caused in
the genome, thereby incapacitating the virus. In an aspect of the invention,
the number of
insertions lowers the probability that the genome may be repaired.
In some embodiments of the invention, at least one deletion is caused by the
CRISPR/gRNA/Cas9 complex. In a preferred embodiment, numerous deletions are
caused in the
genome, thereby incapacitating the virus. In an aspect of the invention, the
number of deletions
lowers the probability that the genome may be repaired. In a highly-preferred
embodiment, the
CRISPR/Cas9/gRNA system of the invention causes significant genomic
disruption, resulting in
effective destruction of the viral genome, while leaving the host genome
intact.
TALENs uses a nonspecific DNA-cleaving nuclease fused to a DNA-binding domain
that
can be to target essentially any sequence. For TALEN technology, target sites
are identified and
expression vectors are made. Linearized expression vectors (e.g., by Notl) may
be used as
template for mRNA synthesis. A commercially available kit may be use such as
the
mMESSAGE mMACHINE SP6 transcription kit from Life Technologies (Carlsbad, CA).
See
Joung & Sander, 2013, TALENs: a widely applicable technology for targeted
genome editing,
Nat Rev Mol Cell Bio 14:49-55.
TALENs and CRISPR methods provide one-to-one relationship to the target sites,
i.e.
one unit of the tandem repeat in the TALE domain recognizes one nucleotide in
the target site,
and the crRNA, gRNA, or sgRNA of CRISPR/Cas system hybridizes to the
complementary
sequence in the DNA target. Methods can include using a pair of TALENs or a
Cas9 protein with
one gRNA to generate double-strand breaks in the target. The breaks are then
repaired via non-
homologous end-joining or homologous recombination (HR).
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FIG. 35 shows ZFN being used to cut viral nucleic acid. Briefly, the ZFN
method
includes introducing into the infected host cell at least one vector (e.g.,
RNA molecule) encoding
a targeted ZFN 305 and, optionally, at least one accessory polynucleotide.
See, e.g., U.S. Pub.
2011/0023144 to Weinstein, incorporated by reference The cell includes target
sequence 311.
The cell is incubated to allow expression of the ZFN 305, wherein a double-
stranded break 317
is introduced into the targeted chromosomal sequence 311 by the ZFN 305. In
some
embodiments, a donor polynucleotide or exchange polynucleotide 321 is
introduced. Swapping a
portion of the viral nucleic acid with irrelevant sequence can fully interfere
transcription or
replication of the viral nucleic acid. Target DNA 311 along with exchange
polynucleotide 321
may be repaired by an error-prone non-homologous end-joining DNA repair
process or a
homology-directed DNA repair process.
Typically, a ZFN comprises a DNA binding domain (i.e., zinc finger) and a
cleavage
domain (i.e., nuclease) and this gene may be introduced as mRNA (e.g., 5'
capped,
polyadenylated, or both). Zinc finger binding domains may be engineered to
recognize and bind
to any nucleic acid sequence of choice. See, e.g., Qu et al., 2013, Zinc-
finger-nucleases mediate
specific and efficient excision of HIV-1 proviral DAN from infected and
latently infected human
T cells, Nucl Ac Res 41(16):7771-7782, incorporated by reference. An
engineered zinc finger
binding domain may have a novel binding specificity compared to a naturally-
occurring zinc
finger protein. Engineering methods include, but are not limited to, rational
design and various
types of selection. A zinc finger binding domain may be designed to recognize
a target DNA
sequence via zinc finger recognition regions (i.e., zinc fingers). See for
example, U.S. Pat. Nos.
6,607,882; 6,534,261 and 6,453,242, incorporated by reference. Exemplary
methods of selecting
a zinc finger recognition region may include phage display and two-hybrid
systems, and are
disclosed in U.S. Pat. 5,789,538; U.S. Pat. 5,925,523; U.S. Pat. 6,007,988;
U.S. Pat. 6,013,453;
U.S. Pat. 6,410,248; U.S. Pat. 6,140,466; U.S. Pat. 6,200,759; and U.S. Pat.
6,242,568, each of
which is incorporated by reference.
A ZFN also includes a cleavage domain. The cleavage domain portion of the ZFNs
may
be obtained from any suitable endonuclease or exonuclease such as restriction
endonucleases and
homing endonucleases. See, for example, Belfort & Roberts, 1997, Homing
endonucleases:
keeping the house in order, Nucleic Acids Res 25(17):3379-3388. A cleavage
domain may be
derived from an enzyme that requires dimerization for cleavage activity. Two
ZFNs may be
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required for cleavage, as each nuclease comprises a monomer of the active
enzyme dimer.
Alternatively, a single ZFN may comprise both monomers to create an active
enzyme dimer.
Restriction endonucleases present may be capable of sequence-specific binding
and cleavage of
DNA at or near the site of binding. Certain restriction enzymes (e.g., Type
ITS) cleave DNA at
sites removed from the recognition site and have separable binding and
cleavage domains. For
example, the Type ITS enzyme FokI, active as a dimer, catalyzes double-
stranded cleavage of
DNA, at 9 nucleotides from its recognition site on one strand and 13
nucleotides from its
recognition site on the other. The FokI enzyme used in a ZFN may be considered
a cleavage
monomer. Thus, for targeted double-stranded cleavage using a FokI cleavage
domain, two ZFNs,
each comprising a FokI cleavage monomer, may be used to reconstitute an active
enzyme dimer.
See Wah, et al., 1998, Structure of FokI has implications for DNA cleavage,
PNAS 95:10564-
10569; U.S. Pat. 5,356,802; U.S. Pat. 5,436,150; U.S. Pat. 5,487,994; U.S.
Pub. 2005/0064474;
U.S. Pub. 2006/0188987; and U.S. Pub. 2008/0131962, each incorporated by
reference.
Virus targeting using ZFN may include introducing at least one donor
polynucleotide
comprising a sequence into the cell. A donor polynucleotide preferably
includes the sequence to
be introduced flanked by an upstream and downstream sequence that share
sequence similarity
with either side of the site of integration in the chromosome. The upstream
and downstream
sequences in the donor polynucleotide are selected to promote recombination
between the
chromosomal sequence of interest and the donor polynucleotide. Typically, the
donor
polynucleotide will be DNA. The donor polynucleotide may be a DNA plasmid, a
bacterial
artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral
vector, a linear piece
of DNA, a PCR fragment, a naked nucleic acid, and may employ a delivery
vehicle such as a
liposome. The sequence of the donor polynucleotide may include exons, introns,
regulatory
sequences, or combinations thereof. The double stranded break is repaired via
homologous
recombination with the donor polynucleotide such that the desired sequence is
integrated into the
chromosome. In the ZFN-mediated process, a double stranded break introduced
into the target
sequence by the ZFN is repaired, via homologous recombination with the
exchange
polynucleotide, such that the sequence in the exchange polynucleotide may be
exchanged with a
portion of the target sequence. The presence of the double stranded break
facilitates homologous
recombination and repair of the break. The exchange polynucleotide may be
physically
integrated or, alternatively, the exchange polynucleotide may be used as a
template for repair of
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the break, resulting in the exchange of the sequence information in the
exchange polynucleotide
with the sequence information in that portion of the target sequence. Thus, a
portion of the viral
nucleic acid may be converted to the sequence of the exchange polynucleotide.
ZFN methods
can include using a vector to deliver a nucleic acid molecule encoding a ZFN
and, optionally, at
least one exchange polynucleotide or at least one donor polynucleotide to the
infected cell.
Meganucleases are endodeoxyribonucleases characterized by a large recognition
site
(double-stranded DNA sequences of 12 to 40 base pairs); as a result this site
generally occurs
only once in any given genome. For example, the 18-base pair sequence
recognized by the I-SceI
meganuclease would on average require a genome twenty times the size of the
human genome to
be found once by chance (although sequences with a single mismatch occur about
three times per
human-sized genome). Meganucleases are therefore considered to be the most
specific naturally
occurring restriction enzymes. Meganucleases can be divided into five families
based on
sequence and structure motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box and PD-
(D/E)XK.
The most well studied family is that of the LAGLIDADG proteins, which have
been found in all
kingdoms of life, generally encoded within introns or inteins although
freestanding members also
exist. The sequence motif, LAGLIDADG, represents an essential element for
enzymatic activity.
Some proteins contained only one such motif, while others contained two; in
both cases the
motifs were followed by ¨75-200 amino acid residues having little to no
sequence similarity with
other family members. Crystal structures illustrates mode of sequence
specificity and cleavage
mechanism for the LAGLIDADG family: (i) specificity contacts arise from the
burial of
extended 13-strands into the major groove of the DNA, with the DNA binding
saddle having a
pitch and contour mimicking the helical twist of the DNA; (ii) full hydrogen
bonding potential
between the protein and DNA is never fully realized; (iii) cleavage to
generate the characteristic
4-nt 3'-OH overhangs occurs across the minor groove, wherein the scissile
phosphate bonds are
brought closer to the protein catalytic core by a distortion of the DNA in the
central "4-base"
region; (iv) cleavage occurs via a proposed two-metal mechanism, sometimes
involving a unique
"metal sharing" paradigm; (v) and finally, additional affinity and/or
specificity contacts can arise
from "adapted" scaffolds, in regions outside the core a/0 fold. See Silva et
al., 2011,
Meganucleases and other tools for targeted genome engineering, Curr Gene Ther
11(1):11-27,
incorporated by reference.
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In some embodiments of the invention, a template sequence is inserted into the
genome.
In order to introduce nucleotide modifications to genomic DNA, a DNA repair
template
containing the desired sequence must be present during homology directed
repair (HDR). The
DNA template is normally transfected into the cell along with the gRNA/Cas9.
The length and
binding position of each homology arm is dependent on the size of the change
being introduced.
In the presence of a suitable template, HDR can introduce significant changes
at the Cas9
induced double strand break.
Some embodiments of the invention may utilize modified version of a nuclease.
Modified
versions of the Cas9 enzyme containing a single inactive catalytic domain,
either RuvC- or
HNH-, are called `nickases'. With only one active nuclease domain, the Cas9
nickase cuts only
one strand of the target DNA, creating a single-strand break or 'nick'.
Similar to the inactive
dCas9 (RuvC- and HNH-), a Cas9 nickase is still able to bind DNA based on gRNA
specificity,
though nickases will only cut one of the DNA strands. The majority of CRISPR
plasmids are
derived from S. pyogenes and the RuvC domain can be inactivated by a DlOA
mutation and the
HNH domain can be inactivated by an H840A mutation.
A single-strand break, or nick, is normally quickly repaired through the HDR
pathway,
using the intact complementary DNA strand as the template. However, two
proximal, opposite
strand nicks introduced by a Cas9 nickase are treated as a double strand
break, in what is often
referred to as a 'double nick' or 'dual nickase' CRISPR system. A double-nick
induced double
strain break can be repaired by either NHEJ or HDR depending on the desired
effect on the gene
target. At these double strain breaks, insertions and deletions are caused by
the CRISPR/Cas9
complex. In an aspect of the invention, a deletion is caused by positioning
two double strand
breaks proximate to one another, thereby causing a fragment of the genome to
be deleted.
iii. Targeting moiety
A nuclease may use the targeting specificity of a guide RNA (gRNA). As
discussed
below, guide RNAs or single guide RNAs are specifically designed to target a
virus genome.
A CRISPR/Cas9 gene editing complex of the invention works optimally with a
guide
RNA that targets the viral genome. Guide RNA (gRNA) (which includes single
guide RNA
(sgRNA), crisprRNA (crRNA), transactivating RNA (tracrRNA), any other
targeting oligo, or
any combination thereof) leads the CRISPR/Cas9 complex to the viral genome in
order to cause
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viral genomic disruption. In an aspect of the invention, CRISPR/Cas9/gRNA
complexes are
designed to target specific viruses within a cell. It should be appreciated
that any virus can be
targeted using the composition of the invention. Identification of specific
regions of the virus
genome aids in development and designing of CRISPR/Cas9/gRNA complexes.
In an aspect of the invention, the CRISPR/Cas9/gRNA complexes are designed to
target
latent viruses within a cell. Once transfected within a cell, the
CRISPR/Cas9/gRNA complexes
cause repeated insertions or deletions to render the genome incapacitated, or
due to number of
insertions or deletions, the probability of repair is significantly reduced.
As an example, the Epstein¨Barr virus (EBV), also called human herpesvirus 4
(HHV-4)
is inactivated in cells by a CRISPR/Cas9/gRNA complex of the invention. EBV is
a virus of the
herpes family, and is one of the most common viruses in humans. The virus is
approximately 122
nm to 180 nm in diameter and is composed of a double helix of DNA wrapped in a
protein
capsid. In this example, the Raji cell line serves as an appropriate in vitro
model. The Raji cell
line is the first continuous human cell line from hematopoietic origin and
cell lines produce an
unusual strain of Epstein-Barr virus while being one of the most extensively
studied EBV
models. To target the EBV genomes in the Raji cells, a CRISPR/Cas9 complex
with specificity
for EBV is needed. The design of EBV-targeting CRISPR/Cas9 plasmids consisting
of a U6
promoter driven chimeric guide RNA (sgRNA) and a ubiquitous promoter driven
Cas9 that were
obtained from Addgene, Inc. Commercially available guide RNAs and Cas9
nucleases may be
used with the present invention. An EGFP marker fused after the Cas9 protein
allowed selection
of Cas9-positive cells.
In an aspect of the invention, guide RNAs are designed, whether or not
commercially
purchased, to target a specific viral genome. The viral genome is identified
and guide RNA to
target selected portions of the viral genome are developed and incorporated
into the composition
of the invention. In an aspect of the invention, a reference genome of a
particular strain of the
virus is selected for guide RNA design.
For example, guide RNAs that target the EBV genome are a component of the
system in
the present example. In relation to EBV, for example, the reference genome
from strain B95-8
was used as a design guide. Within a genome of interest, such as EBV, selected
regions, or genes
are targeted. For example, six regions can be targeted with seven guide RNA
designs for
different genome editing purposes.
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FIG. 7 is a diagram of an EBV genome, with structure-, transformation-, and
latency-
related targets called out. FIG. 7 additionally shows where sgEBV1, sgEBV2,
sgEBV3,
sgEBV4/5, sgEBV6, and 5gEBV7 target the EBV genome.
In relation to EBV, EBNA1 is the only nuclear Epstein-Barr virus (EBV) protein
expressed in both latent and lytic modes of infection. While EBNA1 is known to
play several
important roles in latent infection, EBNA1 is crucial for many EBV functions
including gene
regulation and latent genome replication. Therefore, guide RNAs sgEBV4 and
sgEBV5 were
selected to target both ends of the EBNA1 coding region in order to excise
this whole region of
the genome. These "structural" targets enable systematic digestion of the EBV
genome into
smaller pieces. EBNA3C and LMP 1 are essential for host cell transformation,
and guide RNAs
sgEBV3 and 5gEBV7 were designed to target the 5' exons of these two proteins
respectively.
iv. Introduce to cell
Methods of the invention include introducing into a cell a nuclease and a
sequence-
specific targeting moiety. The nuclease is targeted to viral nucleic acid by
means of the
sequence-specific targeting moiety where it then cleaves the viral nucleic
acid without interfering
with a host genome. Any suitable method can be used to deliver the nuclease to
the infected cell
or tissue. For example, the nuclease or the gene encoding the nuclease may be
delivered by
injection, orally, or by hydrodynamic delivery. The nuclease or the gene
encoding the nuclease
may be delivered to systematic circulation or may be delivered or otherwise
localized to a
specific tissue type. The nuclease or gene encoding the nuclease may be
modified or
programmed to be active under only certain conditions such as by using a
tissue-specific
promoter so that the encoded nuclease is preferentially or only transcribed in
certain tissue types.
In some embodiments, specific CRISPR/Cas9/gRNA complexes are introduced into a
cell. A guide RNA is designed to target at least one category of sequences of
the viral genome. In
addition to latent infections this invention can also be used to control
actively replicating viruses
by targeting the viral genome before it is packaged or after it is ejected.
In some embodiments, a cocktail of guide RNAs may be introduced into a cell.
The guide
RNAs are designed to target numerous categories of sequences of the viral
genome. By targeting
several areas along the genome, the double strand break at multiple locations
fragments the
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genome, lowering the possibility of repair. Even with repair mechanisms, the
large deletions
render the virus incapacitated.
In some embodiments, several guide RNAs are added to create a cocktail to
target
different categories of sequences. For example, two, five, seven or eleven
guide RNAs may be
present in a CRISPR cocktail targeting three different categories of
sequences. However, any
number of gRNAs may be introduced into a cocktail to target categories of
sequences. In
preferred embodiments, the categories of sequences are important for genome
structure, host cell
transformation, and infection latency, respectively.
In some aspects of the invention, in vitro experiments allow for the
determination of the
most essential targets within a viral genome. For example, to understand the
most essential
targets for effective incapacitation of a genome, subsets of guide RNAs are
transfected into
model cells. Assays can determine which guide RNAs or which cocktail is the
most effective at
targeting essential categories of sequences.
For example, in the case of the EBV genome targeting, seven guide RNAs in the
CRISPR
cocktail targeted three different categories of sequences which are identified
as being important
for EBV genome structure, host cell transformation, and infection latency,
respectively. To
understand the most essential targets for effective EBV treatment, Raji cells
were transfected
with subsets of guide RNAs.
FIG. 14 shows relative cell proliferation after targeting various combinations
of regions
in an EBV genome with guide RNAs. Although sgEBV4/5 reduced the EBV genome by
85%,
they could not suppress cell proliferation as effectively as the full cocktail
(FIG. 14). Guide
RNAs targeting the structural sequences (5gEBV1/2/6) could stop cell
proliferation completely,
despite not eliminating the full EBV load (26% decrease). Given the high
efficiency of genome
editing and the proliferation arrest, it was suspect that the residual EBV
genome signature in
sgEBV1/2/6 was not due to intact genomes but to free-floating DNA that has
been digested out
of the EBV genome, i.e. as a false positive.
Once CRISPR/Cas9/gRNA complexes are constructed, the complexes are introduced
into
a cell. It should be appreciated that complexes can be introduced into cells
in an in vitro model or
an in vivo model. In an aspect of the invention, CRISPR/Cas9/gRNA complexes
are designed to
not leave intact genomes of a virus after transfection and complexes are
designed for efficient
transfection.
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Aspects of the invention allow for CRISPR/Cas9/gRNA to be transfected into
cells by
various methods, including viral vectors and non-viral vectors. Viral vectors
may include
retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. It
should be appreciated
that any viral vector may be incorporated into the present invention to
effectuate delivery of the
CRISPR/Cas9/gRNA complex into a cell. Some viral vectors may be more effective
than others,
depending on the CRISPR/Cas9/gRNA complex designed for digestion or
incapacitation. In an
aspect of the invention, the vectors contain essential components such as
origin of replication,
which is necessary for the replication and maintenance of the vector in the
host cell.
In an aspect of the invention, viral vectors are used as delivery vectors to
deliver the
complexes into a cell. Use of viral vectors as delivery vectors are known in
the art. See for
example U.S. Pub. 2009/0017543 to Wilkes et al., the contents of which are
incorporated by
reference.
A retrovirus is a single-stranded RNA virus that stores its nucleic acid in
the form of an
mRNA genome (including the 5' cap and 3' PolyA tail) and targets a host cell
as an obligate
parasite. In some methods in the art, retroviruses have been used to introduce
nucleic acids into a
cell. Once inside the host cell cytoplasm the virus uses its own reverse
transcriptase enzyme to
produce DNA from its RNA genome, the reverse of the usual pattern, thus retro
(backwards).
This new DNA is then incorporated into the host cell genome by an integrase
enzyme, at which
point the retroviral DNA is referred to as a provirus. For example, the
recombinant retroviruses
such as the Moloney murine leukemia virus have the ability to integrate into
the host genome in
a stable fashion. They contain a reverse transcriptase that allows integration
into the host
genome. Retroviral vectors can either be replication-competent or replication-
defective. In some
embodiments of the invention, retroviruses are incorporated to effectuate
transfection into a cell,
however the CRISPR/Cas9/gRNA complexes are designed to target the viral
genome.
In some embodiments of the invention, lentiviruses, which are a subclass of
retroviruses,
are used as viral vectors. Lentiviruses can be adapted as delivery vehicles
(vectors) given their
ability to integrate into the genome of non-dividing cells, which is the
unique feature of
lentiviruses as other retroviruses can infect only dividing cells. The viral
genome in the form of
RNA is reverse-transcribed when the virus enters the cell to produce DNA,
which is then
inserted into the genome at a random position by the viral integrase enzyme.
The vector, now
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called a provirus, remains in the genome and is passed on to the progeny of
the cell when it
divides.
As opposed to lentiviruses, adenoviral DNA does not integrate into the genome
and is not
replicated during cell division. Adenovirus and the related AAV would be
potential approaches
as delivery vectors since they do not integrate into the host's genome. In
some aspects of the
invention, only the viral genome to be targeted is effected by the
CRISPR/Cas9/gRNA
complexes, and not the host's cells. Adeno-associated virus (AAV) is a small
virus that infects
humans and some other primate species. AAV can infect both dividing and non-
dividing cells
and may incorporate its genome into that of the host cell. For example,
because of its potential
use as a gene therapy vector, researchers have created an altered AAV called
self-complementary
adeno-associated virus (scAAV). Whereas AAV packages a single strand of DNA
and requires
the process of second-strand synthesis, scAAV packages both strands which
anneal together to
form double stranded DNA. By skipping second strand synthesis scAAV allows for
rapid
expression in the cell. Otherwise, scAAV carries many characteristics of its
AAV counterpart.
Methods of the invention may incorporate herpesvirus, poxvirus, alphavirus, or
vaccinia virus as
a means of delivery vectors.
In certain embodiments of the invention, non-viral vectors may be used to
effectuate
transfection. Methods of non-viral delivery of nucleic acids include
lipofection, nucleofection,
microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation
or lipid:nucleic
acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of
DNA. Lipofection
is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355, the
contents of each of
which are incorporated by reference in their entirety for all purposes.) and
lipofection reagents
are sold commercially (e.g., Transfectam and Lipofectin). Cationic and neutral
lipids that are
suitable for efficient receptor-recognition lipofection of polynucleotides
include those described
in U.S. Pat. 7,166,298 to Jessee or U.S. Pat. 6,890,554 to Jesse, the contents
of each of which are
incorporated by reference. Delivery can be to cells (e.g. in vitro or ex vivo
administration) or
target tissues (e.g. in vivo administration).
Synthetic vectors are typically based on cationic lipids or polymers which can
complex
with negatively charged nucleic acids to form particles with a diameter in the
order of 100 nm.
The complex protects nucleic acid from degradation by nuclease. Moreover,
cellular and local
delivery strategies have to deal with the need for internalization, release,
and distribution in the
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proper subcellular compartment. Systemic delivery strategies encounter
additional hurdles, for
example, strong interaction of cationic delivery vehicles with blood
components, uptake by the
reticuloendothelial system, kidney filtration, toxicity and targeting ability
of the carriers to the
cells of interest. Modifying the surfaces of the cationic non-virals can
minimize their interaction
with blood components, reduce reticuloendothelial system uptake, decrease
their toxicity and
increase their binding affinity with the target cells. Binding of plasma
proteins (also termed
opsonization) is the primary mechanism for RES to recognize the circulating
nanoparticles. For
example, macrophages, such as the Kupffer cells in the liver, recognize the
opsonized
nanoparticles via the scavenger receptor.
In some embodiments of the invention, non-viral vectors are modified to
effectuate
targeted delivery and transfection. PEGylation (i.e. modifying the surface
with
polyethyleneglycol) is the predominant method used to reduce the opsonization
and aggregation
of non-viral vectors and minimize the clearance by reticuloendothelial system,
leading to a
prolonged circulation lifetime after intravenous (i.v.) administration.
PEGylated nanoparticles
are therefore often referred as "stealth" nanoparticles. The nanoparticles
that are not rapidly
cleared from the circulation will have a chance to encounter infected cells.
However, PEG on the surface can decrease the uptake by target cells and reduce
the
biological activity. Therefore, to attach targeting ligand to the distal end
of the PEGylated
component is necessary; the ligand is projected beyond the PEG "shield" to
allow binding to
receptors on the target cell surface. When cationic liposome is used as gene
carrier, the
application of neutral helper lipid is helpful for the release of nucleic
acid, besides promoting
hexagonal phase formation to enable endosomal escape. In some embodiments of
the invention,
neutral or anionic liposomes are developed for systemic delivery of nucleic
acids and obtaining
therapeutic effect in experimental animal model. Designing and synthesizing
novel cationic
lipids and polymers, and covalently or noncovalently binding gene with
peptides, targeting
ligands, polymers, or environmentally sensitive moieties also attract many
attentions for
resolving the problems encountered by non-viral vectors. The application of
inorganic
nanoparticles (for example, metallic nanoparticles, iron oxide, calcium
phosphate, magnesium
phosphate, manganese phosphate, double hydroxides, carbon nanotubes, and
quantum dots) in
delivery vectors can be prepared and surface-functionalized in many different
ways.
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Guided nuclease systems of the invention may be administered alone or as an
active
ingredient in combination with pharmaceutically acceptable carriers, diluents,
adjuvants and
vehicles. Vectors may be incorporated into topical or intravenous formulations
which may
comprise a liquid or solid filler, diluent, excipient, solvent or
encapsulating material, involved in
carrying or transporting the subject agents from one organ, or portion of the
body, to another
organ, or portion of the body. Each carrier should be compatible with the
other ingredients of the
formulation.
In certain embodiments, compositions of the invention may be encapsulated in
hydrogels.
In another embodiment, composition as disclosed herein can comprise lipid-
based formulations.
Any of the known lipid-based drug delivery systems can be used in the practice
of the invention.
For instance, multivesicular liposomes, multilamellar liposomes and
unilamellar liposomes can
all be used so long as a sustained release rate of the encapsulated active
compound can be
established. Such formulations may be used to modify the release profile of
the guided nuclease
compositions. Methods of making controlled release multivesicular liposome
drug delivery
systems are described in PCT Application Publication Nos: WO 9703652, WO
9513796, and
WO 9423697, the contents of which are incorporated herein by reference.
Synthetic membrane vesicles may comprise a combination of phospholipids,
usually in
combination with steroids, especially cholesterol. Other phospholipids or
other lipids may also
be used. Examples of lipids useful in synthetic membrane vesicle production
include
phosphatidylglycerols, phosphatidylcholines, phosphatidylserines,
phosphatidylethanolamines,
sphingolipids, cerebrosides, and gangliosides, with preferable embodiments
including egg
phosphatidylcholine, dipalmitoylphosphatidylcholine,
distearoylphosphatidyleholine,
dioleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and
dioleoylphosphatidylglycerol.
In preparing lipid-based vesicles containing compositions of the invention,
such variables
as the efficiency of compound encapsulation, labiality of the compound,
homogeneity and size of
the resulting population of vesicles, active compound-to-lipid ratio,
permeability, instability of
the preparation, and pharmaceutical acceptability of the formulation should be
considered.
In another embodiment, guided nuclease systems of the invention can be
delivered in a
vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533),
the contents of
which are incorporated by reference in their entirety for all purposes). In
yet another
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embodiment, guided nuclease systems can be delivered in a controlled release
system. In one
embodiment, a pump may be used (see Langer (1990) supra). In another
embodiment, polymeric
materials can be used (see Howard et al. (1989) J. Neurosurg. 71 : 105, the
contents of which are
incorporated by reference in their entirety for all purposes). In another
embodiment a vector of
the invention can be administered so that it becomes intracellular, e.g., by
use of a retroviral
vector (see, for example, U.S. Pat. No. 4,980,286), or by direct injection, or
by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating
with lipids or cell-
surface receptors or transfecting agents, or by administering it in linkage to
a homeobox-like
peptide which is known to enter the nucleus (see e.g., Joliot et al., 1991,
Proc. Natl. Acad. Sci.
USA 88: 1864-1868), the contents of which are incorporated by reference in
their entirety for all
purposes. Alternatively, a nucleic acid can be introduced intracellularly and
incorporated within
host cell DNA for expression, by homologous recombination.
In some embodiments of the invention, targeted controlled-release systems
responding to
the unique environments of tissues and external stimuli are utilized. Gold
nanorods have strong
absorption bands in the near-infrared region, and the absorbed light energy is
then converted into
heat by gold nanorods, the so-called `photothermal effect'. Because the near-
infrared light can
penetrate deeply into tissues, the surface of gold nanorod could be modified
with nucleic acids
for controlled release. When the modified gold nanorods are irradiated by near-
infrared light,
nucleic acids are released due to thermo-denaturation induced by the
photothermal effect. The
amount of nucleic acids released is dependent upon the power and exposure time
of light
irradiation.
In some embodiments of the invention, liposomes are used to effectuate
transfection into
a cell or tissue. The pharmacology of a liposomal formulation of nucleic acid
is largely
determined by the extent to which the nucleic acid is encapsulated inside the
liposome bilayer.
Encapsulated nucleic acid is protected from nuclease degradation, while those
merely associated
with the surface of the liposome is not protected. Encapsulated nucleic acid
shares the extended
circulation lifetime and biodistribution of the intact liposome, while those
that are surface
associated adopt the pharmacology of naked nucleic acid once they disassociate
from the
liposome.
In some embodiments, the complexes of the invention are encapsulated in a
liposome.
Unlike small molecule drugs, nucleic acids cannot cross intact lipid bilayers,
predominantly due
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to the large size and hydrophilic nature of the nucleic acid. Therefore,
nucleic acids may be
entrapped within liposomes with conventional passive loading technologies,
such as ethanol drop
method (as in SALP), reverse-phase evaporation method, and ethanol dilution
method (as in
SNALP).
In some embodiments, linear polyethylenimine (L-PEI) is used as a non-viral
vector due
to its versatility and comparatively high transfection efficiency. L-PEI has
been used to
efficiently deliver genes in vivo into a wide range of organs such as lung,
brain, pancreas, retina,
bladder as well as tumor. L-PEI is able to efficiently condense, stabilize and
deliver nucleic acids
in vitro and in vivo.
Low-intensity ultrasound in combination with microbubbles has recently
acquired much
attention as a safe method of gene delivery. Ultrasound shows tissue-
permeabilizing effect. It is
non-invasive and site-specific, and could make it possible to destroy tumor
cells after systemic
delivery, while leave nontargeted organs unaffected. Ultrasound-mediated
microbubbles
destruction has been proposed as an innovative method for noninvasive
delivering of drugs and
nucleic acids to different tissues. Microbubbles are used to carry a drug or
gene until a specific
area of interest is reached, and then ultrasound is used to burst the
microbubbles, causing site-
specific delivery of the bioactive materials. Furthermore, the ability of
albumin-coated
microbubbles to adhere to vascular regions with glycocalix damage or
endothelial dysfunction is
another possible mechanism to deliver drugs even in the absence of ultrasound.
See Tsutsui et
al., 2004, The use of microbubbles to target drug delivery, Cardiovasc
Ultrasound 2:23, the
contents of which are incorporated by reference. In ultrasound-triggered drug
delivery, tissue-
permeabilizing effect can be potentiated using ultrasound contrast agents, gas-
filled
microbubbles. The use of microbubbles for delivery of nucleic acids is based
on the hypothesis
that destruction of DNA-loaded microbubbles by a focused ultrasound beam
during their
microvascular transit through the target area will result in localized
transduction upon disruption
of the microbubble shell while sparing non-targeted areas.
Besides ultrasound-mediated delivery, magnetic targeting delivery could be
used for
delivery. Magnetic nanoparticles are usually entrapped in gene vectors for
imaging the delivery
of nucleic acid. Nucleic acid carriers can be responsive to both ultrasound
and magnetic fields,
i.e., magnetic and acoustically active lipospheres (MAALs). The basic premise
is that therapeutic
agents are attached to, or encapsulated within, a magnetic micro- or
nanoparticle. These particles
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may have magnetic cores with a polymer or metal coating which can be
functionalized, or may
consist of porous polymers that contain magnetic nanoparticles precipitated
within the pores. By
functionalizing the polymer or metal coating it is possible to attach, for
example, cytotoxic drugs
for targeted chemotherapy or therapeutic DNA to correct a genetic defect. Once
attached, the
particle/therapeutic agent complex is injected into the bloodstream, often
using a catheter to
position the injection site near the target. Magnetic fields, generally from
high-field, high-
gradient, rare earth magnets are focused over the target site and the forces
on the particles as they
enter the field allow them to be captured and extravasated at the target.
Synthetic cationic polymer-based nanoparticles (-100 nm diameter) have been
developed
that offer enhanced transfection efficiency combined with reduced
cytotoxicity, as compared to
traditional liposomes. The incorporation of distinct layers composed of lipid
molecules with
varying physical and chemical characteristics into the polymer nanoparticle
formulation resulted
in improved efficiency through better fusion with cell membrane and entry into
the cell,
enhanced release of molecules inside the cell, and reduced intracellular
degradation of
nanoparticle complexes.
In some embodiments, the complexes are conjugated to nano-systems for systemic
therapy, such as liposomes, albumin-based particles, PEGylated proteins,
biodegradable
polymer-drug composites, polymeric micelles, dendrimers, among others. See
Davis et al., 2008,
Nanotherapeutic particles: an emerging treatment modality for cancer, Nat Rev
Drug Discov.
7(9):771-782, incorporated by reference. Long circulating macromolecular
carriers such as
liposomes, can exploit the enhanced permeability and retention effect for
preferential
extravasation from tumor vessels. In certain embodiments, the complexes of the
invention are
conjugated to or encapsulated into a liposome or polymerosome for delivery to
a cell. For
example, liposomal anthracyclines have achieved highly efficient
encapsulation, and include
versions with greatly prolonged circulation such as liposomal daunorubicin and
pegylated
liposomal doxorubicin. See Krishna et al., Carboxymethylcellulose-sodium based
transdermal
drug delivery system for propranolol, J Pharm Pharmacol. 1996 Apr; 48(4):367-
70.
Liposomal delivery systems provide stable formulation, provide improved
pharmacokinetics, and a degree of 'passive' or 'physiological' targeting to
tissues. Encapsulation
of hydrophilic and hydrophobic materials, such as potential chemotherapy
agents, are known.
See for example U.S. Pat. No. 5,466,468 to Schneider, which discloses
parenterally
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administrable liposome formulation comprising synthetic lipids; U.S. Pat. No.
5,580,571, to
Hostetler et al. which discloses nucleoside analogues conjugated to
phospholipids; U.S. Pat. No.
5,626,869 to Nyqvist, which discloses pharmaceutical compositions wherein the
pharmaceutically active compound is heparin or a fragment thereof contained in
a defined lipid
system comprising at least one amphiphatic and polar lipid component and at
least one nonpolar
lipid component.
Liposomes and polymerosomes can contain a plurality of solutions and
compounds. In
certain embodiments, the complexes of the invention are coupled to or
encapsulated in
polymersomes. As a class of artificial vesicles, polymersomes are tiny hollow
spheres that
enclose a solution, made using amphiphilic synthetic block copolymers to form
the vesicle
membrane. Common polymersomes contain an aqueous solution in their core and
are useful for
encapsulating and protecting sensitive molecules, such as drugs, enzymes,
other proteins and
peptides, and DNA and RNA fragments. The polymersome membrane provides a
physical
barrier that isolates the encapsulated material from external materials, such
as those found in
biological systems. Polymerosomes can be generated from double emulsions by
known
techniques, see Lorenceau et al., 2005, Generation of Polymerosomes from
Double-Emulsions,
Langmuir 21(20):9183-6, incorporated by reference.
Some embodiments of the invention provide for a gene gun or a biolistic
particle delivery
system. A gene gun is a device for injecting cells with genetic information,
where the payload
may be an elemental particle of a heavy metal coated with plasmid DNA. This
technique may
also be referred to as bioballistics or biolistics. Gene guns have also been
used to deliver DNA
vaccines. The gene gun is able to transfect cells with a wide variety of
organic and non-organic
species, such as DNA plasmids, fluorescent proteins, dyes, etc.
Aspects of the invention provide for numerous uses of delivery vectors.
Selection of the
delivery vector is based upon the cell or tissue targeted and the specific
makeup of the
CRISPR/Cas9/gRNA. For example, in the EBV example discussed above, since
lymphocytes are
known for being resistant to lipofection, nucleofection (a combination of
electrical parameters
generated by a device called Nucleofector, with cell-type specific reagents to
transfer a substrate
directly into the cell nucleus and the cytoplasm) was necessitated for DNA
delivery into the Raji
cells. The Lonza pmax promoter drives Cas9 expression as it offered strong
expression within
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Raji cells. At 24 hours after nucleofection, obvious EGFP signals were
observed from a small
proportion of cells through fluorescent microscopy.
FIG. 6 shows that including an oni-P in the plasmid promoted active plasmid
replication
inside the cells, which increased the transfection efficiency to >60%. The
left panel shows that
untreated cells and cells treated with a Cas9 exhibited similarly low number
of cells with
fluorescent markers. The right panels shows that the oriP promoted higher
transfection.
The EGFP-positive cell population decreased dramatically, however, <10%
transfection
efficiency 48 hours after nucleofection was measured (FIG. 6). A CRISPR
plasmid that included
the EBV origin of replication sequence, oriP yielded a transfection efficiency
>60% (FIG. 6).
Aspects of the invention utilize the CRISPR/Cas9/gRNA complexes for the
targeted
delivery. Common known pathways include transdermal, transmucal, nasal, ocular
and
pulmonary routes. Drug delivery systems may include liposomes, proliposomes,
microspheres,
gels, prodrugs, cyclodextrins, etc. Aspects of the invention utilize
nanoparticles composed of
biodegradable polymers to be transferred into an aerosol for targeting of
specific sites or cell
populations in the lung, providing for the release of the drug in a
predetermined manner and
degradation within an acceptable period of time. Controlled-release technology
(CRT), such as
transdermal and transmucosal controlled-release delivery systems, nasal and
buccal aerosol
sprays, drug-impregnated lozenges, encapsulated cells, oral soft gels,
iontophoretic devices to
administer drugs through skin, and a variety of programmable, implanted drug-
delivery devices
are used in conjunction with the complexes of the invention of accomplishing
targeted and
controlled delivery.
It should be appreciated that the CMV targeting nuclease can be delivered into
a cell,
organ, patient, or fetus by the techniques described herein and by techniques
known in the art.
The CMV targeting nucleases of the invention may be prepared for delivery by
association
(binding, encapsulating, etc.) with vectors and/or guided RNAs.
Aspects of the invention provide for delivering the CMV targeting nuclease
across the
placenta, or trans placental. As the conduit to the fetus, the placenta is
both a drug target and a
drug barrier. Alternatively, the nuclease can be introduced into the amniotic
sac or into the fetus.
For example, the nuclease can be introduced into the amniotic sac or into the
fetus by injection.
Alternatively, the fetus may be treated for CMV infection using the methods of
the invention
after birth, using any known technique in the art for delivering a therapeutic
to an infant.
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v. Cut viral nucleic acid
Once inside the cell, the CRISPR/Cas9/gRNA complexes target the viral genome.
In an
aspect of the invention, the complexes are targeted to viral genomes. In
addition to latent
infections this invention can also be used to control actively replicating
viruses by targeting the
viral genome before it is packaged or after it is ejected. In some
embodiments, methods and
compositions of the invention use a nuclease such as Cas9 to target latent
viral genomes, thereby
reducing the chances of proliferation. The nuclease may form a complex with a
gRNA (e.g.,
crRNA + tracrRNA or sgRNA). The complex cuts the viral nucleic acid in a
targeted fashion to
incapacitate the viral genome. As discussed above, the Cas9 endonuclease
causes a double strand
break in the viral genome. By targeted several locations along the viral
genome and causing not a
single strand break, but a double strand break, the genome is effectively cut
a several locations
along the genome. In a preferred embodiment, the double strand breaks are
designed so that
small deletions are caused, or small fragments are removed from the genome so
that even if
natural repair mechanisms join the genome together, the genome is render
incapacitated.
After introduction into a cell, the CRISPR/Cas9/gRNA complexes act on the
viral
genome, genes, transcripts, or other viral nucleic acid. The double-strand DNA
breaks generated
by CRISPR are repaired with small deletions. These deletions will disrupt the
protein coding and
hence create knockout effects.
The nuclease, or a gene encoding the nuclease, may be delivered into an
infected cell by
transfection. For example, the infected cell can be transfected with DNA that
encodes Cas9 and
gRNA (on a single piece or separate pieces). The gRNAs are designed to
localize the Cas9
endonuclease at one or several locations along the viral genome. The Cas9
endonuclease causes
double strand breaks in the genome, causing small fragments to be deleted from
the viral
genome. Even with repair mechanisms, the deletions render the viral genome
incapacitated.
Cells and tissues treated with a nuclease according to the methods of the
invention are
then provided for transplantation. In some embodiments, organs are treated
with the nuclease to
render the tissue CMV free, prior to transplantation.
In some embodiments of the invention, the nucleases are prepared for use in
organs for
transplant. Organ transplantation is the moving of an organ from one body to
another or from a
donor site to another location on the person's own body, to replace the
recipient's damaged or
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absent organ. Organ can also be created or re-grown from the person's own
cells (stem cells, or
cells extracted from the failing organs) or from cells of another person.
Organs can either be
from a living or cadaveric source. Organs that can be transplanted are the
heart, kidneys, liver,
lungs, pancreas, intestine, and thymus. Tissues include bones, tendons (both
referred to as
musculoskeletal grafts), cornea, skin, heart valves, nerves and veins. Cornea
and musculoskeletal
grafts are commonly transplanted tissues, or organs.
vi. Host genome
It will be appreciated that method and compositions of the invention can be
used to target
viral nucleic acid without interfering with host genetic material. Methods and
compositions of
the invention employ a targeting moiety such as a guide RNA that has a
sequence that hybridizes
to a target within the viral sequence. Methods and compositions of the
invention may further use
a targeted nuclease such as the cas9 enzyme, or a vector encoding such a
nuclease, which uses
the gRNA to bind exclusively to the viral genome and make double stranded
cuts, thereby
removing the viral sequence from the host.
Where the targeting moiety includes a guide RNA, the sequence for the gRNA, or
the
guide sequence, can be determined by examination of the viral sequence to find
regions of about
20 nucleotides that are adjacent to a protospacer adjacent motif (PAM) and
that do not also
appear in the host genome adjacent to the protospacer motif.
Preferably a guide sequence that satisfies certain similarity criteria (e.g.,
at least 60%
identical with identity biased toward regions closer to the PAM) so that a
gRNA/cas9 complex
made according to the guide sequence will bind to and digest specified
features or targets in the
viral sequence without interfering with the host genome. Preferably, the guide
RNA corresponds
to a nucleotide string next to a protospacer adjacent motif (PAM) (e.g., NGG,
where N is any
nucleotide) in the viral sequence. Preferably, the host genome lacks any
region that (1) matches
the nucleotide string according to a predetermined similarity criteria and (2)
is also adjacent to
the PAM. The predetermined similarity criteria may include, for example, a
requirement of at
least 12 matching nucleotides within 20 nucleotides 5' to the PAM and may also
include a
requirement of at least 7 matching nucleotides within 10 nucleotides 5' to the
PAM. An
annotated viral genome (e.g., from GenBank) may be used to identify features
of the viral
sequence and finding the nucleotide string next to a protospacer adjacent
motif (PAM) in the
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viral sequence within a selected feature (e.g., a viral replication origin, a
terminal repeat, a
replication factor binding site, a promoter, a coding sequence, or a
repetitive region) of the viral
sequence. The viral sequence and the annotations may be obtained from a genome
database.
Where multiple candidate gRNA targets are found in the viral genome, selection
of the
sequence to be the template for the guide RNA may favor the candidate target
closest to, or at the
5' most end of, a targeted feature as the guide sequence. The selection may
preferentially favor
sequences with neutral (e.g., 40% to 60%) GC content. Additional background
regarding the
RNA-directed targeting by endonuclease is discussed in U.S. Pub. 2015/0050699;
U.S. Pub.
20140356958; U.S. Pub. 2014/0349400; U.S. Pub. 2014/0342457; U.S. Pub.
2014/0295556; and
U.S. Pub. 2014/0273037, the contents of each of which are incorporated by
reference for all
purposes. Due to the existence of human genomes background in the infected
cells, a set of steps
are provided to ensure high efficiency against the viral genome and low off-
target effect on the
human genome. Those steps may include (1) target selection within viral
genome, (2) avoiding
PAM+target sequence in host genome, (3) methodologically selecting viral
target that is
conserved across strains, (4) selecting target with appropriate GC content,
(5) control of nuclease
expression in cells, (6) vector design, (7) validation assay, others and
various combinations
thereof. A targeting moiety (such as a guide RNA) preferably binds to targets
within certain
categories such as (i) latency related targets, (ii) infection and symptom
related targets, and (iii)
structure related targets.
A first category of targets for gRNA includes latency-related targets. The
viral genome
requires certain features in order to maintain the latency. These features
include, but not limited
to, master transcription regulators, latency-specific promoters, signaling
proteins communicating
with the host cells, etc. If the host cells are dividing during latency, the
viral genome requires a
replication system to maintain genome copy level. Viral replication origin,
terminal repeats, and
replication factors binding to the replication origin are great targets. Once
the functions of these
features are disrupted, the viruses may reactivate, which can be treated by
conventional antiviral
therapies.
A second category of targets for gRNA includes infection-related and symptom-
related
targets. Virus produces various molecules to facilitate infection. Once gained
entrance to the host
cells, the virus may start lytic cycle, which can cause cell death and tissue
damage (HBV). In
certain cases, such as HPV16, cell products (E6 and E7 proteins) can transform
the host cells and
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cause cancers. Disrupting the key genome sequences (promoters, coding
sequences, etc)
producing these molecules can prevent further infection, and/or relieve
symptoms, if not curing
the disease.
A third category of targets for gRNA includes structure-related targets. Viral
genome
may contain repetitive regions to support genome integration, replication, or
other functions.
Targeting repetitive regions can break the viral genome into multiple pieces,
which physically
destroys the genome.
Where the nuclease is a cas protein, the targeting moiety is a guide RNA. Each
cas
protein requires a specific PAM next to the targeted sequence (not in the
guide RNA). This is the
same as for human genome editing. The current understanding the guide
RNA/nuclease complex
binds to PAM first, then searches for homology between guide RNA and target
genome.
Sternberg et al., 2014, DNA interrogation by the CRISPR RNA-guided
endonuclease Cas9,
Nature 507(7490):62-67. Once recognized, the DNA is digested 3-nt upstream of
PAM. These
results suggest that off-target digestion requires PAM in the host DNA, as
well as high affinity
between guide RNA and host genome right before PAM.
It may be preferable to use a targeting moiety that targets portions of the
viral genome
that are highly conserved. Viral genomes are much more variable than human
genomes. In order
to target different strains, the guide RNA will preferably target conserved
regions. As PAM is
important to initial sequence recognition, it is also essential to have PAM in
the conserved
region.
In a preferred embodiment, methods of the invention are used to deliver a
nucleic acid to
cells. The nucleic acid delivered to the cells may include a gRNA having the
determined guide
sequence or the nucleic acid may include a vector, such as a plasmid, that
encodes an enzyme
that will act against the target genetic material. Expression of that enzyme
allows it to degrade or
otherwise interfere with the target genetic material. The enzyme may be a
nuclease such as the
Cas9 endonuclease and the nucleic acid may also encode one or more gRNA having
the
determined guide sequence.
The gRNA targets the nuclease to the target genetic material. Where the target
genetic
material includes the genome of a virus, gRNAs complementary to parts of that
genome can
guide the degredation of that genome by the nuclease, thereby preventing any
further replication
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or even removing any intact viral genome from the cells entirely. By these
means, latent viral
infections can be targeted for eradication.
The host cells may grow at different rate, based on the specific cell type.
High nuclease
expression is necessary for fast replicating cells, whereas low expression
help avoiding off-target
cutting in non-infected cells. Control of nuclease expression can be achieved
through several
aspects. If the nuclease is expressed from a vector, having the viral
replication origin in the
vector can increase the vector copy number dramatically, only in the infected
cells. Each
promoter has different activities in different tissues. Gene transcription can
be tuned by choosing
different promoters. Transcript and protein stability can also be tuned by
incorporating
stabilizing or destabilizing (ubiquitin targeting sequence, etc) motif into
the sequence.
Specific promoters may be used for the gRNA sequence, the nuclease (e.g.,
cas9), other
elements, or combinations thereof. For example, in some embodiments, the gRNA
is driven by a
U6 promoter. A vector may be designed that includes a promoter for protein
expression (e.g.,
using a promoter as described in the vector sold under the trademark
PMAXCLONING by
Lonza Group Ltd (Basel, Switzerland). A vector may be a plasmid (e.g., created
by synthesis
instrument 255 and recombinant DNA lab equipment). In certain embodiments, the
plasmid
includes a U6 promoter driven gRNA or chimeric guide RNA (sgRNA) and a
ubiquitous
promoter-driven cas9. Optionally, the vector may include a marker such as EGFP
fused after the
cas9 protein to allow for later selection of cas9+ cells. It is recognized
that cas9 can use a gRNA
(similar to the CRISPR RNA (crRNA) of the original bacterial system) with a
complementary
trans-activating crRNA (tracrRNA) to target viral sequences complementary to
the gRNA. It has
also been shown that cas9 can be programmed with a single RNA molecule, a
chimera of the
gRNA and tracrRNA. The single guide RNA (sgRNA) can be encoded in a plasmid
and
transcription of the sgRNA can provide the programming of cas9 and the
function of the
tracrRNA. See Jinek, 2012, A programmable dual-RNA-guided DNA endonuclease in
adaptive
bacterial immunity, Science 337:816-821 and especially figure 5A therein for
background.
Using the above principles, methods and compositions of the invention may be
used to
target viral nucleic acid in an infected host without adversely influencing
the host genome.
For additional background see Hsu, 2013, DNA targeting specificity of RNA-
guided
Cas9 nucleases, Nature Biotechnology 31(9):827-832; and Jinek, 2012, A
programmable dual-
RNA-guided DNA endonuclease in adaptive bacterial immunity, Science 337:816-
821, the
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contents of each of which are incorporated by reference. Since the targeted
locations are selected
to be within certain categories such as (i) latency related targets, (ii)
infection and symptom
related targets, or (iii) structure related targets, cleavage of those
sequences inactivates the virus
and removes it from the host. Since the targeting RNA (the gRNA or sgRNA) is
designed to
satisfy according to similarity criteria that matches the target in the viral
genetic sequence
without any off-target matching the host genome, the latent viral genetic
material is removed
from the host without any interference with the host genome.
ii. Apoptotic pathway
In cases where a small number of cells are infected and it would suffice to
ablate the
entire cell (as well as the CMV genome), an aspect of the invention
contemplates administration
of a vector containing a promoter which is active in the latent viral state,
wherein the promoter
drives a cell-killing gene. HSV is a particularly interesting target for this
approach as it has been
estimated that only thousands to tens of thousands neurons are latently
infected. See Hoshino et
al., 2008, The number of herpes simplex virus-infected neurons and the number
of viral genome
copies per neuron correlate with latent viral load in ganglia, Virology
372(1):56-63, incorporated
by reference. Examples of cell-killing genes include both (1) targetable
nucleases that are
targeted to the cell genome; and (2) apoptosis effectors such as BAX and BAK
and proteins that
destroy the integrity of the cell or mitochondrial membrane, such as alpha
hemolysin. (Bayles,
"Bacterial programmed cell death: making sense of a paradox," Nature Reviews
Microbiology
12 pp.63-69 (2014)). Having a promoter that is only activated in latently
infected cells could be
used not only in this context but also be used to increase selectivity of
nuclease therapy by
making activity specific to infected cells; an example of such a promoter is
Latency-Associated
Promoter 1, or "LAP1". (Preston and Efstathiou, "Molecular Basis of HSV
Latency and
Reactivation", in Human Herpesviruses: Biology, Therapy and Immunoprophylaxis
2007.) In
some embodiments, the invention provides methods and therapeutics that can be
used to cause
the death of host cells but only those cells that are infected. For example,
the treatment can
include delivering a gene for a protein that causes cell death, where the gene
is under control of a
viral regulatory element such as a promoter from the genome of the infecting
virus or the gene is
encoded in a vector that includes a viral origin of replication. Where the
virus is present, the gene
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will be expressed and the gene product will cause the death of the cell. The
gene can code for a
protein important in apoptosis, or the gene can code for a nuclease that
digests the host genome.
The apoptotic embodiments may be used to remove infected cells from within a
sample
that contains a mix of infected and uninfected cells. Using a targetable
nuclease, a composition
may be provided that includes a viral-driven promoter, a targetable nuclease,
and guide RNAs
that target the cellular (e.g., human) genome. In the presence of the virus,
the nuclease will kill
the cells. The sample will be left containing only uninfected cells.
An apoptosis protein may be used as the therapeutic. The therapeutic may be
provided
encoded within a vector, in which the vector also encodes a sequence that
causes the therapeutic
to be expressed within a cell that is infected by a virus. The sequence may be
a regulatory
element (e.g., a promoter and an origin of replication) from the genome of the
virus. The
therapeutic may provide a mechanism that selectively causes death of virus-
infected cells. For
example, a protein may be used that restores a deficient apoptotic pathway in
the cell. The gene
may be, for example, BAX, BAK, BCL-2, or alpha-hemolysin. Preferably, the
therapeutic
induces apoptosis in the cell that is infected by the virus and does not
induce apoptosis in an
uninfected cell.
In some embodiments, the invention provides a composition that includes a
viral vector,
plasmid, or other coding nucleic acid that encodes at least one gene that
promotes apoptosis and
at least one promoter associated a viral genome. Apoptosis regulator Bc1-2 is
a family of proteins
that govern mitochondrial outer membrane permeabilization (MOMP) and include
pro-apoptotic
proteins such as Bax, BAD, Bak, Bok, Bcl-rambo, Bcl-xs and BOK/Mtd.
Apoptosis regulator BAX, also known as bc1-2-like protein 4, is a protein that
in humans
is encoded by the BAX gene. BAX is a member of the Bc1-2 gene family. This
protein forms a
heterodimer with BCL2, and functions as an apoptotic activator. This protein
is reported to
interact with, and increase the opening of, the mitochondrial voltage-
dependent anion channel
(VDAC), which leads to the loss in membrane potential and the release of
cytochrome c.
Bc1-2 homologous antagonist/killer is a protein that in humans is encoded by
the BAK1
gene on chromosome 6. This protein localizes to mitochondria, and functions to
induce
apoptosis. It interacts with and accelerates the opening of the mitochondrial
voltage-dependent
anion channel, which leads to a loss in membrane potential and the release of
cytochrome c.
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Human genes encoding proteins that belong to this family include: BAK1, BAX,
BCL2,
BCL2A1, BCL2L1, BCL2L2, BCL2L10, BCL2L13, BCL2L14, BOK, and MCL1.
Incorporation by Reference
References and citations to other documents, such as patents, patent
applications, patent
publications, journals, books, papers, web contents, have been made throughout
this disclosure.
All such documents are hereby incorporated herein by reference in their
entirety for all purposes.
Equivalents
Various modifications of the invention and many further embodiments thereof,
in
addition to those shown and described herein, will become apparent to those
skilled in the art
from the full contents of this document, including references to the
scientific and patent literature
cited herein. The subject matter herein contains important information,
exemplification and
guidance that can be adapted to the practice of this invention in its various
embodiments and
equivalents thereof.
Examples
Example 1: Targeting HSV
T7 in vitro transcription produced the complete guide RNA with scaffold.
Flanking
regions of the genome targets were PCR amplified from HSV2 strain G genomic
DNA (from
ATCC). Cas9 protein (from PNA Bio), guide RNA and target DNA were mixed and
incubated
for in vitro endonuclease assay.
To further test the efficiency against HSV within cells, we subcloned each
HSV2
amplicon mentioned above into an expression vector. The same guide RNA
sequences (RL2,
LATi, LATp, UL9, OriS, and US12) were also cloned into a CRISPR plasmid,
containing CMV
promoter driven cas9 and U6 promoter driven sgRNA scaffold. We transfected
both HSV2
amplicon clones and anti-HSV CRISPR plasmid into 293T cells with Lipofectamine
2000. 72
hours after transfection, cells were harvested for genomic DNA isolation.
FIG. 3 is a gel with lanes showing genomic DNA size bands for cells treated
with the
RL2, LATi, LATp, UL9, OriS, and US12 guide sequences with and without Cas9.
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FIG. 4 shows the results of quantitative PCR assays showing different levels
of
decreasing of HSV DNA in the CRISPR treated samples. In vivo anti-HSV
treatment with a
transient cell model. We used a CRISPR plasmid with a scrambled sgRNA sequence
as control.
DNA sample input was normalized with each control sample. OriS demonstrated
the highest
viral DNA elimination activity followed by RL2, UL9, and LATi.
Example 2: Targeting EBV
Burkitt's lymphoma cell lines Raji, Namalwa, and DG-75 were obtained from ATCC
and
cultured in RPMI 1640 supplemented with 10% FBS and PSA, following ATCC
recommendation. Human primary lung fibroblast IMR-90 was obtained from Coriell
and
cultured in Advanced DMEM/F-12 supplemented with 10% FBS and PSA.
Plasmids consisting of a U6 promoter driven chimeric guide RNA (sgRNA) and a
ubiquitous promoter driven Cas9 were obtained from addgene, as described by
Cong L et al.
(2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:819-
823. An
EGFP marker fused after the Cas9 protein allowed selection of Cas9-positive
cells (FIG. 6). We
adapted a modified chimeric guide RNA design for more efficient Pol-III
transcription and more
stable stem-loop structure (Chen B et al. (2013) Dynamic Imaging of Genomic
Loci in Living
Human Cells by an Optimized CRISPR/Cas System. Cell 155:1479-1491).
We obtained pX458 from Addgene, Inc. A modified CMV promoter with a synthetic
intron (pmax) was PCR amplified from Lonza control plasmid pmax-GFP. A
modified guide
RNA sgRNA(F+E) was ordered from IDT. EBV replication origin oriP was PCR
amplified from
B95-8 transformed lymphoblastoid cell line GM12891. We used standard cloning
protocols to
clone pmax, sgRNA(F+E) and oriP to pX458, to replace the original CAG
promoter, sgRNA and
fl origin. We designed EBV sgRNA based on the B95-8 reference, and ordered DNA
oligos
from IDT. The original sgRNA place holder in pX458 serves as the negative
control.
Lymphocytes are known for being resistant to lipofection, and therefore we
used
nucleofection for DNA delivery into Raji cells. We chose the Lonza pmax
promoter to drive
Cas9 expression as it offered strong expression within Raji cells. We used the
Lonza
Nucleofector II for DNA delivery. 5 million Raji or DG-75 cells were
transfected with 5 ug
plasmids in each 100-ul reaction. Cell line Kit V and program M-013 were used
following Lonza
recommendation. For IMR-90,1 million cells were transfected with 5 ug plasmids
in 100 ul
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Solution V, with program T-030 or X-005. 24 hours after nucleofection, we
observed obvious
EGFP signals from a small proportion of cells through fluorescent microscopy.
The EGFP-
positive cell population decreased dramatically after that, however, and we
measured <10%
transfection efficiency 48 hours after nucleofection (FIG. 6). We attributed
this transfection
efficiency decrease to the plasmid dilution with cell division. To actively
maintain the plasmid
level within the host cells, we redesigned the CRISPR plasmid to include the
EBV origin of
replication sequence, oriP. With active plasmid replication inside the cells,
the transfection
efficiency rose to >60% (FIG. 6).
To design guide RNA targeting the EBV genome, we relied on the EBV reference
genome from strain B95-8. We targeted six regions with seven guide RNA designs
for different
genome editing purposes.
Additional information such as primer design is shown in Wang and Quake, 2014,
RNA-
guided endonuclease provides a therapeutic strategy to cure latent
herpesviridae infection, PNAS
111(36):13157-13162 and in the Supporting Information to that article
published online at the
PNAS website, and the contents of both of those documents are incorporated by
reference for all
purposes.
EBNA1 is crucial for many EBV functions including gene regulation and latent
genome
replication. Guide RNA sgEBV4 and sgEBV5 to both ends of the EBNA1 coding
region in order
to excise this whole region of the genome. Guide RNAs sgEBV1, 2 and 6 fall in
repeat regions,
so that the success rate of at least one CRISPR cut is multiplied. These
"structural" targets enable
systematic digestion of the EBV genome into smaller pieces. EBNA3C and LMP1
are essential
for host cell transformation, and we designed guide RNAs sgEBV3 and 5gEBV7
were designed
to target the 5' exons of these two proteins respectively.
EBV Genome Editing. The double-strand DNA breaks generated by CRISPR are
repaired
with small deletions. FIGS. 8-13 represent CRISPR/Cas9 induced large
deletions.
FIG. 8 shows the genome context around guide RNA sgEBV2 and PCR primer
locations.
FIG. 9 shows the large deletion induced by sgEBV2. Lane 1-3 are before, 5 days
after,
and 7 days after sgEBV2 treatment, respectively.
FIG. 10 shows the genome context around guide RNA sgEBV3/4/5 and PCR primer
locations.
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FIG. 11 shows the large deletions induced by sgEBV3/5 and sgEBV4/5. Lane 1 and
2 are
3F/5R PCR amplicons before and 8 days after sgEBV3/5 treatment. Lane 3 and 4
are 4F/5R PCR
amplicons before and 8 days after sgEBV4/5 treatment.
FIGS. 12 and 13 show that Sanger sequencing confirmed genome cleavage and
repair
ligation 8 days after sgEBV3/5 (FIG. 12) and sgEBV4/5 (FIG. 13) treatment.
Areas 690 and 700
(FIG. 12) and areas 690 and 700 (FIG. 13) indicate the two ends before repair
ligation.
These deletions will disrupt the protein coding and hence create knockout
effects.
SURVEYOR assays confirmed efficient editing of individual sites.
FIG. 32 represents SURVEYOR assay of EBV CRISPR (lanes numbered from left to
right: Lane 1: NEB 100bp ladder; Lane 2: sgEBV1 control; Lane 3: sgEBV1; Lane
4: sgEBV5
control; Lane 5: sgEBV5; Lane 6: 5gEBV7 control; Lane 7: 5gEBV7; Lane 8:
5gEBV4).
Beyond the independent small deletions induced by each guide RNA, large
deletions
between targeting sites can systematically destroy the EBV genome. Guide RNA
sgEBV2 targets
a region with twelve 125-bp repeat units (FIG. 8). PCR amplicon of the whole
repeat region gave
a ¨1.8-kb band (FIG. 9). After 5 or 7 days of sgEBV2 transfection, we obtained
¨0.4-kb bands
from the same PCR amplification (FIG. 9). The ¨1.4-kb deletion is the expected
product of
repair ligation between cuts in the first and the last repeat unit (FIG. 8).
DNA sequences flanking sgRNA targets were PCR amplified with Phusion DNA
polymerase. SURVEYOR assays were performed following manufacturer's
instruction. DNA
amplicons with large deletions were TOPO cloned and single colonies were used
for Sanger
sequencing. EBV load was measured with Taqman digital PCR on Fluidigm BioMark.
A
Taqman assay targeting a conserved human locus was used for human DNA
normalization. 1 ng
of single-cell whole-genome amplification products from Fluidigm Cl were used
for EBV
quantitative PCR.
It is possible to delete regions between unique targets (FIG. 10). Six days
after sgEBV4-5
transfection, PCR amplification of the whole flanking region (with primers
EBV4F and 5R)
returned a shorter amplicon, together with a much fainter band of the expected
2 kb (FIG. 11).
Sanger sequencing of amplicon clones confirmed the direct connection of the
two expected
cutting sites (FIG. 13). A similar experiment with sgEBV3-5 also returned an
even larger
deletion, from EBNA3C to EBNA1 (FIG. 11).
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Additional information such as primer design is shown in Wang and Quake, 2014,
RNA-
guided endonuclease provides a therapeutic strategy to cure latent
herpesviridae infection, PNAS
111(36):13157-13162 and in the Supporting Information to that article
published online at the
PNAS website, and the contents of both of those documents are incorporated by
reference for all
purposes.
Cell Proliferation Arrest With EBV Genome Destruction. Two days after CRISPR
transfection, EGFP-positive cells were flow sorted for further culture and
counted the live cells
daily. FIGS. 14-26 represent cell proliferation arrest with EBV genome
destruction. FIG. 14
shows cell proliferation curves after different CRISPR treatments. Five
independent sgEBV1-7
treatments are shown here. FIGS. 15-20 show flow cytometry scattering signals
before (FIG. 15),
days after (FIG. 16) and 8 days after (FIG. 14) sgEBV1-7 treatments. FIG. 18-
20 show
Annexin V A1exa647 and DAPI staining results before (FIG. 18), 5 days after
(FIG. 19) and 8
days after (FIG. 20) sgEBV1-7 treatments. Regions 300 and 200 correspond to
subpopulation P3
and P4 in (FIGS. 15-17).
FIGS. 21 and 22 show microscopy revealed apoptotic cell morphology after
sgEBV1-7
treatment.
FIG. 23 shows nuclear morphology before sgEBV1-7 treatment.
FIGS. 24-26 show nuclear morphology after sgEBV1-7 treatment.
As expected, cells treated with Cas9 plasmids which lacked oriP or sgEBV lost
EGFP
expression within a few days and proliferated with a rate similar rate to the
untreated control
group (FIG. 14). Plasmids with Cas9-oriP and a scrambled guide RNA maintained
EGFP
expression after 8 days, but did not reduce the cell proliferation rate.
Treatment with the mixed
cocktail sgEBV1-7 resulted in no measurable cell proliferation and the total
cell count either
remained constant or decreased (FIG. 14).
FIG. 15 shows that flow cytometry scattering signals clearly revealed
alterations in the
cell morphology after sgEBV1-7 treatment, as the majority of the cells shrank
in size with
increasing granulation (P4 to P3 shift).
FIG. 19 gives DAPI staining results showing that cells in population P3 also
demonstrated compromised membrane permeability. To rule out the possibility of
CRISPR
cytotoxicity, especially with multiple guide RNAs, the same treatment was
performed on two
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other samples: the EBV-negative Burkitt's lymphoma cell line DG-75 and primary
human lung
fibroblast IMR90.
FIG. 33 shows that the CRISPR treatments were not cytotoxic to the EBV-
negative
Burkitt's lymphoma cell line DG-75
FIG. 34 shows that the CRISPR treatments were not cytotoxic to primary human
lung
fibroblasts IMR90.
Eight and nine days after transfection the cell proliferation rates did not
change from the
untreated control groups, suggesting neglectable cytotoxicity.
Previous studies have attributed the EBV tumorigenic ability to its
interruption of host
cell apoptosis (Ruf IK et al. (1999) Epstein-Barr Virus Regulates c-MYC,
Apoptosis, and
Tumorigenicity in Burkitt Lymphoma. Molecular and Cellular Biology 19:1651-
1660).
Suppressing EBV activities may therefore restore the apoptosis process, which
could explain the
cell death observed in our experiment. Annexin V staining revealed a distinct
subpopulation of
cells with intact cell membrane but exposed phosphatidylserine, suggesting
cell death through
apoptosis (FIG. 18). Bright field microscopy showed obvious apoptotic cell
morphology (FIG.
21) and fluorescent staining demonstrated drastic DNA fragmentation (FIG. 23).
Altogether this
evidence suggests restoration of the normal host cell apoptosis pathway after
EBV genome
destruction.
FIGS. 27-31 represent EBV load quantitation after CRISPR treatment.
FIG. 27 shows EBV load after different CRISPR treatments by digital PCR. Cas9
and
Cas9-oriP had two replicates, and sgEBV1-7 had 5 replicates.
Complete Clearance Of EBV In A Subpopulation.
To study the potential connection between cell proliferation arrest and EBV
genome
editing, the EBV load was quantified in different samples with digital PCR
targeting EBNAL
Another Taqman assay targeting a conserved human somatic locus served as the
internal control
for human DNA normalization. On average, each untreated Raji cell has 42
copies of EBV
genome (FIG. 27). Cells treated with a Cas9 plasmid that lacked oriP or sgEBV
did not have an
obvious difference in EBV load difference from the untreated control. Cells
treated with a Cas9-
plasmid with oriP but no sgEBV had an EBV load that was reduced by ¨50%. In
conjunction
with the prior observation that cells from this experiment did not show any
difference in
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proliferation rate, we interpret this as likely due to competition for EBNA1
binding during
plasmid replication. The addition of the guide RNA cocktail sgEBV1-7 to the
transfection
dramatically reduced the EBV load. Both the live and dead cells have >60% EBV
decrease
comparing to the untreated control.
Although seven guide RNAs were provided at the same molar ratio, the plasmid
transfection and replication process is likely quite stochastic. Some cells
will inevitably receive
different subsets or mixtures of the guide RNA cocktail, which might affect
the treatment
efficiency. To control for such effects, the EBV load was measured at the
single cell level by
employing single-cell whole-genome amplification with an automated
microfluidic system.
Freshly cultured Raji cells were loaded onto the microfluidic chip and
captured 81 single
cells.
For the sgEBV1-7 treated cells, the live cells were flow sorted eight days
after
transfection and captured 91 single cells.
FIGS. 28 shows a single Raji cell as captured on a microfluidic chip.
FIG. 29 shows a single sgEBV1-7 treated cell as captured on the chip.
Following manufacturer's instruction, ¨150 ng amplified DNA was obtained from
each
single cell reaction chamber. For quality control purposes we performed 4-loci
human somatic
DNA quantitative PCR on each single cell amplification product (Wang J, Fan
HC, Behr B,
Quake SR (2012) Genome-wide single-cell analysis of recombination activity and
de novo
mutation rates in human sperm. Cell 150:402-412) and required positive
amplification from at
least one locus.
FIG. 30 is a histogram of EBV quantitative PCR Ct values from single cells
before
treatment. The dash line represents Ct values of one EBV genome per cell. A
log-normal
distribution of EBV load was displayed by the 69 untreated single-cell
products that passed the
quality control, with almost every cell displaying significant amounts of EBV
genomic DNA.
We calibrated the quantitative PCR assay with a subclone of Namalwa Burkitt's
lymphoma cells, which contain a single integrated EBV genome. The single-copy
EBV
measurements gave a Ct of 29.8, which enabled us to determine that the mean Ct
of the 69 Raji
single cell samples corresponded to 42 EBV copies per cells, in concordance
with the bulk
digital PCR measurement. For the sgEBV1-7 treated sample, 71 single-cell
products passed the
quality control and the EBV load distribution was dramatically wider.
CA 03000189 2018-03-27
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FIG. 31 is a histogram of EBV quantitative PCR Ct values from single live
cells 7 days
after sgEBV1-7 treatment. The dash line represents Ct values of one EBV genome
per cell.
While 22 cells had the same EBV load as the untreated cells, 19 cells had no
detectable
EBV and the remaining 30 cells displayed dramatic EBV load decrease from the
untreated
sample.
FIG. 32 represents SURVEYOR assay of EBV CRISPR. Lane 1 (lanes numbered from
left to right): NEB 100bp ladder; Lane 2: sgEBV1 control; Lane 3: sgEBV1; Lane
4: sgEBV5
control; Lane 5: sgEBV5; Lane 6: 5gEBV7 control; Lane 7: 5gEBV7; Lane 8:
sgEBV4. FIG. 33
represents CRISPR cytotoxicity test with EBV-negative Burkitt's lymphoma DG-
75. FIG. 34
represents CRISPR cytotoxicity test with primary human lung fibroblast IMR-90.
Essential Targets For EBV Treatment. The seven guide RNAs in our CRISPR
cocktail
target three different categories of sequences which are important for EBV
genome structure,
host cell transformation, and infection latency, respectively. To understand
the most essential
targets for effective EBV treatment, we transfected Raji cells with subsets of
guide RNAs.
Although sgEBV4/5 reduced the EBV genome by 85%, they could not suppress cell
proliferation
as effectively as the full cocktail (FIG. 14). Guide RNAs targeting the
structural sequences
(5gEBV1/2/6) could stop cell proliferation completely, despite not eliminating
the full EBV load
(26% decrease). Given the high efficiency of genome editing and the
proliferation arrest (FIG.
2), we suspect that the residual EBV genome signature in sgEBV1/2/6 was not
due to intact
genomes but to free-floating DNA that has been digested out of the EBV genome,
i.e. as a false
positive. We conclude that systematic destruction of EBV genome structure
appears to be more
effective than targeting specific key proteins for EBV treatment.
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