U.S. patent application number 16/890666 was filed with the patent office on 2020-09-24 for delivery of polynucleotides using recombinant aav9.
The applicant listed for this patent is NATIONWIDE CHILDREN'S HOSPITAL. Invention is credited to Kevin Foust, Brian K. Kaspar.
Application Number | 20200297872 16/890666 |
Document ID | / |
Family ID | 1000004872107 |
Filed Date | 2020-09-24 |
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United States Patent
Application |
20200297872 |
Kind Code |
A1 |
Kaspar; Brian K. ; et
al. |
September 24, 2020 |
DELIVERY OF POLYNUCLEOTIDES USING RECOMBINANT AAV9
Abstract
The present invention relates to Adeno-associated virus 9
methods and materials useful for systemically delivering
polynucleotides across the blood brain barrier. Accordingly, the
present invention also relates to methods and materials useful for
systemically delivering polynucleotides to the central and
peripheral nervous systems. The present invention also relates to
Adeno-associated virus type 9 methods and materials useful for
intrathecal delivery of polynucleotides. Use of the methods and
materials is indicated, for example, for treatment of lower motor
neuron diseases such as spinal muscle atrophy and amyotrophic
lateral sclerosis as well as Pompe disease and lysosomal storage
disorders. Use of the methods and materials is also indicated, for
example, for treatment of Rett syndrome.
Inventors: |
Kaspar; Brian K.;
(Westerville, OH) ; Foust; Kevin; (Columbus,
OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONWIDE CHILDREN'S HOSPITAL |
Columbus |
OH |
US |
|
|
Family ID: |
1000004872107 |
Appl. No.: |
16/890666 |
Filed: |
June 2, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16159986 |
Oct 15, 2018 |
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16890666 |
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15717158 |
Sep 27, 2017 |
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16159986 |
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14717672 |
May 20, 2015 |
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15717158 |
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13830515 |
Mar 14, 2013 |
9415121 |
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14717672 |
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13270840 |
Oct 11, 2011 |
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13830515 |
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13035777 |
Feb 25, 2011 |
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13270840 |
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PCT/US2009/068818 |
Dec 18, 2009 |
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13035777 |
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61678458 |
Aug 1, 2012 |
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61308884 |
Feb 26, 2010 |
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61139470 |
Dec 19, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2830/008 20130101;
C12N 7/00 20130101; C12N 2750/14132 20130101; C07K 14/4702
20130101; C12N 2750/14121 20130101; C12N 2750/14143 20130101; C07K
14/47 20130101; A61K 48/0075 20130101; A61K 48/005 20130101; C12N
15/86 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07K 14/47 20060101 C07K014/47; C12N 15/86 20060101
C12N015/86; C12N 7/00 20060101 C12N007/00 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with Government support under
R21EY018491 awarded by the National Institutes of Health
(NIH)/National Eye Institute (NEI), under R21NS064328, awarded by
the NIH/National Institute of Neurological Disorders and Stroke
(NINDS) and under RC2 NS69476-01 awarded by the National Institutes
of Health (NIH). The Government has certain rights in the
invention.
Claims
1-20. (canceled)
21. A method of delivering a polynucleotide encoding a therapeutic
peptide for the treatment of mucopolysaccharidosis IIIB (MPS IIIB)
to the central nervous system of a patient suffering from MPS IIIB,
the method comprising administering a recombinant adeno-associated
virus 9 (rAAV9) to the patient by direct intravenous injection,
wherein the rAAV9 comprises the polynucleotide in a
self-complementary genome.
22. The method of claim 21, wherein the rAAV9 is delivered across
the blood brain barrier (BBB) to the patient's central nervous
system.
23. The method of claim 22, wherein the rAAV9 is delivered to the
patient's brain.
24. The method of claim 23, wherein the rAAV9 is delivered to the
patient's spinal cord.
25. A method of treating mucopolysaccharidosis IIIB (MPS IIIB) in a
patient suffering from MPS IIIB, the method comprising
administering a rAAV9 to the patient by direct intravenous
injection, wherein the rAAV9 comprises the polynucleotide in a
self-complementary genome.
26. The method of claim 25, wherein the rAAV9 is delivered across
the blood brain barrier (BBB) to the patient's central nervous
system.
27. The method of claim 26 wherein the rAAV9 is delivered to the
patient's brain.
28. The method of claim 27, wherein the rAAV9 is delivered to the
patient's spinal cord.
Description
[0001] The present application is a continuation-in-part of U.S.
patent application Ser. No. 13/270,840 filed Oct. 11, 2011. U.S.
patent application Ser. No. 13/270,840 is a continuation of U.S.
patent application Ser. No. 13/035,777 filed Feb. 25, 2011. U.S.
patent application Ser. No. 13/035,777 claims the benefit of
priority of U.S. Provisional Application No. 61/308,884, filed Feb.
26, 2010, and is also a continuation-in-part of International
Patent Application No. PCT/US09/68818, filed Dec. 18, 2009.
International Patent Application No. PCT/US09/68818 claims the
benefit of priority of U.S. Provisional Application 61/139,470,
filed Dec. 19, 2008. The present application also claims the
benefit of priority of U.S. Provisional Patent Application No.
61/678,458 filed Aug. 1, 2012.
INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING
[0003] This application contains, as a separate part of disclosure,
a Sequence Listing in computer-readable form (filename:
44125CIP_SeqListing.txt; 4000 bytes--ASCII text file) which is
incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0004] The present invention relates to Adeno-associated virus 9
methods and materials useful for systemically delivering
polynucleotides across the blood brain barrier. Accordingly, the
present invention also relates to methods and materials useful for
systemically delivering polynucleotides to the central and
peripheral nervous systems. The present invention also relates to
Adeno-associated virus type 9 methods and materials useful for
intrathecal delivery (i.e., delivery into the space under the
arachnoid membrane of the brain or spinal cord) of polynucleotides.
Use of the methods and materials is indicated, for example, for
treatment of lower motor neuron diseases such as spinal muscle
atrophy and amyotrophic lateral sclerosis as well as Pompe disease
and lysosomal storage disorders. Use of the methods and materials
is also indicated, for example, for treatment of Rett syndrome.
BACKGROUND
[0005] Large-molecule drugs do not cross the blood-brain-barrier
(BBB) and 98% of small-molecules cannot penetrate this barrier,
thereby limiting drug development efforts for many CNS disorders
[Pardridge, W. M. Nat Rev Drug Discov 1: 131-139 (2002)]. Gene
delivery has recently been proposed as a method to bypass the BBB
[Kaspar, et al., Science 301: 839-842 (2003)]; however, widespread
delivery to the brain and spinal cord has been challenging. The
development of successful gene therapies for motor neuron disease
will likely require widespread transduction within the spinal cord
and motor cortex. Two of the most common motor neuron diseases are
spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis
(ALS), both debilitating disorders of children and adults,
respectively, with no effective therapies to date. Recent work in
rodent models of SMA and ALS involves gene delivery using viruses
that are retrogradely transported following intramuscular injection
[Kaspar et al., Science 301: 839-842 (2003); Azzouz et al., J Clin
Invest 114: 1726-1731 (2004); Azzouz et al., Nature 429: 413-417
(2004); Ralph et al., Nat Med 11: 429-433 (2005)]. However,
clinical development may be difficult given the numerous injections
required to target the widespread region of neurodegeneration
throughout the spinal cord, brainstem and motor cortex to
effectively treat these diseases. Adeno-associated virus (AAV)
vectors have also been used in a number of recent clinical trials
for neurological disorders, demonstrating sustained transgene
expression, a relatively safe profile, and promising functional
responses, yet have required surgical intraparenchymal injections
[Kaplitt et al., Lancet 369: 2097-2105 (2007); Marks et al., Lancet
Neurol 7: 400-408 (2008); Worgall et al., Hum Gene Ther
(2008)].
[0006] SMA is an early pediatric neurodegenerative disorder
characterized by flaccid paralysis within the first six months of
life. In the most severe cases of the disease, paralysis leads to
respiratory failure and death usually by two years of age. SMA is
the second most common pediatric autosomal recessive disorder
behind cystic fibrosis with an incidence of 1 in 6000 live births.
SMA is a genetic disorder characterized by the loss of lower motor
neurons (LMNs) residing along the length of the entire spinal cord.
SMA is caused by a reduction in the expression of the survival
motor neuron (SMN) protein that results in denervation of skeletal
muscle and significant muscle atrophy. SMN is a ubiquitously
expressed protein that functions in U snRNP biogenesis.
[0007] In humans there are two very similar copies of the SMN gene
termed SMN1 and SMN2. The amino acid sequence encoded by the two
genes is identical. However, there is a single, silent nucleotide
change in SMN2 in exon 7 that results in exon 7 being excluded in
80-90% of transcripts from SMN2. The resulting truncated protein,
called SMN.DELTA.7, is less stable and rapidly degraded. The
remaining 10-20% of transcript from SMN2 encodes the full length
SMN protein. Disease results when all copies of SMN1 are lost,
leaving only SMN2 to generate full length SMN protein. Accordingly,
SMN2 acts as a phenotypic modifier in SMA in that patients with a
higher SMN2 copy number generally exhibit later onset and less
severe disease.
[0008] To date, there are no effective therapies for SMA.
Therapeutic approaches have mainly focused on developing drugs for
increasing SMN levels or enhancing residual SMN function. Despite
years of screening, no drugs have been fully effective for
increasing SMN levels as a restorative therapy. A number of mouse
models have been developed for SMA. See, Hsieh-Li et al., Nature
Genetics, 24 (1): 66-70 (2000); Le et al., Hum. Mol. Genet., 14
(6): 845-857 (2005); Monani et al., J. Cell. Biol., 160 (1): 41-52
(2003) and Monani et al., Hum. Mol. Genet., 9 (3): 333-339 (2000).
A recent study express a full length SMN cDNA in a mouse model and
the authors concluded that expression of SMN in neurons can have a
significant impact on symptoms of SMA. See Gavrilina et al., Hum.
Mol. Genet., 17(8):1063-1075 (2008).
[0009] ALS is another disease that results in loss of muscle and/or
muscle function. First characterized by Charcot in 1869, it is a
prevalent, adult-onset neurodegenerative disease affecting nearly 5
out of 100,000 individuals. ALS occurs when specific nerve cells in
the brain and spinal cord that control voluntary movement gradually
degenerate. Within two to five years after clinical onset, the loss
of these motor neurons leads to progressive atrophy of skeletal
muscles, which results in loss of muscular function resulting in
paralysis, speech deficits, and death due to respiratory
failure.
[0010] The genetic defects that cause or predispose ALS onset are
unknown, although missense mutations in the SOD-1 gene occurs in
approximately 10% of familial ALS cases, of which up to 20% have
mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1),
located on chromosome 21. SOD-1 normally functions in the
regulation of oxidative stress by conversion of free radical
superoxide anions to hydrogen peroxide and molecular oxygen. To
date, over 90 mutations have been identified spanning all exons of
the SOD-1 gene. Some of these mutations have been used to generate
lines of transgenic mice expressing mutant human SOD-1 to model the
progressive motor neuron disease and pathogenesis of ALS.
[0011] De novo mutations in the X-linked gene encoding the
transcription factor, Methyl-CpG binding protein 2 (MECP2), are the
most frequent cause of the neurological disorder Rett syndrome
(RTT). Hemizygous males usually die of neonatal encephalopathy.
Heterozygous females survive into adulthood but exhibit severe
symptoms including microcephaly, loss of purposeful hand motions
and speech, and motor abnormalities which appear following a period
of apparently normal development. Both male and female mouse models
exhibit RTT-like behaviors [Guy et al., Nature Genetics, 27:
322-326 (2001); Chen et al., Nature Genetics 27: 327-331 (2001);
and Katz et al., 5: 733-745 (2012)], but most studies have focused
on males because of the shorter latency to and severity in
symptoms. Despite encouraging studies on male mice, no therapeutic
treatment has been shown yet to be effective in females, the more
gender appropriate model.
[0012] AAV is a replication-deficient parvovirus, the
single-stranded DNA genome of which is about 4.7 kb in length
including 145 nucleotide inverted terminal repeat (ITRs). The
nucleotide sequence of the AAV serotype 2 (AAV2) genome is
presented in Srivastava et al., J Virol, 45: 555-564 (1983) as
corrected by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994).
Cis-acting sequences directing viral DNA replication (rep),
encapsidation/packaging and host cell chromosome integration are
contained within the ITRs. Three AAV promoters (named p5, p19, and
p40 for their relative map locations) drive the expression of the
two AAV internal open reading frames encoding rep and cap genes.
The two rep promoters (p5 and p19), coupled with the differential
splicing of the single AAV intron (at nucleotides 2107 and 2227),
result in the production of four rep proteins (rep 78, rep 68, rep
52, and rep 40) from the rep gene. Rep proteins possess multiple
enzymatic properties that are ultimately responsible for
replicating the viral genome. The cap gene is expressed from the
p40 promoter and it encodes the three capsid proteins VP1, VP2, and
VP3. Alternative splicing and non-consensus translational start
sites are responsible for the production of the three related
capsid proteins. A single consensus polyadenylation site is located
at map position 95 of the AAV genome. The life cycle and genetics
of AAV are reviewed in Muzyczka, Current Topics in Microbiology and
Immunology, 158: 97-129 (1992).
[0013] AAV possesses unique features that make it attractive as a
vector for delivering foreign DNA to cells, for example, in gene
therapy. AAV infection of cells in culture is noncytopathic, and
natural infection of humans and other animals is silent and
asymptomatic. Moreover, AAV infects many mammalian cells allowing
the possibility of targeting many different tissues in vivo.
Moreover, AAV transduces slowly dividing and non-dividing cells,
and can persist essentially for the lifetime of those cells as a
transcriptionally active nuclear episome (extrachromosomal
element). The AAV proviral genome is infectious as cloned DNA in
plasmids which makes construction of recombinant genomes feasible.
Furthermore, because the signals directing AAV replication, genome
encapsidation and integration are contained within the ITRs of the
AAV genome, some or all of the internal approximately 4.3 kb of the
genome (encoding replication and structural capsid proteins,
rep-cap) may be replaced with foreign DNA such as a gene cassette
containing a promoter, a DNA of interest and a polyadenylation
signal. The rep and cap proteins may be provided in trans. Another
significant feature of AAV is that it is an extremely stable and
hearty virus. It easily withstands the conditions used to
inactivate adenovirus (56.degree. to 65.degree. C. for several
hours), making cold preservation of AAV less critical. AAV may even
be lyophilized. Finally, AAV-infected cells are not resistant to
superinfection.
[0014] Multiple serotypes of AAV exist and offer varied tissue
tropism. Known serotypes include, for example, AAV1, AAV2, AAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11. AAV9 is
described in U.S. Pat. No. 7,198,951 and in Gao et al., J. Virol.,
78: 6381-6388 (2004). Advances in the delivery of AAV6 and AAV8
have made possible the transduction by these serotypes of skeletal
and cardiac muscle following simple systemic intravenous or
intraperitoneal injections. See Pacak et al., Circ. Res., 99(4):
3-9 (1006) and Wang et al., Nature Biotech., 23(3): 321-8 (2005).
The use of AAV to target cell types within the central nervous
system, though, has required surgical intraparenchymal injection.
See, Kaplitt et al., supra; Marks et al., supra and Worgall et al.,
supra.
[0015] There thus remains a need in the art for methods and vectors
for delivering genes across the BBB.
SUMMARY
[0016] The present invention provides methods and materials useful
for systemically delivering polynucleotides across the BBB. The
present invention also provides methods and materials useful for
intrathecal delivery of polynucleotides to the central nervous
system.
[0017] In one aspect, the invention provides methods of delivering
a polynucleotide across the BBB comprising systemically
administering a recombinant AAV9 (rAAV9) with a genome including
the polynucleotide to a patient. In some embodiments, the rAAV9
genome is a self complementary genome. In other embodiments, the
rAAV9 genome is a single-stranded genome.
[0018] In some embodiments, the methods systemically deliver
polynucleotides across the BBB to the central and/or peripheral
nervous system. Accordingly, a method is provided of delivering a
polynucleotide to the central nervous system comprising
systemically administering a rAAV9 with a self-complementary genome
including the genome to a patient. In some embodiments, the
polynucleotide is delivered to brain. In some embodiments, the
polynucleotide is delivered to the spinal cord. Also provided is a
method of delivering a polynucleotide to the peripheral nervous
system comprising systemically administering a rAAV9 with a
self-complementary genome including the polynucleotide to a patient
is provided. In some embodiments, the polynucleotide is delivered
to a lower motor neuron.
[0019] In another aspect, the invention provides methods of
delivering a polynucleotide to the central nervous system of a
patient in need thereof comprising intrathecal delivery of rAAV9
with a genome including the polynucleotide. In some embodiments,
rAAV9 genome is a self-complementary genome. In some embodiments, a
non-ionic, low-osmolar contrast agent is also delivered to the
patient, for example, iobitridol, iohexol, iomeprol, iopamidol,
iopentol, iopromide, ioversol or ioxilan.
[0020] Embodiments of the invention employ rAAV9 to deliver
polynucleotides to nerve and glial cells. In some aspects, the
glial cell is a microglial cell, an oligodendrocyte or an
astrocyte. In other aspects the rAAV9 is used to deliver a
polynucleotide to a Schwann cell.
[0021] Use of the systemic or intrathecal delivery methods is
indicated, for example, for lower motor neuron diseases such as SMA
and ALS as well as Pompe disease, lysosomal storage disorders,
Glioblastoma multiforme and Parkinson's disease. Lysosomal storage
disorders include, but are not limited to, Activator Deficiency/GM2
Gangliosidosis, Alpha-mannosidosis, Aspartylglucosaminuria,
Cholesteryl ester storage disease, Chronic Hexosaminidase A
Deficiency, Cystinosis, Danon disease, Fabry disease, Farber
disease, Fucosidosis, Galactosialidosis, Gaucher Disease (Type I,
Type II, Type III), GM1 gangliosidosis (Infantile, Late
infantile/Juvenile, Adult/Chronic), I-Cell disease/Mucolipidosis
II, Infantile Free Sialic Acid Storage Disease/ISSD, Juvenile
Hexosaminidase A Deficiency, Krabbe disease (Infantile Onset, Late
Onset), Metachromatic Leukodystrophy, Mucopolysaccharidoses
disorders (Pseudo-Hurler polydystrophy/Mucolipidosis IIIA, MPSI
Hurler Syndrome, MPSI Scheie Syndrome, MPS I Hurler-Scheie
Syndrome, MPS II Hunter syndrome, Sanfilippo syndrome Type A/MPS
III A, Sanfilippo syndrome Type B/MPS III B, Sanfilippo syndrome
Type C/MPS III C, Sanfilippo syndrome Type D/MPS III D, Morquio
Type A/MPS IVA, Morquio Type B/MPS IVB, MPS IX Hyaluronidase
Deficiency, MPS VI Maroteaux-Lamy, MPS VII Sly Syndrome,
Mucolipidosis I/Sialidosis, Mucolipidosis IIIC, Mucolipidosis type
IV), Multiple sulfatase deficiency, Niemann-Pick Disease (Type A,
Type B, Type C), Neuronal Ceroid Lipofuscinoses (CLN6 disease
(Atypical Late Infantile, Late Onset variant, Early Juvenile),
Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease, Finnish Variant
Late Infantile CLN5, Jansky-Bielschowsky disease/Late infantile
CLN2/TPP1 Disease, Kufs/Adult-onset NCL/CLN4 disease, Northern
Epilepsy/variant late infantile CLN8, Santavuori-Haltia/Infantile
CLN1/PPT disease, Beta-mannosidosis, Pompe disease/Glycogen storage
disease type II, Pycnodysostosis, Sandhoff Disease/Adult Onset/GM2
Gangliosidosis, Sandhoff Disease/GM2 gangliosidosis--Infantile,
Sandhoff Disease/GM2 gangliosidosis--Juvenile, Schindler disease,
Salla disease/Sialic Acid Storage Disease, Tay-Sachs/GM2
gangliosidosis, Wolman disease.
[0022] In further embodiments, use of the systemic or intrathecal
delivery methods is indicated for treatment of nervous system
disease such as Rett Syndrome, Alzheimer's Disease, Parkinson's
Disease, Huntington's Disease along with nervous system injury
including spinal cord and brain trauma/injury, stroke, and brain
cancers. In some embodiments, methods of treatment of Rett syndrome
are contemplated where the methods deliver a polynucleotide to the
central nervous system of a patient in need thereof by systemic
delivery of rAAV9 with a genome including the polynucleotide. In
some embodiments, methods of treatment of Rett syndrome are
contemplated where the methods deliver a polynucleotide to the
central nervous system of a patient in need thereof by intrathecal
delivery of rAAV9 with a genome including the polynucleotide.
[0023] In yet another aspect, the invention provides rAAV genomes.
The rAAV genomes comprise one or more AAV ITRs flanking a
polynucleotide encoding a polypeptide (including, but not limited
to, an SMN polypeptide) or encoding short hairpin RNAs directed at
mutated proteins or control sequences of their genes. The
polynucleotide is operatively linked to transcriptional control
DNAs, specifically promoter DNA and polyadenylation signal sequence
DNA that are functional in target cells to form a gene cassette.
The gene cassette may also include intron sequences to facilitate
processing of an RNA transcript when expressed in mammalian
cells.
[0024] In some aspects, the rAAV9 genome encodes a trophic or
protective factor. In various embodiments, use of a trophic or
protective factor is indicated for neurodegenerative disorders
contemplated herein, including but not limited to Alzheimer's
Disease, Parkinson's Disease, Huntington's Disease along with
nervous system injury including spinal cord and brain
trauma/injury, stroke, and brain cancers. Non-limiting examples of
known nervous system growth factors include nerve growth factor
(NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3), neurotrophin-4/5 (NT-4/5), neurotrophin-6 (NT-6), ciliary
neurotrophic factor (CNTF), glial cell line-derived neurotrophic
factor (GDNF), the fibroblast growth factor family (e.g., FGF's
1-15), leukemia inhibitory factor (LIF), certain members of the
insulin-like growth factor family (e.g., IGF-1), the neurturins,
persephin, the bone morphogenic proteins (BMPs), the immunophilins,
the transforming growth factor (TGF) family of growth factors, the
neuregulins, epidermal growth factor (EGF), platelet-derived growth
factor (PDGF), vascular endothelial growth factor family (e.g. VEGF
165), follistatin, Hif1, and others. Also generally contemplated
are zinc finger transcription factors that regulate each of the
trophic or protective factors contemplated herein. In further
embodiments, methods to modulate neuro-immune function are
contemplated, including but not limited to, inhibition of
microglial and astroglial activation through, for example, NFkB
inhibition, or NFkB for neuroprotection (dual action of NFkB and
associated pathways in different cell types.) by siRNA, shRNA,
antisense, or miRNA. In still further embodiments, the rAAV9 genome
encodes an apoptotic inhibitor (e.g., bcl2, bclxL). Use of a rAAV9
encoding a trophic factor or spinal cord injury modulating protein
or a suppressor of an inhibitor of axonal growth (e.g., a
suppressor of Nogo [Oertle et al., The Journal of Neuroscience,
23(13):5393-5406 (2003)] is also contemplated for treating spinal
cord injury.
[0025] In some embodiments, use of materials and methods of the
invention is indicated for neurodegenerative disorders such as
Parkinson's disease. In various embodiments, the rAAV9 genome may
encode, for example, Aromatic acid dopa decarboxylase (AADC),
Tyrosine hydroxylase, GTP-cyclohydrolase 1 (gtpch1), apoptotic
inhibitors (e.g., bcl2, bclxL), glial cell line-derived
neurotrophic factor (GDNF), the inhibitory neurotransmitter-amino
butyric acid (GABA), and enzymes involved in dopamine biosynthesis.
In further embodiments, the rAAV9 genome may encode, for example,
modifiers of Parkin and/or synuclein.
[0026] In some embodiments, use of materials and methods of the
invention is indicated for neurodegenerative disorders such as
Alzheimer's disease. In further embodiments, methods to increase
acetylcholine production are contemplated. In still further
embodiments, methods of increasing the level of a choline
acetyltransferase (ChAT) or inhibiting the activity of an
acetylcholine esterase (AchE) are contemplated.
[0027] In some embodiments, the rAAV9 genome may encode, for
example, methods to decrease mutant Huntington protein (htt)
expression through siRNA, shRNA, antisense, and/or miRNA for
treating a neurodegenerative disorder such as Huntington's
disease.
[0028] In some embodiments, use of materials and methods of the
invention is indicated for neurodegenerative disorders such as ALS.
In some aspects, treatment with the embodiments contemplated by the
invention results in a decrease in the expression of molecular
markers of disease, such as TNF.alpha., nitric oxide,
peroxynitrite, and/or nitric oxide synthase (NOS).
[0029] In other aspects, the vectors could encode short hairpin
RNAs directed at mutated proteins such as superoxide dismutase for
ALS, or neurotrophic factors such as GDNF or IGF1 for ALS or
Parkinson's disease.
[0030] In some embodiments, use of materials and methods of the
invention is indicated for preventing or treating
neurodevelopmental disorders such as Rett Syndrome. For embodiments
relating to Rett Syndrome, the rAAV9 genome may encode, for
example, methyl cytosine binding protein 2 (MECP2).
[0031] The rAAV genomes of the invention lack AAV rep and cap DNA.
AAV DNA in the rAAV genomes (e.g., ITRs) may be from any AAV
serotype for which a recombinant virus can be derived including,
but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4,
AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11. The
nucleotide sequences of the genomes of the AAV serotypes are known
in the art. For example, the complete genome of AAV-1 is provided
in GenBank Accession No. NC_002077; the complete genome of AAV-2 is
provided in GenBank Accession No. NC 001401 and Srivastava et al.,
J. Virol., 45: 555-564 {1983); the complete genome of AAV-3 is
provided in GenBank Accession No. NC_1829; the complete genome of
AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5
genome is provided in GenBank Accession No. AF085716; the complete
genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at
least portions of AAV-7 and AAV-8 genomes are provided in GenBank
Accession Nos. AX753246 and AX753249, respectively; the AAV-9
genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004);
the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006);
and the AAV-11 genome is provided in Virology, 330(2): 375-383
(2004).
[0032] In another aspect, the invention provides DNA plasmids
comprising rAAV genomes of the invention. The DNA plasmids are
transferred to cells permissible for infection with a helper virus
of AAV (e.g., adenovirus, E1-deleted adenovirus or herpesvirus) for
assembly of the rAAV genome into infectious viral particles.
Techniques to produce rAAV particles, in which an AAV genome to be
packaged, rep and cap genes, and helper virus functions are
provided to a cell are standard in the art. Production of rAAV
requires that the following components are present within a single
cell (denoted herein as a packaging cell): a rAAV genome, AAV rep
and cap genes separate from (i.e., not in) the rAAV genome, and
helper virus functions. The AAV rep and cap genes may be from any
AAV serotype for which recombinant virus can be derived and may be
from a different AAV serotype than the rAAV genome ITRs, including,
but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4,
AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11. Production of
pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is
incorporated by reference herein in its entirety. In various
embodiments, AAV capsid proteins may be modified to enhance
delivery of the recombinant vector. Modifications to capsid
proteins are generally known in the art. See, for example, US
20050053922 and US 20090202490, the disclosures of which are
incorporated by reference herein in their entirety.
[0033] A method of generating a packaging cell is to create a cell
line that stably expresses all the necessary components for AAV
particle production. For example, a plasmid (or multiple plasmids)
comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and
cap genes separate from the rAAV genome, and a selectable marker,
such as a neomycin resistance gene, are integrated into the genome
of a cell. AAV genomes have been introduced into bacterial plasmids
by procedures such as GC tailing (Samulski et al., 1982, Proc.
Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers
containing restriction endonuclease cleavage sites (Laughlin et
al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation
(Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The
packaging cell line is then infected with a helper virus such as
adenovirus. The advantages of this method are that the cells are
selectable and are suitable for large-scale production of rAAV.
Other examples of suitable methods employ adenovirus or baculovirus
rather than plasmids to introduce rAAV genomes and/or rep and cap
genes into packaging cells.
[0034] General principles of rAAV production are reviewed in, for
example, Carter, 1992, Current Opinions in Biotechnology, 1533-539;
and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol.,
158:97-129). Various approaches are described in Ratschin et al.,
Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad.
Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251
(1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski
et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989,
J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95/13365 and
corresponding U.S. Pat. No. 5,658,776; WO 95/13392; WO 96/17947;
PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298
(PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243
(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine
13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615;
Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Pat. Nos.
5,786,211; 5,871,982; and 6,258,595. Single-stranded rAAV are
specifically contemplated. The foregoing documents are hereby
incorporated by reference in their entirety herein, with particular
emphasis on those sections of the documents relating to rAAV
production.
[0035] The invention thus provides packaging cells that produce
infectious rAAV. In one embodiment packaging cells may be stably
transformed cancer cells such as HeLa cells, 293 cells and PerC.6
cells (a cognate 293 line). In another embodiment, packaging cells
are cells that are not transformed cancer cells such as low passage
293 cells (human fetal kidney cells transformed with E1 of
adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells
(human fetal fibroblasts), Vero cells (monkey kidney cells) and
FRhL-2 cells (rhesus fetal lung cells).
[0036] In still another aspect, the invention provides rAAV (i.e.,
infectious encapsidated rAAV particles) comprising a rAAV genome of
the invention. In some embodiments, the rAAV genome is a
self-complementary genome.
[0037] In some embodiments, the invention includes, but is not
limited to, the exemplified rAAV named "rAAV SMN." The rAAV SMN
genome has in sequence an AAV2 ITR, the chicken .beta.-actin
promoter with a cytomegalovirus enhancer, an SV40 intron, the SMN
coding DNA set out in SEQ ID NO: 1 (GenBank Accession Number
NM_000344.2), a polyadenylation signal sequence from bovine growth
hormone and another AAV2 ITR. Conservative nucleotide substitutions
of SMN DNA are also contemplated (e.g., a guanine to adenine change
at position 625 of GenBank Accession Number NM_000344.2). The
genome lacks AAV rep and cap DNA, that is, there is no AAV rep or
cap DNA between the ITRs of the genome. SMN polypeptides
contemplated include, but are not limited to, the human SMN1
polypeptide set out in NCBI protein database number NP_000335.1.
Also contemplated is the SMN1-modifier polypeptide plastin-3 (PLS3)
[Oprea et al., Science 320(5875): 524-527 (2008)]. Sequences
encoding other polypeptides may be substituted for the SMN DNA.
[0038] Other rAAV9 are provided such as a rAAV9 named "scAAV9
MECP2." Its genome has in sequence an AAV2 ITR missing the terminal
resolution site, an approximately 730 bp murine MECP2 promoter
fragment, SV40 intron sequences, murine MECP2 coding sequences, a
bovine growth hormone polyadenylation signal sequence and an AAV2
ITR. The scAAV9 MECP2 genome lacks AAV rep and cap DNA, that is,
there is no AAV rep or cap DNA between the ITRs of the genome. Yet
another rAAV9 provided is a rAAV9 named "scAAV9 hMECP2." Its genome
has in sequence an AAV2 ITR missing the terminal resolution site,
an approximately 730 bp murine MECP2 promoter fragment, SV40 intron
sequences, human MECP2.alpha. coding sequences, a bovine growth
hormone polyadenylation signal sequence and an AAV2 ITR. The scAAV9
hMECP2 genome lacks AAV rep and cap DNA, that is, there is no AAV
rep or cap DNA between the ITRs of the genome. Substitution of
human MECP2 promoter sequences for the corresponding murine MECP2
promoter sequences is specifically contemplated.
[0039] The rAAV of the invention may be purified by methods
standard in the art such as by column chromatography or cesium
chloride gradients. Methods for purifying rAAV vectors from helper
virus are known in the art and include methods disclosed in, for
example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999);
Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); U.S. Pat.
No. 6,566,118 and WO 98/09657.
[0040] In another aspect, the invention contemplates compositions
comprising rAAV of the present invention. In one embodiment,
compositions of the invention comprise a rAAV encoding a SMN
polypeptide. In another embodiment, compositions of the invention
comprise a rAAV encoding a MECP2 polypeptide. In other embodiments,
compositions of the present invention may include two or more rAAV
encoding different polypeptides of interest.
[0041] Compositions of the invention comprise rAAV in a
pharmaceutically acceptable carrier. The compositions may also
comprise other ingredients such as diluents and adjuvants.
Acceptable carriers, diluents and adjuvants are nontoxic to
recipients and are preferably inert at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, or other organic acids; antioxidants such as ascorbic
acid; low molecular weight polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as Tween, pluronics or polyethylene glycol
(PEG).
[0042] Titers of rAAV to be administered in methods of the
invention will vary depending, for example, on the particular rAAV,
the mode of administration, the treatment goal, the individual, and
the cell type(s) being targeted, and may be determined by methods
standard in the art. Titers of rAAV may range from about
1.times.10.sup.6, about 1.times.10.sup.7, about 1.times.10.sup.8,
about 1.times.10.sup.9, about 1.times.10.sup.10, about
1.times.10.sup.11, about 1.times.10.sup.12, about 1.times.10.sup.13
to about 1.times.10.sup.14 or more DNase resistant particles (DRP)
per ml. Dosages may also be expressed in units of viral genomes
(vg). Dosages may also vary based on the timing of the
administration to a human. These dosages of rAAV may range from
about 1.times.10.sup.11 vg/kg, about 1.times.10.sup.12, about
1.times.10.sup.13, about 1.times.10.sup.14, about
1.times.10.sup.15, about 1.times.10.sup.16 or more viral genomes
per kilogram body weight in an adult. For a neonate, the dosages of
rAAV may range from about 1.times.10.sup.11, about
1.times.10.sup.12, about 3.times.10.sup.12, about
1.times.10.sup.13, about 3.times.10.sup.13, about
1.times.10.sup.14, about 3.times.10.sup.14, about
1.times.10.sup.15, about 3.times.10.sup.15, about
1.times.10.sup.16, about 3.times.10.sup.16 or more viral genomes
per kilogram body weight.
[0043] Methods of transducing nerve or glial target cells with rAAV
are contemplated by the invention. The methods comprise the step of
administering an intravenous or intrathecal effective dose, or
effective multiple doses, of a composition comprising a rAAV of the
invention to an animal (including a human being) in need thereof.
If the dose is administered prior to development of a
disorder/disease, the administration is prophylactic. If the dose
is administered after the development of a disorder/disease, the
administration is therapeutic. In embodiments of the invention, an
effective dose is a dose that alleviates (eliminates or reduces) at
least one symptom associated with the disorder/disease state being
treated, that slows or prevents progression to a disorder/disease
state, that slows or prevents progression of a disorder/disease
state, that diminishes the extent of disease, that results in
remission (partial or total) of disease, and/or that prolongs
survival. Examples of disease states contemplated for treatment by
methods of the invention are listed herein above.
[0044] Combination therapies are also contemplated by the
invention. Combination as used herein includes both simultaneous
treatment or sequential treatments. Combinations of methods of the
invention with standard medical treatments (e.g., riluzole in ALS)
are specifically contemplated, as are combinations with novel
therapies.
[0045] Route(s) of administration and serotype(s) of AAV components
of rAAV (in particular, the AAV ITRs and capsid protein) of the
invention may be chosen and/or matched by those skilled in the art
taking into account the infection and/or disease state being
treated and the target cells/tissue(s).
[0046] In some embodiments, administration of the rAAV9 to the
patient is contemplated to occur at postnatal day 1 (P1). In some
embodiments, administration is contemplated to occur at P2, P3, P4,
P5, P6, P7, P8, P9, P10, P11, P12, P13, P14, P15, P16, P17, P18,
P19, P20, P21, P22, P23, P24, P25, P26, P27, P28, P29, P30, P31,
P32, P33, P34, P35, P36, P37, P38, P39, P40, P41, P42, P43, P44,
P45, P46, P47, P48, P49, P50, P51, P52, P53, P54, P55, P56, P57,
P58, P59, P60, P61, P62, P63, P64, P65, P66, P67, P68, P69, P70,
P71, P72, P73, P74, P75, P76, P77, P78, P79, P80, P81, P82, P83,
P84, P85, P86, P87, P88, P89, P90, P91, P92, P93, P94, P95, P96,
P97, P98, P99, P100, P110, P120, P130, P140, P150, P160, P170,
P180, P190, P200, P250, P300, P350, 1 year, 1.5 years, 2 years, 2.5
years, 3 years or older. While delivery to an individual in need
thereof after birth is contemplated, intrauteral delivery and
delivery to the mother are also contemplated.
[0047] Compositions suitable for systemic or intrathecal use
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid to the extent that easy syringability exists. It must
be stable under the conditions of manufacture and storage and must
be preserved against the contaminating actions of microorganisms
such as bacteria and fungi. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycol and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating such as lecithin, by the maintenance of the
required particle size in the case of a dispersion and by the use
of surfactants. The prevention of the action of microorganisms can
be brought about by various antibacterial and antifungal agents,
for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal and the like. In many cases it will be preferable to
include isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of the injectable compositions can be brought
about by use of agents delaying absorption, for example, aluminum
monostearate and gelatin, and Tween family of products (e.g., Tween
20).
[0048] Sterile injectable solutions are prepared by incorporating
rAAV in the required amount in the appropriate solvent with various
other ingredients enumerated above, as required, followed by filter
sterilization. Generally, dispersions are prepared by incorporating
the sterilized active ingredient into a sterile vehicle which
contains the basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and the freeze
drying technique that yield a powder of the active ingredient plus
any additional desired ingredient from the previously
sterile-filtered solution thereof.
[0049] Transduction with rAAV may also be carried out in vitro. In
one embodiment, desired target cells are removed from the subject,
transduced with rAAV and reintroduced into the subject.
Alternatively, syngeneic or xenogeneic cells can be used where
those cells will not generate an inappropriate immune response in
the subject.
[0050] Suitable methods for the transduction and reintroduction of
transduced cells into a subject are known in the art. In one
embodiment, cells can be transduced in vitro by combining rAAV with
the cells, e.g., in appropriate media, and screening for those
cells harboring the DNA of interest using conventional techniques
such as Southern blots and/or PCR, or by using selectable markers.
Transduced cells can then be formulated into pharmaceutical
compositions, and the composition introduced into the subject by
various techniques, such as by injection into the spinal cord.
[0051] Transduction of cells with rAAV of the invention results in
sustained expression of polypeptide. The present invention thus
provides methods of administering/delivering rAAV (e.g., encoding
SMN protein or MECP2 protein) of the invention to an animal or a
human patient. These methods include transducing nerve and/or glial
cells with one or more rAAV of the present invention.
[0052] Transduction may also be carried out with gene cassettes
comprising tissue specific control elements. For example, promoters
that allow expression specifically within neurons or specifically
within astrocytes. Examples include neuron specific enolase and
glial fibrillary acidic protein promoters. Inducible promoters
under the control of an ingested drug may also be developed (e.g.,
rapamycin). By way of non-limiting example, it is understood that
systems such as the tetracycline (TET on/off) system [see, for
example, Urlinger et al., Proc. Natl. Acad. Sci. USA
97(14):7963-7968 (2000) for recent improvements to the TET system]
and Ecdysone receptor regulatable system [Palli et al., Eur J.
Biochem 270: 1308-1315 (2003] may be utilized to provide inducible
polynucleotide expression. It will also be understood by the
skilled artisan that combinations of any of the methods and
materials contemplated herein may be used for treating a
neurodegenerative disease.
[0053] The term "transduction" is used to refer to the
administration/delivery of a polynucleotide (e.g., SMN DNA or MECP2
DNA) to a recipient cell either in vivo or in vitro, via a
replication-deficient rAAV of the invention resulting in expression
of a functional polypeptide (e.g., SMN or MECP2) by the recipient
cell.
[0054] Thus, the invention provides methods of administering an
effective dose (or doses, administered essentially simultaneously
or doses given at intervals) of rAAV of the invention to a patient
in need thereof.
[0055] In still another aspect, methods of the invention may be
used to deliver polynucleotides to a vascular endothelial cell
rather than across the BBB.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] FIG. 1 depicts GFP expression in the gastrocnemius muscle of
AAV9-GFP or PBS treated mice.
[0057] FIG. 2 depicts widespread neuron and astrocyte AAV9-GFP
transduction in CNS and PNS 10-days-post-intravenous injection of
P1 mice. (A-B) GFP and ChAT immunohistochemistry of cervical (A)
and lumbar (B) spinal cord. (C) High-power magnification shows
extensive co-localization of GFP and ChAT positive cells. (arrow
indicates a GFP-positive astrocyte). (D) Neurons and astrocytes
transduced in the hippocampus. (E) Pyramidal cells in the cortex
were GFP positive. (F) Clusters of GFP positive astrocytes were
observed throughout the brain. Scale bars (A-B) 200 .mu.m, (C) 50
.mu.m, (D-F) 50 .mu.m.
[0058] FIG. 3 shows that intravenous injection of AAV9 leads to
widespread neonatal spinal cord transduction. Cervical (a-c) and
lumbar (e-k) spinal cord sections ten-days following facial-vein
injection of 4.times.10.sup.11 particles of scAAV9-CB-GFP into
postnatal day-1 mice. GFP-expression (a,e,i) was predominantly
restricted to lower motor neurons (a,e,i) and fibers that
originated from dorsal root ganglia (a,e). GFP-positive astrocytes
(i) were also observed scattered throughout the tissue sections.
Lower motor neuron and astrocyte expression were confirmed by
co-localization using choline acetyl transferase (ChAT) (b,f,j) and
glial fibrillary acidic protein (GFAP) (c,g,k), respectively. A
z-stack image (i-k) of the area within the box in h, shows the
extent of motor neuron and astrocyte transduction within the lumbar
spinal cord. Scale bars, 200 .mu.m (d,h), 20 .mu.m (1).
[0059] FIG. 4 shows that intravenous injection of AAV9 leads to
widespread and long term neonatal spinal cord transduction in
lumbar motor neurons. Z-series confocal microscopy showing
GFP-expression in 21-day-old mice that received 4.times.10.sup.11
particles of scAAV9-CB-GFP intravenous injections on postnatal
day-1. Z-stack images of GFP (a), ChAT (b), GFAP (c) and merged (d)
demonstrating persistent GFP-expression in motor neurons and
astrocytes (d) for at least three-weeks following scAAV9-CB-GFP
injection. Scale bar, 20 .mu.m (d).
[0060] FIG. 5 depicts in situ hybridization of spinal cord sections
from neonate and adult injected animals demonstrates that cells
expressing GFP are transduced with scAAV9-CB-GFP. Negative control
animals injected with PBS (a-b) showed no positive signal. However,
antisense probes for GFP demonstrated strong positive signals for
both neonate (c) and adult (e) sections analyzed. No positive
signals were found for the sense control probe in neonate (d) or
adult (f) spinal cord sections. Tissues were counterstained with
Nuclear Fast Red for contrast while probe hybridization is in
black.
[0061] FIG. 6 depicts cervical (A), thoracic (B) and lumbar (C)
transverse sections from mouse spinal cord labeled for GFP and
ChAT. The box in (C) denotes the location of (D-F). GFP (D), ChAT
(E) and merged (F) images of transduced motor neurons in the lumbar
spinal cord. In addition to motor neuron transductions, GFP
positive fibers are seen in close proximity and overlapping motor
neurons (D and F). Scale bars=(A-C) 200 .mu.m and (F) 50 .mu.m.
[0062] FIG. 7 depicts GFP (A), ChAT (B) and merged (C) images of a
transverse section through lumbar spinal cord of a P10 mouse that
had previously been injected at one day old with scAAV9 GFP. (D)
represents a z-stack merged image of the ventral horn from (C). (E)
shows that the scAAV9 vector resulted in more transduced motor
neurons when compared to ssAAV9 vector in the lumbar spinal cord.
Scale bars=(C) 100 .mu.m and (D) 50 .mu.m.
[0063] FIG. 8 depicts AAV9-GFP targeting of astrocytes in the
spinal cord of adult-mice. (A-B) GFP immunohistochemistry in
cervical (A) and lumbar (B) spinal cord demonstrating astrocyte
transduction following tail-vein injection. (hatched-line indicates
grey-white matter interface). (C) GFP and GFAP immunohistochemistry
from lumbar spinal cord indicating astrocyte transduction. Scale
bars (A-B) 100 .mu.m, (C) 20 .mu.m.
[0064] FIG. 9 shows that intravenous injection of AAV9 leads to
widespread predominant astrocyte transduction in the spinal cord
and brain of adult mice. GFP-expression in the cervical (a-c) and
lumbar (e-g) spinal cord as well as the brain (m-o) of adult mice
7-weeks after tail vein injection of 4.times.10.sup.12 particles of
scAAV9-CB-GFP. In contrast to postnatal day-1 intravenous
injections, adult tail vein injection resulted in almost
exclusively astrocyte transduction. GFP (a,e), ChAT (b,f) and GFAP
(c,g) demonstrate the abundance of GFPexpression throughout the
spinal grey matter, with lack of co-localization with lower motor
neurons and white matter astrocytes. Co-localization of GFP (i),
excitatory amino acid transporter 2 (EAAT2) (j), and GFAP (k)
confirm that transduced cells are astrocytes. Tail vein injection
also resulted in primarily astrocyte transduction throughout the
brain as seen in the cortex (m-n), thalamus (o) and midbrain.
Neuronal GFP-expression in the brain was restricted to the
hippocampus and dentate gyrus (m-n, FIG. 11e-f).
[0065] FIG. 10 depicts diagrams of coronal sections throughout the
mouse brain corresponding to the approximate locations shown in
(FIG. 9m-o). The box in (a) corresponds to the location shown in
(FIG. 9m). The smaller box in (b) corresponds to (FIG. 9n) and the
larger box to (FIG. 9o).
[0066] FIG. 11 depicts high-magnification of merged GFP and dapi
images of brain regions following neonate (a-d) or adult (e-f)
intravenous injection of scAAV9-CB-GFP. Astrocytes and neurons were
easily detected in the striatum (a), hippocampus (b) and dentate
gyrus (c) following postnatal day-1 intravenous injection of
4.times.10.sup.11 particles of scAAV9-CB-GFP. Extensive
GFP-expression within cerebellar Purkinje cells (d) was also
observed. Pyramidal cells of the hippocampus (e) and granular cells
of the dentate gyrus (f) were the only neuronal transduction within
the brain following adult tail vein injection. In addition to
astrocyte and neuronal transduction, widespread vascular
transduction (f) was also seen throughout all adult brain sections
examined. Scale bars, 200 .mu.m (e); 100 .mu.m (f), 50 .mu.m
(a-d).
[0067] FIG. 12 depicts widespread GFP-expression 21-days following
intravenous injection of 4.times.10.sup.11 particles of
scAAV9-CB-GFP to postnatal day-1 mice. GFP localized in neurons and
astrocytes throughout multiple structures of the brain as depicted
in: (a) striatum (b) cingulate gyrus (c) fornix and anterior
commissure (d) internal capsule (e) corpus callosum (f) hippocampus
and dentate gyrus (g) midbrain and (h) cerebellum. All panels show
GFP and DAPI merged images. Schematic representations depicting the
approximate locations of each image throughout the brain are shown
in (FIG. 13). Higher magnification images of select structures are
available in (FIG. 11, 14). Scale bars, 200 .mu.m (a); 50 .mu.m
(e); 100 .mu.m (b-d,f-h).
[0068] FIG. 13 depicts diagrams of coronal sections throughout the
mouse brain. corresponding to the approximate locations shown in
FIG. 12(a-h) for postnatal day-1 injected neonatal mouse brains.
The box in (a) corresponds to the location of (FIG. 12a). The
smaller box in (b) corresponds to (FIG. 12b) and the larger box to
(FIG. 12c). The larger box in (c) corresponds to (FIG. 12d) while
the smaller box in (c) represents (FIG. 12e). Finally, (d-f)
correspond to (FIG. 12f-h) respectively.
[0069] FIG. 14 depicts co-localization of GFP positive cells with
GAD67. Immunohistochemical detection of GFP (a,d,g,j) and GAD67
(b,e,h,k) expression within select regions of mouse brain 21-days
following postnatal day-1 injection of 4.times.10.sup.11 particles
of scAAV9-CB-GFP. Merged images (c,f,i,l) show limited
co-localization of GFP and GAD67 signals in the cingulate gyrus
(a-c), the dentate gyrus (d-f) and the hippocampus (g-i), but
numerous GFP/GAD67 Purkinje cells within the cerebellum (l). Scale
bars, 100 .mu.m (c), 50 .mu.m (a-b,d-l).
[0070] FIG. 15 depicts gel electrophoresis and silver staining of
various AAV9-CBGFP vector preparations demonstrates high purity of
research grade virus utilized in studies. Shown are 2 vector
batches at varying concentrations demonstrating the predominant 3
viral proteins (VP); VP1, 2, 3 as the significant components of the
preparation. 1 .mu.l, 5 .mu.l, and 10 .mu.l were loaded of each
respective batch of virus.
[0071] FIG. 16 depicts direct injection of scAAV9-CB-GFP into the
brain and demonstrates predominant neuronal transduction. Injection
of virus into the striatum (a) and hippocampus (b) resulted in the
familiar neuronal transduction pattern as expected. Co-labeling for
GFP and GFAP demonstrate a lack of astrocyte transduction in the
injected structures with significant neuronal cell transduction.
Scale bars, 50 .mu.m (a), 200 .mu.m (b).
[0072] FIG. 17 is a schematic of scAAV9/MECP2 vector.
[0073] FIG. 18 shows that systemic injection of MECP2B.sup.null/y
mice with scAAV9/MECP2 virus results in MECP2 expression in
different cell types in brain. (a) Experimental paradigm. (b) MECP2
expression is expressed preferentially in brainstem of injected
mice (n=3). (c) Expression of MECP2 in neurons and non neuronal
cells varies with brain region (n=3). In panels b and c *P<0.05,
**P<0.01 and ***P<0.001 by one way ANOVA (Newman-Keuls
multiple comparison test). Data are means.+-.s.e.m.
[0074] FIG. 19 shows MECP2 expressed from virus binds to DNA,
restores normal neuronal somal size and improves survival. (f)
Kaplan-Meier survival curve. (g) Observational scores.
MECP2Bnull/y-scAAV9/MECP2 (n=5), MECP2Bnull/y-AAV9/Control (n=6),
MECP2+/y (n=6). Data are means.+-.s.e.m. (h) Field pixel
intensities of MECP2-Cy3 immunofluorescence measured from brainstem
sections of non-injected and scAAV9/MECP2-injected males (left) and
females (right). n=10 fields each condition. ALU, Arbitrary Linear
Unit.
[0075] FIG. 20 shows systemic delivery of scAAV9/MECP2 virus into
Mecp2.sup.Bnull/+ mice prevents progression, or reverses aberrant
behaviors. (a) Experimental paradigm. Mice were analyzed five
months post injection. (b) Average observational scores of
Mecp2.sup.Bnull/+ mice injected with scAAV9/MeCP2 (n=8),
scAAV9/Control (n=5). Non-injected (Mecp2.sup.+/+) mice (n=8).
Arrow indicates time of behavioral analysis. (c) Rotorod activity
on third day of test. (d) Inverted grid test. (e) Platform test.
scAAV9/MeCP2 (n=8), scAAV9/Control (n=5). Mecp2.sup.+/+ (n=8). (f)
Nesting ability. scAAV9/MeCP2 (n=8), scAAV9/Control (n=5).
Mecp2.sup.+/+ (n=8). *P<0.05, **P<0.01, ***P<0.001 and
ns=not significant by one way ANOVA (Newman-Keuls multiple
comparison test for panel c and one way ANOVA (Dunn's multiple
comparison test for panels d-f. Data are means.+-.s.e.m.
[0076] FIG. 21 is a Kaplan-Meier survival curve showing that
Mecp2.sup.Bnull/+ mice injected with scAAV9/MECP2 do not die
prematurely compared to non-injected Mecp2.sup.+/+ mice. P>0.05
by Gehan-Breslow-Wilcoxon test.
[0077] FIG. 22 shows the sequence of the genome of the exemplary
rAAV9 named "scAAV9 MECP2." Its genome has in sequence an AAV2 ITR
missing the terminal resolution site (nucleotides 662-767), an
approximately 730 bp murine MECP2 promoter fragment (nucleotides
859-1597), SV40 late 19s and late 16s intron sequences (1602-1661),
murine MECP2 coding sequences (nucleotides 1799-3304), a bovine
growth hormone polyadenylation signal sequence (nucleotides
3388-3534) and an AAV2 ITR (nucleotides 3614-3754). The scAAV9
MECP2 genome lacks AAV rep and cap DNA, that is, there is no AAV
rep or cap DNA between the ITRs of the genome.
[0078] FIG. 23 shows the sequence of the genome of the exemplary
rAAV9 named "scAAV9 hMECP2." Its genome has in sequence an AAV2 ITR
missing the terminal resolution site (nucleotides 662-767), an
approximately 730 bp murine MECP2 promoter fragment (nucleotides
859-1597), SV40 late 19s and late 16s intron sequences (nucleotides
1602-1661), human MECP2.alpha. coding sequences (nucleotides
1765-3261), a bovine growth hormone polyadenylation signal sequence
(nucleotides 3314-3460) and an AAV2 ITR (nucleotides 3540-3680).
The scAAV9 hMECP2 genome lacks AAV rep and cap DNA, that is, there
is no AAV rep or cap DNA between the ITRs of the genome.
DETAILED DESCRIPTION
[0079] The present invention is illustrated by the following
examples relating to a novel rAAV9 and its ability to efficiently
deliver genes to the spinal cord via intravenous delivery in both
neonatal animals and in adult mice. Example 1 describes experiments
showing that rAAV9 can transduce and express protein in mouse
skeletal muscle. Example 2 describes experiments in which the
expression of the rAAV9 transgene was examined. Example 3 describes
the ability of rAAV9 to transduce and express protein in lumbar
motor neurons (LMNs). Example 4 describes the evaluation of vectors
that do not require second-strand synthesis. Example 5 describes
experiments focused on examining whether rAAV9 vectors were
enhanced for retrograde transport to target dorsal root ganglion
(DRG) and LMNs or could easily pass the blood-brain-barrier (BBB)
in neonates. Example 6 describes the evaluation of optimal delivery
of rAAV9 expressing SMN for postnatal gene replacement in a mouse
model of Type 2 SMA for function and survival. Example 7 describes
the examination of the brains of mice following postnatal day-one
intravenous injection of scAAV9-CBGFP. Example 8 describes the
investigation of whether astrocyte transduction is related to
vector purity or delivery route. Example 9 describes administration
of scAAV9-GFP in a nonhuman primate. Example 10 describes
experiments demonstrating that self complementary rAAV9 bearing
MECP2 cDNA under control of a fragment of its own promoter
(scAAV9/MECP2), was capable of significantly stabilizing or
reversing disease phenotypes when administered systemically into
female RTT mouse models.
Example 1
[0080] The ability of AAV9 to target and express protein in
skeletal muscle was evaluated in an in vivo model system.
[0081] Intravenous administration of 1.times.10.sup.11 particles of
scAAV9-GFP was performed in a total volume of 50 .mu.l to postnatal
day 1 mice and the extent of muscle transduction was evaluated. The
rAAV GFP genome included in sequence an AAV2 ITR, the chicken
.beta.-actin promoter with a cytomegalovirus enhancer, an SV40
intron, the GFP DNA, a polyadenylation signal sequence from bovine
growth hormone and another AAV2 ITR. The ability of the AAV9
vectors to transduce skeletal muscle was evaluated using a GFP
expressing vector. AAV9-GFP expressed at high levels in the
skeletal muscles that were analyzed. Ten days following injections,
animals were euthanized and gastrocnemius muscles were rapidly
isolated, frozen using liquid nitrogen chilled isopentane, and
sectioned on a cryostat at 15 .mu.m. Analysis of muscle sections
using a Zeiss Axiovert microscope equipped with GFP fluorescence
demonstrated that AAV9-GFP expressed at very high levels, with over
90% of the analyzed gastrocnemius muscle transduced (FIG. 1). No
GFP expression was detected in PBS control treated animals (FIG.
1). These results showed that AAV9 was effective at targeting and
expressing in skeletal muscles.
Example 2
[0082] Transgene expression following intravenous injection in
neonatal animals prior to the closure of the BBB and in adult
animals was examined.
[0083] Mice used were C57Bl/6 littermates. The mother (singly
housed) of each litter to be injected was removed from the cage.
The postnatal day 1 (P1) pups were rested on a bed of ice for
anesthetization. For neonate injections, a light microscope was
used to visualize the temporal vein (located just anterior to the
ear). Vector solution was drawn into a 3/10 cc 30 gauge insulin
syringe. The needle was inserted into the vein and the plunger was
manually depressed. Injections were in a total volume of 1000 of a
phosphate buffered saline (PBS) and virus solution. A total of
1.times.10.sup.11 DNase resistant particles of scAAV9 CB GFP
(Virapur LLC, San Diego) were injected. One-day-old wild-type mice
received temporal vein injections of 1.times.10.sup.11 particles of
a self-complementary (sc) AAV9 vector [McCarty et al., Gene
therapy, 10: 2112-2118 (2003)] that expressed green fluorescent
protein (GFP) under control of the chicken-.beta.-actin hybrid
promoter (CB). A correct injection was verified by noting blanching
of the vein. After the injection pups were returned to their cage.
When the entire litter was injected, the pups were rubbed with
bedding to prevent rejection by the mother. The mother was then
reintroduced to the cage. Neonate animals were sacrificed ten days
post injection, spinal cords and brains were extracted, rinsed in
PBS, then immersion fixed in a 4% paraformaldehyde solution.
[0084] Adult tail vein injections were performed on .about.70 day
old C57Bl/6 mice. Mice were placed in restraint that positioned the
mouse tail in a lighted, heated groove. The tail was swabbed with
alcohol then injected intravenously with a 100 .mu.l viral solution
containing a mixture of PBS and 5.times.10.sup.11 DNase resistant
particles of scAAV9 CB GFP. After the injection, animals were
returned to their cages. Two weeks post injection, animals were
anesthetized then transcardially perfused first with 0.9% saline
then 4% paraformaldehyde. Brains and spinal cords were harvested
and immersion fixed in 4% paraformaldehyde for an additional 24-48
hours.
[0085] Neonate and adult brains were transferred from
paraformaldehyde to a 30% sucrose solution for cryoprotection. The
brains were mounted onto a sliding microtome with Tissue-Tek O.C.T.
compound (Sakura Finetek USA, Torrance, Calif.) and frozen with dry
ice. Forty micron thick sections were divided into 5 series for
histological analysis. Tissues for immediate processing were placed
in 0.01 M PBS in vials. Those for storage were placed in antifreeze
solution and transferred to -20.degree. C. Spinal cords were cut
into blocks of tissue 5-6 mm in length, then cut into 40 micron
thick transverse sections on a vibratome. Serial sections were kept
in a 96 well plate that contained 4% paraformaldehyde and were
stored at 4.degree. C.
[0086] Brains and spinal cords were both stained as floating
sections. Brains were stained in a 12-well dish, and spinal cords
sections were stained in a 96-well plate to maintain their
rostral-caudal sequence. Tissues were washed three times for 5
minutes each in PBS, then blocked in a solution containing 10%
donkey serum and 1% Triton X-100 for two hours at room temperature.
After blocking, antibodies were diluted in the blocking solution at
1:500. The primary antibodies used were as follows: goat anti-ChAT
and mouse anti-NeuN (Chemicon), rabbit anti-GFP (Invitrogen) and
guinea pig anti-GFAP (Advanced Immunochemical). Tissues were
incubated in primary antibody at 4.degree. C. for 48-72 hours then
washed three times with PBS. After washing, tissues were incubated
for 2 hours at room temperature in the appropriate secondary
antibodies (1:125 Jackson Immunoresearch) with DAPI. Tissues were
then washed three times with PBS, mounted onto slides then
coverslipped. All images were captured on a Zeiss laser-scanning
confocal microscope.
[0087] Spinal cords had remarkable GFP expression throughout all
levels with robust GFP expression in fibers that ascended in the
dorsal columns and fibers that innervated the spinal gray matter,
indicating dorsal root ganglia (DRG) transduction. GFP positive
cells were also found in the ventral region of the spinal cord
where lower motor neurons reside (FIG. 2A-B). Labeling of choline
acetyl transferase (ChAT) positive cells with GFP demonstrated a
large number of ChAT positive cells expressing GFP throughout all
cervical and lumbar sections examined, indicating widespread LMN
transduction (FIG. 2C). Approximately 56% of ChAT positive cells
strongly expressed GFP in sections analyzed of the lumbar spinal
cord (598 GFP+/1058 ChAT+, n=4)(Table 1, below). This is the
highest proportion of LMNs transduced by a single injection of AAV
reported. Stereology for total number of neurons in a given area
and total number of GFP+ cells was performed on a Nikon E800
fluorescent microscope with computer-assisted microscopy and image
analysis using StereoInvestigator software (MicroBrightField, Inc.,
Williston, Vt.) with the optical dissector principle to avoid
oversampling errors and the Cavalieri estimation for volumetric
measurements. Coronal 40 .mu.m sections, 240 .mu.m apart covering
the regions of interest in its rostro-caudal extension was
evaluated. The entire dentate gyrus, caudal retrosplenial/cingulate
cortex; containing the most caudal extent of the dentate gyrus;
extending medially to the subiculum and laterally to the occipital
cortex, and the purkinje cell layer was sampled using .about.15-25
optical dissectors in each case. Fluorescent microscopy using a
60.times. objective for NeuN and GFP were utilized and cells within
the optical dissector were counted on a computer screen. Neuronal
density and positive GFP density were calculated by multiplying the
total volume to estimate the percent of neuronal transduction in
each given area as previously described [Kempermann et al.,
Proceedings of the National Academy of Sciences of the United
States of America 94: 10409-10414 (1997)].
[0088] For motor neuron quantification, serial 40 .mu.m thick
lumbar spinal cord sections, each separated by 480 .mu.m, were
labeled as described for GFP and ChAT expression. Stained sections
were serially mounted on slides from rostral to caudal, then
coverslipped. Sections were evaluated using confocal microscopy
(Zeiss) with a 40.times. objective and simultaneous FITC and Cy3
filters. FITC was visualized through a 505-530 nm band pass filter
to avoid contaminating the Cy3 channel. The total number of ChAT
positive cells found in the ventral horns with defined soma was
tallied by careful examination through the entire z-extent of the
section. GFP labeled cells were quantified in the same manner,
while checking for co-localization with ChAT. The total number of
cells counted per animal ranged from approximately 150-366 cells
per animal. For astrocyte quantification, as with motor neurons,
serial sections were stained for GFP, GFAP and EAAT2, then mounted.
Using confocal microscopy with a 63.times. objective and
simultaneous FITC and Cy5 filters, random fields in the ventral
horns of lumbar spinal cord sections from tail vein injected
animals were selected. The total numbers of GFP and GFAP positive
cells were counted from a minimum of at least 24-fields per animal
while focusing through the entire z extent of the section.
[0089] In addition to widespread DRG and motor neuron transduction,
GFP-positive glial cells were observed throughout the spinal gray
matter (FIG. 2C; arrow). The brains were next examined following P1
intravenous injection of AAV9-CB-GFP and revealed extensive GFP
expression in all regions analyzed, including the hippocampus (FIG.
2D), cortex (FIG. 2E), striatum, thalamus, hypothalamus and choroid
plexus, with predominant neuronal transduction. However, transduced
astrocytes were also found in all regions of the brain examined
(FIG. 2F).
[0090] The remarkable pattern of GFP expression observed following
P1 administration suggests two independent modes of viral entry
into the central nervous system (CNS). Due to the ubiquitous GFP
expression throughout the brain, the virus likely crossed the
developing BBB. However the GFP expression pattern in the neonate
spinal cord is defined with respect to the specific DRG and LMN
transduction. The DRG and the LMN have projections into the
periphery which suggests retrograde transport may be the mechanism
of transduction. In support of retrograde transport as the method
of spinal cord neuronal transduction, there were no GFP positive
interneurons observed in any section examined. Alternatively, the
virus may have a LMN tropism after crossing the BBB, but this
appears unlikely as ChAT positive cells still migrating from the
central canal to the ventral horn were largely untransduced (FIG.
2A-B).
TABLE-US-00001 TABLE 1 Neonate GFP (mean +/- s.e.m) .sup. NeuN
(mean +/- s.e.m.) .sup. % (mean +/- s.e.m.) Brain
Retrosplenial/Cingulate 142,658.30 +/- 11124.71.sup. 762,104.30 +/-
38397.81 15.84 +/- 1.93 Dentate Gyrus 42,304.33 +/- 15613.33
278,043.70 +/- 11383.56 14.82 +/- 4.89 Purkinje cells 52,720.33 +/-
1951.33.sup. 73,814 66 +/- 5220.80 71.88 +/- 3.65 GFP (mean +/- s.e
m.) ChAT (mean +/- s.e.m.) .sup. % (mean +/- s.e.m.) Lumbar 10 days
post injection 149.5 +/- 31.65 .sup. 264.5 +/- 53.72 55.16 +/- 1.95
spinal cord 21 days post injection 83.33 +/- 10.33 .sup. 140.0+/-
31.76 60 79 +/- 2.96 Adult GFP (mean +/- s e m) .sup. GFAP (mean
+/- s.e.m.) % (mean +/- s e m) .sup. Lumbar % GFP colabeled w/GFAP
48.00 +/- 10.12 43.00 +/- 7.00 91 44 +/- 4.82 spinal cord % GFAP +
transduced 41 33 +/- 5 55 64 33 +/- 8 67 64 23 +/- 0.96 (grey
matter)
[0091] Additional experiments were done on one-day-old wild-type
mice where they were administered temporal vein injections of
4.times.10.sup.11 particles of a self-complementary (sc) AAV9
vector [McCarty et al., Gene therapy 10: 2112-2118 (2003)] that
expressed green fluorescent protein (GFP) under control of the
chicken-.beta.-actin hybrid promoter (CB).
[0092] Histological processing was performed as above. Brains and
spinal cords were both stained as floating sections. Brains were
stained in a 12-well dish, and spinal cords sections were stained
in a 96-well plate to maintain their rostral-caudal sequence.
Tissues were washed three-times for 5-minutes each in PBS, then
blocked in a solution containing 10% donkey serum and 1% Triton
X-100 for two hours at room temperature. After blocking, antibodies
were diluted in the blocking solution at 1:500. The primary
antibodies used were as follows: goat anti-ChAT and mouse anti-NeuN
(Millipore, Billerica, Mass.), rabbit anti-GFP (Invitrogen,
Carlsbad, Calif.), guinea pig anti-GFAP (Advanced Immunochemical,
Long Beach, Calif.) and goat anti-GAD67 (Millipore, Billerica,
Mass.). Tissues were incubated in primary antibody at 4.degree. C.
for 48-72 hours then washed three times with PBS. After washing,
tissues were incubated for 2 hours at room temperature in the
appropriate secondary antibodies (1:125 Jackson Immunoresearch,
Westgrove, Pa.) with DAPI. Tissues were then washed three times
with PBS, mounted onto slides then coverslipped. All images were
captured on a Zeiss-laser-scanning confocal microscope.
[0093] Animals were sacrificed 10- or 21-days post-injection, and
brains and spinal cords were evaluated for transgene expression.
Robust GFP-expression was found in heart and skeletal muscles as
expected. Strikingly, spinal cords had remarkable GFP-expression
throughout all levels, with robust GFP-expression in fibers that
ascended in the dorsal columns and fibers that innervated the
spinal grey matter, indicating dorsal root ganglia (DRG)
transduction. GFP-positive cells were also found in the ventral
region of the spinal cord where lower motor neurons reside (FIGS.
3a and e). Co-labeling for choline acetyl transferase (ChAT) and
GFP-expression within the spinal cord demonstrated a large number
of ChAT positive cells expressing GFP throughout all cervical and
lumbar sections examined, indicating widespread LMN transduction
(FIG. 4). Approximately 56% of ChAT positive cells strongly
expressed GFP in sections analyzed of the lumbar spinal cord of 10
day-old animals and .about.61% of 21 day-old animals, demonstrating
early and persistent transgene expression in lower motor neurons
(Table 1). Similar numbers of LMN expression were seen in cervical
and thoracic regions of the spinal cord. This is the highest
proportion of LMNs transduced by a single injection of AAV
reported. In addition to widespread DRG and motor neuron
transduction, we observed GFP-positive glial cells throughout the
spinal grey matter, indicating that AAV9 could express in
astrocytes with the CB promoter. The remarkable pattern of
GFP-expression observed following postnatal day-one administration
suggests two independent modes of viral entry into the CNS. Due to
the ubiquitous GFP-expression throughout the brain, the virus
likely crossed the developing BBB. However the GFP-expression
pattern in the neonate spinal cord is defined with respect to the
specific DRG and LMN transduction. The DRG and the LMN have
projections into the periphery which suggests retrograde transport
may be the mechanism of transduction. In support of retrograde
transport as the method of spinal cord neuronal transduction, there
were no GFP-positive interneurons observed in any section examined.
Alternatively, the virus may have a LMN tropism after crossing the
BBB, but this appears unlikely as ChAT positive cells still
migrating from the central canal to the ventral horn were largely
untransduced.
[0094] In situ hybridization confirmed that viral transcription,
and not protein uptake, was responsible for the previously unseen
transduction pattern (FIG. 5).
Example 3
[0095] The ability of AAV9 to transduce and express protein in LMN
was evaluated. LMN transduction in the lumbar ventral horn was
evaluated following intravenous administration of 1.times.10.sup.11
particles of ss or scAAV9 GFP to postnatal day 1 mice in an effort
to effectively deliver a transgene to spinal cord motor neurons.
Both single-stranded and self-complementary AAV9-GFP vectors were
produced via transient transfection production methods and were
purified two times on CsCl gradients. The AAV9 GFP genomes are
identical with the exception that scAAV genomes have a mutation in
one ITR to direct packaging of specifically self-complementary
virus. The single stranded AAV constructs do not contain the ITR
mutation and therefore package predominantly single stranded virus.
Viral preps were titered simultaneously using TAQMAN Quantitative
PCR. P1 mice (n=5/group) were placed on an ice-cold plates to
anesthetize and virus was delivered using 0.3 cc insulin syringes
with 31 gauge needles that were inserted into the superficial
facial vein. Virus was delivered in a volume of 50 .mu.l. Animals
recovered quickly after gene delivery with no adverse events noted.
Animals were injected with a xylazine/ketamine mixture and were
decapitated 10-days following injection and spinal cords were
harvested then post-fixed in 4% paraformaldehyde, sectioned using a
Vibratome and immunohistochemistry was performed using co-labeling
for ChAT and GFP. Analysis of GFP expression was performed using a
Zeiss Confocal Microscope.
[0096] Intravenous injection of single stranded AAV9-GFP resulted
in widespread DRG transduction as evidenced by GFP positive fibers
innervating the spinal grey matter and ascending in the dorsal
columns (FIG. 6A-C). Numerous sections showed strong GFP staining
in motor neurons as assessed by co-labeling GFP with Choline
acetyltransferase (ChAT) (FIG. 3E-F). Counting the total number of
motor neurons in treated animals demonstrated approximately 8% of
total motor neurons residing in the lumbar region of the spinal
cord were transduced. This finding was remarkable given that motor
neuron transduction has typically been very low (less than 1% of
total motor neurons), particularly by remote delivery approaches
such as retrograde transport.
Example 4
[0097] Self-complementary scAAV9 vectors that do not require
second-strand synthesis (a rate limiting step of AAV vectors) which
would allow for greater efficiencies of expression in motor
neurons, were evaluated.
[0098] Viral particles were prepared as in Example 3. Intravenous
injections into the facial vein of P1 pups were performed as
described above and the animals as described above 10 days
post-injection. As with ssAAV9 injections significant transduction
of DRG was observed throughout the spinal cord. Remarkably,
significant motor neuron transduction in treated animals was found
in the two areas of the spinal cord that were evaluated including
the cervical and lumbar spinal cord. Quantification of GFP+/ChAT+
double labeled cells expressed as a percentage of total ChAT+ cells
within the lumbar spinal cord showed that .about.45% of LMN were
transduced by dsAAV9 compared with .about.8% of ssAAV9 (FIG. 7E).
Indeed, some regions of the spinal cord showed >90% motor neuron
transduction (FIG. 7D) and other regions may have greater amounts
of GFP positive motor neurons, given that dim GFP positive cells
were not counted due to a conservative GFP positive scoring used in
the counting. This amount of LMN transduction following a single
injection of AAV has not previously been reported.
Example 5
[0099] Further investigation focused on whether AAV9 vectors were
enhanced for retrograde transport to target DRG and LMNs or could
easily pass the BBB in neonates.
[0100] The pattern of transduction was examined to determine if it
was consistent between neonates and adult animals. Adult mice were
injected via tail vein delivery using 4.times.10.sup.11 to
5.times.10.sup.11 particles of scAAV9-CB-GFP. A strikingly
different transduction pattern was seen in adult treated animals
compared to the treated neonates. Most noticeably, there was an
absence of GFP positive DRG fibers and a marked decrease in LMN
transduction in all cervical and lumbar spinal cord sections
examined. GFP-positive astrocytes were easily observed throughout
the entire dorsal-ventral extent of the grey matter in all regions
of the spinal cord (FIG. 8a-b and FIGS. 9a-c and e-g) with the
greatest GFP-expression levels found in the higher dosed animals.
Co-labeling of GFP-positive cells with astroglial markers
excitatory amino acid transporter 2 (EAAT2) and glial fibrillary
acidic protein (GFAP) (FIG. 8C) demonstrated that approximately 90%
of the GFP-positive cells were astrocytes. Counts of total
astrocytes in the lumbar region of the spinal cord by z-series
collected confocal microscopy showed over 64% of total astrocytes
were positive for GFP (FIG. 9i-k and Table 1). FIG. 10 depicts
diagrams of coronal sections throughout the mouse brain
corresponding to the approximate locations shown in (FIG. 9m-o).
The box in (a) corresponds to the location shown in (FIG. 9m). The
smaller box in (b) corresponds to (FIG. 9n) and the larger box to
(FIG. 9o).
[0101] Viral transcription was again confirmed in adult tissues
with in situ hybridization (FIG. 5). Furthermore, whereas neonate
intravenous injection resulted in indiscriminate astrocyte and
neuronal transduction throughout the brain, adult tail-vein
injections produced isolated and localized neuronal expression only
in the hippocampus and dentate gyrus (FIG. 9m-n and FIG. 11e-f) in
both low and high dose animals. Low-dose animals had isolated
patches of transduced astrocytes scattered throughout the entire
brain. Of significance, high-dose animals had extensive astrocyte
and vascular transduction throughout the entire brain (FIG. 9m-o
and FIG. 11e-f) that persisted for at least seven-weeks
post-injection (n=5), suggesting a dose-response of transduction,
without regional specificity.
[0102] To date, efficient glial transduction has not been reported
for any AAV serotype indicating that AAV9 has a unique transduction
property in the CNS following intravenous delivery. An occasional
neuron transduced in the spinal cord, although these events were
scarce in adult animals. Furthermore, whereas neonate intravenous
injection resulted in indiscriminate transduction throughout the
brain, adult tail vein injections produced isolated and localized
neuronal expression in the hippocampus with isolated patches of
glial transduction scattered throughout the entire brain. The
scarcity of LMN and DRG transduction seen in the adult paradigm
suggests there is a developmental period in which access by
circulating virus to these cell populations becomes restricted.
Assuming a dependence on retrograde transport for DRG and LMN
transduction following intravenous injection, Schwann cell or
synapse maturation may be an important determinant of successful
rAAV9 LMN and DRG transduction.
[0103] The results demonstrate the striking capacity of AAV9 to
efficiently target neurons, and in particular motor neurons in the
neonate and astrocytes in the adult following intravenous delivery.
A simple intravenous injection of AAV9 as described here is
clinically relevant for both SMA and ALS. In the context of SMA,
data suggests that increased expression of survival motor neuron
(SMN) gene in LMNs may hold therapeutic benefit [Azzouz et al., The
Journal of Clinical Investigation, 114: 1726-1731 (2004) and
Baughan et al., Mol. Ther. 14: 54-62 (2006)]. The importance of the
results presented here is that with a single injection SMN
expression levels are effectively restored in LMN. Additionally,
given the robust neuronal populations transduced throughout the CNS
in neonatal animals, this approach also allows for overexpressing
or inhibiting genes using siRNA [see, for example, Siegel et al.,
PLoS Biology, 2: e419 (2004)]. The results also demonstrated
efficient targeting of astrocytes in adult-treated animals and this
finding is relevant for treating ALS where the non-cell autonomous
nature of disease progression has recently been discovered and
astrocytes have been specifically linked to disease progression
[Yamanaka et al., Nature Neuroscience, 11: 251-253 (2008)].
Targeting these cells with trophic factors or to circumvent
aberrant glial activity is useful in treating ALS [Dodge et al.,
Mol. Ther., 16(6):1056-64 (2008)].
Example 6
[0104] Optimal delivery of AAV9 expressing SMN is described for
postnatal gene replacement in a mouse model of Type 2 SMA.
[0105] Studies of the SMA patient population and the various SMA
animal models have established a positive correlation between
amounts of full-length SMN protein produced and lessened disease
severity. Histone deacetylase (HDAC) inhibitors and small molecules
are currently being investigated for their ability to increase
transcript production or alter exon 7 inclusion from the remaining
SMN2 gene [Avila et al., J. Clin. Invest., 117(3):659-71 (2007) and
Chang et al., Proc. Natl. Acad. Sci. USA, 98(17):9808-9813 (2001)].
Data presented herein demonstrates that a large percentage of LMNs
can be targeted with a scAAV9 vector, and SMN gene replacement to
treat SMA animals is therefore contemplated.
[0106] Mendelian inheritance predicts 25% of the pups in the
litters of SMA breeders to be affected. Affected SMA mice are
produced by interbreeding SMN2.sup.+/+, SMN.DELTA.7.sup.+/+,
Smn.sup.+/- mice. Breeders are maintained as homozygotes for both
transgenes and heterzygotes for the knockout allele. Mice were
genotyped by PCR following extraction of total genomic DNA from a
tail snip (see below). One primer set was used to confirm the
presence of the knockout allele while the second primer set
detected an intact mouse Smn allele. Animals were treated with
either scAAV9 SMN or scAAV9 GFP as controls.
[0107] SMA parent mice (Smn.sup.+/-, SMN2.sup.+/+,
SMN.DELTA.7.sup.+/+) were time mated [Monani et al., Human
Molecular Genetics 9: 333-339 (2000)]. Cages were monitored 18-21
days after visualization of a vaginal plug for the presence of
litters. Once litters were delivered, the mother was separated out,
pups were given tattoos for identification and tail samples were
collected. Tail samples were incubated in lysis solution (25 mM
NaOH, 0.2 mM EDTA) at 90.degree. C. for one hour. After incubation,
tubes were placed on ice for ten minutes and then received an equal
volume of neutralization solution (40 mM Tris pH5). After the
neutralization buffer, the extracted genomic DNA was added to two
different PCR reactions for the mouse Smn allele (Forward 1:
5'-TCCAGCTCCGGGATATTGGGATTG (SEQ ID NO: 2), Reverse 1:
5'-AGGTCCCACCACCTAAGAAAGCC (SEQ ID NO: 3), Forward 2:
5'-GTGTCTGGGCTGTAGGCATTGC (SEQ ID NO: 4), Reverse 2:
5'-GCTGTGCCTTTTGGCTTATCTG (SEQ ID NO: 5)) and one reaction for the
mouse Smn knockout allele (Forward: 5'-GCCTGCGATGTCGGTTTCTGTGAGG
(SEQ ID NO: 6), Reverse: 5'-CCAGCGCGGATCGGTCAGACG (SEQ ID NO: 7)).
After analysis of the genotyping PCR, litters were culled to three
animals. Affected animals (Smn.sup.-/-, SMN2.sup.+/+,
SMN.DELTA.7.sup.+/+) were injected as previously described with
5.times.10.sup.11 particles of self complementary AAV9 SMN or GFP
[Foust et al., Nat Biotechnol 27: 59-65 (2009)].
[0108] AAV9 was produced by transient transfection procedures using
a double stranded AAV2-ITR based CB-GFP vector, with a plasmid
encoding Rep2Cap9 sequence as previously described [Gao et al.,
Journal of Virology 78: 6381-6388 (2004)] along with an adenoviral
helper plasmid; pHelper (Stratagene, La Jolla, Calif.) in 293
cells. The serotype 9 sequence was verified by sequencing and was
identical to that previously described [Gao et al., Journal of
Virology 78: 6381-6388 (2004)]. Virus was purified by two cesium
chloride density gradient purification steps, dialyzed against
phosphate-buffered-saline (PBS) and formulated with 0.001%
Pluronic-F68 to prevent virus aggregation and stored at 4.degree.
C. All vector preparations were titered by quantitative-PCR using
Taq-Man technology. Purity of vectors was assessed by 4-12%
SDS-Acrylamide gel electrophoresis and silver staining (Invitrogen,
Carlsbad, Calif.).
[0109] To determine transduction levels in SMA mice (SMN2.sup.+/+;
SMN.DELTA.7.sup.+/+; Smn.sup.-/-), 5.times.10.sup.11 genomes of
scAAV9-GFP or -SMN (n=4 per group) under control of the
chicken-.beta.-actin hybrid promoter were injected into the facial
vein at P1. Forty-two.+-.2% of lumbar spinal motoneurons were found
to express GFP 10 days post injection. The levels of SMN in the
brain, spinal cord and muscle in scAAV9-SMN-treated animals are
shown in. SMN levels were increased in brain, spinal cord and
muscle in treated animals, but were still below controls
(SMN2.sup.+/+; SMN.DELTA.7.sup.+/+; Smn.sup.+/-) in neural tissue.
Spinal cord immunohistochemistry demonstrated expression of SMN
within choline acetyl transferase (ChAT) positive cells after
scAAV9-SMN injection.
[0110] Pups were weighed daily and tested for righting reflex every
other day from P5-P13. Righting reflex is analyzed by placing
animals on a flat surface on their sides and timing 30 seconds to
evaluate if the animals return to a upright position [Butchbach et
al., Neurobiology of Disease 27: 207-219 (2007)]. Every five days
between P15 and P30, animals were tested in an open field analysis
(San Diego Instruments, San Diego, Calif.). Animals were given
several minutes within the testing chamber prior to the beginning
of testing then activity was monitored for five minutes. Beam
breaks were recorded in the X, Y and Z planes, averaged across
groups at each time point and then graphed.
[0111] Whether scAAV9-SMN treatment of SMA animals improved motor
function was then evaluated. SMA animals treated with scAAV9-SMN or
-GFP were evaluated for the ability of the animals to right
themselves compared to control and untreated animals (n=10 per
group). Control animals were found to right themselves quickly,
whereas the SMN- and GFP-treated SMA animals showed difficulty at
P5. By P13, however, 90% of SMN treated animals could right
themselves compared to 20% of GFP-treated controls and 0% of
untreated SMA animals, demonstrating that SMN-treated animals
improved. Evaluating animals at P18 showed SMN-treated animals were
larger than GFP-treated but smaller than controls. Locomotive
ability of the SMN-treated animals were nearly identical to
controls as assayed by x, y and z plane beam breaks (open field
testing) and wheel running. Age-matched untreated SMA animals were
not available as controls for open field or running wheel analysis
due to their short lifespan.
[0112] Survival in SMN-treated SMA animals (n=11) compared to
GFP-treated SMA animals (n=11) was then evaluated using Kaplan
Meier survival analysis. No GFP-treated control animals survived
past P22, with a median lifespan of 15.5 days. The body weight in
treated SMN- or GFP-treated animals compared to wild-type
littermates was analyzed. The GFP-treated animal's weight peaked at
P10 and then precipitously declined until death. In contrast,
SMN-treated animals showed a steady weight gain to approximately
P40, where the weight stabilized at 17 grams, half of the weight of
controls. No deaths occurred in the SMN-treated group until P97.
Furthermore, this death appeared to be unrelated to SMA. The mouse
died after trimming of long extensor teeth. Four animals (P90-99)
were euthanized for electrophysiology of neuromuscular junctions
(NMJ). The remaining six animals remain alive, surpassing 250 days
of age.
[0113] For electrophysiology analysis, a recording chamber was
continuously perfused with Ringer's solution containing the
following (in mmol/1): 118 NaCl, 3.5 KCl, 2 CaCl.sub.2), 0.7
MgSO.sub.4, 26.2 NaHCO.sub.3, 1.7 NaH.sub.2PO.sub.4, and 5.5
glucose, pH 7.3-7.4 (20-22.degree. C., equilibrated with 95%
O.sub.2 and 5% CO.sub.2). Endplate recordings were performed as
follows. After dissection, the tibialis anterior muscle was
partially bisected and folded apart to flatten the muscle. After
pinning, muscle strips were stained with 10 .mu.M 4-Di-2ASP
[4-(4-diethylaminostyryl)-Nmethylpyridinium iodide] (Molecular
Probes) and imaged with an upright epifluorescence microscope. At
this concentration, 4-Di-2ASP staining enabled visualization of
surface nerve terminals as well as individual surface muscle
fibers. All of the endplates were imaged and impaled within 100
.mu.m. Two-electrode voltage clamp were used to measure endplate
current (EPC) and miniature EPC (MEPC) amplitude. Muscle fibers
were crushed away from the endplate band and voltage clamped to -45
mV to avoid movement after nerve stimulation.
[0114] To determine whether the reduction in endplate currents
(EPCs) was corrected with scAAV9-SMN, EPCs were recorded from the
tibialis anterior (TA) muscle [Wang et al., J Neurosci 24,
10687-10692 (2004)]. P9-10 animals were evaluated to ensure the
presence of the reported abnormalities within our mice. Control
mice had an EPC amplitude of 19.1.+-.0.8 nA versus 6.4.+-.0.8 nA in
untreated SMA animals (p=0.001) confirming published results [Kong
et al., J Neurosci 29, 842-851 (2009)]. Interestingly, P10
scAAV9-SMN-treated SMA animals had a significant improvement
(8.8.+-.0.8 vs. 6.4.+-.0.8 nA, p<0.05) over age-matched
untreated SMA animals. Gene therapy treatment, however, had not
restored normal EPC at P10 (19.1.+-.0.8 vs. 8.8.+-.0.8 nA,
p=0.001). At P90-99, there was no difference in EPC amplitude
between controls and SMA mice that had been treated with scAAV-SMN.
Thus, treatment with scAAV9-SMN fully corrected the reduction in
synaptic current. Importantly, P90-99 age-matched untreated SMA
animals were not available as controls due to their short
lifespan.
[0115] The number of synaptic vesicles released following nerve
stimulation (quantal content) and the amplitude of the muscle
response to the transmitter released from a single vesicle (quantal
amplitude) determine the amplitude of EPCs. Untreated SMA mice have
a reduction in EPC due primarily to reduced quantal content [Kong
et al., J Neurosci 29, 842-851 (2009)]. In our P9-10 cohort,
untreated SMA animals had a reduced quantal content when compared
with wild-type controls (5.7.+-.0.6 vs. 12.8.+-.0.6, p<0.05),
but scAAV9-SMN treated animals were again improved over the
untreated animals (9.5.+-.0.6 vs. 5.7.+-.0.6, p<0.05), but not
to the level of wild-type animals (9.5.+-.0.6 vs. 12.8.+-.0.6,
p<0.05). At P90-99, when quantal content was measured in treated
SMA mice, a mild reduction was present (control=61.3.+-.3.5,
SMA-treated=50.3.+-.2.6, p<0.05), but was compensated for by a
statistically significant increase in quantal amplitude
(control=1.39.+-.0.06, SMA treated=1.74.+-.0.08, p<0.05).
Quantal amplitudes in young animals had no significant differences
(control=1.6.+-.0.1, untreated SMA=1.3.+-.0.1, treated
SMA=1.1.+-.0.1 nA, p=0.28).
[0116] The reduction in vesicle release in untreated SMA mice was
due to a decrease in probability of vesicle release, demonstrated
by increased facilitation of EPCs during repetitive stimulation
[Kong et al., J Neurosci 29: 842-851 (2009)]. Both control and
treated SMA EPCs were reduced by close to 20% by the 10th pulse of
a 50 Hz train of stimuli (22.+-.3% reduction in control vs 19.+-.1%
reduction in treated SMA, p=0.36). This demonstrates that the
reduction in probability of release was corrected by replacement of
SMN. During electrophysiologic recording, no evidence of
denervation was noted. Furthermore, all adult NMJs analyzed showed
normal morphology and full maturity. P9-10 transverse abdominis
immunohistochemistry showed the typical neurofilament accumulation
in untreated SMA NMJs[Kong et al., J Neurosci 29: 842-851 (2009)],
whereas treated SMA NMJs showed a marked reduction in neurofilament
accumulation.
[0117] A recent study using an HDAC inhibitor to extend survival of
SMA mice reported necrosis of the extremities and internal tissues
[Narver et al., Ann Neurol 64: 465-470 (2008)]. In the studies
described herein, mice developed necrotic pinna between P45-70.
Pathological examination of the pinna noted vascular necrosis, but
necrosis was not found elsewhere.
[0118] To explore the therapeutic window in SMA mice, systemic
scAAV9-GFP injections were performed at varying postnatal time
points to evaluate the pattern of transduction of motor neurons and
astrocytes. scAAV9-GFP systemic injections in mice on P2, P5 or P10
showed distinct differences in the spinal cord. There was a shift
from neuronal transduction in P2-treated animals toward
predominantly glial transduction in older, P10 animals, consistent
with previous studies and knowledge of the developing blood-brain
barrier in mice [Foust et al., Nat. Biotechnol. 27: 59-65 (2009);
Saunders et al., Nat. Biotechnol. 27: 804-805, author reply 805
(2009)].
[0119] To determine the therapeutic effect of SMN delivery at these
various time points, small cohorts of SMA-affected mice were
injected with scAAV9-SMN on P2, P5 and P10 and evaluated for
changes in survival and body weight. P2-injected animals were
rescued and indistinguishable from animals injected with scAAV9-SMN
on P1. However, P5-injected animals showed a more modest increase
in survival of approximately 15 days, whereas P10-injected animals
were indistinguishable from GFP-injected SMA pups. These findings
support previous studies demonstrating the importance of increasing
SMN levels in neurons of SMA mice [Gavrilina et al., Hum. Mol.
Genet. 17: 1063-1075 (2008)]. Furthermore, these results suggest a
period during development in which intravenous injection of scAAV9
can target neurons in sufficient numbers for benefit in SMA.
[0120] The above results demonstrate robust, postnatal rescue of
SMA mice with correction of motor function, neuromuscular
electrophysiology, and increased survival following a one-time gene
delivery of SMN. Intravenous scAAV9 treats neurons, muscle and
vascular endothelium. Vascular delivery of scAAV9 SMN in the mouse
was safe, and well tolerated.
Example 7
[0121] The brains of mice were examined following postnatal day-one
intravenous injection of scAAV9-CBGFP and extensive GFP-expression
was found in all regions analyzed, including the striatum, cortex,
anterior commisure, internal capsule, corpus callosum, hippocampus
and dentate gyrus, midbrain and cerebellum (FIG. 12a-h,
respectively, FIG. 11). GFP-positive cells included both neurons
and astrocytes throughout the brain. To further characterize the
transduced neurons, brains were co-labeled for GFP and GAD67, a GAB
Aergic marker. FIG. 13 depicts diagrams of coronal sections
throughout the mouse brain corresponding to the approximate
locations shown in FIG. 12a-h for postnatal day-1 injected neonatal
mouse brains. The box in (13a) corresponds to the location of (FIG.
12a). The smaller box in (13b) corresponds to (FIG. 12b) and the
larger box to (FIG. 12c). The larger box in (13c) corresponds to
(FIG. 12d) while the smaller box in (13c) represents (FIG. 12e).
Finally, (13d-f) correspond to (FIG. 12f-h) respectively.
[0122] The cortex, hippocampus and dentate had very little
colocalization between GFP and GAD67 labeled cells (FIG. 14a-i),
while Purkinje cells in the cerebellum were extensively co-labeled
(FIG. 14j-1). Finally, unbiased-estimated stereological
quantification of transduction showed that 18.8+/-1.9% within the
retrosplenial/cingulate cortex, 14.8+/-4.8% within the dentate
gyrus and 71.8+/-3.65% within the Purkinje layer of total neurons
were transduced following a one-time administration of virus (Table
1).
Example 8
[0123] Efficient astrocyte transduction by an AAV8-, but not an
AAV9-vector, following direct brain injection has been previously
reported. Astrocyte transduction, however, was suggested to be
related to viral purification [Klein et al., Mol Ther 16: 89-96
(2008)]. To investigate whether AAV9 astrocyte transduction was
related to vector purity or delivery route, multiple AAV9
preparations were evaluated for vector purity by silver-stain and
8.times.10.sup.10 particles of the same scAAV9-CB-GFP vector
preparations from the intravenous experiments were injected into
the striatum and dentate gyrus of adult mice. Silver-staining
showed that vector preparations were relatively pure and of
research grade quality (FIG. 15). Two-weeks post-intracranial
injection, we observed significant neuronal transduction within the
injected regions using these vector preparations. However, no
evidence for colocalization was found between GFP and GFAP labeling
throughout the injected brains (n=3) (FIG. 16), as previously
reported [Cearley et al., Mol Ther 16: 1710-1718 (2008)],
suggesting the astrocyte transduction in this work may be injection
route- and serotype-dependent and not due to vector purity.
[0124] The scarcity of LMN and DRG transduction seen in the adult
paradigm suggests there is a developmental period in which access
by circulating virus to these cell populations becomes restricted.
Assuming a dependence on retrograde transport for DRG and LMN
transduction following intravenous injection, Schwann cell or
synapse maturation may be an important determinant of successful
AAV9 LMN and DRG transduction. Direct intramuscular injection of
AAV9 into adults did not result in readily detectable expression in
motor neurons by retrograde transport. These results suggest that
AAV9 escapes brain vasculature in a similar manner as skeletal and
cardiac muscle vasculature. Once free of the vasculature, these
data suggest that AAV9 infects the astrocytic-perivascular-endfeet
that surround capillary endothelial cells [Abbott et al., Nat Rev
Neurosci 7: 41-53 (2006)].
[0125] In summary, these results demonstrate the unique capacity of
AAV9 to efficiently target cells within the CNS, and in particular
widespread neuronal and motor neuron transduction in the neonate,
and extensive astrocyte transduction in the adult following
intravenous delivery. A simple intravenous injection of AAV9 as
described herein may be clinically relevant for both SMA and ALS.
In the context of SMA, data suggest that increased expression of
survival motor neuron (SMN) gene in LMNs may hold therapeutic
benefit [Azzouz et al., The Journal of Clinical Investigation 114:
1726-1731 (2004); Baughan et al., Mol Ther 14: 54-62 (2006)]. The
importance of the results presented here is that a single injection
may be able to effectively restore SMN expression levels in LMNs.
Additionally, given the robust neuronal populations transduced
throughout the CNS in neonatal animals, this approach may also
allow for rapid, relatively inexpensive generation of chimeric
animals for gene overexpression, or gene knock-down [Siegel et al.,
PLoS Biology 2: e419 (2004)]. Additionally, constructing AAV9 based
vectors with neuronal or astrocyte specific promoters may allow
further specificity, given that AAV9 targets multiple non-neuronal
tissues following intravenous delivery [Inagaki et al., Mol Ther
14: 45-53 (2006); Pacak et al., Circulation Research 99: e3-9
(2006)]. The results also demonstrate efficient targeting of
astrocytes in adult-treated animals, and this finding is relevant
for treating ALS, where the non-cell autonomous nature of disease
progression has recently been discovered, and astrocytes have been
specifically linked to disease progression [Yamanaka et al., Nature
Neuroscience 11: 251-253 (2008)]. The ability to target astrocytes
for producing trophic factors, or to circumvent aberrant glial
activity may be beneficial for treating ALS24. In sum, these data
highlight a relatively non-invasive method to efficiently deliver
genes to the CNS and are useful in basic and clinical neurology
studies.
Example 9
[0126] The ability of scAAV9 to traverse the blood-brain barrier in
nonhuman primates [Kota et al., Sci. Transl. Med 1: 6-15 (2009)]
was also investigated. A male cynomolgus macaque was intravenously
injected on P1 with 1.times.10.sup.14 particles
(2.2.times.10.sup.11 particles/g of body weight) of scAAV9-GFP and
euthanized it 25 days after injection. Examination of the spinal
cord revealed robust GFP expression within the dorsal root ganglia
and motor neurons along the entire neuraxis, as seen in P1-injected
mice. This finding demonstrated that early systemic delivery of
scAAV9 efficiently targets motor neurons in a nonhuman primate.
Example 10
[0127] Self complementary (sc) rAAV9 bearing MECP2 cDNA under
control of a fragment of its own promoter (scAAV9/MECP2), was shown
to be capable of significantly stabilizing or reversing disease
phenotypes when administered systemically into female RTT mouse
models.
[0128] To counteract possible over-expression and better mimic the
expression pattern of virally-mediated MECP2, a rAAV9 containing
MECP2 (E1) cDNA under control of an .about.730 bp fragment of its
own promoter was constructed [Rastegar et al., PloS One, 4: e6810
(2009)] (scAAV9 MECP2; FIG. 17).
[0129] Mouse MECP2-.alpha. polynucleotide was cloned in a plasmid
downstream of a 730 bp fragment of MECP2 promoter. Recombinant AAV9
was produced by transient transfection procedures using a
double-stranded AAV2-ITR-based MECP2 minimal promoter--MECP2 (E1)
vector, with a plasmid encoding Rep2Cap9 sequence as previously
described along with an adenoviral helper plasmid pHelper
(Stratagene) in 293 cells [Gao et al., J. Virol. 78: 6381-6388
(2004) and Fu et al., Mol Ther., 8(6): 911-917 (2003)]. Virus was
purified by cesium chloride density gradient purification steps as
previously described, dialyzed against PBS and formulated with
0.001% Pluronic-F68 to prevent virus aggregation and stored at
4.degree. C. [Ayuso et al., Gene Ther., 17(4):503-510 (2010)]. All
vector preparations were titered by quantitative PCR using Taq-Man
technology. Purity of vectors was assessed by 4-12% SDS-acrylamide
gel electrophoresis and silver staining (Invitrogen). The resulting
rAAV9 was named "scAAV9/MECP2." The sequence of its genome is shown
in FIG. 22 and has in sequence: a mutated AAV2 ITR lacking the
terminal resolution site, an approximately 730 bp murine MECP2
promoter fragment, SV40 intron sequences, murine MECP2.alpha. cDNA,
a bovine growth hormone polyadenylation signal sequence and an AAV2
ITR.
[0130] Mice were group housed with littermates in standard housing
on a 12:12 h light:dark cycle. MECP2.sup.Stop (Stock number:
006849) [Guy et al., Science, 315: 1143-1147 (2007)] and
MECP2.sup.Bird.knockout (Stock number: 003890; MECP2.sup.Bnull)
[Guy et al., Nature Genetics, 27: 322-326 (2001)] mice were
obtained from Jackson Laboratories and were on a C57BL/6
background. The wild type male mice were crossed to female
MECP2.sup.+/Stop and MECP2.sup.+/Bnull mice to yield male and
female MECP2.sup.Stop and MECP2.sup.Bnull genotypes. The floxed
Stop sequence was identified from tail biopsies using the following
primers: common 5'-AACAGTGCCAGCTGCTCTTC-3', WT
5'-CTGTATCCTTGGGTCAAGCTG-3', and mutant 5'-GCCAGAGGCCACTTGTGTAG-3'.
For Bird null following primers were used
5'-CCACCCTCCAGTTTGGTTTA-3' and 5'-GACCCCTTGGGACTGAAGTT-3' [Lioy et
al., Nature, 475: 497-500 (2011)].
[0131] Mice were placed in a restraint that positioned the mouse
tail in a lighted, heated groove. The tail was swabbed with alcohol
then injected intravenously with a 300 .mu.l viral solution
containing 3.times.10.sup.12 DNase-resistant particles of scAAV9 in
PBS (FIG. 20, panel A). After the injection, mice were returned to
their cages. All animal procedures were approved by Oregon Health
and Science University Institutional Animal Care and Use
Committee.
[0132] For phenotype scoring, mice were removed from their home
cage and placed onto a metal laminar flow hood for observation.
[0133] For mobility: 0=wild type; 1=reduced movement when compared
to wild type, with extended freezing periods or extended delay to
movement when first placed on the surface; 2=complete loss of
movement when placed on the surface.
[0134] For gait: 0=wild type; 1=hind limbs spread wider than wild
type when ambulating and/or a lowered pelvis when ambulating;
2=lack of full strides by hind limbs resulting in a dragging of
hindquarters.
[0135] For hind limb clasping: 0=WT; hind limbs splay outward when
suspended by the tail; 1=one hind limb is pulled into the body or
forelimbs are stiff and splayed outward without motion; 2=one hind
limb is pulled into the body and forelimbs are stiff and splayed
outward without motion and might form a widened bowl shape, or both
hind limbs are pulled into the body with or without abnormal
forelimb posture.
[0136] For tremor: 0=no tremor; 1=intermittent mild tremor;
2=continuous tremor or intermittent violent tremor.
[0137] For general condition: 0=shiny coat, clear and opened eyes,
normal body stance; 1=dull or squinty eyes, dull or ungroomed coat,
somewhat hunched stance; 2=piloerection, hunched stance.
[0138] For behavioral testing, all tests were performed at the same
time of day (12.00 to 18.00 hrs) and in the same dedicated
observation room. Mice were never subjected to multiple tasks on
the same day.
[0139] Open field activity--Mice were placed singly into the center
of an open field arena (14.times.14 inches) equipped to record live
images from the top. Activity was recorded for 20 minutes using
StereoScan Software (Clever Systems) on a Dell computer fitted with
a window operating system. Software calculated the total distance
travelled and average velocity of the movements from recorded
movies. The mice could not see the experimenter during
recordings.
[0140] Rotorod--Mice were placed on an elevated rotating rod
(diameter: 7 cm, elevated: 45 cm, Economex, Columbus Instruments,
Columbus, Ohio, USA), initially rotating at 5.0 rpm. The rod
accelerated 5.0 rpm/s. The latency to fall (s) was recorded
manually by using individual mouse specific stopwatches. Each mouse
receives three trials per day, with no delay between trials, on
three consecutive days.
[0141] Platform test--Performed as described in Grady et al., J.
Neuroscience, 26: 2841-2851 (2006) with some modifications. Each
mouse was timed for how long it remained on an elevated, circular
platform (3.0 cm in diameter) with rounded edges. A maximum score
of 60 s was assigned if the mouse remained on the platform for the
entire test trial without falling. Two trials were administered for
each test with 4 h intervening between trials, and means were
calculated across the trials for each mouse.
[0142] Inverted screen test--Performed as described in Grady et
al., 2006 with some modifications. Each mouse was placed in the
middle of wire grid (parallel metal wires 0.5 cm apart) that was
inverted to 180.degree.. A mouse was timed for how long it remained
upside down on the screen, with a maximum score of 60 s being given
if the animal did not fall. Two trials were administered for each
test with 4 h intervening between trials, and means were calculated
across the trials for each mouse.
[0143] Nesting ability-Mice were placed in individual cages and
provided with a nest building material (5 cm.times.5 cm.times.0.5
cm). The material was placed in top left corner of cage and nesting
ability was scored over night based on the interaction of
individual mouse with nesting material. The score of 0, 1, 2 and 3
were assigned. The score 0 was assigned to mouse that not at all
interacted with material, score 3 was assigned to mouse that
completely used the material to build a nest.
[0144] Novel Object recognition test--Test is conducted in open
field arena used to evaluate motor activity. The two objects (a
sphere and a box) were selected based on similar volume and
unbiased interaction of wild type mice. During habituation, the
mice were allowed to explore an empty arena for 5 minutes.
Twenty-four hours after habituation, the mice were exposed to the
familiar arena with two identical objects (sphere) placed at an
equal distance for 5 minutes. The next day, same exercise was
repeated. On third day of the test, the mice are allowed to explore
the open field in the presence of the familiar and a novel object
(Box) for 5 minutes to test cognition. The time spent exploring
each object on second and final day of test was recorded to
estimate the extent of novel object recognition by calculating
discrimination index (DI)=(Tn-Tf)/(Tn+Tf). Tn; time with novel
object and Tf; time with familiar object. The DI value can vary
between +1 and -1, where a positive score indicates more time spent
with the novel object, a negative score indicates more time spent
with the familiar object, and a zero score indicates a null
preference.
[0145] After phenotypic scoring and behavioral testing, mice were
anaesthetized by intraperitoneal injection of Avertin (2-2-2
Tribromoethanol) and sacrificed by transcardial perfusion of 4%
parafomaldehyde in phosphate-buffered saline. Brains were
equilibrated in 30% sucrose overnight at 4.degree. C. Sagittal
sections (40 .mu.m) were cut at -20.degree. C. using a cryostat
(Leica) and stored at -20.degree. C. Sections were immunolabeled
overnight at 4.degree. C. using the following primary antibodies:
rabbit-MECP2 (1:500, Covance), mouse-GFAP (1:500, Abcam),
chicken-GFAP (1:200, Abcam), mouse-NeuN (1:200, Millipore).
Appropriate Alexa/Dylight Fluor secondary antibodies (1:500,
Molecular Probes) were used for 1 h at room temperature. DAPI was
present in the ProLong Gold Antifade (Invitrogen) mounting reagent.
Nissl staining (at either 594 nm or 640 nm) was performed as
instructed by the manufacturer (NeuroTrace, Invitrogen). All images
were collected on a Zeiss confocal laser scanning LSM 510
microscope.
[0146] MECP2 expressing cells were identified as described in Lioy
et al. (2011) with some modifications: nuclei of astrocytes (GFAP+
at 555 nm or 640 nm; NeuN- at 555 nm or 640 nm) and neurons (NeuN+
at 555 nm or 640 nm) were first identified by DAPI staining. Cells
with clearly identified nuclei were then assessed for MECP2
expression by analyzing 505 nm signal (excitation: 488 nm) in the
nucleus.
[0147] The following measurements were analyzed using one-way ANOVA
followed, when appropriate (P<0.05), by Newman-Keuls post-hoc
test: anatomical and cell-type expression patterns of transduced
MECP2, whole body and brain weights, respiratory parameters, open
field activity and time on rotarod. The following measurements were
analyzed using Kruskal-Wallis test followed, when appropriate
(P<0.05), by Dunn's multiple comparisons test: phenotype
severity scores, nesting scores, time on an inverted grid, time on
a platform, and novel object recognition. Survival curves were
compared using the Log-Rank method. All statistics were performed
using PRISM 5.0 software.
[0148] The scAAV9/MECP2 construct is expressed in both neurons and
glia in vitro, and in MECP2Bnull/y mice, virally-expressed MECP2
was detected immunochemically in heterochromatic puncta of both
cell types, indicating wild type DNA binding function. Notably,
MECP2-positive neurons in the CA3 region of scAAV9/MECP2-injected
males had significantly larger somal sizes than MECP2-negative
neurons.
[0149] The MECP2 expressed from scAAV9/MECP2 was detected
throughout the brain. However, with the exception of cerebellum,
MECP2 expression was not over represented in astrocytes, (FIG. 18).
This could reflect, in part, the specific cell specific regulatory
elements in the cloned promoter fragment because MECP2 is expressed
generally at lower levels in astocytes than neurons [Ballas et al.,
Nature Neuroscience, 12: 311-317 (2009) and Skene et al., Molecular
Cell, 37: 457-468 (2010)]. Consistent with all of these metrics,
the injected male mice had prolonged lifespans and improved
observational scores compared to control injected mice (FIG. 19,
panels a and b).
[0150] A potential concern with virally-mediated gene transfer of
MECP2 is over-expression, because MECP2 duplication gives rise to a
neurological disease [del Gaudio et al., Genetics in Medicine, 8:
784-792 (2006) and Friez et al., Pediatrics, 118: e1687-1695
(2006)]. To assess this issue, in an unbiased manner, the average
MECP2 expression level was determined in transduced brains by
recording field pixel intensities of MECP2-Cy3 fluorescence in
hindbrain sections selected randomly. The results indicated that
scAAV9/MECP2 injection resulted in physiological levels of MECP2
protein (FIG. 19, panel c). Interestingly, WT brains showed two
peaks of MECP2 fluorescence that were precisely recapitulated in
the MECP2 transduced brains, although the cellular nature of the
fluorescence is not identified by this method of analysis.
[0151] Having established that scAAV9/MECP2 programmed MECP2
expression to approximately physiological levels in multiple cell
types in brain, rescue parameters were examined in 10 to 12
month-old symptomatic MECP2Bnull/+ mice that were systemically
injected with scAAV9/MECP2 or control virus (FIG. 21). Like the
males, there was no evidence for over-expression of MECP2 and viral
therapy did not compromise survival (FIG. 19, panel c; FIG. 21).
The observational scores increased initially from two to three.
Strikingly, by 12-weeks, scAAV9/MECP2 injected females stabilized
at an improved score of one until the end of scoring at 24-weeks,
while females injected with control virus progressed to a score of
nearly six (FIG. 20, panel b). The scAAV9/MECP2 injected
MECP2Bnull/+ mice also performed significantly better than
scAAV9/control females in rotorod, inverted grid and platform
tests, and nesting ability (FIG. 20, panels c-f). None of the
injected females exhibited seizures, unlike the females injected
with control virus (2/5).
[0152] Previous gene therapy work has shown modest, but
encouraging, improvement of symptoms in male mouse models of RTT
[Gadalla et al., Mol. Ther., 21: 18-30 (2013)]. However, the
disease initiates and progresses differently in females and males,
due to the mosaic nature of MECP2 loss of function in females.
Therefore, therapeutics designed especially for affected females
are required. The results presented herein are important because
they suggest, for the first time, that symptoms in human RTT female
patients may be reversible by ectopic expression of MECP2 in a
rAAV9 virus that infects peripheral tissue and multiple cell types
within the CNS. Interestingly, the experiments also indicate that
not every cell needs to be repaired with MECP2 in order to
stabilize or reverse phenotypes in female mice, consistent with the
finding that an .about.5% increase in MECP2 levels over WT levels
is sufficient to mediate longer lifespans [Robinson et al., Brain,
135: 2699-2710 (2012) and Lioy et al. (2011).
[0153] While the present invention has been described in terms of
various embodiments and examples, it is understood that variations
and improvements will occur to those skilled in the art. Therefore,
only such limitations as appear in the claims should be placed on
the invention.
[0154] All documents referred to in this application, including
priority documents, are hereby incorporated by reference in their
entirety with particular attention to the content for which they
are referred.
Sequence CWU 1
1
711621DNAHomo sapiens 1ccacaaatgt gggagggcga taaccactcg tagaaagcgt
gagaagttac tacaagcggt 60cctcccggcc accgtactgt tccgctccca gaagccccgg
gcggcggaag tcgtcactct 120taagaaggga cggggcccca cgctgcgcac
ccgcgggttt gctatggcga tgagcagcgg 180cggcagtggt ggcggcgtcc
cggagcagga ggattccgtg ctgttccggc gcggcacagg 240ccagagcgat
gattctgaca tttgggatga tacagcactg ataaaagcat atgataaagc
300tgtggcttca tttaagcatg ctctaaagaa tggtgacatt tgtgaaactt
cgggtaaacc 360aaaaaccaca cctaaaagaa aacctgctaa gaagaataaa
agccaaaaga agaatactgc 420agcttcctta caacagtgga aagttgggga
caaatgttct gccatttggt cagaagacgg 480ttgcatttac ccagctacca
ttgcttcaat tgattttaag agagaaacct gtgttgtggt 540ttacactgga
tatggaaata gagaggagca aaatctgtcc gatctacttt ccccaatctg
600tgaagtagct aataatatag aacagaatgc tcaagagaat gaaaatgaaa
gccaagtttc 660aacagatgaa agtgagaact ccaggtctcc tggaaataaa
tcagataaca tcaagcccaa 720atctgctcca tggaactctt ttctccctcc
accacccccc atgccagggc caagactggg 780accaggaaag ccaggtctaa
aattcaatgg cccaccaccg ccaccgccac caccaccacc 840ccacttacta
tcatgctggc tgcctccatt tccttctgga ccaccaataa ttcccccacc
900acctcccata tgtccagatt ctcttgatga tgctgatgct ttgggaagta
tgttaatttc 960atggtacatg agtggctatc atactggcta ttatatgggt
ttcagacaaa atcaaaaaga 1020aggaaggtgc tcacattcct taaattaagg
agaaatgctg gcatagagca gcactaaatg 1080acaccactaa agaaacgatc
agacagatct ggaatgtgaa gcgttataga agataactgg 1140cctcatttct
tcaaaatatc aagtgttggg aaagaaaaaa ggaagtggaa tgggtaactc
1200ttcttgatta aaagttatgt aataaccaaa tgcaatgtga aatattttac
tggactcttt 1260tgaaaaacca tctgtaaaag actggggtgg gggtgggagg
ccagcacggt ggtgaggcag 1320ttgagaaaat ttgaatgtgg attagatttt
gaatgatatt ggataattat tggtaatttt 1380atggcctgtg agaagggtgt
tgtagtttat aaaagactgt cttaatttgc atacttaagc 1440atttaggaat
gaagtgttag agtgtcttaa aatgtttcaa atggtttaac aaaatgtatg
1500tgaggcgtat gtggcaaaat gttacagaat ctaactggtg gacatggctg
ttcattgtac 1560tgtttttttc tatcttctat atgtttaaaa gtatataata
aaaatattta attttttttt 1620a 1621224DNAArtificial SequenceSynthetic
nucleotide 2tccagctccg ggatattggg attg 24323DNAArtificial
SequenceSynthetic nucleotide 3aggtcccacc acctaagaaa gcc
23422DNAArtificial SequenceSynthetic nucleotide 4gtgtctgggc
tgtaggcatt gc 22522DNAArtificial SequenceSynthetic nucleotide
5gctgtgcctt ttggcttatc tg 22625DNAArtificial SequenceSynthetic
nucleotide 6gcctgcgatg tcggtttctg tgagg 25721DNAArtificial
SequenceSynthetic nucleotide 7ccagcgcgga tcggtcagac g 21
* * * * *