Note: Descriptions are shown in the official language in which they were submitted.
WO 2021/146711
PCT/ITS2021/013955
- 1 -
ADENO-ASSOCIATED VIRAL VECTOR, COMPOSITIONS, METHODS OF
PROMOTING MUSCLE REGENERATION, AND TREATMENT METHODS
[00011 This application claims the priority benefit of U.S.
Provisional Patent Application
Serial No. 62/962,712, filed January 17, 2020, and U.S. Provisional Patent
Application Serial
No. 63/128,047, filed December 19, 2020, which are hereby incorporated by
reference in their
entirety.
[00021 This invention was made with government support under RO1
AR074430-01
awarded by the National Institutes of Health. The government has certain
rights in the invention.
FIELD
[00031 The present application relates to adeno-associated viral (AAV)
vectors and
lentiviral vectors comprising a nucleic acid molecule encoding an AU-rich mRNA
binding factor
1 (AUF1) protein or a functional fragment thereof, as well as compositions and
methods of use
thereof.
BACKGROUND
[00041 Muscle wasting diseases represent a major source of human disease.
They can be
genetic in origin (primarily muscular dystrophies), related to aging
(sarcopenia), or the result of
traumatic muscle injury, among others. There are few treatment options
available for individuals
with myopathies, or those who have suffered severe muscle trauma, or the loss
of muscle mass
with aging (known as sarcopenia). The physiology of myopathies is well
understood and
founded on a common pathogenesis of relentless cycles of muscle degeneration
and
regeneration, typically leading to functional exhaustion of muscle stem
(satellite) cells and their
progenitor cells that fail to reactivate, and at times their loss as well
(Carlson & Conboy, "Loss
of Stem Cell Regenerative Capacity Within Aged Niches,- Aging Cell 6(3):371-82
(2007);
Shefer et al., "Satellite-cell Pool Size Does Matter: Defining the Myogenic
Potency of Aging
Skeletal Muscle," Dev. Biol. 294(1):50-66 (2006); Bernet et al., "p38 MAPK
Signaling
Underlies a Cell-autonomous Loss of Stem Cell Self-renewal in Skeletal Muscle
of Aged Mice,"
Nat. Med. 20(3):265-71 (2014); and Dumont et al., "Intrinsic and Extrinsic
Mechanisms
Regulating Satellite Cell Function," Development 142(9):1572-1581 (2015)).
[00051 Age-related skeletal muscle loss and atrophy is
characterized by the progressive
loss of muscle mass, strength, and endurance with age. It can be a significant
source of frailty,
increased fractures, and mortality in the elderly population (Vermeiren et
al., "Frailty and the
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 2 -
Prediction of Negative Health Outcomes: A Meta-Analysis," J. Am. Med. Dir.
Assoc.
17(12):1163.e1-1163.e17 (2016) and Buford, T. W., "Sarcopenia: Relocating the
Forest among
the Trees," Toxicol. Pathot 45(7):957-960 (2017)). Although different
strategies have been
investigated to counter muscle loss and atrophy, regular resistance exercise
is the most effective
in slowing muscle loss and atrophy, but compliance and physical limitations
are significant
barriers (Wilkinson et al., "The Age-Related loss of Skeletal Muscle Mass and
Function:
Measurement and Physiology of Muscle Fibre Atrophy and Muscle Fibre Loss in
Humans,-
Ageing Res. Rev. 47:123-132 (2018)). Consequently, with an aging global
population,
therapeutic strategies need to be developed to reverse age-related muscle
decline.
[0006] Muscle regeneration is initiated by skeletal muscle stem (satellite)
cells that reside
between striated muscle fibers (myofibers), which are the contractile cellular
bundles, and the
basal lamina that surrounds them (Carlson & Conboy, "Loss of Stem Cell
Regenerative Capacity
within Aged Niches," Aging Cell 6(3):371-382 (2007) and Schiaffino & Reggiani,
"Fiber Types
in Mammalian Skeletal Muscles," Physiot Rev. 91(4):1447-1531 (2011)). Upon
physical injury
to muscle, the anatomical niche is disrupted, normally quiescent satellite
cells become activated
and proliferate asymmetrically. Some satellite cells reconstitute the stem
cell population while
most others differentiate and fuse to form new myofibers (Hindi et al.,
"Signaling Mechanisms in
Mammalian Myoblast Fusion," Sci. Signal. 6(272):re2 (2013)). Studies have
demonstrated the
singular importance of the satellite cell/myoblast population in muscle
regeneration (Shefer et
al., "Satellite-cell Pool Size Does Matter: Defining the Myogenic Potency of
Aging Skeletal
Muscle," Dev. Biol. 294(1):50-66 (2006); Dumont et al., "Intrinsic and
Extrinsic Mechanisms
Regulating Satellite Cell Function," Development 142(9):1572-1581 (2015);
Briggs & Morgan,
"Recent Progress in Satellite Cell/Myoblast Engraftment -- Relevance for
Therapy, FEBS J.
280(17):4281-93 (2013); Morgan & Zammit, "Direct Effects of the Pathogenic
Mutation on
Satellite Cell Function in Muscular Dystrophy," Exp. Cell Res. 316(18):3100-8
(2010); and
Relaix & Zammit, "Satellite Cells are Essential for Skeletal Muscle
Regeneration: The Cell on
the Edge Returns Centre Stage," Development 139(16):2845-56 (2012)).
[0007] Myofibers are divided into two types that display
different contractile and
metabolic properties: slow-twitch (Type I) and fast-twitch (Type II). Slow-
and fast-twitch
myofibers are defined according to their contraction speed, metabolism, and
type of myosin gene
expressed (Schiaffino & Reggiani, "Fiber Types in Mammalian Skeletal Muscles,-
Physiot Rev.
91(4):1447-1531 (2011) and Bassel-Duby & Olson, "Signaling Pathways in
Skeletal Muscle
Remodeling," Annu. Rev. Biochem. 75:19-37 (2006)). Slow-twitch myofibers are
rich in
mitochondria, preferentially utilize oxidative metabolism, and provide
resistance to fatigue at the
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 3 -
expense of speed of contraction. Fast-twitch myofibers more readily atrophy in
response to
nutrient deprivation, traumatic damage, advanced age-related loss
(sarcopenia), and cancer-
mediated cachexia, whereas slow-twitch myofibers are more resilient (Wang &
Pessin,
"Mechanisms for Fiber-Type Specificity of Skeletal Muscle Atrophy," Curr.
Op/n. Cl/n. Nutr.
Metab. Care 16(3):243-250 (2013); Tonkin et al., "SIRT1 Signaling as Potential
Modulator of
Skeletal Muscle Diseases," Curr. Op/n. PharmacoL 12(3):372-376 (2012); and
Arany, Z, "PGC-
1 Coactivators and Skeletal Muscle Adaptations in Health and Disease,- Cum
Op/n. Genet. Dev.
18(5):426-434 (2008)). Peroxisome proliferator-activated receptor gamma co-
activator 1-alpha
(PGCla or Ppargcl) is a major physiological regulator of mitochondrial
biogenesis and Type I
myofiber specification (Lin et al., -Transcriptional Co-Activator PGC-1 Alpha
Drives the
Formation of Slow-Twitch Muscle Fibres," Nature 418 (6899):797-801 (2002))
PGCla
stimulates mitochondrial biogenesis and oxidative metabolism through increased
expression of
nuclear respiratory factors (NRFs) such as NRF1 and 2 that stimulate
mitochondrial
biosynthesis, mitochondria transcription factor A (Tfam), and in addition to
mitochondrial
biosynthesis, also promote slow myofiber formation through increased
expression of Mef2
proteins (Lin et al., "Transcriptional Co-Activator PGC-1 Alpha Drives the
Formation of Slow-
Twitch Muscle Fibres," Nature 418 (6899):797-801 (2002); Lai et al., "Effect
of Chronic
Contractile Activity on mRNA Stability in Skeletal Muscle," Am. J. Physiol.
Cell. Physiol.
299(1):C155-163 (2010); Ekstrand et al., "Mitochondrial Transcription Factor A
Regulates
mtDNA Copy Number in Mammals," Hum. Mol. Genet. 13(9):935-944 (2004); and
Scarpulla,
RC, "Transcriptional Paradigms in Mammalian Mitochondrial Biogenesis and
Function,"
Physiol. Rev. 88(2): 611-638 (2008)). Importantly, PGC la protects muscle from
atrophy due to
disuse, certain myopathies, starvation, sarcopenia, cachexia, and other causes
(Wiggs, M. P.,
"Can Endurance Exercise Preconditioning Prevention Disuse Muscle Atrophy?,"
Front. Physiol.
6:63 (2015); Wing et al., "Proteolysis in Illness-Associated Skeletal Muscle
Atrophy: From
Pathways to Networks," Crit. Rev. Clin. Lab. Sci. 48(2).49-70 (2011); Bost &
Kaminski, "The
Metabolic Modulator PGC-lalpha in Cancer," Am. J. Cancer Res. 9(2):198-211
(2019); and Dos
Santos et al., "The Effect of Exercise on Skeletal Muscle Glucose Uptake in
type 2 Diabetes: An
Epigenetic Perspective," Metabolism 64(12):1619-1628 (2015)).
[0008] Skeletal muscle can remodel between slow- and fast-twitch myofibers
in response
to physiological stimuli, load bearing, atrophy, disease, and injury (Bassel-
Duby & Olson,
"Signaling Pathways in Skeletal Muscle Remodeling," Anntt. Rev. Biochem. 75:19-
37 (2006)),
involving transcriptional, metabolic, and post-transcriptional control
mechanisms (Schiaffino &
Reggiani, "Fiber Types in Mammalian Skeletal Muscles," Physiol. Rev.
91(4):1447-1531 (2011)
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 4 -
and Robinson & Dilworth, "Epigenetic Regulation of Adult Myogenesis," Curr.
Top Dev. Biol.
126:235-284 (2018)). The ability to selectively promote slow-twitch muscle has
been along-
standing goal, because endurance slow-twitch Type I myofibers provide greater
resistance to
muscle atrophy (Talbot & Mayes, "Skeletal Muscle Fiber Type: Using Insights
from Muscle
Developmental Biology to Dissect Targets for Susceptibility and Resistance to
Muscle Disease,"
Wiley Interdiscip. Rev. Dev. Biol. 5(4):518-534 (2016)), and could be an
effective therapy for
sarcopenia, Duchenne Muscular Dystrophy, cachexia, and other muscle wasting
diseases (Selsby
et al., "Rescue of Dystrophic Skeletal Muscle By PGC-lalpha Involves A Fast To
Slow Fiber
Type Shift In The Mdx Mouse," PLoS One 7(1):e30063 (2012); von Maltzahn et
al., "Wnt7a
Treatment Ameliorates Muscular Dystrophy," Proc. Natl. Acad. Sci. USA
109(50):20614-20619
(2012); and Ljubicic et al., "The Therapeutic Potential Of Skeletal Muscle
Plasticity In
Duchenne Muscular Dystrophy: Phenotypic Modifiers As Pharmacologic Targets,"
FASEB
28(2):548-568 (2014)).
[0009] Duchenne Muscular Dystrophy ("DMD") is one of the most
severe disorders of
muscle degeneration known as myopathies. Inherited in an X-linked recessive
manner, the
disorder is caused by mutations in the dystrophin gene, resulting in a near-
absence of expression
of the protein, which plays a key role in stabilization of muscle cell
membranes (Bonilla et al.,
"Duchenne Muscular Dystrophy: Deficiency of Dystrophin at the Muscle Cell
Surface," Cell
54(4):447-452 (1988) and Hoffman et al., "Dystrophin: The Protein Product of
the Duchenne
Muscular Dystrophy Locus," Cell 51(6):919-928 (1987)). Consequently, only
males with the
mutation are afflicted with DMD, which affects 1 in 3500 live births. There
are no cures for
DMD, and currently approved approaches involve limited use of corticosteroids
to dampen
inflammatory immune responses, a secondary exacerbating effect of muscle
atrophy. While the
inflammatory response is generally beneficial in normal muscle wound repair
and regeneration,
in DMD the response is no longer self-limiting due to the chronic nature of
muscle damage. This
results in exacerbation of necrosis of existing muscle and depletion of muscle
fibers (myofibers)
with replacement by connective and adipose tissue (Camwath & Shotton,
"Muscular Dystrophy
in the mdx Mouse: Histopathology of the Soleus and Extensor Digitorum Longus
Muscles,"
Neurol. Sci. 80(1):39-54 (1987); Tanabe et al., "Skeletal Muscle Pathology in
X Chromosome-
Linked Muscular Dystrophy (mdx) Mouse," Acta Neuropathol. 69(1-2):91-95
(1986); and
Fairclough et al., "Pharmacologically Targeting the Primary Defect and
Downstream Pathology
in Duchenne Muscular Dystrophy," CUM Gene Ther. 12(3):206-244 (2012)). While
steroids can
provide short-term increased muscle strength, long-term treatment is
ultimately ineffective and
can exacerbate disease. Steroids do not target the underlying cause of
disease. There is therefore
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 5 -
an urgent need for pharmacologic approaches that address the primary
underlying cause of
D1VID: loss of muscle fiber strength, loss of muscle stem cells, loss of
muscle regenerative
capacity, and attenuation of the exacerbating destructive effects of the
pathological immune
response on muscle health and integrity (Fairclough et al., "Pharmacologically
Targeting the
Primary Defect and Downstream Pathology in Duchenne Muscular Dystrophy," Curr.
Gene
Ther. 12(3):206-244 (2012)).
[0010] Dystrophin functions to assemble the dystroglycan complex
at the sarcolemma,
which connects the extracellular matrix to the cytoplasmic intermediate
filaments of the muscle
cell, providing physical strength and structural integrity to muscle fibers
which are readily
damaged in the absence of dystrophin (Yiu & Kornberg, "Duchenne Muscular
Dystrophy,"
1Veurol. India 56(3).236-247 (2008)). Dystrophin-defective myofibers are very
easily damaged
by minor stresses and micro-tears in DMD. This triggers continuous cycles of
muscle repair and
regeneration, depletes the muscle stem cell population, and provokes a
destructive immune
response that increases with age (Yiu & Kornberg, "Duchenne Muscular
Dystrophy," Neurol
India 56(3):236-247 (2008); Smythe et al., "Age Influences The Early Events of
Skeletal Muscle
Regeneration: Studies of Whole Muscle Grafts Transplanted Between Young (8
Weeks) and Old
(13-21 Months) Mice," Exp. Gerontol. 43(6):550-562 (2008); Heslop et al.,
"Evidence for a
Myogenic Stem Cell that is Exhausted in Dystrophic Muscle," I Cell Sci .
113(Pt 12):2299-
32208 (2000); Cros et al., "Muscle Hypertrophy in Duchenne Muscular Dystrophy.
A
Pathological and Morphometric Study," I. Neurol 236(1):43-47 (1989); and Abdel-
Salam et al.,
"Markers of Degeneration and Regeneration in Duchenne Muscular Dystrophy,"
Acta Myol.
28(3):94-100 (2009)). In this regard, it has been shown that the progressive
loss of muscle and
its regenerative capacity in MID results from exhaustion (inability to
activate) and depletion of
the muscle stem cell population (i.e., satellite cells) (Carlson 8z Conboy,
"Loss of Stem Cell
Regenerative Capacity Within Aged Niches," Aging Cell 6(3):371-82 (2007);
Kudryashova et
al., "Satellite Cell Senescence Underlies Myopathy in a Mouse Model of Limb-
girdle Muscular
Dystrophy 2H," 1 Clin. Invest. 122(5):1764-76 (2012); Gopinath & Rando, "Stem
Cell Review
Series: Aging of the Skeletal Muscle Stem Cell Niche," Aging Cell 7(4):590-8
(2008); Morgan &
Zammit, "Direct Effects of the Pathogenic Mutation on Satellite Cell Function
in Muscular
Dystrophy," Exp. Cell Res. 316(18):3100-8 (2010); and Collins et al., "Stem
Cell Function, Self-
renewal, and Behavioral Heterogeneity of Cells From the Adult Muscle Satellite
Cell Niche,"
Cell 122(2):289-301 (2005)). Typically, in normal muscle, the small pool of
satellite cells that
do not differentiate following injury repopulate muscle and re-enter the
quiescent state in their
niche, only to be activated again upon muscle damage to differentiate and fuse
into myofibers.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 6 -
The niche is defined both structurally and morphologically as sites where
satellite cells reside
adjacent to muscle fibers, in which quiescence is maintained by the structural
integrity of the
micro-environment, identified by laminin and other structural proteins
(Carlson & Conboy,
"Loss of Stem Cell Regenerative Capacity Within Aged Niches," Aging Cell
6(3):371-82 (2007);
Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell
Function,"
Development 142(9):1572-1581 (2015); Gopinath & Rando, "Stem Cell Review
Series: Aging of
the Skeletal Muscle Stem Cell Niche,- Aging Cell 7(4):590-8 (2008); Seale &
Rudnicki, "A New
Look at the Origin, Function, and "Stem-cell" Status of Muscle Satellite
Cells," Dev. Biol.
218(2):115-24 (2000); Briggs & Morgan, "Recent Progress in Satellite
Cell/N1yoblast
Engraftment -- Relevance for Therapy, FEBS J280(17).4281-93 (2013); Collins et
al., "Stem
Cell Function, Self-renewal, and Behavioral Heterogeneity of Cells From the
Adult Muscle
Satellite Cell Niche," Cell 122(2):289-301 (2005); and Murphy et al.,
"Satellite Cells,
Connective Tissue Fibroblasts and Their Interactions are Crucial for Muscle
Regeneration,"
Development 138(17):3625-37 (2011)). Studies suggest that it is the continuous
damage to
muscle in DMD that destroys this satellite cell niche, preventing these stem
cells from renewing
and ultimately leading to their functional exhaustion and cessation of muscle
repair.
[0011] The myogenesis program is controlled by genes that encode
myogenic regulatory
factors (MRFs) (Mok & Sweetman, "Many Routes to the Same Destination: Lessons
From
Skeletal Muscle Development," Reproduction 141(3):301-12 (2011)), which
orchestrate
differentiation of the activated satellite cell to become myoblasts, arrest
their proliferation, cause
them to differentiate, and fuse with multi-nucleated myofibers (Mok &
Sweetman, "Many
Routes to the Same Destination: Lessons From Skeletal Muscle Development,"
Reproduction
141(3):301-12 (2011)). Unique expression markers identify and stage skeletal
muscle
regeneration. PAX7 is a transcription factor expressed by quiescent and early
activated satellite
cells (Brack, A.S., "Pax7 is Back," Sk-elet. Muscle 4(1):24 (2014) and
Gunther, S., et al., "Myf5-
positive Satellite Cells Contribute to Pax7-dependent Long-term Maintenance of
Adult Muscle
Stem Cells," Cell Stem Cell 13(5):590-601 (2013)).
[0012] As satellite cells age, they lose their ability to
maintain a quiescent population
(Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell
Function,"
Development 142(9):1572-1581 (2015)), and become depleted or functionally
exhausted, a
primary cause of sarcopenia (muscle loss) with aging and in myopathic diseases
(Bernet et al.,
"p38 MAPK Signaling Underlies a Cell-autonomous Loss of Stem Cell Self-renewal
in Skeletal
Muscle of Aged Mice," Nat. Med. 20(3):265-71 (2014); Dumont et al., "Intrinsic
and Extrinsic
Mechanisms Regulating Satellite Cell Function," Development 142(9): 1572-
1581(2015);
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 7 -
Kudryashova et al., "Satellite Cell Senescence Underlies Myopathy in a Mouse
Model of Limb-
girdle Muscular Dystrophy 2H," I Cl/n. Invest. 122(5):1764-76 (2012); and
Silva et al.,
"Inhibition of Stat3 Activation Suppresses Caspase-3 and the Ubiquitin-
proteasome System,
Leading to Preservation of Muscle Mass in Cancer Cachexia,"1 Biol. Chem.
290(17):11177-87
(2015)).
[0013] Thus, there remains an urgent need for effective
therapeutic options that address
the primary underlying cause myopathic diseases (e.g., sarcopenia, Duchenne
muscular
dystrophy, traumatic muscle injury), which include, e.g., loss of muscle fiber
strength, loss of
muscle stem cells, loss of muscle regenerative capacity, and attenuation of
the exacerbating
destructive effects of the pathological immune response on muscle health and
integrity.
[0014] The present application is directed to overcoming these
and other deficiencies in
the art.
SUMMARY
[0015] One aspect of the present application relates to an adeno-
associated viral (AAV)
vector comprising a muscle cell-specific promoter and a nucleic acid molecule
encoding an AU-
rich mRNA binding factor 1 (AIJF1) protein or a functional fragment thereof,
where the nucleic
acid molecule is heterologous to and operatively coupled to the muscle cell-
specific promoter.
[0016] Another aspect of the present application relates to a
composition comprising an
adeno-associated viral (AAV) vector as described herein.
[0017] A further aspect of the present application relates to a
pharmaceutical composition
comprising an adeno-associated viral (AAV) vector described herein and a
pharmaceutically-
acceptable carrier.
[0018] Another aspect of the present application relates to a
method of promoting muscle
regeneration. This method involves contacting muscle cells with an adeno-
associated viral
(AAV) vector described herein or a composition described herein under
conditions effective to
express exogenous AUF1 in the muscle cells to increase muscle cell mass,
increase muscle cell
endurance, and/or reduce serum markers of muscle atrophy.
[0019] A further aspect of the present application relates to a
method of treating
degenerative skeletal muscle loss in a subject. This method involves selecting
a subject in need
of treatment for skeletal muscle loss and administering to the selected
subject an adeno-
associated viral (AAV) vector described herein or a composition described
herein under
conditions effective to cause skeletal muscle regeneration in the selected
subject
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-8-
100201 Yet a further aspect of the present application relates
to a method of preventing
traumatic muscle injury in a subject. This method involves selecting a subject
at risk of
traumatic muscle injury and administering to the selected subject an adeno-
associated viral
(AAV) vector described herein, a composition described herein, or a lentiviral
vector comprising
a muscle cell specific promoter and a nucleic acid molecule encoding an AU-
rich mRNA binding
factor 1 (AUF1) protein or a functional fragment thereof, where the nucleic
acid molecule is
heterologous to and operatively coupled to the muscle cell-specific promoter.
[0021] Still another aspect of the present application relates
to a method of treating
traumatic muscle injury in a subject. This method involves selecting a subject
having traumatic
muscle injury and administering to the selected subject an adeno-associated
viral (AAV) vector
described herein, a composition described herein, or alentiviral vector
comprising a muscle cell
specific promoter and a nucleic acid molecule encoding an AU-rich mRNA binding
factor 1
(AUF1) protein or a functional fragment thereof, where the nucleic acid
molecule is heterologous
to and operatively coupled to the muscle cell-specific promoter.
[0022] Prior studies have demonstrated that supplying ALTF1 to an animal
model in
which AUF1 had been experimentally deleted could result in new muscle
regeneration (see PCT
Publication No. WO 2016/196350, which is hereby incorporated by reference in
its entirety).
The present application is based, in part, on the surprising discovery that
AUF1 supplementation
by gene delivery restores muscle regeneration and function in degenerative
muscle diseases such
as Duchenne Muscular Dystrophy when there is no mutation or limitation of AUF1
expression.
This is particularly surprising in view of the fact that providing
supplementary AUF1 has no
impact on normal muscle and does not induce regeneration of normal muscle.
[0023] While not wishing to be bound by any theory as to how the
mechanism works, the
data presented herein demonstrate, inter alia, that in animal models of
degenerative muscle
diseases: (i) AUF1 gene transfer in Duchenne Muscular Dystrophy compensates
for loss of
mutated dystrophin by upregulating the dystrophin homolog utrophin, restoring
muscle function;
(ii) AUF1 gene delivery does not activate regeneration of normal muscle; (iii)
AUF1
supplementation by gene transfer accelerates regeneration of wounded muscle
and promotes
muscle function despite normal levels of AUF1 expression in wounded muscle;
and (iv) AUF1
supplementation restores muscle regeneration, muscle mass, and function in
aging muscle.
[0024] As described in the Examples, infra, AUF1 supplementation
by gene transfer
restores muscle regeneration, muscle mass, and muscle function in degenerative
muscle diseases
such as Duchenne Muscular Dystrophy in age-related loss of muscle mass and
function and in
traumatic muscle injury.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-9-
100251 The Examples disclosed herein demonstrate that loss of
expression of AUF1
occurs naturally during aging in skeletal muscle, and underlies age-related
muscle loss and
atrophy in sedentary animals, but can be reversed by AAV8-AUF1 skeletal muscle
gene transfer.
Mice receiving AUF1 gene therapy regain significant and durable skeletal
muscle mass and
exercise endurance; an increase in Pax7+ activated satellite cells and
myoblasts, a key indicator
of sustainable muscle regeneration; increased expression of PGCla through
stabilization of its
mRNA; increased mitochondrial biogenesis; and decreased markers of muscle
degeneration.
The Examples disclosed herein further demonstrate that muscle cell-specific
AUF1 gene therapy
restores skeletal muscle mass and function in a mouse model of Duchenne
muscular dystrophy.
AUF1 gene therapy (e.g., by lentivirus vector delivery directly to muscle or
systemic delivery of
AUF1 by AAV8 vector) is also shown to be effective to: (1) activate muscle
stem (satellite)
cells; (2) reduce expression of established biomarkers of muscle atrophy; (3)
accelerated the
regeneration of mature muscle fibers (myofibers); (4) enhanced expression of
muscle
regeneration factors; (5) strongly accelerate the regeneration of injured
muscle; (6) increase
regeneration of both major types of muscle (i.e., slow-twitch (Type I) or fast-
twitch (Type II)
fibers); and restore muscle mass, muscle strength, and create normal muscle.
[0026] AAV8-AUF1 gene therapy may provide a potential long-term
therapeutic
intervention for debilitating human muscle loss and atrophy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGs. IA-IL show AUF1 supplementation in skeletal muscle improves
exercise
endurance in 12 and 28 month old mice. FIG. 1A is a pair of photographic
images showing
representative staining of AAV GFP control and AAV AUF1/GFP positive myofibers
in TA
muscle 40 d post-administration. FIG. 1B is a graph showing quantification of
GFP positive
myofibers in TA muscle 40 d post-AAV administration. n=5 mice. FIG. 1C is a
pair of graphs
showing relative fold increased expression of aufl mRNA in gastrocnemius, TA,
EDL, and
soleus muscles 40 d post-AAV administration. n=8-9 mice. FIGs. 1D-H are graphs
showing
strength and exercise endurance in 3 and 12 month old mice and 40 d post-AAV
administration:
grip strength time (FIG. ID), maximum speed (FIG. 1E), work performance (FIG.
IF), time to
exhaustion (FIG. 1G), and distance to exhaustion (FIG. IH). n=5-9 mice. FIGs.
1I-L are graphs
showing strength and exercise endurance 6 months post-AAV administration in 18
month old
mice: maximum speed (FIG 1I), work performance (FIG 1J), time to exhaustion
(FIG. 1K), and
distance to exhaustion (FIG. IL). n=4 mice. Mean SEM from 5 or more
independent studies
*P<0.05, **P<0.01 by unpaired Mann-Whitney U test.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-10-
100281 FIGs. 2A-2J show AUF1 gene therapy induces muscle mass
along with an
increase in myofiber capacity. FIGs. 2A-B are graphs showing muscle weight
relative to total
body weight 40 d post-AAV administration for gastrocnemius and TA muscles,
respectively.
n=8-9 mice. FIGs. 2C-D are graphs showing frequency distribution of
gastrocnemius myofiber
CSA and mean area at 40 d post-AAV administration. n=6 mice/group. FIGs. 2E-F
are graphs
showing frequency distribution of TA muscle CSA and mean area at 40 d post-AAV
administration. n=5 mice. FIG. 2G is a pair of photographic images showing
representative
immunostain of slow myofiber (red) and nuclei (DAPI blue) in gastrocnemius
muscle at 40 d
post-therapy. Scale bar: 200 p.m. FIG. 2H is a pair of graphs showing slow
myofibers per field
and mean CSA of slow and fast myofibers in gastrocnemius muscle at 40 d post-
AAV
administration. FIG. 21 is a pair of photographic images showing
representative immunostain of
slow myofiber (red) and nuclei (blue) in soleus muscle 40 d after AAV AUF1-GFP
or AAV GFP
administration. Scale bar: 200 ium. FIG. 2J is a graph showing slow-twitch
soleus muscle
myofiber 40 d after AAV AUF1 or AAV GFP administration. Mean cross surface
area (CSA).
n=3 mice per group.
[0029] FIGs. 3A-3J show molecular markers of skeletal muscle
myogenesis in AAV8
AUF1-GFP gene transferred mice. FIGs. 3A-B are graphs showing relative myh7
mRNA levels
in gastrocnemius (FIG. 3A) and soleus (FIG. 3B) muscles normalized to
invariant nuclear
TATA-box binding protein (tbp) mRNA at 40 d post-gene transfer. FIGs. 3C-D are
graphs
showing relative fast myosin mRNA levels in gastrocnemius (FIG. 3C) and soleus
(FIG. 3D)
muscles normalized to tbp mRNA at 40 d gene transfer. FIG. 3E is a graph
showing expression
levels of mRNAs as indicated in gastrocnemius muscle at 40 d post-gene
transfer. FIG. 3F is a
graph showing DNA mitochondrial content in gastrocnemius muscle 40 d or 6
months post gene
transfer. FIG. 3G is a graph showing mil and nrf2 mRNA levels in gastrocnemius
muscle 40 d
after gene transfer. FIG. 3H is a graph showing nrfl and nrf2 mRNA levels in
the soleus muscle
40 d after gene transfer. FIG. 31 is a pair of graphs showing mitochondrial
DNA content in the
gastrocnemius muscle 40 d and 6 months after gene transfer. FIG. 3J is a graph
showing
mitochondrial DNA content in the soleus muscle 40 d after gene transfer. Mean
SEM from 3
or more independent studies. *P<0.05; **P<0.01 by unpaired Mann-Whitney U
test.
[0030] FIGs. 4A-4H show AUF1 is highly expressed in slow-twitch-enriched
soleus
muscle and stabilizes pgc la mRNA. FIG. 4A is a pair of graphs showing
relative anfl mRNA
expression in 3 and 12 month old WT mice in TA, gastrocnemius, EDL, and soleus
muscles.
n=5-7 mice. FIG. 4B is a representative immunoblot of AUF1 protein level and
quantification in
TA, gastrocnemius, EDL, and soleus muscle in 3 month old mice. FIG 4C is a
graph showing
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 11 -
relative myh7 mRNA expression in 3 month old mouse TA, gastrocnemius, EDL, and
soleus
muscles. FIG. 4D shows relative pgcla mRNA expression and protein levels in WT
C2C12
myoblasts and AUF1 KO myoblasts. FIG. 4E is a pair of graphs showing relative
pgcla mRNA
expression in TA, gastrocnemius, and EDL muscles 40 d post-treatment, and in
gastrocnemius at
6 months. FIG. 4F is a representative immunoblot of two AAV8-GFP control and
AAV8-AUF1
GFP animals (left) and quantification of AUF1 and PGC1ct in three animals per
group (right) at 6
months after treatment. FIG. 4G is a graph showing Pgcla mRNA
immunoprecipitation with
endogenous AUF1 protein in myoblasts 48 h after myotube induction of
differentiation in WT
C2C12 cells. n=3. FIG. 4H is a graph showing Pgcla mRNA decay rate in WT and
AUF1 KO
C2C12 cells. Mean SEM from 3 or more independent studies. Panels A and B:
****P<0.001
by Kruskall -Wallis test. All other panels *P<0.05, **P<0.01, ***P<0.001 by
unpaired Mann¨
Whitney U test.
100311 FIGs. 5A-5H show loss of AUF1 expression induces atrophy
of slow-twitch
myofibers. FIG. 5A is a graph showing body weight of WT and AUF1 KO mice at 3
months.
FIG. 5B shows TA, gastrocnemius, EDL, and soleus muscle mass in 3 month old WT
and AUF1
KO mice. Representative image of WT and AUF1 KO soleus muscles shown. FIG. 5C
shows
photographic images of a representative immunostain of slow (top) or fast
(bottom) myosin (red)
and laminin (green) in the soleus muscle from 3 month old WT and AUF1 KO mice.
Scale bar:
200 lam. FIGs. 5D-E are graphs showing slow-twitch myofibers per field of
percentage and
number, respectively, in 3 month old WT and AUF1 KO mice. FIGs. 5F-G are
graphs showing
fast-twitch myofibers per field of percentage and number, respectively, in 3
month old WT and
AUF1 KO mice. FIG. 5H is a graph showing mean soleus slow- and fast-twitch
myofiber CSA
in 3 month old WT and AUF1 KO mice, n=6-7 mice.
100321 FIGs. 6A-6I show AUF1 deletion induces slow- and fast-
twitch muscle atrophy at
6 months of age. FIG. 6A is a graph showing body weight of WT and AUF1 KO mice
at 6
months, n=5-6 mice. FIG. 6B shows TA, EDL, gastrocnemius, and soleus muscle
weight in 6
month old WT and AUF1 KO mice. FIG. 6C shows representative photographic
images of
excised muscles from 6 month old WT and AUF1 KO mice. FIG. 6D are photographic
images
showing representative immunostain of slow myosin (red) and laminin (green) in
soleus muscle
from 6 month old WT and AUF1 KO mice. Scale bar: 500 lam. FIG. 6E is a graph
showing
mean CSA of slow- and fast-twitch myofibers in soleus muscle of 6 month old WT
and AUF1
KO mice. FIG. 6F is a graph showing percentage of slow-twitch myofibers in 6
month old WT
and AUF1 KO mice in soleus muscle. FIG. 6G is a pair of photographic images
showing
representative staining of slow myosin (red) and laminin (green) in 6 month
old WT and AUF1
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 12 -
KO gastrocnemius muscle. Nuclei were stained by DAPI (blue), scale bar, 200
pm. FIG. 6H is
a graph showing the number of slow-twitch myofibers per field in gastrocnemius
muscle of 6
month old WT and AUF1 KO mice. n=4 mice per group. FIG. 61 is a graph showing
mean
gastrocnemius myofiber CSA of slow- and fast-twitch myofibers in 6 month old
WT and AUF1
KO mice. n=4 mice per group. Mean SEM from 4 or more independent studies.
*P<0.05,
**P<0.01 by unpaired Mann-Whitney U test.
[0033] FIGs. 7A-7G show AUF1 supplementation in skeletal muscle
improves exercise
endurance in 12-month old (middle-aged) and 18 month old mice. FIG. 7A is a
graph showing
relative expression of aufl mRNA in the TA, gastrocnemius, EDL, and soleus
muscles
normalized to invariant TBP mRNA at 3 and 12 months of age in WT mice. FIG. 7B
shows
representative immunoblot and quantification of AUF1 protein levels in the TA
muscle of WT
mice with age at 3, 12, and 18 months. GAPDH is a loading control. n=3 mice
per group per
lane. FIG. 7C are graphs showing TA, gastrocnemius, EDL muscle mass, and
soleus in 3, 12,
and 18 month old WT mice normalized to total body weight. FIG. 7D is an
immunoblot of
AUF1 andp-tubulin in TA muscle as in FIG. 7A, 40 d after AAV8 administration.
FIG. 7E is a
graph showing cuff] mRNA expression normalized to invariant gapdh mRNA in
various organs
of 12 month old mice, 40 d after AAV8 AUF1-GFP or AAV8 GFP control
administration. FIG.
7F shows representative Pax7 staining in TA muscle in 12 month old mice 40 d
after AAV8
AUF1-GFP or AAV8 GFP control vector administration. Scale bar, 100 pm.
Quantification of
Pax7 mRNA expression normalized to invariant TBP mRNA, in TA muscle of 12
month old
mice 40 d after AAV8 AUF1-GFP or AAV8 GFP control vector administration. n=8-9
per mice
group. FIG. 7G is a graph showing relative expression of Trim63 and Fbxo32
mRNAs in TA
muscle normalized to TBP mRNA 40 d after AAV administration. Mean SEM from 3
or more
independent studies. FIG. 7A-B: *P<0.05, **P<0.01 by Kruskall-Wallis test. All
other panels
*P<0.05, **P<0.01 by unpaired Mann-Whitney U test.
[0034] FIG. 8A-8B show AUF1 controls myosin and MEF2C
expression. The graphs of
FIG. 8A show relative expression of fast and slow myosin mRNAs normalized to
gapdh mRNA
in differentiating (48 h) WT myotubes and AUF1 KO C2C12 cells. n=5 mice per
group. FIG.
8B is a graph showing mef2c mRNA expression normalized to TBP mRNA in
gastrocnemius
muscle 6 months after AAV AUF1-GFP or AAV GFP injection. n=5 mice per group.
Mean
SEM from 5 or more independent studies. *P<0.05 by unpaired Mann-Whitney U
test.
[0035] FIGs. 9A-9G show AUF1 deletion induces slow-twitch muscle
atrophy at a young
age. FIG. 9A shows representative photographic images of TA, EDL, and
gastrocnemius
muscles in 3 month old WT and AUF1 KO mice. FIG. 9B shows representative
immunostain
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 13 -
images of slow and fast myosin (red) myofibers in the soleus of WT and AUF1 KO
mice. DAPI
stain (blue) of nuclei, laminin (green) stain of extracellular matrix. Scale
bar: 500 lam. FIG. 9C
shows photographic images of representative stains of slow myosin (red) and
laminin (green) in
3 month old WT and AUF1 KO gastrocnemius muscle (scale bar, 200 pm). FIGs. 9D-
E are
graphs showing percentage and number, respectively, of slow-twitch myofibers
per field in
gastrocnemius muscle of 3 month old WT and AUF1 KO mice. FIG. 9F is a graph
showing
mean gastrocnemius muscle area of slow- and fast-twitch myofibers in 3 month
old WT and
AUF1 KO mice. n=4 mice per group. FIG. 9G shows levels of PGC la, AUF1, and
control
GAPDH protein in gastrocnemius and soleus muscles of 3 month old WT and AUF1
KO mice.
Each lane corresponds to one mouse. Lower band in AUF1 gastrocnemius muscle
lanes is a
non-specific protein. Mean SEM from 3 or more independent studies. *13<0.05
by unpaired
Mann-Whitney U test. ns, (not significant).
100361 FIGs. 10A-10C illustrate the development of AAV8
expression vectors. FIG.
10A is a schematic illustration of the development of AAV8 expression vectors.
The cDNA of
the murine p40AuFI cDNA was cloned into an AAV8 vector under the tMCK promoter
(AAV8-
tMCK-AUF1-1RES-eGFP) (Vector Biolabs). The tMCK promoter was generated by the
addition of a triple tandem of 2RS5 enhancer sequences (3-Ebox) ligated to the
truncated
regulation region of the MCK (muscle creatine kinase) promoter, which induces
high muscle
specificity (Blankinship et al., "Efficient Transduction of Skeletal Muscle
Using Vectors Based
on Adeno-associated Virus Serotype 6," Mol. Ther. 10(4):671-8 (2004), which is
hereby
incorporated by reference in its entirety). AAV8 vectors express AUF1 and GFP
(AUF1-GFP,
with GFP translated from the same mRNA by the HCV IRES), or as a control only
GFP.
Expression of both genes is controlled by the creatine kinase tMCK promoter
that is selectively
active in skeletal muscle cells. The AAV8-tMCK-1RES-eGFP construct was used as
a control
vector. FIG. 10B shows the amino acid sequence of the encoded p40AuF1 isoform
(SEQ ID
NO:27) expressed in transduced cells by the AAV8 vector in FIG. 10A. FIG. 10C
shows the
nucleotide sequence (SEQ ID NO:28) of the coding region of the p40Aun isoform.
100371 FIGs. 11A-11B show AAV8 transduction frequency in mdx
mice. AAV8 AUF1-
GFP and AAV8 GFP control vector-treated mdx mice displayed similar vector
transduction and
retention rates, shown by tibialis anterior (TA) muscle GFP staining. FIG. 11A
shows
representative photographic images of GFP immunofluorescence staining of TA
muscle (green)
to highlight AAV8 transduction efficiency and laminin-a2 staining (red) to
highlight muscle fiber
architecture and integrity. FIG. 11B is a graph showing quantification of 3
animals per condition
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 14 -
for AAV8 GFP transduction in TA muscle. There is no statistical difference
(ns) in transduction
efficiency between control AAV8 GFP and treatment AAV8 AUF1 GFP groups.
[0038] FIGs. 12A-12F show AUF1 gene therapy enhances muscle mass
and endurance in
mcbc mice. One month old C57BL/10ScSn male DMD mice (herein mdx mice, JACS)
were
administered 2x1011 genome copies of AAV8 AUF1-GFP or control AAV8 GFP as a
single
retro-orbital injection of 50 ul containing 2.5x1011 AAV particles. Two months
following
AAV8 administration, mdx mice transduced with AAV8 AUF1-GFP or AAV8 GFP as a
control were tested by standard procedures for exercise performance (see
Examples, infra).
FIG. 12A is a graph showing Inc& control mice receiving only AAV8 GFP at three
months old
had an average body weight of 29 gm compared to 30 gm for wild type (WT) C57BL
mice.
In contrast, when compared to control AAV8 GFP treated mcbc mice, AAV8 AUF1-
GFP
supplemented mcbc mice had an average body weight of 31 gm, a significant
increase
compared to control /H&c mice. FIG. 12B is a graph showing when normalized to
body weight
and at 2 months post-gene therapy transduction, AAV8 AUF1-GFP treated mdx mice
demonstrated a 10% increase in tibialis anterior (TA) muscle mass, an 11%
increase in
extensor digitorum longus (EDL) muscle mass, and an 8.5% increase in
gastrocnemius muscle
mass. There was no difference in soleus muscle mass. Compared to control AAV8
GFP
treated 111dX mice, AUF1 supplemented inclx mice showed a ¨40% improvement in
grid hanging
time (FIG. 12C), a measure of limb-girdle skeletal muscle strength and
endurance. When tested
by treadmill, AAV AUF1-GFP mcbc mice displayed 16% higher maximum speed (FIG.
12D), a
35% greater time to exhaustion (FIG 12E), and a 37% increased distance to
exhaustion (FIG.
12F). These data demonstrate a substantial and statistically significant
increase in exercise
performance and endurance in nictx mice as a result of AUF1 gene transfer. All
results are
expressed as the mean+ SEM. Two group comparisons were analyzed by the
unpaired Mann-
Whitney test. *, P<0.05.
[0039] FIGs. 13A-13D show AUF1 gene therapy does not increase WT
muscle mass
or endurance. Normal WT C57BL mice, the same background as mdx mice, were
administered at 1 month of age AAV8 GFP control or AAV8 AUF1-GFP at 2x1011
genome
copies by retro-orbital injection as described in FIGs. 12A-12F. Mice were
analyzed at 3 months
post-gene transfer. These data are in contrast to the significant increase in
muscle mass and
exercise endurance found in mcbc mice. Rather, WT mice administered with AAV8
AUF1-GFP
compared to control AAV8 GFP mice of the same genetic background, show no
statistically
significant increase in body weight (FIG. 13A), treadmill time to exhaustion
(FIG. 13B),
maximum speed (FIG. 13C), and distance to exhaustion (FIG. 13D). All results
are expressed
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 15 -
as the mean SEM. Two group comparisons were analyzed by the unpaired Mann-
Whitney test.
No results were found to be significantly different at P<0.05.
[0040] FIG. 14 shows AAV8 AUF1 gene therapy reduces serum
creatine kinase levels
in mdx mice. mdx mice at 1 month old were administered AAV8 AUF1-GFP or
control
AAV8 GFP as described in FIGs. 12A-12F. At 3 months, mice were tested for
levels of serum
creatine kinase (CK) activity, a measure of sarcolemma leakiness and muscle
atrophy. Top:
Raw data showing serum CD activity results for WT control, mdx mice treated
with AAV8
GFP vector alone, and mdx mice treated with AAV8 AUF1 GFP. Bolton':
Quantification of
three replicate studies of 3 mice each. Control AAV8 GFP "mix mice displayed
high levels of
serum CK activity, mt.& mice that received AAV8 AUF1-GFP gene therapy were
reduced in
serum CK activity by more than 4-fold, a highly significant reduction. WT
C57BL mice had
no detectable level of serum CK activity. ND, not detected. All results are
expressed as the
mean SEM. Two group comparisons were analyzed by the unpaired Mann-Whitney
test. ",
P<0.01, *** P<0.001.
[0041] FIGs. 15A-15B show AAV8 AUF1 gene therapy reduces muscle necrosis
and
fibrosis in mdx mouse diaphragm. mcbc mice at 1 month old were administered
AAV8 AUF1-
GFP or control AAV8 GFP as described in FIGs. 12A-12F. At 3 months, diaphragms
were
reduced from AAV8 GFP control and AAV8 AUF1-GFP mice, embedded FFPE and
stained
with H&E (FIG. 15A). The percent degenerative diaphragm muscle was scored and
found to
be reduced by 74% by AUF1 gene transfer. WT C57BL mouse diaphragm served as a
control. Diaphragm muscle from mdx mice was stained with Masson Trichome to
quantify
muscle fibrosis (FIG. 15B). Shown are representative muscle sections. AUF1
gene transfer
reduced fibrosis by 2-fold compared to control AAV8 GFP treated animals. All
results are
expressed as the mean SEM. Two group comparisons were analyzed by the
unpaired Mann-
Whitney test. **, P<0.01. Otherwise analyzed by Fisher Exact test as
indicated.
[0042] FIGs. 16A-16B show AAV8 AUF1 gene therapy reduces muscle
immune cell
invasion. mdx mice at 1 month old were administered AAV8 AUF1-GFP or control
AAV8
GFP as described in FIGs. 12A-12F. At 3 months, diaphragms were resected from
AAV8
GFP control and AAV8 AUF1-GFP treated mice, embedded in FFPE, and stained with
an
antibody to the macrophage biomarker CD68 coupled with the red fluorescence
marker Alexa
Fluor 555. Representative images show strong reduction in macrophage CD68
staining in
AAV8 AUF1-GFP treated animals compared to AAV8 GFP controls (FIG. 16A).
Quantification of 5 fields per specimen from 3 mice per group for CD68
staining (FIG. 16B).
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 16 -
All results are expressed as the mean SEM. Two group comparisons were
analyzed by the
unpaired Mann-Whitney test *, P<0.05.
[0043] FIGs. 17A-17E show AAV8 AUF1 gene therapy suppresses
expression of
embryonic myosin heavy chain (eMHC) in mdx mice. eMHC is a clinical marker of
muscle
degeneration in DMD. mdx mice at 1 month old were administered AAV8 AUF1-GFP
or
control AAV8 GFP as described in FIGs. 12A-12F. At 3 months, diaphragm muscle
was
removed, fixed in FFPE, and stained with antibodies to eMHC (green), nuclei
(DAPI, blue),
and laminin (red). Immunotluorescence was carried out and representative
images shown
compared to WT C57BL6 mice (FIG. 17A). AAV8 AUF1-GFP gene transfer strongly
reduced eHMC expression in diaphragm. High magnification of diaphragm stained
as in FIG.
17A showing strong reduction in eMHC expression by AUF1 gene transfer (FIG.
17B).
Quantification of eMHC staining in myofibers, showing a 75% reduction in eMHC
expression
by AUF1 gene transfer (FIG. 17C). The percent of centro-nuclei per
myofiber/field was
quantified, a measure of normal muscle fiber maturation (FIG. 17D). AUF1 gene
transfer
reduced the percentage of centro-nuclei by 52% compared to AAV8 GFP controls.
Myofiber
cross sectional area (CSA) was quantified (FIG. 17E). AUF1 gene transfer
strongly increased
the CSA of the larger myofibers, indicative of mature regenerative muscle. All
results are
expressed as the mean SEM. Two group comparisons were analyzed by the
unpaired Mann-
Whitney test. Multiple group comparisons were performed using one-way analysis
of variance
(ANOVA). The non-parametric Kruskal¨Wallis test followed by the Dunn's
comparison of
pairs was used to analyze groups when suitable. *, P<0.05; *** P<0.001.
[0044] FIGs. 18A-18C show AAV8 AUF1 gene transfer increases
expression of
endogenous utrophin-A in mdx mice. mdx mice at 1 month old were administered
AAV8
AUF1-GFP or control AAV8 GFP as described in FIGs. 12A-12F. The gastrocnemius
muscle
was removed at 3 months, fixed in FFPE, and stained with DAPI (blue for
nuclei, antibodies
to utrophin (red) and laminin (green) (FIG. 18A). Representative images from 3
mice for
each group are shown. AUF1 gene therapy strongly increased expression of
utrophin and
showed evidence for normalization of myofiber integrity (laminin staining).
Immunoblot
analysis for utrophin, AUF1, and GAPDH (invariant control) proteins was
conducted on the
gastrocnemius muscle of 3 AAV8 GFP and 3 AAV8 AUF1-GFP mdx mice at 3 months
(FIG.
18B). Gastrocnemius utrophin protein levels were increased by an average of 20-
fold in
animals receiving AUF1 gene therapy. AUF1 protein levels were increased an
average of 3-4
fold. Utrophin mRNA levels were quantified by qRT-PCR and normalized to
invariant TBP
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 17 -
mRNA (FIG. 18C). There was no statistically significant difference between
samples n=3
animals for each condition_
[0045] FIGs. 19A-19C show AAV8 AUF1 gene transfer increases
expression of
satellite cell activation gene Pax7, key muscle regeneration genes pgc I a and
mef2c, slow
twitch determination genes and mitochondrial DNA content in mdx mice. mdx mice
at 1
month old were administered AAV8 AUF1-GFP or control AAV8 GFP as described in
FIGs.
12A-12F. The gastrocnemius muscle was removed at 3 months, mRNA extracted and
quantified by qRT-PCR relative to invariant tbp mRNA. AUF1 gene therapy
increased
expression of pgc I a, mef2c, and Pax7 mRNAs in the gastrocnemius of mdx mice
relative to
controls receiving vector alone (FIG. 19A). Wild type non-mdx animals (WT)
served as a
control for normal muscle levels in age-matched animals. AAV8 AUF1 gene
therapy restored
near WT levels or exceeded WT levels of gene expression. AUF1 gene therapy
increased
expression of slow-twitch lineage determination myosin mRNAs in the
gastrocnemius muscle
in mdx animals relative to controls receiving vector alone (FIG. 19B) AAV8
AUF1 gene
therapy restored near WT levels or exceeded WT levels of gene expression. AUF1
gene
therapy increased expression of mitochondrial DNA in the gastrocnemius muscle
of mdx
mice, consistent with increased slow-twitch muscle mass (FIG 19C). All results
are expressed
as the mean+ SEM. Two group comparisons were analyzed by the unpaired Mann-
Whitney test.
*, P<0.05; **, P<0.01; *** P<0.001.
[0046] FIG. 20 shows genome-wide transcriptomic and translatomic studies
demonstrate
AUF1 activation of C2C12 myoblast muscle fiber development. Proliferating
C2C12 mouse
cardiac myoblasts were transduced with lentivirus control vectors or
lentivirus vectors
expressing p45 AUF1, and induced to differentiate into myotubes by culturing
in differentiation
medium as described in the Examples infra. Proliferating myoblasts were used
because they are
activated in p38 MAPK and other signaling pathways that promote myogenesis,
which is
representative of the activated state and population of muscle cells following
muscle damage
from wounding, or the state of muscle in myogenic diseases, such as chronic
regenerative
attempts that occur in Duchene Muscular Dystrophy (DMD). Overview of the
experimental
approach. At 48 h, when myotubes begin to form, polyribosomes were separated
by sucrose
sedimentation corresponding to poorly translated (2 & 3 ribosome) fraction and
well translated
(>4 polysome) fractions, total mRNA and mRNA in polyribosome fractions were
independently
purified (polyA+ fraction devoid of rRNA), bacterial libraries were generated
and subjected to
deep sequencing using RNAseq, in two independent studies. Genome-wide mRNA
abundance
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 18 -
used 10g2 ratios of translated/total mRNA. Procedures and bioinformatic
pipeline used for
analysis are described in the Examples infra
[0047] FIGs. 21A-21B show AUF1 supplementation stimulates
expression of major
muscle development pathways and decreases expression of inflammatory cytokine,
inflammation, cell proliferation, cell death, and anti-muscle regeneration
pathways. Data from
FIG. 20 genome-wide mRNA expression and translation analysis. Major
upregulated pathways
at the levels of transcription, translation, or both with AUF1 supplementation
in C2C12
myoblasts (FIG. 21A). Analyzed by KEGG. Major downregulated pathways at the
levels of
transcription, translation, or both with AUF1 supplementation in C2C12
myoblasts (FIG. 21B).
Analyzed by KEGG.
[0048] FIGs. 22A-22B show AUF1 supplementation of C2C12
myoblasts upregulates
pathways for major biological processes and molecular functions in muscle
development and
regeneration. Data from FIG. 20 genome-wide mRNA expression and translation
analysis.
Major upregulated biological processes at the levels of transcription,
translation, or both with
AUF1 supplementation in C2C12 myoblasts (FIG. 22A). Analyzed by KEGG. Major
upregulated molecular functions at the levels of transcription, translation,
or both with AUF1
supplementation in C2C12 myoblasts (FIG. 22B). Analyzed by KEGG.
[0049] FIGs. 23A-23B show AUF1 supplementation of C2C12
myoblasts decreases
muscle inflammation, inflammatory cytokine, and signaling pathways that oppose
muscle
regeneration. Data from FIG. 20 genome-wide mRNA expression and translation
analysis.
Major downregulated biological processes at the levels of transcription,
translation, or both with
AUF1 supplementation in C2C12 myoblasts (FIG. 23A). Analyzed by KEGG. Major
downregulated molecular functions at the levels of transcription, translation,
or both with AUF1
supplementation in C2C12 myoblasts (FIG. 23B). Analyzed by KEGG.
[0050] FIG. 24 shows AUF1 supplementation of C2C12 myoblasts decreases
expression
of muscle genes associated with development of fibrosis. Data from FIG. 20
genome-wide
mRNA expression and translation analysis. Major downregulated pathways and
functions at the
levels of transcription, translation, or both with AUF1 supplementation in
C2C12 myoblasts.
Analyzed by KEGG.
[0051] FIGs. 25A-25D show lentivirus transduction of injured TA muscle with
p45
AUF1 in mice activates satellite cells and reduces biomarkers of muscle
atrophy. A lentivirus
vector was developed expressing cDNA for p45 AUF1 under control of the CMV
promoter
(Abbadi et al., "Muscle Development and Regeneration Controlled by AUF1-
mediated Stage-
specific Degradation of Fate-determining Checkpoint mRNAs," Proc. Nat'l. Acad.
Sci. USA
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 19 -
116:11285-90 (2019), which is hereby incorporated by reference in its
entirety). Three month
old male mice were administered an intramuscular injection of 50 ill of
filtered 1.2% BaC12 in
sterile saline with control lentivirus vector or with lentivirus AUF1 vector
(1x108 genome
copies) (total volume 100 111) into the left Tibialis Anterior (TA) muscle
(FIG. 25A). The right
TA muscle remained uninjured as a control. Mice were sacrificed at 7 days post-
injection. TA
muscles were excised, weighed, and normalized to mouse body weight in grams.
TA injury
reduced TA weight by 27% which was restored to near-uninjured levels by
concurrent AUF1
gene therapy. In FIG. 25B, immunoblot analysis of AUF1 normalized to invariant
GAPDH
protein for TA muscle at 7 days post-lentivirus p45 AUF1 administration as in
FIG. 25A.
Shown is a representative uninjured, two injured, and injured TA muscles with
concurrent p45
AUF1 gene therapy from independent animals. Lentivirus p45 AUF1 gene transfer
strongly
increased levels of the p45 AUF1 isoform but not p42 AUF1 and p40 AUF1 that
were not encoded (p37
AUF1 =
is undetectable). In FIG. 25C, TA muscles as in FIG. 25A were probed by qRT-
PCR for
Pax7 mRNA levels, a biomarker of muscle satellite (stem) cell activation, and
normalized to
invariant TATA-box binding protein (TBP) mRNA. AUF1 gene therapy increased
Pax7
expression by >3-fold. In FIG. 25D, TA muscles as in FIG. 25A were probed by
qRT-PCR for
expression of muscle atrophy biomarker genes TRIM63 and Fbxo32, normalized to
TBP mRNA.
TA muscle injury strongly induced expression of TRIM63 and Fbxo32 mRNA, which
were
downregulated to uninjured TA muscle levels by p45 Au" gene therapy,
indicating strong
cessation of muscle injury due to AUF1 intramuscular administration. No
statistical difference
(ns). All results are expressed as the mean + SEM with at least three
independent trials of 3 or
more animals per condition. Two group comparisons were analyzed by the
unpaired Mann-
Whitney test. *, P<0.05; **, P<0.01; ***, P<0.001.
[0052] FIGs. 26A-26D show p45 AUF1 lentivirus transduction
enhances expression of
muscle regeneration factors (MRFs) following TA muscle injury. Three month old
male mice
were injured in the TA muscle with BaC12 and administered with an
intramuscular injection of
control lentivirus vector or lentivirus AUF1 vector (see FIGs. 25A-D). Mice
were sacrificed at 7
days post-injection. TA muscles were probed by qRT-PCR for identified mRNAs
normalized to
invariant TBP mRNA. In FIG. 26A, myogenin and MyoD mRNA levels, biomarkers of
myoblast activation, differentiation, and muscle regeneration (Zammit,
"Function of the
Myogenic Regulatory Factors Myf5, MyoD, Myogenin and MRF4 in Skeletal Muscle,
Satellite
Cells and Regenerative Myogenesis," Semin. Cell. Dev. Biol. 72:19-32 (2017),
which is hereby
incorporated by reference in its entirety), were increased ¨2-fold by AUF1
gene therapy relative
to injured control vector specimens. In FIG. 25B, myh8 mRNA, an embryonic
myosin only
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 20 -
expressed in adult muscle during muscle regeneration and a marker of co-
expression of utrophin
(Guiraud et al., "Embryonic Myosin is a Regeneration Marker to Monitor
Utrophin-based
Therapies for DMD," HUM. Mol. Genet. 28:307-19 (2019), which is hereby
incorporated by
reference in its entirety), was increased in expression by 5-fold in injured
muscle with AUF1
gene therapy relative to injured control vector specimens. In FIG. 26C, myh7
mRNA, a myosin
that specifies slow-twitch muscle (Zammit, "Function of the Myogenic
Regulatory Factors
Myf5, MyoD, Myogenin and MRF4 in Skeletal Muscle, Satellite Cells and
Regenerative
Myogenesis," Seinin. Cell. Dev. Biol. 72:19-32 (2017), which is hereby
incorporated by
reference in its entirety), was increased in expression by -2-fold in injured
muscle with AUF1
gene therapy relative to injured control vector specimens. In FIG. 26D, myh4
mRNA, a myosin
that specifies fast-twitch muscle (Zammit, -Function of the Myogenic
Regulatory Factors Myf5,
MyoD, Myogenin and MRF4 in Skeletal Muscle, Satellite Cells and Regenerative
Myogenesis,"
Semin. Cell. Dev. Biol. 72:19-32 (2017), which is hereby incorporated by
reference in its
entirety), was increased in expression by -2-fold in injured muscle with AUF1
gene therapy
relative to injured control vector specimens. All results are expressed as the
mean SEM with at
least three independent trials of 3 or more animals per condition. Two group
comparisons were
analyzed by the unpaired Mann-Whitney test. *, P<0.05; **, P<0.01.
[00531 FIGs. 27A-27D show p45 AUF1 lentivirus gene therapy
promotes rapid
regeneration of injured muscle. Three month old male mice were injured in the
TA muscle with
BaC12, and administered with an intramuscular injection of control lentivirus
vector or lentivirus
AUF1 vector, as in FIGs. 25A-25D. Mice were sacrificed at 3 days and 7 days
post-injury. FIG.
27A shows photographic images of muscle fibers provide evidence for
accelerated but normal
muscle regeneration of myofibers in animals administered lentiviral AUF1 gene
therapy. TA
muscle in OCT was sectioned and stained for immunofluorescence microscopy
analysis for
Laminin alpha 2 (red), Nuclei are stained with DAN (blue). Note the disrupted
myofiber
architecture and high level of central nuclei in the injured TA muscle treated
with vector alone
compared to the injured TA muscle administered lentiviral AUF1 gene therapy,
consistent with
accelerated muscle regeneration and mature myofibers. Scale bar, 2001.1m. FIG.
27B is a graph
showing the percent muscle loss (atrophy) or gain (increase in mass)
determined for the injured
TA muscle compared to uninjured control or injured muscle receiving control
lentivirus vector or
lentivirus p45 AUF1, measured at sacrifice at 3 days and 7 days post-injury.
Injured TA muscle
receiving sham gene therapy sustained a 20% loss in mass by day 3 following
injury, which only
very slightly improved by day 7. In contrast, injured TA muscle receiving AUF1
gene therapy
showed a trend to less atrophy by day 3, which was almost fully recovered by
day 7,
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 21 -
demonstrating near normal mass. FIG 27C is a graph showing high levels of
myotube central
nuclei are a marker of immature myofiber development (Yin et al., "Satellite
Cells and the
Muscle Stem Cell Niche," Physiol. Rev. 93:23-67 (2013) and Schiaffino &
Reggiani, "Fiber
Types in Mammalian Skeletal Muscles," Physiol. Rev. 91:1447-531 (2011), which
are hereby
incorporated by reference in their entirety). TA muscle analyzed at day 7 post-
injury
administered p45 AUF1 gene therapy were reduced by half in the percent of
myofibers with
central nuclei compared to vector only control injured muscle. This is
consistent with
accelerated muscle regeneration provided by AUF1 gene transfer. FIG. 27D is a
graph showing
a wider cross-sectional area of myofibers (cross-sectional area, CSA) with low
numbers of
central nuclei are indicative of mature myofiber development (Yin et al.,
"Satellite Cells and the
Muscle Stem Cell Niche," Physiol. Rev. 93:23-67 (2013) and Schiaffino &
Reggiani, "Fiber
Types in Mammalian Skeletal Muscles," Physiol. Rev. 91:1447-531 (2011), which
are hereby
incorporated by reference in their entirety). AUF1 gene transfer in injured TA
muscle produced
a striking increase in CSA with reduced central nuclei per myofiber,
consistent with generation
of mature myofibers. All results are expressed as the mean+ SEM with at least
three
independent trials of 3 or more animals per condition. Two group comparisons
were analyzed by
the unpaired Mann-Whitney test. *, P<0.05; ", P<0.01, ***, P<0.001.
[00541 FIGs. 28A-28F show AUF1 is essential to promote repair of
injured muscle, and
can provide injury protection benefit when delivered by AAV8 gene transfer.
FIG. 28A is a
schematic illustration of an AUF1 conditional knockout mouse developed as
party of the
technology described herein. Shown is a schematic of the exon 3 LoxP site
insertions in the
AUF1 gene. Lox sites were cloned to flank exon 3 of AUF1, which is maintained
in all 4 AUF1
isoforms and contains the RNA binding domain. AUFIFloVFlox mice were derived,
syblings
mated to homogeneic purity generated, then mated with a Pax7cre ERT2 (B6;129-
Pax7tm21(cre/ERT2)Fan/J mouse) (Jackson Labs). This provides cre recombinase
induction by
tamoxifen administration only in PAX7 expressing muscle satellite and myoblast
cells. FIG.
28B is a graph showing results of three month old mice induced for cre
expression with 5 daily
i.p. injections of tamoxifen (3 mg/kg). There was no change in body weight of
cre-induced mice.
FIG. 28C is a graph showing weight of non-injured skeletal muscles in mice
were not
significantly different in uninduced and tamoxifen induced cre mice. FIG. 28D
shows
tamoxifen induction of cre for 3 months specifically deletes the aufl gene in
skeletal muscle and
abolishes skeletal muscle AUF1 protein expression. A representative immunoblot
is shown for
AUF1 levels in TA skeletal muscle and kidney, normalized to invariant GAPDH in
control
AUF1Hox/Flox and AUF1Hox/Hox x )7C"ERT2 mice after 5 days of cre induction and
analyzed
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 22 -
at day 7. There is no evidence for expression of AUF1 after Pax7-specific cre
induction in
muscle, whereas abundant AUF1 is present in kidney. FIG 28E is a graph showing
one month
old AUF1F1'/Fl" x PAX7c"ERT2 mice were either sham injected or injected with
tamoxifen for
days as above, then maintained on a diet that included oral tamoxifen for 5
months daily at 500
5 mg/kg (Envigo). Wild type (WT) BL6 mice and AUF1Flox/Flox x pAx-,cre
ERT2 mice were either
not induced for cre-expression (labeled AUF 1iFfl/Fax7) or induced for 5
months and deleted in the
AUF1 gene (labeled AAUF lfuflIF'). One set of AAUF 1 filfl/Pax7 mice induced
for cre expression
for 5 months were also administered at 1 month of age with 2.0x10" AAV8 AUF1
particles
(2x10" genome copies) by single retro-orbital injection of 50 pl. All mice
were then injured
by 1.2% BaC12 injection in the TA muscle, as described in FIGs. 25A-D. AUF1 is
controlled by
the creatine kinase tMCK promoter that is selectively active in skeletal
muscle cells. TA muscle
was excised at 7 days post-BaC12 injection and the percent of muscle atrophy
determined by
weight. TA muscle of AUF1Fl0x/Fl0x x p crc
ERT2 mice expressing AUF1 and WT mice
expressing AUF1 (not induced for cre) showed 16-18% atrophy that was not
statistically
different. In contrast, deletion of the AUF1 gene caused strongly increased
atrophy of the TA
muscle, doubling atrophy levels to 35%. However, animals deleted for the AUF1
gene but
prophylactically administered AAV8 AUF1 gene therapy demonstrated dramatically
reduced
levels of TA muscle atrophy, averaging ¨3%. FIG. 28F is a graph showing AUF1
control and
cre-induced skeletal muscle AUF1 deleted mice were tested at 5 months for grip
strength, a
measure of limb-girdle skeletal muscle strength and endurance. AUF1 deleted
mice showed a
¨50% reduction in grip strength. Collectively, these data demonstrate that
AUF1 is essential for
maintenance of muscle strength and muscle regeneration following injury, and
that AUF1 gene
therapy provides a remarkable ability to promote muscle regeneration and
protect muscle from
extensive damage despite traumatic injury. All results are expressed as the
mean SEM with at
least three independent trials of 3 or more animals per condition. Two group
comparisons were
analyzed by the unpaired Mann-Whitney test. *, P<0.05.
DETAILED DESCRIPTION
[0055] One aspect of the present application relates to an adeno-
associated viral (AAV)
vector, comprising a muscle cell-specific promoter and a nucleic acid molecule
encoding an AU-
rich mRNA binding factor 1 (AUF1) protein or a functional fragment thereof,
where the nucleic
acid molecule is heterologous to and operatively coupled to the muscle cell-
specific promoter.
[0056] The term "vector" is used interchangeably with
"expression vector." The term
vector" may refer to viral or non-viral, prokaryotic or eukaryotic, DNA or RNA
sequences that
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/ITS2021/013955
- 23 -
are capable of being transfected into a cell, referred to as "host cell," so
that all or a part of the
sequences are transcribed. It is not necessary for the transcript to be
expressed. It is also not
necessary for a vector to comprise a transgene having a coding sequence.
Vectors are frequently
assembled as composites of elements derived from different viral, bacterial,
or mammalian
genes. Vectors contain various coding and non-coding sequences, such as
sequences coding for
selectable markers, sequences that facilitate their propagation in bacteria,
or one or more
transcription units that are expressed only in certain cell types. For
example, mammalian
expression vectors often contain both prokaryotic sequences that facilitate
the propagation of the
vector in bacteria and one or more eukaryotic transcription units that are
expressed only in
eukaryotic cells. It will be appreciated by those skilled in the art that the
design of the
expression vector can depend on such factors as the choice of the host cell to
be transformed, the
level of expression of protein desired, etc.
[0057] The term "promoter" is used interchangeably with
"promoter element" and
"promoter sequence." Likewise, the term "enhancer" is used interchangeably
with "enhancer
element" and -enhancer sequence." The term "promoter" refers to a minimal
sequence of a
transgene that is sufficient to initiate transcription of a coding sequence of
the transgene.
Promoters may be constitutive or inducible. A constitutive promoter is
considered to be a strong
promoter if it drives expression of a transgene at a level comparable to that
of the
cytomegalovirus promoter (CMV) (Boshart et al., "A Very Strong Enhancer is
Located
Upstream of an Immediate Early Gene of Human Cytomegalovirus," Ce// 41:521
(1985), which
is hereby incorporated by reference in its entirety). Promoters may be
synthetic, modified, or
hybrid promoters. Promoters may be coupled with other regulatory
sequences/elements which,
when bound to appropriate intracellular regulatory factors, enhance
("enhancers-) or repress
("repressors") promoter-dependent transcription. A promoter, enhancer, or
repressor, is said to
be "operably linked" to a transgene when such element(s) control(s) or
affect(s) transgene
transcription rate or efficiency. For example, a promoter sequence located
proximally to the 5'
end of a transgene coding sequence is usually operably linked with the
transgene. As used
herein, the term "regulatory elements" is used interchangeably with
"regulatory sequences" and
refers to promoters, enhancers, and other expression control elements, or any
combination of
such elements.
[0058] Promoters are positioned 5 (upstream) to the genes that
they control. Many
eukaryotic promoters contain two types of recognition sequences: TATA box and
the upstream
promoter elements. The TATA box, located 25-30 bp upstream of the
transcription initiation
site, is thought to be involved in directing RNA polymerase II to begin RNA
synthesis at the
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 24 -
correct site. In contrast, the upstream promoter elements determine the rate
at which
transcription is initiated These elements can act regardless of their
orientation, but they must be
located within 100 to 200 bp upstream of the TATA box.
[0059] Enhancer elements can stimulate transcription up to 1000-
fold from linked
homologous or heterologous promoters. Enhancer elements often remain active
even if their
orientation is reversed (Li et al., "High Level Desmin Expression Depends on a
Muscle-Specific
Enhancer,- I Bto. Chem. 266(10):6562-6570 (1991), which is hereby incorporated
by reference
in its entirety). Furthermore, unlike promoter elements, enhancers can be
active when placed
downstream from the transcription initiation site, e.g., within an intron, or
even at a considerable
distance from the promoter (Yutzey et al., "An Internal Regulatory Element
Controls Troponin I
Gene Expression," Mol. Cell. Bio. 9(4):1397-1405 (1989), which is hereby
incorporated by
reference in its entirety).
[0060] The term "muscle cell-specific" refers to the capability
of regulatory elements,
such as promoters and enhancers, to drive expression of an operatively linked
nucleic acid
molecule (e.g., a nucleic acid molecule encoding an AU-rich mRNA binding
factor 1 (AUF1)
protein or a functional fragment thereof) exclusively or preferentially in
muscle cells or muscle
tissue.
[0061] Adeno-associated viral (AAV) vectors disclosed herein
comprise a muscle cell-
specific promoter. In some embodiments, the muscle cell-specific promoter
mediates cell-
specific and/or tissue-specific expression of an AUF1 protein or fragment
thereof. The promoter
may be a mammalian promoter. For example, the promoter may be selected from
the group
consisting of a human promoter, a murine promoter, a porcine promoter, a
feline promoter, a
canine promoter, an ovine promoter, a non-human primate promoter, an equine
promoter, a
bovine promoter, and the like.
[0062] In some embodiments, the muscle cell-specific promoter is selected
from the
group consisting of a muscle creatine kinase (MCK) promoter, a C5-12 promoter,
a CK6-CK9
promoter, a dMCK promoter, a tMCK promoter, a smooth muscle 22 (SM22)
promoter, a myo-3
promoter, a Spc512 promoter, a creatine kinase (CK) 8 promoter, a creatine
kinase (CK) 8e
promoter, a U6 promoter, a H1 promoter, a desmin promoter, a Pitx3 promoter, a
skeletal alpha-
actin promoter, a MHCK7 promoter, and a Sp-301 promoter. Suitable muscle cell-
specific
promoter sequences are well known in the art and are provided in Table 1 below
(Malerba et al.,
"PABPN1 Gene Therapy for Oculopharyngeal Muscular Dystrophy," Nat.
C01111111112. 8:14848
(2017); Wang et al., "Construction and Analysis of Compact Muscle-Specific
Promoters for
AAV Vectors," Gene. Ther. 15:1489-1499 (2008); Piekarowicz etal., "A Muscle
Hybrid
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
-25 -
Promoter as a Novel Tool for Gene Therapy," Mol. Ther. Methods Cl/n. Dev.
15:157-169 (2019);
Salva et al., "Design of Tissue-Specific Regulatory Cassettes for High-Level
rAAV-Mediated
Expression in Skeletal and Cardiac Muscle," Mol. Ther. 15(2):320-329 (2007);
Lui et al.,
"Synthetic Promoter for Efficient and Muscle-Specific Expression of Exogenous
Genes,"
Plasmid 106:102441 (2019), which are hereby incorporated by reference in their
entirety.).
Table 1: Muscle Specific-Promoter Sequences
Promoter Sequence*
SEQ
ID NO:
Human AGCCAGCCTCAGTTTCCCCTCCACTCAGTCCCTAGGAGGAAGGGGCGCCC 1
muscle AAGCGCGGGTTTCTGGGGTTAGACTGCCCTCCATTGCAATTGGTCCTTCT
creatine CCCGGCCTCTGCTTCCTCCAGCTCACAGGGTATCTGCTCCTCCTGGAGCC
kinase ACACCTTGGTTCCCCGAGGTGCCGCTGGGACTCGGGTAGGGGTGAGGGCC
CAGGGGGCACAGGGGGAGCCGAGGGCCACAGGAAGGGCTGGTGGCTGAAG
(MCK)
GAGACTCAGGGGCCAGGGGACGGIGGCTICTACGTGCTTGGGACGTTCCC
AGCCACCGTCCCATGTTCCCGGCGGGGGGCCAGCTGTCCCCACCGCCAGC
CCAACTCAGCACTIGGTCAGGGTATCAGCTTGGIGGGGGGGCGTGAGCCC
AGCCCCTGGGGCGGCTCAGCCCATACAAGGCCATGGGGCTGGGCGCAAAG
CATGCCTGGGTTCAGGGTGGGTATGGTGCGGGAGCAGGGAGGTGAGAGGC
TCAGCTGCCCTCCAGAACTCCTCCCTGGGGACAACCCCTCCCAGCCAATA
GCACAGCCTAGGTCCCCCTATATAAGGCCACGGCTGCTGGCCCTTCCTTT
(NCBI sequence ID No. 1158)
Human CTGAGGCTCAGGGCTAGCTCGCCCATAGACATACATGGCAGGCAGGCTTT 2
desmin GGCCAGGATCCCTCCGCCTGCCAGGCGTCTCCCTGCCCTCCCTTCCTGCC
TAGAGACCCCCACCCTCAAGCCTGGCTGGTCTTTGCCTGAGACCCAAACC
TCTTCGACTTCAAGAGAATATTTAGGAACAAGGTGGTTTAGGGCCTTTCC
TGGGAACAGGCCTTGACCCITTAAGAAATGACCCAAAGTCTCTCCTTGAC
CAAAAAGGGGACCCTCAAACTAAAGGGAAGCCTCTCTTCTGCTGICTCCC
CTGACCCCACTCCCCCCCACCCCAGGACGAGGAGATAACCAGGGCTGAAA
GAGGCCCGCCTGGGGGCTGCAGACATGCTTGCTGCCTGCCCTGGCGAAGG
ATTGGCAGGCTTGCCCGTCACAGGACCCCCGCTGGCTGACTCAGGGGCGC
AGGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCACGGCCACGGGCCGC
CCTTTCCTGGCAGGACAGCGGGATCTTGCAGCTGTCAGGGGAGGGGAGGC
GGGGGCTGATGTCAGGAGGGATACAAATAGTGCCGACGGCTGGGGGCCCT
(NCBI sequence ID No. 1674)
Human GGAGTTCCAGGGGCGTAAAGGAGAGGGAGTTCGCCTICCITCCCITCCTG
3
skeletal ACACTCAGGAGTGACTGCTICTCCAATCCTCCCAACCCCACCACTCCACA
muscle CGACTCCCTCTTCCCGGTAGTCGCAAGTGGGAGITTGGGGATCTGAGCAA
AGAACCCGAAGAGGAGTTGAAATATTGGAAGTCAGCAGTCAGGCACCTIC
alpha
CCGAGCGCCCAGGGCGCTCAGAGTGGACATGGTTGGGGAGGCCTITGGGA
actin actal
CAGGTGCGGTTCCCGGAGCGCAGGCGCACACATGCACCCACCGGCGAACG
CGGTGACCCTCGCCCCACCCCATCCCCTCCGGCGGGCAACTGGGICGGGT
CAGGAGGGGCAAACCCGCTAGGGAGACACTCCATATACGGCCCGGCCCGC
GTTACCTGGGACCGGGCCAACCCGCTCCTICTTTGGTCAACGCAGGGGAC
CCGGGCGGGGGCCCAGGCCGCGAACCGGCCGAGGGAGGGGGCTCTAGTGC
CCAACACCCAAATATGGCTCGAGAAGGGCAGCGACATTCCTGCGGGGTGG
CGCGGAGGGAATGCCCGCGGGCTATATAAAACCTGAGCAGAGGGACAAGC
(NCBI sequence ID No. 58)
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-26 -
Promoter Sequence*
SEQ
ID NO:
Mouse AGAAACCTGTGGTCTAGAGGCGGGGCGGGGCCGATGGAGGCAACGCACGC
4
muscle CCCCGCAGGCGCCCAGGCCACGCCCTCTGCCGCAGCATTCGGTGAAACCT
creatine GCGTTCCGAGAACTTCTGAAAACTTTATCTGGGGGCCTTCGAGAAGGCTC
kinase AGACAGTAAGGGTGCATGCTGCCAATCCTGAGGAGCTGAGTTCGATCCCT
(MCK) GAGACCTTCAGGGTGGACAGAGACGGACTCCCACATGTTGTTTTCTGACT
TCTACATGTGICCAGTCATACATACACAAATATGGAATAAACAGATGGCT
CATCAGGTAAGAGTGCTGGCTGCTTTTGCAGAGGACCCAGGTTCGATTTC
CAGAACCCACATGTCGGCTCAAAATCATCTGTAATTCCAGTTCCAGGGAG
ATCCAGCACTTTCTICCAGGGCCTCCACAGACACACATAAAATAAAGATA
AAAATCTCCAAAAAATATTGTTTTAATAATTACAACCTGAAGACCTTGCA
CAACTATTCCTGGCTGAGAAGATGGTAAGGGCGCTAGCTGCCAAGCTTGA
CAGCCTGAGITTCATCTCCAAGAACCATGAAAACTGACTCCTGGGAATTA
(NCBI sequence ID No. 12715)
Molise GGAAGCAGAAGGCCAACATTCCTCCCAAGGGAAACTGAGGCTCAGAGTTA
5
desmin AAACCCAGGTATCAGTGATATGCATGTGCCCCGGCCAGGGTCACTCTCTG
ACTAACCGGTACCTACCCTACAGGCCTACCTAGAGACTCTTTTGAAAGGA
TGGTAGAGACCTGTCCGGGCTITGCCCACAGTCGTTGGAAACCTCAGCAT
TTTCTAGGCAACTTGTGCGAATAAAACACTTCGGGGGTCCTTCTTGTTCA
TTCCAATAACCTAAAACCTCTCCTCGGAGAAAATAGGGGGCCTCAAACAA
ACGAAATTCTCTAGCCCGCTTTCCCCAGGATAAGGCAGGCATCCAAATGG
AAAAAAAGGGGCCGGCCGGGGGTCTCCTGICAGCTCCTTGCCCTGTGAAA
CCCAGCAGGCCTGCCTGTCTICTGICCTCTTGGGGCTGTCCAGGGGCGCA
GGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCTCGCCTGTGGCCGCCC
TTTTCCTGGCAGGACAGAGGGATCCTGCAGCTGTCAGGGGAGGGGCGCCG
GGGGGTGATGTCAGGAGGGCTACAAATAGTGCAGACAGCTAAGGGGCTCC
(NCBI sequence ID No. 13346)
Mouse GGGGTGATGTGTGICAGATCTCTGGATTGGGGGAGCTTCAAAGTGGGAAA
6
skeletal GAAAATGGAGTTCAAATGIGGGGCTTATTTTCCATCCCTACCTGGAGCCC
muscle ATGACTCCTCCCGGCTCACCTGACCACAGGGCTACCTCCCCTGAGCTTAA
GCATCAAGGCTTAGTAGTCTGAGTTAAGdAACCCATAAATGGGGTGCATT
alpha
GTGGCAGGTCAGCAATCGTGIGTCCAGGIGGGCAGAACTGGGGAGACCTT
actin actal
TCAAACAGGTAAATCTTGGGAAGTACAGACCAGCAGTCTGCAAAGCAGTG
ACCTTTGGCCCAGCACAGCCCTTCCGTGAGCCTTGGAGCCAGTTGGGAGG
GGCAGACAGCTGGGGATACTCTCCATATACGGCCTGGTCCGGTCCTAGCT
ACCTGGGCCAGGGCCAGTCCTCTCCTTCTTTGGTCAGTGCAGGAGACCCG
GGCGGGGACCCAGGCTGAGAACCAGCCGAAGGAAGGGACTCTAGTGCCCG
ACACCCAAATATGGCTTGGGAAGGGCAGCAACATTCCTTCGGGGCGGTGT
GGGGAGAGCTCCCGGGACTATATAAAAACCTGTGCAAGGGGACAGGCGGT
(NCBI sequence ID No. 11459)
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-27 -
Promoter Sequence*
SEQ
ID NO:
MCK7 C TAGAAGCTGCAT GT CTAAGC TAGACC CT TCA GAT
TAAAAATAACTGAGG 7
TAAGGGCCTGGGTAGGGGAGGTGGT GT GAGACGCT CC TGTCTCTCCT CTA
T CT GCCCATCGGCCCT T TGGGGAGGAGGAAT GT GCCCAAGGACTAAAAAA
AGGCCATGGAGCCAGAGGGGCGAGGGCAACAGACCTT TCATGGGCAAACC
T TGGGGCCCTGCT GT CTAGCATGCCCCACTACGGGTC TAGGCT GCCCAT G
TAAGGAGGCAAGGCCT GGGGACACCCGAGAT GCCT GGT TATAAT TAACCC
AGACATGTGGC TGCCCCCCCCCCCCCAACACCT GCTGCCTCTAAAAATAA
CCCTGTCCCTGGTGGATCCCCTGCATGCGAAGATCTT CGAACAAGGCT GT
GGGGGACTGAGGGCAGGCTGTAACAGGCTTGGGGGCCAGGGCT TATACGT
GCCTGGGACTCCCAAAGTATTACTGTT CCAT GT TCCCGGCGAAGGGCCAG
CTGTCCCCCGCCAGCTAGACT CAGCACTTAGT T TAGGAACCAGTGAGCAA
GTCAGCCCTIGGGGCAGCCCATACAAGGCCAT GGGGC TGGGCAAGCT GCA
CGCCT GGGTCC GGGGT GGGCACGGT GC CCGGGCAACGAGCT GAAAGCT CA
T CT GCTCTCAGGGGCCCCTCCCT GGGGACAGCCCCTC CTGGCTAGTCACA
CCCTGTAGGCT CCICTATATAACCCAGGGGCACAGGGGCTGCCCT CAT T C
TACCACCACCT CCACAGCAC
Spc5-12 CGAGCTCCACC GCGGT GGCGGCCGT CC GCCCT CGGCACCAT CCTCACGAC
8
ACCCAAATATGGCGACGGGTGAGGAAT GGTGGGGAGT TAT T TT TAGAGCG
GTGAGGAAGGT GGGCAGGCAGCAGGTGT TGGCGCT CT AAAAATAACT CCC
GGGAGT TAT T T TTAGAGCGGAGGAATGGTGGACACCCAAATATGGCGACG
GT T CCTCACCC GT CGCCATAT TTGGGT GTCCGCCCTCGGCCGGGGCCGCA
T TCCT GGGGGC CGGGCGGTGCTCCCGC CCGCCT CGAT AAAAGGCT CCGGG
GCCGGCGGCGGCCCACGAGCT ACCCGGAGGAGCGGGAGGCGCCAAGCT CT
AGAACTAGTGGATCCCCCGGGCTGCAGGAAT IC
*See Malerba et al., "PABPN1 Gene Therapy for Owl pilau fieal Muscular
Dystrophy,- Nat.
Coimnun. 8:14848 (2017); Wang et al., "Construction and Analysis of Compact
Muscle-Specific
Promoters for AAV Vectors," Gene. /her. 15:1489-1499 (2008); Piekarowicz et
al., "A Muscle
Hybrid Promoter as a Novel Tool for Gene Therapy," Mol. 'her. Methods ('fin.
Dev. 15:157-169
(2019); and Salva et al., "Design of Tissue-Specific Regulatory Cassettes for
High-Level rAAV-
Mediated Expression in Skeletal and Cardiac Muscle," Mol. Ther. 15(2):320-329
(2007), which
are hereby incorporated by reference in their entirety.
[0063] In some embodiments, the muscle cell-specific promoter is
a muscle creatine-
kinase ("MCK") promoter. The muscle creatine kinase (MCK) gene is highly
active in all
striated muscles. Creatine kinase plays an important role in the regeneration
of ATP within
contractile and ion transport systems. It allows for muscle contraction when
neither glycolysis
nor respiration is present by transferring a phosphate group from
phosphocreatine to ADP to
form ATP. There are four known isoforms of creatine kinase: brain creatine
kinase (CKB),
muscle creatine kinase (MCK), and two mitochondrial forms (CKMi). MCK is the
most
abundant non-mitochondrial mRNA that is expressed in all skeletal muscle fiber
types and is also
highly active in cardiac muscle. The MCK gene is not expressed in myoblasts,
but becomes
transcriptionally active when myoblasts commit to terminal differentiation
into myocytes. MCK
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-28 -
gene regulatory regions display striated muscle-specific activity and have
been extensively
characterized in vivo and in vitro The major known regulatory regions in the
MCK gene include
a muscle-specific enhancer located approximately 1.1 kb 5' of the
transcriptional start site in
mouse and a 358-bp proximal promoter. Additional sequences that modulate MCK
expression
are distributed over 3.3 kb region 5' of the transcriptional start site and in
the 3.3-kb first intron.
Mammalian MCK regulatory elements, including human and mouse promoter and
enhancer
elements, are described in Hauser et al., "Analysis of Muscle Creatine Kinase
Regulatory
Elements in Recombinant Adenoviral Vectors," Mol. Therapy 2:16-25 (2000),
which is hereby
incorporated by reference in its entirety. Suitable muscle creatine kinase
(MCK) promoters
include, without limitation, a wild type MCK promoter, a dMCK promoter, and a
tMCK
promoter (Wang et al., "Construction and Analysis of Compact Muscle-Specific
Promoters for
AAV Vectors," Gene Ther. 15(22):1489-1499 (2008), which is hereby incorporated
by reference
in its entirety).
[0064] Genes involved in rapid response to cell stimuli are
highly regulated and typically
encode mRNAs that are selectively and rapidly degraded to quickly terminate
protein expression
and reprogram the cell (Moore et al., "Physiological Networks and Disease
Functions of RNA-
binding Protein AUF1," Wiky Interdiscip. Rev. RNA 5(4):549-64 (2014), which is
hereby
incorporated by reference in its entirety). These include growth factors,
inflammatory cytokines
(Moore et al., "Physiological Networks and Disease Functions of RNA-binding
Protein AUF1,"
Wiley Interdiscip Rev RNA 5(4):549-64 (2014) and Zhang et al., "Purification,
Characterization,
and cDNA Cloning of an AU-rich Element RNA-binding Protein, AUF1," Mol. Cell.
Biol.
13(12):7652-65 (1993), which are hereby incorporated by reference in their
entirety), and tissue
stem cell fate-determining mRNAs (Chenette et al., "Targeted mRNA Decay by RNA
Binding
Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle
Integrity,"
Cell Rep. 16(5):1379-90 (2016), which is hereby incorporated by reference in
its entirety) that
have very short half-lives of 5-30 minutes.
[0065] Short-lived mRNAs typically contain an AU-rich element
("ARE") in the 3'
untranslated region ("3'UTR") of the mRNA, having the repeated sequence AUUUA
(Moore et
al., "Physiological Networks and Disease Functions of RNA-binding Protein
AUF1," Wiley
Interdiscip Rev. RNA 5(4):549-64 (2014), which is hereby incorporated by
reference in its
entirety), which confers rapid decay. The ARE serves as a binding site for
regulatory proteins
known as AU-rich binding proteins (AUBPs) that control the stability and in
some cases the
translation of the mRNA (Moore et al., "Physiological Networks and Disease
Functions of RNA-
binding Protein AUF1," Wiley Interdiscip. Rev. RNA 5(4):549-64 (2014); Zhang
et al.,
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-29 -
"Purification, Characterization, and cDNA Cloning of an AU-rich Element RNA-
binding
Protein, AUF'1,"Mol. Cell. Biol. 13(12)-7652-65 (1993); and Halees et al.,
"ARED Organism:
Expansion of ARED Reveals AU-rich Element Cluster Variations Between Human And
Mouse,"
Nucleic Acids Res 36(Database issue):D137-40 (2008), which are hereby
incorporated by
reference in their entirety).
[0066] AU-rich mRNA binding factor 1 (AUF1; HNRNPD) binds with
high affinity to
repeated AU-rich elements ("AREs") located in the 3' untranslated region ("3'
UTR") found in
approximately 5% of mRNAs. Although AUF1 typically targets ARE-mRNAs for rapid
degradation, while not as well understood, it can oppositely stabilize and
increase the translation
of some ARE-mRNAs (Moore et al., "Physiological Networks and Disease Functions
of RNA-
Binding Protein AUF1,- Wiley Interdiscip. Rev. RNA 5(4):549-564 (2014), which
is hereby
incorporated by reference in its entirety). It was previously reported that
mice with AUF1
deficiency undergo an accelerated loss of muscle mass due to an inability to
carry out the
myogenesis program (Chenette et al., "Targeted mRNA Decay by RNA Binding
Protein AUF1
Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity,"
Cell Rep.
16(5):1379-90 (2016), which is hereby incorporated by reference in its
entirety). It was also
found that AUF1 expression is severely reduced with age in skeletal muscle,
and this
significantly contributes to loss and atrophy of muscle, loss of muscle mass,
and reduced
strength (Abbadi et al., "Muscle Development and Regeneration Controlled by
AUF1-mediated
Stage-specific Degradation of Fate-determining Checkpoint mRNAs," Proc. Natl.
Acad. Sci.
USA 116(23):11285-11290 (2019), which is hereby incorporated by reference in
its entirety). It
was also found that AUF1 controls all major stages of skeletal muscle
development, starting with
satellite cell activation and lineage commitment, by selectively targeting for
rapid degradation
the major differentiation checkpoint mRNAs that block entry into each next
step of muscle
development.
[0067]
AUF1 has four related protein isoforms identified by their molecular
weight
(p37AuFi, p40 AUFi p42'1, p45 AUF I) derived by differential splicing of a
single pre-mRNA
(Moore et al., "Physiological Networks and Disease Functions of RNA-Binding
Protein AUF1,"
Wiley Interdiscip. Rev. RNA 5(4):549-564 (2014); Chen & Shyu, "AU-Rich
Elements:
Characterization and Importance in mRNA Degradation," Trends Biochem. Sci.
20(11):465-470
(1995); and Kim et al., "Emerging Roles of RNA and RNA-Binding Protein Network
in Cancer
Cells," Bil4B Rep. 42(3):125-130 (2009), which are hereby incorporated by
reference in their
entirety). Each of these four isoforms include two centrally-positioned,
tandemly arranged RNA
recognition motifs ("RRIVIO which mediate RNA binding (DeMaria et al.,
"Structural
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 30 -
Determinants in AUF 1 Required for High Affinity Binding to A+U-rich
Elements," I Biol.
Chem. 272.27635-27643 (1997), which is hereby incorporated by reference in its
entirety)
[0068] The general organization of an RRM is a 13-a-3-p-a-13 RNA
binding platform of
anti-parallel 3-sheets backed by the a-helices (Zucconi & Wilson, "Modulation
of Neoplastic
Gene Regulatory Pathways by the RNA-binding Factor AUF1," Front. Biosci.
16:2307-2325
(2013); Nagai et al., "The RN? Domain: A Sequence-specific RNA-binding Domain
Involved in
Processing and Transport of RNA," Trends Biochem. Sci. 20:235-240 (1995),
which are hereby
incorporated by reference in their entirety). Structures of individual AUF1
RRNI domains
resolved by NMR are largely consistent with this overall tertiary fold
(Zucconi & Wilson,
"Modulation of Neoplastic Gene Regulatory Pathways by the RNA-binding Factor
AUF1,"
Front. Biosci. 16:2307-2325 (2013); Nagata et al., "Structure and Interactions
with RNA of the
N-terminal UUAG-specific RNA-binding Domain of hnRNP DO," I. Mol. Biol.
287:221-237
(1999); and Katahira et al., "Structure of the C-terminal RNA-binding Domain
of hnRNF' DO
(AUF1), its Interactions with RNA and DNA, and Change in Backbone Dynamics
Upon
Complex Formation with DNA," I. Mol. Biol. 311:973-988 (2001), which are
hereby
incorporated by reference in their entirety).
[0069] Mutations and/or polymorphisms in AUF1 are linked to
human limb girdle
muscular dystrophy (LGMD) type 1G (Chenette et al., "Targeted mRNA Decay by
RNA
Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal
Muscle
Integrity," Cell Rep. 16(5):1379-1390 (2016), which is hereby incroproated by
reference in its
entirety), suggesting a critical requirement for AUF1 in post-natal skeletal
muscle regeneration
and maintenance.
[0070] The term "fragment- or "portion- when used herein with
respect to a given
polypeptide sequence (e.g., AUF1), refers to a contiguous stretch of amino
acids of the given
polypeptide' s sequence that is shorter than the given polypeptide's full-
length sequence. A
fragment of a polypeptide may be defined by its first position and its final
position, in which the
first and final positions each correspond to a position in the sequence of the
given full-length
polypeptide. The sequence position corresponding to the first position is
situated N-terminal to
the sequence position corresponding to the final position. The sequence of the
fragment or
portion is the contiguous amino acid sequence or stretch of amino acids in the
given polypeptide
that begins at the sequence position corresponding to the first position and
ends at the sequence
position corresponding to the final position. Functional or active fragments
are fragments that
retain functional characteristics, e.g., of the native sequence or other
reference sequence.
Typically, active fragments are fragments that retain substantially the same
activity as the wild-
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 31 -
type protein. A fragment may, for example, contain a functionally important
domain, such as a
domain that is important for receptor or ligand binding_
[0071] Accordingly, in certain embodiments, functional fragments
of AUF1 as described
herein include at least one RNA recognition domain ("RRM") domain. In certain
embodiments,
functional fragments of AUF1 as described herein include two RRM domains.
[0072] AUF1 or functional fragments thereof as described herein
may be derived from a
mammalian AUF1. In one embodiment, the AUF1 or functional fragment thereof is
a human
AUF1 or functional fragment thereof. In another embodiment, the AUF1 or
functional fragment
thereof is a murine AUF1 or a functional fragment thereof. The AUF1 protein
according to
embodiments described herein may include one or more of the AUF1 isoforms
p37A1JF1 ,p40AUF%
p42Au", and p45AuFl. The GenBank accession numbers corresponding to the
nucleotide and
amino acid sequences of each human and mouse isoform is found in Table 2
below, each of
which is hereby incorporated by reference in its entirety.
Table 2: Summary of GenBank Accession Numbers of AUF1 Sequences
Isoform Human Mouse
Nucleotide Amino Acid Nucleotide Amino
Acid
p37AuFt NM 001003810.2 NP 001003810.1 NM 001077267.2 NP 001070735.1
(SEQ ID NO:9) (SEQ ID NO:10) (SEQ ID NO: 11) (SEQ ID
NO:12)
p 40AuF1 NM 002138.3 NP 002129.2 NM 007516.3 NP
031542.2
(SEQ ID NO: 13) (SEQ ID NO:14) (SEQ ID NO: 15) (SEQ ID
NO:16)
p42AuFt NM 031369.2 NP 112737.1 NM 001077266.2 NP
001070734.1
(SEQ ID NO: 17) (SEQ ID NO:18) (SEQ ID NO: 19) (SEQ ID
NO:20)
p45AUFl NM _031370.2 NP 112738.1 NM 001077265.2 NP
001070733.1
(SEQ ID NO:21) (SEQ ID NO:22) (SEQ ID NO:23) (SEQ ID
NO:24)
[0073] The sequences referred to in Table 2 are reproduced
below.
[0074] The human p37AuF1 nucleotide sequence of GenBank
Accession No.
NM 001003810.1 (SEQ ID NO:9) is as follows:
CTTCCGTCGG CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA
60
GCGGCCGCCG CTGGTGCTTA TTCTTTTTTA GTGCAGCGGG AGAGAGCGGG AGTGTGCGCC 120
GCGCGAGAGT GGGAGGCGAA GGGGGCAGGC CAGGGAGAGG CGCAGGAGCC TTTGCAGCCA
180
CGCGCGCGCC TTCCCTGTCT TGTGTGCTTC GCGAGGTAGA GCGGGCGCGC GGCAGCGGCG
240
GGGATTACTT TGCTGCTAGT TTCGGTTCGC GGCAGCGGCG GGTGTAGTCT CGGCGGCAGC
300
GGCGGAGACA CTAGCACTAT GTCGGAGGAG CAGTTCGGCG GGGACGGGGC GGCGGCAGCG
360
GCAACGGCGG CGGTAGGCGG CTCGGCGGGC GAGCAGGAGG GAGCCATGGT GGCGGCGACA 420
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 32 -
CAGGGGGCAG CGGCGGCGGC GGGAAGCGGA GCCGGGACCG GGGGCGGAAC CGCGTCTGGA
480
GGCACCGAAG GGGGCAGCGC CGAGTCGGAG GGGGCGAAGA TTGACGCCAG TAAGAACGAG
540
GAGGATGAAG GGAAAATGTT TATAGGAGGC CTTAGCTGGG ACACTACAAA GAAAGATCTG
600
AAGGACTACT TTTCCAAATT TGGTGAAGTT GTAGACTGCA CTCTGAAGTT AGATCCTATC
660
ACAGGGCGAT CAAGGGGTTT TGGCTTTGTG CTATTTAAAG AATCGGAGAG TGTAGATAAG
720
GTCATGGATC AAAAAGAACA TAAATTGAAT GGGAAGGTGA TTGATCCTAA AAGGGCCAAA
780
GCCATGAAAA CAAAAGAGCC GGTTAAAAAA ATTTTTGTTG GTGGCCTTTC TCCAGATACA
840
CCTGAAGAGA AAATAAGGGA GTACTTTGGT GGTTTTGGTG AGGTGGAATC CATAGAGCTC
900
CCCATGGACA ACAAGACCAA TAAGAGGCGT GGGTTCTGCT TTATTACCTT TAAGGAAGAA
960
GAACCAGTGA AGAAGATAAT GGAAAAGAAA TACCACAATG TTGGTCTTAG TAAATGTGAA 1020
ATAAAAGTAG CCATGTCGAA GGAACAATAT CAGCAACAGC AACAGTGGGG ATCTAGAGGA
1080
GGATTTGCAG GAAGAGCTCG TGGAAGAGGT GGTGACCAGC AGAGTGGTTA TGGGAAGGTA
1140
TCCAGGCGAG GTGGTCATCA AAATAGCTAC AAACCATACT AAATTATTCC ATTTGCAACT
1200
TATCCCCAAC AGGTGGTGAA GCAGIATTTT CCAATTTGAA GATTCATTTG AAGGTGGCTC
1260
CTGCCACCTG CTAATAGCAG TTCAAACTAA ATTTTTTGTA TCAAGTCCCT GAATGGAAGT 1320
ATGACGTTGG GTCCCTCTGA AGTTTAATTC TGAGTTCTCA TTAAAAGAAA TTTGCTTTCA
1380
TTGTTTTATT TCTTAATTGC TATGCTTCAG AATCAATTTG TGTTTTATGC CCTTTCCCCC
1440
AGTATTGTAG AGCAAGTCTT GTGTTAAAAG CCCAGTGTGA CAGTGTCATG ATGTAGTAGT
1500
GTCTTACTGG TTTTTTAATA AATCCTTTTG TATAAAAATG TATT GGCT CT TTTATCAT CA
1560
GAATAGGAAA AATTGTCATG GATTCAAGTT ATTAAAAGCA TAAGTTTGGA AGACAGGCTT 1620
GCCGAAATTG AGGACATGAT TAAAATTGCA GTGAAGTTTG AAATGTTTTT AGCAAAATCT
1680
AATTTTTGCC ATAATGTGTC CTCCCTGTCC AAATTGGGAA TGACTTAATG TCAATTTGTT
1740
TGTTGGTTGT TTTAATAATA CTTCCTTATG TAGCCATTAA GATTTATATG AATATTTTCC
1800
CAAATGCCCA GTTTTTGCTT AATATGTATT GTGCTTTTTA GAACAAATCT GGATAAATGT
1860
GCAAAAGTAC CCCTTTGCAC AGATAGTTAA TGTTTTATGC TTCCATTAAA TAAAAAGGAC 1920
TTAAAATCTG TTAATTATAA TAGAAATGCG GCTAGTTCAG AGAGATTTTT AGAGCTGTGG
1980
TGGACTTCAT AGATGAATTC AAGTGTTGAG GGAGGATTAA AGAAATATAT ACCGTGTTTA
2040
TGTGTGTGTG CTT
[0075] The human p37AuF1 amino acid sequence of GenBank
Accession No.
NP 001003810.1 (SEQ ID NO:10) is as follows:
MSEEQFGGDG AAAAATAAVG GSAGEQEGAM VAATQGAAAA AGSGAGTGGG TASGGTEGGS
60
AESEGAKIDA SKNEEDEGKM FIGGLSWDTT KKDLKDYFSK FGEVVDCTLK LDPITGRSRG
120
FGFVLFKESE SVDKVMDQKE HKLNGKVIDP KRAKAMKTKE PVKKIFVGGL SPDTPEEKIR
180
EYFGGFGEVE SIELPMDNKT NKRRGFCFIT FKEEEPVKKI MEKKYHNVGL SKCEIKVAMS
240
KEQYQQQQQW GSRGGFAGRA RGRGGDQQSG YGKVSRRGGH QNSYKPY
[0076] The human p40AuF1 nucleotide sequence of GenBank
Accession No.
NM 002138.3 (SEQ ID NO:13) is as follows:
CTTCCGTCGG CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA
60
GCGGCCGCCG CTGGTGCTTA TTCTTTTTTA GTGCAGCGGG AGAGAGCGGG AGTGTGCGCC
120
GCGCGAGAGT GGGAGGCGAA GGGGGCAGGC CAGGGAGAGG CGCAGGAGCC TTTGCAGCCA 180
CGCGCGCGCC TTCCCTGTCT TGTGTGCTTC GCGAGGTAGA GCGGGCGCGC GGCAGCGGCG
240
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 33 -
GGGATTACTT TGCTGCTAGT TTCGGTTCGC GGCAGCGGCG GGTGTAGTCT CGGCGGCAGC
300
GGCGGAGACA CTAGCACTAT GTCGGAGGAG GAGTTCGGCG GGGACGGGGC GGCGGCAGCG
360
GCAACGGCGG CGGTAGGCGG CTCGGCGGGC GAGCAGGAGG GAGCCATGGT GGCGGCGACA
420
CAGGGGGCAG CGGCGGCGGC GGGAAGCGGA GCCGGGACCG GGGGCGGAAC CGCGTCTGGA
480
GGCACCGAAG GGGGCAGCGC CGAGTCGGAG GGGGCGAAGA TTGACGCCAG TAAGAACGAG
540
GAGGATGAAG GCCATTCAAA CTCCTCCCCA CGACACTCTG AAGCAGCGAC GGCACAGCGG
600
GAAGAATGGA AAATGTTTAT AGGAGGCCTT AGCTGGGACA CTACAAAGAA AGATCTGAAG
660
GACTACTTTT CCAAATTTGG TGAAGTTGTA GACTGCACTC TGAAGTTAGA TCCTATCACA
720
GGGCGATCAA GGGGTTTTGG CTTTGTGCTA TTTAAAGAAT CGGAGAGTGT AGATAAGGTC
780
ATGGATCAAA AAGAACATAA ATTGAATGGG AAGGTGATTG ATCCTAAAAG GGCCAAAGCC 840
ATGAAAACAA AAGAGCCGGT TAAAAAAATT TTTGTTGGTG GCCTTTCTCC AGATACACCT
900
GAAGAGAAAA TAAGGGAGTA CTTTGGTGGT TTTGGTGAGG TGGAATCCAT AGAGCTCCCC
960
ATGGACAACA AGACCAATAA GAGGCGTGGG TTCTGCTTTA TTACCTTTAA GGAAGAAGAA
1020
CCAGTGAAGA AGATAATGGA AAAGAAATAC CACAATGTTG GTCTTAGTAA ATGTGAAATA
1080
AAAGTAGCCA TGTCGAAGGA ACAATATCAG CAACAGCAAC AGTGGGGATC TAGAGGAGGA 1140
TTTGCAGGAA GAGCTCGTGG AAGAGGTGGT GACCAGCAGA GTGGTTATGG GAAGGTATCC
1200
AGGCGAGGTG GTCATCAAAA TAGCTACAAA CCATACTAAA TTATTCCATT TGCAACTTAT
1260
CCCCAACAGG TGGTGAAGCA GTATTTTCCA ATTTGAAGAT TCATTTGAAG GTGGCTCCTG
1320
CCACCTGCTA ATAGCAGTTC AAACTAAATT TTTTGTATCA AGTCCCTGAA TGGAAGTATG
1380
ACGTTGGGTC CCTCTGAAGT TTAATTCTGA GTTCTCATTA AAAGAAATTT GCTTTCATTG 1440
TTTTATTTCT TAATTGCTAT GCTTCAGAAT CAATTTGTGT TTTATGCCCT TTCCCCCAGT
1500
ATTGTAGAGC AAGTCTTGTG TTAAAAGCCC AGTGTGACAG TGTCATGATG TAGTAGTGTC
1560
TTACTGGTTT TTTAATAAAT CCTTTTGTAT AAAAATGTAT TGGCTCTTTT ATCATCAGAA
1620
TAGGAAAAAT TGTCATGGAT TCAAGTTATT AAAAGCATAA GTTTGGAAGA CAGGCTTGCC
1680
GAAATTGAGG ACATGATTAA AATTGCAGTG AAGTTTGAAA TGTTTTTAGC AAAATCTAAT 1740
TTTTGCCATA ATGTGTCCTC CCTGTCCAAA TTGGGAATGA CTTAATGTCA ATTTGTTTGT
1800
TGGTTGTTTT AATAATACTT CCTTATGTAG CCATTAAGAT TTATATGAAT ATTTTCCCAA
1860
ATGCCCAGTT TTTGCTTAAT ATGTATTGTG CTTTTTAGAA GAAATCTGGA TAAATGTGCA
1920
AAAGTACCCC TTTGCACAGA TAGTTAATGT TTTATGCTTC CATTAAATAA AAAGGACTTA
1980
AAATCTGTTA ATTATAATAG AAATGCGGCT AGTTCAGAGA aATTTTTAGA GCTGTGGTGG 2040
ACTTCATAGA TGAATTCAAG TGTTGAGGGA GGATTAAAGA AATATATACC GTGTTTATGT
2100
GTGTGTGCTT
[0077] The human p40Aun amino acid sequence of GenBank Accession
No.
NP 002129.2 (SEQ ID NO:14) is as follows:
MSEEQFGGDG AAAAATAAVG GSAGEQEGAM VAATQGAAAA AGSGAGTGGG TASGGTEGGS 60
AESEGAKIDA SKNEEDEGHS NSSPRHSEAA TAQREEWKMF IGGLSWDTTK KDLKDYFSKF
120
GEVVDCTLKL DPITGRSRGF GFVLFKESES VDKVMDQKEH KLNGKVIDPK RAKAMKTKEP
180
VKKIFVGGLS PDTPEEKIRE YFGGFGEVES IELPMDNKTN KRRGFCFITF KEEEPVKKIM
240
EKKYHNVGLS KCEIKVAMSK EQYQQQQQWG SRGGFAGRAR GRGGDQQSGY GKVSRRGGHQ
300
NSYKPY
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 34 -
[0078] The human p42AuF1 nucleotide sequence of GenBank
Accession No.
NM 031369.2 (SEQ ID NO:17) is as follows:
CTTCCGTCGG CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA 60
GCGGCCGCCG CTGGTGCTTA TTCTTTTTTA GTGCAGCGGG AGAGAGCGGG AGTGTGCGCC 120
5 GCGCGAGAGT GGGAGGCGAA GGGGGCAGGC CAGGGAGAGG CGCAGGAGCC TTTGCAGCCA 180
CGCGCGCGCC TTCCCTGTCT TGTGTGCTTC GCGAGGTAGA GCGGGCGCGC GGCAGCGGCG 240
GGGATTACTT TGCTGCTAGT TTCGGTTCGC GGCAGCGGCG GGTGTAGTCT CGGCGGCAGC 300
GGCGGAGACA CTAGCACTAT GTCGGAGGAG CAGTTCGGCG GGGACGGGGC GGCGGCAGCG 360
GCAACGGCGG CGGTAGGCGG CTCGGCGGGC GAGCAGGAGG GAGCCATGGT GGCGGCGACA 420
10 CAGGGGGCAG CGGCGGCGGC GGGAAGCGGA GCCGGGACCG GGGGCGGAAC CGCGTCTGGA 480
GGCACCGAAG GGGGCAGCGC CGAGTCGGAG GGGGCGAAGA TTGACGCCAG TAAGAACGAG 540
GAGGATGAAG GGAAAATGTT TATAGGAGGC CTTAGCTGGG ACACTACAAA GAAAGATCTG 600
AAGGACTACT TTTCCAAATT TGGTGAAGTT GTAGACTGCA CTCTGAAGTT AGATCCTATC 660
ACAGGGCGAT CAAGGGGTTT TGGCTTTGTG CTATTTAAAG AATCGGAGAG TGTAGATAAG 720
15
GTCATGGATC AAAAAGAACA TAAATTGAAT GGGAAGGTGA TTGATCCTAA AAGGGCCAAA 780
GCCATGAAAA CAAAAGAGCC GGTTAAAAAA ATTTTTGTTG GTGGCCTTTC TCCAGATACA 840
CCTGAAGAGA AAATAAGGGA GTACTTTGGT GGTTTTGGTG AGGTGGAATC CATAGAGCTC 900
CCCATGGACA ACAAGACCAA TAAGAGGCGT GGGTTCTGCT TTATTACCTT TAAGGAAGAA 960
GAACCAGTGA AGAAGATAAT GGAAAAGAAA TACCACAATG TTGGTCTTAG TAAATGTGAA 1020
20 ATAAAAGTAG CCATGTCGAA GGAACAATAT CAGCAACAGC AACAGTGGGG ATCTAGAGGA 1080
GGATTTGCAG GAAGAGCTCG TGGAAGAGGT GGTGGCCCCA GTCAAAACTG GAACCAGGGA 1140
TATAGTAACT ATTGGAATCA AGGCTATGGC AACTATGGAT ATAACAGCCA AGGTTACGGT 1200
GGTTATGGAG GATATGACTA CACTGGTTAC AACAACTACT ATGGATATGG TGATTATAGC 1260
AACCAGaAGA GTGGTTATGG GAAGGTATCC AGGCGAGGTG GTCATCAAAA TAGCTACAAA 1320
25 CCATACTAAA TTATTCCATT TGCAACTTAT CCCCAACAGG TGGTGAAGCA GTATTTTCCA 1380
ATTTGAAGAT TCATTTGAAG GTGGCTCCTG CCACCTGCTA ATAGCAGTTC AAACTAAATT 1440
TTTTGTATCA AGTCCCTGAA TGGAAGTATG ACGTTGGGTC CCTCTGAAGT TTAATTCTGA 1500
GTTCTCATTA AAAGAAATTT GCTTICATTG TTTTATTTCT TAATTGCTAT GCTTCAGAAT 1560
CAATTTGTGT TTTATGCCCT TTCCCCCAGT ATTGTAGAGC AAGTCTTGTG TTAAAAGCCC 1620
30 AGTGTGACAG TGTCATGATG TAGTAGTGTC TTACTGGTTT TTTAATAAAT CCTTTTGTAT 1680
AAAAATGTAT TGGCTCTTTT ATCATCAGAA TAGGAAAAAT TGTCATGGAT TCAAGTTATT 1740
AAAAGCATAA GTTTGGAAGA CAGGCTTGCC GAAATTGAGG ACATGATTAA AATTGCAGTG 1800
AAGTTTGAAA TGTTITTAGC AAAATCTAAT TTTTGCCATA ATGTGTCCTC CCTGTCCAAA 1860
TTGGGAATGA CTTAATGTCA ATTTGTTTGT TGGTTGTTTT AATAATACTT CCTTATGTAG 1920
35
CCATTAAGAT TTATATGAAT ATTTTCCCAA ATGCCCAGTT TTTGCTTAAT ATGTATTGTG 1980
CTTTTTAGAA CAAATCTGGA TAAATGTGCA AAAGTACCCC TTTGCACAGA TAGTTAAT GT 2040
TTTATGCTTC CAT TAAATAA AAAGGACTTA AAAT CT GTTA AT TATAATAG AAATGCGGCT 2100
AGTTCAGAGA GAT TTTTAGA GCT GT GGT GG ACTT CATAGA TGAATTCAAG TGTTGAGGGA 2160
GGATTAAAGA AATATATACC GTGTTTAT GT GTGTGTGCTT
40 [0079] The human p42AuF1 amino acid sequence of GenBank
Accession No.
NP 112737.1 (SEQ ID NO:18) is as follows:
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 35 -
MSEEQFGGDG AAAAATAAVG GSAGEQEGAM VAATQGAAAA AGSGAGTGGG TASGGTEGGS 61
AESEGAKIDA SKNEEDEGKM FIGGLSWDTT KKDLKDYFSK FGEVVDCTLK LDPITGRSRG 121
FGFVLFKESE SVDKVMDQKE HKLNGKVIDP KRAKAMKTKE PVKKIFVGGL SPDTPEEKIR 181
EYFGGFGEVE SIELPMDNKT NKRRGFCFIT FKEEEPVKKI MEKKYHNVGL SKCEIKVAMS 241
5 KEQYQQQQQW GSRGGFAGRA RGRGGGPSQN WNQGYSNYWN QGYGNYGYNS QGYGGYGGYD 301
YTGYNNYYGY GDYSNQQSGY GKVSRRGGHQ NSYKPY
WOW The human p45AuFl nucleotide sequence of GenBank
Accession No.
INM 031370.2 (SEQ ID NO:21) is as follows:
CTTCCGTCGG CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA 60
GCGGCCGCCG CTGGTGCTTA TTCTTTTTTA GTGCAGCGGG AGAGAGCGGG AGTGTGCGCC 120
GCGCGAGAGT GGGAGGCGAA GGGGGCAGGC aAGGGAGAGG CGCAGGAGCC TTTGCAGCCA 180
CGCGCGCGCC TTCCCTGTCT TGTGTGCTTC GCGAGGTAGA GCGGGCGCGC GGCAGCGGCG 240
GGGATTACTT TGCTGCTAGT TTCGGTTCGC GGCAGCGGCG GGTGTAGTCT CGGCGGCAGC 300
GGCGGAGACA CTAGCACTAT GTCGGAGGAG aAGTTCGGCG GGGACGGGGC GGCGGCAGCG 360
15 GCAACGGCGG CGGTAGGCGG CTCGGCGGGC GAGCAGGAGG GAGCCATGGT GGCGGCGACA 420
CAGGGGGCAG CGGCGGCGGC GGGAAGCGGA GCCGGGACCG GGGGCGGAAC CGCGTCTGGA 480
GGCACCGAAG GGGGCAGCGC CGAGTCGGAG GGGGCGAAGA TTGACGCCAG TAAGAACGAG 540
GAGGATGAAG GCCATTCAAA CTCCTCCCCA CGACACTCTG AAGCAGCGAC GGCACAGCGG 600
GAAGAATGGA AAATGTTTAT AGGAGGCCTT AGCTGGGACA CTACAAAGAA AGATCTGAAG 660
20 GACTACTTTT CCAAATTTGG TGAAGTTGTA GACTGCACTC TGAAGTTAGA TCCTATCACA 720
GGGCGATCAA GGGGTTTTGG CTTTGTGCTA TTTAAAGAAT CGGAGAGTGT AGATAAGGTC 780
ATGGATCAAA AAGAACATAA ATTGAATGGG AAGGTGATTG ATCCTAAAAG GGCCAAAGCC 840
ATGAAAACAA AAGAGCCGGT TAAAAAAATT TTTGTTGGTG GCCTTTCTCC AGATACACCT 900
GAAGAGAAAA TAAGGGAGTA CTTTGGTGGT TTTGGTGAGG TGGAATCCAT AGAGCTCCCC 960
25
ATGGACAACA AGACCAATAA GAGGCGTGGG TTCTGCTTTA TTACCTTTAA GGAAGAAGAA 1020
CCAGTGAAGA AGATAATGGA AAAGAAATAC aACAATGTTG GTCTTAGTAA ATGTGAAATA 1080
AAAGTAGCCA TGTCGAAGGA ACAATATCAG CAACAGCAAC AGTGGGGATC TAGAGGAGGA 1140
TTTGCAGGAA GAGCTCGTGG AAGAGGTGGT GGCCCCAGTC AAAACTGGAA CCAGGGATAT 1200
AGTAACTATT GGAATCAAGG CTATGGCAAC TATGGATATA ACAGCCAAGG TTACGGTGGT 1260
30 TATGGAGGAT ATGACTACAC TGGTTACAAC AACTACTATG GATATGGTGA TTATAGCAAC 1320
CAGCAGAGTG GTTATGGGAA GGTATCCAGG CGAGGTGGTC ATCAAAATAG CTACAAACCA 1390
TACTAAATTA TTCCATTTGC AACTTATCCC aAACAGGTGG TGAAGCAGTA TTTTCCAATT 1440
TGAAGATTCA TTTGAAGGTG GCTCCTGCCA CCTGCTAATA GCAGTTCAAA CTAAATTTTT 1500
TGTATCAAGT CCCTGAATGG AAGTATGACG TTGGGTCCCT CTGAAGTTTA ATTCTGAGTT 1560
35 CTCATTAAAA GAAATTTGCT TTCATTGTTT TATTTCTTAA TTGCTATGCT TCAGAATCAA 1620
TTTGTGTTTT ATGCCCTTTC CCCCAGTATT GTAGAGCAAG TCTTGTGTTA AAAGCCCAGT 1680
GTGACAGTGT CATGATGTAG TAGTGTCTTA CTGGTTTTTT AATAAATCCT TTTGTATAAA 1740
AATGTATTGG CTCTTTTATC ATCAGAATAG GAAAAATTGT aATGGATTCA AGTTATTAAA 1800
AGCATAAGTT TGGAAGACAG GCTTGCCGAA ATTGAGGACA TGATTAAAAT TGCAGTGAAG 1860
40
TTTGAAATGT TTTTAGCAAA ATCTAATTTT TGCCATAATG TGTCCTCCCT GTCCAAATTG 1920
GGAATGACTT AATGTCAATT TGTTTGTTGG TTGTTTTAAT AATACTTCCT TATGTAGCCA 1980
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 36 -
TTAAGATTTA TATGAATATT TTCCCAAATG CCCAGTTTTT GCTTAATATG TATTGTGCTT 2040
TTTAGAACAA ATCTGGATAA ATGTGCAAAA GTACCCCTTT GCACAGATAG TTAATGTTTT 2100
ATGCTTCCAT TAAATAAAAA GGACTTAAAA TCTGTTAATT ATAATAGAAA TGCGGCTAGT 2160
TCAGAGAGAT TTTTAGAGCT GTGGTGGACT TCATAGATGA ATTCAAGTGT TGAGGGAGGA 2220
TTAAAGAAAT ATATACCGTG TTTATGTGTG TGTGCTT
[0081] The human p45AuF1 amino acid sequence of GenBank
Accession No.
NP 112738.1 (SEQ ID NO:22) is as follows:
MSEEQFGGDG AAAAATAAVG GSAGEQEGAM VAATQGAAAA AGSGAGTGGG TASGGTEGGS 60
AESEGAKIDA SKNEEDEGHS NSSPRHSEAA TAQREEWKMF IGGLSWDTTK KDLKDYFSKF 120
10 GEVVDCTLKL DPITGRSRGF GFVLFKESES VDKVMDQKEH KLNGKVIDPK RAKAMKTKEP 180
VKKIFVGGLS PDTPEEKIRE YFGGFGEVES IELPMDNKTN KRRGFCFITF KEEEPVKKIM 240
EKKYHNVGLS KCEIKVAMSK EQYQQQQQWG SRGGFAGRAR GRGGGPSQNW NQGYSNYWNQ 300
GYGNYGYNSQ GYGGYGGYDY TGYNNYYGYG DYSNQQSGYG KVSRRGGHQN SYKPY
[0082] The mouse p37AuF1 nucleotide sequence of GenBank
Accession No.
NM 001077267.2 (SEQ ID NO: 11) is as follows:
CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA GTGGCCGCCG 60
CTGCTACTTC ATTCTTTTTT TTTTCAGTGC AGCCGGGGAG AGCGAGAGAG CGCGCTGCGC 120
GAGAGTGGGA GGCGAGGGGG GCAGGCCGGG GAGAGGCGCA GGAGCCCTTG CAGCCACGCG 180
CGCGCCTTGT CTAGGGTGCC TCGCGAGGTA GAGCGGGCAT CGCGCGGCGG CGGCGGGGAT 240
20 TACTTTGCTG CTAGTTTCGG TTCGCGGCGG CGGCGGCGTC GGCGGGTGTC GTCTTCGGCG .. 300
GCGGCAGTAG CAGTATGTCG GAGGAGCAGT TCGGAGGGGA CGGGGCGGCG GCGGCGGCAA 360
CGGCGGCGGT AGGCGGCTCG GCGGGCGAGC AGGAGGGAGC CATGGTGGCG GCGGCGGCGC 420
AGGGGCCGGC GGCGGCGGCG GGAAGCGGGA GCGGCGGCGG CGGCTCTGCG GCCGGAGGCA 480
CCGAAGGAGG CAGCGCCGAG GCAGAGGGAG CCAAGATCGA CGCCAGTAAG AACGAGGAGG 540
ATGAAGGGAA AATGTTTATA GGAGGCCTTA GCTGGGACAC CACAAAGAAA aATCTGAAGG 600
ACTACTTTTC CAAATTTGGT GAAGTTGTAG ACTGCACTCT GAAGTTAGAT CCTATCACAG 660
GGCGATCAAG GGGTTTTGGC TTTGTGCTAT TTAAAGAGTC GGAGAGTGTA GATAAGGTCA 720
TGGATCAGAA AGAACATAAA TTGAATGGGA AAGTCATTGA TCCTAAAAGG GCCAAAGCCA 780
TGAAAACAAA AGAGCCTGTC AAAAAAATTT TTGTTGGTGG CCTTTCTCCA aACACACCTG 840
30 AAGAAAAAAT AAGAGAGTAC TTTGGTGGTT TTGGTGAGGT TGAATCCATA GAGCTCCCTA 900
TGGACAACAA GACCAATAAG AGGCGTGGGT TCTGTTTTAT TACCTTTAAG GAAGAGGAGC 960
CAGTGAAGAA GATAATGGAA AAGAAATACC ACAATGTTGG TCTTAGTAAA TGTGAAATAA 1020
AAGTAGCCAT GTCAAAGGAA CAGTATCAGC AGCAGCAGCA GTGGGGATCT AGAGGAGGGT 1080
TTGCAGGCAG AGCTCGCGGA AGAGGTGGAG ATCAGCAGAG TGGTTATGGG AAAGTATCCA 1140
35 GGCGAGGTGG ACATCAAAAT AGCTACAAAC CATACTAAAT TATTCCATTT GCAACTTATC 1200
CCCAACAGGT GGTGAAGCAG TATTITCCAA TTTGAAGATT CATTTGAAGG TGGCTCCTGC 1260
CACCTGCTAA TAGCAGTTCA AACTAAATTT TTTCTATCAA GTTCCTGAAT GGAAGTATGA 1320
CGTTGGGTCC CTCTGAAGTT TAATTCTGAG TTCTCATTAA AAGAATTTGC TTTCATTGTT 1380
TTATTTCTTA ATTGCTATGC TTCAGTATCA ATTTGTGTTT TATGCCCCCC CTCCCCCCCA 1440
40
GTATTGTAGA GCAAGTCTTG TGTTAAAAAA AGCCCAGTGT GACAGTGTCA TGATGTAGTA 1500
GTGTCTTACT GGTTTTTTAA TAAATCCTTT TGTATAAAAA TGTATTGGCT CTTTTATCAT 1560
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 37 -
CAGAATAGGA GGAAGTGAAA TACTACAAAT GTTTGTCTTG GATTCAAGTC ACTAGAAGCA 1620
TAAATTTGAG GGGATAAAAA CAACGGTAAA CTTTGTCTGA AAGAGGGCAT GGTTAAAAAT 1680
GTAGTGAATT TTAAATGTTT TTAGCAAAAT TTGATTTTGC CCAAGAATCC CTGTCTGAAT 1740
TGGAAATGAC TTAATGTAGT CAATGTGCTT GTTGGTTGTC TTAATATTAC TTCTGTAGCC 1800
5 ATTAAGTTTT ATGAGTAACT TCCCAAATAC CCACGTTTTT CTTTATATGT ATTGTGCTTT 1860
TTAAAAACAA ATCTGGAAAA ATGGGCAAGA ACATTTGCAG ACAATTGTTT TTAAGCTTCC 1920
ATTAAATAAA AAAAATGTGG ACTTAAGGAA ATCTATTAAT TTAAATAGAA CTGCAGCTAG 1980
TTTAGAGAGT ATTTTTTTCT TAAAGCTTTG GTGTAATTAG GGAAGATTTT AAAAAATGCA 2040
TAGTGTTTAT TTGTATGTGT GCTCTTTTTT TAAGTCAATT TTTGGGGGGT TGGTCTGTTA 2100
10 ACTGAGTCTA GGATTTAAAG GTAAGATGTT CCTAGAAATC TTGTCATCCC AAAGGGGCGG 2160
GCGCTAAGGT GAAACTTCAG GGTTCAGTCA GGGTCACTGC TTTATGTGTG AAATCACTCA 2220
AATTGGTAAG TCTCTTATGT TAGCATTCAG GACATTGATT TCAACTTGGA TGGACAATTT 2280
ATAGTTACTA CTGAATTGTG TGTTAATGTG TTCAGTCCTG GTAAGTTTTC AGTTTGATCA 2340
GTTAGTTGGA AGCAGACTTG AAGAGCTGTT AGTCACGTGA GCCATGGGTG CAGTCGATCT 2400
15
GTGGTCAGAT GCCTGAGTCT GTGATAGTGA ATTGTGTCTA AAGACATTTT AATGATAAAA 2460
GTCAGTGCTG TAAAGTTGAA AGTTCATGAG AGACATACAA TGAGGGCTGC AGCCCATTTT 2520
TAAAAACATT ATAATACAAA AGTATGCACA TTTGTTTACA TATCCCTGCC TTTGTATTAC 2580
AGTGGCAGGT TTGTGTACTT AAACTGGGAA AGCCTCAGAT CTATGATTAC CTGGCCTATC 2640
ATAGAAAGTG TCTAAATAAA TCACTCTGTC AATTGAATAC ATTAGTATTA GCTAGCATAC 2700
20 TTCATTATGC CTGTTTTCCA TAAATACCAC ACCAAAAACT TGCTTGGGGC AGTTTGAGCC 2760
TAGTTCATGA GCTGCTATCA GATTGGTCTT GATCCTATAT AATAGGCCAA ATGTCTGTAA 2820
ACAGCTGTGC TGGTGGAATG TAGAAAGTCA CTGCACTCAG ATTCAACTTC CTGATTGGAA 2880
GTCATCACAG TGTGATTAAA CATTITCACA AAGAATAGTA GATAAATAAC TTGGTTTTTA 2940
ATGTTAACTT TGTTTCCATT AAGTCACATT TAAAAACTTA TCCTCACGCC TACCTGAGTT 3000
25 AATTATCTGT TGACCTAGAT ATCTTTCTGG CCACTCACTG ACTTATTTCT TGAACTTTTG 3060
CCATTTGCAT AAATCTTGTC AGCTTTGTTC TTGATTATGC ATTGTCCAGG CTGAGCTAGT 3120
TGTCTTTCCA GGAATCCCTT TGTCTCTGAA TTAGGTCCTT TGTTTCCTAA ATCATCCTGC 3180
TTGTTTGGCA CAAGTCTTCC CAGGCCAGTG AGACCTCCGT GTCCTCTCAG CACCATAGGG 3240
GTAGGTAACC CTGGTTAGGC TGGACAGGGG TTTGCTGAGG GAGTTTGTTC ATTTGAATCT 3300
30 AGGTCTTACA TGACGTCTTT CAAATAGGGT TTTTACCTTG ACACTAAACT GTCCAGTCTA 3360
AGCAGTTCTG CAAAATGTGA GGGAATTATG AACTTCTTCC TGCAGTGGGT TTTTATGGTT 3420
TTGGTTTGTT TTTTGTTGTT TTGGTTCTTT GTTGAGCCCT GGACAAAAAC TTCCCTAGTT 3480
CTGGTTTCTA CAATTTAAAT TAAAAACAGA ATTCATCTTA GAATTTTTCA CCCTCTTCCC 3540
CAACTATTCT AATCAATCTT AAGTATGCCC TTCATCTTTT TTCCTTCCTA AGGCTTTTAC 3600
35
TGATAGTGTA ATTCCGTACT CTTCAACCCT GGGAAGGCTG AAGTGGATTC TTGAGCTCAT 3660
TTCAAGGCTG ACCTGGGTGT TGGCAAGAAC CCAGCTTAGA ACAAACACAT GCAAGGCCAT 3720
CTTACCTTAC ATCCTGTTGC TTGGACTTCT TCCTGCTCAA AGTTTTTAGT GGATGCTAAG 3780
TGATCTTTGC TTCCACTGAG GAGTGGAACA CTTTAGAATG AACCTCTAGA TAGATATTTT 3840
TATTGTCTGG TGAGGGTTAC TGGAGTTTCC CACCCTGCCT GAAGGGTGAA TCTGGCTTAC 3900
40 AGT
GTT CT CA T CT CAAAGGG AAGAAGGCAG AT GGCT GT GT CCAGAGAGAG CCATCACAGT 3960
TTGCTTCAGA GACACTAGAA TGGGCTGGAA GATCTAGTGG TCTTAATCAG ACTTGAAACC 4020
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 38 -
TGGCCTTTCT TCATTACCCA TATGTCTACC AGTACTTGGG CTAACACTTA AGCCATTAGG 4080
GCCTTTGTAG GGGTGTTTTG AGACCCCCTC aATGCTAACA AATATACAGG TTTCTTAACA 4140
TTTGCTCATA AACTTGTAAA GCTTACTTTC TCTTAATCCA CCCCACATTT AACAAGCCCT 4200
GGTACTTAGA ATTTCAGAAG AGTAATGGCA GGTAGGTGTG TGTGTGTGTG TGTGTGTGTG 4260
TGTGTGTGTG TGTGTGTGAG AGAGAGAGAG AGAGAGAGAG AGAGAGAGAG AGAGAGAGAG 4320
AGAGAGAGAG AGAAGTTTGT GGAAAATCAG GTAATGACAG CTCATCCTTT TAGAATTGTA 4380
CTTCAGAATA GAAACATTTG GTGGGCTGTT AGGTAGCTTT GATTACTTGT GGGTAGACCT 4440
GCTAGTATTG CCAGTCCTCA AGCAATGAGC TTTCTGTATC TTGTTTACTA GATATATACT 4500
ACCAGGTGAG TCATTTCCTG GGGTTCTGTT TTCTTTTAAA ATCTTTCCCT AAACTTAATA __ 4560
TGTATTAAAA AGTCTGGCTT TTCAGTCCAT TCTTTGTGCA CTGGGATGGC AATTGCTTCA 4620
TTATATGACA ATTGCTGTTC CCAAGTCAGA ATTCAGTGTG CTGATTTGAC ATCAGTTCGT __ 4680
CCCGAATAAG TTCCTGTTAC CAGGATTTAC ATTCAGCACA TTAGAAACTT GTTGGTGTGC 4740
TTTTATTCTT GGAGCATTTT CCTTAGACTA CCTTCCACTT TGAGTGCTCT GTTTAGGATG 4800
TTGAGGTGTT AGGATTCTTG ACAGCCAGAA AGACTGAACC aACTATCTGG GCACAGTGTT 4860
15 CGTGTTGCTC TATAAATGTA TGCTTTTTTT GATTTGGGGT TGTTTTACCT ACATTGTCAA 4920
ACTAGATCCA TGCTTAACAG TGATAATGAA GGCTTTTTGT TTGTTTTGTT TGTGGGTCCT 4980
CCCCCCCCCC CCAAGACAGG GTTTCTCTGT AGGCTGTCCT AGAACTTGTT CTTTTTTAAC 5040
CAAAATTTGG CAAGGCTGAA AATGGAATCC TATAATCAAT GCTGGCCACA TTAAAGTTAA 5100
TAGTTGAGAA GTCTTGTCTG AATTTCCTTG GGCAAAAAGA TTCTAGCCAG TTCAATACCC 5160
TGTTGTGCAA ATTCAATTTG CTGTTATAAT TTGCTCTCAG TTATCAGTTG GAAGGAGGTT 5220
AATTCTAATG TACTTGGAAG AGGCCTGTAG ACCATCTATA ACTGCATCAG TTGTACAGCG 5280
TTGTTGCCTG GGATTCTCTA GTTCACATAA ACTCCCAAGT CTTAGCCGTG GTGATGGCTA 5340
CAGTGTGGAA GAIGGTGAGC ATTCTAGTGA GTATCGCGAT GACGGCAGTA AAGAGCAGCA 5400
GGCAGCCGTG GCTGGGCTCA CTGACCGTGG CTGTAAGTTA CGGAGGCAGC ACACACTTCT 5460
25 GTACACACCT CTCATCAGTT ACCGGAGTCA TTGCATTGCG GACTAACTGG CTGACTCAAG __ 5520
TTGTCTTGCT ACTGAAGTCT TGAGTTGGTC TCATGCATTT ACCCTGTTGA CTTGAGCACC 5580
TTAAAGTCGA AAGGATGTCT GGTTGTGGCT TTATTGTAAA aAGCCTTAGG TAAAGAGGGG 5640
AGTATATCGG TTAGGAAGGT GAAAAATGAT ACTTCCAAGT TCAGTGGGAA ACCCTGGGTT 5700
TATCCCCCAG CTTAAGAAAG AATGCCTAAC AATGTTTCAG AATTAGATTC TGTGGAAGGT 5760
GAGGGTGTTA GAACAGTCCA AATTTGTTAT TGTAGACTTG aAGTGGGAGG AATTTTTAAA 5820
TATACAGATC AGTCGACACT CATTAACTTC ACTGATAAAG GTGGAAACGG ATGTGGCAAC 5880
ACTTCTAAGT TCATTTGTAT ATGTTTGTAA TTTGATTGGT TGTATTCTGT TGCACTCTAG 5940
AATTTGAAGG CAAGGTTACC TCTGCTTTTT AATTTTTTTT TTTTTAAAGA AACAAAAAAC 6000
ACTGAAAGAA ACTTCAAAAG ATCTGTTAAT GCTAATACCT GAATGTGGCA TTTAACATGT 6060
CATGGAAACT GCTTTGAATA AATACTTGAG AAAAGGAATG AAATAATTGC CGTTTTTGTT 6120
GTTGAGTGAA TGGGTGTGGT TTAATGAGCG TAATCATTTT TATAAAACAG CTGTGAGACT 6100
GAAGTGGAAT CCTTATTAAA TGTGGAAAAT GGCCTTTGAG GATTACAGTA GAGATTCAAC 6240
TAAGAGAGTA AATAAAGCTT GAAACTAATT CGTTGTAAAT TGCTTCTACA ATCATTGCTC 6300
TATATAGCAT GCTATTGCCA ATCAGTTTTA TGTATTAAGA CCTATCAGCA TGTCTTTTTT 6360
40 AGGTTGACCT CATTTTAAAT TATAAGATGC TCTCTGTACC GTTTTAACAT TTCCAGGATT 6420
TATTCTTTCT AGGCAAATTC CACTGGACTG TTTCCATTGT AGAAGCTTCC TTATAGATTC 6480
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 39 -
TTCAAATGAA GCTTACAGTG TGCTTTCTTG GGGTTTTGAT TTGCACTAAA TTTTATTTTC
6540
TGAAAGATCA CTTATGTTTA TAATGTAGTG CTTTGTCTTA ACAATTAAAC TTTCCAGCAC
6600
TCATGCA
[0083] The mouse p37AuF1 amino acid sequence of GenBank
Accession No.
NP 001070735.1 (SEQ ID NO:12) is as follows:
MSEEQFGGDG AAAAATAAVG GSAGEQEGAM VAAAAQGPAA AAGSGSGGGG SAAGGTEGGS
60
AEAEGAKIDA SKNEEDEGKM FIGGLSWDTT KKDLKDYFSK FGEVVDCTLK LDPITGRSRG
120
FGFVLFKESE SVDKVMDQKE HKLNGKVIDP KRAKAMKTKE PVKKIFVGGL SPDTPEEKIR
180
EYFGGFGEVE SIELPMDNKT NKRRGFCFIT FKEEEPVKKI MEKKYHNVGL SKCEIKVAMS
240
KEQYQQQQQW GSRGGFAGRA RGRGGDQQSG YGKVSRRGGH QNSYKPY
[0084] The mouse p40AuF1 nucleotide sequence of GenBank
Accession No.
NM 007516.3 (SEQ ID NO:15) is as follows:
CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA GTGGCCGCCG
60
CTGCTACTTC ATTCTTTTTT TTTTCAGTGC AGCCGGGGAG AGCGAGAGAG CGCGCTGCGC
120
GAGAGTGGGA GGCGAGGGGG GCAGGCCGGG GAGAGGCGCA GGAGCCCTTG CAGCCACGCG 180
CGCGCCTTGT CTAGGGTGCC TCGCGAGGTA GAGCGGGCAT CGCGCGGCGG CGGCGGGGAT
240
TACTTTGCTG CTAGTTTCGG TTCGCGGCGG CGGCGGCGTC GGCGGGTGTC GTCTTCGGCG
300
GCGGCAGTAG CACTATGTCG GAGGAGCAGT TCGGAGGGGA CGGGGCGGCG GCGGCGGCAA
360
CGGCGGCGGT AGGCGGCTCG GCGGGCGAGC AGGAGGGAGC aATGGTGGCG GCGGCGGCGC
420
AGGGGCCGGC GGCGGCGGCG GGAAGCGGGA GCGGCGGCGG CGOCTCTGCG GCCGGAGGCA 480
CCGAAGGAGG CAGCGCC GAG GCAGAGGGAG CCAAGATCGA CGCCAGTAAG AACGAGGAGG
540
ATGAAGGCCA TTCAAACTCC TCCCCACGAC ACACTGAAGC AGCGGCGGCA CAGCGGGAAG
600
AATGGAAAAT GTTTATAGGA GGCCTTAGCT GGGACACCAC AAAGAAAGAT CTGAAGGACT
660
ACTTTTCCAA ATTTGGTGAA GTTGTAGACT GCACTCTGAA GTTAGATCCT ATCACAGGGC
720
GATCAAGGGG TTTTGGCTTT GTGCTATTTA AAGAGTCGGA GAGTGTAGAT AAGGTCATGG 780
ATCAGAAAGA ACATAAATTG AATGGGAAAG TCATTGATCC TAAAAGGGCC AAAGCCATGA
840
AAACAAAAGA GCCTGTCAAA AAAATTTTTG TTGGTGGCCT TTCTCCAGAC ACACCTGAAG
900
AAAAAATAAG AGAGTACTTT GGTGGTTTTG GTGAGGTTGA ATCCATAGAG CTCCCTATGG
960
ACAACAAGAC CAATAAGAGG CGTGGGTTCT GTTTTATTAC CTTTAAGGAA GAGGAGCCAG
1020
TGAAGAAGAT AATGGAAAAG AAATACCACA ATGTTGGTCT TAGTAAATGT GAAATAAAAG 1090
TAGCCATGTC AAAGGAACAG TATCAGCAGC AGCAGCAGTG GGGATCTAGA GGAGGGTTTG
1140
CAGGCAGAGC TCGCGGAAGA GGTGGAGATC AGCAGAGTGG TTATGGGAAA GTATCCAGGC
1200
GAGGTGGACA TCAAAATAGC TACAAACCAT ACTAAATTAT TCCATTTGCA ACTTATCCCC
1260
AACAGGTGGT GAAGCAGTAT TTTCCAATTT GAAGATTCAT TTGAAGGTGG CTCCTGCCAC
1320
CTGCTAATAG CAGTTCAAAC TAAATTTTTT CTATCAAGTT CCTGAATGGA AGTATGACGT 1330
TGGGTCCCTC TGAAGTTTAA TTCTGAGTTC TCATTAAAAG AATTTGCTTT CATTGTTTTA
1440
TTTCTTAATT GCTATGCTTC AGTATCAATT TGTGTTTTAT GCCCCCCCTC CCCCCCAGTA
1500
TTGTAGAGCA AGTCTTGTGT TAAAAAAAGC CCAGTGTGAC AGTGTCATGA TGTAGTAGTG
1560
TCTTACTGGT TTTTTAATAA ATCCTTTTGT ATAAAAATGT ATTGGCTCTT TTATCATCAG
1620
AATAGGAGGA AGTGAAATAC TACAAATGTT TGTCTTGGAT TCAAGTCACT AGAAGCATAA 1680
ATTTGAGGGG ATAAAAACAA CGGTAAACTT TGTCTGAAAG AGGGCATGGT TAAAAATGTA
1740
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-40 -
GTGAATTTTA AATGITTTTA GCAAAATTTG ATTTTGCCCA AGAATCCCTG TCTGAATTGG 1800
AAATGACTTA ATGTAGTCAA TGTGCTTGTT GGTTGTCTTA ATATTACTTC TGTAGCCATT 1860
AAGTTTTATG AGTAACTTCC CAAATACCCA CGTTTTTCTT TATATGTATT GTGCTTTTTA 1920
AAAACAAATC TGGAAAAATG GGCAAGAACA TTTGCAGACA ATTGTTTTTA AGCTTCCATT 1980
AAATAAAAAA AATGTGGACT TAAGGAAATC TATTAATTTA AATAGAACTG CAGCTAGTTT 2040
AGAGAGTATT TTTTTCTTAA AGCTTTGGTG TAATTAGGGA AGATTTTAAA AAATGCATAG 2100
TGTTTATTTG TATGTGTGCT CTTTTTTTAA GTCAATTTTT GGGGGGTTGG TCTGTTAACT 2160
GAGTCTAGGA TTTAAAGGTA AGATGTTCCT AGAAATCTTG TCATCCCAAA GGGGCGGGCG 2220
CTAAGGTGAA ACTTCAGGGT TCAGICAGGG TCACTGCTTT ATGTGTGAAA TCACTCAAAT 2280
TGGTAAGTCT CTTATGTTAG CATTCAGGAC ATTGATTTCA ACTTGGATGG ACAATTTATA 2340
GTTACTACTG AATTGTGTGT TAATGTGTTC AGTCCTGGTA AGTTTTCAGT TTGATCAGTT 2400
AGTTGGAAGC AGACTTGAAG AGCTGTTAGT CACGTGAGCC ATGGGTGCAG TCGATCTGTG 2460
GTCAGATGCC TGAGTCTGTG ATAGTGAATT GTGTCTAAAG ACATTTTAAT GATAAAAGTC 2520
AGTGCTGTAA AGTTGAAAGT TCATGAaAGA CATACAATGA GGGCTGCAGC CCATTTTTAA 2560
AAACATTATA ATACAAAAGT ATGCACATTT GTTTACATAT CCCTGCCTTT GTATTACAGT 2640
GGCAGGTTTG TGTACTTAAA CTGGGAAAGC CTCAGATCTA TGATTACCTG GCCTATCATA 2700
GAAAGTGTCT AAATAAATCA CTCTGTCAAT TGAATACATT AGTATTAGCT AGCATACTTC 2760
ATTATGCCTG TTTTCCATAA ATACCACACC AAAAACTTGC TTGGGGCAGT TTGAGCCTAG 2820
TTCATGAGCT GCTATCAGAT TGGTCTTGAT CCTATATAAT AGGCCAAATG TCTGTAAACA 2880
GCTGTGCTGG TGGAATGTAG AAAGTCACTG CACTCAGATT CAACTTCCTG ATTGGAAGTC 2940
ATCACAGTGT GATTAAACAT TTTCACAAAG AATAGTAGAT AAATAACTTG GTTTTTAATG 3000
TTAACTTTGT TTCCATTAAG TCACATTTAA AAACTTATCC TCACGCCTAC CTGAGTTAAT 3060
TATCTGTTGA CCTAGATATC TTTCTGGCCA CTCACTGACT TATTTCTTGA ACTTTTGCCA 3120
TTTGCATAAA TCTTGTCAGC TTTGTTCTTG ATTATGCATT GTCCAGGCTG AGCTAGTTGT 3180
CTTTCCAGGA ATCCCTTTGT CTCTGAATTA GGTCCTTTGT TTCCTAAATC ATCCTGCTTG 3240
TTTGGCACAA GTCTTCCCAG GCCAGTGAGA CCTCCGTGTC CTCTCAGCAC CATAGGGGTA 3300
GGTAACCCTG GTTAGGCTGG ACAGGGGTTT GCTGAGGGAG TTTGTTCATT TGAATCTAGG 3360
TCTTACATGA CGTCTTTCAA ATAGGGTTTT TACCTTGACA CTAAACTGTC CAGTCTAAGC 3420
AGTTCTGCAA AATGTGAGGG AATTATGAAC TTCTTCCTGC AGTGGGTTTT TATGGTTTTG 3480
30 GTTTGTTTTT TGTTGTTTTG GTTCTTTGTT aAGCCCTGGA CAAAAACTTC CCTAGTTCTG 3540
GTTTCTACAA TTTAAATTAA AAACAGAATT CATCTTAGAA TTTTTCACCC TCTTCCCCAA 3600
CTATTCTAAT CAATCTTAAG TATGCCCTTC ATCTTTTTTC CTTCCTAAGG CTTTTACTGA 3660
TAGTGTAATT CCGTACTCTT CAACCCTGGG AAGGCTGAAG TGGATTCTTG AGCTCATTTC 3720
AAGGCT GAG C T GG GT GT T GG CAAGAAC C CA GC T TAGAACA AACACAT G CA AGGC CAT
CT T 3780
35 ACCTTACATC CTGTTGCTTG GACTTCTTCC TGCTCAAAGT TTTTAGTGGA TGCTAAGTGA 3840
T CT T T GCT TC CACTGAGGAG TGGAACACTT TAGAATGAAC CT CTAGATAG ATATTTTTAT
3900
TGTCTGGTGA GGGTTACTGG AGTTTCCCAC CCTGCCTGAA GGGTGAATCT GGCTTACAGT 3960
GTTCTCATCT CAAAGGGAAG AAGGCAGATG GCTGTGTCCA GAGAGAGCCA TCACAGTTTG 4020
CTTCAGAGAC ACTAGAATGG GCTGGAAGAT CTAGTGGTCT TAATCAGACT TGAAACCTGG 4080
40
CCTTTCTTCA TTACCCATAT GTCTACCAGT ACTTGGGCTA ACACTTAAGC CATTAGGGCC 4140
TTTGTAGGGG TGTTTTGAGA CCCCCTCCAT GCTAACAAAT ATACAGGTTT CTTAACATTT 4200
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-41 -
GCTCATAAAC TTGTAAAGCT TACTTTCTCT TAATCCACCC aACATTTAAC AAGCCCTGGT 4260
ACTTAGAATT TCAGAAGAGT AATGGCAGGT AGGTGTGTGT GTGTGTGTGT GTGTGTGTGT 4320
GTGTGTGTGT GTGTGAGAGA GAGAGAGAGA GAGAGAGAGA GAGAGAGAGA GAGAGAGAGA 4330
GAGAGAGAGA AGTTTGTGGA AAATCAGGTA ATGACAGCTC ATCCTTTTAG AATTGTACTT 4440
CAGAATAGAA ACATTTGGTG GGCTGTTAGG TAGCTTTGAT TACTTGTGGG TAGACCTGCT 4500
AGTATTGCCA GTCCTCAAGC AATGAGCTTT CTGTATCTTG TTTACTAGAT ATATACTACC 4560
AGGTGAGTCA TTTCCTGGGG TTCTGTTTTC TTTTAAAATC TTTCCCTAAA CTTAATATGT 4620
ATTAAAAAGT CTGGCTTTTC AGTCCATTCT TTGTGCACTG GGATGGCAAT TGCTTCATTA 4680
TATGACAATT GCTGTTCCCA AGTCAGAATT CAGTGTGCTG ATTTGACATC AGTTCGTCCC 4740
10 GAATAAGTTC CTGTTACCAG GATTTACATT CAGCACATTA GAAACTTGTT GGTGTGCTTT 4800
TATTCTTGGA GCATTTTCCT TAGACTACCT TCCACTTTGA GTGCTCTGTT TAGGATGTTG 4860
AGGTGTTAGG ATTCTTGACA GCCAGAAAGA CTGAACCCAC TATCTGGGCA aAGTGTTCGT 4920
GTTGCTCTAT AAATGTATGC TTTTTTTGAT TTGGGGTTGT TTTACCTACA TTGTCAAACT 4980
AGATCCATGC TTAACAGTGA TAATGAAGGC TTTTTGTTTG TTTTGTTTGT GGGTCCTCCC 5040
CCCCCCCCCA AGACAGGGTT TCTCTGTAGG CTGTCCTAGA ACTTGTTCTT TTTTAACCAA 5100
AATTTGGCAA GGCTGAAAAT GGAATCCTAT AATCAATGCT GGCCACATTA AAGTTAATAG 5160
TTGAGAAGTC TTGTCTGAAT TTCCTTGGGC AAAAAGATTC TAGCCAGTTC AATACCCTGT 5220
TGTGCAAATT CAATTTGCTG TTATAATTTG CTCTCAGTTA TCAGTTGGAA GGAGGTTAAT 5280
TCTAATGTAC TTGGAAGAGG CCTGTAGACC ATCTATAACT GCATCAGTTG TACAGCGTTG 5340
20 TTGCCTGGGA TTCTCTAGTT CACATAAACT CCCAAGTCTT AGCCGTGGTG ATGGCTACAG 5400
TGTGGAAGAT GGTGAGCATT CTAGTGAGTA TCGCGATGAC GGCAGTAAAG AGCAGCAGGC 5460
AGCCGTGGCT GGGCTCACTG ACCGTGGCTG TAAGTTACGG AGGaAGCACA aACTTCTGTA 5520
CACACCTCTC ATCAGTTACC GGAGICATTG CATTGCGGAC TAACTGGCTG ACTCAAGTTG 5580
TCTTGCTACT GAAGTCTTGA GTTGGTCTCA TGCATTTACC CTGTTGACTT aAGCACCTTA 5640
AAGTCGAAAG GATGTCTGGT TGTGGCTTTA TTGTAAACAG CCTTAGGTAA AGAGGGGAGT 5700
ATATCGGTTA GGAAGGTGAA AAATGATACT TCCAAGTTCA GTGGGAAACC CTGGGTTTAT 5760
CCCCCAGCTT AAGAAAGAAT GCCTAACAAT GTTTCAGAAT TAGATT CT GT GGAAGGT GAG 5820
GGTGTTAGAA CAGTCCAAAT TTGTTAT T GT AGACTTGCAG TGGGAGGAAT TTTTAAATAT 5880
ACAGATCAGT CGACACT CAT TAACTTCACT GATAAAGGTG GAAACGGATG TGGCAACACT 5940
TCTAAGTTCA TTTGTATATG TTTGTAATTT GATTGGTTGT ATTCTGTTGC ACTCTAGAAT 6000
TTGAAGGCAA GGTTACCTCT GCTTTTTAAT TTTTTTTTTT TTAAAGAAAG AAAAAACACT 6060
GAAAGAAACT TCAAAAGATC TGTTAATGCT AATACCTGAA TGTGGCATTT AACATGTCAT 6120
GGAAACTGCT TTGAATAAAT ACTTGAGAAA AGGAATGAAA TAATTGCCGT TTTTGTTGTT 6180
GAGTGAATGG GTGTGGTTTA ATGAGCGTAA TCATTTTTAT AAAACAGCTG TGAGACTGAA 6240
35 GTGGAATCCT TATTAAATGT GGAAAATGGC CTTTGAGGAT TACAGTAGAG ATTCAACTAA 6300
GAGAGTAAAT AAAGCTTGAA ACTAATTCGT TGTAAATTGC TTCTACAATC ATTGCTCTAT 6360
ATAGCATGCT ATTGCCAATC AGTTTTATGT ATTAAGACCT ATCAGCATGT CTTTTTTAGG 6420
TTGACCTCAT TTTAAATTAT AAGATGCTCT CTGTACCGTT TTAACATTTC CAGGATTTAT 6480
TCTTTCTAGG CAAATTCCAC TGGACTGTTT CCATTGTAGA AGCTTCCTTA TAGATTCTTC 6540
40 AAATGAAGCT TACAGTGTGC TTTCTTGGGG TTTTGATTTG CACTAAATTT TATTTTCTGA 6600
AAGATCACTT ATGTTTATAA TGTAGTGCTT TGTCTTAACA ATTAAACTTT CCAGCACTCA 6660
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-42 -
TGCA
[0085] The mouse p40AuF1 amino acid sequence of GenBank
Accession No.
NP 031542.2 (SEQ ID NO:16) is as follows:
MSEEQFGGDG AAAAATAAVG GSAGEQEGAM VAAAAQGPAA AAGSGSGGGG SAAGGTEGGS 60
AEAEGAKIDA SKNEEDEGHS NSSPRHTEAA AAQREEWKMF IGGLSWDTTK KDLKDYFSKF 120
GEVVDCTLKL DPITGRSRGF GFVLFKESES VDKVMDQKEH KLNGKVIDPK RAKAMKTKEP 180
VKKIFVGGLS PDTPEEKIRE YFGGFGEVES IELPMDNKTN KRRGFCFITF KEEEPVKKIM 240
EKKYHNVGLS KCEIKVAMSK EQYQQQQQWG SRGGFAGRAR GRGGDQQSGY GKVSRRGGHQ 300
NSYKPY
[0086] The mouse p42AuF1 nucleotide sequence of GenBank Accession No.
NM 001077266.2 (SEQ ID NO: 19) is as follows:
CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA GTGGCCGCCG 60
CTGCTAETTC ATTCTTTTTT TTTTCAGTGC AGCCGGGGAG AGCGAGAGAG CGCGCTGCGC 120
GAGAGTGGGA GGCGAGGGGG GCAGGCCGGG GAGAGGCGCA GGAGCCCTTG aAGCCACGCG 180
15 CGCGCCTTGT CTAGGGTGCC TCGCGAGGTA GAGCGGGCAT CGCGCGGCGG CGGCGGGGAT 240
TACTTTGCTG CTAGTTTCGG TTCGCGGCGG CGGCGGCGTC GGCGGGTGTC GTCTTCGGCG 300
GCGGCAGTAG CACTATGTCG GAGGAGaAGT TCGGAGGGGA CGGGGCGGCG GCGGCGGCAA 360
CGGCGGCGGT AGGCGGCTCG GCGGGCGAGC AGGAGGGAGC CATGGTGGCG GCGGCGGCGC 420
AGGGGCCGGC GGCGGCGGCG GGAAGCGGGA GCGGCGGCGG CGGCTCTGCG GCCGGAGGCA 480
20 CCGAAGGAGG CAGCGCCGAG GCAGAGGGAG CCAAGATCGA CGCCAGTAAG AACGAGGAGG 540
ATGAAGGGAA AATGTTTATA GGAGGCCTTA GCTGGGACAC CACAAAGAAA GATCTGAAGG 600
ACTACTTTTC CAAATTTGGT GAAGTTGTAG ACTGCACTCT GAAGTTAGAT CCTATCACAG 660
GGCGATCAAG GGGTTTTGGC TTTGTGCTAT TTAAAGAGTC GGAGAGTGTA GATAAGGTCA 720
TGGATCAGAA AGAACATAAA TTGAATGGGA AAGTCATTGA TCCTAAAAGG GCCAAAGC CA 780
25 TGAAAACAAA
AGAGCCTGTC AAAAAAATTT TTGTTGGTGG CCTTTCTCCA aACACACCTG 840
AAGAAAAAAT AAGAGAGTAC TTTGGTGGTT TTGGTGAGGT TGAATCCATA GAGCTCCCTA 900
TGGACAACAA GACCAATAAG AGGCGTGGGT TCTGTITTAT TACCTTTAAG GAAGAGGAGC 960
CAGTGAAGAA GATAATGGAA AAGAAATACC ACAATGTTGG TCTTAGTAAA TGTGAAATAA 1020
AAGTAGCCAT GTCAAAGGAA CAGTATCAGC AGCAGCAGCA GTGGGGATCT AGAGGAGGGT 1080
30 TTGCAGGCAG
AGCTCGCGGA AGAGGTGGAG GCCCCAGTCA AAACTGGAAC CAGGGATATA 1140
GTAACTATTG GAATCAAGGC TATGGCAACT ATGGATATAA CAGCCAAGGT TACGGAGGTT 1200
ATGGAGGATA TGACTACACT GGTTACAACA ACTACTATGG ATATGGTGAT TATAGCAATC 1260
AGCAGAGTGG TTATGGGAAA GTATCCAGGC GAGGTGGACA TCAAAATAGC TACAAACCAT 1320
ACTAAATTAT TCCATTTGCA ACTTATCCCC AACAGGTGGT GAAGCAGTAT TTTCCAATTT 1380
35 GAAGATTCAT TTGAAGGTGG CTCCTGCCAC CTGCTAATAG CAGTTCAAAC TAAATTTTTT 1440
CTATCAAGTT CCTGAATGGA AGTATGACGT TGGGTCCCTC TGAAGTTTAA TTCTGAGTTC 1500
TCATTAAAAG AATTTGCTTT CATTGTTTTA TTTCTTAATT GCTATGCTTC AGTATCAATT 1560
TGTGTTTTAT GCCCCCCCTC CCCCCCAGTA TTGTAGAGCA AGTCTTGTGT TAAAAAAAGC 1620
CCAGTGTGAC AGTGTCATGA TGTAGTAGTG TCTTACTGGT TTTTTAATAA ATCCTTTTGT 1680
40 ATAAAAATGT
ATTGGCTCTT TTATCATCAG AATAGGAGGA AGTGAAATAC TACAAATGTT 1740
TGTCTTGGAT TCAAGTCACT AGAAGCATAA ATTTGAGGGG ATAAAAACAA CGGTAAACTT 1800
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-43 -
TGTCTGAAAG AGGGCATGGT TAAAAATGTA GTGAATTTTA AATGTTTTTA GCAAAATTTG 1860
ATTTTGCCCA AGAATCCCTG TCTGAATTGG AAATGACTTA ATGTAGTCAA TGTGCTTGTT 1920
GGTTGTCTTA ATATTACTTC TGTAGCCATT AAGTTTTATG AGTAACTTCC CAAATACCCA 1980
CGTTTTTCTT TATATGTATT GTGCTTTTTA AAAACAAATC TGGAAAAATG GGCAAGAACA 2040
TTTGCAGACA ATTGTTTTTA AGCTTCCATT AAATAAAAAA AATGTGGACT TAAGGAAATC 2100
TATTAATTTA AATAGAACTG CAGCTAGTTT AGAGAGTATT TTTTTCTTAA AGCTTTGGTG 2160
TAATTAGGGA AGATTTTAAA AAATGCATAG TGTTTATTTG TATGTGTGCT CTTTTTTTAA 2220
GTCAATTTTT GGGGGGTTGG TCTGTTAACT GAGTCTAGGA TTTAAAGGTA AGATGTTCCT 2280
AGAAATCTTG TCATCCCAAA GGGGCGGGCG CTAAGGTGAA ACTTCAGGGT TCAGTCAGGG 2340
10 TCACTGCTTT ATGTGTGAAA TCACTCAAAT TGGTAAGTCT CTTATGTTAG CATTCAGGAC 2400
ATTGATTTCA ACTTGGATGG ACAATTTATA GTTACTACTG AATTGTGTGT TAATGTGTTC 2460
AGTCCTGGTA AGTTTTCAGT TTGATCAGTT AGTTGGAAGC AGACTTGAAG AGCTGTTAGT 2520
CACGTGAGCC ATGGGTGCAG TCGATCTGTG GTCAGATGCC TGAGTCTGTG ATAGTGAATT 2580
GTGTCTAAAG ACATITTAAT GATAAAAGTC AGTGCTGTAA AGTTGAAAGT TCATGAGAGA 2640
CATACAATGA GGGCTGaAGC CCATTTTTAA AAACATTATA ATACAAAAGT ATGCACATTT 2700
GTTTACATAT CCCTGCCTTT GTATTACAGT GGCAGGTTTG TGTACTTAAA CTGGGAAAGC 2760
CTCAGATCTA TGATTACCTG GCCTATCATA GAAAGTGTCT AAATAAATCA CTCTGTCAAT 2820
TGAATACATT AGTATTAGCT AGCATACTTC ATTATGCCTG TTTTCCATAA ATACCACACC 2880
AAAAACTTGC TTGGGGCAGT TTGAGCCTAG TTCATGAGCT GCTATCAGAT TGGTCTTGAT 2940
20 CCTATATAAT AGGCCAAATG TCTGTAAACA GCTGTGCTGG TGGAATGTAG AAAGTCACTG 3000
CACTCAGATT CAACTTCCTG ATTGGAAGTC ATCACAGTGT GATTAAACAT TTTCACAAAG 3060
AATAGTAGAT AAATAACTTG GTTTTTAATG TTAACTTTGT TTCCATTAAG TCACATTTAA 3120
AAACTTATCC TCACGCCTAC CTGAGTTAAT TATCTGTTGA CCTAGATATC TTTCTGGCCA 3180
CTCACTGACT TATTTCTTGA ACTTTTGCCA TTTGaATAAA TCTTGTCAGC TTTGTTCTTG 3240
ATTATGCATT GTCCAGGCTG AGCTAGTTGT CTTTCCAGGA ATCCCTTTGT CTCTGAATTA 3300
GGTCCTTTGT TTCCTAAATC ATCCTGCTTG TTTGGCACAA GTCTTCCCAG GCCAGTGAGA 3360
CCTCCGTGTC CTCTCAGCAC CATAGGGGTA GGTAACCCTG GTTAGGCTGG ACAGGGGTTT 3420
GCTGAGGGAG TTTGTTCATT TGAATCTAGG TCTTACATGA CGTCTTTCAA ATAGGGTTTT 3480
TACCTTGACA CTAAACTGTC CAGTCTAAGC AGTTCTGCAA AATGTGAGGG AATTATGAAC 3540
30 TTCTTCCTGC AGTGGGTTTT TATGGTTTTG GTTTGTTTTT TGTTGTTTTG GTTCTTTGTT 3600
GAGCCCTGGA CAAAAACTTC CCTAGTTCTG GTTTCTACAA TTTAAATTAA AAACAGAATT 3660
CATCTTAGAA TTTTTCACCC TCTTCCCCAA CTATTCTAAT aAATCTTAAG TATGCCCTTC 3720
ATCTTTTTTC CTTCCTAAGG CTTTTACTGA TAGTGTAATT CCGTACTCTT CAACCCTGGG 3780
AAGGCTGAAG TGGATTCTTG AGCTCATTTC AAGGCTGACC TGGGTGTTGG CAAGAACCCA 3840
GCTTAGAACA AACACATGCA AGGCCATCTT ACCTTACATC CTGTTGCTTG GACTTCTT CC 3900
TGCTCAAAGT TTTTAGTGGA TGCTAAGTGA TCTTTGCTTC CACTGAGGAG TGGAACACTT 3960
TAGAATGAAC CTCTAGATAG ATATTTTTAT TGTCTGGTGA GGGTTACTGG AGTTTCCCAC 4020
CCTGCCTGAA GGGTGAATCT GGCTTACAGT GTTCTCATCT CAAAGGGAAG AAGGCAGATG 4080
GCTGTGTCCA GAGAGAGCCA TCACAGTTTG CTTCAGAGAC ACTAGAATGG GCTGGAAGAT 4140
40 CTAGTGGTCT TAATCAGACT TGAAACCTGG CCTTTCTTCA TTACCCATAT GTCTACCAGT 4200
ACTTGGGCTA ACACTTAAGC CATTAGGGCC TTTGTAGGGG TGTTTTGAGA CCCCCTCCAT 4260
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-44 -
GCTAACAAAT ATACAGGTTT CTTAACATTT GCTCATAAAC TTGTAAAGCT TACTTTCTCT 4320
TAATCCACCC CAGATTTAAC AAGCCCTGGT ACTTAGAATT TCAGAAGAGT AATGGCAGGT .. 4380
AGGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGAGAGA GAGAGAGAGA 4440
GAGAGAGAGA GAGAGAGAGA GAGAGAGAGA GAGAGAGAGA AGTTTGTGGA AAATCAGGTA 4500
ATGACAGCTC ATCCTTTTAG AATTGTACTT CAGAATAGAA ACATTTGGTG GGCTGTTAGG 4560
TAGCTTTGAT TACTTGTGGG TAGACCTGCT AGTATTGCCA GTCCTCAAGC AATGAGCTTT 4620
CTGTATCTTG TTTACTAGAT ATATACTACC AGGTGAGTCA TTTCCTGGGG TTCTGTTTTC 4680
TTTTAAAATC TTTCCCTAAA CTTAATATGT ATTAAAAAGT CTGGCTTTTC AGTCCATTCT 4740
TTGTGCACTG GGATGGCAAT TGCTICATTA TATGACAATT GCTGTTCCCA AGTCAGAATT 4800
CAGTGTGCTG ATTTGACATC AGTTCGTCCC GAATAAGTTC CTGTTACCAG GATTTACATT 4860
CAGCACATTA GAAACTTGTT GGTGTGCTTT TATTCTTGGA GCATTTTCCT TAGACTACCT 4920
TCCACTTTGA GTGCTCTGTT TAGGATGTTG AGGTGTTAGG ATTCTTGACA GCCAGAAAGA 4980
CTGAACCCAC TATCTGGGCA CAGTGTTCGT GTTGCTCTAT AAATGTATGC TTTTTTTGAT 5040
TTGGGGTTGT TTTACCTACA TTGTCAAACT AGATCCATGC TTAACAGTGA TAATGAAGGC 5100
TTTTTGTTTG TTTTGTTTGT GGGTCCTCCC CCCCCCCCCA AGACAGGGTT TCTCTGTAGG 5160
CTGTCCTAGA ACTTGTTCTT TTTTAACCAA AATTTGGCAA GGCTGAAAAT GGAATCCTAT 5220
AATCAATGCT GGCCACATTA AAGTTAATAG TTGAGAAGTC TTGTCTGAAT TTCCTTGGGC 5280
AAAAAGATTC TAGCCAGTTC AATACCCTGT TGTGCAAATT CAATTTGCTG TTATAATTTG 5340
CTCTCAGTTA TCAGTTGGAA GGAGGTTAAT TCTAATGTAC TTGGAAGAGG CCTGTAGACC 5400
20 ATCTATAACT GCATCAGTTG TACAGCGTTG TTGCCTGGGA TTCTCTAGTT CACATAAACT 5460
CCCAAGTCTT AGCCGTGGTG ATGGCTACAG TGTGGAAGAT GGTGAGCATT CTAGTGAGTA .. 5520
TCGCGATGAC GGCAGTAAAG AGGAGGAGGC AGCCGTGGCT GGGCTCACTG ACCGTGGCTG 5580
TAAGTTACGG AGGCAGCACA CACTICTGTA CACACCTCTC ATCAGTTACC GGAGTCATTG 5640
CATTGCGGAC TAACTGGCTG ACTCAAGTTG TCTTGCTACT GAAGTCTTGA GTTGGTCTCA 5700
TGCATTTACC CTGTTGACTT GAGCACCTTA AAGTCGAAAG GATGTCTGGT TGTGGCTTTA 5760
TTGTAAACAG CCTTAGGTAA AGAGGGGAGT ATATCGGTTA GGAAGGTGAA AAATGATACT 5820
TCCAAGTTCA GTGGGAAACC CTGGGTTTAT CCCCCAGCTT AAGAAAGAAT GCCTAACAAT 5880
GTTTCAGAAT TAGATTCTGT GGAAGGTGAG GGTGTTAGAA aAGTCCAAAT TTGTTATTGT 5940
AGACTTGCAG TGGGAGGAAT TTTTAAATAT ACAGATCAGT CGACACTCAT TAACTTCACT 6000
30 GATAAAGGTG GAAACGaATG TGGCAACACT TCTAAGTTCA TTTGTATATG TTTGTAATTT 6060
GATTGGTTGT ATTCTGTTGC ACTCTAGAAT TTGAAGGCAA GGTTACCTCT GCTTTTTAAT 6120
TTTTTTTTTT TTAAAGAAAG AAAAAACACT GAAAGAAACT TCAAAAGATC TGTTAATGCT 6180
AATACCTGAA TGTGGCATTT AACATGTCAT GGAAACTGCT TTGAATAAAT ACTTGAGAAA 6240
AGGAATGAAA TAATTGCCGT TTTTGTTGTT aAGTGAATGG GTGTGGTTTA ATCAGCGTAA 6300
35 TCATTTTTAT AAAACAGCTG TGAGACTGAA GTGGAATCCT TATTAAATGT GGAAAATGGC 6360
CTTTGAGGAT TACAGTAGAG ATTCAACTAA GAGAGTAAAT AAAGCTTGAA ACTAATTCGT 6420
TGTAAATTGC TTCTACAATC ATTGCTCTAT ATAGCATGCT ATTGCCAATC AGTTTTATGT 6480
ATTAAGACCT ATCAGCATGT CTTTTTTAGG TTGACCTCAT TTTAAATTAT AAGATGCTCT 6540
CTGTACCGTT TTAACATTTC CAGGATTTAT TCTTTCTAGG CAAATTCCAC TGGACTGTTT 6600
40 CCATTGTAGA
AGCTTCCTTA TAGATTCTTC AAATGAAGCT TACAGTGTGC TTTCTTGGGG 6660
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-45 -
TTTTGATTTG CACTAAATTT TATTITCTGA AAGATCACTT ATGTTTATAA TGTAGTGCTT
6720
TGTCTTAACA ATTAAACTTT CCAGCACTCA TGCA
[0087] The mouse p42AuF1 amino acid sequence of GenBank
Accession No.
NP 001070734.1 (SEQ ID NO:20) is as follows:
MSEEQFGGDG AAAAATAAVG GSAGEQEGAM VAAAAQGPAA AAGSGSGGGG SAAGGTEGGS 60
AEAEGAKIDA SKNEEDEGKM FIGGLSWDTT KKDLKDYFSK FGEVVDCTLK LDPITGRSRG
120
FGFVLFKESE SVDKVMDQKE HKLNGKVIDP KRAKAMKTKE PVKKIFVGGL SPDTPEEKIR
180
EYFGGFGEVE SIELPMDNKT NKRRGFCFIT FKEEEPVKKI MEKKYHNVGL SKCEIKVAMS
240
KEQYQQQQQW GSRGGFAGRA RGRGGGPSQN WNQGYSNYWN QGYGNYGYNS QGYGGYGGYD
300
YTGYNNYYGY GDYSNQQSGY GKVSRRGGHQ NSYKPY
[0088] The mouse p45AuF1 nucleotide sequence of GenBank
Accession No.
NM 001077265.2 (SEQ ID NO:23) is as follows:
CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA GTGGCCGCCG
60
CTGCTACTTC ATTCTTTTTT TTTTCAGTGC AGCCGGGGAG AGCGAGAGAG CGCGCTGCGC
120
GAGAGTGGGA GGCGAGGGGG GCAGGCCGGG GAGAGGCGCA GGAGCCCTTG CAGCCACGCG 180
CGCGCCTTGT CTAGGGTGCC TCGCGAGGTA GAGCGGGCAT CGCGCGGCGG CGGCGGGGAT
240
TACTTTGCTG CTAGTTTCGG TTCGCGGCGG CGGCGGCGTC GGCGGGTGTC GTCTTCGGCG
300
GCGGCAGTAG CACTATGTCG GAGGAGCAGT TCGGAGGGGA CGGGGCGGCG GCGGCGGCAA
360
CGGCGGCGGT AGGCGGCTCG GCGGGCGAGC AGGAGGGAGC aATGGTGGCG GCGGCGGCGC
420
AGGGGCCGGC GGCGGCGGCG GGAAGCGGGA GCGGCGGCGG CGGCTCTGCG GCCGGAGGCA 480
CCGAAGGAGG CAGCGCC GAG GCAGAGGGAG CCAAGATCGA CGCCAGTAAG AACGAGGAGG
540
ATGAAGGCCA TTCAAACTCC TCCCCACGAC ACACTGAAGC AGCGGCGGCA CAGCGGGAAG
600
AATGGAAAAT GTTTATAGGA GGCCTTAGCT GGGACACCAC AAAGAAAGAT CTGAAGGACT
660
ACTTTTCCAA ATTTGGTGAA GTTGTAGACT GCACTCTGAA GTTAGATCCT ATCACAGGGC
720
GATCAAGGGG TTTTGGCTTT GTGCTATTTA AAGAGTCGGA GAGTGTAGAT AAGGTCATGG 780
ATCAGAAAGA ACATAAATTG AATGGGAAAG TCATTGATCC TAAAAGGGCC AAAGCCATGA
840
AAACAAAAGA GCCTGTCAAA AAAATTTTTG TTGGTGGCCT TTCTCCAGAC ACACCTGAAG
900
AAAAAATAAG AGAGTACTTT GGTGGTTTTG GTGAGGTTGA ATCCATAGAG CTCCCTATGG
960
ACAACAAGAC CAATAAGAGG CGTGGGTTCT GTTTTATTAC CTTTAAGGAA GAGGAGCCAG
1020
TGAAGAAGAT AATGGAAAAG AAATACCACA ATGTTGGTCT TAGTAAATGT GAAATAAAAG 1080
TAGCCATGTC AAAGGAACAG TATCAGCAGC AGCAGCAGTG GGGATCTAGA GGAGGGTTTG
1140
CAGGCAGAGC TCGCGGAAGA GGTGGAGGCC CCAGTCAAAA CTGGAACCAG GGATATAGTA
1200
ACTATTGGAA TCAAGGCTAT GGCAACTATG GATATAACAG CCAAGGTTAC GGAGGTTATG
1260
GAGGATATGA CTACACTGGT TACAACAACT ACTATGGATA TGGTGATTAT AGCAATCAGC
1320
AGAGTGGTTA TGGGAAAGTA TCCAGGCGAG GTGGACATCA AAATAGCTAC AAACCATACT 1330
AAATTATTCC ATTTGCAACT TATCCCCAAC AGGTGGTGAA GCAGTATTTT CCAATTTGAA
1440
GATTCATTTG AAGGTGGCTC CTGCCACCTG CTAATAGCAG TTCAAACTAA ATTTTTTCTA
1500
TCAAGTTCCT GAATGGAAGT ATGACGTTGG GTCCCTCTGA AGTTTAATTC TGAGTTCTCA
1560
TTAAAAGAAT TTGCTTTCAT TGTTTTATTT CTTAATTGCT ATGCTTCAGT ATCAATTTGT
1620
GTTTTATGCC CCCCCTCCCC CCCAGTATTG TAGAGCAAGT CTTGTGTTAA AAAAAGCCCA 1680
GTGTGACAGT GTCATGATGT AGTAGTGTCT TACTGGTTTT TTAATAAATC CTTTTGTATA
1740
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-46 -
AAAATGTATT GGCTCTTTTA TCATCAGAAT AGGAGGAAGT GAAATACTAC AAATGTTTGT 1800
CTTGGATTCA AGTCACTAGA AGCATAAATT TGAGGGGATA AAAACAACGG TAAACTTTGT 1860
CTGAAAGAGG GCATGGTTAA AAATGTAGTG AATTTTAAAT GTTTTTAGCA AAATTTGATT 1920
TTGCCCAAGA ATCCCTGTCT GAATTGGAAA TGACTTAATG TAGTCAATGT GCTTGTTGGT 1980
TGTCTTAATA TTACTTCTGT AGCCATTAAG TTTTATGAGT AACTTCCCAA ATACCCACGT 2040
TTTTCTTTAT ATGTATTGTG CTTTTTAAAA ACAAATCTGG AAAAATGGGC AAGAACATTT 2100
GCAGACAATT GTTTTTAAGC TTCCATTAAA TAAAAAAAAT GTGGACTTAA GGAAATCTAT 2160
TAATTTAAAT AGAACTGCAG CTAGTTTAGA GAGTATTTTT TTCTTAAAGC TTTGGTGTAA 2220
TTAGGGAAGA TTTTAAAAAA TGCATAGTGT TTATTTGTAT GTGTGCTCTT TTTTTAAGTC 2280
10 AATTTTTGGG GGGTTGGTCT GTTAACTGAG TCTAGGATTT AAAGGTAAGA TGTTCCTAGA 2340
AATCTTGTCA TCCCAAAGGG GCGGGCGCTA AGGTGAAACT TCAGGGTTCA GTCAGGGTCA 2400
CTGCTTTATG TGTGAAATCA CTCAAATTGG TAAGTCTCTT ATGTTAGCAT TCAGGACATT 2460
GATTTCAACT TGGATGGACA ATTTATAGTT ACTACTGAAT TGTGTGTTAA TGTGTTCAGT 2520
CCTGGTAAGT TTTCAGTTTG ATCAGTTAGT TGGAAGCAGA CTTGAAGAGC TGTTAGTCAC 2580
GTGAGCCATG GGTGCAGTCG ATCTGTGGTC AGATGCCTGA GTCTGTGATA GTGAATTGTG 2640
TCTAAAGACA TTTTAATGAT AAAAGTCAGT GCTGTAAAGT TGAAAGTTCA TGAGAGACAT 2700
ACAATGAGGG CTGCAGCCCA TTTTTAAAAA aATTATAATA CAAAAGTATG CACATTTGTT 2760
TACATATCCC TGCCTTTGTA TTACAGTGGC AGGTTTGTGT ACTTAAACTG GGAAAGCCTC 2820
AGATCTATGA TTACCTGGCC TATCATAGAA AGTGTCTAAA TAAATCACTC TGTCAATTGA 2880
20 ATACATTAGT ATTAGCTAGC ATACTTCATT ATGCCTGTTT TCCATAAATA CCACACCAAA 2940
AACTTGCTTG GGGCAGTTTG AGCCTAGTTC AT GAGCT GCT AT CAGAT T GG T CT T GAT C CT
3000
ATATAATAGG CCAAATGTCT GTAAACAGCT GTGCTGGTGG AATGTAGAAA GTCACTGCAC 3060
TCAGATTCAA CTTCCTGATT GGAAGTCATC ACAGTGTGAT TAAACATTTT CACAAAGAAT 3120
AGTAGATAAA TAACTTGGTT TTTAATGTTA ACTTTGTTTC aATTAAGTCA CATTTAAAAA 3180
CTTATCCTCA CGCCTACCTG AGTTAATTAT CTGTTGACCT AGATATCTTT CTGGCCACTC 3240
ACTGACTTAT TTCTTGAACT TTTGCCATTT GCATAAATCT TGTCAGCTTT GTTCTTGATT 3300
ATGCATTGTC CAGGCTGAGC TAGTTGTCTT TCCAGGAATC CCTTTGTCTC TGAATTAGGT 3360
CCTTTGTTTC CTAAATCATC CTGCTTGTTT GGCACAAGTC TTCCCAGGCC AGTGAGACCT 3420
CCGTGTCCTC TCAGCACCAT AGGGGTAGGT AACCCTGGTT AGGCTGGACA GGGGTTTGCT 3480
GAGGGAGTTT GTTCATTTGA ATCTAGGTCT TACATGACGT CTTTCAAATA GGGTTTTTAC 3540
CTTGACACTA AACTGTCCAG TCTAAGCAGT TCTGCAAAAT GTGAGGGAAT TATGAACTTC 3600
TTCCTGCAGT GGGTTTTTAT GGTTTTGGTT TGTTTTTTGT TGTTTTGGTT CTTTGTTGAG 3660
CCCTGGACAA AAACTTCCCT AGTTCTGGTT TCTACAATTT AAATTAAAAA aAGAATTCAT 3720
CTTAGAATTT TTCACCCTCT TCCCCAACTA TTCTAATCAA TCTTAAGTAT GCCCTTCATC 3780
35 TTTTTTCCTT CCTAAGGCTT TTACTGATAG TGTAATTCCG TACTCTTCAA CCCTGGGAAG 3840
GCTGAAGTGG ATTCTTGAGC TCATTTCAAG GCTGACCTGG GTGTTGGCAA GAACCCAGCT 3900
TAGAACAAAC ACATGCAAGG CCATCTTACC TTACATCCTG TTGCTTGGAC TTCTTCCTGC 3960
T CAAAGT T TT TAGT GGAT GC TAAGT GAT CT TTGCTTCCAC TGAGGAGT GG AACACT T TAG
4020
AATGAACCTC TAGATAGATA T TT TTAT T GT CT GGTGAGGG TTACTGGAGT T T CCCACC CT
4080
GCCTGAAGGG T GAAT CT GGC TTACAGT GTT CT CATCT CAA AGGGAAGAAG GCAGATGGCT
4140
GT GT CCAGAG AGAGCCAT CA CAGTTTGCTT CAGAGACACT AGAATGGGCT GGAAGAT C TA 4200
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-47 -
GTGGTCTTAA TCAGACTTGA AACCTGGCCT TTCTTCATTA CCCATATGTC TACCAGTACT 4260
TGGGCTAACA CTTAAGCCAT TAGGGCCTTT GTAGGGGTGT TTTGAGACCC CCTCCATGCT 4320
AACAAATATA CAGGTTTCTT AACATTTGCT CATAAACTTG TAAAGCTTAC TTTCTCTTAA 4330
TCCACCCCAC ATTTAACAAG CCCTGGTACT TAGAATTTCA GAAGAGTAAT GGCAGGTAGG 4440
5 TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGAGAGAGAG AGAGAGAGAG 4500
AGAGAGAGAG AGAGAGAGAG AGAGAGAGAG AGAGAGAAGT TTGTGGAAAA TCAGGTAATG 4560
ACAGCTCATC CTTTTAGAAT TGTACTTCAG AATAGAAACA TTTGGTGGGC TGTTAGGTAG 4620
CTTTGATTAC TTGTGGGTAG ACCTGCTAGT ATTGCCAGTC CTCAAGCAAT GAGCTTTCTG 4680
TATCTTGTTT ACTAGATATA TACTACCAGG TGAGTCATTT CCTGGGGTTC TGTTTTCTTT 4740
10 TAAAATCTTT CCCTAAACTT AATATGTATT AAAAAGTCTG GCTTTTCAGT CCATTCTTTG 4800
TGCACTGGGA TGGCAATTGC TTCATTATAT GACAATTGCT GTTCCCAAGT CAGAATTCAG 4860
TGTGCTGATT TGACATCAGT TCGTCCCGAA TAAGTTCCTG TTACCAGGAT TTACATTCAG 4920
CACATTAGAA ACTTGTTGGT GTGCTTTTAT TCTTGGAGCA TTTTCCTTAG ACTACCTTCC 4980
ACTTTGAGTG CTCTGTTTAG GATGTTGAGG TGTTAGGATT CTTGACAGCC AGAAAGACTG 5040
15 AACCCACTAT CTGGGCACAG TGTTCGTGTT GCTCTATAAA TGTATGCTTT TTTTaATTTG 5100
GGGTTGTTTT ACCTACATTG TCAAACTAGA TCCATGCTTA ACAGTGATAA TGAAGGCTTT 5160
TTGTTTGTTT TGTTTGTGGG TCCTCCCCCC CCCCCCAAGA CAGGGTTTCT CTGTAGGCTG 5220
TCCTAGAACT TGTTCTTTTT TAACCAAAAT TTGGCAAGGC TGAAAATGGA ATCCTATAAT 5280
CAATGCTGGC CAGATTAAAG TTAATAGTTG AGAAGTCTTG TCTGAATTTC CTTGGGCAAA 5340
20 AAGATTCTAG CCAGTTCAAT ACCCTGTTGT GCAAATTCAA TTTGCTGTTA TAATTTGCTC 5400
TCAGTTATCA GTTGGAAGGA GGTTAATTCT AATGTACTTG GAAGAGGCCT GTAGACCATC 5460
TATAACTGCA TCAGTTGTAC AGCGTTGTTG CCTGGGATTC TCTAGTTCAE ATAAACTCCC 5520
AAGTCTTAGC CGTGGTGATG GCTACAGTGT GGAAGATGGT GAGCATTCTA GTGAGTATCG 5580
CGATGACGGC AGTAAAGAGC AGCAGGCAGC CGTGGCTGGG CTCACTGACC GTGGCTGTAA 5640
25 GTTACGGAGG CAGCACACAC TTCTGTACAC ACCTCTCATC AGTTACCGGA GTCATTGCAT 5700
TGCGGACTAA CTGGCTGACT CAAGTTGTCT TGCTACTGAA GTCTTGAGTT GGTCTCATGC 5760
ATTTACCCTG TTGACTTGAG CACCTTAAAG TCGAAAGGAT GTCTGGTTGT GGCTTTATTG 5820
TAAACAGCCT TAGGTAAAGA GGGGAGTATA TCGGTTAGGA AGGTGAAAAA TGATACTT CC 5880
AAGTTCAGTG GGAAACCCTG GGTTTATCCC CCAGCTTAAG AAAGAATGCC TAACAATGTT 5940
30
TCAGAATTAG ATTCTGTGGA AGGTGAGGGT GTTAGAACAG TCCAAATTTG TTATTGTAGA 6000
CTTGCAGTGG GAGGAATTTT TAAATATACA GATCAGTCGA CACTCATTAA CTTCACTGAT 6060
AAAGGTGGAA ACGGATGTGG CAACACTTCT AAGTTCATTT GTATATGTTT GTAATTTGAT 6120
TGCTTGTATT CTCTTGCACT CTAGAATTTG AAGGCAAGGT TACCTCTGCT TTTTAATTTT 6180
TTTTTTTTTA AAGAAACAAA AAACACTGAA AGAAACTTCA AAAGATCTCT TAATCCTAAT 6240
35
ACCTGAATGT GGCATTTAAC ATGTCATGGA AACTGCTTTG AATAAATACT TGAGAAAAGG 6300
AATGAAATAA TTGCCGTTTT TGTTGTTGAG TGAATGGGTG TGGTTTAATG AGCGTAATCA 6360
TTTTTATAAA ACAGCTGTGA GACTGAAGTG GAATCCTTAT TAAATGTGGA AAATGGCCTT 6420
TGAGGATTAC AGTAGAGATT CAACTAAGAG AGTAAATAAA GCTTGAAACT AATTCGTTGT 6480
AAATTGCTTC TAGAATCATT GCTCTATATA GCATGCTATT GCCAATCAGT TTTATGTATT 6540
40
AAGACCTATC AGCATGTCTT TTTTAGGTTG ACCTCATTTT AAATTATAAG ATGCTCTCTG 6600
TACCGTTTTA ACATTTCCAG GATTTATTCT TTCTAGGCAA ATTCCACTGG ACTGTTTCCA 6660
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-48 -
TTGTAGAAGC TTCCTTATAG ATTCTTCAAA TGAAGCTTAC AGTGTGCTTT CTTGGGGTTT
6720
TGATTTGCAC TAAATTTTAT TTTCTGAAAG ATCACTTATG TTTATAATGT AGTGCTTTGT
6760
CTTAACAATT AAACTTTCCA GCACTCATGC A
[0089] The mouse p45AuF1 amino acid sequence of GenBank
Accession No.
NP 001070733.1 (SEQ ID NO:24) is as follows:
MSEEQFGGDG AAAAATAAVG GSAGFQEGAM VAAAAQGPAA AAGSGSGGGG SAAGGTEGGS
60
AEAEGAKIDA SKNEEDEGHS NSSPRHTEAA AAQRFEWKMF IGGLSWDTTK KDLKDYFSKF
120
GEVVDCTLKL DPITGRSRGF GFVLFKESES VDKVMDQKEH KLNGKVIDPK RAKAMKTKEP
180
VKKIFVGGLS PDTPEEKIRE YFGGFGEVES IELPMDNKTN KRRGFCFITF KEEEPVKKIM
240
EKKYHNVGLS KCEIKVAMSK EQYQQQQQWG SRGGFAGRAR GRGGGPSQNW NQGYSNYWNQ 300
GYGNYGYNSQ GYGGYGGYDY TGYNNYYGYG DYSNQQSGYG KVSRRGGHQN SYKPY
[0090] It is noted that the sequences described herein may be
described with reference to
accession numbers that include, e.g., a coding sequence or protein sequence
with or without
additional sequence elements or portions (e.g., leader sequences, tags,
immature portions,
regulatory regions, etc.). Thus, reference to such sequence accession numbers
or corresponding
sequence identification numbers refers to either the sequence fully described
therein or some
portion thereof (e.g., that portion encoding a protein or polypepti de of
interest to the technology
described herein (e.g., AUF1 or a functional fragment thereof); the mature
protein sequence that
is described within a longer amino acid sequence; a regulatory region of
interest (e.g., promoter
sequence or regulatory element) disclosed within a longer sequence described
herein; etc).
Likewise, variants and isoforms of accession numbers and corresponding
sequence identification
numbers described herein are also contemplated.
[0091] Accordingly, in certain embodiments, the AUF1 protein
referred to herein has an
amino acid sequence as set forth in Table 2 and the sequences disclosed
herein, or is a functional
fragment thereof. In one embodiment, the functional fragment as referred to
herein includes an
amino acid sequence that has at least 80%, at least 85%, at least 90%, at
least 95%, at least 97%,
or at least 99% amino acid sequence identity to an amino acid sequence
disclosed herein.
[0092] In some embodiments, the AAV vector described herein
includes a nucleic acid
molecule encoding a nucleotide sequence set forth in Table 2 (or described
herein), or portions
thereof that encode a functional fragment of an AUF1 protein as described
supra.
[0093] As described in more detail below, compositions according
to the present
application may be useful in gene therapy, which includes both ex vivo and in
vivo techniques.
Thus, host cells can be genetically engineered ex vivo with a nucleic acid
molecule (or
polynucleotide), with the engineered cells then being provided to a patient to
be treated.
Delivery of the active agent of a composition described herein in vivo may
involve a process that
effectively introduces a molecule of interest (e.g., AUF1 protein or a
functional fragment
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-49 -
thereof) into the cells or tissue being treated. In the case of polypeptide-
based active agents, this
can be carried out directly or, alternatively, by transfecting
transcriptionally active DNA into
living cells such that the active polypeptide coding sequence is expressed and
the polypeptide is
produced by cellular machinery. Transcriptionally active DNA may be delivered
into the cells or
tissue, e.g., muscle, being treated using transfection methods including, but
not limited to,
electroporation, microinjection, calcium phosphate coprecipitation, DEAE
dextran facilitated
transfection, cationic liposomes, and retroviruses. In certain embodiments,
the DNA to be
transfected is cloned into a vector.
[0094] Alternatively, cells can be engineered in vivo by
administration of the
polynucleotide using techniques known in the art. For example, by direct
injection of a "naked"
polynucleotide (Feigner et al., "Gene Therapeutics,- Nature 349:351-352
(1991); U.S. Patent
No. 5,679,647; Wolff et al., "The Mechanism of Naked DNA Uptake and
Expression,- Adv.
Genet. 54:3-20 (2005), which are hereby incorporated by reference in their
entirety) or a
polynucleotide formulated in a composition with one or more other targeting
elements which
facilitate uptake of the polynucleotide by a cell.
[0095] Host cells that can be used with the vectors described
herein include, without
limitation, myocytes. The term "myocyte," as used herein, refers a cell that
has been
differentiated from a progenitor myoblast such that it is capable of
expressing muscle-specific
phenotype under appropriate conditions. Terminally differentiated myocytes
fuse with one
another to form myotubes, a major constituent of muscle fibers. The term
"myocyte" also refers
to myocytes that are de-differentiated. The term includes cells in vivo and
cells cultured ex vivo
regardless of whether such cells are primary or passaged. Myocytes are found
in all muscle
types, e.g., skeletal muscle, cardiac muscle, smooth muscle, etc. Myocytes are
found and can be
isolated from any vertebrate species, including, without limitation, human,
orangutan, monkey,
chimpanzee, dog, cat, rat, rabbit, mouse, horse, cow, pig, elephant, etc.
Alternatively, the host
cell can be a prokaryotic cell, e.g., a bacterial cell such as E. coil, that
is used, for example, to
propagate the vectors.
[0096] It may be desirable in certain circumstances to utilize
myocyte progenitor cells
such as mesenchymal precursor cells or myoblasts rather than fully
differentiated myoblasts.
Examples of tissue from which such cells can be isolated include placenta,
umbilical cord, bone
marrow, skin, muscle, periosteum, or perichondrium. Myocytes can be derived
from such cells,
for example, by inducing their differentiation in tissue culture. The present
application
encompasses not only myocyte precursor/progenitor cells, but also cells that
can be trans-
differentiated into myocytes, e.g., adipocytes and fibroblasts.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 50 -
[0097] Also encompassed are expression systems comprising
nucleic acid molecules
described herein Generally, the use of recombinant expression systems involves
inserting a
nucleic acid molecule encoding the amino acid sequence of a desired peptide
into an expression
system to which the molecule is heterologous (i.e., not native or not normally
present). One or
more desired nucleic acid molecules encoding a peptide described herein (e.g.,
AUF1) may be
inserted into the vector. When multiple nucleic acid molecules are inserted,
the multiple nucleic
acid molecules may encode the same or different peptides. The heterologous
nucleic acid
molecule is inserted into the expression system or vector in proper sense
(5'3') orientation
relative to the promoter and any other 5' regulatory molecules, and correct
reading frame.
[0098] The preparation of the nucleic acid constructs can be carried out
using standard
cloning procedures well known in the art as described by Joseph Sambrook et
al., MOLECULAR
CLONING: A LABORATORY MANUAL (Cold Springs Harbor 2012), which is hereby
incorporated
by reference in its entirety. U.S. Patent No. 4,237,224 to Cohen and Boyer,
which is hereby
incorporated by reference in its entirety, describes the production of
expression systems in the
form of recombinant plasmids using restriction enzyme cleavage and ligation
with DNA ligase.
These recombinant plasmids are then introduced by means of transformation and
replicated in a
suitable host cell.
[0099] A nucleic acid molecule encoding an AUF1 protein or
functional fragment thereof
and that is operatively coupled to a muscle-cell specific promoter (e.g.,
muscle creatine kinase
(MCK) promoter) may include an additional elements including, without
limitation, a leader
sequence, a suitable 3' regulatory region to allow transcription in the host
or a certain medium,
and/or any additional desired component, such as reporter or marker genes.
Such additional
elements may be cloned into the vector of choice using standard cloning
procedures in the art,
such as described in Joseph Sambrook et al., MOLECULAR CLONING: A LABORATORY
MANUAL
(Cold Springs Harbor 2012); Frederick M. Ausubel, SHORT PROTOCOLS IN MOLECULAR
BIOLOGY
(Wiley 2002); and U.S. Patent No. 4,237,224 to Cohen and Boyer, which are
hereby
incorporated by reference in their entirety.
[01001 In some embodiments, the adeno-associated viral vector
comprises a nucleic acid
molecule encoding a reporter protein. The reporter protein may be selected
from the group
consisting of, e.g., I3-galactosidase, chloramphenicol acetyl transferase,
luciferase, and
fluorescent proteins.
[01011 In certain embodiments, the reporter protein is a
fluorescent protein. Suitable
fluorescent proteins include, without limitation, green fluorescent proteins
(e.g., GFP, GFP-2,
tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP,
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-51 -
AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine,
Venus, YPet,
PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite,
mKalamal, GFPuv,
Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet,
AmCyanl,
Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed
monomer, mCherry,
mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2,
mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO,
Kusabira-
Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), or any other
suitable fluorescent
protein. In certain embodiments, the reporter protein is a fluorescent protein
selected from the
group consisting of green fluorescent protein (GFP), enhanced green
fluorescent protein (EGFP),
and yellow fluorescent protein (YFP).
[0102] In some embodiments, the reporter protein is luciferase.
As used herein, the term
"luciferase" refers to members of a class of enzymes that catalyze reactions
that result in
production of light. Luciferases have been identified in and cloned from a
variety of organisms
including fireflies, click beetles, sea pansy (Rendla), marine copepods, and
bacteria among
others. Examples of luciferases that may be used as reporter proteins include,
e.g., Rendla (e.g.,
Renilla reniformis) luciferase, Gaussia (e.g., Gaussia princeps) luciferase),
Metridia luciferase,
firefly (e.g., Photinus pyrahs luciferase), click beetle (e.g., Pyrearinus
termitillunnnans)
luciferase, deep sea shrimp (e.g., Oplophorus gracihrostris) luciferase).
Luciferase reporter
proteins include both naturally occurring proteins and engineered variants
designed to have one
or more altered properties relative to the naturally occurring protein, such
as increased
photostability, increased pH stability, increased fluorescence or light
output, reduced tendency to
dimerize, oligomerize, aggregate or be toxic to cells, an altered emission
spectrum, and/or altered
substrate utilization.
[0103] Purine-rich element binding protein f3 (Purr.) is a
transcriptional repressor of
smooth muscle a-actin (SMA) gene expression in growth-activated vascular
smooth muscle
cells. In some embodiments, the adeno-associated viral vector comprises a
nucleic acid
molecule encoding a purine-rich element binding protein 13 (Purfl) inhibitor.
[0104] siRNAs are double stranded synthetic RNA molecules
approximately 20-25
nucleotides in length with short 2-3 nucleotide 3' overhangs on both ends. The
double stranded
siRNA molecule represents the sense and anti-sense strand of a portion of the
target mRNA
molecule, in this case a portion of a Purfl mRNA. The sequence of Pur13 mRNA
is readily
known in the art and accessible to one of skill in the art for purposes of
designing siRNA and
shRNA oligonucleotides.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 52 -
[0105] siRNA molecules are typically designed to target a region
of the mRNA target
approximately 50-100 nucleotides downstream from the start codon Methods and
online tools
for designing suitable siRNA sequences based on the target mRNA sequences are
readily
available in the art (see, e.g., Reynolds et al., "Rational siRNA Design for
RNA Interference,"
Nat. Biotech. 2:326-330 (2004); Chalk et al., "Improved and Automated
Prediction of Effective
siRNA," Bioehem. Biophys. Res. Comm. 319(1):264-274 (2004), Zhang et al.,
"Weak Base
Pairing in Both Seed and 3' Regions Reduces RNAi Off-targets and Enhances
si/shRNA
Designs," Nucleic Acids Res. 42(19):12169-76 (2014), which are hereby
incorporated by
reference in their entirety). Upon introduction into a cell, the siRNA complex
triggers the
endogenous RNA interference (RNAi) pathway, resulting in the cleavage and
degradation of the
target mRNA molecule.
[0106] Short or small hairpin RNA ("shRNA") molecules are
similar to siRNA
molecules in function, but comprise longer RNA sequences that make a tight
hairpin turn.
shRNA is cleaved by cellular machinery into siRNA and gene expression is
silenced via the
cellular RNA interference pathway. Methods and tools for designing suitable
shRNA sequences
based on the target mRNA sequences (e.g., Purf3 mRNA sequences) are readily
available in the
art (see e.g., Taxman et al., "Criteria for Effective Design, Constructions,
and Gene Knockdown
shRNA Vectors," BMC Biotech. 6:7 (2006) and Taxman et al., "Short Hairpin RNA
(shRNA):
Design, Delivery, and Assessment of Gene Knockdown," Meth. Mol. Biol. 629: 139-
156 (2010),
which are hereby incorporated by reference in their entirety).
[0107] Other suitable agents that can be encoded by the
recombinant construct disclosed
herein for purposes of inhibiting Pur13 include microRNAs ("miRNAs"). miRNAs
are small,
regulatory, noncoding RNA molecules that control the expression of their
target mRNAs
predominantly by binding to the 3' untranslated region (UTR). A single UTR may
have binding
sites for many miRNAs or multiple sites for a single miRNA, suggesting a
complex post-
transcriptional control of gene expression exerted by these regulatory RNAs
(Shulka et al.,
"MicroRNAs: Processing, Maturation, Target Recognition and Regulatory
Functions," Mol.
Cell. Pharmacol. 3(3):83-92 (2011), which is hereby incorporated by reference
in its entirety).
Mature miRNA are initially expressed as primary transcripts known as a pri-
miRNAs which are
processed, in the cell nucleus, to 70-nucleotide stem-loop structures called
pre-miRNAs by the
microprocessor complex. The dsRNA portion of the pre-miRNA is bound and
cleaved by Dicer
to produce a mature 22 bp double-stranded miRNA molecule that can be
integrated into the
RISC complex; thus, miRNA and siRNA share the same cellular machinery
downstream of their
initial processing.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 53 -
[0108] microRNAs known to inhibit the expression of Pur13
molecules are known in the
art and suitable for incorporation into the recombinant genetic const.ruct
described herein For
example, miR-22,miR-208b, and miR-499 are known to modulate expression of
Pur13 (see, e.g.,
Gurha et al., "Targeted Deletion of MicroRNA-22 Promotes Stress-Induced
Cardiac Dilation and
Contractile Dysfunction," Circulation 125(22):2751-2761 (2012) and Simionescu-
Bankston &
Kumar, "Noncoding RNAs in the Regulation of Skeletal Muscle Biology in Health
and Disease,"
Mol Med. 94(8):853-866 (2017), which are hereby incorporated by reference in
their entirety).
[0109] A variety of genetic signals and processing events that
control many levels of
gene expression (e.g., DNA transcription and messenger RNA ("mRNA")
translation) can be
incorporated into the nucleic acid construct to maximize protein production.
For the purposes of
expressing a cloned nucleic acid sequence encoding a desired protein, it is
advantageous to use
strong promoters to obtain a high level of transcription.
[0110] There are other specific initiation signals required for
efficient gene transcription
and translation in eukaryotic cells that can be included in the nucleic acid
construct to maximize
protein production. Depending on the vector system and host utilized, any
number of suitable
transcription and/or translation elements, including constitutive, inducible,
and repressible
promoters, as well as minimal 5' promoter elements, enhancers or leader
sequences may be used.
[0111] In some embodiments, the Puri3 inhibitor is a
polypeptide. In a more specific
embodiment, the Pur13 inhibitor is an antibody. As used herein, the term
"antibody" is meant to
include intact immunoglobulins derived from natural sources or from
recombinant sources, as
well as immunoreactive portions (i.e. antigen binding portions) of intact
immunoglobulins.
Antibodies may exist in a variety of forms including, for example, polyclonal
antibodies,
monoclonal antibodies, intracellular antibodies, antibody fragments (e.g. Fv,
Fab, and F(ab)2),
single chain antibodies (scFv), single-domain antibodies, chimeric antibodies,
and humanized
antibodies (Ed Harlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL
(Cold
Spring Harbor Laboratory Press, 1999); Houston et al., "Protein Engineering of
Antibody
Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain
Fv Analogue
Produced in Escherichia coli," Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);
Bird et al,
"Single-Chain Antigen-Binding Proteins," Science 242:423-426 (1988), which are
hereby
incorporated by reference in their entirety).
[0112] Adeno-associated viral vectors and recombinant adeno-
associated virus (AAV)
vectors are well known delivery vehicles that can be constructed and used to
deliver a nucleic
acid molecule to cells, as described in Shi et al., "Therapeutic Expression of
an Anti-Death
Receptor-5 Single-Chain Fixed Variable Region Prevents Tumor Growth in Mice,"
Cancer Res.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 54 -
66:11946-53 (2006); Fukuchi etal., "Anti-A13 Single-Chain Antibody Delivery
via Adeno-
Associated Vim s for Treatment of Alzheimer's Disease," Neurohiol. Dis. 23:502-
511 (2006);
Chatterjee et al., "Dual-Target Inhibition of HIV-1 In Vitro by Means of an
Adeno-Associated
Virus Antisense Vector," Science 258:1485-1488 (1992); Ponnazhagan et al.,
"Suppression of
Human Alpha-globin Gene Expression Mediated by the Recombinant Adeno-
associated Virus 2-
based Antisense Vectors," J. Exp. Med. 179:733-738 (1994), which are hereby
incorporated by
reference in their entirety. In vivo use of these vehicles is described in
Flotte etal., "Stable In
Vivo Expression of the Cystic Fibrosis Transmembrane Conductance Regulator
With an Adeno-
Associated Virus Vector," Proc. Nat'l. Acad. Sci. 90:10613-10617 (1993), which
is hereby
incorporated by reference in its entirety.
[0113] Recombinant adeno-associated virus (AAV) vectors provide
the ability to stably
transduce and express genes with very long-term (many years) duration in
skeletal muscle, and
depending on the AAV vector serotype and its modification, to do so with high
muscle-tropism
and selectivity whether using local intramuscular injection or systemic routes
of delivery
(Phillips et al., "Systemic Gene Transfer to Skeletal Muscle Using
Reengineered AAV Vectors,"
Methods MoL Biol. 709:141-51(2011) and Muraine et al., "Transduction
Efficiency of Adeno-
Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal
Muscle," Hum.
Gene Ther. 31(3-4):233-240 (2020), which are hereby incorporated by reference
in their
entirety). Moreover, for certain AAV serotypes and engineered variants,
particularly AAV8 and
its engineered variants, studies in mice have been shown to be predictive of
human skeletal
muscle transduction and gene expression, as found in clinical trials for
skeletal muscle
transmission and expression (Phillips et al., "Systemic Gene Transfer to
Skeletal Muscle Using
Reengineered AAV Vectors,- Methods Mot Biol. 709:141-51(2011) and Muraine et
al.,
"Transduction Efficiency of Adeno-Associated Virus Serotypes After Local
Injection in Mouse
and Human Skeletal Muscle," Hum. Gene Ther. 31(3-4):233-240 (2020), which are
hereby
incorporated by reference in their entirety).
[0114] The AAV vector described herein may comprise a sequence
isolated or derived
from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5
(AAV5), 6
(AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11) or any
combination
thereof.
[0115] In some embodiments, the adeno-associated viral (AAV)
vector is a recombinant
vector.
[0116] In one particular embodiment, the AAV vector is AAV8.
AAV8 derived from
macaques is very poorly immunogenic, resulting in long-term expression of the
encoded
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 55 -
transgene (for many years), and efficiently transduce skeletal muscle with
high tropism and
selectivity in both human and mouse (Phillips et a!, "Systemic Gene Transfer
to Skeletal Muscle
Using Reengineered AAV Vectors," Methods Mol. Biol. 709:141-51 (2011); Muraine
et al.,
"Transduction Efficiency of Adeno-Associated Virus Serotypes After Local
Injection in Mouse
and Human Skeletal Muscle," Hum. Gene Ther. 31(3-4):233-240 (2020);
Blankinship et al.,
"Efficient Transduction of Skeletal Muscle Using Vectors Based on Adeno-
associated Virus
Serotype 6," Mol. Ther. 10(4):671-8 (2004), and Gregorevic et al., "Viral
Vectors for Gene
Transfer to Striated Muscle," Curr. Opin. Mol. Ther. 6(5):491-8 (2004), which
are hereby
incorporated by reference in their entirety). AAV8 shows essentially no liver
tropism, is largely
specific for skeletal fibers and satellite cells, and has been shown to
transduce skeletal muscles
throughout the body (Wang et al., "Construction and Analysis of Compact Muscle-
specific
Promoters for AAV Vectors," Gene Ther. 15(22):1489-99 (2008), which is hereby
incorporated
by reference in its entirety).
[0117]
According to one embodiment, the adeno-associated viral (AAV) vector is an
AAV8 vector with the nucleotide sequence of SEQ ID NO:25.
AAV8 AtTF1 Construct Sequence (SEQ ID NO:25)
CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC
60
GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGaAGAGAG GGAGTGGCCA 120
ACTCCATCAC TAGGGGTTCC TGCGGCCTAA GGCAATTGGC CACTACGGGT CTAGGCTGCC
180
CAT GTAAGGA GGCAAGGCCT GGGGACACCC GAGATGCCTG GT TATAAT TA ACCCCAACAC
240
CTGCTGCCCC CCCCCCCCAA CACCTGCTGC CTGAGCCTGA GCGGTTACCC CACCCCGGTG
300
CCTGGGTCTT AGGCTCTGTA CACCATGGAG GAGAAGCTCG CTCTAAAAAT AACCCTGTCC
360
CTGGTGGATC GCCACTACGG GTCTAGGCTG CCCATGTAAG GAGGCAAGGC CTGGGGACAC 420
CCGAGATGCC TGGTTATAAT TAACCCCAAC ACCTGCTGCC CCCCCCCCCC AACACCTGCT
480
GCCTGAGCCT GAGCGGTTAC CCCACCCCGG TGCCTGGGTC TTAGGCTCTG TACACCATGG
540
AGGAGAAGCT CGCTCTAAAA ATAACCCTGT CCCTGGTGGA TCGCCACTAC GGGTCTAGGC
600
TGCCCATGTA AGGAGGCAAG GCCTGGGGAC ACCCGAGATG CCTGGTTATA ATTAACCCCA
660
ACACCTGCTG CCCCCCCCCC CCAACACCTG CTGCCTGAGC CTGAGCGGTT ACCCCACCCC 720
GGTGCCTGGG TCTTAGGCTC TGTACACCAT GGAGGAGAAG CTCGCTCTAA AAATAACCCT
780
GTCCCTGGTG GATCCCTCCC TGGGGAaAGC CCCTCCTGGC TAGTCACACC CTGTAGGCTC
840
CTCTATATAA CCCAGGGGCA CAGGGGCTGC CCCCGGGTCA CCGCTAGCCA AAGCTTCTCG
900
AGGCTGGCTA GTTAAGCTAT CAACAAGTTT GTACAGAAAA GCAGGCTTTA AAGGAACCAA
960
TTCAGTCGAC GCTAGCAAGC TTGGTACCGG ATCCGAATTC CACCATGTCG GAGGAGCAGT 1020
TCGGAGGGGA CGGGGCGGCG GCGGCGGCAA CGGCGGCGGT AGGCGGCTCG GCGGGCGAGC
1080
AGGAGGGAGC CATGGTGGCG GCGGCGGCGC AGGGGCCGGC GGCGGCGGCG GGAAGCGGGA
1140
GCGGCGGCGG CGGCTCTGCG GCCGGAGGCA CCGAAGGAGG aAGCGCCaAG GCAGAGGGAG
1200
CCAAGATCGA CGCCAGTAAG AACGAGGAGG ATGAAGGCCA TTCAAACTCC TCCCCACGAC
1260
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 56 -
ACACTGAAGC AGCGGCGGCA CAGCGGGAAG AATGGAAAAT GTTTATAGGA GGCCTTAGCT 1320
GGGACACCAC AAAGAAAGAT CTGAAGGACT ACTTTTCCAA ATTTGGTGAA GTTGTAGACT 1380
GCACTCTGAA GTTAGATCCT ATCACAGGGC GATCAAGGGG TTTTGGCTTT GTGCTATTTA 1440
AAGAGTCGGA GAGTGTAGAT AAGGTCATGG ATCAGAAAGA ACATAAATTG AATGGGAAAG 1500
TCATTGATCC TAAAAGGGCC AAAGCCATGA AAACAAAAGA GCCTGTCAAA AAAATTTTTG 1560
TTGGTGGCCT TTCTCCAGAC ACACCTGAAG AAAAAATAAG AGAGTACTTT GGTGGTTTTG 1620
GTGAGGTTGA ATCCATAGAG CTCCCTATGG ACAACAAGAC CAATAAGAGG CGTGGGTTCT 1680
GTTTTATTAC CTTTAAGGAA GAGGAGCCAG TGAAGAAGAT AATGGAAAAG AAATACCACA 1740
ATGTTGGTCT TAGTAAATGT GAAATAAAAG TAGCCATGTC AAAGGAACAG TATCAGCAGC 1800
10 AGCAGCAGTG GGGATCTAGA GGAGGGTTTG aAGGCAGAGC TCGCGGAAGA GGTGGAGATC .. 1860
AGCAGAGTGG TTATGGGAAA GTATCCAGGC GAGGTGGACA TCAAAATAGC TACAAACCAT 1920
ACTAAGATAT CGCGGCCGCC TCGAGGACTA CAAGGATGAC GATGACAAGG ATTACAAAGA 1980
CGACGATGAT AAGGACTATA AGGATGATGA CGACAAATAA TAGCAATTCC TCGACGACTG 2040
CATAGGGTTA CCCCCCTCTC CCTCCCCCCC CCCTAACGTT ACTGGCCGAA GCCGCTTGGA 2100
15 ATAAGGCCGG TGTGCGTTTG TCTATATGTT ATTTTCCACC ATATTGCCGT CTTTTGGCAA 2160
TGTGAGGGCC CGGAAACCTG GCCCTGTCTT CTTGACGAGC ATTCCTAGGG GTCTTTCCCC 2220
TCTCGCCAAA GGAATGCAAG GTCTGTTGAA TGTCGTGAAG GAAGCAGTTC CTCTGGAAGC 2280
TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG aAGCGGAACC CCCCACCTGG 2340
CGACAGGTGC CTCTGCGGCC AAAAGCCACG TGTATAAGAT ACACCTGCAA AGGCGGCACA 2400
20 ACCCCAGTGC CACGTTGTGA GTTGGATAGT TGTGGAAAGA GTCAAATGGC TCTCCTCAAG 2460
CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC aATTGTATGG GATCTGATCT 2520
GGGGCCTCGG TGCACATGCT TTACATGTGT TTAGTCGAGG TTAAAAAACG TCTAGGCCCC 2580
CCGAACCACG GGGACGTGGT TTTCCTTTGA AAAACACGAT GATAATGGCC ACAACTAGTG 2640
CCACCATGGT GAGCAAGGGC GAGGAGCTGT TCACCGGGGT GGTGCCCATC CTGGTCGAGC 2700
25 TGGACGGCGA CGTAAACGGC CACAAGTTCA GCGTGTCCGG CGAGGGCGAG GGCGATGCCA 2760
CCTACGGCAA GCTGACCCTG AAGTTCATCT GCACCACCGG CAAGCTGCCC GTGCCCTGGC 2820
CCACCCTCGT GACCACCCTG ACCTACGGCG TGCAGTGCTT aAGCCGCTAC CCCGACCACA 2880
TGAAGCAGCA CGACTTCTTC AAGTCCGCCA TGCCCGAAGG CTACGTCCAG GAGCGCACCA 2940
TCTTCTTCAA GGACGACGGC AACTACAAGA CCCGCGCCGA GGTGAAGTTC GAGGGCGACA 3000
30 CCCTGGTGAA CCGCATCGAG CTGAAGGGCA TCGACTTCAA GGAGGACGGC AACATCCTGG 3060
GGCACAAGCT GGAGTACAAC TACAACAGCC ACAACGTCTA TATCATGGCC GACAAGCAGA 3120
AGAACGGCAT CAAGGTGAAC TTCAAGATCC GCCACAACAT CGAGGACGGC AGCGTGCAGC 3180
TCGCCGACCA CTACCAGCAG AACACCCCCA TCGGCGACGG CCCCGTGCTG CTGCCCGACA 3240
ACCACTACCT GAGCACCCAG TCCGCCCTGA GCAAAGACCC aAACGAGAAG CGCGATCACA 3300
35 TGGTCCTGCT GGAGTTCGTG ACCGCCGCCG GGATCACTCT CGGCATGGAC GAGCTGTACA 3360
AGTAAGTTTA AACTCTAGAC CCAGCTTTCT TGTACAAAGT GGTTGATCTA GAGGGCCCGT 3420
AACTAGTTGA GCGGCCGCAA CTCGAGACTC TAGAGGTTAA TCGATAATCA ACCTCTGGAT .. 3480
TACAAAATTT GTGAAAGATT GACTGGTATT CTTAACTATG TTGCTCCTTT TACGCTATGT 3540
GGATACGCTG CTTTAATGCC TTTGTATCAT GCTATTGCTT CCCGTATGGC TTTCATTTTC 3600
40 TCCTCCTTGT ATAAATCCTG GTTGCTGTCT CTTTATGAGG AGTTGTGGCC CGTTGTCAGG 3660
CAACGTGGCG TGGTGTGCAC TGTGTTTGCT GACGCAACCC CCACTGGTTG GGGCATTGCC 3720
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
VITI 2021/146711
PCT/US2021/013955
- 57 -
ACCACCTGTC AGCTCCTTTC CGGGACTTTC GCTTTCCCCC TCCCTATTGC CACGGCGGAA 3780
CTCATCGCCG CCTGCCTTGC CCGCTGCTGG ACAGGGGCTC GGCTGTTGGG CACTGACAAT 3840
TCCGTGGTGT TGTCGGGGAA ATCATCGTCC TTTCCTTGGC TGCTCGCCTG TGTTGCCACC 3900
TGGATTCTGC GCGGGACGTC CTTCTGCTAC GTCCCTTCGG CCCTCAATCC AGCGGACCTT 3960
CCTTCCCGCG GCCTGCTGCC GGCTCTGCGG CCTCTTCCGC GTCTTCGCCT TCGCCCTCAG 4020
ACGAGTCGGA TCTCCCTTTG GGCCGCCTCC CCGCATCGAA ACCCGCTGAC TAGACGACTG 4080
TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG 4140
AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA 4200
GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA aAGCAAGGGG GAGGATTGGG 4260
10 AAGACAATAG CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGCCGCGGGC CGCAGGAACC 4320
CCTAGTGATG GAGTTGGCCA CTCCCTCTCT GCGCGCTCGC TCGCTCACTG AGGCCGGGCG 4380
ACCAAAGGTC GCCCGACGCC CGGGCTTTGC CCGGGCGGCC TCAGTGAGCG AGCGAGCGCG 4440
CAGCTGCCTG CAGGGGCGCC TGATGCGGTA TTTTCTCCTT ACGCATCTGT GCGGTATTTC 4500
ACACCGCATA CGTCAAAGCA ACCATAGTAC GCGCCCTGTA GCGGCGCATT AAGCGCGGCG 4560
15 GGTGTGGTGG TTACGCGCAG CGTGACCGCT ACACTTGCCA GCGCCCTAGC GCCCGCTCCT 4620
TTCGCTTTCT TCCCTTCCTT TCTCGCCACG TTCGCCGGCT TTCCCCGTCA AGCTCTAAAT 4680
CGGGGGCTCC CTTTAGGGTT CCGATTTAGT GCTTTACGGC ACCTCGACCC CAAAAAACTT 4740
GATTTGGGTG ATGGTTCACG TAGTGGGCCA TCGCCCTGAT AGACGGTTTT TCGCCCTTTG 4800
ACGTTGGAGT CCACGTTCTT TAATAGTGGA CTCTTGTTCC AAACTGGAAC AACACTCAAC 4860
20 CCTATCTCGG GCTATTCTTT TGATTTATAA GGGATTTTGC CGATTTCGGC CTATTGGTTA 4920
AAAAATGAGC TGATTTAACA AAAATTTAAC GCGAATTTTA ACAAAATATT AACGTTTACA 4980
ATTTTATGGT GCACTCTCAG TACAATCTGC TCTGATGCCG CATAGTTAAG CCAGCCCCGA 5040
CACCCGCCAA CACCCGCTGA CGCGCCCTGA CGGGCTTGTC TGCTCCCGGC ATCCGCTTAC 5100
AGACAAGCTG TGACCGTCTC CGGGAGCTGC ATGTGTCAGA GGTTTTCACC GTCATCACCG 5160
25 AAACGCGCGA GACGAAAGGG CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA 5220
ATAATGGTTT CTTAGACGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG AACCCCTATT 5280
TGTTTATTTT TCTAAATACA TTCAAATATG TATCCGCTCA TGAGACAATA ACCCTGATAA 5340
ATGCTTCAAT AATATTGAAA AAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT 5400
ATTCCCTTTT TTGCGGCATT TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA 5460
30 GTAAAAGATG CTGAAGATCA GTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC 5520
AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC GAAGAACGTT TTCCAATGAT GAGCACTTTT 5580
AAAGTTCTGC TATGTGGCGC GGTATTATCC CGTATTGACG CCGGGCAAGA GCAACTCGGT 5640
CGCCGCATAC ACTATTCTCA GAATGACTTG GTTGAGTACT CACCAGTCAC AGAAAAGCAT 5700
CTTACGGATG GCATCACAGT AAGACAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC 5760
35
ACTGCGGCCA ACTTACTTCT GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG 5820
CACAACATGG GGGATCATGT AACTCGCCTT GATCGTTGGG AACCGGAGCT GAATGAAGCC 5880
ATACCAAACG ACGAGCGTGA CACCACGATG CCTGTAGCAA TGGCAACAAC GTTGCGCAAA 5940
CTATTAACTG GCGAACTACT TACTCTAGCT TCCCGGCAAC AATTAATAGA CTGGATGGAG 6000
GCGGATAAAG TTGCAGGACC ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT 6060
40 GATAAATCTG GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT 6120
GGTAAGCCCT CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA 6180
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 58 -
CGAAATAGAC AGATCGCTGA GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC
6240
CAAGTTTACT CATATATACT TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC
6300
TAGGTGAAGA TCCTTTTTGA TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC
6360
CACTGAGCGT CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG
6420
CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG 6480
GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA
6540
AATACTGTCC TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG
6600
CCTACATACC TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG
6660
TGTCTTACCG GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA
6720
ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC 6730
CTACAGCGTG AGCTATGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT
6840
CCGGTAAGCG GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC
6900
TGGTATCTTT ATAGICCTGT CGGGTTTCGC aACCTCTGAC TTGAGCGTCG ATTTTTGTGA
6960
TGCTCGTCAG GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC
7020
CTGGCCTTTT GCTGGCCTTT TGCTCACATG T 7051
[0118] Another aspect of the present application relates to a
composition comprising an
adeno-associated viral (AAV) vector as described herein.
[0119] In some embodiments, the composition of the present
application further
comprises a buffer solution.
[0120] The composition of the present application may further comprise one
or more
targeting elements. Suitable targeting elements include, without limitation,
agents such as
saponins or cationic polyamides (see, e.g.,U U.S. Patent Nos. 5,739,118 and
5,837,533, which are
hereby incorporated by reference in their entirety); microparticles,
microcapsules, liposomes, or
other vesicles; lipids; cell-surface receptors; transfecting agents; peptides
(e.g., one known to
enter the nucleus); or ligands (such as one subject to receptor-mediated
endocytosis). Suitable
means for using such targeting elements include, without limitation:
microparticle bombardment;
coating the polynucleotide with lipids, cell-surface receptors, or
transfecting agents;
encapsulation of the polynucleotide in liposomes, microparticles, or
microcapsules;
administration of the polynucleotide linked to a peptide which is known to
enter the nucleus; or
administration of the polynucleotide linked to a ligand subject to receptor-
mediated endocytosis
(see, e.g., Wu et al., "Receptor-Mediated in vitro Gene Transformation by a
Soluble DNA
Carrier System," I. Biol. Chem. 262:4429-4432 (1987), which is hereby
incorporated by
reference in its entirety), which can be used to target cell types
specifically expressing the
receptors. Alternatively, a polynucleotide-ligand complex can be formed
allowing the
polynucleotide to be targeted for cell specific uptake and expression in vivo
by targeting a
specific receptor (see, e.g., PCT Application Publication Nos. WO 92/06180, WO
92/22635, WO
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 59 -
92/203167, WO 93/14188, and WO 93/20221, which are hereby incorporated by
reference in
their entirety)
[0121] Thus, in some embodiments, the composition further
includes a transfection
reagent. The transfection reagent may be a positively charged transfection
reagent. Suitable
transfection reagents are well known in the art and include, e.g.,
Lipofectamine" RNAiMAX
(Invitrogen"), Lipofectamine 2000 (Invitrogen"), Lipofectamine" 3000
(Invitrogen"),
Invivofectamine 3.0 (Invitrogen"), Lipofectamine" MessengerMAX" (Invitrogen"),
Lipofectin' (Invitrogen'), siLentFet" (Bio-Rad), DharmaFECT" (Dharmacon),
HiPerFect
(Qiagen), TransIT-X2' (Mirus), jetMESSENGER" (Polyplus), Trans-Hi', JetPEI"
(Polyplus),
and ViaFectTM (Promega).
[0122] In some embodiments, the composition is an aqueous
composition. Aqueous
compositions of the present application comprise an effective amount of the
vector, dissolved or
dispersed in a pharmaceutically acceptable carrier or aqueous medium.
[0123] A further aspect of the present application relates to a
pharmaceutical composition
comprising an adeno-associated viral (AAV) vector described herein and a
pharmaceutically-
acceptable carrier.
[0124] The term "pharmaceutically acceptable carrier" refers to
a carrier that does not
cause an allergic reaction or other untoward effect in patients to whom it is
administered and are
compatible with the other ingredients in the formulation. Pharmaceutically
acceptable carriers
include, for example, pharmaceutical diluents, excipients, or carriers
suitably selected with
respect to the intended form of administration, and consistent with
conventional pharmaceutical
practices. For example, solid carriers/diluents include, but are not limited
to, a gum, a starch
(e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose, mannitol,
sucrose, dextrose), a
cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g.,
polymethylacrylate),
calcium carbonate, magnesium oxide, talc, or mixtures thereof.
Pharmaceutically acceptable
carriers may further comprise minor amounts of auxiliary substances such as
wetting or
emulsifying agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the
nucleic acid molecule described herein.
[0125] The vector(s) (i.e., adeno-associated viral (AAV) vector
and/or lentiviral vectors
disclosed herein) and/or pharmaceutical composition(s) disclosed herein can be
formulated
according to any available conventional method. Examples of preferred dosage
forms include a
tablet, a powder, a subtle granule, a granule, a coated tablet, a capsule, a
syrup, a troche, an
inhalant, a suppository, an injectable, an ointment, an ophthalmic ointment,
an eye drop, a nasal
drop, an ear drop, a cataplasm, a lotion and the like. In the formulation,
generally used additives
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 60 -
such as a diluent, a binder, a disintegrant, a lubricant, a colorant, a
flavoring agent, and if
necessary, a stabilizer, an emulsifier, an absorption enhancer, a surfactant,
a pH adjuster, an
antiseptic, an antioxidant, and the like can be used.
[0126] In addition, formulating a pharmaceutical composition can
be carried out by
combining compositions that are generally used as a raw material for
pharmaceutical
formulation, according to conventional methods. Examples of these compositions
include, for
example, (1) an oil such as a soybean oil, a beef tallow and synthetic
glyceride; (2) hydrocarbon
such as liquid paraffin, squalene, and solid paraffin; (3) ester oil such as
octyldodecyl myristic
acid and isopropyl myristic acid; (4) higher alcohol such as cetostearyl
alcohol and behenyl
alcohol, (5) a silicon resin, (6) a silicon oil, (7) a surfactant such as
polyoxyethylene fatty acid
ester, sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene
sorbitan fatty acid ester,
a solid polyoxyethylene castor oil and polyoxyethylene polyoxypropylene block
co-polymer; (8)
water soluble macromolecule such as hydroxyethyl cellulose, polyacrylic acid,
carboxyvinyl
polymer, polyethyleneglycol, polyvinylpyrrolidone and methylcellulose;
(9)lower alcohol such
as ethanol and isopropanol; (10) multivalent alcohol such as glycerin,
propyleneglycol,
dipropyleneglycol and sorbitol; (11) a sugar such as glucose and cane sugar;
(12) an inorganic
powder such as anhydrous silicic acid, aluminum magnesium silicicate, and
aluminum silicate;
(13) purified water, and the like.
[0127] Additives for use in the above formulations may include,
for example, (1) lactose,
corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, and
silicon dioxide as the
diluent; (2) polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl
cellulose, gum arabic,
tragacanth, gelatine, shellac, hydroxypropyl cellulose, hydroxypropylmethyl
cellulose,
polyvinylpyrrolidone, polypropylene glycol-poly oxyethylene-block co-polymer,
meglumine,
calcium citrate, dextrin, pectin, and the like as the binder; (3) starch,
agar, gelatine powder,
crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate,
dextrin, pectic,
carboxymethylcellulose/calcium, and the like as the disintegrant, (4)
magnesium stearate, talc,
polyethyleneglycol, silica, condensed plant oil, and the like as the
lubricant; (5) any colorant
whose addition is pharmaceutically acceptable is adequate as the colorant; (6)
cocoa powder,
menthol, aromatizer, peppermint oil, cinnamon powder as the flavoring agent;
(7) antioxidants
whose addition is pharmaceutically accepted such as ascorbic acid or alpha-
tophenol.
[0128] For parenteral administration in an aqueous solution, for
example, the solution
generally is suitably buffered and the liquid diluent first rendered isotonic
for example with
sufficient saline or glucose. Such aqueous solutions may be used, for example,
for intravenous,
intramuscular, subcutaneous and intraperitoneal administration. Preferably,
sterile aqueous
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 61 -
media are employed as is known to those of skill in the art, particularly in
light of the present
application By way of illustration, a single dose may be dissolved in 1 ml of
isotonic NaCl
solution and either added to 1000 ml of hypodermoclysis fluid or injected at
the proposed site of
infusion, (see, for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-
1038 and 1570-1580, which is hereby incorporated by reference in its
entirety). Some variation
in dosage will necessarily occur depending on the condition of the subject
being treated. The
person responsible for administration will, in any event, determine the
appropriate dose for the
individual subject. Moreover, for human administration, preparations should
meet sterility,
pyrogenicity, general safety, and purity standards as required by FDA Office
of Biologies
standards.
Age-Related Muscle Loss and Sarcopenia Muscle Atrophy
[0129] As described herein, advancing age and sedentary life-
style promotes significant
muscle loss that becomes largely irreversible with advancing age, including
very severe muscle
loss and atrophy with age (sarcopenia). Sarcopenia and age-related muscle loss
is a significant
source of morbidity and mortality in the aging and the elderly population.
Only physical
exercise is considered an effective strategy to improve muscle maintenance and
function, but it
must begin well before the onset of disease. There are few effective
therapeutic options.
[0130] The Examples of the present application demonstrate that
skeletal muscle
expression of the AUF1 gene is downregulated with age in mice. It is
hypothesized that skeletal
muscle expression of the AUF1 gene is also downregulated with age in humans,
thereby possibly
contributing to muscle loss with age. The results presented herein demonstrate
that AUF1
skeletal muscle gene transfer: (1) strongly enhances exercise endurance in
middle-aged (12
month; equivalent to 50-60 year old humans) and old mice (18 months;
equivalent to >70 years
of age humans) to levels of performance displayed by young mice (3 months old;
equivalent to
late teens, early 20's in humans); (2) stimulates both fast and slow muscle,
but specifically
specifies slow muscle lineage by increasing levels of expression of the gene
pgcla (Peroxisome
proliferator-activated receptor gamma co-activator 1-alpha), a major activator
of mitochondrial
biogenesis and slow-twitch myofiber specification; (3) significantly increases
skeletal muscle
mass and normal muscle fiber formation in middle age and old mice in age-
related muscle loss;
and (4) reduces expression of established biomarkers of muscle atrophy and
muscle
inflammation in age-related muscle loss.
[0131] Thus, another aspect of the present application relates
to a method of promoting
muscle regeneration. This method involves contacting muscle cells with an
adeno-associated
viral (AAV) vector or a composition described herein under conditions
effective to express
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 62 -
exogenous AUF1 in the muscle cells to increase muscle cell mass, increase
muscle cell
endurance, and/or reduce seam markers of muscle atrophy
[0132] As used herein, the terms "promote," "promotion," and
"promoting" refer to an
increase in an activity, response, condition, or other biological parameter,
including the
production, presence, expression, or function of cells, biomolecules or
bioactive molecules. The
terms "promote," "promotion," and "promoting include, but are not limited to,
initiation of an
activity, response, or condition, as well as initiation of the production,
presence, or expression of
cells, biomolecules, or bioactive molecules. The terms "promote," "promotion,"
and
"promoting" may also include measurably increasing an activity, response, or
condition, or
measurably increasing the production, presence, expression, or function of
cells, biomolecules,
or bioactive molecules, as compared to a native or control level.
[0133] Suitable cells for use according to the methods of the
present application include,
without limitation, mammalian cells such as rodent (mouse or rat) cells, cat
cells, dog cells,
rabbit cells, horse cells, sheep cells, pig cells, cow cells, and non-human
primate cells. In some
embodiments the cells are human cells.
[0134] In some embodiments, the muscle cells are selected from
the group consisting of a
myocyte, a myoblast, a skeletal muscle cell, a cardiac muscle cell, a smooth
muscle cell, and a
muscle stem cells (e.g., a satellite cell).
[0135] The method may be carried out in vitro or ex vivo.
[0136] In some embodiments, the method further involves culturing the
muscle cells ex
vivo under conditions effective to express exogenous AUF1.
[0137] In some embodiments, the method is carried out in vivo.
[0138] In some embodiments, the method further involves
contacting the muscle cells
with a purine-rich element binding protein 13 (Pur13) inhibitor. The Pur13
inhibitor may be a
nucleic acid molecule, a polypeptide, or a small molecule. In some
embodiments, the nucleic
acid molecule is selected from the group consisting of siRNA, shRNA, and
miRNA. Suitable
nucleic acid molecules are described in detail supra.
[0139] Contacting, according to the methods of the present
application, may be carried
out by oral administration, topical administration, transdermal
administration, parenteral
administration, subcutaneous administration, intravenous administration,
intramuscular
administration, intraperitoneal administration, by intranasal instillation
administration, by
intracavitary or intravesical instillation, intraocular administration,
intraarterial administration,
intralesional administration, or by application to mucous membranes. Thus, in
some
embodiments, the contacting is carried out by intramuscular administration,
intravenous
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 63 -
administration, subcutaneous administration, oral administration, or
intraperitoneal
administration to a subject In specific embodiments, the administering is
carried out by
intramuscular injection.
[0140] A further aspect of the present application relates to a
method of treating
degenerative skeletal muscle loss in a subject. This method involves selecting
a subject in need
of treatment for skeletal muscle loss and administering to the selected
subject an adeno-
associated viral (AAV) vector described herein or a composition described
herein under
conditions effective to cause skeletal muscle regeneration in the selected
subject.
[0141] In carrying out the methods of the present application,
"treating" or "treatment"
includes inhibiting, preventing, ameliorating or delaying onset of a
particular condition. Treating
and treatment also encompasses any improvement in one or more symptoms of the
condition or
disorder. 'Treating and treatment encompasses any modification to the
condition or course of
disease progression as compared to the condition or disease in the absence of
therapeutic
intervention.
[0142] Suitable subjects for treatment according to the methods of the
present application
include, without limitation, domesticated and undomesticated animals such as
rodents (mouse or
rat), cats, dogs, rabbits, horses, sheep, pigs, and non-human primates. In
some embodiments the
subject is a human subject. Exemplary human subjects include, without
limitation, infants,
children, adults, and elderly subjects.
[0143] In some embodiments, the subject has a degenerative muscle
condition. As used
herein, the term "degenerative muscle condition" refers to conditions,
disorders, diseases and
injuries characterized by one or more of muscle loss, muscle degeneration or
wasting, muscle
weakness, and defects or deficiencies in proteins associated with normal
muscle function, growth
or maintenance. In certain embodiments, a degenerative muscle condition is
sarcopenia or
cachexia. In other embodiments, a degenerative muscle condition is one or more
of muscular
dystrophy, muscle injury, including acute muscle injury, resulting in loss of
muscle tissue,
muscle atrophy, wasting or degeneration, muscle overuse, muscle disuse
atrophy, muscle disuse
atrophy, denervation muscle atrophy, dysferlinopathy, AIDS/HIV, diabetes,
chronic obstructive
pulmonary disease, kidney disease, cancer, aging, autoimmune disease,
polymyositis, and
dermatomyositis. Thus, in some embodiments, the subject has a degenerative
muscle condition
selected from the group consisting of sarcopenia or myopathy.
[0144] The compositions and methods described herein may be used
in combination with
other known treatments or standards of care for given diseases, injury, or
conditions. For
example, in the context of muscular dystrophy, a composition of the invention
for promoting
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 64 -
muscle satellite cell expansion can be administered in conjunction with such
compounds as CT-
1, pregnisone, or myostatin The treatments (and any combination treatments
provided herein)
may be administered together, separately or sequentially.
[0145] The subject may have a muscle disorder mediated by
functional AUF1 deficiency
or a muscle disorder not mediated by functional AUF deficiency.
[0146] In some embodiments, the subject has an adult-onset
myopathy or muscle
disorder.
[0147] As used herein, the term "muscular dystrophy" includes,
for example, Duchenne,
Becker, Limb-girdle, Congenital, Facioscapulohumeral, Myotonic,
Oculopharyngeal, Distal, and
Emery-Dreifuss muscular dystrophies. In particular embodiments, the muscular
dystrophy is
characterized, at least in part, by a deficiency or dysfunction of the protein
dystrophin. Such
muscular dystrophies may include Duchenne muscular dystrophy (DMD) and Becker
muscular
dystrophy (DMD). In other embodiments, the muscular dystrophy is associated
with
degenerative muscle conditions such as muscle disuse atrophy, denervation
muscle atrophy,
dysferlinopathy, AIDS/HIV, diabetes, chronic obstructive pulmonary disease,
kidney disease,
cancer, aging, autoimmune disease, polymyositis, and dermatomyositis.
[0148] In some embodiments of the methods disclosed herein, the
subject has Duchenne
Muscular Dystrophy (DMD). As described above, DMD is an X-linked muscle
wasting disease
that is quite common (1/3500 live births), generally but not exclusively found
in males, caused
by mutations in the dystrophin gene that impair its expression for which there
are few therapeutic
options that have been shown to be effective. Muscle satellite cells are
unresponsive in DMD
and are said to be functionally exhausted, thereby limiting or preventing new
muscle
development and regeneration. DMD typically presents in the second year after
birth and
progresses over the next two to three decades to death in young men.
[0149] In some embodiments of the methods disclosed herein, the subject has
Becker
muscular dystrophy. As described above, Becker muscular dystrophy is a less
severe form of
the disease that also involves mutations that impair dystrophin function or
expression but less
severely. There are few therapeutic options that have been shown to be
effective for Becker
muscular dystrophy. There are no cures for DMD or Becker disease.
[0150] In some embodiments of the methods disclosed herein, the subject has
traumatic
muscle injury. As used herein, the term "traumatic muscle injury" refers to a
condition resulting
from a wide variety of incidents, ranging from, e.g., everyday accidents,
falls, sporting accidents,
automobile accidents, to surgical resections to injuries on the battlefield,
and many more. Non-
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 65 -
limiting examples of traumatic muscle injuries include battlefield muscle
injuries, auto accident-
related muscle injuries, and sports-related muscle injuries
[0151] In some embodiments, the administering is effective to
treat a subject having
degenerative skeletal muscle loss For example, the administering may be
effective to activate
muscle stem cells, accelerate the regeneration of mature muscle fibers
(myofibers), enhance
expression of muscle regeneration factors, accelerate the regeneration of
injured skeletal muscle,
increase regeneration of slow-twitch (Type I) and/or fast-twitch (Type II)
fibers), and/or restore
muscle mass, muscle strength, and create normal muscle following in the
selected subject.
[0152] In some embodiments, the administering is effective to
transduce skeletal muscle
cells (e.g-., cardiac diaphragm cells) and/or provide long-term (e.g., lasting
at least 1 month, 2
months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months,
10 months, 11
months, or more) muscle cell-specific AUF1 expression in the selected subject.
[0153] In other embodiments, the administering is effective to
(i) activate high levels of
satellite cells and myoblasts; (ii) significantly increase skeletal muscle
mass and normal muscle
fiber formation; and/or (iii) significantly enhanced exercise endurance in the
selected subject as
compared to when the administering is not carried out.
[0154] In further embodiments, the administering is effective to
reduce (i) biomarkers of
muscle atrophy and muscle cell death; (ii) inflammatory immune cell invasion
in skeletal muscle
(including diaphragm); and/or (iii) muscle fibrosis and necrosis in skeletal
muscle (including
diaphragm) in the selected subject, as compared to when the administering is
not carried out.
[0155] In certain embodiments, the administering is effective to
(i) increase expression of
endogenous utrophin in DMD muscle cells and/or (ii) suppress expression of
embryonic
dystrophin, a marker of muscle degeneration in DMD in the selected subject, as
compared to
when the administering is not carried out. In some embodiments of the methods
disclosed
herein, said administering is effective to upregulate endogenous utrophin
protein expression in
the selected subject, as compared to when the administering is not carried
out. In some
embodiments of the methods disclosed herein, said administering is effective
to upregulate
endogenous utrophin protein expression in said muscle cells, as compared to
when the
administering is not carried out.
[0156] In some embodiments, the administering is effective to (i) increase
normal
expression of genes involved in muscle development and regeneration and/or
(ii) suppress genes
involved in muscle cell fibrosis, death, and muscle-expressed inflammatory
cytokines in the
selected subject, as compared to when the administering is not carried out.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 66 -
[0157] In further embodiments, the administering does not
increase muscle mass,
endurance, or activate satellite cells in normal skeletal muscle
[0158] In some embodiments, the administering is effective to
accelerate muscle gain in
the selected subject, as compared to when said administering is not carried
out.
[0159] In certain embodiments, the administering is effective to reduce
expression of
established biomarkers of muscle atrophy in a subject having degenerative
skeletal muscle loss.
Suitable biomarkers of muscle atrophy include, without limitation, TRIM63 and
Fbxo32 mRNA.
In some embodiments, the administering is effective to enhance expression of
established
biomarkers of muscle myoblast activation, differentiation, and muscle
regeneration in the
selected subject. Suitable biomarkers of muscle atrophy include, without
limitation, myogenin
and MyoD mRNA levels, biomarkers of myoblast activation, differentiation and
muscle
regeneration (Zammit, "Function of the Myogenic Regulatory Factors Myf5, MyoD,
Myogenin
and MRF4 in Skeletal Muscle, Satellite Cells and Regenerative Myogenesis,-
Semin. Cell. Dev.
Biol. 72:19-32 (2017), which is hereby incorporated by reference in its
entirety).
[0160] In some embodiments, the method further involves administering a
purine-rich
element binding protein 3 (Pur13) inhibitor. The Pur13 inhibitor may be a
nucleic acid molecule, a
polypeptide, or a small molecule. In some embodiments, the nucleic acid
molecule is selected
from the group consisting of siRNA, shRNA, and miRNA. Suitable nucleic acid
molecules are
describe in detail supra.
[0161] Administering, according to the methods of the present application,
may be
carried out orally, topically, transdermally, parenterally, subcutaneously,
intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical
instillation, intraocularly, intraarterially, intralesionally, or by
application to mucous membranes.
Thus, in some embodiments, the administering is carried out intramuscularly,
intravenously,
subcutaneously, orally, or intraperitoneally. In specific embodiments, the
administering is
carried out by intramuscular injection. In some embodiments, an adeno-
associated virus (AAV)
vector is administered by intramuscular injection.
[0162] In some embodiments, the administering is carried out
by intramuscular
injection.
Traumatic Muscle Injury
[0163] A further aspect of the present application relates to a
method of preventing
traumatic muscle injury in a subject. This method involves selecting a subject
at risk of
traumatic muscle injury and administering to the selected subject an adeno-
associated viral
(AAV) vector described herein, a composition described herein, or a lentiviral
vector comprising
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 67 -
a muscle cell specific promoter and a nucleic acid molecule encoding an AU-
rich mRNA binding
factor 1 (AUF 1) protein or a functional fragment thereof, where the nucleic
acid molecule is
heterologous to and operatively coupled to the muscle cell-specific promoter.
[0164] Still another aspect of the present application relates
to a method of treating
traumatic muscle injury in a subject. This method involves selecting a subject
having traumatic
muscle injury and administering to the selected subject an adeno-associated
viral (AAV) vector
described herein, a composition described herein, or a lentiviral vector
comprising a muscle cell
specific promoter and a nucleic acid molecule encoding an AU-rich mRNA binding
factor 1
(AUF1) protein or a functional fragment thereof, where the nucleic acid
molecule is heterologous
to and operatively coupled to the muscle cell-specific promoter.
[0165] Suitable subjects for treatment according to the methods
of the present application
include, without limitation, domesticated and undomesticated animals such as
rodents (mouse or
rat), cats, dogs, rabbits, horses, sheep, pigs, and non-human primates. In
some embodiments the
subject is a human subject. Exemplary human subjects include, without
limitation, infants,
children, adults, and elderly subjects.
[0166] As described supra, the term "traumatic muscle injury"
refers to a condition
resulting from a wide variety of incidents, ranging from, e.g., everyday
accidents, falls, sporting
accidents, automobile accidents, to surgical resections to injuries on the
battlefield, and many
more. Non-limiting examples of traumatic muscle injuries include battlefield
muscle injuries,
auto accident-related muscle injuries, and sports-related muscle injuries.
[0167] In some embodiments, the subject is at risk of developing
or is in need of
treatment for a traumatic muscle injury selected from the group consisting of
a laceration, a blunt
force contusion, a shrapnel wound, a muscle pull, a muscle tear, a bum, an
acute strain, a chronic
strain, a weight or force stress injury, a repetitive stress injury, an
avulsion muscle injury, and
compartment syndrome.
[0168] In some embodiments, the subject is at risk of developing
or is in need of
treatment for a traumatic muscle injury that involves volumetric muscle loss
("VML"). The
terms "volumetric muscle loss" or "VML" refer to skeletal muscle injuries in
which endogenous
mechanisms of repair and regeneration are unable to fully restore muscle
function in a subject.
The consequences of VML are substantial functional deficits in joint range of
motion and
skeletal muscle strength, resulting in life-long dysfunction and disability.
[0169] In some embodiments, the administering is carried out in
a subject at risk of
developing a traumatic muscle injury and a prophylactically effective amount
of the adeno-
associated viral (AAV) vector, composition, or lentiviral vector of the
present application is
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 68 -
administered. The term "prophylactically effective amount" refers to an amount
effective, at
dosages and for periods of time necessary, to achieve the desired prophylactic
result
[0170] In some embodiments, the administering is carried to
treat a subject having
traumatic muscle injury and said administering is carried out immediately
after the traumatic
muscle injury (for example, within one minute, 2 minutes, 3 minutes, 4
minutes, 5 minutes, 6
minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes,
13 minutes, 14
minutes, 15 minutes, 60 minutes, or any amount of time there between) of the
traumatic muscle
injury. In certain embodiments, said administering is carryout out within 1
hour, 2 hours, 3
hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11
hours, 12 hours, 13
hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours,
21 hours, 22 hours,
23 hours, or 24 hours of the traumatic muscle injury. In other embodiments,
said administering
is carried out within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8
days, 9 days, 10 days,
11 days, 12 days, 13 days, or 14 days of the traumatic muscle injury. In
further embodiments,
said administering may be carried out within 1 week, 2 weeks, 3 weeks, 4
weeks, 5 weeks, 6
weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14
weeks, 15
weeks, 52 weeks, or any amount of time there between of the traumatic muscle
injury.
[0171] Adeno-associated virus (AAV) vectors and lentiviral
vectors are currently the
recombinant gene delivery system of choice for the transfer of exogenous genes
in vivo,
particularly into humans. These vectors provide efficient delivery of genes
into cells, and the
transferred polynucleotides are stably integrated into the chromosomal DNA of
the host.
[0172] The adeno-associated viral (AAV) vector and/or the
lentiviral vector for use in the
methods disclosed herein may encode AUF1 isoform p37AuF1, p40AUF1, p42AuF1
,
or p45AUF1.
Suitable AUF isoform nucleic acid and amino acid sequences are identified
supra. In certain
embodiments, the adeno-associated viral (AAV) vector and/or the lentiviral
vector for use in the
methods disclosed herein encodes AUF isoform p45AuF1
.
[0173] In some embodiments, the adeno-associated virus (AAV)
vector is AAV8-0/ICK-
AUF1 or another human AAV including but not limited to AAV1, AAV2, AAV5, AAV6,
or
AAV9 vector encoding AUF1 (e.g., AUF 1 isoforms 37 AUF1 p40AUF1, p42Aun,
and/or p45AuF1).
In other embodiments, the AAV is a human novel AAV capsid variant engineered
for enhanced
muscle-specific tropism including but not limited to AAV2i8 or AAV2.5. In yet
other
embodiments, the AAV vector is a non-human primate AAV vector including but
not limited to
AAVrh.8, AAVrh.10, AAVrh.43, or AAVrh.74.
[0174] In some embodiments, the lentiviral vector is a
lentivirus p45 AUF1 vector, or a
lentivirus expressing another AUF1 isoform (e.g.,
p 37 ALA', p40 AUF1 or p42 Aum) or
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 69 -
combinations thereof (Abbadi et al., "Muscle Development and Regeneration
Controlled by
AUF1-mediated Stage-specific Degradation of Fate-determining Checkpoint
mRNAs," Proc.
Nat'l. Acad. Sci. USA 116:11285-90 (2019), which is hereby incorporated by
reference in its
entirety). Other embodiments include expression of p37 AUF1, p40 AUF1, p42
AUF1, p45 MT", or
combinations thereof from non-human lentivirus vectors including but not
limited to simian,
feline, and other mammalian lentivirus gene transfer vectors.
[0175] In one particular embodiment, the AUF1 p45 lentivirus vector has
the following
nucleotide sequence:
AUF1 p45 Lentivirus Vector Shuttle Plasmid (SEQ ID NO:26)
10 CGAAAAGTGC CACCTGCAGC CTGAATATGG GCCAAACAGG ATATCTGTGG TAAGCAGTTC 60
CTGCCCCGGC TCAGGGCCAA GAACAGATGG AACAGCTGAA TATGGGCCAA ACAGGATATC 120
TGTGGTAAGC AGTTCCTGCC CCGGCTCAGG GCCAAGAACA GATGGTCCCC AGATGCGGTC 180
CAGCCCTCAG CAGTTTCTAG AGAACCATCA GATGTTTCCA GGGTGCCCCA AGGACCTGAA 240
ATGACCCTGT GCCTTATTTG AACTAACCAA TCAGTTCGCT TCTCGCTTCT GTTCGCGCGC 300
TTCTGCTCCC CGAGCTCAAT AAAAGAGCCC ACAACCCCTC ACTCGGGGCG CCAGTCCTCC 360
GATTGACTGA GTCGCCCGGG TACCCGTGTA TCCAATAAAC CCTCTTGaAG TTGCATCCGA 420
CTTGTGGTCT CGCTGTTCCT TGGGAGGGTC TCCTCTGAGT GATTGACTAC CCGTCAGCGG 480
GGGTCTTTCA TTTGGGGGCT CGTCCGGGAT CGGGAGACCC CTGCCCAGGG ACCACCGACC 540
CACCACCGGG AGGCAAGCTG GCCAGCAACT TATCTGTGTC TGTCCGATTG TCTAGTGTCT 600
ATGACTGATT TTATGCGCCT GCGTCGGTAC TAGTTAGCTA ACTAGCTCTG TATCTGGCGG 660
ACCCGTGGTG GAACTGACGA GTTCTGAACA CCCGGCCGCA ACCCTGGGAG ACGTCCCAGG 720
GACTTTGGGG GCCGTTTTTG TGGCCCGACC TGAGGAAGGG AGTCGATGTG GAATCCGACC 780
CCGTCAGGAT ATGTGGTTCT GGTAGGAGAC GAGAACCTAA AACAGTTCCC GCCTCCGTCT 840
GAATTTTTGC TTTCGGTTTG GAACCGAAGC CGCGCGTCTT GTCTGCTGCA GCGCTGCAGC 900
25 ATCGTTCTGT GTTGTCTCTG TCTGACTGTG TTTCTGTATT TGTCTGAAAA TTAGGGCCAG 960
ACTGTTACCA CTCCCTTAAG TTTGACCTTA GGTCACTGGA AAGATGTCGA GCGGATCGCT 1020
CAGAACCAGT CGGTAGATGT CAAGAAGAGA CGTTGGGTTA CCTTCTGCTC TGCAGAATGG 1080
CCAACCTTTA ACGTCGGATG GCCGCGAGAC GGCACCTTTA ACCGAGACCT CATCACCCAG 1140
GTTAAGATCA AGGTCTTTTC ACCTGGCCCG CATGGACACC CAGACCAGGT CCCCTACATC 1200
30 GTGACCTGGG AAGCCTTGGC TTTTGACCCC CCTCCCTGGG TCAAGCCCTT TGTACACCCT 1260
AAGCCTCCGC CTCCTCTTCC TCCATCCGCC CCGTCTCTCC CCCTTGAACC TCCTCGTTCG 1320
ACCCCGCCTC GATCCTCCCT TTATCCAGCC CTCACTCCTT CTCTAGGCGC CGGCCGGATC 1380
CATGTCGGAG GAGCAGTTCG GCGGGGACGG GGCGGCGGCA GCGGCAACGG CGGCGGTAGG 1440
CGGCTCGGCG GGCGAGCAGG AGGGAGCCAT GGTGGCGGCG ACAaAGGGGG CAGCGGCGGC 1500
35 GGCGGGAAGC
GGAGCCGGGA CCGGGGGCGG AACCGCGTCT GGAGGCACCG AAGGGGGCAG 1560
CGCCGAGTCG GAGGGGGCGA AGATTGACGC aAGTAAGAAC GAGGAGGATG AAGGCCATTC 1620
AAACTCCTCC CCACGACACT CTGAAGCAGC GACGGCACAG CGGGAAGAAT GGAAAATGTT 1680
TATAGGAGGC CTTAGCTGGG ACACTAGAAA GAAAGATCTG AAGGACTACT TTTCCAAATT 1740
TGGTGAAGTT GTAGACTGCA CTCTGAAGTT AGATCCTATC ACAGGGCGAT CAAGGGGTTT 1800
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 70 -
T GGCT T T GT G CTATTTAAAG AATCGGAGAG TGTAGATAAG GTCATGGATC AAAAAGAACA 1860
TAAATTGAAT GGGAAGGTGA TTGATCCTAA AAGGGCCAAA GCCATGAAAA CAAAAGAGCC 1920
GGTTAAAAAA ATTTTTGTTG GTGGCCTTTC TCCAGATACA CCTGAAGAGA AAATAAGGGA 1980
GTACTTTGGT GGTTTTGGTG AGGTGGAATC CATAGAGCTC CCCATGGACA ACAAGACCAA 2040
TAAGAGGCGT GGGTTCTGCT TTATTACCTT TAAGGAAGAA GAACCAGTGA AGAAGATAAT 2100
GGAAAAGAAA TACCACAATG TTGGTCTTAG TAAATGTGAA ATAAAAGTAG CCATGTCGAA 2160
GGAACAATAT CAGCAACAGC AACAGTGGGG ATCTAGAGGA GGATTTGCAG GAAGAGCTCG 2220
TGGAAGAGGT GGTGGCCCCA GTCAAAACTG GAACCAGGGA TATAGTAACT ATTGGAATCA 2280
AGGCTATGGC AACTATGGAT ATAACAGCCA AGGTTACGGT GGTTATGGAG GATATGACTA 2340
10 CACTGGTTAC AACAACTACT ATGGATATGG TGATTATAGC AACCAGCAGA GTGGTTATGG 2400
GAAGGTATCC AGGCGAGGTG GTCATCAAAA TAGCTACAAA CCATACGACT ACAAGGACGA 2460
CGATGACAAG TGAGTCGACC AATTCCGGTT ATTTTCCACC ATATTGCCGT CTTTTGGCAA 2520
TGTGAGGGCC CGGAAACCTG GCCCTGTCTT CTTGACGAGC ATTCCTAGGG GTCTTTCCCC 2580
TCTCGCCAAA GGAATGCAAG GTCTGTTGAA TGTCGTGAAG GAAGCAGTTC CTCTGGAAGC 2640
15 TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG CAGCGGAACC CCCCACCTGG 2700
CGACAGGTGC CTCTGCGGCC AAAAGCCACG TGTATAAGAT ACACCTGCAA AGGCGGCACA 2760
ACCCCAGTGC CACGTTGTGA GTTGGATAGT TGTGGAAAGA GTCAAATGGC TCTCCTCAAG 2820
CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC aATTGTATGG GATCTGATCT 2880
GGGGCCTCGG TGCACATGCT TTACATGTGT TTAGTCGAGG TTAAAAAACG TCTAGGCCCC 2940
20
CCGAACCACG GGGACGTGGT TTTCCTTTGA AAAACACGAT GATAATACCA TGAAAAAGCC 3000
TGAACTCACC GCGACGTCTG TCGAGAAGTT TCTGATCGAA AAGTTCGACA GCGTCTCCGA 3060
CCTGATGCAG CTCTCGGAGG GCGAAGAATC TCGTGCTTTC AGCTTCGATG TAGGAGGGCG 3120
TGGATATGTC CTGCGGGTAA ATAGCTGCGC CGATGGTTTC TACAAAGATC GTTATGTTTA 3180
TCGGCAETTT GCATCGGCCG CGCTCCCGAT TCCGGAAGTG CTTGACATTG GGGAATTTAG 3240
25 CGAGAGCCTG ACCTATTGCA TCTCCCGCCG TGCACAGGGT GTCACGTTGC AAGACCTGCC 3300
TGAAACCGAA CTGCCCGCTG TTCTGCAGCC GGTCGCGGAG GCCATGGATG CGATCGCTGC 3360
GGCCGATCTT AGCCAGACGA GCGGGTTCGG CCCATTCGGA CCGCAAGGAA TCGGTCAATA 3420
CACTACATGG CGTGATTTCA TATGCGCGAT TGCTGATCCC aATGTGTATC ACTGGCAAAC 3480
TGTGATGGAC GACACCGTCA GTGCGTCCGT CGCGCAGGCT CTCGATGAGC TGATGCTTTG 3540
30 GGCCGAGGAC TGCCCCGAAG TCCGGCACCT CGTGCACGCG GATTTCGGCT CCAACAATGT 3600
CCTGACGGAC AATGGCCGCA TAACAGCGGT aATTGACTGG AGCGAGGCGA TGTTCGGGGA 3660
TTCCCAATAC GAGGTCGCCA ACATCTTCTT CTGGAGGCCG TGGTTGGCTT GTATGGAGCA 3720
GCAGACGCGC TACTTCGAGC GGAGGCATCC GGAGCTTGCA GGATCGCCGC GGCTCCGGGC 3780
GTATATGCTC CGCATTGGTC TTGACCAACT CTATCAGAGC TTGGTTGACG GCAATTTCGA 3840
35 TGATGCAGCT TGGGCGCAGG GTCGATGCGA CGCAATCGTC CGATCCGGAG CCGGGACTGT 3900
CGGGCGTACA CAAATCGCCC GCAGAAGCGC GGCCGTCTGG ACCGATGGCT GTGTAGAAGT 3960
ACTCGCCGAT AGTGGAAACC GACGCCCCAG aACTCGTCCG AGGGCAAAGG AATAGAGTAG 4020
ATGCCGACCG GGATCTATCG ATAAAATAAA AGATTTTATT TAGTCTCCAG AAAAAGGGGG 4080
GAATGAAAGA CCCCACCTGT AGGTTTGGCA AGCTAGCTTA AGTAACGCCA TTTTGCAAGG 4140
40
CATGGAAAAA TACATAACTG AGAATAGAGA AGTTCAGATC AAGGTCAGGA ACAGATGGAA 4200
CAGCTGAATA TGGGCCAAAC AGGATATCTG TGGTAAGCAG TTCCTGCCCC GGCTCAGGGC 4260
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 71 -
CAAGAACAGA TGGAACAGCT GAATATGGGC CAAACAGGAT ATCTGTGGTA AGCAGTTCCT 4320
GCCCCGGCTC AGGGCCAAGA ACAGATGGTC CCCAGATGCG GTCCAGCCCT CAGCAGTTTC 4380
TAGAGAACCA TCAGATGTTT CCAGGGTGCC CCAAGGACCT GAAATGACCC TGTGCCTTAT 4440
TTGAACTAAC CAATCAGTTC GCTTCTCGCT TCTGTTCGCG CGCTTCTGCT CCCCGAGCTC 4500
5 AATAAAAGAG CCCACAACCC CTCACTCGGG GCGCCAGTCC TCCGATTGAC TGAGTCGCCC 4560
GGGTACCCGT GTATCCAATA AACCCTCTTG CAGTTGCATC CGACTTGTGG TCTCGCTGTT 4620
CCTTGGGAGG GTCTCCTCTG AGTGATTGAC TACCCGTCAG CGGGGGTCTT TCACATGCAG 4680
CATGTATCAA AATTAATTTG GTTTTTTTTC TTAAGTATTT ACATTAAATG GCCATAGTTG 4740
CATTAATGAA TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT 4600
10 TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC 4860
TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA 4920
GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT 4980
AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC 5040
CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT 5100
15
GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG 5160
CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG 5220
GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT 5280
CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG aAGCAGCCAC TGGTAACAGG 5340
ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC 5400
20 GGCTACACTA GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA 5460
AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT 5520
GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT 5580
TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA 5640
TTATCAAAAA GGATCTTCAC CTAGATCCTT TTGCGGCCGC AAATCAATCT AAAGTATATA 5700
25 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT 5760
CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG 5820
GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC GCTCACCGGC 5880
TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC 5940
AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC 6000
30
GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC 6060
GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC 6120
CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG TCAGAAGTAA 6180
GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT 6240
GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA 6300
35 GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA 6360
TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG 6420
GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA ACTGATCTTC 6480
AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC 6540
AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA 6600
40 TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TAGATATTTG AATGTATTTA 6660
GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCC
6697
CA 03164622 2022- 7- 13
SUBSTITUTE SHEET (RULE 26)
WO 2021/146711
PCT/US2021/013955
- 72 -
[0176] In some embodiments, the administering is effective to
prevent muscle atrophy
and/or muscle loss following traumatic muscle injury to the selected subject.
In other
embodiments, the administering is effective to activate muscle stem cells
following traumatic
muscle injury to the selected subject. In further embodiments, the
administering is effective to
accelerate the regeneration of mature muscle fibers (myofibers), enhance
expression of muscle
regeneration factors, accelerate the regeneration of injured muscle, increased
regeneration of
slow-twitch (Type I) and/or fast-twitch (Type II) fibers), and/or restore
muscle mass, muscle,
strength and create normal muscle following traumatic muscle injury in the
selected subject.
[0177] In some embodiments, the administering is effective to
accelerate muscle gain
following traumatic muscle injury in the selected subject, as compared to when
said
administering is not carried out.
[0178] In certain embodiments, the administering is effective to
reduce expression of
established biomarkers of muscle atrophy following traumatic muscle injury to
the selected
subject. Suitable biomarkers of muscle atrophy include, without limitation,
TRIM63 and Fbxo32
mRNA. In some embodiments, the administering is effective to enhance
expression of
established biomarkers of muscle myoblast activation, differentiation and
muscle regeneration
following traumatic muscle injury to the selected subject. Suitable biomarkers
of muscle atrophy
include, without limitation, myogenin and MyoD mRNA levels, biomarkers of
myoblast
activation, differentiation and muscle regeneration (Zammit, "Function of the
Myogenic
Regulatory Factors Myf5, MyoD, Myogenin and MRF4 in Skeletal Muscle, Satellite
Cells and
Regenerative Myogenesis," Semin. Cell. Dev. Biol. 72:19-32 (2017), which is
hereby
incorporated by reference in its entirety).
[0179] In some embodiments, the administering is effective to
deliver the vector or
pharmaceutical composition described herein to a specific tissue in the
subject. The tissue may
be muscle tissue. For example, the muscle tissue may be all types of skeletal
muscle, smooth
muscle, or cardiac muscle.
[0180] Administering, according to the methods of the present
application, may be
carried out orally, topically, transdermally, parenterally, subcutaneously,
intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical
instillation, intraocularly, intraarterially, intralesionally, or by
application to mucous membranes.
Thus, in some embodiments, the administering is carried out intramuscularly,
intravenously,
subcutaneously, orally, or intraperitoneally. In specific embodiments, the
administering is
carried out by intramuscular injection. In some embodiments, an adeno-
associated virus (AAV)
vector is administered by intramuscular injection.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 73 -
[0181] In other embodiments, the administering is carried out by
systemic
administration. Thus, in some embodiments, a lentiviral vector comprising a
muscle cell specific
promoter and a nucleic acid molecule encoding an AU-rich mRNA binding factor 1
(AUF1)
protein or a functional fragment thereof, where the nucleic acid molecule is
heterologous to and
operatively coupled to the muscle cell-specific promoter is administered
systemically. In some
embodiments, the lentiviral vectors administered systemically is a lentivirus
expressing p37 AUF1,
p40 AUF1, 02 AUF1, and/or p45 AUF1 AUF1 vector (Abbadi et al., "Muscle
Development and
Regeneration Controlled by AUF1-mediated Stage-specific Degradation of Fate-
determining
Checkpoint mRNAs," Proc. Nat 'I. Acad. Sci. USA 116:11285-90 (2019), which is
hereby
incorporated by reference in its entirety).
[0182] Formulations for injection may be presented in unit
dosage form, e.g., in
ampoules or in multi-dose containers, with an added preservative. The
compositions may take
such forms as suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain
formulatory agents such as suspending, stabilizing and/or dispersing agents.
[0183] Suitable regimens for initial contacting and further doses or for
sequential
contacting steps may all be the same or may be variable. Appropriate regimens
can be
ascertained by the skilled artisan, from the disclosure of the present
application, the documents
cited herein, and the knowledge in the art.
[0184] A dosage unit to be administered in methods of the
present application will vary
depending on the vector used, the route of administration, the type of tissue
and cell being
targeted, and the purpose of treatment, among other parameters. Dosage for
treatment can be
determined by a skilled person who would know how to determine dose using
methods standard
in the art. A dosage unit, corresponding to genome copy number, for example,
could range from
about, lx101 to lx1011, 1x102 to lx10", 1x103 to lx10", 1x104 to lx1011, 1x105
to lx10", 1x106
to lx1011, 1x107 to lx1011, 1x108 to 1x1011, 1x109 to lx1011, lx101 to
lx1011, lx101 to lx101 ,
1x102 to lx101 , 1x103 to lx101 , 1x104 to lx101 , 1x105 to lx101 , 1x106 to
lx101 , 1x107 to
1x1010, 1x108 to 1x1010, 1x109 to lx10", lx101 to 1x109, 1x102 to 1x109, 1x103
to 1x109, 1x104
to lx109, 1X105 to lx109, lx106 to lx109, lx107 to lx109, lx1OS to lx109,
lx101 to lx108, lx102
to 1x108, 1)(103 to 1x108, 1x104 to 1x108, 1x105 to 1x108, 1x106 to 1x108, or
1x107 to 1x108
genome copies of a vector disclosed herein. In some embodiments, a dosage
unit, corresponding
to genome copy number, for example, is administered in the range of lx101 to
lx1012, 1x102 to
lx1012, 1x103 to lx1012, 1X104 to 1X1012, 1X105 to lx1012, 1X106 to lx1012,
1x107 to lx1012,
1x108 to lx1012, 1X109 to 1X1012, 1X101 to lx1012, or lx1011 to lx1012genome
copies; lx101 to
lx1013, lx102 to lx1013, lx103 to 1x1013, lx104 to lx1013, lx105 to 1x1013,
lx106 to lx1013,
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 74 -1x107 to lx1013, 1x108 to lx1013, 1x109 to lx1 013, lx101 to lx1013,
lx10" to lx1013, or 1x1012
to lx1013 genome copies; lx101 to lx1014, 1X102 to 1x1014, 1x103 to 1x1014,
1x104 to 1x1014,
1)(105 to 1x1014, 1x106 to 1x1014, 1x107 to 1x1011, 1x108 to 1x1014, 1x109 to
1x1014, 1x101 to
1x1011, 1x1011 to 1x1011, 1x1012 to 1x1011, or lx1013 to 1x1011 genome copies;
lx101 to lx1015,
1x102 to 1x1015, 1x103 to lx1015, 1x104 to 1x1015, 1x105 to lx1015, 1x106 to
lx1015, 1x107 to
1x1015, 1x108 to 1x1015, 1x109 to 1x105, 1x1019 to 1x1015, 1x1011 to 1x1015,
1x1012 to 1x1015,
1x1013 to lx1015, or lx1014 to 1x1015 genome copies; 1x101 to 1x1016, 1x102 to
1x1016, 1x103 to
1x1016, 1x104 to 1x1016, 1x105 to 1x1016, 1x106 to 1x1016, 1x107 to 1x1016,
1x108 to 1x1016,
1x109 to 1x1016, 1x1019 to 1x106, 1x1011 to 1x1016, 1x1012 to 1x1016, 1x1013
to 1x1016, 1x1014 to
1x1016, or 1x10" to 1x1016 genome copies; 1x101 to 3x1016, 1x102 to 3x1016,
1x103 to 3x1016,
lx 104 to 3x 1016, lx1 05 to 3x1016, lx 1 06 to 3x1 016, 1 x 107 to 3x1016, lx
108 to 3x 1016, lx 1 09 to
3x1016, 1x1010 to 3x1016, 1x1011 to 3)(101-6, 1x1012 to 3x1016, 1x1013 to
3x1016, 1x1014 to 3x1016,
or lx 1015 to 3x1016 genome copies; and any amount there between. Dosage will
depend on route
of administration, type of tissue and cells to receive the vector, timing of
administration to
human subjects, whether dosage is determined based on total genome copies to
be delivered, and
whether administration is determined by genome copies per kilogram body
weight.
101851 In some embodiments, a subject is administered a vector
or pharmaceutical
composition described herein in one dose. In other embodiments, the subject is
administered the
vector or pharmaceutical composition described herein in a series of two or
more doses in
succession. In some other embodiments, where the subject is administered the
vector or
pharmaceutical composition described herein in a single dose, in two doses,
and/or more than
two doses, the doses may be the same or different, and they are administered
with equal or with
unequal intervals between them.
101861 A subject may be administered the vector or
pharmaceutical composition
described herein in many frequencies over a wide range of times. In some
embodiments, the
subject is administered the vector or pharmaceutical composition described
herein over a period
of less than one day. In other embodiments, the subject is contacted over two,
three, four, five,
or six days. In some embodiments, the contacting is carried out one or more
times per week,
over a period of weeks. In other embodiments, the contacting is carried out
over a period of
weeks for one to several months. In various embodiments, the contacting is
carried out over a
period of months. In others, the contacting may be carried out over a period
of one or more
years. Generally, lengths of treatment will be proportional to the length of
the ischemic disease
process, the effectiveness of the therapies being applied, and the condition
and response of the
subject being treated. According to some embodiments, the contacting is
carried out daily.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 75 -
[0187] The choice of formulation for administered the vector or
pharmaceutical
composition described herein will depend on a variety of factors Prominent
among these will be
the species of subject, the nature of the disorder, dysfunction, or disease
being treated and its
state and distribution in the subject, the nature of other therapies and
agents that are being
administered, the optimum route for administration, survivability via the
route, the dosing
regimen, and other factors that will be apparent to those skilled in the art.
In particular, for
instance, the choice of suitable carriers and other additives will depend on
the exact route of
contacting and the nature of the particular dosage form.
[0188] In the methods described herein, rather than
administering a vector, other means
of administering AUF can be carried out including by direct injection of: (i)
encoding p37AuF1
,
p40 AUF1, p42 AUF1, and/or p45 AUF1 DNA by plasmid; (ii) mRNA encoding p37
AUF1, p 40 AUF1,
p42 AUF1, and/or p45 AUF1; and/or (iii) nanoparticle incorporation of AUF1
encoding DNA or
mRNA.
EXAMPLES
[0189] The examples below are intended to exemplify the practice
of embodiments of the
present application but are by no means intended to limit the scope thereof.
Materials and Methods for Examples 1 ¨ 8
Mice
[0190] All animal studies were approved by the NYU School of Medicine
Institutional
Animal Care and Use Committee (IACUC) and conducted in accordance with IACUC
guidelines. All ant- KO mice and WT mice are of the 129/B6-background, bred at
the F3 and
F4 generations from aut- heterozygous mice (Pont et al., "mRNA Decay Factor
AUF1
Maintains Normal Aging, Telomere Maintenance, and Suppression of Senescence by
Activation
of Telomerase Transcription," Molecular Cell 47(1):5-15 (2012) and Lu et al.,
"Endotoxic Shock
in AUF1 Knockout Mice Mediated by Failure to Degrade Proinflammatory Cytokine
mRNAs,"
Genes Dev. 20(22):3174-3 184 (2006), which are hereby incorproated by
reference in their
entirety). 12 month old C57BL6 mice (Jackson) for AUF1 supplementation during
AAV
experiments. One month old C57BL10 and C57BL/10ScSn-Dmc1nth/J mice (Jackson)
were used
for A AV experiments in Example S.
Cells
[0191] C2C12 cells were obtained from the American Type Culture
Collection (ATCC),
authenticated by STR profiling and routinely checked for mycoplasma
contamination. C2C12
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 76 -
cells were maintained in DMEM (Corning), 10% FBS (Gibco), and 1% penicillin
streptomycin
(Life Technologies). To differentiate cells, media was switched to DMEM
(Corning), 2% Horse
Serum (Gibco), and 1% penicillin streptomycin (Life Technologies) during 96
hours (Panda et
al., "RNA-Binding Protein AUF1 Promotes Myogenesis by Regulating MEF2C
Expression
Levels," Mol. Cell Biol. 34(16): 3106-3119 (2014), which is hereby
incorporated by reference in
its entirety). aufl KO C2C12 cells were created with Crispr-Cas9 methods
(Abbadi et al.,
"Muscle Development and Regeneration Controlled by AUF I-Mediated Stage-
Specific
Degradation of Fate-Determining Checkpoint mRNAs," Proc. Natl. Acad. Sci. USA
116(23):11285-11290 (2019), which is hereby incorporated by refernce in its
entirety). For
assays performed in the presence of actinomycin D to determine mRNA stability,
C2C12
myoblasts cells were treated with 0.2 jig/ml of actinomycin D (Sigma). RNA
immune-
precipitation experiments were done in WT C2C12 before and 48 hours of
differentiation using a
normal IgG rabbit control or a rabbit-anti AUF1 antibody (07-260, Millipore).
Immunofluorescence
[0192] Mice had skeletal muscles removed as indicated in the text, put in
OCT, frozen in
dry ice-cooled isopentane (Tissue-Tek), fixed in 4% paraformaldehyde, and
blocked in 3% BSA
in TBS. C2C12 cells were fixed in 4% paraformaldehyde and blocked in 3% BSA in
PBS.
Samples were immunostained overnight with antibodies: AUF1 (07-260,
Millipore), Slow
myosin (N0Q7 5 4D, Sigma), Fast myosin (MY-32, Sigma), Laminin alpha 2 (4T-T8-
2, Sigma),
and GFP (2956, Cell signaling). Slow and fast myosin staining were done using
MOM kit
(Vector biolabs). Alexa Fluor donkey 488 and 555 secondary antibodies were
used at 1:300 and
incubated for 1 hour at room temperature. Slides were sealed with Vectashield
with DAPI
(Vector). Images were processed using ImageJ.
Microscopy, Image Processing, and Analysis
[0193] Images were acquired using a Zeiss LSM 700 confocal microscope,
primarily
with the 20X lens. Images were processed using ImageJ. If needed, color
balance was adjusted
linearly for the entire image and all images in experimental sets.
Immunoblot Studies
[0194] C2C12 cells or muscle tissues were lysed using lysis
buffer (50 mmol/L Tris-HC1,
pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% TritonX100)
supplemented
with complete protease inhibitor cocktail (Complete mini, ROCHE). Equal
amounts of total
protein were loaded on a polyacrylamide gel, resolved and transferred to PVDF
membrane.
Membrane was blocked with 5% nonfat milk in TBS-Tween 20 (0.1%) for 1 hour and
probed
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 77 -
with Antibody against AUF1 (07-260, Millipore) or against PGClalpha (Novus
biologicals
NBP1-04676). Bands were detected by peroxidase conjugated secondary antibodies
(GE
healthcare) and visualized with the ECL chemiluminescence system. The
immunoblots were
also probed with a rabbit antibody to 13-tubulin (Cell Signaling 2146S) or
GAPDH (Cell
Signaling 2118S) as a control for loading. Quantification was performed by
ImageJ.
Real-Time PCR Analysis
[0195] RNA was extracted using Trizol (Invitrogen) according to
the manufacturer's
instructions. DNase treatment was systematically performed. Quantification of
extracted RNA
was assessed using Nanodrop. The cDNA was synthesized using High Capacity cDNA
Reverse
Transcription Kit (Applied Biosystems). mRNA was analyzed by real-time PCR
using the iTaq
Universal SYBR Green Supermix (Bio Rad) probe. Relative quantification was
determined
using the comparative CT method with data normalized to housekeeping gene and
calibrated to
the average of control groups.
AA V-AUF1 Expression/AAV AUF1 Gene Transfer
[0196] AUF1 was integrated into an AAV8 vector under the tMCK promoter
(AAV8-
tMCK-AUF1-1RES-eGFP) (Vector Biolabs) (FIG. 10). AAV8-tMCK-IRES-eGFP was used
as a
control vector. This promoter was generated by the addition of a triple tandem
of 2RS5 enhancer
sequences (3-Ebox) ligated to the truncated regulation region of the MCK
(muscle creatine
kinase) promoter, which induced high muscle specificity (Wang et al., -
Construction and
Analysis of Compact Muscle-Specific Promoters for AAV Vectors," Gene Ther.
15(22):1489-
1499 (2008), which is hereby incorporated by reference in its entirety).
C57B16 mice were
injected with a single retro-orbital injection of 50 n1 (final concentration:
2.5x10" particles).
Muscle Function Tests
[0197] Grid hanging time. Mice were placed in the center of a
grid, 30 cm above soft
bedding to prevent injury should they fall. The grid was then inverted. Grid
hanging time was
measured as the amount of time mice held on before dropping off the grid. Each
mouse was
analyzed twice with 5 repetitions per mouse.
[0198] Time, distance to exhaustion, and maximum speed. After 1
week of acclimation,
mice were placed on a treadmill and the speed was increased by 1 m/min every 3
minutes and the
slope was increased every 9 minutes by 5 cm to a maximum of 15 cm. Mice were
considered to
be exhausted when they stayed on the electric grid more than 10 seconds. Based
on their
weight and running performance, work performance was calculated in Joules (J).
Each mouse
was analyzed twice with 5 repetitions per mouse.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 78 -
[0199] Strength by grip test (Examples 8 and 9): In this test,
mice grasp a horizon tall
grid connected to a dynamometer and are pulled backwards five times by tugging
on the tail.
The force applied to the grid each time before the animal loses its grip is
recorded in Newtons.
The average of the five tests is then normalized to the whole-body weight of
each mouse. Mice
are typically analyzed twice with 5 repetitions per mouse.
Dexa Muscle Mass Non-Invasive Quantitative Analysis (Example 7)
[0200] Dual energy X-ray absorptiometry (DEXA) was used to
record lean muscle mass
and changes in muscle mass upon injury or age previously published (Chenette
et al., "Targeted
mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate,
Promoting Skeletal Muscle Integrity,- Cell Rep. 16(5):1379-1390 (2016), which
is hereby
incorporated by reference in its entirety).
Quantification of satellite cells (Example 7)
[0201] Muscles are excised and digested in collagenase type I.
Cell numbers are
quantified by flow cytometry gating for Sdc4+ CD45- CD31- Scal- satellite cell
populations
(Shefer et al," Satellite-Cell Pool Size Does Matter. Defining the Myogenic
Potency of Aging
Skeletal Muscle," Dev. Biol. 294(1):50-66 (2006) and Brack et al., "Pax7 is
Back," Skelet.
Muscle 4(1):24 (2014), which are hereby incorporated by reference in their
entirety).
Muscle Fiber Type Analysis (Example 7)
[0202] Skeletal muscles were removed, put in OCT compound, fixed
in 4%
paraformaldehyde, and immunostained with antibodies to AUF1 (07-260,
Millipore), slow
myosin (N0Q7 5 4D, Sigma), fast myosin (MY-32, Sigma), and laminin alpha 2
membrane
component (4H8-2, Sigma).
Histological Studies and Biochemical Analysis of Muscle Tissues (Examples 7
and 8)
[0203] Muscles were removed and frozen in OCT compound, fixed in
4%
paraformaldehyde, and blocked in 3% BSA in TBS. Immunofluorescence or
immunochemistry
(Hematoxylin and Eosin, Masson Trichome) was performed. Fibrosis was assessed
by staining
of muscle sections with Masson trichrome to visualize areas of collagen
deposition and
quantified using ImageJ software. Immunofluorescence images were acquired
using a Zeiss
LSM 700 confocal microscope. Images and morphometric analysis (Feret diameter,
Cross
sectional area) were processed using ImageJ as recently described (Abbadi et
al., "Muscle
Development and Regeneration Controlled by AUF1-Mediated Stage-Specific
Degradation of
Fate-Determining Checkpoint mRNAs," Proc. Natl. Acad. Sci. USA 116(23):11285-
11290
(2019), which is hereby incorporated by reference in its entirety). Muscles
were harvested for
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 79 -
biochemical analysis including immunoblot, RNAseq, and RT-PCR analysis.
Evan Blue Dye Analysis (Example 7)
[0204] Evan Blue dye was used as an in vivo marker of muscle
damage. It identifies
permeable skeletal myofibers that have become damaged (Wooddell et al.,
"Myofiber Damage
Evaluation by Evans Blue Dye Injection," Curr. Protoc. Mouse Biol. 1(4):463-
488 (2011), which
is hereby incorporated by reference in its entirety).
Serum Creatine Kinase (CK) Activity (Example 7)
[0205] Serum CK was evaluated at 37 C by standard
spectrophotometric analysis using a
creatine kinase activity assay kit (abcam). The results are expressed in
mU/mL.
Blood Harvesting (Example 7)
102061 Peripheral blood was harvested to quantify creatine
kinase levels, and levels of
cytokines, cells and inflammatory markers.
Quantification and Statistical Analysis
[0207] All results are expressed as the mean SEM. Two group
comparisons were
analyzed by the unpaired Mann-Whitney test. Multiple group comparisons were
performed
using one-way analysis of variance (ANOVA). The non-parametric Kruskal¨Wallis
test
followed by the Dunn's comparison of pairs was used to analyze groups when
suitable. P-values
of <0.05 were considered significant. All statistical analyses were performed
using GraphPad
Prism (version 7) software.
Genome- Wide Transcriptomic and Trans latomic Studies and Bioinformatic Data
Analysis (Example 7)
[0208] Polysome fractionation and inRNA isolation. Polysome
isolation was performed
by separation of ribosome-bound mRNAs by sucrose gradient centrifugation using
cytoplasmic
extracts as previously described (de la Parra et al., "A Widespread Alternate
form of Cap-
Dependent mRNA Translation Initiation," Nat. Commun. 9(1):3068 (2018) and
Badura et al.,
"DNA Damage and eIF4G1 in Breast Cancer Cells Reprogram Translation for
Survival and
DNA Repair mRNAs," Proc. Natl. Acad.
USA 109(46):18767-72 (2012), which are hereby
incorporated by reference in their entirety). Post-fractionation samples were
pooled based on
enriched for mRNAs bound to 2-3 ribosomes and >4 ribosomes corresponding to
poorly
translated and well translated fractions respectively, and used for RNA
sequencing (RNAseq).
RNA quality was measured by a Bioanalyzer (Agilent Technologies).
[0209] RNA sequencing and data analysis. Paired-end RNA-seq was
carried out by the
New York University School of Medicine Genome Technology Core using the
Illumina HiSeq
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 80 -
4000 single read. The low-quality reads (less than 20) were trimmed with
Trimmomatic (Bolger
et al., "Trimmomatic: A Flexible Trimmer for Illumina Sequence Data,"
Bioinformatics,
30(15):2114-20 (2014), which is hereby incorporated by reference in its
entirety) (version 0.36)
with the reads lower than 35 nt being excluded. The resulted sequences were
aligned with STAR
(Dobin et al., "STAR: Ultrafast Universal RNA-Seq Aligner," Bioinformatics
29(1):15-21
(2013), which is hereby incorporated by reference in its entirety) (version
2.6.0a) to the hg38
reference genome in the single-end mode. The alignment results were sorted
with SAMtools (Li
et al., "The Sequence Alignment/Map format and SAMtools," Biontfarmatics
25(16):2078-2079
(2009), which is hereby incorporated by reference in its entirety) (version
1.9), after which
supplied to HT Seq (Anders et al., -HTSeq--A Python Framework to Work with
High-
Throughput Sequencing Data," BioinfOrmatics 31(2):166-9 (2015), which is
hereby incorporated
by reference in its entirety) (version 0.10.0) to obtain the feature counts.
The feature counts
tables from different samples were concatenated with a custom R script. To
examine differences
in transcription and translation, total mRNA and polysome mRNA were quantile-
normalized
separately. Regulation by transcription and translation and accompanying
statistical analysis was
performed using RIVET (Ernlund et al., "RIVET: Comprehensive Graphic User
Interface for
Analysis and Exploration of Genome-Wide Translatomics Data," BMC Genomics
19(1):809
(2018), which is hereby incorporated by reference in its entirety), where
significant genes were
identified as P<0.05 and >1 log fold change. Reactome pathway analysis was
performed on
genes that were up- and down-regulated by transcription and translation using
Metascape (Zhou
et al., "Metascape Provides a Biologist-Oriented Resource for the Analysis of
Systems-Level
Datasets," Nat. Commun. 10(1):1523 (2019), which is hereby incorporated by
reference in its
entirety). Pathway analysis and enrichment plots of the top 100 genes that
were the most
regulated by transcription and/or translation were generated using DAVID
(Huang da et al.,
"Systematic and Integrative Analysis of Large Gene Lists Using DAVID
Bioinformatics
Resources," Nat. Protoc. 4(1):44-57 (2009), which is hereby incorporated by
reference in its
entirety) and Metascape. Prediction of transcription factors of the same list
of 100 genes was
performed using Enrichr (Chen et al., "Enrichr: Interactive and Collaborative
HTML5 Gene List
Enrichment Analysis Tool," BMC Bioinformatics 14:128 (2013), which is hereby
incorporated
by reference in its entirety) (TRANSFAC and JASPER PWM program) and PASTAA
(Roider et
al., "Predicting Transcription Factor Affinities to DNA from a Biophysical
Model,-
Bioilfformatics 23(2):134-41 (2007), which is hereby incorporated by reference
in its entirety)
online tool. Genes enriched in TFH cells was determined from GSE16697
(Johnston et al.,
"Bc16 and Blimp-1 are Reciprocal and Antagonistic Regulators of T Follicular
Helper Cell
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-81 -
Differentiation," Science 325(5943):1006-1010 (2009), which is hereby
incorporated by
reference in its entirety) and similar genes between datasets were determined
using Venny.
Traumatic Injury Animal Model (Example 8)
[0210] Three month old male mice, unless otherwise noted,
were administered an
intramuscular injection of 50 IA of filtered 1.2% BaC17 in sterile saline with
control or with
lentivirus AUF1 vector (Ix108 genome copy number/ml) (total volume 100 til)
into the left
tibialis anterior (TA) muscle. The right TA muscle remained uninjured as a
control. Mice were
sacrificed at 3 or 7 days post-injection. Muscles were weighed and frozen in
OCT for
immunofluorescence staining or put in Trizol for mRNA extraction.
Example 1 ¨ Skeletal Muscle AUF1 Expression is Downregulated with Age
[0211]
Because mice deleted in the cuff/ gene undergo an accelerated loss of
muscle
mass (Chenette et al., "Targeted mRNA Decay by RNA Binding Protein AUF1
Regulates Adult
Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity," Cell Rep.
16(5):1379-1390
(2016); Abbadi et al., "Muscle Development and Regeneration Controlled by AUF1-
Mediated
Stage-Specific Degradation of Fate-Determining Checkpoint mRNAs," Proc. Natl.
Acad. Sci.
USA 116(23):11285-11290 (2019); and Pont et al., "mRNA Decay Factor AUF1
Maintains
Normal Aging, Telomere Maintenance, and Suppression of Senescence by
Activation of
Telomerase Transcription," Molecular Cell 47(1):5-15 (2012), which are hereby
incorporated by
reference in their entreity), whether reduced expression of AUF1 with age
occurs in wild type
animals and is involved in age-related muscle atrophy was investigated. The
expression of
AUF1 in limb skeletal muscles of young (3 month), middle-aged (12 month) and
older mice (18
month) was analyzed. Compared to 3 month young mice, aufl mRNA expression was
strongly
downregulated by 12 months of age in non-exercised animals, shown in the
tibialis anterior
(TA), gastrocnemius, extensor digitorum longus (EDL) and soleus muscles (FIG.
7A). In all
studies test mRNAs were normalized to gapdh or tbp mRNAs which were unchanged
in
abundance regardless of AUF1 expression. As shown in the TA muscle, AUF1
protein levels
tracked mRNA levels, demonstrating reduction by 60% at 12 months and 80% at 18
months
(FIG. 7B). Reduced skeletal muscle AUF1 expression with age in non-exercised
animals was
associated with a significant loss of muscle mass in limb muscles, shown in
the TA, EDL,
gastrocnemius and soleus muscles in 12 and 18 month old mice compared to 3
month old
animals (FIG. 7C). Importantly, by 18 months of age, loss of muscle mass began
to plateau from
12 month values. The TA muscle was reduced in mass by almost 50%, the EDL by
30%, the
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 82 -
soleus by almost 50% and the gastrocnemius by 25%. These data clearly show
that by 12-18
months of age, sedentary mice have undergone a significant reduction in
skeletal muscle mass
consistent with muscle loss and atrophy typically observed in the absence of
exercise and with
aging.
Example 2 ¨ AUF1 Skeletal Muscle Gene Transfer Enhances Exercise Endurance in
Middle-Aged and Old Mice
[0212]
Whether loss of skeletal muscle mass with age in mice is a result of
reduced
expression of AUF1 in skeletal muscle was investigated. An AAV8 (adeno-
associated virus type
8) vector was developed to deliver and selectively express AUF1 in skeletal
muscle. AAV
vectors express AUF1 and GFP (AUF1-GFP, with GFP translated from the same mRNA
by the
HCV IRES), or as a control only GFP. Expression of both genes is controlled by
the creatine
kinase tMCK promoter that is selectively active in skeletal muscle cells Wang
et al.,
"Construction and Analysis of Compact Muscle-Specific Promoters for AAV
Vectors," Gene
Ther. 15(22).1489-1499 (2008), which is hereby incorporated by reference in
its entirety). Mice
ages 3 and 12 months were administered a single retro-orbital injection of
either AAV AUF1-
GFP or control AAV GFP vectors (2.0 x 1011 genome copies). When analyzed
starting at 40
days post-administration of AAV vectors, as shown in 12 month old mice, both
AAV AUF1-
GFP and AAV GFP control vector-treated animals displayed similar vector
transduction and
retention rates, shown by TA muscle GFP staining (FIGs. 1A-1B). cliff] mRNA
expression in
skeletal muscle was increased at this time over that of endogenous levels by
AAV8 AUF1-GFP
administration, on average 2.5-fold in EDL, 6-fold in TA, 2.5-fold in
gastrocnemius and slightly
in soleus muscle which has a high endogenous level, as shown later (FIG. 1C).
AUF1 protein
levels in gene transferred animals in skeletal muscle, as shown in the TA
muscle, demonstrated
4-6 fold increased expression over endogenous levels, corresponding to mRNA
levels (FIG. 7D).
There was no evidence for increased expression of AUFI in non-muscle tissues
compared to
control mice (kidney, lung, spleen, liver) (FIG. 7E), demonstrating strong
tissue specificity for
skeletal muscle expression of AUF1 by the tMCK promoter. Importantly, Pax7
expression, a
key marker for activation of muscle satellite cells and proliferating
myoblasts, was also increased
3-4 fold with AAV AUF1-GFP administration (FIG. 7F). Correspondingly, markers
of muscle
atrophy such as trim63 and fbro32 (Nilwik et al., "The Decline in Skeletal
Muscle Mass with
Aging is Mainly Attributed to a Reduction in type II muscle Fiber Size," Exp.
Geronlol.
48(5):492-498 (2013), which is hereby incorporated by reference in its
entirety), were
downregulated 3-fold in the TA muscle of animals administered with AAV AU1F1-
GFP (FIG.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 83 -
7G). Collectively, these data indicate that only moderate levels of AUF1 gene
transfer into
skeletal muscle was sufficient to reduce markers of muscle atrophy coincident
with activation of
satellite cells and myoblasts.
102131 It was therefore investigated whether AUF1 gene transfer
can increase physical
endurance in middle aged and older sedentary mice, using a number of well-
established criteria.
Twelve month old sedentary mice were administered AAV8 AUF1-GFP or control
AAV8 GFP,
then tested at 40 days post-administration. AUF1 supplemented mice showed a
¨50%
improvement in grid hanging time (FIG. ID), a measure of limb-girdle skeletal
muscle strength
and endurance. When tested by treadmill, AAV AUF1-GFP mice displayed 25%
higher
maximum speed (FIG. 1E) and 50% increase in work performance (FIG. 1F)
compared to AAV
GFP control mice, as well as 25% greater time to exhaustion and 30% increased
distance to
exhaustion (FIG. 1G, FIG. 1H). When compared to 3 month old mice receiving
control AAV
GFP, 12 month old mice gained equivalent physical endurance capacity to the
level of young
mice (FIGs. 1E-1H). Physical endurance was also tested 6 months post AAV-AUF1
injection of
12 month old mice that were 18 months at the time and kept non-exercised until
the time of
testing. Maximum speed (FIG. 1I), work performed (FIG. 1J), as well as time
and distance to
exhaustion (FIG. 1K, FIG. 1L) were all significantly higher in AUF1-AAV
treated animals,
similar to 12 month old mice at 40 days post-treatment. These results
demonstrate that the
enhancement of exercise endurance in older mice with muscle loss and atrophy
by
supplementation with AUF1 is durable at 6 months post-treatment, with no
evidence for
diminution. It was therefore next investigated whether the biological and
molecular
characteristics of AUF1 restored skeletal muscle.
Example 3 ¨ AUF1 Gene Therapy Increases Muscle Mass and Greater Slow-Twitch
than
Fast-Twitch Myofibers
[0214] Skeletal muscles vary in slow- and fast-twitch myofiber composition
(Type I or
II, respectively). TA, EDL, and gastrocnemius muscles are composed mostly of
Type II fast-
twitch myofibers (nearly 99% fast, 1% slow), whereas the soleus muscle is
highly enriched in
Type I slow-twitch myofibers (nearly 40% slow, 60% fast) (Augusto et at.,
"Skeletal Muscle
Fiber Types in C57BL6J mice," J. Alorphot Sci. 21(2):89-94 (2004), which is
hereby
incorporated by reference in its entirety). Analysis of the gastrocnemius and
TA muscles
showed that 12 month sedentary old mice gained an average total increase of
¨20% in muscle
mass relative to body weight in animals administered AAV AUF1-GFP compared to
AAV GFP
controls (FIG. 2A, FIG. 2B). Increased muscle fiber size (myofiber cross-
sectional area, CSA)
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 84 -
and number are established hallmarks of muscle regeneration (Schiaffino &
Reggiani, "Fiber
Types in Mammalian Skeletal Muscles," Physiol. Rev. 91(4):1447-1531 (2011) and
Yin et al.,
"Satellite Cells and the Muscle Stem Cell Niche," Physiol. Rev. 93(1):23-67
(2013), which are
hereby incorporated by reference in their entirety). Compared to GFP control
mice, as shown in
the TA and gastrocnemius muscles of 12 month old AAV AUF1-GFP mice, there was
a
significant increase in the percentage of larger myofibers, which was
particularly pronounced for
larger myofibers (>3200 tm2) (FIGs. 2C-2F). Increased myofiber size can be
indicative of
vigorous and mature muscle regeneration. It was also investigated whether
supplemental AUF1
expression promotes slow-twitch, fast-twitch or both types of myofibers. AUF1
supplementation
increased the number and size of slow-twitch myofibers per field by nearly 60%
compared to
fast-twitch fibers, as shown in the gastrocnemius muscle (FIGs. 2G-2H). In the
soleus muscle,
which is composed primarily of slow-twitch muscle, the myofiber area was
similarly increased
with AUF1 supplementation (FIG. 21, FIG. 2J).
[0215] Expression levels of different myosin type mRNAs also
support that AUF1 gene
transfer resulted in real gain in skeletal muscle mass. The major slow-twitch
myosin mRNA,
myh7, was increased 6-fold in gastrocnemius and 2-fold in soleus muscle with
AUF1 gene
transfer (FIG. 3A, FIG. 3B), whereas fast myosin mRNAs such as myh 1 , rnyh2
and myh4 were
not statistically changed (FIG. 3C, FIG. 3D).
[0216] Further evidence was obtained for increased muscle
generation by AUF1 is
supported by measuring the mRNA levels of several genes whose expression are
hallmarks of
increased myofiber regeneration, oxidative processes and mitochondrial
biogenesis. Slow-twitch
myofibers in particular are enriched in oxidative mitochondria (Schiaffino &
Reggiani, "Fiber
Types in Mammalian Skeletal Muscles," Physiol. Rev. 91(4):1447-1531 (2011),
which is hereby
incorporated by reference in its entirety). The focus of these studies was on
the gastrocnemius
muscle because it demonstrated a median response to AUF1 gene therapy and it
is not biased
toward enrichment of slow-twitch myofibers. While AUF1 gene transfer had no
effect on
gastrocnemius mRNA levels of non-mitochondrial genes such as ppara (peroxisome
proliferator-activated receptor alpha) or sic] (Sineoculis homeobox homolog
1), it increased
levels of mitochondrial mRNAs for tfam (mitochondria transcription factor A)
by 4-fold, acadvl
(acyl-CoA dehydrogenase very long chain) by 6-fold, nrfl by 3-fold and nrf2 by
2-fold (nuclear
respiratory factor) (FIGs. 3E-3H). The ratio of mitochondrial to nuclear DNA
was also increased
in the gastrocnemius with AUF1 gene transfer, indicative of increased
mitochondrial content at
both 40 days and 6 months post-gene transfer (FIG. 31, FIG. 3J). Collectively,
these results show
that AUF1 promotes transition from fast to slow twitch myofiber.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 85 -
Example 4 ¨ AUF1 Stimulates Slow-Twitch Muscle Development in Part by
Increasing
PGCla Expression
[0217] Increased levels slow-twitch Type I muscle fibers are
particularly sought for
combating muscle loss with age because it is associated with increased muscle
endurance. A key
feature of slow muscle is that it confers exercise endurance because slow-
twitch myofibers have
much higher oxidative capacity than fast-twitch fibers (Cartee et al.,
"Exercise Promotes Healthy
Aging of Skeletal Muscle," Cell Metab. 23(6):1034-1047 (2016) and Yoo et al.,
"Role of
Exercise in Age-Related Sarcopenia," I Exerc. Rehabil. 14(4):551-558 (2018),
which are hereby
incorporated by reference in their entirety). Therefore, the level of AUF1
expression in different
muscles with varying proportions of slow- and fast myofibers was
characterized. There was a
notable 2-4 fold higher level of expression of aufl mRNA and AUF1 protein
levels in the soleus
muscle of 3 month and sedentary 12 month old untreated mice compared to other
muscle types
with fewer slow-twitch myofibers (FIG. 4A, FIG. 4B). Accordingly, of the lower
limb skeletal
muscles, the soleus muscle is the most endurant, the most enriched in slow-
twitch myofibers
(Schiaffino & Reggiani, "Fiber Types in Mammalian Skeletal Muscles," Physiol.
Rev.
91(4):1447-1531 (2011) and Augusto et al., "Skeletal Muscle Fiber Types in
C57BL6J mice," J.
Morphol. Sci. 21(2):89-94 (2004), which are hereby incorporated by reference
in their entirety),
and expresses much higher levels of myh7 (FIG. 4C), the main slow-twitch
myofiber myosin.
Therefore, the role of AUF1 in expression of different levels of myosin mRNAs
was assessed by
deletion of AUF1 in C2C12 mouse myoblasts. Deletion of AUF1 increased the
expression of
fast-twitch nlyh2 mRNA levels, while slow myosin mRNAs, such as ugh 7 or
ntyI2, were
decreased (FIG. 8A), consistent with AUF1 greater specification of slow-twitch
myofiber
development. Importantly, expression of the myocyte enhancer factor 2 (nief2c)
gene, a key
transcriptional regulator of overall skeletal muscle development, was also
increased by AUF1
supplementation (FIG. 8B). MEF2c can activate or repress different myogenic
transcriptional
programs and its increased expression is also consistent with increased
generation of Type
slow-twitch muscle (Lin et al., "Transcriptional Co-Activator PGC-1 Alpha
Drives the
Formation of Slow-Twitch Muscle Fibres,- Nature 418 (6899):797-801 (2002),
which is hereby
incorproated by reference in its entirety), suggesting involvement in AUF1-
mediated
specification of slow-twitch muscle.
[0218] The MEF2c protein stimulates expression of PGCla
(Peroxisome proliferator-
activated receptor gamma coactivator 1 alpha) which drives the specification
and development of
slow-twitch myofibers (Lin et al., "Transcriptional Co-Activator PGC-1 Alpha
Drives the
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 86 -
Formation of Slow-Twitch Muscle Fibres," Nature 418 (6899):797-801 (2002),
which is hereby
incorproated by reference in its entirety). Deletion of the mill gene in C2C12
myoblasts induced
to differentiate to myotubes decreased pgcla mRNA levels by half and protein
levels by 4-fold
(FIG. 4D), suggesting that AUF1 acts to increase PGC ice protein and mRNA
expression.
Accordingly, AAV8-AUF1 gene transfer in mice showed that pgcla mRNA levels
were
increased 2-3 fold in the gastrocnemius and EDL muscles, and trended toward
upregulation in
the TA muscle in 12 month old mice (FIG. 4E). AUF1 gene transfer in 18 month
old sedentary
mice also strongly increasedpgc/a mRNA levels ¨2.5-fold, as shown in the
gastrocnemius
muscle (FIG. 4E), which corresponded to an average 5-fold increase in PGCla
protein levels
(FIG. 4F).
[0219] The pgcice mRNA contains a 3' UTR with multiple ARE
motifs that could be
potential AUF1-binding sites (Lai et al., "Effect of Chronic Contractile
Activity on mRNA
Stability in Skeletal Muscle," Am. J. Physiol Cell. Physiol. 299(1):C155-163
(2010), which is
hereby incorporated by reference in its entirety). Therefore, AUF1 was
immunoprecipitated
from WT C2C12 myoblasts 48 hours after differentiation when AUF1 is expressed,
with control
IgG or anti-AUF1 antibodies, followed by qRT-PCR to quantify the levels of
bound pgcla
mRNA (FIG. 4G). AUF1 bound strongly to the pgcla mRNA in differentiating C2C12
cells.
The effect of AUF1 expression on the pgcla mRNA half-life was then determined
using WT and
AUF1 KO C2C12 cells by addition of actinomycin D to block new transcription
(FIG. 4H).
Surprisingly, in the absence of AUF1, pgcla mRNA displayed an almost 3-fold
reduced
stability. The pgcla mRNA therefore belongs to the class of ARE-mRNAs that are
stabilized
rather than destabilized by AUF1, accounting in part for increased levels of
PGClcc protein and
increased specification of slow-twitch fiber formation by AUF1. Therefore, the
impact of AUF1
expression specifically on slow-twitch muscle loss and atrophy was
investigated.
Example 5 ¨ Loss of AUF1 Expression Selectively Accelerates Atrophy of Slow-
Twitch
Muscle in Young Mice
[0220] To better understand the role of AUF1 gene therapy in the
formation and
maintenance of slow-twitch myofibers, slow-twitch myofibers in WT and A UF1 KO
mice were
investigated at 3 months of age, before the onset of dystrophy (Chenette et
al., "Targeted mRNA
Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate,
Promoting
Skeletal Muscle Integrity," Cell Rep. 16(5):1379-1390 (2016), which is hereby
incorproated by
reference in its entirety). At 3 months, WT and ctufl KO mice have similar
body weights (FIG.
5A). While deletion of aufl did not change the size, color (mitochondrial
density, myoglobin
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 87 -
content) or weight of the TA, EDL or gastrocnemius muscles, it did reduce the
size and weight
of the soleus muscle by half at 3 months, which was much paler, indicative of
loss of
mitochondrial and myoglobin-rich Type I myofibers (FIG. 5B; FIG. 9A). The
proportion and
number per field of slow myosin myofibers in the AUF1 KO mouse soleus muscle
was reduced
40-50% (FIGs. 5C-5E; FIG. 9B). In contrast, both the proportion and number of
fast-myosin-
expressing myofibers was increased by 25% or more in the absence of AUF 1
expression (FIG.
5C, FIG. 5F, FIG. 5G; FIG. 9B). Reduced expression of slow myosin was also
seen in the
gastrocnemius muscle with aull deletion in cuff] KO mice (FIGs. 9C-9E). In
addition, the mean
CSA was reduced by 2-fold in slow-twitch myofibers, as shown in the soleus and
gastrocnemius
muscles, but was unchanged in fast-twitch myofibers (FIG. 5H; FIG. 9F).
Consistent with these
data, AUF1 KO mice at 3 months expressed 3-4 fold lower levels of PGC la
protein than WT
mice, as shown in the gastrocnemius and soleus muscles (FIG. 9G). AUF1
therefore specifies
regeneration and maintenance of slow-twitch muscle.
Example 6 ¨ Loss of AUF1 in Older Mice Accelerates Atrophy and Loss of both
Slow-
Twitch and Fast-Twitch Muscle
[0221]
At 6 months of age, auf I KO mice show a 20% loss of body weight, which is
largely a result of loss of skeletal muscle mass (FIG. 6A). Unlike 3 month old
mice where the
slow-twitch rich soleus muscle was the only muscle showing significant atrophy
in the absence
of AUF1 expression, in 6 month old mice both fast-twitch rich and slow-twitch
rich muscles
demonstrate significant atrophy. The size and weight of the TA, EDL and
gastrocnemius
muscles were reduced by ¨25% in aufl KO compared to WT animals, and the soleus
muscle was
reduced by almost 50% (FIG. 6B). In addition, aufl KO mouse skeletal muscles
were paler than
control WT mice, consistent with greater loss of mitochondri al-dense, slow-
twitch myofibers
(FIG. 6C). Accordingly, the mean CSA of both slow- and fast-twitch myofibers,
as shown in the
soleus and gastrocnemius muscles, showed a striking reduction at 6 months in
auf/ KO mice
compared to WT, indicative of overall myofiber atrophy (FIG. 6D, FIG. 6E). As
seen in young
mice, AUF1 deficiency reduced by half the percentage and number of slow-twitch
myofibers per
field in the soleus and gastrocnemius muscles (FIGs. 6F-6I). Thus, while AUF1
specifies
development of slow-twitch muscle, its additional activities are essential for
maintenance and
regeneration of both slow- and fast-twitch muscle, consistent with the ability
of AUF1 gene
transfer to promote increased overall muscle mass and function in sedentary
animals that have
undergone muscle loss and atrophy during aging.
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 88 -
Discussion of Examples 1 ¨ 6
[0222] This work reports three important sets of findings: (1)
AUF1 expression in
skeletal muscle is lost with aging in sedentary mice, which contributes to the
development of
age-related muscle atrophy; (2) AUF1 gene therapy is a promising therapeutic
intervention to
delay or reverse the loss of muscle mass and strength with age; and (3) AUF1
is required to form
both slow and fast myofiber, but also promotes transition from fast to slow
muscle phenotype by
increasing PGCla levels through stabilization of its mRNA. AUF I generally
promotes rapid
decay of ARE-containing mRNAs but can stabilize a subset of other ARE-mRNAs
(Moore et al.,
"Physiological Networks and Disease Functions of RNA-Binding Protein AUF1,"
Wiley
Interdiscip. Rev. RNA 5(4):549-564 (2014), which is hereby incorporated by
reference in its
entirety). During muscle regeneration, AUF1 therefore regulates satellite cell
maintenance and
differentiation in part by programming each stage of myogenesis through
selective degradation
of short-lived myogenic checkpoint ARE-mRNAs (Chenette et al., "Targeted mRNA
Decay by
RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting
Skeletal Muscle
Integrity," Cell Rep. 16(5):1379-1390 (2016) and Abbadi et al., "Muscle
Development and
Regeneration Controlled by AUF1-Mediated Stage-Specific Degradation of Fate-
Determining
Checkpoint mRNAs," Proc. Natl. Acad. Sci. USA 116(23): 11285-11290 (2019),
which are
hereby incorporated by reference in their entirety). In addition, as shown
here, by increasing
AUF1 expression levels in sedentary mice using gene transfer, AUF1 increases
myosin and
oxidative mitochondrial gene expression that promotes slow myofiber formation
and oxidative
phenotype. There is also evidence for reduced AUF1 expression in human
skeletal muscle with
aging (Masuda et al., "Tissue- and Age-Dependent Expression of RNA-Binding
Proteins that
Influence mRNA Turnover and Translation," Aging (Albany NY) 1:681-698 (2009),
which is
hereby incorporated by reference in its entirety), although the general
inability to obtain serial
age-related but otherwise normal muscle specimens limits the ability to expand
this finding.
[0223] Gene therapy of skeletal muscle with AUF1 by AAV8-AUF1
significantly
promoted new muscle mass and exercise endurance in middle aged non-exercised
mice that had
significant muscle loss and atrophy. Notably, in a rat model designed to
characterize skeletal
muscle markers of increased physical exercise endurance, two major factors
that were found to
be increased in expression were AUF1 and PGCla (Lai et al., "Effect of Chronic
Contractile
Activity on mRNA Stability in Skeletal Muscle," Am. J. Physiol. Cell. Physiol.
299(1):C155-163
(2010), which is hereby incorporated by reference in its entirety). Moreover,
an exercise study in
mice found that while one week of exercise induced increased levels of PGCla,
after four weeks
of exercise AUF1 increased as much as 50% without changes in other ARE-binding
proteins
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 89 -
(Matravadia et al., "Exercise Training Increases the Expression and Nuclear
Localization of
mRNA Destabilizing Proteins in Skeletal Muscle,- Am. I Physiol. Regul Integr.
Comp. Physiol.
305(7):R822-831 (2013), which is hereby incorporated by refernece by its
entirety).
[0224] Interestingly, pgc I a, tfam and nrf2 mRNAs all contain
AREs in their 3'UTRs,
which may be subject to regulation by ARE-binding proteins, including AUF1 (D'
Souza et al.,
"mRNA Stability as a Function of Striated Muscle Oxidative Capacity," AM. J.
Physiol. Regul.
Integr. Comp. Physiol. 303(4):R408-417 (2012), which is hereby incorporated by
reference in its
entirety). While perplexing at the time, AUF1 was then only known to cause ARE-
mRNA
decay, not stabilization. These findings, when combined with the results
disclosed herein,
suggests that AUF1 programs a feed-forward mechanism to promote muscle
regeneration
through stabilization ofpgc/amRNA and, through other AUF1 activities as well
(Chenette et al.,
"Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem
Cell
Fate, Promoting Skeletal Muscle Integrity,- Cell Rep. 16(5):1379-1390 (2016)
and Abbadi et al.,
"Muscle Development and Regeneration Controlled by AUF1-Mediated Stage-
Specific
Degradation of Fate-Determining Checkpoint mRNAs," Proc. Natl. Acad. Sci. USA
116(23):11285-11290 (2019), which are hereby incorporated by reference in
their entirety).
Consistent with this conclusion, the AUF1 KO mice used herein present at a
young age a
reduction of slow twitch myofiber size and a decreased level of PGC la
expression.
[0225] That AUF1 muscle supplementation increased PGCla protein
levels is important.
PGC la activates expression of downstream factors such as NRFs and Tfam that
promote
mitochondrial biogenesis, which are essential for the formation of slow-twitch
muscle fibers,
reduced fatigability of muscle and greater oxidative metabolism (Lin et al.,
"Transcriptional Co-
Activator PGC-1 Alpha Drives the Formation of Slow-Twitch Muscle Fibres,"
Nature
418(6899):797-801 (2002), which is hereby incorproated by reference in its
entirety). These
findings, along with enhanced mitochondrial DNA content observed with AUF1
supplementation, suggest that AUF1 is responsible for key activities in slow-
twitch myofiber
maintenance and increased exercise endurance in mice. Previous studies have
shown the benefit
of increased PGCla expression in muscle damage repair and angiogenesis (Wiggs,
M. P., "Can
Endurance Exercise Preconditioning Prevention Disuse Muscle Atrophy?,- Front.
Physiol. 6:63
(2015); Wing et al., "Proteolysis in Illness-Associated Skeletal Muscle
Atrophy: From Pathways
to Networks," Crit. Rev. Clin. Lab. Sci. 48(2):49-70 (2011); Bost & Kaminski,
"The Metabolic
Modulator PGC-lalpha in Cancer," Am. J. Cancer Res. 9(2):198-211 (2019), Dos
Santos et al.,
"The Effect of Exercise on Skeletal Muscle Glucose Uptake in type 2 Diabetes:
An Epigenetic
Perspective," Metabolism 64(12):1619-1628 (2015); Haralampieva et al., "Human
Muscle
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 90 -
Precursor Cells Overexpressing PGC-lalpha Enhance Early Skeletal Muscle Tissue
Formation,"
Cell Transplant 26(6):1103-1114 (2017); and Janice Sanchez et al., "Depletion
of HuR in
Murine Skeletal Muscle Enhances Exercise Endurance and Prevents Cancer-Induced
Muscle
Atrophy," Nat. Commun. 10(1):4171 (2019), which are hereby incorporated by
reference in their
entriety).
[0226] AUF1 skeletal muscle gene transfer is therefore
beneficial in countering muscle
loss and atrophy because it is required to enable multiple key steps in
myogenesis. AUF1
stimulates greater muscle development and physical exercise capacity in aging
sedentary muscle,
which in turn likely further stimulates AUF1 expression as a result of
exercise itself Moreover,
the effects of AUF1 gene transfer appear to be long-lasting. Improved exercise
endurance in the
studies disclosed herein was found to be sustained for at least 6 months
beyond the time of gene
transfer (the last time point tested) with no evidence for reduction in AUF1
expression or
efficacy. In this regard, AUF1 supplementation also increased levels of Pax7+
activated satellite
cells and myoblasts, suggesting gene transfer into muscle stem cells and an
active myogenesis
process.
[0227] Apart from AUF1, other ARE RNA-binding proteins have also
been shown to be
involved in the myogenesis process. Of particular relevance to the studies
disclosed herein, HuR
was recently found to destabilize pgela mRNA, leading to the formation of type
II myofibers
(Janice Sanchez et al., "Depletion of HuR in Murine Skeletal Muscle Enhances
Exercise
Endurance and Prevents Cancer-Induced Muscle Atrophy," Nat. Commun. 10(1):4171
(2019),
which is hereby incorporated by reference in its entriety). It is noteworthy
that AUF1 and HuR
often have opposite effects on ARE-mRNA stability, in accord with the findings
disclosed
herein, and both are essential for the maintenance of myofiber specification.
AUF1 can also
interact with HuR although the potential functional consequence is unknown,
and AUF1 can also
compete for binding to AREs with TIA-1, which blocks AUF1-mediated mRNA decay
ARE-
mRNA translation (Pullmann et al., "Analysis of Turnover and Translation
Regulatory RNA-
Binding Protein Expression Through Binding to Cognate mRNAs,"Mol. Cell Biol.
27(18):6265-
6278 (2007), which is hereby incorporated by reference in its entirety).
Clearly, the role of
ARE-binding proteins in myogenesis is complex and further investigation into
their combined
activities is needed to better understand this complexity. How muscle
homeostasis is regulated
by AUF1 with the other ARE-binding proteins remains to be discovered.
[0228] Finally, it is important to note that while AUF1
specifies Type I slow-twitch
myofiber development, it also promotes and reprograms the overall myogenesis
regeneration
program (Abbadi et al., "Muscle Development and Regeneration Controlled by
AUF1-Mediated
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-91 -
Stage-Specific Degradation of Fate-Determining Checkpoint mRNAs," Proc. Natl.
Acad. Sci.
USA 116(23):11285-11290 (2019), which is hereby incorporated by reference in
its entirety),
evidenced by the fact that AUF1 skeletal muscle gene transfer did not result
in abnormal muscle
development, abnormal balance of muscle fiber types or muscle overgrowth.
Example 7 ¨ AUF1 Restores Skeletal Muscle Mass and Function in Duchenne
Muscular
Dystrophy (DMD) Mice
[0229] To examine the effect of AUF1 gene therapy on skeletal
muscle mass and
function in a mouse model of Duchenne muscular dystrophy (DMD), the cDNA for
full-length
p45 AUF1 isoform, which carries out all AUF1 functions, was cloned into an
AAV8 vector
under the control of the tMCK promoter (AAV8-tMCK-AUF1-IRES-eGFP) (Vector
Biolabs),
with the AAV8-tMCK-1RES-eGFP 2RS5 enhancer sequences (3-Ebox) ligated to the
truncated
regulation region of the MCK (muscle creatine kinase) promoter, which provides
high skeletal
muscle specificity.
[0230] The transduction frequency of AAV8 AUF1-GFP and AAV8 GFP
control vectors
was evaluated in mdx mice by tibialis and muscle GFP staining (FIG. 11A). No
statistical
differences in transduction efficiency was observed between control AAV8 GFP
and treatment
AAV8 AUF1 GFP groups (FIG. 11B).
[0231] To determine whether AUF1 supplementation enhances muscle
mass and/or
endurance in indx mice, one month old C57B110 and mdx mice were administered
AAV8-AUF1-
GFP or control AAV8-GFP vectors at 2x1011 genome copies by retro-orbital
injection (FIGs.
12A-12F and FIGs. 13A-13D). Mice were weighted and monitored for 2 months.
AAV8
AUF1-GFP supplemented mdx mice had a significant increase in average body
weight, as
compared to control mdx mice (FIG. 12A). Moreover, AAV8 AUF1-GFP treated mdx
mice
demonstrated a 10% increase in tibialis anterior (TA) muscle mass and an 11%
increase in
extensor digitorum longus (EDL) muscle mass (FIG. 12B), as compared to control
AAV8 GFP
treated mcbc mice. Compared to control AAV8 GFP treated mdx mice, AUF1
supplemented
mdx mice showed a ¨40% improvement in grid hanging time, a measure of limb-
girdle skeletal
muscle strength and endurance (FIG. 12C). When tested by treadmill, AAV AUF1-
GFP mdx
mice displayed 16% higher maximum speed (FIG. 12D), a 35% greater time to
exhaustion (FIG.
12E), and a 37% increased distance to exhaustion (FIG. 12F). These data
demonstrate a
substantial and statistically significant increase in exercise performance and
endurance in mdx
mice as a result of AUF1 gene transfer. In contrast to the mdx mice, there was
no significant
increase in body weight (FIG. 13A), treadmill time to exhaustion (FIG. 13B),
maximum speed
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 92 -
(FIG. 13C), or distance to exhaustion (FIG. 13D) in AAV8 AUF1-GFP treated WT
mice as
compared to control AAV8 GFP treated mice of the same genetic background.
[0232] AUF1 overexpression in mdx mice also ameliorated the
diaphragm dystrophic
phenotype (FIGs. 15A-15B). The percent degenerative diaphragm muscle was
reduced by
74% in AAV8 AUF1-GFP treated mdx mice as compared to control AAV8 GFP treated
mdx
mice (FIG. 15A). AUF1 gene transfer also significantly reduced diaphragm
fibrosis (FIG. 15B)
and macrophage infiltration (FIGs. 16A-16B) in AAV8 AUF1-GFP treated mdx mice,
as
compared to control AAV8 GFP treated mdx mice.
[0233] Histological signs of muscular dystrophy, including
myofiber centro-nucleation
and embryonic myosin heavy chain (eMHC) expression were tested. The percent of
centro-
nuclei and eMHC positive fibers found increased in mdx mice were highly
downregulated upon
AUF1 supplementation (FIGs 17A-17D). The size of centro-nuclei myofibers was
also
increased upon AUF1 supplementation (FIG. 17E).
[0234] Serological level of creatine kinase (CK) activity, a
measure of sarcolemma
leakiness used to aid diagnosis of DMD is found increased in control mdx mice,
however CK
activity was highly decreased upon AUF1 supplementation in mdx mice (FIG. 14).
[0235] Utrophin expression was also assessed in vitro and in
vivo. In vitro, only WT
C2C12 myoblasts differentiated into myotubes present an increase of utrophin
mRNA and
protein. AUF1 gene therapy strongly increased expression of utrophin and
showed evidence
for normalization of myofiber integrity in mdx mice, relative to control mdx
mice receiving
vector alone (FIGs. 18A-18C). AAV8 AUF1 gene transfer increased expression of
satellite
cell activation gene l'ax7 (FIG. 19A), key muscle regeneration genes pgc 1 a
and mef2c (FIG.
19A), slow twitch determination genes (FIG. 19B), and mitochondrial DNA
content (FIG.
19C) in mdx mice, relative to control mdx mice receiving vector alone.
[0236] Genome-wide transcriptomic and translatomic studies were carried out
to evaluate
whether AUF1 activation of C2C12 activates myoblast muscle fiber development
(FIG. 20).
These studies demonstrate that AUF1 supplementation (i) stimulates expression
of major muscle
development pathways and decreases expression of inflammatory cytokine,
inflammation, cell
proliferation, cell death, and anti-muscle regeneration pathways (FIGs. 21A-
21B); (ii)
upregulates pathways for major biological processes and molecular functions in
muscle
development and regeneration (FIGs. 22A-22B); (iii) decreases muscle
inflammation,
inflammatory cytokine, and signaling pathways that oppose muscle regeneration
(FIGs. 23A-
23B); and (iv) decreases expression of muscle genes associated with
development of fibrosis
(FIG. 24).
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 93 -
Discussion of Example 7
Dystrophin Gene Therapy
[0237] As described above, DMD is caused by mutations in the
dystrophin gene,
resulting in a near-absence of expression of the protein, which plays a key
role in stabilization of
muscle cell membranes (Bonilla et al., "Duchenne Muscular Dystrophy:
Deficiency of
Dystrophin at the Muscle Cell Surface," Cell 54(4):447-452 (1988) and Hoffman
et al.,
"Dystrophin: The Protein Product of the Duchenne Muscular Dystrophy Locus,"
Cell 51(6):919-
928 (1987), which is hereby incorporated by reference in its entirety). Since
the dystrophin gene
is very large, it is impossible to reintroduce the entire gene by gene
therapy. Thus, current gene
therapy attempts involve introducing by gene transfer "mini" and "micro"
dystrophin genes, i.e.,
small pieces of the dystrophin gene packaged in AAV vectors. To date, none
have been shown
to be very effective and there is evidence that because mini and micro
dystrophin genes are
different than an individual's dystrophin gene, they evoke an immune response
against the
therapeutic gene. Since dystrophin is mutated in DMD, there is currently
intense interest in
finding ways to increase expression of the dystrophin homolog known as
utrophin that has
overlapping function. To date, this has not been achieved at therapeutic
levels that can be shown
to be effective.
DMD mdx Mouse Model
[0238] The most widely used DMD niclic mouse (C57BL/10
background) has a
spontaneous genetic mutation resulting in a nonsense mutation (premature stop
codon) in exon
23 of the very large dystrophin mRNA, similar to the occurrence in roughly 13%
of DMD males
(Bulfield et al., "X Chromosome-Linked Muscular Dystrophy (mdx) in the Mouse,"
Proc. Natl.
Acad. Sci. USA 81(4):1189-1192 (1984), which is hereby incorporated by
refernce in its
entirety). This mdx mouse model has been used extensively for DMD
investigations and
therapeutics research, and is considered the "gold standard" animal model for
study of DMD.
The C57BL/10 mdx mice are as susceptible to physical muscle damage as are
humans and
reflects human disease in certain tissues (diaphragm, cardiac muscles),
although they are less
susceptible to damage in skeletal muscle (Moens et al., "Increased
Susceptibility of EDL
Muscles from mdx Mice to Damage Induced by Contractions with Stretch," J.
Muscle Res. Cell.
Moth. 14(4):446-451 (1993), which is hereby incorporated by refernce in its
entirety). As in
humans, the disease progresses in skeletal muscle with age in mdx mice (Moens
et al., "Increased
Susceptibility of EDL Muscles from mdx Mice to Damage Induced by Contractions
with
Stretch," J. Muscle Res. Cell. Moth. 14(4):446-451 (1993), which is hereby
incorporated by
reference in its entirety). Equally important, the diaphragm as a target for
myo-pathogenesis in
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 94 -
inch mice has been shown to very precisely reproduce the level and rate of
damage seen in
humans and is an excellent readout for effectiveness of therapeutic
intervention (Stedman et al.,
"The mdx Mouse Diaphragm Reproduces the Degenerative Changes of Duchenne
Muscular
Dystrophy," Nature 352(6335):536-539 (1991), which is hereby incorporated by
reference in its
entirety), and will be studied here.
[0239] Importantly, both mdx mice and DMD patients deplete their
satellite cells after
cycles of necrosis and regeneration of myofibers which promotes disease
progression (Manning
& O'Malley, "What has the mdx Mouse Model of Duchenne Muscular Dystrophy
Contributed to
our Understanding of this Disease?" J. Muscle Res. Cell Motif. 36(2):155-167
(2015) and Coley
et al., "Effect of Genetic Background on the Dystrophic Phenotype in mdx
Mice," Hum. Mol.
Genet. 25(1).130-145 (2016), which are hereby incorporated by reference in
their entirety).
Moreover, mdx mice and DMD patients both develop an inflammatory response that
increases
with disease progression (Manning & O'Malley, "What has the mdx Mouse Model of
Duchenne
Muscular Dystrophy Contributed to our Understanding of this Disease?" J.
Muscle Res. Cell
Moth. 36(2):155-167 (2015) and Coley et al., "Effect of Genetic Background on
the Dystrophic
Phenotype in mdx Mice,- Hum. Mol. Genet. 25(1):130-145 (2016), which are
hereby
incorporated by reference in their entirety).
[0240] Despite the fact that skeletal muscle dystrophic disease
is generally milder in the
mcbc mouse than in humans, it still provides a predictive model for
pharmacologic response,
particularly when coupled with progression of disease in diaphragm. Thus, the
mcbc mouse
provides a reliable, well-established and predictive model in which to follow
disease progression
and treatment response in animals that has been proven to be useful in
development of strategies
for interventional agents for DMD clinical trial (Fairclough et al., "Davies,
Pharmacologically
Targeting the Primary Defect and Downstream Pathology in Duchenne Muscular
Dystrophy,"
Cum Gene Ther. 12(3):206-244 (2012) and Stedman et al., "The mdx Mouse
Diaphragm
Reproduces the Degenerative Changes of Duchenne Muscular Dystrophy," Nature
352(6335):536-539 (1991), which are hereby incorporated by reference in their
entirety).
Moreover, studies have also shown that allowing marx mice to participate in
voluntary exercise
(wheel running, treadmill) increases skeletal muscle disease due to the
introduction of micro-
tears from physical stress, similar to human (Smythe et al., "Voluntary Wheel
Running in
Dystrophin-Deficient (mdx) Mice: Relationships Between Exercise Parameters and
Exacerbation
of the Dystrophic Phenotype," PLoS Curr. 3:RRN1295 (2011); Nakae et al.,
"Quantitative
Evaluation of the Beneficial Effects in the mdx Mouse of Epigallocatechin
Gallate, an
Antioxidant Polyphenol from Green Tea," Histochem. Cell Biol. 137(6):811-27
(2012); and
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 95 -
Archer et at., "Persistent and Improved Functional Gain in mdx Dystrophic Mice
after Treatment
with L-Arginine and Deflazacort,- FASEB I 20(6):738-740 (2006), which are
hereby
incorporated by reference in their entirety). Thus, there are readily
available methods for
producing a representative human skeletal muscle form of disease in mdx mice
that constitute a
model for therapeutic assessment and clinical development.
AUF1 Gene Therapy
[0241] The results of Example 7 demonstrate that muscle cell-
specific AUF1 gene
therapy restores skeletal muscle mass and function in a mouse model of
Duchenne muscular
dystrophy. In particular, evaluation of muscle cell-specific gene therapy in
the DMD mdx mdoel
provided evidenced that AAV8 vectored AUF1 gene therapy. (1) efficiently
transduced skeletal
muscle including cardiac diaphragm and to provide long-duration AUF1
expression without
evidence of loss of expression over 6 months (the longest time point tested);
(2) activated high
levels of satellite cells and myoblasts; (3) significantly increased skeletal
muscle mass and
normal muscle fiber formation; (4) significantly enhanced exercise endurance;
(5) strongly
reduced biomarkers or muscle atrophy and muscle cell death in DMD mice; (6)
strongly reduced
inflammatory immune cell invasion in skeletal muscle including diaphragm; (7)
strongly reduced
muscle fibrosis and necrosis in skeletal muscle including diaphragm; (8)
strongly increased
expression of endogenous utrophin in DMD muscle cells while suppressing
expression of
embryonic dystrophin, a marker of muscle degeneration in DMD; (9) increased
normal
expression of a large group of genes all of which are involved in muscle
development and
regeneration, and to suppress genes involved in muscle cell fibrosis, death
and muscle-expressed
inflammatory cytokines; and (10) did not increase muscle mass, endurance or
activate satellite
cells in normal skeletal muscle. No aberrant effects of AUF1 skeletal muscle
specific gene
therapy were observed.
Example 8 ¨ AUF1 Gene Therapy Accelerates Skeletal Muscle Regeneration In
Muscle-
Injured Mice
[0242] A mouse model of BaC12 induced necrosis (Garry et al.,
"Cardiotoxin Induced
Injury and Skeletal Muscle Regeneration," Methods !Vol. Biol. 1460:61-71
(2016) and Tierney et
al., "Inducing and Evaluating Skeletal Muscle Injury by Notexin and Barium
Chloride," Methods
Mol. Biol. 1460:53-60 (2016), which are hereby incorporated by reference in
their entirety) was
used to examine whether AUF I gene therapy accelerates skeletal muscle
regeneration.
[0243] In this study, three month old male mice were
administered an intramuscular
injection of 50 IA of filtered 1.2% BaC12 in sterile saline with control
lentivirus vector or with
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 96 -
lentivirus p45 AUF1 vector (Abbadi et al., "Muscle Development and
Regeneration Controlled
by AUF1-mediated Stage-specific Degradation of Fate-determining Checkpoint
mRNAs,- Proc.
Nat'l. Acad. Sci. USA 116:11285-90 (2019), which is hereby incorporated by
reference in its
entirety) into the left tibialis anterior (TA) muscle. The right TA muscle
remained uninjured as a
control.
[0244] Muscle atrophy was determined by weight of excised TA
muscle. In mice
sacrificed at 7 days post-injection, TA injury reduced TA weight by 27% which
was restored to
near-uninjured levels by concurrent AUF1 gene therapy (FIG. 25A). p45 AUF I
gene transfer
increased AUF1 expression by several fold in lentivirus transduced muscle
(FIG. 25B), which
was associated with reduced expression of TRIM63 and Fbxo32, two established
biomarkers of
muscle atrophy, that were strongly increased following muscle injury but
reduced to near non-
injured levels with AUF1 gene transfer (FIG. 25D). Strong muscle regeneration
correlated with
strong activation of the PAX7, gene consistent with satellite cell activation
in the TA muscle
(FIG. 25C). p45 AUF1 gene transfer also significantly enhanced expression of
muscle
regeneration factors (MRFs) such as MyoD and myogenin (FIG. 26A), myh8 (FIG.
26B), myh7
(FIG. 26C), and myh4 (FIG. 26D).
[0245] Images of muscle fibers provide further evidence for
accelerated but normal
muscle regeneration of myofibers in animals administered lentiviral AUF1 that
was not seen in
control vector mice. A disrupted myofiber architecture and high level of
central nuclei in the
vector alone TA muscle was observed compared to lenti-AUF1 supplementation
(FIG. 27A).
Likewise, injured TA muscle receiving sham gene therapy sustained a 20% loss
in mass by day 3
following injury, which only very slightly improved by day 7 (FIG. 27B). In
contrast, injured
TA muscle receiving AUF1 gene therapy showed a trend to less atrophy by day 3,
which was
almost fully recovered by day 7, demonstrating near normal mass (FIG. 27B).
Accelerated
muscle regeneration produced mature myofibers, as shown by the striking
increase in CSA and
reduced central nuclei per myofiber (FIGs. 27C-27D).
[0246] Finally, using an inducible AUF1 conditional knockout
mouse (FIGs. 28A-28D)
developed as party of the technology described herein, selective AUF1 deletion
only in skeletal
muscle demonstrated the essential requirement for AUF1 expression to promote
regeneration of
muscle following traumatic injury (FIG. 28E), and the ability to protect
muscle from extensive
injury when delivered as AAV8 AUF1 gene therapy (FIG. 28E). In particular, TA
muscle from
mice injured by 1.2% BaC12 injection were evaluated for muscle atrophy at 7
days injection. TA
muscle of AUF1Flox/Flox x PAX7c"ERT2 mice expressing AUF1 and WT mice
expressing AUF1
(not induced for cre) showed 16-18% atrophy that was not statistically
different (FIG. 28E). In
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 97 -
contrast, deletion of the AUF1 gene caused strongly increased atrophy of the
TA muscle,
doubling atrophy levels to 35% (FIG. 28E). However, animals deleted for the
AUFI gene but
prophylactically administered AAV8 AUFI gene therapy demonstrated dramatically
reduced
levels of TA muscle atrophy, averaging ¨3% (FIG. 28E). AUF1 deleted mice were
tested at 5
months for grip strength, a measure of limb-girdle skeletal muscle strength
and endurance.
AUF I deleted mice showed a ¨50% reduction in grip strength (FIG. 28F).
[0247]
Collectively, these data demonstrate that AUF1 is essential for
maintenance of
muscle strength and muscle regeneration following injury, and that AUF1 gene
therapy provides
a remarkable ability to promote muscle regeneration and protect muscle from
extensive damage
despite traumatic injury.
Discussion of Example 8
[0248]
Large, severe, or traumatic muscle injuries can result in volumetric
muscle
loss (VML) in which the conventional muscle repair mechanisms of the body that
innately repair
and regenerate muscle are overwhelmed, resulting in permanent muscle injury,
poor ability to
repair muscle, muscle loss, and functional impairment (Grogan et al.,
"Volumetric Muscle Loss,"
J. Am. Acad. Orthop. Surg. 19(Suppl 1):S35-7 (2011); Sicherer et al., "Recent
Trends in Injury
Models to Study Skeletal Muscle Regeneration and Repair," Bioengineering
(Basel) 7 (2020);
Qazi et al., "Cell Therapy to Improve Regeneration of Skeletal Muscle
Injuries," J. Cachexia
Sarcopenia Muscle 10:501-16 (2019); and Garg et al., "Volumetric Muscle Loss:
Persistent
Functional Deficits Beyond Frank Loss of Tissue," J. Or/hop. Res. 33:40-6
(2015), which are
hereby incorporated by reference in their entirety). Traumatic skeletal muscle
injuries are the
most common injuries whether in military service, sports or just accidents in
everyday life
(Copland et al., "Evidence-Based Treatment of Hamstring Tears," Curr. Sports
/vied. Rep. 8:308-
14 (2009), which is hereby incorporated by reference in its entirety).
Traumatic injuries typically
result in muscle necrosis and chronic inflammation, and if they proceed to
ViVIL, they can
irreparably deplete muscle by 20% or more, which is replaced by fibrotic scar
tissue and sets in
and persistently long-term disability (Copland et al., "Evidence-Based
Treatment of Hamstring
Tears," Curr. Sports Med. Rep. 8:308-14 (2009) and Jarvinen et al., "Muscle
Injuries: Biology
and Treatment,- Am. J. Sports Med. 33:745-64 (2005), which are hereby
incorporated by
reference in their entirety). In fact, open bone fractures resulting from
accidents or military
injuries, of which there are more than 150,000 a year in the civilian
population alone in the
United States, are responsible for the majority (65%) of severe and poorly
healing muscle
injuries, in many cases resulting in permanent functional disabilities in as
much as 8% of the
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 98 -
population (Owens et at., "Characterization of Extremity Wounds in Operation
Iraqi Freedom
and Operation Enduring Freedom,- J. Orthop. Trauma 21:254-7 (2007); Corona et
al.,
"Volumetric Muscle Loss Leads to Permanent Disability Following Extremity
Trauma,"
RehabiL Res. Dev. 52:785-92 (2015); and Court-Brown et al., "The Epidemiology
of Tibial
Fractures," J. Bone ,Joint Surg. Br. 77:417-21 (1995), which are hereby
incorporated by reference
in their entirety).
[0249] With skeletal muscle injury, normally quiescent muscle
satellite cells are released
from their niche in the basal lamina, become activated and begin proliferating
(Dumont et al.,
"Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function,"
Development
142:1572-81 (2015), which is hereby incorporated by reference in its
entirety). Typically,
activation of quiescent satellite cells results from micro-damage to muscle
fibers (Murphy et al.,
"Satellite Cells, Connective Tissue Fibroblasts and their Interactions are
Crucial for Muscle
Regeneration," Development 138:3625-37 (2011); Carlson et al., "Loss of Stem
Cell
Regenerative Capacity within Aged Niches," Aging Cell 6:371-82 (2007); Collins
et al., "Stem
Cell Function, Self-Renewal, and Behavioral Heterogeneity of Cells from the
Adult Muscle
Satellite Cell Niche,- Cell 122:289-301 (2005); Gopinath et al., "Stem Cell
Review Series:
Aging of the Skeletal Muscle Stem Cell Niche," Aging Cell 7:590-8 (2008);
Seale et al., "A New
Look at the Origin, Function, and "Stem-Cell" Status of Muscle Satellite
Cells," Dev Biol
218:115-24 (2000); and Dumont et al., "Intrinsic and Extrinsic Mechanisms
Regulating Satellite
Cell Function,- Development 142:1572-81(2015), which are hereby incorporated
by reference in
their entirety) but with extensive damage there is chronic release and
activation of satellite cells
which can become functionally exhausted and even depleted in such
circumstances.
[0250] Satellite cells are a small population of muscle cells
comprising ¨2-4% of adult
skeletal muscle cells. Only a small number of satellite cells self-renew and
return to quiescence,
while the rest differentiate into muscle progenitor cells called myoblasts.
Myoblasts undergo
myogenesis (muscle development), a program that includes fusing with existing
damaged muscle
fibers (myofibers), thereby repairing and regenerating new muscle (Gunther et
al., "Myf5-
Positive Satellite Cells Contribute to Pax7-Dependent Long-Term Maintenance of
Adult Muscle
Stem Cells," Cell Stem Cell 13:590-601 (2013), which is hereby incorporated by
reference in its
entirety). However, traumatic muscle injury can easily exceed the ability of
the myogenesis
program to repair injured muscle fibers.
[0251] The newly generated myofibers fall into one of two
categories: slow-twitch (Type
I) or fast-twitch (Type II) fibers, defined according to their speed of
movement, type of
metabolism, and myosin gene expression. Type II myofibers are the first to
atrophy in response
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 99 -
to traumatic damage, whereas slow-twitch myofibers are more resilient (Arany,
Z. "PGC-1
Coactivators and Skeletal Muscle Adaptations in Health and Disease," Curr.
Op/n. Genet. Dev.
18:426-34 (2008) and Wang et al., "Mechanisms for Fiber-Type Specificity of
Skeletal Muscle
Atrophy," Curr. Op/n. Chit. Nutr. _IVIetab. Care 16:243-50 (2013), which are
hereby incorporated
by reference in their entirety). The ability to stimulate skeletal muscle
regeneration in general,
and to selectively promote more resilient slow-twitch muscle in particular,
has been a long-
standing goal of regenerative muscle biology and clinical practice, as it
could potentially be an
effective therapy for traumatic muscle injury and various forms of muscular
dystrophies
(Ljubicic et al., "The Therapeutic Potential of Skeletal Muscle Plasticity in
Duchenne Muscular
Dystrophy: Phenotypic Modifiers as Pharmacologic Targets," FASEB J. 28:548-68
(2014),
which is hereby incorporated by reference in its entirety). As satellite cells
age, or with
traumatic muscle injuries that result in chronic cycles of necro-regeneration,
satellite cells lose
their regenerative capacity and are difficult to reactivate (Bernet et al.,
"p38 MAPK Signaling
Underlies a Cell-Autonomous Loss of Stem Cell Self-Renewal in Skeletal Muscle
of Aged
Mice," Nat. Med. 20:265-71 (2014); Dumont et al., "Intrinsic and Extrinsic
Mechanisms
Regulating Satellite Cell Function," Development 142:1572-81 (2015);
Kudryashova et al.,
"Satellite Cell Senescence Underlies Myopathy in a Mouse Model of Limb-Girdle
Muscular
Dystrophy 2H," J. Cl/n. Invest. 122:1764-76 (2012); and Silva et al.,
"Inhibition of Stat3
Activation Suppresses Caspase-3 and the Ubiquitin-Proteasome System, Leading
to Preservation
of Muscle Mass in Cancer Cachexia," J. Biol. Chem. 290: 1177-87 (2015), which
are hereby
incorporated by reference in their entirety).
[0252] The cycles of muscle degeneration and regeneration in
large or traumatic injuries
can lead to functional exhaustion and even loss of muscle stem cells that are
essential for muscle
regeneration and repair (Carlson et al., -Loss of Stem Cell Regenerative
Capacity within Aged
Niches," Aging Cell 6:371-82 (2007); Shefer et al., "Satellite-Cell Pool Size
does Matter:
Defining the Myogenic Potency of Aging Skeletal Muscle," Dev. Biol. 294:50-66
(2006); Bernet
et al., "p38 MAPK Signaling Underlies a Cell-Autonomous Loss of Stem Cell Self-
Renewal in
Skeletal Muscle of Aged Mice," Nat. Med. 20:265-71 (2014); and Dumont et al.,
"Intrinsic and
Extrinsic Mechanisms Regulating Satellite Cell Function," Development 142:1572-
81 (2015),
which are hereby incorporated by reference in their entirety), resulting in
severe loss of muscle
regenerative capacity, permanent muscle loss and chronic disability (Brack, A.
S., "Pax7 is
Back," Skelet Muscle 4:24 (2014), which is hereby incorporated by reference in
its entirety).
Consequently, there are few therapeutic options to increase de novo muscle
regeneration, mass
and strength available for individuals with severe skeletal muscle injuries,
and little evidence
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 100 -
that any approaches are very particularly effective (Corona et al.,
"Pathophysiology of
Volumetric Muscle Loss Injury,- Cells Tissues Organs 202:180-88 (2016), which
is hereby
incorporated by reference in its entirety).
[0253] Physical rehabilitation approaches have not been found to
be effective in
increasing existing muscle mass, muscle regeneration or strength in
individuals who have VML
injuries (Garg et al., "Volumetric Muscle Loss: Persistent Functional Deficits
Beyond Frank
Loss of Tissue,- J. Orthop. Res. 33:40-6 (2015) and Mase et al., "Clinical
Application of an
Acellular Biologic Scaffold for Surgical Repair of a Large, Traumatic
Quadriceps Femoris
Muscle Defect," Orthopedics 33:511(2010), which are hereby incorporated by
reference in their
entirety). Muscle regeneration approaches that are focused on attenuating the
underlying
inflammatory response resulting from injury fail to promote effective
regeneration of new
muscle mass or strength (Corona et al., "Pathophysiology of Volumetric Muscle
Loss Injury,"
Cells Tissues Organs 202:180-88 (2016) and Qazi et al., "Cell Therapy to
Improve Regeneration
of Skeletal Muscle Injuries," J. Cachexia Sarcopenia Muscle 10:501-16 (2019),
which are
hereby incorporated by reference in their entirety).
[0254] Surgical treatments for individuals with chronic muscle
injury are also not very
effective and have significant limitations. Surgical intervention normally
involves surgical
reconstruction of injured muscle using autologous muscle transplant and
engraftment from
healthy muscle elsewhere in the body, which has a high rate of graft
degeneration and failure, re-
injury, and itself can cause traumatic injury of the resident healthy donor
muscle and loss of
function (Whiteside, L. A., "Surgical Technique: Gluteus Maximus and Tensor
Fascia Lata
Transfer for Primary Deficiency of the Abductors of the Hip," Cl/n. Orthop.
Re/at. Res. 472:645-
53 (2014); Dziki et at, "An Acellular Biologic Scaffold Treatment for
Volumetric Muscle Loss:
Results of a 13-Patient Cohort Study," A'PJ Regen. Med. 1:16008 (2016); Sicari
et al., "An
Acellular Biologic Scaffold Promotes Skeletal Muscle Formation in Mice and
Humans with
Volumetric Muscle Loss," Sci. Transl. Med. 6:234ra58 (2014); Hurtgen et al.,
"Autologous
Minced Muscle Grafts Improve Endogenous Fracture Healing and Muscle Strength
after
Musculoskeletal Trauma," Physiol. Rep. 5 (2017); and Qazi et al., "Cell
Therapy to Improve
Regeneration of Skeletal Muscle Injuries," J. Cachexia Sarcopenia Muscle
10:501-16 (2019),
which are hereby incorporated by reference in its entirety). Other surgical
approaches that use
experimental scaffolds and muscle organoids to promote increased muscle
regeneration are
technically complex and have also not shown consistent efficacy in model
systems (Gholobova
et al., "Vascularization of Tissue-Engineered Skeletal Muscle Constructs,"
Biomaterials
235:119708 (2020) and Sicherer et al., "Recent Trends in Injury Models to
Study Skeletal
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
-101 -
Muscle Regeneration and Repair," Bioengineering (Basel) 7 (2020), which are
hereby
incorporated by reference in their entirety).
[0255] Molecular approaches to treat skeletal traumatic injuries
generally consist of
growth factor therapies, including intramuscular administration or release
from implanted
biomaterials of hepatocyte growth factor (HGF), insulin-like growth factor
(IGF), vascular
endothelial growth factor (VEGF) and fibroblast growth factor (FGF) among
others (Syverud et
al., "Growth Factors for Skeletal Muscle Tissue Engineering,- Cells Tissues
Organs 202:169-79
(2016); Pawlikowski et al., "Regulation of Skeletal Muscle Stem Cells by
Fibroblast Growth
Factors," Dev. Dyn. 246:359-67 (2017); Menetrey et al., "Growth Factors
Improve Muscle
Healing in vivo," J. Bone Joint Sttrg. Br. 82:131-7 (2000); Rodgers et al.,
"mTORC1 Controls
the Adaptive Transition of Quiescent Stem Cells from GO to G(Alert)," Nature
510.393-6
(2014); Allen et al., "Hepatocyte Growth Factor Activates Quiescent Skeletal
Muscle Satellite
Cells in vitro," J. Cell Physiol. 165:307-12 (1995); Miller et al.,
"Hepatocyte Growth Factor
Affects Satellite Cell Activation and Differentiation in Regenerating Skeletal
Muscle," Am. J.
Physiol. Cell Physiol. 278:C174-81 (2000); Grasman et al., "Bi omim eti c
Scaffolds for
Regeneration of Volumetric Muscle Loss in Skeletal Muscle Injuries," Acta
Biomater. 25:2-15
(2015); and Cezar et al., "Timed Delivery of Therapy Enhances Functional
Muscle
Regeneration," Adv. Healthc. Mater. 6 (2017), which are hereby incorporated by
reference in
their entirety). These approaches suffer from the limitation of administration
of a single muscle
growth promoting factor, and that these factors are short-lived, whereas
muscle regeneration is
complex and requires many factors that must act in concert with each other in
a precise spatial
and temporal manner over time to effect muscle repair and regeneration. It is
therefore not
surprising that administration of growth factors, even in combinations, have
not shown
significant muscle regenerative effects even in experimental models of
traumatic muscle injury
(Pumberger et al., "Synthetic Niche to Modulate Regenerative Potential of MSCs
and Enhance
Skeletal Muscle Regeneration," Biomaterials 99:95-108 (2016), which is hereby
incorporated by
reference in its entirety).
[0256] Most cellular therapies attempt to repopulate muscle
regenerative stem (satellite)
cells, and reduce necro-inflammation by using transplanted muscle satellite
cells or other cells of
myogenic origin. However, there are significant impediments to this approach.
First, the cells
employed must be freshly isolated allogeneic, which means harvesting them from
existing
surgically removed healthy muscle, in the case of individuals with traumatic
and VML injuries.
Second, the stem and myogenic cells need to be cultured and expanded, which is
technically
difficult and not scalable given the magnitude of unmet need. Thus, autologous
muscle cell
CA 03164622 2022- 7- 13
WO 2021/146711
PCT/US2021/013955
- 102 -
therapies are not clinically feasible for treatment of the majority of
patients in need (Qazi et al.,
"Cell Therapy to Improve Regeneration of Skeletal Muscle Injuries,- I Cacheria
Sareopenia
Muscle 10:501-16 (2019), which is hereby incorporated by reference in its
entirety).
[0257] The therapeutic options currently available for the
treatment of large and/or
traumatic muscle injury (e.g., cell therapies, surgical therapies, growth
factor and hormonal
therapies, molecular therapies, and gene therapies) aim to increase muscle
regeneration, muscle
mass, and muscle strength for severe skeletal muscle injuries. However, most
of the available
treatment options work only very poorly, if at all. The results of Example 8
demonstrate that
AUF1 gene therapy (e.g., by lentivirus vector delivery directly to muscle or
systemic delivery of
AUF1 by AAV8 vector) is effective to: (1) activate muscle stem (satellite)
cells; (2) reduce
expression of established biomarkers of muscle atrophy; (3) accelerated the
regeneration of
mature muscle fibers (myofibers); (4) enhanced expression of muscle
regeneration factors; (5)
strongly accelerate the regeneration of injured muscle; (6) increase
regeneration of both major
types of muscle (i.e., slow-twitch (Type I) or fast-twitch (Type II) fibers);
and restore muscle
mass, muscle strength, and create normal muscle.
[0258] Although preferred embodiments have been depicted and
described in detail
herein, it will be apparent to those skilled in the relevant art that various
modifications, additions,
substitutions, and the like can be made without departing from the spirit of
the invention and
these are therefore considered to be within the scope of the invention as
defined in the claims
which follow.
CA 03164622 2022- 7- 13
