U.S. patent application number 17/335251 was filed with the patent office on 2022-04-28 for methods and materials for activating an internal ribosome entry site in exon 5 of the dmd gene.
The applicant listed for this patent is RESEARCH INSTITUTE AT NATIONWIDE CHILDREN'S HOSPITAL, UNIVERSITY OF WESTERN AUSTRALIA. Invention is credited to Kevin Flanigan, Nicolas Sebastien Wein, Stephen Wilton.
Application Number | 20220127607 17/335251 |
Document ID | / |
Family ID | 1000006075436 |
Filed Date | 2022-04-28 |
![](/patent/app/20220127607/US20220127607A1-20220428-D00001.png)
![](/patent/app/20220127607/US20220127607A1-20220428-D00002.png)
![](/patent/app/20220127607/US20220127607A1-20220428-D00003.png)
![](/patent/app/20220127607/US20220127607A1-20220428-D00004.png)
![](/patent/app/20220127607/US20220127607A1-20220428-D00005.png)
![](/patent/app/20220127607/US20220127607A1-20220428-D00006.png)
![](/patent/app/20220127607/US20220127607A1-20220428-D00007.png)
![](/patent/app/20220127607/US20220127607A1-20220428-D00008.png)
![](/patent/app/20220127607/US20220127607A1-20220428-D00009.png)
![](/patent/app/20220127607/US20220127607A1-20220428-D00010.png)
![](/patent/app/20220127607/US20220127607A1-20220428-D00011.png)
View All Diagrams
United States Patent
Application |
20220127607 |
Kind Code |
A1 |
Flanigan; Kevin ; et
al. |
April 28, 2022 |
METHODS AND MATERIALS FOR ACTIVATING AN INTERNAL RIBOSOME ENTRY
SITE IN EXON 5 OF THE DMD GENE
Abstract
The present invention relates to the delivery of oligomers for
treating patients with a 5' mutation in their DMD gene other than a
DMD exon 2 duplication. The invention provides methods and
materials for activating an internal ribosome entry site in exon 5
of the DMD gene resulting in translation of a functional truncated
isoform of dystrophin. The methods and materials can be used for
the treatment of muscular dystrophies arising from 5' mutations in
the DMD gene such as Duchenne Muscular Dystrophy or Becker Muscular
Dystrophy.
Inventors: |
Flanigan; Kevin; (Columbus,
OH) ; Wein; Nicolas Sebastien; (Columbus, OH)
; Wilton; Stephen; (Perth, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RESEARCH INSTITUTE AT NATIONWIDE CHILDREN'S HOSPITAL
UNIVERSITY OF WESTERN AUSTRALIA |
Columbus
Crawley |
OH |
US
AU |
|
|
Family ID: |
1000006075436 |
Appl. No.: |
17/335251 |
Filed: |
June 1, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15502702 |
Feb 8, 2017 |
11053494 |
|
|
PCT/US15/44366 |
Aug 7, 2015 |
|
|
|
17335251 |
|
|
|
|
62035395 |
Aug 9, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0019 20130101;
C12N 2750/14143 20130101; C12N 2840/203 20130101; C12N 2310/3513
20130101; C12N 15/113 20130101; C12N 2310/315 20130101; C12N
2310/3519 20130101; C12N 2320/33 20130101; C12N 2320/31 20130101;
C12N 2310/346 20130101; C12N 2310/11 20130101; C12N 2310/3231
20130101; A61K 31/58 20130101; C12N 2330/51 20130101; A61K 31/573
20130101; A61K 31/712 20130101; C12N 2310/3233 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 9/00 20060101 A61K009/00; A61K 31/573 20060101
A61K031/573; A61K 31/58 20060101 A61K031/58; A61K 31/712 20060101
A61K031/712 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with Government support under
NS043264 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1-23. (canceled)
24. A method of ameliorating Duchenne Muscular Dystrophy or Becker
Muscular Dystrophy in a patient with a 5' mutation in a DMD gene
but without a DMD exon 2 duplication, the method comprising
administering to the patient a recombinant adeno-associated virus
(rAAV) comprising a DMD exon 5 internal ribosome entry site
(IRES)-activating oligomer construct comprising: (a) the nucleotide
sequence set forth in SEQ ID NO: 5, 7, or 8; or (b) a nucleotide
sequence that expresses an RNA transcript comprising the nucleotide
sequence set forth in SEQ ID NO: 9, 11, or 12.
25. The method of claim 24, wherein the progression of a dystrophic
pathology is inhibited in the patient following administration of
the rAAV to the patient.
26. The method of claim 24, wherein muscle function is improved in
the patient following administration of the rAAV to the
patient.
27. The method of claim 26, wherein the improvement in muscle
function is an improvement in muscle strength or an improvement in
stability in standing and walking.
28. The method of claim 24, wherein the genome of the rAAV lacks
adeno-associated virus rep and cap DNA.
29. The method of claim 24, wherein the genome of the rAAV is a
self-complementary genome.
30. The method of claim 24, wherein the genome of the rAAV is a
single-stranded genome.
31. The method of claim 24, wherein the rAAV comprises one or more
capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,
AAV8, AAV9, AAV10, AAV11, or AAVrh74.
32. The method of claim 31, wherein the rAAV comprises one or more
capsid proteins from AAV1, AAV6, AAV8, AAV9, or AAVrh74.
33. The method of claim 24, wherein the DMD exon 5 IRES-activating
oligomer construct comprises the nucleotide sequence set forth in
SEQ ID NO: 5.
34. The method of claim 24, wherein the DMD exon 5 IRES-activating
oligomer construct comprises the nucleotide sequence set forth in
SEQ ID NO: 7.
35. The method of claim 24, wherein the DMD exon 5 IRES-activating
oligomer construct comprises the nucleotide sequence set forth in
SEQ ID NO: 8.
36. The method of claim 24, wherein the DMD exon 5 IRES-activating
oligomer construct comprises a nucleotide sequence that expresses
an RNA comprising the nucleotide sequence set forth in SEQ ID NO:
9.
37. The method of claim 24, wherein the DMD exon 5 IRES-activating
oligomer construct comprises a nucleotide sequence that expresses
an RNA comprising the nucleotide sequence set forth in SEQ ID NO:
11.
38. The method of claim 24, wherein the DMD exon 5 IRES-activating
oligomer construct comprises a nucleotide sequence that expresses
an RNA comprising the nucleotide sequence set forth in SEQ ID NO:
12.
39. The method of claim 24, further comprising administering a
glucocorticoid to the patient.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/502,702, now U.S. Pat. No. 11,053,494, issued Jul. 6, 2021,
which is a U.S. national stage application of PCT/US2015/044366,
filed Aug. 7, 2015, which claims the benefit of the filing date of
U.S. Provisional Patent Application No. 62/035,395 filed Aug. 9,
2014, which is incorporated by reference herein in its
entirety.
INCORPORATION BY REFERENCE OF THE DEQUENCE LISTING
[0003] This application contains, as a separate part of disclosure,
a Sequence Listing in computer-readable form (filename:
48873A_Seqlisting.txt; 20,279 bytes--ASCII text file; created May
25, 2021) which is incorporated by reference herein in its
entirety.
FIELD OF THE INVENTION
[0004] The present invention relates to the delivery of oligomers
for treating patients with a 5' mutation in their DMD gene other
than a DMD exon 2 duplication. The invention provides methods and
materials for activating an internal ribosome entry site in exon 5
of the DMD gene resulting in a functional truncated isoform of
dystrophin. The methods and materials can be used for the treatment
of muscular dystrophies arising from 5' mutations in the DMD gene
such as Duchenne Muscular Dystrophy or Becker Muscular
Dystrophy.
BACKGROUND
[0005] Muscular dystrophies (MDs) are a group of genetic diseases.
The group is characterized by progressive weakness and degeneration
of the skeletal muscles that control movement. Some forms of MD
develop in infancy or childhood, while others may not appear until
middle age or later. The disorders differ in terms of the
distribution and extent of muscle weakness (some forms of MD also
affect cardiac muscle), the age of onset, the rate of progression,
and the pattern of inheritance.
[0006] One form of MD is Duchenne Muscular Dystrophy (DMD). It is
the most common severe childhood form of muscular dystrophy
affecting 1 in 5000 newborn males. DMD is caused by mutations in
the DMD gene leading to absence of dystrophin protein (427 KDa) in
skeletal and cardiac muscles, as well as GI tract and retina.
Dystrophin not only protects the sarcolemma from eccentric
contractions, but also anchors a number of signaling proteins in
close proximity to sarcolemma. Many clinical cases of DMD are
linked to deletion mutations in the DMD gene. Despite many lines of
research following the identification of the DMD gene, treatment
options are limited. Corticosteroids are clearly beneficial but
even with added years of ambulation the benefits are offset by
long-term side effects. The original controlled, randomized,
double-blind study reported more than 20 years ago showed benefits
using prednisone [Mendell et al., N. Engl. J. Med., 320: 1592-1597
(1989)]. Subsequent reports showed equal efficacy using
deflazacort, a sodium-sparing steroid [Biggar et al., J. Pediatr.,
138: 45-50 (2001)]. Recent studies also demonstrate efficacy by
exon skipping, prolonging walking distance on the 6MWT. Thus far,
published clinical studies have reported benefit for only mutations
where the reading frame is restored by skipping exon 51 [Cirak et
al., Lancet, 378: 595-605 (2011) and Goemans et al., New Engl. J.
Med. 364: 1513-1522 (2011)]. In the only report of a double blind,
randomized treatment trial promising results were demonstrated with
eteplirsen, a phosphorodiamidate morpholino oligomer (PMO) [Mendell
et al., Annals Neurology, 74(5): 637-647 (2013)]. In all of these
exon-skipping trials, the common denominator of findings has been a
plateau in walking ability after an initial modest improvement.
Another exon-skipping article is Greer et al., Molecular
Therapy--Nucleic Acids, 3: 3155 (2014).
[0007] See also, U.S. Patent Application Publication Nos.
2012/0077860 published Mar. 29, 2012; 2013/0072541 published Mar.
21, 2013; and 2013/0045538 published Feb. 21, 2013.
[0008] In contrast to the deletion mutations, DMD exon duplications
account for around 5% of disease-causing mutations in unbiased
samples of dystrophinopathy patients [Dent et al., Am. J. Med.
Genet., 134(3): 295-298 (2005)], although in some catalogues of
mutations the number of duplications is higher [including that
published by the United Dystrophinopathy Project in Flanigan et
al., Hum. Mutat., 30(12): 1657-1666 (2009), in which it was
11%].
[0009] Mutations in the DMD gene result in either the more severe
DMD or the milder Becker muscular dystrophy (BMD). The phenotype
generally depends upon whether the mutation results in the complete
absence of the protein product dystrophin (in DMD) or preserves a
reading frame that allows translation of a partially functional
dystrophin protein (in BMD) [Monaco, Trends in Biochemical
Sciences, 14: 412-415 (1989)]. We previously identified a
particular BMD founder allele (c.9T>G; p.Trp3X) that did not
follow this reading frame rule [Flanigan et al., Neuromuscular
Disorders: NMD, 19: 743-748 (2009) and Flanigan et al., Human
Mutation, 30: 1657-1666 (2009)]. Although this nonsense mutation is
predicted to result in no protein translation, muscle biopsy
revealed significant amounts (.about.21%) of dystrophin expression
of minimally decreased size and the clinical phenotype is one of a
very mild dystrophinopathy [Flanigan et al., Neuromuscular
Disorders: NMD, 19: 743-748 (2009)]. In cellulo and in vitro
translation studies demonstrated that in p.Trp3X patients
translation is initiated from AUGs in exon 6, suggesting alternate
translation initiation as a mechanism of phenotypic amelioration
[Gurvich et al., Human Mutation, 30: 633-640 (2009)]. Noting that
most truncating mutations reported in 5' exons were in fact
associated with BMD rather than DMD, we proposed that altered
translation initiation may be a general mechanism of phenotypic
rescue for 5' mutations in this gene, a prediction supported by
subsequent reports [Witting and Vissing, Neuromuscular Disorders:
NMD, 23: 25-28 (2013) and Flanigan et al., Neuromuscular Disorders:
NMD, 23: 192 (2013)]. The canonical actin-binding domain 1 (ABD1)
was previously proposed to be essential for protein function
[Gimona et al., FEBS Letters, 513: 98-106 (2002).
[0010] Translation initiation is commonly understood to occur by
cap-dependent initiation. Internal ribosome entry sites (IRESs) are
RNA regulatory sequences that govern cap-independent translation
initiation in eukaryotic cells, which is activated when
cap-dependent translation is compromised (e.g., during cell
stress). Ribosomes are recruited directly to these IRESs on the
mRNA and can then continue scanning in a 5' to 3' direction for
alternative initiation codons. They were first described in
viruses, and among the earliest characterized was the
encephalomyocarditis virus (EMCV) IRES. Almost 85 cellular IRESs
have been described to date and are mainly located in 5'UTR
regions; for example, the 5'UTR of utrophin A, an autosomal
homologue of dystrophin, contains an IRES that is both particularly
active in regenerating muscle and inducible by exposure to
glucocorticoid (the mainstay of therapy for DMD) [Miura et al., J.
Biol. Chem., 280: 32997-33005 (2005) and Miura et al., PloS One, 3:
e2309 (2008)]. However, other eukaryotic IRESs have been described
within coding sequences, and some have also been implicated in the
modulation of pathology. These include an IRES in the APC gene
linked to a mild version of familial adenomatous polyposis coli in
which patients with certain 5' mutations still produce a partially
functional protein through the use of a downstream initiation
codon.
[0011] Adeno-associated virus (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).
There are multiple serotypes of AAV. The nucleotide sequences of
the genomes of the AAV serotypes are known. 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 (see also U.S. Pat. Nos.
7,282,199 and 7,790,449 relating to AAV-8); 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). The
sequence of the AAV rh.74 genome is provided herein. Cis-acting
sequences directing viral DNA replication (rep),
encapsidation/packaging and host cell chromosome integration are
contained within the AAV 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).
[0012] 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. 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 (56o 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.
[0013] There remains a need in the art for treatments for muscular
dystrophies including DMD and BMD.
SUMMARY
[0014] The present disclosure contemplates methods and products for
preventing disease, delaying the progression of disease, and/or
treating patients with one or more 5' mutations of the DMD gene.
The methods are based on the identification of a
glucocorticoid-inducible IRES in exon 5 of the DMD gene, the
activation of which can generate a functional N-terminally
truncated dystrophin isoform
[0015] The disclosure contemplates methods of ameliorating Duchenne
Muscular Dystrophy or Becker Muscular Dystrophy in a patient with a
5' mutation in the DMD gene comprising the step of administering a
DMD exon 5 IRES-activating oligomer construct to the patient,
wherein the patient does not have a DMD exon 2 duplication.
[0016] In some embodiments of the methods, the DMD exon 5
IRES-activating oligomer construct targets one of the following
portions of exon 2 of the DMD gene:
TABLE-US-00001 (SEQ ID NO: 1) 5' TCAAAAGAAAACATTCACAAAATGGGTA 3',
(SEQ ID NO: 2) 5' GCACAATTTTCTAAGGTAAGAAT 3', (SEQ ID NO: 3) 5'
TAGATGAAAGAGAAGATGTTCAAAAGAAAAC 3', or (SEQ ID NO: 4) 5'
TAGATGAAAGAGAAGATGTTC 3'.
[0017] In some embodiments of the methods, the DMD exon 5
IRES-activating oligomer construct is a U7snRNA polynucleotide
construct in the genome of a recombinant adeno-associated virus. In
some of these embodiments, the genome of the recombinant
adeno-associated virus lacks adeno-associated virus rep and cap
DNA. In some of these embodiments, the virus genome is a
self-complementary genome. In some of these embodiments the
recombinant adeno-associated virus is a recombinant AAV1 virus, a
recombinant AAV6 virus, a recombinant AAV9 virus or a recombinant
AAV rh74 virus. In some embodiments, the U7snRNA polynucleotide
construct comprises: the U7-B antisense polynucleotide
TACCCATTTTGCGAATGTTTTCTTTTGA (SEQ ID NO: 5), the U7-C antisense
polynucleotide ATTCTTACCTTAGAAAATTGTGC (SEQ ID NO: 6), the U7-AL
antisense polynucleotide GTTTTCTTTTGAAGATCTTCTCTTTCATCTA (SEQ ID
NO: 7), or the U7-AS antisense polynucleotide GAAGATCTTCTCTTTCATCTA
(SEQ ID NO: 8).
[0018] In some embodiments of the methods, the DMD exon 5
IRES-activating oligomer construct is an antisense oligomer. In
some embodiments, the antisense oligomer is an exon 2-targeting
antisense oligomer: B antisense oligomer
UACCCAUUUUGCGAAUGUUUUCUUUUGA (SEQ ID NO: 9), C antisense oligomer
AUUCUUACCUUAGAAAAUUGUGC (SEQ ID NO: 10), AL antisense oligomer
GUUUUCUUUUGAACAUCUUCUCUUUCAUCUA (SEQ ID NO: 11) or AS antisense
oligomer GAACAUCUUCUCUUUCAUCUA (SEQ ID NO:12). In some of these
embodiments, the exon 2-targeting antisense oligomer: is a
phosphorodiamidate morpholino oligomer (PMO), is a cell penetrating
peptide-conjugated PMO (PPMO), is a PMO internalizing peptide
(PIP), comprises tricyclo-DNA (tcDNA) or comprises
2'O-methyl-phosphorothioate modifications.
[0019] In some embodiments of the methods, the progression of a
dystrophic pathology is inhibited in the patient.
[0020] In some embodiments of the methods, muscle function is
improved in the patient. The improvement in muscle function can be
an improvement in muscle strength or an improvement in stability in
standing and walking.
[0021] In some embodiments, the contemplated methods further
comprise administering a glucocorticoid to the patient.
[0022] The disclosure contemplates a recombinant adeno-associated
virus (AAV) comprising a DMD exon 5 IRES-activating oligomer
construct, wherein the DMD exon 5 IRES-activating oligomer
construct is a U7snRNA polynucleotide construct comprising:
TABLE-US-00002 U7-B antisense sequence (SEQ ID NO: 5)
TACCCATTTTGCGAATGTTTTCTTTTGA, U7-C antisense sequence (SEQ ID NO:
6) ATTCTTACCTTAGAAAATTGTGC, U7-AL antisense polynucleotide (SEQ ID
NO: 7) GTTTTCTTTTGAAGATCTTCTCTTTCATCTA, or U7-AS antisense
polynucleotide (SEQ ID NO: 8) GAAGATCTTCTCTTTCATCTA.
In some embodiments, the genome of the recombinant AAV lacks AAV
rep and cap DNA. In some embodiments, the recombinant AAV genome is
a self-complementary genome. In some embodiments, the recombinant
adeno-associated virus is a recombinant AAV1 virus, a recombinant
AAV6 virus, a recombinant AAV9 virus or a recombinant AAV rh74
virus. In some embodiments, the self-complementary genome comprises
the DMD exon 5 IRES-activating U7 snRNA polynucleotide construct
U7_ACCA (FIG. 15A shows the genome insert 3' to 5' while FIG. 15B
shows the reverse complement of the sequence of FIG. 15A).
[0023] The disclosure contemplates a DMD exon 5 IRES-activating
oligomer construct, wherein the DMD exon 5 IRES-activating oligomer
construct is an exon 2-targeting antisense oligomer: B antisense
oligomer UACCCAUUUUGCGAAUGUUUUCUUUUGA (SEQ ID NO: 9), C antisense
oligomer AUUCUUACCUUAGAAAAUUGUGC (SEQ ID NO: 10), AL antisense
oligomer GUUUUCUUUUGAACAUCUUCUCUUUCAUCUA (SEQ ID NO: 11) or AS
antisense oligomer GAACAUCUUCUCUUUCAUCUA (SEQ ID NO:12). In some
embodiments, the exon 2-targeting antisense oligomer: is a
phosphorodiamidate morpholino oligomer (PMO), is a cell penetrating
peptide-conjugated PMO (PPMO), is a PMO internalizing peptide
(PIP), comprises tricyclo-DNA (tcDNA) or comprises
2'O-methyl-phosphorothioate modifications.
BRIEF DESCRIPTION OF THE DRAWING
[0024] FIGS. 1A-1F. Human biopsy samples corroborate translation
from exon 6. FIG. 1A shows immunoblot analysis of muscle from an
asymptomatic individual with a deletion of exon 2 (DEL2) resulting
in a frameshift and premature stop codon (p.Tyr11PhefsX7)
demonstrates expression of dystrophin of minimally decreased size.
Antibodies: NCL-DYS1 (rod domain), NCL-DYS2 (C-terminal). FIG. 1B
shows sequences from mass spectrometric analysis of dystrophin
peptides from muscle biopsy of the deletion exon 2 individual (SEQ
ID NO: 28) results in the identification of no peptides encoded
prior to M124 (in exon 6), whereas peptides encoded within exon 2,
3, 4 were readily identified in control muscle (Control) (SEQ ID
NO: 29). The control peptides of FIG. 1B are found at positions
24-61, 83-104, 125-141, and 196-226 of the dystrophin reference
sequence, i.e., UniProt accession number P11532, set out in SEQ ID
NO: 27. FIG. 1C shows immunoblot analysis of dystrophin expression
of muscle from a BMD patient with a truncating frameshift (FS)
mutation in exon 2 (c.40_41del), from a normal control (WT), and
from a DMD patient with a duplication of exon 2 (DUP2). In the
presence of the premature stop codon induced by the frameshifting
mutation, a dystrophin protein of diminished size and amount can be
detected using a C-terminal antibody (PA1-21011, Thermo, Inc.; red)
but not using an antibody detected to epitopes encoded within exon
1 (Manex1A, green). In contrast, dystrophin is entirely absent in
the Dup2 patient. FIG. 1D shows ribosome profiling data was used to
compute a translation efficiency (TE) metric for each of the 1000
most abundant transcripts (by mRNA mass) from patient FS
(c.40_41del) and normal control muscle. TE value for each gene was
calculated from the normalized number of ribosome footprint
sequence reads divided by the number of RNA-Seq reads mapped within
the coding (CDS) sequence. The rank transcript abundance of the top
1000 genes was computed from the total number of mapped reads per
transcript. The subset of genes classified as `sarcomeric` by Gene
Ontology annotation are colored red and the location of the DMD
gene is circled. FIG. 1E shows RNA-Seq read depth from muscle total
RNA mapped to the 5' region of the DMD gene (hg19,
chrX:32,737,599-33,487,390). Read depth for Dp427m exons 1 through
7 was truncated at 40 reads per nucleotide; the exonic read depth
ranged from 67 to 91 (FS, c.40_41del) and 58 to 89 (normal) reads
per nucleotide. FIG. 1F shows ribosome footprints mapped to the 5'
region (nt. 1 to 1500) of the Dp427m (NM_004006.2) transcript. The
locations of the exon 1 Dp427m start codon and the c.40_41del
mutations are shown, with the short ORF (p.Glu14Argfs*17) as the
first CDS segment (green) separated from the remainder of the CDS
(green) beginning at the exon 6 alternate AUG (green) initiation
codons. Asterisks show the locations of the 9 out-of-frame AUG
codons in exons 1 through 5.
[0025] FIGS. 2A-2F. Dystrophin exon 5 can induce cap-independent
translation. (FIG. 2A) Induction of translation of the downstream
(FLuc) cistron in an in vitro transcription/translation system
(rabbit reticulocyte lysate [RRL], left) and following transfection
into C2C12 cells (right) in a dicistronic dual luciferase reporter.
Results are expressed as the ratio of Firefly:Renilla luciferase
(F/L), and normalized to the empty vector (set as 1). (FIG. 2B)
Formaldehyde electrophoresis of the T7 transcription products used
in the RRL assay confirms RNA integrity. (FIG. 2C) Mapping of the
exon 5 IRES: dicistronic mapping constructs (left) used to map
cap-independent translation activity. In each case, numbering is
based upon the Dp427m cDNA sequence; the full-length construct
pRdEF+4+369 (exon 1 to 6) begins at the +4 position to exclude the
native AUG initiation codon. Exon 6 was preserved, and AUG2 (M124)
and AUG3 (M128) were cloned in-frame with the downstream FLuc
reporter. FLuc luminescence (cap-independent) is expressed as a
percentage of RLuc luminescence (cap-dependent) after transfection
of the dicistronic constructs in C2C12 cells (right). All results
were normalized to the exon 6 alone vector, the FLuc:RLuc ratio of
which was set at a value of 1. Statistical analysis was performed
using a Kruskal-Wallis test, comparing the results for each
construct versus the exon 6 alone vector, which resulted in levels
of expression comparable to an entirely empty vector (p>0.99).
Significantly increased translation of the downstream reporter was
demonstrated with the exon 1 to 6 (p<0.0001), exon 2 to 6
(p=0.0175), exon 3 to 6 (p=0.0009), exon 3* to 6 (p=0.0078), exon 4
to 6 (p=0.0078), or exon 5 to 6 (p=0.0019). In contrast, deletion
of exon 5 (either in whole or in part) resulted in no significant
difference for all three in comparison to exon 6 alone. (FIG. 2D)
RT-PCR products amplified from RNA derived from transfected C2C12
cells, using primers located as depicted as arrows on the scheme in
panel (FIG. 2C), shows no evidence of altered splicing. (FIG. 2E)
Northern blot analysis of C2C12 transfected cells using a P.sup.32
radiolabeled probe targeting the FLuc cistron shows no evidence of
RNA strand breakage to explain the increased signal in the presence
of dystrophin exons 1-5. (A non-specific band of approximately 3 kb
is detected in every transfection condition, including the empty
vector, and is therefore unrelated to the increase in FLuc or EMCV
signals compared to empty vector. (FIG. 2F) IRES activity is
abrogated by the presence of a duplicated exon 2, but not by a
deletion of exon 2. Error bars represent s.d.
[0026] FIGS. 3A-3D. Out-of-frame exon-skipping stimulates IRES
activity in patient-derived cell lines. (FIG. 3A) Schematic
representation of the human DMD exon 1-10 reading frame (blue) and
5'UTR (red). Blue numbers above each exon indicate cDNA positions;
red numbers at the base of each exon indicate the amino acid
position. The canonical actin binding domain 1 is represented,
along with the predicted (via ScanProsite) CH and ABS domains.
(FIG. 3B) Schematic representation of exon 2 (SEQ ID NO: 30). The
selected targeted sequences are indicated below, affecting either
splice acceptor (S.A.), splice donor (S.D.), or exon splice
enhancer (E.S.E.) sequences (as predicted using Human Splicing
Finder or ESE finder 3.0). (FIG. 3C) Two copies each of U7-C and
U7-AL were cloned into the same AAV plasmid, as they were the most
efficient in skipping exon 2 (see FIGS. 10A-10B). The resulting
construct is referred to as U7-ACCA. RT-PCR results after infection
of U7-ACCA vector (1E11 vg) or H2A antisense oligonucleotide (AON
H2A) into either wt or duplicated exon 2 FibroMyoD (FM) cells.
These are derived from patient fibroblast lines stably infected
with hTERT and a tet-inducible MyoD lentivirus; treatment with
doxycycline results in transdifferentiation into a myogenic
lineage, with subsequent dystrophin mRNA expression. (FIG. 3D)
Immunoblot performed 14 days after infection of FM cells with
U7-ACCA shows expression of the smaller N-truncated dystrophin
protein (arrow). Antibody: C-terminal dystrophin (PA1-21011,
Thermo, Inc.). A smaller band of approximately 390 kDa is detected
in every lane, but is non-specific (as seen in the untreated
sample) and does not correspond to the IRES-driven isoform. (The
image was assembled for clarity; complete images are included as
FIGS. 11A-11B).
[0027] FIGS. 4A-4F. Intramuscular delivery of U7-ACCA results in
significant N-truncated dystrophin expression in Dup2 mice,
restoring localization of dystrophin-associated proteins. (FIGS.
4A-4B) RT-PCR results performed 4 weeks after TA intramuscular
injection of 1e11vg U7-ACCA show nearly complete skipping of both
copies of exon 2 in both (FIG. 4A) Dup2 and (FIG. 4B) control BI6
mice (PDN: methylprednisolone 1 mg/kg/day intraperitoneal). In Dup2
animals (FIG. 4A), quantification revealed the Dup2 transcript to
be 5.1% of total, whereas the wild type was 8.6% and the Del2
transcript was 86.3%. In wild type BI6 animals (FIG. 4B), the wild
type transcript was 14.2% and Del2 transcript was 85.8%. (FIG. 4C)
RNA-Seq read depth using a tibialis anterior muscle total RNA
library from Dup2 U7-ACCA treated (upper) and Dup2 untreated
(lower) mice, mapped to the 5' region of the mouse Dmd gene (mm9,
chrX:80,150,000-81,050,000). (FIG. 4D) Immunoblot performed a month
after infection shows significant expression of the N-truncated
isoform (asterisk) in both Dup2 and control BI6 mice. The protein
induced in BI6 males injected with U7-ACCA is of the same size as
that expressed in the Dup2 treated animals, confirming the size
difference between this protein and the full-length isoform.
(C-terminal antibody: PA1-21011,Thermo, Inc). Coomassie staining of
the same samples demonstrates no difference in migration behavior.
(FIG. 4E) Immunofluorescent staining of dystrophin (C-terminal
antibody: PA1-21011,Thermo, Inc), .beta.-dystroglycan (Beta-DG;
MANDAG2); and neuronal nitric oxide synthetase (nNOS; sc-648; Santa
Cruz). (FIG. 4F) Evans blue (EBD) protection assay in Dup2 mice one
month after intramuscular injection with 1e11vg shows stabilization
of muscle membranes. Evans blue uptake (red) is seen only in fibers
without positive dystrophin expression (green, C-terminal antibody:
PA1-21011,Thermo, Inc). (Dup2 mice used for these panel; n=5)
[0028] FIGS. 5A-5F. Glucorticoid activation of the dystrophin IRES.
(FIG. 5A) Dual luciferase assay performed on lysates from C2C12
cells transfected with the pRDEF vector carrying the exon 5-6 IRES
construct. Methylprednisolone (PDN) increases IRES activity in a
dose-dependent fashion. Error bars representate s.d. (FIG. 5B) Dup2
FM cells treated with both U7-ACCA and PDN (6.4 .mu.M) show
increased dystrophin expression. The image intensity for the
wild-type lane was lowered to allow identification of bands.
MHC=myosin heavy chain (loading control). (FIG. 5C) Representative
immunoblot demonstrates increased expression of dystrophin in Dup2
mice injected with 1e11vg U7-ACCA after treatment with PDN (1
mg/kg/day). % Dys: intensity ratio of dystrophin:.alpha.-actinin,
normalized to control muscle. (FIG. 5D) Quantification of the
dystrophin/.alpha.-actinin signal in U7-ACCA treated muscles in the
presence or absence of PDN. Five animals treated with U7-ACCA in
the tibialis anterior muscles were injected with either PBS or PDN
(1 mg/kg/day). Immunoblot was performed on each muscle in
duplicate, and the signals for both dystrophin and .alpha.-actinin
from the resulting 5 lanes were quantified using ImageJ.
Significantly more dystrophin was present in muscles from
PDN-treated animals (P=0.0159, two tailed Mann-Whitney test, error
bars represent s.d.). (FIG. 5E) Representative western blot
demonstrates an increased level of utrophin in Dup2 compared to BI6
mouse. Treatment with PDN (1 mg/kg/day) does not increase
expression of utrophin. (FIG. 5F) Quantification of the
utrophin/a-actinin signal in treated muscles in the presence or
absence of U7-ACCA and PDN. Five animals treated with U7-ACCA in
the tibialis anterior muscles were injected with either PBS or PDN
(1 mg/kg/day). The signals for both utrophin and .alpha.-actinin
from the resulting 5 lanes were quantified using ImageJ. No
significance was detected between the four (Kruskal-Wallis, error
bars represent s.d.).
[0029] FIGS. 6A-6E. Expression of the IRES-driven isoform improves
muscle membrane integrity and protects Dup2 muscle from
contraction-induced damage. Dup2 tibialis anterior muscles were
treated by intramuscular injection of 5e11vg U7-ACCA alone or with
methylprednisolone (PDN: 1 mg/kg/day intraperitoneal) and analyzed
at 4 weeks post-injection. (FIG. 6A) Central nucleation in
untreated Dup2 animals (73.0.+-.1.6% of myofibers) was
significantly reduced by treatment with U7-ACCA alone
(65.2.+-.2.2%, ***p=0.0002). No significant difference was observed
between Dup2 and Dup2+PDN. (FIG. 6B) The percentage of Evans blue
dye (EBD)-positive fibers in untreated Dup2 muscle (14.7.+-.6.6%;
one outlier is represented as a dot) is reduced by treatment with
U7-ACCA alone (2.8.+-.1.8%, *p=0.0310) or in combination with
prednisone (0.65.+-.0.5%, ***p=0.0005). No significant difference
was observed betweenDup2 and Dup2+PDN. EBD-positive fibers were
quantified as a percent out of a total of 5,000 fibers counted per
animal. (FIG. 6C) Normalized maximum hindlimb (Norm max HL) grip
strength in untreated Dup2 mice (2.22.+-.0.26 kg force/kg mass of
animal, or kgf/kg) is significantly lower than B16 (3.36.+-.0.37
kgf/kg, ***p<0.0001). Significantly improved strength follows
treatment with either U7-ACCA alone (3.35.+-.0.32 kgf/kg,
***p<0.0001) or in combination with prednisone (3.17.+-.0.28
kgf/kg, ***p=0.0002), both of which restore strength to a level not
significantly different from that seen in BI6. No significant
difference was observed between Dup2 and Dup2+PDN. (FIG. 6D)
Normalized specific force following tetanic contraction in
untreated Dup2 animals (170.9.+-.14.3 mN/mm.sup.2) is significantly
less than in BI6 (274.0.+-.12.1 mN/mm.sup.2,**p=0.0061).
Significantly increased force follows treatment with U7-ACCA alone
(236.04.+-.19.4 mN/mm.sup.2, *p=0.0350) or with prednisone
(251.2.+-.10.4 mN/mm.sup.2, **p=0.0025), both of which restore
specific force to a level not significantly different from that
seen in BI6. No significant difference was observed between Dup2
and Dup2+PDN. (FIG. 6E) Treatment significantly protects Dup2
muscle from loss of force following repetitive eccentric
contractions. Two-way analysis of variance demonstrates significant
improvement in decay curves versus untreated Dup2 (*p<0.05 and
***p<0.001), and Bonferroni post-hoc analysis demonstrates that
the combination of both treatment showed no significant difference
from control BI6 in force retention following contractions #3 to
#10 (*p<0.05 and ***p<0.001). No significant difference was
observed between Dup2 and Dup2+PDN (p<0.99). Two way ANOVA
demonstrates significant difference between Dup2+U7 and Dup2+U7+PDN
(*p<0.05) (FIGS. 6A-6C) n=4 animals studied for each condition
and when applied 2000 fibers count/mouse, two tailed
Kruskal-Wallis, error bar as s.d.); (FIGS. 6D-6E) n=5 muscles from
at least 3 animals, error bar as s.e.m.
[0030] FIGS. 7A-7G. Mutational analysis in the deletion exon 2
asymptomatic boy. (FIG. 7A) H&E-stained muscle section from the
patient with deletion of exon 2 (DEL2) reveals an absence of
dystrophic features. (FIG. 7B) Immunohistochemical staining of
muscle sections from the same patient using NCL-DYS3 antibody
(exons 10-12). Manex1A staining (exon 1 specific) was not performed
at that time, and tissue is no longer available. (FIG. 7C) CGH
profile of the genomic context (top panel) and of the entire X
chromosome (bottom panel) of the 12.983 bp deletion including exon
2 (shown in the overlay track at the bottom of the top panel).
(FIG. 7D) Alignment of the sequenced junction with the reference
genome sequence (NCBI hg18) (SEQ ID NOs: 31-33, respectively).
Proximal and distal reference sequences are colored differently and
the junction is in black. Vertical bars between the sequences
represent sequence homology. A microhomology of 5 bp (CTGTG, shown
a box) is found at the junction between the distal and proximal
sequences, characteristic for non-homologous end joining. (FIG. 7E)
Genomic sequences of the breakpoint with the microhomology sequence
underlined in blue (SEQ ID NO: 34). (FIGS. 7F-7G) RT-PCR and
sequencing results confirm the deletion of exon 2 at RNA level.
[0031] FIGS. 8A-8D. Immunofluorescent analysis of muscle from the
frameshift (c.40_41del) patient. (FIG. 8A) Immunostaining using a
dystrophin antibody (Abcam 15277, C-terminal) shows dystrophin at
the sarcolemmal membrane in both control and patient muscle
biopsies whereas Manex1A staining is absent in the patient sample,
confirming the lack of expression of the epitope encoded by exon 1.
(FIG. 8B) Ribosome Profiling of the DMD muscle-isoform transcript.
The normalized average reads (read depth per nt. versus the average
read depth on NM_004006) for RNA-Seq reads are plotted every 25
nucleotides using an averaged normalized average read depth per
nucleotide calculated from a 500 bp. sliding window. Reads from
patient FS(c.40_41del) are shown in red and from control muscle in
grey, with regression lines shown for each set of averages. (FIG.
8C) as in (FIG. 8B) except using RPF-Seq reads, with the linear
regression line calculated for the CDS region only. (FIG. 8D) The
exon structure of the NM_004006 transcript is drawn to the same
scale as the x-axis from (FIG. 8B) and (FIG. 8C). The arrow
indicates the location of the alternate translation initiation
sites in exon 6. Since the experiment used total RNA, the RNA-Seq
reads mapping to NM_004006 are derived from both nascent and mature
transcripts. The 5' to 3' gradient of RNA-Seq reads shown in (FIG.
8B) agrees with an original estimate from human skeletal muscle of
the relative excess of 5' exons in nascent RNA due to the transit
time (.about.16 hrs.) for RNA polymerase to transcribe across the
.about.2.2 Mb of chr. X region containing the 79 DMD exons of the
muscle isoform. Regression analysis of the RPF-Seq reads does not
indicate a 5' to 3' gradient, inferring that ribosomes are equally
distributed across the length of the mature transcript.
[0032] FIGS. 9A-9C. The dystrophin RES is not ubiquitously active.
(FIG. 9A) Dual luciferase assays demonstrate activation in two
myogenic cell lines (C2C12, and a commercial human skeletal muscle
myoblast line [hSKMM]), but not in HEK209K cells, suggesting
preferential activation in cells of a myogenic lineage. (FIG. 9B)
Northern blot from transfected C2C12 and 293k using a probe against
Firefly luciferase demonstrates the presence of the transcript as
well as the previously described (FIGS. 2A-2F) nonspecific band.
Notably, this band is present in all conditions, including
following transfection with the exon 6 alone construct, and
therefore is unrelated to the the fold change seen with exon 5
containing constructs. (FIG. 9C) RT-PCR products amplified from RNA
derived from transfected 293k cells shows no evidence of altered
splicing. Error bars represent s.d.
[0033] FIGS. 10A-10B. Optimization of AAV mediated U7
exon-skipping. (FIG. 10A)
Four different target sequences (AS, AL, B or C) were cloned into
AAV under the control of U7. Infection of these AAV either alone
(FIG. 10A) or in combination (FIG. 10B) were performed in both
control and duplicated exon 2 patient derived-FibroMyoD. 3 days
post AAV infection, RT-PCR results demonstrated that in U7-C is
able to induce exon-skipping in both control and duplicated exon 2
patient FibroMyoD whereas U7-AL is only able to induce skipping in
the patient cell lines. Two copies constructs U7-C and U7-AL were
cloned into a same AAV plasmid (U7-ACCA). (c) Transfection of a
Dup2 patient's cultured MyoD transformed fibroblasts and primary
myoblasts, using an AON (AONH2A) which targets an internal exon 2
sequence, gave similar results, but at lower efficiency than
U7-mediated skipping.
[0034] FIGS. 11A-11B. Western blot from patient-derived cell lines.
(FIG. 11A) The original western blot from FIG. 3d seen in two
different imaging intensities, low (upper panel) and high (lower
panel). Lane 1 from the upper panel and lanes 2 and 4 from the
lower panel were used to assemble FIG. 3d. FM=FibroMyoD derived
control cell lines; FM Dup=FibroMyoD patient-derived cell lines
from an exon 2 duplicated patient; FS=protein from muscle biopsy of
c.40_41del. (FIG. 11B) Coomassie staining of the same samples as
seen in FIG. 4c demonstrates no significant difference in migration
behavior.
[0035] FIGS. 12A-12B. Glucocorticoid increases IRES activity but
cannot force its activation. (FIG. 12A) Dual luciferase assay
results after transfection of 3 constructs in 293k treated with
glucocorticoid demonstrate that IRES activity cannot be induced by
this compound. Error bars represent s.d. (FIG. 12B) Genomic qPCR of
AAV copy number confirm that increase of dystrophin level detected
by western blot in PDN treated mice is not due an increased number
of AAV vector in the PDN treated animals. N=4 animals per group.
Error bars represent s.d.
[0036] FIG. 13 is the rh74 genome sequence (SEQ ID NO: 14) wherein
nucleotides 210-2147 are the Rep 78 gene open reading frame,
882-208 are the Rep52 open reading frame, 2079-2081 are the Rep78
stop, 2145-2147 are the Rep78 stop, 1797-1800 are a splice donor
site, 2094-2097 are a splice acceptor site, 2121-2124 are a splice
acceptor site, 174-181 are the p5 promoter+1 predicted, 145-151 are
the p5 TATA box, 758-761 are the p19 promoter+1 predicted, 732-738
are the p19 TATA box, 1711-1716 are the p40 TATA box, 2098-4314 are
the VP1 Cap gene open reading frame, 2509-2511 are the VP2 start,
2707-2709 are the VP3 start and 4328-4333 are a polyA signal.
[0037] FIG. 14 shows a map of a plasmid with an AAV genome insert
of an exemplary exon 2-targeted U7snRNA.
[0038] FIG. 15A shows the AAV genome insert (3' to 5') (SEQ ID NO:
15 is the same sequence in the 5' to 3' direction) of the plasmid
of FIG. 14. FIG. 15B shows the reverse complement (SEQ ID NO: 26)
of the sequence in FIG. 15A.
[0039] U7 encode for a U7snRNP that share some features with
spliceosomal snRNPs. Although it is not involved in pre-mRNA
splicing, it processes the 3' ends of histone mRNA (Muer and
Schumperli 1997; Dominski and Marzluff 1999). Nucleotides 1-113 of
SEQ ID NO: 15 correspond to the 3' ITR, nucleotides 114-220 of SEQ
ID NO: 15 correspond to the 3' untranslated region (UTR) (reverse
orientation sequence). Nucleotides 221-251 of SEQ ID NO: 15
correspond to SmOPT (reverse orientation sequence). SmOPT is a
modification of the original Sm-binding site of U7 snRNA with a
consensus sequence derived from spliceosomal snRNAs (Grimm et al.
1993; Stefanovic et al. 1995a). Nucleotides 252-262: of SEQ ID NO:
15 correspond to a loop (reverse orientation sequence). Nucleotides
263-295 correspond to U7-Along (reverse orientation sequence),
which is an antisense sequence that targets the acceptor site of
exon 2. Nucleotides 296-551 of SEQ ID NO: 15 correspond to U7
(reverse orientation sequence), nucleotides 558-664 of SEQ ID NO:
15 correspond to 3' UTR (reverse orientation sequence), nucleotides
665-695 of SEQ ID NO: 15 correspond to SmOPT (reverse orientation
sequence), and nucleotides 696-706 of SEQ ID NO: 15 correspond to a
loop (reverse orientation sequence). Nucleotides 707-731 of SEQ ID
NO: 15 correspond to U7-C (reverse orientation sequence), which is
an antisense sequence that targets the donor site of exon 2.
Nucleotides 732-987 of SEQ ID NO: 15 correspond to U7 (reverse
orientation sequence), nucleotides 994-1100 of SEQ ID NO: 15
correspond to 3' UTR (reverse orientation sequence), nucleotides
1111-1131 of SEQ ID NO: 15 correspond to SmOPT (reverse orientation
sequence), nucleotides 1132-1142 of SEQ ID NO: 15 correspond to a
loop (reverse orientation sequence), nucleotides 1143-1167 of SEQ
ID NO: 15 correspond to U7-C (reverse orientation sequence),
nucleotides 1168-1423 of SEQ ID NO: 15 correspond to U7 (reverse
orientation sequence), nucleotides 1430-1536 of SEQ ID NO: 15
correspond to 3' UTR (reverse orientation sequence), nucleotides
1537-1567 of SEQ ID NO: 15 correspond to SmOPT (reverse orientation
sequence), nucleotides 1568-1578 of SEQ ID NO: 15 correspond to a
loop (reverse orientation sequence), nucleotides 1579-1611 of SEQ
ID NO: 15 correspond to U7-Along (reverse orientation sequence),
nucleotides 1612-1867 of SEQ ID NO: 15 correspond to U7 (reverse
orientation sequence) and nucleotides 1920-2052 of of SEQ ID NO: 15
correspond to the ITR.
[0040] FIG. 16 shows a schematic of a vector used in creation of a
mdx.sup.dup2 (Dup2) mouse.
[0041] FIGS. 17A-17F shows (FIG. 17A) RT-PCR performed on 5
different Dup2 mouse muscles one month after tail vein injection of
AAV9.U7-ACCA (3.3E12 vg/kg). As demonstrated by the presence of
multiple transcripts (here labeled Dup2, wt, and Del2), U7-ACCA
treatment is able to force skipping of one or both copies of exon 2
in all muscles tested. (TA: tibialis anterior; Gas: gastrocnemius;
: heart; Tri: triceps; dia: diaphragm.) (FIG. 17B) Western blot
performed on 5 different muscles one month after injection
demonstrates the presence of dystrophin in all tested muscles.
(FIG. 17C) Immunostaining of dystrophin on the same samples
confirms dystrophin expression and its proper localization at the
sarcolemma. (FIG. 17D) Evaluation of both forelimb and hindlimb
grip strength demonstrates a complete correction of grip strength
in Dup2 animals treated with AAV9.U7-ACCA. (FIG. 17E) Normalized
specific and total forces following tetanic contraction show
improvement in muscle force in comparison to untreated Dup2
animals. (FIG. 17F) Cardiac papillary muscles demonstrate
improvements in length-dependent force generation in treated
animals.
[0042] FIGS. 18A-18G. (FIG. 18A) IM study design. Escalating doses
of the AAV9.U7snRNA-ACCA vector were delivered to the tibialis
anterior muscle at 2 months, and muscle analyzed at 3 months by
mRNA, protein, and electrophysiology studies. (FIG. 18B)
Quantification of mRNA by RT-PCR at ascending dose levels of IM
injection. Transcripts contain either two (Dup2), one (WT), or zero
copies (.DELTA.2) of exon 2. Expression of the N-truncated
dystrophin following ascending dose levels of IM injection. Protein
expression by (FIG. 18C) immunofluorescence or (FIG. 18D)
immunoblot demonstrates a dose response. (FIG. 18E) Quantification
of the immunoblot suggests maximal protein expression at 3.1E11 vg.
Amelioration of deficits in absolute force (FIG. 18F), specific
force (FIG. 18G), in response to eccentric contraction following IM
injection into the tibialis anterior muscle of 3.1 E11 vg.
[0043] FIGS. 19A-19E. (FIG. 19A) IV study design. Escalating doses
of the AAV9.U7snRNA-ACCA vector were delivered systemically at 2
months, and muscle analyzed at 3 months by mRNA, protein, and
electrophysiology studies. (FIG. 19B) Quantification of mRNA by
RT-PCR at ascending dose levels of IV injection. Transcripts
contain either two (Dup2), one (WT), or zero copies (.DELTA.2) of
no exon 2. (FIG. 19C) Quantification of dystrophin by immunoblot
following IV injection. Expression follows a dose response, with
expression in triceps lagging that in heart and diaphragm. (FIG.
19D) Immunostaining of dystrophin from BI6 and Dup2. (FIG. 19E)
expression following IV injection. A dose response is seen, with
significant dystrophin expression in the heart and diaphragm at
higher doses.
[0044] FIG. 20. Early injection of AAV9.U7-ACCA prevents the muscle
pathology in the Dup2 mouse. Immunostaining of dystrophin
demonstrates production and localization of N-terminally truncated
dystrophin at the plasma membrane. No centronucleation was observed
following hematoxylin and eosin staining. By 6 months of age,
untreated Dup2 mice typically demonstrate 60% of their fibers with
central nuclei (data not shown).
[0045] FIG. 21. Generation of alternative N-terminally truncated
dystrophins in human cell lines derived from patients carrying
mutations within the first nine exons. RT-PCR results after
skipping of exon 2 using either AAV1.U7-ACCA vector (1.times.10E11
vector genomes) or H2A antisense oligonucleotide (AON H2A) in
various patient cell lines carrying mutation within exon 1 to 4.
This results in approximately 90% of transcript lacking exon 2
(quantification not shown). FM=FibroMyoD cells derived from healthy
human subject. Immunoblot performed 14 d after infection of
FibroMyod cells with AAV1.U7-ACCA shows expression of the
N-terminally trucated dystrophin protein. A smaller band of
approximately 390 kDa is detected in every lane but is nonspecific
(as seen in the untreated sample) and does not correspond to the
IRES-driven isoform. (The image was assembled for clarity, with
wild-type contrast altered to clearly show bands.)
[0046] FIGS. 22A-22C. Expression of the N-truncated dystrophin
following treatment with PPMO antisense oligonucleotide. (FIG. 22A)
transfection in C2C12 mouse myoblasts or (FIG. 22B) intramuscular
injection into Dup2 mouse tibialis anterior muscles of AL-PPMO.
RT-PCR results from treated cells or muscles demonstrate an
efficient skipping of exon 2. (FIG. 22C) immunofluorescence of
dystrophin shows expression of a plasma membrane protein following
intramuscular injection of AL-PPMO antisense oligonucleotide.
DESCRIPTION
[0047] As noted above, the present disclosure contemplates methods
and products for preventing, delaying the progression of, and/or
treating patients with one or more 5' mutations of the DMD gene
that are based on the activation of a glucocorticoid-inducible IRES
in exon 5 of the DMD gene. The activation of the inducible IRES in
exon 5 of the DMD gene generates a functional N-terminally
truncated dystrophin isoform.
[0048] As used herein, a "5' mutation of the DMD gene" is a
mutation within or affecting exon 1, 2, 3 or 4 of the DMD gene. In
the methods of the invention, the patients treated do not have a
DMD exon 2 duplication, but a "mutation affecting exon 1, 2, 3 or
4" as contemplated herein can be a duplication other than a DMD
exon 2 duplication.
[0049] In one aspect, the methods involve using an "DMD exon 5
IRES-activating oligomer construct." As used herein, a DMD exon 5
IRES-activating oligomer construct targets exon 2 to induce altered
splicing that results in the exclusion of exon 2 from the mature
RNA causing a frameshift in the DMD gene reading frame and inducing
utilization of the IRES in exon 5 for translational initiation.
[0050] In some embodiments, the DMD exon 5 IRES-activating oligomer
construct targets one of the following portions (shown 5' to 3') of
exon 2 of the DMD gene.
TABLE-US-00003 B: (SEQ ID NO: 1) TCAAAAGAAAACATTCACAAAATGGGTA (+17
+ 44) C: (SEQ ID NO: 2) GCACAATTTTCTAAGGTAAGAAT (+48 - 8) AL: (SEQ
ID NO: 3) TAGATGAAAGAGAAGATGTTCAAAAGAAAAC (-3 + 28) AS: (SEQ ID NO:
4) TAGATGAAAGAGAAGATGTTC (-3 + 18)
[0051] In some embodiments, a rAAV is used to deliver a U7 small
nuclear RNA polynucleotide construct that is targeted to DMD exon 2
by an antisense polynucleotide. In some embodiments, the U7 small
nuclear RNA is a human U7 small nuclear RNA. In some embodiments,
the polynucleotide construct is inserted in the genome of a rAAV9,
the genome of a rAAV6 or the genome of a rAAVrh74. In some
embodiments, the U7 small nucleotide RNA construct comprises
exemplary targeting antisense polynucleotides including, but not
limited to the following where, for example, the "U7-AL antisense
polynucleotide" is respectively complementary to and targets the
"AL" exon 2 sequence in the preceding paragraph.
TABLE-US-00004 U7-B antisense polynucleotide: (SEQ ID NO: 5)
TACCCATTTTGCGAATGTTTTCTTTTGA U7-C antisense polynucleotide: (SEQ ID
NO: 6) ATTCTTACCTTAGAAAATTGTGC U7-AL antisense polynucleotide: (SEQ
ID NO: 7) GTTTTCTTTTGAAGATCTTCTCTTTCATCTA U7-AS antisense
polynucleotide: (SEQ ID NO: 8) GAAGATCTTCTCTTTCATCTA
[0052] In some embodiments, the DMD exon 5 IRES-activating oligomer
construct is an exon 2-targeting antisense oligomer. In some
embodiments, the antisense oligomers are contemplated to include
modifications compared to the native phosphodiester
oligodeoxynucleotide polymer to limit their nuclease sensitivity.
Contemplated modifications include, but are not limited to,
phosphorodiamidate morpholino oligomers (PPOs), cell penetrating
peptide-conjugated PMOs (PPMOs), PMO internalizing peptides (PIP)
[(Betts et al., Sci. Rep., 5: 8986 (2015)], tricyclo-DNA (tcDNA)
[Goyenvalle et al., Nat. Med., 21: 270-275 (2015)] and
2'O-methyl-phosphorothioate modifications. Exemplary DMD exon 5
IRES-activating oligomer constructs that are exon 2-targeting
antisense oligomers include, but are not limited to, the following
antisense oligomers (shown 5' to 3') where, for example, the "B
antisense oligomer" respectively targets the "B" exon 2 target in
paragraph [0032].
TABLE-US-00005 B antisense oligomer: (SEQ ID NO: 9)
UACCCAUUUUGCGAAUGUUUUCUUUUGA C antisense oligomer: (SEQ ID NO: 10)
AUUCUUACCUUAGAAAAUUGUGC AL antisense oligomer: (SEQ ID NO: 11)
GUUUUCUUUUGAACAUCUUCUCUUUCAUCUA AS antisense oligomer: (SEQ ID NO:
12) GAACAUCUUCUCUUUCAUCUA H2A (+12 + 41): (SEQ ID NO: 13)
CCAUUUUGUGAAUGUUUUCUUUUGAACAUC
[0053] In another aspect, a method of ameliorating a muscular
dystrophy (such as DMD or BMD) in a patient with a 5' mutation of
the DMD gene is provided. In some embodiments, the method comprises
the step of administering a rAAV to the patient, wherein the genome
of the rAAV comprises a DMD exon 5 IRES-activating oligomer
construct. In some embodiments, the method comprises the step of
administering a DMD exon 5 IRES-activating oligomer construct that
is an exon 2-targeting antisense oligomer. In some embodiments, the
patient is also treated with a glucocorticoid.
[0054] In yet another aspect, the invention provides a method of
inhibiting the progression of dystrophic pathology associated with
a muscular dystrophy (such as DMD or BMD). In some embodiments, the
method comprises the step of administering a rAAV to a patient with
a 5' mutation of the DMD gene, wherein the genome of the rAAV
comprises a DMD exon 5 IRES-activating oligomer construct. In some
embodiments, the method comprises the step of administering a DMD
exon 5 IRES-activating oligomer construct that is an exon
2-targeting antisense oligomer. In some embodiments, the patient is
also treated with a glucocorticoid.
[0055] In still another aspect, a method of improving muscle
function in a patient with a 5' mutation of the DMD gene is
provided. In some embodiments, the method comprises the step of
administering a rAAV to the patient, wherein the genome of the rAAV
comprises a DMD exon 5 IRES-activating oligomer construct. In some
embodiments, the method comprises the step of administering a DMD
exon 5 IRES-activating oligomer construct that is an exon
2-targeting antisense oligomer. In some embodiments, the
improvement in muscle function is an improvement in muscle
strength. The improvement in muscle strength is determined by
techniques known in the art such as the maximal voluntary isometric
contraction testing (MVICT). In some instances, the improvement in
muscle function is an improvement in stability in standing and
walking. The improvement in stability strength is determined by
techniques known in the art such as the 6-minute walk test (6MWT)
or timed stair climb. In some embodiments, the patient is also
treated with a glucocorticoid.
[0056] In another aspect, the invention provides a method of
delivering a DMD exon 5 IRES-activating oligomer construct to an
animal (including, but not limited to, a human) with a 5' mutation
of the DMD gene. In some embodiments, the method comprises the step
of a rAAV to the patient, wherein the genome of the rAAV comprises
a DMD exon 5 IRES-activating oligomer construct. In some
embodiments, the method comprises the step of administering a DMD
exon 5 IRES-activating oligomer construct that is an exon
2-targeting antisense oligomer. In some embodiments, the animal is
also treated with a glucocorticoid.
[0057] Cell transduction efficiencies of the methods of the
invention described herein may be at least about 60, about 65,
about 70, about 75, about 80, about 85, about 90 or about 95
percent.
[0058] In some embodiments of the foregoing methods of the
invention, the virus genome is a self-complementary genome. In some
embodiments of the methods, the genome of the rAAV lacks AAV rep
and cap DNA. In some embodiments of the methods, the rAAV is a SC
rAAV U7_ACCA comprising the exemplary genome set out in FIG. 15. In
some embodiments, the rAAV is a rAAV6. In some embodiments, the
rAAV is a rAAV9. In some embodiments the rAAV is a rAAV rh74 (FIG.
13).
[0059] In yet another aspect, the invention provides a rAAV
comprising the AAV rAAV9 capsid and a genome comprising the
exemplary DMD exon 5 IRES-activating U7 snRNA polynucleotide
construct U7_ACCA. In some embodiments, the genome of the rAAV
lacks AAV rep and cap DNA. In some embodiments, the rAAV comprises
a self-complementary genome. In some embodiments of the methods,
the rAAV is a SC rAAV U7_ACCA comprising the exemplary genome is
set out in FIGS. 15A-15B. In some embodiments, the rAAV is a rAAV6.
In some embodiments, the rAAV is a rAAV9. In some embodiments the
rAAV is a rAAV rh74 (FIG. 13).
[0060] Recombinant AAV genomes of the invention comprise one or
more AAV ITRs flanking at least one DMD exon 5 IRES-activating U7
snRNA polynucleotide construct. Genomes with DMD exon 5
IRES-activating U7 snRNA polynucleotide constructs comprising each
of the targeting antisense sequences set out in paragraph [0033]
are specifically contemplated, as well as genomes with DMD exon 5
IRES-activating U7 snRNA polynucleotide constructs comprising each
possible combination of two or more of the targeting antisense
sequences set out in paragraph [0033]. In some embodiments,
including the exemplified embodiments, the U7 snRNA polynucleotide
includes its own promoter. AAV DNA in the rAAV genomes 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, AAV-11 and AAV
rh.74. As noted in the Background section above, the nucleotide
sequences of the genomes of various AAV serotypes are known in the
art. In some embodiments of the invention, the promoter DNAs are
muscle-specific control elements, including, but not limited to,
those derived from the actin and myosin gene families, such as from
the myoD gene family [See Weintraub et al., Science, 251: 761-766
(1991)], the myocyte-specific enhancer binding factor MEF-2
[Cserjesi and Olson, Mol. Cell. Biol., 11: 4854-4862 (1991)],
control elements derived from the human skeletal actin gene [Muscat
et al., Mol. Cell. Biol., 7: 4089-4099 (1987)], the cardiac actin
gene, muscle creatine kinase sequence elements [Johnson et al.,
Mol. Cell. Biol., 9:3393-3399 (1989)] and the murine creatine
kinase enhancer (MCK) element, desmin promoter, control elements
derived from the skeletal fast-twitch troponin C gene, the
slow-twitch cardiac troponin C gene and the slow-twitch troponin I
gene: hypoxia-inducible nuclear factors [Semenza et al., Proc.
Natl. Acad. Sci. USA, 88: 5680-5684 (1991)], steroid-inducible
elements and promoters including the glucocorticoid response
element (GRE) [See Mader and White, Proc. Natl. Acad. Sci. USA, 90:
5603-5607 (1993)], and other control elements.
[0061] DNA plasmids of the invention comprise 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 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, AAV-11 and AAV rh74. Use of cognate components is
specifically contemplated. Production of pseudotyped rAAV is
disclosed in, for example, WO 01/83692 which is incorporated by
reference herein in its entirety.
[0062] 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., Proc. Natl.
Acad. S6. USA, 79:2077-2081 (1982)], addition of synthetic linkers
containing restriction endonuclease cleavage sites [Laughlin et
al., Gene, 23:65-73 (1983)] or by direct, blunt-end ligation
[Senapathy & Carter, J. Biol. Chem., 259:4661-4666 (1984)]. 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.
[0063] General principles of rAAV production are reviewed in, for
example, Carter, Current Opinions in Biotechnology, 1533-1539
(1992); and Muzyczka, Curr. Topics in Microbial. and Immunol.,
158:97-129 (1992). 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., Mol. Cell. Biol., 7:349 (1988). Samulski et al.,
J. Virol., 63:3822-3828 (1989); 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., Vaccine, 13:1244-1250
(1995); Paul et al., Human Gene Therapy, 4:609-615 (1993); Clark et
al., Gene Therapy, 3:1124-1132 (1996); U.S. Pat. Nos. 5,786,211;
5,871,982; and 6,258,595. 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.
[0064] 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).
[0065] The rAAV 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.
[0066] In another embodiment, the invention contemplates
compositions comprising a DMD exon 5 IRES-activating oligomer
construct of the present invention in a viral delivery vector or
other delivery vehicle. Compositions of the invention comprise a
pharmaceutically acceptable carrier. The compositions may also
comprise other ingredients such as diluents. Acceptable carriers
and diluents 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-formig counterions such as sodium;
and/or nonionic surfactants such as Tween, pluronics or
polyethylene glycol (PEG).
[0067] Sterile injectable solutions are prepared by incorporating
the active ingredient 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.
[0068] 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) (i.e., 1.times.10.sup.1 vg, 1.times.10.sup.8 vg,
1.times.10.sup.9 vg, 1.times.10.sup.10 vg, 1.times.10.sup.11 vg,
1.times.10.sup.12 vg, 1.times.10.sup.13 vg, 1.times.10.sup.14 vg,
respectively).
[0069] Methods of transducing a target cell (e.g., a skeletal
muscle) of a patient with a 5' mutation of the DMD gene with a rAAV
of the invention, in vivo or in vitro, are contemplated herein. The
methods comprise the step of administering an effective dose, or
effective multiple doses, of a composition comprising a rAAV of the
invention to an animal (including a human being) with a 5' mutation
of the DMD gene. If the dose is administered prior to development
of DMD, the administration is prophylactic. If the dose is
administered after the development of DMD, 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 DMD being treated, that slows or prevents
progression to DMD, 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.
[0070] Administration of an effective dose of the compositions may
be by routes standard in the art including, but not limited to,
intramuscular, parenteral, intravenous, oral, buccal, nasal,
pulmonary, intracranial, intraosseous, intraocular, rectal, or
vaginal. 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). In some embodiments,
the route of administration is intramuscular. In some embodiments,
the route of administration is intravenous.
[0071] Combination therapies are also contemplated by the
invention. Combination therapy as used herein includes simultaneous
treatment or sequential treatments. Combinations of methods of the
invention with standard medical treatments (e.g., corticosteroids
and/or immunosuppressive drugs) are specifically contemplated, as
are combinations with other therapies such as those mentioned in
the Background section above. In some embodiments, the
corticosteroid is a glucocorticoid such as prednisone, deflazacort
or Medrol (6-methyl-prednisolone; PDN).
EXAMPLES
[0072] Aspects and embodiments of the invention are illustrated by
the following examples.
[0073] Most mutations that truncate the reading frame of the DMD
gene cause loss of dystrophin expression and lead to DMD. However,
amelioration of disease severity can result from alternate
translation initiation beginning in DMD exon 6 that leads to
expression of a highly functional N-truncated dystrophin. This
novel isoform results from usage of an IRES within exon 5 that is
glucocorticoid-inducible. IRES activity was confirmed in patient
muscle by both peptide sequencing and ribosome profiling as
described below. Generation of a truncated reading frame upstream
of the IRES by exon skipping led to synthesis of a functional
N-truncated isoform in both patient-derived cell lines and in a DMD
mouse model, where expression protects muscle from
contraction-induced injury and corrects muscle force to the same
level as control mice. These results support a novel therapeutic
approach for patients with mutations within the 5' exons of the DMD
gene. See also, Wein et al., Abstracts/Neuromuscular Disorders, 23:
738-852 (2013).
Example 1
Evidence for IRES-Induced Translation from Human Muscle Samples
[0074] We previously published that nonsense and frameshifting
mutations leading to a stop codon within at least the first two DMD
exons should result in the mild BMD phenotype via exon 6
translation initiation [Gurvich et al., Human Mutation, 30: 633-640
(2009)]. However, duplication of exon 2--which is the most common
single exon duplication and results in a premature stop codon
within the duplicated exon 2 sequence--would seem to be an
exception to this prediction, as it is usually associated with DMD
[White et al., Human Mutation, 27: 938-945 (2006)]. However, a
deletion of exon 2, which also results in a premature stop codon,
has not been described, either in our large cohort [Flanigan et
al., Human Mutation, 30: 1657-1666 (2009)] or in other large
publicly available catalogues (www.dmd.nl). We interpreted this
lack of reported cases to mean that the clinical features in
patients with exon 2 deletions are either asymptomatic or
exceedingly mild due to expression of the N-truncated isoform.
[0075] This interpretation was confirmed by the detection of a
deletion of exon 2 (DEL2) in an Italian boy who first presented at
age 6 years for evaluation of an incidentally detected elevation of
serum creatine kinase (550 iu/l; normal value<200 iu/l). Normal
early motor milestones were reported and no muscle dystrophy was
ever reported in the family. His neurological examination was
entirely normal at 15 years of age. Muscle biopsy showed slight
fiber size variability (FIG. 7A), and in some sections an increased
number of central nuclei along with some densely stained
hypercontracted fibers. Immunofluorescent analysis using a
C-terminal antibody showed the presence of dystrophin at the
membrane (FIG. 7B). Interestingly, western blot revealed that the
detected dystrophin had a smaller molecular weight (.about.410 kDa)
(FIG. 1a), and mutational analysis revealed a deletion of exon 2
(FIGS. 7C-G). Subsequent peptide sequencing using tandem mass
spectrometry (LC-MS/MS).sup.20 confirmed the absence of any
residues encoded by exons 1 through 5 among the 99 unique peptides
detected and matched to dystrophin, consistent with translation
initiation within exon 6 (FIG. 1B and Table 1).
TABLE-US-00006 TABLE 1 Peptide spectrum match in human muscle.
Dystrophin peptides encoded in exons 1-10. (N) represents the
number of times a peptide sequence was detected in normal control
muscle or in muscle from the patient with a deletion of exon 2.
Dystrophin Peptide Spectrum Match (N) Peptide Normal SEQ se- MW
control Del2 ID quence [Da] Exon muscle Muscle NO WVN 9,795,009 2 1
0 16 AQF SK QHI 16,718,084 3 1 0 17 ENL FSD LQD GR LLD 12,997,520 4
1 0 18 LLE GLT GQK VLQ 24,782,521 4-5 2 0 19 NNN VDL VNI GST DIV
DGN HK NLM 14,496,990 6 3 0 20 AGL QQT NSE K LEH 10,705,737 7 1 0
21 AFN IAR YQL 8,504,665 7 1 1 22 GIE K LLD 17,488,606 7-8 2 3 23
PED VDT TYP DKK SYA 18,948,820 9 3 2 24 YTQ AAY VTT SDP TR SPF
15,817,538 9-10 3 1 25 PSQ HLE APE DK
[0076] In a complementary approach, we examined DMD translation
efficiency, promoter usage, and alternate splicing using muscle RNA
isolated from a mild BMD patient with an exon 2 frameshift mutation
(c.40_41del [p.Glu14ArgfsX17], referred to as FS) whose western
blot also revealed expression of the same smaller molecular weight
dystrophin (.about.410 kDa) which lacked the N-terminal epitope
(FIG. 1C; FIG. 8A). To confirm our western blot results, muscle
homogenate from the same FS patient was used to construct RNA-Seq
libraries for ribosome-protected fragments (i.e., ribosome
footprints isolated after RNase digestion) and for total RNA. We
compared the mRNA translation efficiency in normal versus patient
muscle using the ratio of reads from ribosome-protected fragments
(RPFs) to reads from RNA-Seq. Among the top 1000 most abundant
muscle mRNAs, DMD displayed the greatest change in translation
efficiency (FIG. 1D), indicating a .about.5-fold reduction in the
amount of ribosomes translating the DMD muscle transcript in the
frameshifted patient FS. This decreased amount of translation is
consistent with both the expected reduction in dystrophin level
given the patient's mild BMD phenotype, and with the amount of
dystrophin seen in p.Trp3X patients.sup.4 and other 5' mutation
alleles (FIG. 1C).
[0077] The saw-tooth RNA-Seq pattern observed in DMD introns 1
through 8 (FIG. 1E) confirmed that the major transcription start
was located at the dystrophin muscle-specific promoter (Dp427m) and
that DMD exons 1 through 7 underwent efficient co-transcriptional
splicing [Ameur et al., Nature Structural & Molecular Biology,
18: 1435-1440 (2011)] in both the control and FS patient samples.
Two alternate 427 kD isoforms of dystrophin (Dp427p and Dp427c) are
expressed primarily in the central nervous system, and differ from
Dp427m only in the use of alternate exon 1 sequences. The lack of a
strong nascent RNA signal from either the Dp427p or Dp427c
promoters confirmed that up-regulation of alternate promoters does
not contribute to alternate AUG usage in exon 6 (FIG. 1E). In both
samples, RNA-Seq reads spanning exon-exon junctions mapped
exclusively to the known junctions between Dp427m exon 1 and exon
11, indicating that splicing of novel 5' UTRs from alternate
promoters did not contribute to exon 6 AUG usage. The distribution
of ribosome footprints mapped on Dp427m exons 1 through 11 revealed
normal levels of exon 1 AUG initiation, followed by premature
termination in exon 2 and resumption of translation following the
exon 6 in-frame AUG codons (FIG. 1F) that continued into the body
of the DMD transcript (FIGS. 8B-8D), consistent with efficient
alternate translation initiation.
Example 2
In Vitro Transcription/Translation Studies
[0078] Having demonstrated new evidence for efficient alternate
translation initiation using both ribosome profiling and protein
analysis directly in patient muscle, we sought to characterize the
elements contributing to the high translation efficiency. To
determine whether exons 1 through 5 of DMD contain an IRES, we
cloned the 5' portion of the cDNA encompassing exons 1 through part
of exon 6, beginning at the +4 position to exclude the native AUG
initiation codon (c.4_c.369, referred as exon 1 to 6), into the
dicistronic dual luciferase reporter vector pRDEF. This vector
contains an upstream cap-dependent renilla luciferase (RLuc) open
reading frame (ORF) under control of an SV40 promoter and a
downstream cap-independent firefly luciferase (FLuc) ORF under the
control of the sequences of interest, with the two ORFs separated
by a secondary structure element (dEMCV) that prevents ribosomal
scanning (FIG. 2C). We used the EMCV IRES sequence as a positive
control, and normalized all values to the empty vector. In each
case we included 49 nucleotides from exon 6 that placed the exon 6
AUGs in-frame with the downstream FLuc reporter. This sequence
corresponds to the first 39 nt, inclusive of the two in-frame AUGs
(M124 and M128), and 10 additional nucleotides used for cloning
purposes. T7 mediated RNA were generated from the different
constructs and were used to perform rabbit reticulocyte lysate
(RRL) translation assays (FIG. 2A, left panel). Size and integrity
of the corresponding RNAs were checked using a formaldehyde agarose
gel (FIG. 2B). Cap-independent translation activity (represented as
the ratio of downstream FLuc to the RLuc luminescence) of the exons
1-5 of DMD results in a 1.5-1.7 fold increase in FLuc signal, less
than the 3.4-3.8 increase seen with the control EMCV IRES but
consistent with IRES activity (FIG. 2A, left panel).
Example 3
IRES Activity in Cell Cultures
[0079] RRL-based translation may underestimate IRES activity of
either viral or eukaryotic cellular IRESs, possibly due to the
limiting amounts of RNA binding proteins in this specialized
extract or due to the lack of tissue-specific IRES trans-acting
factors (ITAFs). Therefore, the assay was performed in C2C12
myoblasts which express dystrophin, and we observed that the
presence of the exon 1 to 6 construct leads to .about.8 fold higher
FLuc expression relative to exon 6 alone vector (FIG. 2A, right
panel). This represents .about.50% of the activity of the control
EMCV IRES, suggesting the presence of a relatively strong IRES
within exons 1-5. To map the position of the IRES, deletion
constructs consisting of the 5' portion of the DMD gene (exons 1-5)
or appropriate controls were cloned into pRDEF (FIG. 2C). Deletion
of the first 300 nucleotides (nt) of this sequence did not
significantly change the FLuc expression, whereas removal or
inversion of the last 71 nt (representing nearly all of exon 5)
completely abrogates expression of the FLuc reporter, and further
deletions within exon 5 result in greatly reduced FLuc expression.
To test the hypothesis that the putative IRES required muscle
specific factors, we repeated the experiments in HEK293K cells,
which do not endogenously express dystrophin, and in a commercial
human myoblast cell line (hSKMM). Unlike the ECMV IRES, the
putative DMD IRES did not stimulate FLuc expression in 293K cells
whereas the level of stimulation in hSKMM cells replicated the
C2C12 results (FIG. 9A), suggesting that the IRES is preferentially
active in muscle.
[0080] Control experiments were performed to exclude the
possibility of aberrant splicing events, cryptic promoter
activities, or other potential artifacts leading to
misinterpretation of the dicistronic assay. We removed the upstream
SV40 promoter to generate a promoterless version of the pRDEF
vector containing the exon 1 to 6 (c.4_c.369) DMD sequence.
Transfection of this construct into C2C12 myoblasts showed only
minimal background luminescence from both RLuc and FLuc, strongly
arguing against any cryptic promoter activity in the DMD coding
sequence (data not shown). No aberrant splicing was detected by
RT-PCR (FIG. 2D and FIG. 9C), and RNA integrity was confirmed by a
northern blot (FIGS. 2e and 9b).
[0081] Notably, although either duplication or deletion of exon 2
results in an interrupted reading frame, the disparate associated
clinical phenotypes led to the hypothesis that IRES activity may be
diminished in the presence of an exon 2 duplication. We tested this
hypothesis in C2C12 cells and showed that IRES activation was
equivalent between the full length (exons 1-6) and deletion 2
cDNAs, but was markedly reduced in the presence of an exon 2
duplication (FIG. 2F) confirming that duplication but not deletion
of exon 2 ablates IRES activity.
Example 4
Out-of-Frame Exon-Skipping is Able to Drive an IRES Mediated
Dystrophin In Vitro
[0082] In considering skipping of exons prior to the exon 5 IRES,
only the removal of exon 2 will disrupt the reading frame and
result in a premature stop codon (FIG. 3A). We contemplated that
deletion of this exon could be used therapeutically to increase
activation of the IRES, whether by use of antisense
oligonucleotides (AONs) [Wood et al., Brain: A Journal of
Neurology, 133: 957-972 (2010); van Deutekom et al., New England
Journal of Medicine, 357: 2677-2686 (2007) and Kinali et al.,
Lancet Neurology, 8: 918-928 (2009)] or by use of AAV-U7 mediated
antisense delivery [Goyenvalle et al., Science, 306: 1796-1799
(2004) and Vulin et al., Molecular Therapy: Journal of the American
Society of Gene Therapy, 20: 2120-2133 (2012)]. We selected four
different sequences (respectively labeled "B", "AL", "AS" and "C"
in FIG. 3B) for U7snRNA targeting and cloned each into AAV1 to
assess exon-skipping efficiency in myoblasts generated from either
a wild type or an exon 2 duplication fibroblast cell lines that
expresses a doxycycline-inducible MyoD (referred as FibroMyoD)
[Chaouch et al., Human Gene Therapy, 20: 784-790 (2009)]. All
constructs were able to skip either one or two copies of exon 2
(FIGS. 10A-10B). Subsequently, in order to increase skipping
efficiency, two copies of each of the U7-C and U7-AL targeting
antisense sequences were cloned into the single self-complementary
(sc) AAV1 vector (and designated AAV1.U7-ACCA), the genome of which
is shown in FIGS. 15A-15B in the 3' to 5' orientation. U7-C and
U7-AL were used to avoid any possible overlap in the antisense
sequence between AL and B. A known antisense sequence (AON H2A) was
used as a positive control of skipping [Tennyson and Worton,
Nucleic Acids Res., 24: 3059-3064 (1996)]. Infection of FibroMyoD
cells resulted in 88.6% of the DMD transcript with complete
skipping of exon 2 leading to the production of N-terminally
truncated dystrophin (FIGS. 3C-3D and FIG. 12A).
Example 5
IRES Driven N-Truncated Dystrophin is Expressed After Out-of-Frame
Exon-Skipping in a Novel Mouse Model Harboring a Duplication of
Exon 2
[0083] We tested the ability of the U7-ACCA vector to skip exon 2
in vivo in a mouse model carrying a duplication of exon 2 on a
C57BL/6 background (the Dup2 mouse; described in Example 8 below).
The resulting DMD mRNA contains two copies of exon 2, disrupting
the reading frame and resulting in nearly complete absence of
dystrophin expression. AAV1.U7-ACCA (1e11vg) was injected directly
into the tibialis anterior muscle in six to eight week-old Dup2
mice (n=5) or BI6 control mice. Four weeks later, RT-PCR analysis
from injected muscles demonstrates nearly complete exon-skipping of
exon 2 in Dup2 or BI6 (FIGS. 4A-4B). Consistent with the RT-PCR
results, the saw-tooth RNA-Seq pattern observed in Dmd introns 1
and 2 confirmed the suppression of co-transcriptional splicing of
the duplicated exon 2 as well as the high-efficiency of
co-transcriptional splicing of exon 1 to exon 3 in the treated mice
(FIG. 4C). Western blot and immunostaining demonstrate expression
of the N-truncated protein. Sarcolemmal staining is restored for
.beta.-dystroglycan and nNOS (FIGS. 4D-4E), suggesting the presence
of a functional dystroglycan complex.
[0084] We also performed a dose escalation study using
intramuscular injection (IM) into the tibialis anterior (TA) of
Dup2 mice in order to assess the degree of dose response for exon
skipping and protein expression. IM escalating doses are set out in
FIG. 18A. As seen in FIG. 18B, the degree of skipped transcript
shows an expected dose response. FIG. 18B shows a similar expected
dose response in protein expression, maximal at 3.1E11 vg per
injection, with significant correction of physiologic force defects
(FIG. 18C).
Example 6
Glucocorticoid Increases Activation of the Dystrophin IRES
[0085] We examined the effect of glucocorticoid exposure on IRES
activity as a muscle-specific IRES found in the 5' UTR of utrophin,
an analog of dystrophin, was found to be glucocorticoid-activated
[Miura et al., PloS One, 3: e2309 (2008)]. Furthermore, treatment
with the glucocorticoids prednisone and deflazacort are standard
treatment for DMD. We assayed exon 5 IRES activity using the exon 5
to 6 construct in C2C12 cells in the presence of increasing
concentrations of 6-methyl-prednisolone (PDN) and found that
downstream FLuc activity increased in a dose-dependent fashion from
around 7 fold change in the absence of PDN to over 20 fold at 6.4
.mu.M PDN (FIG. 5A).This glucocorticoid activation was not seen
after transfection of the exon 6 alone or the inverted exon 5
control constructs or in 293K (FIG. 5A). An increase in dystrophin
expression was seen in Dup2 FibroMyoD cells treated with 6.4 .mu.M
PDN (FIG. 5B) and co-treatment of Dup2 mice (n=5) with both U7-ACCA
and PDN resulted in an increase in dystrophin expression over
U7-ACCA alone (FIGS. 5C-5D), consistent with glucocorticoid
inducibility. An increase to less than 3% compared to untreated
Dup2 was seen with PDN alone in rare samples (represented in FIG.
5C), suggesting some leakiness of the IRES in the Dup2 model. In
all cases, this increase of dystrophin expression was not due to a
difference in the AAV vector genome copy number (data not shown).
Because utrophin translation may be regulated by corticosteroids
and overexpression can compensate for absent dystrophin, we
assessed utrophin levels in the same injected muscles (FIG. 5E). In
untreated Dup2 animals, utrophin levels were increased in
comparison to B16, similar to what has been reported in mdx, the
standard dystrophinopathy mouse model. Comparison of the four
groups reveals no statistically significant difference in utrophin
levels between PDN treated and untreated animals (FIG. 5F),
excluding utrophin upregulation as a cause of functional rescue
following PDN treatment.
Example 7
Local IRES Driven N-Truncated Dystrophin Expression Stabilizes
Muscle Membrane and Corrects Force Deficits in Dup2 Mouse
Muscle
[0086] We examined whether expression of the IRES-driven isoform
improved muscle integrity and physiology in the Dup2 mouse. Similar
to the case in mdx mice, dystrophic changes in Dup2 mice are
quantifiable at 4 weeks of age as widespread muscle regeneration
characterized by centralized nuclei (Vulin et al., manuscript in
press). One month after intramuscular injection of AAV1.U7-ACCA
into the tibialis anterior muscle of 4-week old Dup2 mice,
expression of the IRES driven isoform results in a significant
reduction of centralized nuclei (FIG. 6A). To demonstrate that this
isoform restores membrane integrity, treated and untreated Dup2
mice were subjected to a downhill running protocol and injected
with Evans blue dye (EBD), which enters skeletal muscle fibers that
have been permeabilized by membrane damage. Following
intraperitoneal injection of EBD, uptake is found only in fibers
without dystrophin staining, suggesting the N-truncated protein
stabilizes the sarcolemma and provides further evidence for the
functionality of this protein in vivo (FIG. 4F). Quantification of
the number of EBD positive fiber confirms that expression of the
IRES driven isoform results in protection of muscle fibers in these
mice (FIG. 6B). Importantly, this membrane protection is associated
with restoration of hindlimb grip strength (FIG. 6C) and muscle
specific force (FIG. 6D) to the levels seen in BI6 control mice.
Dup2 muscles injected with U7-ACCA with or without prednisone were
significantly more resistant to contraction-induced injury than
untreated Dup2 muscle, and the combination of both treatments
showed no significant difference from BI6 controls (FIG. 6E),
Despite the minimal (<3%) expression of dystrophin seen in some
Dup2 muscles by PDN (FIG. 5C), treatment of the Dup2 muscles by PDN
alone does not result in a significant amelioration of the muscle
physiology (FIGS. 6A-6E).
Example 8
DMD Models
[0087] Examples of models of the DMD exon 2 duplication include in
vivo and in vitro models as follows.
mdx.sup.dup2 Mouse Model
[0088] Mice carrying a duplication of exon 2 within the DMD locus
were developed. The exon 2 duplication mutation is the most common
human duplication mutation and results in relatively severe
DMD.
[0089] A map of the insertion vector is shown in FIG. 16 In the
map, the numbers indicate the relative positions of cloning sites
and exons and restriction sites. The neo cassette is in the same
direction of the gene and the insertion point is precisely at
32207/32208 bp in the intron2. At least 150 bp extra intronic
sequences are kept on each side of inserted exon 2, E2 region is
1775-2195 bp. Sizes of exon 2 and intron 2 are 62 bp and 209572 bp
respectively.
[0090] Male C57BL/6 ES cells were transfected with the vector (FIG.
16) carrying an exon2 construct and then insertion was checked by
PCR. One good clone was found, amplified and injected in dozens of
albino BL/6 blastocysts. Injected blastocysts were implanted into
recipient mice. The dystrophin gene from chimeric males was checked
by PCR and then by RT-PCR. The colony was expanded and includes
some female mice bred to homozygosity. Dystrophin expression in
muscles from a 4 week old hemizygous mdxdup2 mouse was essentially
absent.
Immortalized and Conditionally Inducible FibroMyoD Cell Lines
[0091] Expression of the MyoD gene in mammalian fibroblasts results
in transdifferentiation of cells into the myogenic lineage. Such
cells can be further differentiated into myotubes, and they express
muscle genes, including the DMD gene.
[0092] Immortalized cell lines that conditionally express MyoD
under the control of a tetracycline-inducible promoter were
generated. This is achieved by stable transfection of the primary
fibroblast lines of a lentivirus the tet-inducible MyoD and
containing the human telomerase gene (TER). The resultant stable
line allows MyoD expression to be initiated by treatment with
doxycycline. Such cell lines were generated from patients with DMD
who carry a duplication of exon 2.
[0093] Using the line, duplication skipping using 2'-O-methyl
antisense oligomers (AONs) provided by Dr. Steve Wilton (Perth,
Australia) was demonstrated. Multiple cell lines were tested.
Transiently MyoD-Transfected Primary Cell Lines
[0094] Proof-of-principle experiments using primary fibroblast
lines transiently transfected with adenovirus-MyoD were conducted.
The adenovirus constructs were not integrated in the cell genomes,
yet MyoD was transiently expressed. The resulting DMD expression
was sufficient to perform exon skipping experiments (although
reproducibility favors the stably transfected lines.)
Example 9
Intravenous Injection of AAV9-U7_ACCA in the Dup2 Mouse Model
Results in Significant Expression of the N-Truncated Isoform and
Correction of Strength Deficit
[0095] We tested the ability of an AAV9-U7-ACCA genome to skip exon
2 in vivo in Dup2 mice upon intravenous injection. The U7-ACCA
genome was cloned into a rAAV9 vector (designated AAV9-U7_ACCA
herein) for administration to the mice. AAV9-U7_ACCA was injected
into the tail vein (3.3E12 vg/kg) of five Dup2 mice. One month
after injection, treated animals were examined.
[0096] Results of the experiment are shown in FIGS. 17A-17F.
[0097] We also carried out dose escalation studies of intravenous
dosing (FIG. 19A). As seen in FIG. 19B, the degree of skipped
transcript shows an expected dose response, as was seen in the IM
studies. At the highest level, the majority of transcript consists
of either wild-type transcript, which is translated into
full-length dystrophin, or exon 2-deleted transcript, which is
translated into the N-truncated isoform; importantly, either
isoform provides a functional benefit to the mouse (as to humans).
FIG. 19C shows a similar expected dose response in protein
expression. Quite importantly, in terms of clinical utility, at the
higher doses there is unquestionable and abundant expression of
dystrophin in the diaphragm and heart muscles. Quantification of
protein expression on immunoblot (FIG. 19D) confirms the dose
escalation response.
[0098] Newborn screening (NBS) for DMD in human newborns is now
feasible, therefore we tested the benefits of early expression of
the N-trucated isoform by delivery of AAV9.U7-ACCA vector
(8.times.10.sup.11 vg) results at postnatal day 1 (P1) in Dup2
mice. This single injection results in widespread expression of the
N-truncated isoform in all muscles, with sustained protection of
muscle fibers through one and six months post treatment (FIG.
20).
Example 10
[0099] PPMOs having following sequences (shown 5' to 3') are
administered to Dup2 mice.
TABLE-US-00007 C antisense oligomer: (SEQ ID NO: 10)
AUUCUUACCUUAGAAAAUUGUGC AL antisense oligomer: (SEQ ID NO: 11)
GUUUUCUUUUGAACAUCUUCUCUUUCAUCUA
[0100] We transfected the AL-PPMO into wild type C2C12 mouse
myoblasts (FIG. 22). Three days following transfection, an RT-PCR
was performed and demonstrated an efficient exon 2 skipping (FIG.
22A). A similar experiment was performed in the Dup2 mouse model.
Intramuscular injection of the AL-PPMO into the tibialis anterior
(TA) of Dup2 mice was performed in order to assess the degree of
exon 2 skipping and protein expression. As seen in FIG. 22B, exon 2
skipping was achieved efficiently. FIG. 22C was obtained using the
same treated TA muscles. Immunostaining of dystrophin was carried
out and of dystophrin he results demonstrated efficient production
and localization to the plasma membrane protein.
[0101] In another experiment, systemic injections are given in the
tail vein of another cohort of mice of three doses weekly at 12
mg/kg. We will evaluate skipping and dystrophin restoration at 4
weeks after the first injection.
Example 11
[0102] Patients harboring a nonsense mutation within exon 1 or 2
still express the highly functional N-terminally truncated
dystrophin isoform. This is due to the presence of IRES in exon 5
that allow re-entry of the ribosome and translation from exon 6.
Therefore we hypothesize that creation of a nonsense mutation
should force activation of the IRES in human patient cell lines
carrying either missense mutation or in frame deletion duplication,
within exon 1 to 4. Only removal of exon 2 generates a stop codon
in exon 3. Therefore complete skipping of exon 2 in patient
carrying the above mentioned mutation, would induce a stop codon in
exon 3, and thereby production of the IRES-mediated N-terminally
truncated isoform.
[0103] We collected cells from human patients carrying mutation in
these exons. The cells were then infected with a lentivirus
expressing an inducible MyoD that forces conversion of fibroblasts
to myoblasts which can then be further differentiated into
myotubes, the cell type that expresses dystrophin (referred to
hereafter as "myofibroblasts"). Despite aiming to collect cells
from patients harboring missense mutation or in frame deletion or
duplication within exon 1 to 4, only cells from patient carrying a
nonsense mutation were available. These cells were derived from BMD
patients, and as they carry a nonsense mutation they already
naturally expressed the N-terminally truncated dystrophin isoform.
However, treatment with AAV1.U7-ACCA at differentiation resulted in
higher expression of the IRES-initiated isoform by day 14 (FIG.
21).
Discussion of Results in the Examples
[0104] We have demonstrated the presence of a
glucocorticoid-responsive IRES within DMD exon 5 that can drive the
expression of an N-truncated but functional dystrophin. Ribosome
profiling from a BMD patient with an exon 2 frameshifting mutation
demonstrated a mild reduction in dystrophin translation efficiency
and a ribosome footprint pattern consistent with ribosome loading
beginning in exons 5 and 6. The relevance of this IRES-induced
isoform to the amelioration of disease severity, which we first
described in patients with exon 1 nonsense mutations [Flanigan et
al., Neuromuscular Disorders: NMD, 19: 743-748 (2009)], is also
confirmed by the mass spectrometric data from the first ever
reported case of an exon 2 deletion, found in an entirely
asymptomatic subject. Finally, in a novel therapeutic approach, we
have induced out-of-frame exon-skipping to generate a premature
stop codon and consequently force activation of the IRES in both
patient-derived cell lines and in a novel DMD mouse model, in which
we restored components of the dystrophin complex and corrected the
pathologic and physiologic features of muscle injury.
[0105] Most eukaryotic mRNAs are monocistronic and possess a
specialized cap structure at their 5' terminus, which is required
for translation initiation as this is where scanning by the 40S
ribosomal subunit begins. Despite clear evidence for the
cap-dependent 5.fwdarw.3' scanning model of initiation,
bioinformatic analysis has suggested that .about.50% of human
transcripts contain 5'UTR short upstream open reading frames
(uORFs) that may mediate transcript-specific translation efficiency
and control. uORFs may function by modulating either leaky scanning
or termination-dependent reinitiation, although uORFs can also
dynamically regulate access to IRES elements as shown for the
mammalian cationic amino acid transporter 1 gene, CAT1/SLC7A1.
Recognizing the cautions raised regarding IRES identification via
reporter assays, all control experiments performed in this
study--including assessment of RNA integrity by RT-PCR and Northern
blot, use of a promoterless plasmid, and use of an appropriate
positive IRES control - were consistent with cap-independent
initiation due to IRES activity. We mapped a minimal region
harboring a DMD IRES activity to 71 nt, of a small length compared
to EMCV (588 nt) but similar in size to that identified in the
c-myc 5'UTR (50 nt). This is an important feature as such small
IRESs can be used in dicistronic vectors, where space is limited
when packaged into viral vectors such as AAV.
[0106] Although the precise molecular mechanism by which cellular
IRESs modulate translation has not been defined in the literature,
the requirement of ITAFs has been strongly suggested. These
cellular proteins act in trans to augment IRES activity. Almost all
ITAFs have been shown to harbor RNA binding domains and have been
hypothesized to act as RNA chaperones, helping the IRES primary
sequence attain appropriate conformational state intrinsic to its
activity. This is likely relevant to the loss of dystrophin IRES
activity in the presence of an exon 2 duplication, which may ablate
IRES function by formation of a complex secondary structure or
cause the formation of an inhibitory uORF that interferes with ITAF
access to the exon 5 IRES.
[0107] Our results provide a molecular explanation for the rescue
of 5' truncating mutations via a heretofore undescribed mechanism
of post-transcriptional regulation of dystrophin expression. The
identification of this new cellular IRES and the resultant
dystrophin isoform has significant implications for understanding
the basic biology of muscle and dystrophin. We note that exon 5 of
DMD is highly conserved, with 87% identity to human found in the
dog, mouse, horse, and chicken DMD genes, and 67% among 39 species
including D. rerio and X. tropicalis. The presence of an IRES
within such a highly conserved region strongly suggests selective
pressure favoring a programmed role for alternate translation
initiation. The role of the IRES under normal conditions is
unclear, but ongoing efforts to understand the relevant cell
lineage-specific and/or conditional activation signals will shed
light on underlying mechanisms of IRES control and elucidate
potentially novel functions of dystrophin.
[0108] An intriguing question is how the N-truncated isoform
remains functional. A key cellular role for dystrophin is presumed
to be transmitting the force of contraction across the sarcolemma
to extracellular structures by serving as an important
architectural bridge role between the F-actin cytoskeleton and the
muscle plasma membrane. Two regions within dystrophin are
responsible for F-actin binding: ABD1 (actin binding domain,
spanning residues 15-237) and ABD2 (spanning residues 1468-2208). A
number of studies have shown a lack of stability of dystrophin in
the setting of deletions within the ABD1 domain. However, we note
that most of these studies were performed with microdystrophin
constructs lacking the ABD2 domain, which has been shown to enhance
the interaction between ABD1 and actin. Such miniproteins bind
actin and modify actin dynamics in a different manner compared to
the full length version. Although results with such constructs show
that absence of ABD2 does not completely abrogate binding of
dystrophin to actin, it is unlikely that absence of ABD1 completely
disrupts the interaction between dystrophin and actin. Expression
of transgenes deleted for ABD1 lessens the mdx phenotype and
restores the costameric pattern of the M band and Z lines,
suggesting that the link between dystrophin and the subsarcolemmal
cytoskeleton involves more than an interaction with ABD1. In
agreement with this, other members of the cytoskeleton have been
shown to interact with the dystrophin spectrin-repeat.
[0109] Although some series suggest that BMD due to mutations
affecting ABD1 is more severe [Beggs et al., American Journal of
Human Genetics, 49: 54-67 (1991)], our clinical and experimental
observations--as well reports of other BMD patients lacking part or
all of the ABD1 domain [Winnard et al., Human Molecular Genetics,
2: 737-744 (1993); Winnard et al., American Journal of Human
Genetics, 56: 158-166 (1995) and Heald et al., Neurology, 44:
2388-2390 (1994)]--clearly indicate the significant functionality
of the IRES-driven N-truncated isoform despite lacking the first
half of the canonical ABD1 (FIG. 3a). This is of particular
interest since forcing expression of this isoform by generating an
out-of-frame transcript in order to induce IRES activity holds
substantial therapeutic potential. This novel out-of-frame strategy
could be combined with glucocorticoid treatment, a drug already
used in DMD/BMD patients, which should increase IRES activation.
Significantly, rather than being a personalized exon-skipping
approach for patients with exon 2 duplications (who represent
nearly 2% of DMD patients in one large series), out-of-frame
skipping of exon 2 to induce expression of such a protein is
contemplated for treatment of all patients who harbor mutations at
the 5' end of the DMD gene (up to 6% in the same cohort) [Flanigan
et al., Neuromuscular Disorders: NMD, 19: 743-748 (2009)].
[0110] While the present invention has been described in terms of
specific embodiments, it is understood that variations and
modifications will occur to those skilled in the art. Accordingly,
only such limitations as appear in the claims should be placed on
the invention.
[0111] All documents referred to in this application are hereby
incorporated by reference in their entirety with particular
attention to the content for which they are referred.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 34 <210> SEQ ID NO 1 <211> LENGTH: 28 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
1 tcaaaagaaa acattcacaa aatgggta 28 <210> SEQ ID NO 2
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 2 gcacaatttt ctaaggtaag aat 23
<210> SEQ ID NO 3 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3
tagatgaaag agaagatgtt caaaagaaaa c 31 <210> SEQ ID NO 4
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 4 tagatgaaag agaagatgtt c 21
<210> SEQ ID NO 5 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Adeno-associated virus <400>
SEQUENCE: 5 tacccatttt gcgaatgttt tcttttga 28 <210> SEQ ID NO
6 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Adeno-associated virus <400> SEQUENCE: 6 attcttacct
tagaaaattg tgc 23 <210> SEQ ID NO 7 <211> LENGTH: 31
<212> TYPE: DNA <213> ORGANISM: Adeno-associated virus
<400> SEQUENCE: 7 gttttctttt gaagatcttc tctttcatct a 31
<210> SEQ ID NO 8 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Adeno-associated virus <400>
SEQUENCE: 8 gaagatcttc tctttcatct a 21 <210> SEQ ID NO 9
<211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 9 uacccauuuu gcgaauguuu ucuuuuga
28 <210> SEQ ID NO 10 <211> LENGTH: 23 <212>
TYPE: RNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
10 auucuuaccu uagaaaauug ugc 23 <210> SEQ ID NO 11
<211> LENGTH: 31 <212> TYPE: RNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 11 guuuucuuuu gaacaucuuc
ucuuucaucu a 31 <210> SEQ ID NO 12 <211> LENGTH: 21
<212> TYPE: RNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12 gaacaucuuc ucuuucaucu a 21 <210> SEQ
ID NO 13 <211> LENGTH: 30 <212> TYPE: RNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 13 ccauuuugug
aauguuuucu uuugaacauc 30 <210> SEQ ID NO 14 <211>
LENGTH: 4472 <212> TYPE: DNA <213> ORGANISM:
Adeno-associated virus rh74 <400> SEQUENCE: 14 ctccatcact
aggggtaacc gcgaagcgcc tcccacgctg ccgcgtcagc gctgacgtaa 60
attacgtcat aggggagtgg tcctgtatta gctgtcacgt gagtgctttt gcgacatttt
120 gcgacaccac gtggccattc atggtatata tggccgagtg agcgagcagg
atctccattt 180 tgaccgcgaa atttgaacga gcagcagcca tgccgggctt
ctacgagatc gtgcttaagg 240 tgccgagcga cctggacgag cacctgccgg
gcatttctga ctcgtttgtg aactgggtgg 300 cagagaagga atgggagctg
cccccggatt ctgacatgga tcggaatctg attgagcagg 360 cacccctgac
cgtggccgag aagctacagc gcgacttcct ggtccaatgg cgccgcgtga 420
gtaaggcccc ggaggccctc ttctttgttc agttcgagaa gggcgagtcc tacttccacc
480 tccatattct ggtagagacc acgggggtca aatccatggt gctgggccgc
ttcctgagtc 540 agattcggga caagctggtg cagaccatct accgcgggat
cgagccgacc ctgcccaact 600 ggttcgcggt gacaaagacg cgtaatggcg
ccggaggggg gaacaaggtg gtggacgagt 660 gctacatccc caactacctg
ctgcccaaga ctcagcccga gctgcagtgg gcgtggacta 720 acatggagga
gtatataagc gcgtgcttga acctggccga gcgcaaacgg ctcgtggcgc 780
agcacctgac ccacgtcagc cagacccagg agcagaacaa ggagaatctg aacccgaatt
840 ctgacgcgcc tgtcatccgg tcaaaaacct ccgcgcgcta catggagctg
gtcgggtggc 900 tggtggaccg gggcatcacc tccgagaagc agtggatcca
ggaggaccag gcctcgtaca 960 tctccttcaa cgccgcctcc aactcgcggt
ctcagatcaa ggccgcgctg gacaatgccg 1020 gcaagatcat ggcgctgacc
aaatccgcgc ccgactacct ggtaggcccc gctctgcccg 1080 cggacattaa
atccaaccgc atctaccgca tcctggagct gaatggctac gaccctgcct 1140
acgccggttc cgtctttctc ggctgggccc agaaaaagtt tggcaaaagg aacaccatct
1200 ggctgtttgg gccggccacc acgggcaaga ccaacatcgc ggaagccatc
gcccacgccg 1260 tgcccttcta cggctgcgtc aactggacca atgagaactt
tcccttcaac gattgcgtcg 1320 acaagatggt gatctggtgg gaggagggca
agatgacggc caaggtcgtg gagtccgcca 1380 aggccattct cggcggcagc
aaggtgcgcg tggaccaaaa gtgcaagtcg tccgcccaga 1440 tcgatcccac
ccccgtgatc gtcacctcca acaccaacat gtgcgccgtg attgacggga 1500
acagcaccac cttcgagcac cagcagccgt tgcaggaccg gatgttcaaa tttgaactta
1560 cccgccgtct ggagcacgac tttggcaagg tgacaaagca ggaagtcaaa
gagttcttcc 1620 gctgggcgca ggatcacgtg accgaggtgg cgcatgagtt
ctacgtcaga aagggtggag 1680 ctaacaaaag acccgccccc gatgacgcgg
atataagcga gcccaagcgg gcctgcccct 1740 cagtcgcgga tccatcgacg
tcagacgcgg aaggagctcc ggtggacttt gccgacaggt 1800 accaaaacaa
atgttctcgt cacgcgggca tgcttcagat gctgtttccc tgcaaaacat 1860
gcgagagaat gaatcagaat ttcaacattt gcttcacgca cgggaccaga gactgttcag
1920 aatgtttccc tggcgtgtca gaatctcaac cggtcgtcag aaaaaagacg
tatcggaaac 1980 tctgtgcgat tcatcatctg ctggggcggg cacccgagat
tgcttgctcg gcctgcgacc 2040 tggtcaacgt ggacctggat gactgtgttt
ctgagcaata aatgacttaa accaggtatg 2100 gctgccgatg gttatcttcc
agattggctc gaggacaacc tctctgaggg cattcgcgag 2160 tggtgggacc
tgaaacctgg agccccgaaa cccaaagcca accagcaaaa gcaggacaac 2220
ggccggggtc tggtgcttcc tggctacaag tacctcggac ccttcaacgg actcgacaag
2280 ggggagcccg tcaacgcggc ggacgcagcg gccctcgagc acgacaaggc
ctacgaccag 2340 cagctccaag cgggtgacaa tccgtacctg cggtataatc
acgccgacgc cgagtttcag 2400 gagcgtctgc aagaagatac gtcttttggg
ggcaacctcg ggcgcgcagt cttccaggcc 2460 aaaaagcggg ttctcgaacc
tctgggcctg gttgaatcgc cggttaagac ggctcctgga 2520 aagaagagac
cggtagagcc atcaccccag cgctctccag actcctctac gggcatcggc 2580
aagaaaggcc agcagcccgc aaaaaagaga ctcaattttg ggcagactgg cgactcagag
2640 tcagtccccg accctcaacc aatcggagaa ccaccagcag gcccctctgg
tctgggatct 2700 ggtacaatgg ctgcaggcgg tggcgctcca atggcagaca
ataacgaagg cgccgacgga 2760 gtgggtagtt cctcaggaaa ttggcattgc
gattccacat ggctgggcga cagagtcatc 2820 accaccagca cccgcacctg
ggccctgccc acctacaaca accacctcta caagcaaatc 2880 tccaacggga
cctcgggagg aagcaccaac gacaacacct acttcggcta cagcaccccc 2940
tgggggtatt ttgacttcaa cagattccac tgccactttt caccacgtga ctggcagcga
3000 ctcatcaaca acaactgggg attccggccc aagaggctca acttcaagct
cttcaacatc 3060 caagtcaagg aggtcacgca gaatgaaggc accaagacca
tcgccaataa ccttaccagc 3120 acgattcagg tctttacgga ctcggaatac
cagctcccgt acgtgctcgg ctcggcgcac 3180 cagggctgcc tgcctccgtt
cccggcggac gtcttcatga ttcctcagta cgggtacctg 3240 actctgaaca
atggcagtca ggctgtgggc cggtcgtcct tctactgcct ggagtacttt 3300
ccttctcaaa tgctgagaac gggcaacaac tttgaattca gctacaactt cgaggacgtg
3360 cccttccaca gcagctacgc gcacagccag agcctggacc ggctgatgaa
ccctctcatc 3420 gaccagtact tgtactacct gtcccggact caaagcacgg
gcggtactgc aggaactcag 3480 cagttgctat tttctcaggc cgggcctaac
aacatgtcgg ctcaggccaa gaactggcta 3540 cccggtccct gctaccggca
gcaacgcgtc tccacgacac tgtcgcagaa caacaacagc 3600 aactttgcct
ggacgggtgc caccaagtat catctgaatg gcagagactc tctggtgaat 3660
cctggcgttg ccatggctac ccacaaggac gacgaagagc gattttttcc atccagcgga
3720 gtcttaatgt ttgggaaaca gggagctgga aaagacaacg tggactatag
cagcgtgatg 3780 ctaaccagcg aggaagaaat aaagaccacc aacccagtgg
ccacagaaca gtacggcgtg 3840 gtggccgata acctgcaaca gcaaaacgcc
gctcctattg taggggccgt caatagtcaa 3900 ggagccttac ctggcatggt
gtggcagaac cgggacgtgt acctgcaggg tcccatctgg 3960 gccaagattc
ctcatacgga cggcaacttt catccctcgc cgctgatggg aggctttgga 4020
ctgaagcatc cgcctcctca gatcctgatt aaaaacacac ctgttcccgc ggatcctccg
4080 accaccttca ctaaggccaa gctggcttct ttcatcacgc agtacagtac
cggccaggtc 4140 agcgtggaga tcgagtggga gctgcagaag gagaacagca
aacgctggaa cccagagatt 4200 cagtacactt ccaactacta caaatctaca
aatgtggact ttgctgtcaa tactgagggt 4260 acttattccg agcctcgccc
cattggcacc cgttacctca cccgtaatct gtaattacat 4320 gttaatcaat
aaaccggtta attcgtttca gttgaacttt ggtctcctgt ccttcttatc 4380
ttatcggtta ccatagaaac tggttactta ttaactgctt ggtgcgcttc gcgataaaag
4440 acttacgtca tcgggttacc cctagtgatg ga 4472 <210> SEQ ID NO
15 <211> LENGTH: 2052 <212> TYPE: DNA <213>
ORGANISM: Adeno-associated virus <400> SEQUENCE: 15
cgaccgcgcg agcgagcgag tgactccggc gggcccgttt cgggcccgca gcccgctgga
60 aaccagcggg ccggagtcac tcgctcgctc gcgcgtctct ccctcaccgg
ttgaggtagt 120 gatccccaag gaacatcaat tactaattgg gcggtacgat
gaatagatgc atcggtacga 180 gatctattgt tgtatcctcg acactaaccg
acaaaagtcg gttagtcgtg actgagtaaa 240 cgtatcggaa atgttcgcca
gtgtttgagt tctttgctcg ccaaaattat cagaaaatct 300 tataacaaat
agcttggctt attccttgac acgaaacact aagtgtatag tcacctcccc 360
acacctttac cgtggaacta gagtgggagt agctttcacc tcaactacag gaagggaccg
420 agcgatgtct gcgtgaaggc gttcaaaaga aaacttctag aagagaaagt
agatttaaaa 480 acctcgtcca aaagactgaa gccagccttt tggggagggt
taaagtgacc agatgttact 540 ttcgttttgt caagagaagg ggcgaggggc
cacacactct ccccgaaact aggaagagac 600 caaaggatcc tttgcgcata
caccgatcta ttgttgtatc ctcgacacta accgacaaaa 660 gtcggttagt
cgtgactgag taaacgtatc ggaaatgttc gccagtgttt gagttctttg 720
ctcgccaaaa ttatcagaaa atcttataac aaatagcttg gcttattcct tgacacgaaa
780 cactaagtgt atagtcacct ccccacacct ttaccgtgga actagagtgg
gagtagcttt 840 cacctcaact acaggaaggg accgagcgat gtctgcgtga
aggcgtttaa gaatggaatc 900 ttttaacacg ttaaaaacct cgtccaaaag
actgaagcca gccttttggg gagggttaaa 960 gtgaccagat gttactttcg
ttttgtcaag agaaggggcg aggggccaca cactctcccc 1020 gaaactagga
agagaccaaa ggatcctttg cgcatacacc gatctattgt tgtatcctcg 1080
acactaaccg acaaaagtcg gttagtcgtg actgagtaaa cgtatcggaa atgttcgcca
1140 gtgtttgagt tctttgctcg ccaaaattat cagaaaatct tataacaaat
agcttggctt 1200 attccttgac acgaaacact aagtgtatag tcacctcccc
acacctttac cgtggaacta 1260 gagtgggagt agctttcacc tcaactacag
gaagggaccg agcgatgtct gcgtgaaggc 1320 gtttaagaat ggaatctttt
aacacgttaa aaacctcgtc caaaagactg aagccagcct 1380 tttggggagg
gttaaagtga ccagatgtta ctttcgtttt gtcaagagaa ggggcgaggg 1440
gccacacact ctccccgaaa ctaggaagag accaaaggat cctttgcgca tacaccgatc
1500 tattgttgta tcctcgacac taaccgacaa aagtcggtta gtcgtgactg
agtaaacgta 1560 tcggaaatgt tcgccagtgt ttgagttctt tgctcgccaa
aattatcaga aaatcttata 1620 acaaatagct tggcttattc cttgacacga
aacactaagt gtatagtcac ctccccacac 1680 ctttaccgtg gaactagagt
gggagtagct ttcacctcaa ctacaggaag ggaccgagcg 1740 atgtctgcgt
gaaggcgttc aaaagaaaac ttctagaaga gaaagtagat ttaaaaacct 1800
cgtccaaaag actgaagcca gccttttggg gagggttaaa gtgaccagat gttactttcg
1860 ttttgtcaag agaaggggcg aggggccaca cactctcccc gaaactagga
agagaccaaa 1920 ggatcctttg cgcatacaca tgttggggtg agggagagac
gcgcgagcga gcgagtgact 1980 ccggcccgct ggtttccagc gggctgcggg
cccgaaacgg gcccgccgga gtcactcgct 2040 cgctcgcgcg tc 2052
<210> SEQ ID NO 16 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 16 Trp
Val Asn Ala Gln Phe Ser Lys 1 5 <210> SEQ ID NO 17
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 17 Gln His Ile Glu Asn Leu Phe
Ser Asp Leu Gln Asp Gly Arg 1 5 10 <210> SEQ ID NO 18
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 18 Leu Leu Asp Leu Leu Glu Gly
Leu Thr Gly Gln Lys 1 5 10 <210> SEQ ID NO 19 <211>
LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 19 Val Leu Gln Asn Asn Asn Val Asp Leu Val
Asn Ile Gly Ser Thr Asp 1 5 10 15 Ile Val Asp Gly Asn His Lys 20
<210> SEQ ID NO 20 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 Asn
Leu Met Ala Gly Leu Gln Gln Thr Asn Ser Glu Lys 1 5 10 <210>
SEQ ID NO 21 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 21 Leu Glu
His Ala Phe Asn Ile Ala Arg 1 5 <210> SEQ ID NO 22
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 22 Tyr Gln Leu Gly Ile Glu Lys 1
5 <210> SEQ ID NO 23 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 23 Leu
Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp Lys Lys 1 5 10 15
<210> SEQ ID NO 24 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 24 Ser
Tyr Ala Tyr Thr Gln Ala Ala Tyr Val Thr Thr Ser Asp Pro Thr 1 5 10
15 Arg <210> SEQ ID NO 25 <211> LENGTH: 14 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
25 Ser Pro Phe Pro Ser Gln His Leu Glu Ala Pro Glu Asp Lys 1 5 10
<210> SEQ ID NO 26 <211> LENGTH: 2052 <212> TYPE:
DNA <213> ORGANISM: Adeno-associated virus <400>
SEQUENCE: 26 gctggcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt
cgggcgacct 60 ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
gggagtggcc aactccatca 120 ctaggggttc cttgtagtta atgattaacc
cgccatgcta cttatctacg tagccatgct 180 ctagataaca acataggagc
tgtgattggc tgttttcagc caatcagcac tgactcattt 240 gcatagcctt
tacaagcggt cacaaactca agaaacgagc ggttttaata gtcttttaga 300
atattgttta tcgaaccgaa taaggaactg tgctttgtga ttcacatatc agtggagggg
360 tgtggaaatg gcaccttgat ctcaccctca tcgaaagtgg agttgatgtc
cttccctggc 420 tcgctacaga cgcacttccg caagttttct tttgaagatc
ttctctttca tctaaatttt 480 tggagcaggt tttctgactt cggtcggaaa
acccctccca atttcactgg tctacaatga 540 aagcaaaaca gttctcttcc
ccgctccccg gtgtgtgaga ggggctttga tccttctctg 600 gtttcctagg
aaacgcgtat gtggctagat aacaacatag gagctgtgat tggctgtttt 660
cagccaatca gcactgactc atttgcatag cctttacaag cggtcacaaa ctcaagaaac
720 gagcggtttt aatagtcttt tagaatattg tttatcgaac cgaataagga
actgtgcttt 780 gtgattcaca tatcagtgga ggggtgtgga aatggcacct
tgatctcacc ctcatcgaaa 840 gtggagttga tgtccttccc tggctcgcta
cagacgcact tccgcaaatt cttaccttag 900 aaaattgtgc aatttttgga
gcaggttttc tgacttcggt cggaaaaccc ctcccaattt 960 cactggtcta
caatgaaagc aaaacagttc tcttccccgc tccccggtgt gtgagagggg 1020
ctttgatcct tctctggttt cctaggaaac gcgtatgtgg ctagataaca acataggagc
1080 tgtgattggc tgttttcagc caatcagcac tgactcattt gcatagcctt
tacaagcggt 1140 cacaaactca agaaacgagc ggttttaata gtcttttaga
atattgttta tcgaaccgaa 1200 taaggaactg tgctttgtga ttcacatatc
agtggagggg tgtggaaatg gcaccttgat 1260 ctcaccctca tcgaaagtgg
agttgatgtc cttccctggc tcgctacaga cgcacttccg 1320 caaattctta
ccttagaaaa ttgtgcaatt tttggagcag gttttctgac ttcggtcgga 1380
aaacccctcc caatttcact ggtctacaat gaaagcaaaa cagttctctt ccccgctccc
1440 cggtgtgtga gaggggcttt gatccttctc tggtttccta ggaaacgcgt
atgtggctag 1500 ataacaacat aggagctgtg attggctgtt ttcagccaat
cagcactgac tcatttgcat 1560 agcctttaca agcggtcaca aactcaagaa
acgagcggtt ttaatagtct tttagaatat 1620 tgtttatcga accgaataag
gaactgtgct ttgtgattca catatcagtg gaggggtgtg 1680 gaaatggcac
cttgatctca ccctcatcga aagtggagtt gatgtccttc cctggctcgc 1740
tacagacgca cttccgcaag ttttcttttg aagatcttct ctttcatcta aatttttgga
1800 gcaggttttc tgacttcggt cggaaaaccc ctcccaattt cactggtcta
caatgaaagc 1860 aaaacagttc tcttccccgc tccccggtgt gtgagagggg
ctttgatcct tctctggttt 1920 cctaggaaac gcgtatgtgt acaaccccac
tccctctctg cgcgctcgct cgctcactga 1980 ggccgggcga ccaaaggtcg
cccgacgccc gggctttgcc cgggcggcct cagtgagcga 2040 gcgagcgcgc ag 2052
<210> SEQ ID NO 27 <211> LENGTH: 240 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <300> PUBLICATION
INFORMATION: <308> DATABASE ACCESSION NUMBER:
UniProtKB/Swiss-Prot: P11532.3 <309> DATABASE ENTRY DATE:
2015-07-22 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(240)
<400> SEQUENCE: 27 Met Leu Trp Trp Glu Glu Val Glu Asp Cys
Tyr Glu Arg Glu Asp Val 1 5 10 15 Gln Lys Lys Thr Phe Thr Lys Trp
Val Asn Ala Gln Phe Ser Lys Phe 20 25 30 Gly Lys Gln His Ile Glu
Asn Leu Phe Ser Asp Leu Gln Asp Gly Arg 35 40 45 Arg Leu Leu Asp
Leu Leu Glu Gly Leu Thr Gly Gln Lys Leu Pro Lys 50 55 60 Glu Lys
Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Lys Ala 65 70 75 80
Leu Arg Val Leu Gln Asn Asn Asn Val Asp Leu Val Asn Ile Gly Ser 85
90 95 Thr Asp Ile Val Asp Gly Asn His Lys Leu Thr Leu Gly Leu Ile
Trp 100 105 110 Asn Ile Ile Leu His Trp Gln Val Lys Asn Val Met Lys
Asn Ile Met 115 120 125 Ala Gly Leu Gln Gln Thr Asn Ser Glu Lys Ile
Leu Leu Ser Trp Val 130 135 140 Arg Gln Ser Thr Arg Asn Tyr Pro Gln
Val Asn Val Ile Asn Phe Thr 145 150 155 160 Thr Ser Trp Ser Asp Gly
Leu Ala Leu Asn Ala Leu Ile His Ser His 165 170 175 Arg Pro Asp Leu
Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala 180 185 190 Thr Gln
Arg Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly 195 200 205
Ile Glu Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp 210
215 220 Lys Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu
Pro 225 230 235 240 <210> SEQ ID NO 28 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 28 Tyr Gln Leu Gly Ile Glu Lys Leu Leu Asp
Pro Glu Asp Val Asp Thr 1 5 10 15 Thr Tyr Pro Asp Lys Lys 20
<210> SEQ ID NO 29 <211> LENGTH: 112 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 29 Trp
Val Asn Ala Gln Phe Ser Lys Phe Gly Lys Gln His Ile Glu Asn 1 5 10
15 Leu Phe Ser Asp Leu Gln Asp Gly Arg Arg Leu Leu Asp Leu Leu Glu
20 25 30 Gly Leu Thr Gly Gln Lys Val Leu Gln Asn Asn Asn Val Asp
Leu Val 35 40 45 Asn Ile Gly Ser Thr Asp Ile Val Asp Gly Asn His
Lys Asn Ile Met 50 55 60 Ala Gly Leu Gln Gln Thr Asn Ser Glu Lys
Ile Leu Leu Leu Glu His 65 70 75 80 Ala Phe Asn Ile Ala Arg Tyr Gln
Leu Gly Ile Glu Lys Leu Leu Asp 85 90 95 Pro Glu Asp Val Asp Thr
Thr Tyr Pro Asp Lys Lys Ser Ile Leu Met 100 105 110 <210> SEQ
ID NO 30 <211> LENGTH: 75 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 30 tagatgaaag
agaagatgtt caaaagaaaa cattcacaaa atgggtaaat gcacaatttt 60
ctaaggtaag aatgg 75 <210> SEQ ID NO 31 <211> LENGTH: 80
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 31 aactccattt atcaattaac caaaaactcc
caaataactg tgtaggttcc atgagttcag 60 gagattccac ttcaactaat 80
<210> SEQ ID NO 32 <211> LENGTH: 80 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 32
aactccattt atcaattaac caaaaactcc caaataactg tggtgtatat ttatctattt
60 ttatgggttg caaaatactt 80 <210> SEQ ID NO 33 <211>
LENGTH: 80 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 33 caaattctcc cctgaaaatt taaaaaaata
cattgttctg tggtgtatat ttatctattt 60 ttatgggttg caaaatactt 80
<210> SEQ ID NO 34 <211> LENGTH: 55 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 34
aattaaccaa aaactcccaa ataactgtgg tgtatattta tctattttta tgggt 55
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 34 <210>
SEQ ID NO 1 <211> LENGTH: 28 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 1
tcaaaagaaa acattcacaa aatgggta 28 <210> SEQ ID NO 2
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 2 gcacaatttt ctaaggtaag aat 23
<210> SEQ ID NO 3 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3
tagatgaaag agaagatgtt caaaagaaaa c 31 <210> SEQ ID NO 4
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 4 tagatgaaag agaagatgtt c 21
<210> SEQ ID NO 5 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Adeno-associated virus <400>
SEQUENCE: 5 tacccatttt gcgaatgttt tcttttga 28 <210> SEQ ID NO
6 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Adeno-associated virus <400> SEQUENCE: 6 attcttacct
tagaaaattg tgc 23 <210> SEQ ID NO 7 <211> LENGTH: 31
<212> TYPE: DNA <213> ORGANISM: Adeno-associated virus
<400> SEQUENCE: 7 gttttctttt gaagatcttc tctttcatct a 31
<210> SEQ ID NO 8 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Adeno-associated virus <400>
SEQUENCE: 8 gaagatcttc tctttcatct a 21 <210> SEQ ID NO 9
<211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 9 uacccauuuu gcgaauguuu ucuuuuga
28 <210> SEQ ID NO 10 <211> LENGTH: 23 <212>
TYPE: RNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
10 auucuuaccu uagaaaauug ugc 23 <210> SEQ ID NO 11
<211> LENGTH: 31 <212> TYPE: RNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 11 guuuucuuuu gaacaucuuc
ucuuucaucu a 31 <210> SEQ ID NO 12 <211> LENGTH: 21
<212> TYPE: RNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12 gaacaucuuc ucuuucaucu a 21 <210> SEQ
ID NO 13 <211> LENGTH: 30 <212> TYPE: RNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 13 ccauuuugug
aauguuuucu uuugaacauc 30 <210> SEQ ID NO 14 <211>
LENGTH: 4472 <212> TYPE: DNA <213> ORGANISM:
Adeno-associated virus rh74 <400> SEQUENCE: 14 ctccatcact
aggggtaacc gcgaagcgcc tcccacgctg ccgcgtcagc gctgacgtaa 60
attacgtcat aggggagtgg tcctgtatta gctgtcacgt gagtgctttt gcgacatttt
120 gcgacaccac gtggccattc atggtatata tggccgagtg agcgagcagg
atctccattt 180 tgaccgcgaa atttgaacga gcagcagcca tgccgggctt
ctacgagatc gtgcttaagg 240 tgccgagcga cctggacgag cacctgccgg
gcatttctga ctcgtttgtg aactgggtgg 300 cagagaagga atgggagctg
cccccggatt ctgacatgga tcggaatctg attgagcagg 360 cacccctgac
cgtggccgag aagctacagc gcgacttcct ggtccaatgg cgccgcgtga 420
gtaaggcccc ggaggccctc ttctttgttc agttcgagaa gggcgagtcc tacttccacc
480 tccatattct ggtagagacc acgggggtca aatccatggt gctgggccgc
ttcctgagtc 540 agattcggga caagctggtg cagaccatct accgcgggat
cgagccgacc ctgcccaact 600 ggttcgcggt gacaaagacg cgtaatggcg
ccggaggggg gaacaaggtg gtggacgagt 660 gctacatccc caactacctg
ctgcccaaga ctcagcccga gctgcagtgg gcgtggacta 720 acatggagga
gtatataagc gcgtgcttga acctggccga gcgcaaacgg ctcgtggcgc 780
agcacctgac ccacgtcagc cagacccagg agcagaacaa ggagaatctg aacccgaatt
840 ctgacgcgcc tgtcatccgg tcaaaaacct ccgcgcgcta catggagctg
gtcgggtggc 900 tggtggaccg gggcatcacc tccgagaagc agtggatcca
ggaggaccag gcctcgtaca 960 tctccttcaa cgccgcctcc aactcgcggt
ctcagatcaa ggccgcgctg gacaatgccg 1020 gcaagatcat ggcgctgacc
aaatccgcgc ccgactacct ggtaggcccc gctctgcccg 1080 cggacattaa
atccaaccgc atctaccgca tcctggagct gaatggctac gaccctgcct 1140
acgccggttc cgtctttctc ggctgggccc agaaaaagtt tggcaaaagg aacaccatct
1200 ggctgtttgg gccggccacc acgggcaaga ccaacatcgc ggaagccatc
gcccacgccg 1260 tgcccttcta cggctgcgtc aactggacca atgagaactt
tcccttcaac gattgcgtcg 1320 acaagatggt gatctggtgg gaggagggca
agatgacggc caaggtcgtg gagtccgcca 1380 aggccattct cggcggcagc
aaggtgcgcg tggaccaaaa gtgcaagtcg tccgcccaga 1440 tcgatcccac
ccccgtgatc gtcacctcca acaccaacat gtgcgccgtg attgacggga 1500
acagcaccac cttcgagcac cagcagccgt tgcaggaccg gatgttcaaa tttgaactta
1560 cccgccgtct ggagcacgac tttggcaagg tgacaaagca ggaagtcaaa
gagttcttcc 1620 gctgggcgca ggatcacgtg accgaggtgg cgcatgagtt
ctacgtcaga aagggtggag 1680 ctaacaaaag acccgccccc gatgacgcgg
atataagcga gcccaagcgg gcctgcccct 1740 cagtcgcgga tccatcgacg
tcagacgcgg aaggagctcc ggtggacttt gccgacaggt 1800 accaaaacaa
atgttctcgt cacgcgggca tgcttcagat gctgtttccc tgcaaaacat 1860
gcgagagaat gaatcagaat ttcaacattt gcttcacgca cgggaccaga gactgttcag
1920 aatgtttccc tggcgtgtca gaatctcaac cggtcgtcag aaaaaagacg
tatcggaaac 1980 tctgtgcgat tcatcatctg ctggggcggg cacccgagat
tgcttgctcg gcctgcgacc 2040 tggtcaacgt ggacctggat gactgtgttt
ctgagcaata aatgacttaa accaggtatg 2100 gctgccgatg gttatcttcc
agattggctc gaggacaacc tctctgaggg cattcgcgag 2160 tggtgggacc
tgaaacctgg agccccgaaa cccaaagcca accagcaaaa gcaggacaac 2220
ggccggggtc tggtgcttcc tggctacaag tacctcggac ccttcaacgg actcgacaag
2280 ggggagcccg tcaacgcggc ggacgcagcg gccctcgagc acgacaaggc
ctacgaccag 2340 cagctccaag cgggtgacaa tccgtacctg cggtataatc
acgccgacgc cgagtttcag 2400 gagcgtctgc aagaagatac gtcttttggg
ggcaacctcg ggcgcgcagt cttccaggcc 2460 aaaaagcggg ttctcgaacc
tctgggcctg gttgaatcgc cggttaagac ggctcctgga 2520 aagaagagac
cggtagagcc atcaccccag cgctctccag actcctctac gggcatcggc 2580
aagaaaggcc agcagcccgc aaaaaagaga ctcaattttg ggcagactgg cgactcagag
2640 tcagtccccg accctcaacc aatcggagaa ccaccagcag gcccctctgg
tctgggatct 2700 ggtacaatgg ctgcaggcgg tggcgctcca atggcagaca
ataacgaagg cgccgacgga 2760 gtgggtagtt cctcaggaaa ttggcattgc
gattccacat ggctgggcga cagagtcatc 2820 accaccagca cccgcacctg
ggccctgccc acctacaaca accacctcta caagcaaatc 2880 tccaacggga
cctcgggagg aagcaccaac gacaacacct acttcggcta cagcaccccc 2940
tgggggtatt ttgacttcaa cagattccac tgccactttt caccacgtga ctggcagcga
3000 ctcatcaaca acaactgggg attccggccc aagaggctca acttcaagct
cttcaacatc 3060 caagtcaagg aggtcacgca gaatgaaggc accaagacca
tcgccaataa ccttaccagc 3120 acgattcagg tctttacgga ctcggaatac
cagctcccgt acgtgctcgg ctcggcgcac 3180 cagggctgcc tgcctccgtt
cccggcggac gtcttcatga ttcctcagta cgggtacctg 3240
actctgaaca atggcagtca ggctgtgggc cggtcgtcct tctactgcct ggagtacttt
3300 ccttctcaaa tgctgagaac gggcaacaac tttgaattca gctacaactt
cgaggacgtg 3360 cccttccaca gcagctacgc gcacagccag agcctggacc
ggctgatgaa ccctctcatc 3420 gaccagtact tgtactacct gtcccggact
caaagcacgg gcggtactgc aggaactcag 3480 cagttgctat tttctcaggc
cgggcctaac aacatgtcgg ctcaggccaa gaactggcta 3540 cccggtccct
gctaccggca gcaacgcgtc tccacgacac tgtcgcagaa caacaacagc 3600
aactttgcct ggacgggtgc caccaagtat catctgaatg gcagagactc tctggtgaat
3660 cctggcgttg ccatggctac ccacaaggac gacgaagagc gattttttcc
atccagcgga 3720 gtcttaatgt ttgggaaaca gggagctgga aaagacaacg
tggactatag cagcgtgatg 3780 ctaaccagcg aggaagaaat aaagaccacc
aacccagtgg ccacagaaca gtacggcgtg 3840 gtggccgata acctgcaaca
gcaaaacgcc gctcctattg taggggccgt caatagtcaa 3900 ggagccttac
ctggcatggt gtggcagaac cgggacgtgt acctgcaggg tcccatctgg 3960
gccaagattc ctcatacgga cggcaacttt catccctcgc cgctgatggg aggctttgga
4020 ctgaagcatc cgcctcctca gatcctgatt aaaaacacac ctgttcccgc
ggatcctccg 4080 accaccttca ctaaggccaa gctggcttct ttcatcacgc
agtacagtac cggccaggtc 4140 agcgtggaga tcgagtggga gctgcagaag
gagaacagca aacgctggaa cccagagatt 4200 cagtacactt ccaactacta
caaatctaca aatgtggact ttgctgtcaa tactgagggt 4260 acttattccg
agcctcgccc cattggcacc cgttacctca cccgtaatct gtaattacat 4320
gttaatcaat aaaccggtta attcgtttca gttgaacttt ggtctcctgt ccttcttatc
4380 ttatcggtta ccatagaaac tggttactta ttaactgctt ggtgcgcttc
gcgataaaag 4440 acttacgtca tcgggttacc cctagtgatg ga 4472
<210> SEQ ID NO 15 <211> LENGTH: 2052 <212> TYPE:
DNA <213> ORGANISM: Adeno-associated virus <400>
SEQUENCE: 15 cgaccgcgcg agcgagcgag tgactccggc gggcccgttt cgggcccgca
gcccgctgga 60 aaccagcggg ccggagtcac tcgctcgctc gcgcgtctct
ccctcaccgg ttgaggtagt 120 gatccccaag gaacatcaat tactaattgg
gcggtacgat gaatagatgc atcggtacga 180 gatctattgt tgtatcctcg
acactaaccg acaaaagtcg gttagtcgtg actgagtaaa 240 cgtatcggaa
atgttcgcca gtgtttgagt tctttgctcg ccaaaattat cagaaaatct 300
tataacaaat agcttggctt attccttgac acgaaacact aagtgtatag tcacctcccc
360 acacctttac cgtggaacta gagtgggagt agctttcacc tcaactacag
gaagggaccg 420 agcgatgtct gcgtgaaggc gttcaaaaga aaacttctag
aagagaaagt agatttaaaa 480 acctcgtcca aaagactgaa gccagccttt
tggggagggt taaagtgacc agatgttact 540 ttcgttttgt caagagaagg
ggcgaggggc cacacactct ccccgaaact aggaagagac 600 caaaggatcc
tttgcgcata caccgatcta ttgttgtatc ctcgacacta accgacaaaa 660
gtcggttagt cgtgactgag taaacgtatc ggaaatgttc gccagtgttt gagttctttg
720 ctcgccaaaa ttatcagaaa atcttataac aaatagcttg gcttattcct
tgacacgaaa 780 cactaagtgt atagtcacct ccccacacct ttaccgtgga
actagagtgg gagtagcttt 840 cacctcaact acaggaaggg accgagcgat
gtctgcgtga aggcgtttaa gaatggaatc 900 ttttaacacg ttaaaaacct
cgtccaaaag actgaagcca gccttttggg gagggttaaa 960 gtgaccagat
gttactttcg ttttgtcaag agaaggggcg aggggccaca cactctcccc 1020
gaaactagga agagaccaaa ggatcctttg cgcatacacc gatctattgt tgtatcctcg
1080 acactaaccg acaaaagtcg gttagtcgtg actgagtaaa cgtatcggaa
atgttcgcca 1140 gtgtttgagt tctttgctcg ccaaaattat cagaaaatct
tataacaaat agcttggctt 1200 attccttgac acgaaacact aagtgtatag
tcacctcccc acacctttac cgtggaacta 1260 gagtgggagt agctttcacc
tcaactacag gaagggaccg agcgatgtct gcgtgaaggc 1320 gtttaagaat
ggaatctttt aacacgttaa aaacctcgtc caaaagactg aagccagcct 1380
tttggggagg gttaaagtga ccagatgtta ctttcgtttt gtcaagagaa ggggcgaggg
1440 gccacacact ctccccgaaa ctaggaagag accaaaggat cctttgcgca
tacaccgatc 1500 tattgttgta tcctcgacac taaccgacaa aagtcggtta
gtcgtgactg agtaaacgta 1560 tcggaaatgt tcgccagtgt ttgagttctt
tgctcgccaa aattatcaga aaatcttata 1620 acaaatagct tggcttattc
cttgacacga aacactaagt gtatagtcac ctccccacac 1680 ctttaccgtg
gaactagagt gggagtagct ttcacctcaa ctacaggaag ggaccgagcg 1740
atgtctgcgt gaaggcgttc aaaagaaaac ttctagaaga gaaagtagat ttaaaaacct
1800 cgtccaaaag actgaagcca gccttttggg gagggttaaa gtgaccagat
gttactttcg 1860 ttttgtcaag agaaggggcg aggggccaca cactctcccc
gaaactagga agagaccaaa 1920 ggatcctttg cgcatacaca tgttggggtg
agggagagac gcgcgagcga gcgagtgact 1980 ccggcccgct ggtttccagc
gggctgcggg cccgaaacgg gcccgccgga gtcactcgct 2040 cgctcgcgcg tc 2052
<210> SEQ ID NO 16 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 16 Trp
Val Asn Ala Gln Phe Ser Lys 1 5 <210> SEQ ID NO 17
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 17 Gln His Ile Glu Asn Leu Phe
Ser Asp Leu Gln Asp Gly Arg 1 5 10 <210> SEQ ID NO 18
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 18 Leu Leu Asp Leu Leu Glu Gly
Leu Thr Gly Gln Lys 1 5 10 <210> SEQ ID NO 19 <211>
LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 19 Val Leu Gln Asn Asn Asn Val Asp Leu Val
Asn Ile Gly Ser Thr Asp 1 5 10 15 Ile Val Asp Gly Asn His Lys 20
<210> SEQ ID NO 20 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 Asn
Leu Met Ala Gly Leu Gln Gln Thr Asn Ser Glu Lys 1 5 10 <210>
SEQ ID NO 21 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 21 Leu Glu
His Ala Phe Asn Ile Ala Arg 1 5 <210> SEQ ID NO 22
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 22 Tyr Gln Leu Gly Ile Glu Lys 1
5 <210> SEQ ID NO 23 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 23 Leu
Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp Lys Lys 1 5 10 15
<210> SEQ ID NO 24 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 24 Ser
Tyr Ala Tyr Thr Gln Ala Ala Tyr Val Thr Thr Ser Asp Pro Thr 1 5 10
15 Arg <210> SEQ ID NO 25 <211> LENGTH: 14 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
25 Ser Pro Phe Pro Ser Gln His Leu Glu Ala Pro Glu Asp Lys 1 5 10
<210> SEQ ID NO 26 <211> LENGTH: 2052 <212> TYPE:
DNA <213> ORGANISM: Adeno-associated virus <400>
SEQUENCE: 26 gctggcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt
cgggcgacct 60 ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
gggagtggcc aactccatca 120 ctaggggttc cttgtagtta atgattaacc
cgccatgcta cttatctacg tagccatgct 180 ctagataaca acataggagc
tgtgattggc tgttttcagc caatcagcac tgactcattt 240
gcatagcctt tacaagcggt cacaaactca agaaacgagc ggttttaata gtcttttaga
300 atattgttta tcgaaccgaa taaggaactg tgctttgtga ttcacatatc
agtggagggg 360 tgtggaaatg gcaccttgat ctcaccctca tcgaaagtgg
agttgatgtc cttccctggc 420 tcgctacaga cgcacttccg caagttttct
tttgaagatc ttctctttca tctaaatttt 480 tggagcaggt tttctgactt
cggtcggaaa acccctccca atttcactgg tctacaatga 540 aagcaaaaca
gttctcttcc ccgctccccg gtgtgtgaga ggggctttga tccttctctg 600
gtttcctagg aaacgcgtat gtggctagat aacaacatag gagctgtgat tggctgtttt
660 cagccaatca gcactgactc atttgcatag cctttacaag cggtcacaaa
ctcaagaaac 720 gagcggtttt aatagtcttt tagaatattg tttatcgaac
cgaataagga actgtgcttt 780 gtgattcaca tatcagtgga ggggtgtgga
aatggcacct tgatctcacc ctcatcgaaa 840 gtggagttga tgtccttccc
tggctcgcta cagacgcact tccgcaaatt cttaccttag 900 aaaattgtgc
aatttttgga gcaggttttc tgacttcggt cggaaaaccc ctcccaattt 960
cactggtcta caatgaaagc aaaacagttc tcttccccgc tccccggtgt gtgagagggg
1020 ctttgatcct tctctggttt cctaggaaac gcgtatgtgg ctagataaca
acataggagc 1080 tgtgattggc tgttttcagc caatcagcac tgactcattt
gcatagcctt tacaagcggt 1140 cacaaactca agaaacgagc ggttttaata
gtcttttaga atattgttta tcgaaccgaa 1200 taaggaactg tgctttgtga
ttcacatatc agtggagggg tgtggaaatg gcaccttgat 1260 ctcaccctca
tcgaaagtgg agttgatgtc cttccctggc tcgctacaga cgcacttccg 1320
caaattctta ccttagaaaa ttgtgcaatt tttggagcag gttttctgac ttcggtcgga
1380 aaacccctcc caatttcact ggtctacaat gaaagcaaaa cagttctctt
ccccgctccc 1440 cggtgtgtga gaggggcttt gatccttctc tggtttccta
ggaaacgcgt atgtggctag 1500 ataacaacat aggagctgtg attggctgtt
ttcagccaat cagcactgac tcatttgcat 1560 agcctttaca agcggtcaca
aactcaagaa acgagcggtt ttaatagtct tttagaatat 1620 tgtttatcga
accgaataag gaactgtgct ttgtgattca catatcagtg gaggggtgtg 1680
gaaatggcac cttgatctca ccctcatcga aagtggagtt gatgtccttc cctggctcgc
1740 tacagacgca cttccgcaag ttttcttttg aagatcttct ctttcatcta
aatttttgga 1800 gcaggttttc tgacttcggt cggaaaaccc ctcccaattt
cactggtcta caatgaaagc 1860 aaaacagttc tcttccccgc tccccggtgt
gtgagagggg ctttgatcct tctctggttt 1920 cctaggaaac gcgtatgtgt
acaaccccac tccctctctg cgcgctcgct cgctcactga 1980 ggccgggcga
ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga 2040
gcgagcgcgc ag 2052 <210> SEQ ID NO 27 <211> LENGTH: 240
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION
NUMBER: UniProtKB/Swiss-Prot: P11532.3 <309> DATABASE ENTRY
DATE: 2015-07-22 <313> RELEVANT RESIDUES IN SEQ ID NO:
(1)..(240) <400> SEQUENCE: 27 Met Leu Trp Trp Glu Glu Val Glu
Asp Cys Tyr Glu Arg Glu Asp Val 1 5 10 15 Gln Lys Lys Thr Phe Thr
Lys Trp Val Asn Ala Gln Phe Ser Lys Phe 20 25 30 Gly Lys Gln His
Ile Glu Asn Leu Phe Ser Asp Leu Gln Asp Gly Arg 35 40 45 Arg Leu
Leu Asp Leu Leu Glu Gly Leu Thr Gly Gln Lys Leu Pro Lys 50 55 60
Glu Lys Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Lys Ala 65
70 75 80 Leu Arg Val Leu Gln Asn Asn Asn Val Asp Leu Val Asn Ile
Gly Ser 85 90 95 Thr Asp Ile Val Asp Gly Asn His Lys Leu Thr Leu
Gly Leu Ile Trp 100 105 110 Asn Ile Ile Leu His Trp Gln Val Lys Asn
Val Met Lys Asn Ile Met 115 120 125 Ala Gly Leu Gln Gln Thr Asn Ser
Glu Lys Ile Leu Leu Ser Trp Val 130 135 140 Arg Gln Ser Thr Arg Asn
Tyr Pro Gln Val Asn Val Ile Asn Phe Thr 145 150 155 160 Thr Ser Trp
Ser Asp Gly Leu Ala Leu Asn Ala Leu Ile His Ser His 165 170 175 Arg
Pro Asp Leu Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala 180 185
190 Thr Gln Arg Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly
195 200 205 Ile Glu Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr
Pro Asp 210 215 220 Lys Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe
Gln Val Leu Pro 225 230 235 240 <210> SEQ ID NO 28
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 28 Tyr Gln Leu Gly Ile Glu Lys
Leu Leu Asp Pro Glu Asp Val Asp Thr 1 5 10 15 Thr Tyr Pro Asp Lys
Lys 20 <210> SEQ ID NO 29 <211> LENGTH: 112 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
29 Trp Val Asn Ala Gln Phe Ser Lys Phe Gly Lys Gln His Ile Glu Asn
1 5 10 15 Leu Phe Ser Asp Leu Gln Asp Gly Arg Arg Leu Leu Asp Leu
Leu Glu 20 25 30 Gly Leu Thr Gly Gln Lys Val Leu Gln Asn Asn Asn
Val Asp Leu Val 35 40 45 Asn Ile Gly Ser Thr Asp Ile Val Asp Gly
Asn His Lys Asn Ile Met 50 55 60 Ala Gly Leu Gln Gln Thr Asn Ser
Glu Lys Ile Leu Leu Leu Glu His 65 70 75 80 Ala Phe Asn Ile Ala Arg
Tyr Gln Leu Gly Ile Glu Lys Leu Leu Asp 85 90 95 Pro Glu Asp Val
Asp Thr Thr Tyr Pro Asp Lys Lys Ser Ile Leu Met 100 105 110
<210> SEQ ID NO 30 <211> LENGTH: 75 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 30
tagatgaaag agaagatgtt caaaagaaaa cattcacaaa atgggtaaat gcacaatttt
60 ctaaggtaag aatgg 75 <210> SEQ ID NO 31 <211> LENGTH:
80 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 31 aactccattt atcaattaac caaaaactcc
caaataactg tgtaggttcc atgagttcag 60 gagattccac ttcaactaat 80
<210> SEQ ID NO 32 <211> LENGTH: 80 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 32
aactccattt atcaattaac caaaaactcc caaataactg tggtgtatat ttatctattt
60 ttatgggttg caaaatactt 80 <210> SEQ ID NO 33 <211>
LENGTH: 80 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 33 caaattctcc cctgaaaatt taaaaaaata
cattgttctg tggtgtatat ttatctattt 60 ttatgggttg caaaatactt 80
<210> SEQ ID NO 34 <211> LENGTH: 55 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 34
aattaaccaa aaactcccaa ataactgtgg tgtatattta tctattttta tgggt 55
* * * * *