U.S. patent application number 13/287583 was filed with the patent office on 2013-10-31 for gene therapy for neurodegenerative disorders.
This patent application is currently assigned to GENZYME CORPORATION. The applicant listed for this patent is Seng H. CHENG, Marco A. PASSINI, Lamya SHIHABUDDIN. Invention is credited to Seng H. CHENG, Marco A. PASSINI, Lamya SHIHABUDDIN.
Application Number | 20130287736 13/287583 |
Document ID | / |
Family ID | 43050324 |
Filed Date | 2013-10-31 |
United States Patent
Application |
20130287736 |
Kind Code |
A1 |
PASSINI; Marco A. ; et
al. |
October 31, 2013 |
GENE THERAPY FOR NEURODEGENERATIVE DISORDERS
Abstract
Compositions and methods for treating disorders affecting motor
function, such as motor function affected by disease or injury to
the brain and/or spinal cord, are disclosed.
Inventors: |
PASSINI; Marco A.;
(Bridgewater, NJ) ; SHIHABUDDIN; Lamya;
(Bridgewater, NJ) ; CHENG; Seng H.; (Bridgewater,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PASSINI; Marco A.
SHIHABUDDIN; Lamya
CHENG; Seng H. |
Bridgewater
Bridgewater
Bridgewater |
NJ
NJ
NJ |
US
US
US |
|
|
Assignee: |
GENZYME CORPORATION
Cambridge
MA
|
Family ID: |
43050324 |
Appl. No.: |
13/287583 |
Filed: |
November 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2010/001239 |
Apr 27, 2010 |
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13287583 |
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61174982 |
May 2, 2009 |
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61268059 |
Jun 8, 2009 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/320.1 |
Current CPC
Class: |
A61K 48/005 20130101;
C12N 7/00 20130101; A61K 38/1709 20130101; A61P 21/00 20180101;
A61K 48/00 20130101; C12N 2750/14133 20130101; A61P 21/02 20180101;
A61K 48/0075 20130101; C12N 2750/14121 20130101; A61K 48/0008
20130101; A61K 31/7088 20130101; C07K 14/4702 20130101; C12N
2750/14143 20130101; C12N 15/86 20130101; C12N 2750/14171 20130101;
A61P 25/00 20180101 |
Class at
Publication: |
424/93.2 ;
435/320.1; 435/235.1 |
International
Class: |
C12N 15/86 20060101
C12N015/86 |
Claims
1. A self-complementary adeno-associated virus (scAAV) vector
comprising a polynucleotide encoding a protein that modulates motor
function in a subject with a motor neuron disorder.
2. The scAAV vector of claim 1, wherein the motor neuron disorder
is selected from spinal muscular atrophy (SMA), amytrophic lateral
sclerosis (ALS), spinal bulbar muscular atrophy, spinal cerebellar
ataxia, primary lateral sclerosis (PLS), or traumatic spinal cord
injury.
3. The scAAV vector of claim 2, wherein the motor neuron disorder
is SMA.
4. The scAAV vector of claim 3, wherein the polynucleotide encodes
a survival motor neuron (SMN) protein.
5. The scAAV vector of claim 4, wherein the SMN protein is encoded
by human SMN-1.
6. The scAAV vector of claim 5, wherein the SMN protein comprises
an amino acid sequence with at least 90% sequence identity to SEQ
ID NO:2.
7. The scAAV vector of claim 6, wherein the SMN protein comprises
an amino acid sequence of SEQ ID NO:2.
8. A recombinant AAV virion, comprising the scAAV vector of claim
1.
9. A composition comprising a recombinant AAV virion according to
claim 8 and a pharmaceutically acceptable excipient.
10. A method of modulating motor function in a subject with a motor
neuron disorder comprising administering a therapeutically
effective amount of the composition of claim 9 to cells of the
subject.
11. A method of providing SMN protein to a subject with spinal
muscular atrophy (SMA) comprising administering a recombinant AAV
virion comprising an AAV vector according to claim 4 to cells of a
subject in need thereof.
12. The method of claim 10, wherein the composition is administered
via administration into at least one region of the deep cerebellar
nuclei of the cerebellum.
13. The method of claim 10, wherein the composition is administered
via direct spinal cord injection.
14. The method of claim 10, wherein the composition is administered
via intracerebroventricular injection
15. The method of claim 14, wherein the composition is administered
into at least one cerebral lateral ventricle.
16. The method of claim 10, wherein the composition is administered
via both intracerebroventricular injection and direct spinal cord
injection.
17. The method of claim 10, wherein the composition is administered
via intrathecal injection.
18-19. (canceled)
20. The method of claim 11, wherein the composition is administered
via administration into at least one region of the deep cerebellar
nuclei of the cerebellum.
21. The method of claim 11, wherein the composition is administered
via direct spinal cord injection.
22. The method of claim 11, wherein the composition is administered
via intracerebroventricular injection
23. The method of claim 22, wherein the composition is administered
into at least one cerebral lateral ventricle.
24. The method of claim 11, wherein the composition is administered
via both intracerebroventricular injection and direct spinal cord
injection.
25. The method of claim 11, wherein the composition is administered
via intrathecal injection.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of PCT/US2010/001239
filed Apr. 27, 2010, which claims the benefit under 35 USC
.sctn.119(e)(1) of U.S. Provisional Application Nos. 61/174/982,
filed May 2, 2009 and 61/268,059, filed Jun. 8, 2009, which
applications are incorporated herein by reference in their
entireties.
TECHNICAL FIELD
[0002] The present invention relates generally to gene delivery
methods. In particular, the invention relates to compositions and
methods for treating disorders affecting motor function, such as
motor function affected by disease or injury to the brain and/or
spinal cord.
DESCRIPTION OF THE INVENTION
[0003] Gene therapy is an emerging treatment modality for disorders
affecting the central nervous system (CNS). CNS gene therapy has
been facilitated by the development of viral vectors capable of
effectively infecting post-mitotic neurons. The central nervous
system is made up of the spinal cord and the brain. The spinal cord
conducts sensory information from the peripheral nervous system to
the brain and conducts motor information from the brain to various
effectors. For a review of viral vectors for gene delivery to the
central nervous system, see Davidson et al., Nature Rev. (2003)
4:353-364.
[0004] Adeno-associated virus (AAV) vectors are considered useful
for CNS gene therapy because they have a favorable toxicity and
immunogenicity profile, are able to transduce neuronal cells, and
are able to mediate long-term expression in the CNS (Kaplitt et
al., Nat. Genet. (1994) 8:148-154; Bartlett et al., Hum. Gene Ther.
(1998) 9:1181-1186; and Passini et al., J. Neurosci. (2002)
22:6437-6446).
[0005] One useful property of AAV vectors lies in the ability of
some AAV vectors to undergo retrograde and/or anterograde transport
in neuronal cells. Neurons in one brain region are interconnected
by axons to distal brain regions thereby providing a transport
system for vector delivery. For example, an AAV vector may be
administered at or near the axon terminals of neurons. The neurons
internalize the AAV vector and transport it in a retrograde manner
along the axon to the cell body. Similar properties of adenovirus,
HSV, and pseudo-rabies virus have been shown to deliver genes to
distal structures within the brain (Soudas et al., FASEB J. (2001)
15:2283-2285; Breakefield et al., New Biol. (1991) 3:203-218; and
deFalco et al., Science (2001) 291:2608-2613).
[0006] Several experimenters have reported that the transduction of
the brain by AAV serotype 2 (AAV2) is limited to the intracranial
injection site (Kaplitt et al., Nat. Genet. (1994) 8:148-154;
Passini et al., J. Neurosci. (2002) 22:6437-6446; and Chamberlin et
al., Brain Res. (1998) 793:169-175). There is also evidence that
retrograde axonal transport of neurotrophic viral vectors,
including AAV and lentiviral vectors, can also occur in select
circuits of the normal rat brain (Kaspar et al., Mol. Ther. (2002)
5:50-56; Kasper et al., Science (2003) 301:839-842 and Azzouz et
al., Nature (2004) 429:413-417. Roaul et al., Nat. Med. (2005)
11(4):423-428 and Ralph et al., Nat. Med. (2005) 11(4):429-433
report that intramuscular injection of lentivirus expressing
silencing human Cu/Zn superoxide dismutase (SODI) interfering RNA
retarded disease onset of amyotrophic lateral sclerosis (ALS) in a
therapeutically relevant rodent model of ALS.
[0007] Cells transduced by AAV vectors may express a therapeutic
transgene product, such as an enzyme or a neurotrophic factor, to
mediate beneficial effects intracellularly. These cells may also
secrete the therapeutic transgene product, which may be
subsequently taken up by distal cells where it may mediate its
beneficial effects. This process has been described as
cross-correction (Neufeld et al., Science (1970) 169:141-146).
[0008] A property of the recombinant AAV vectors described above is
the requirement that the single-stranded DNA (ssDNA) AAV genome
must be converted into double-stranded DNA (dsDNA) prior to
expression of the encoded transgene. This step can be circumvented
by the use of self-complementary vectors which package an inverted
repeat genome that folds into dsDNA without requiring DNA synthesis
or base-pairing between multiple vector genomes, thereby increasing
efficiency of AAV-mediated gene transfer. For a review of
self-complementary AAV vectors, see e.g., McCarty, D. M. Molec.
Ther. (2008) 16:1648-1656.
[0009] Spinal muscular atrophy (SMA) is an autosomal recessive
neuromuscular disorder caused by mutations in the survival motor
neuron 1 (SMN1) gene and loss of encoded SMN protein (Lefebvre et
al., Cell (1995) 80:155-165). The lack of SMN results in motor
neuron degeneration in the ventral (anterior) horn of the spinal
cord, which leads to weakness of the proximal muscles responsible
for crawling, walking, neck control and swallowing, and the
involuntary muscles that control breathing and coughing (Sumner C.
J., NeuroRx (2006) 3:235-245). Consequently, SMA patients present
with increased tendencies for pneumonia and other pulmonary
problems such as restrictive lung disease. The onset of disease and
degree of severity are determined in part by the phenotypic
modifier gene SMN2, which is capable of making a small amount of
SMN (Monani et al., Hum. Mol. Genet. (1999) 8:1177-1183; Lorson et
al., Proc. Natl. Acad. Sci. USA (1999) 96:6307-6311). Thus,
patients with a high SMN2 copy number (3-4 copies) exhibit a less
severe form of the disease (referred to as Types II or III),
whereas 1-2 copies of SMN2 typically result in the more severe Type
I disease (Campbell et al., Am. J. Hum. Genet. (1997) 61:40-50;
Lefebvre et al., Nat. Genet. (1997) 16:265-269). Currently, there
are no effective therapies for SMA.
[0010] A fundamental strategy for treating this monogenic disorder
is to increase SMN levels in SMA patients. One approach to
accomplish this is to modulate the endogenous SMN2 gene with small
molecules that activate the SMN2 promoter or correct the SMN2
pre-mRNA splicing pattern. The alteration of SMN2 splicing also can
be realized with antisense oligonucleotides and trans-splicing
RNAs. However, while modulating SMN2 in vitro increased SMN levels
and reconstituted nuclear gems in SMA cell lines, efficacy studies
with small molecule drugs have not translated to measurable
improvements in the clinic (Oskoui et al., Nerotherapeutics (2008)
5:499-506).
SUMMARY OF THE INVENTION
[0011] The present invention is based on the discovery that both
conventional recombinant AAV (rAAV) virions, as well as recombinant
self-complementary AAV vectors (scAAV), are able to deliver genes
to the CNS with successful expression in the CNS and treatment of
neurodegenerative disease. This therapy approach for the delivery
of genes encoding therapeutic molecules that result in at least
partial correction of neuropathologies provides a highly desirable
method for treating a variety of neurodegenerative disorders,
including SMA.
[0012] Thus in one embodiment, the invention is directed to a
self-complementary adeno-associated virus (scAAV) vector comprising
a polynucleotide encoding a protein that modulates motor function
in a subject with a motor neuron disorder. In certain embodiments,
the motor neuron disorder is selected from spinal muscular atrophy
(SMA), amytrophic lateral sclerosis (ALS), spinal bulbar muscular
atrophy, spinal cerebellar ataxia, primary lateral sclerosis (PLS),
or traumatic spinal cord injury.
[0013] In additional embodiments, the polynucleotide present in the
scAAV vector encodes a survival motor neuron (SMN) protein. In
certain embodiments, the SMN protein is human SMN-1. In further
embodiments, the SMN-1 comprises an amino acid sequence with at
least 90% sequence identity to the sequence depicted in FIG. 9B. In
additional embodiments, the SMN-1 comprises an amino acid sequence
as depicted in FIG. 9B.
[0014] In yet further embodiments, the invention is directed to a
recombinant AAV virion, comprising an scAAV vector as described
above.
[0015] In additional embodiments, the invention is directed to a
composition comprising a recombinant AAV virion as above and a
pharmaceutically acceptable excipient.
[0016] In further embodiments, the invention is directed to a
method of modulating motor function in a subject with a motor
neuron disorder comprising administering a therapeutically
effective amount of the composition above to cells of the subject.
In certain embodiments, the composition is administered to cells in
vitro to transduce the cells and the transduced cells are
administered to the subject. In alternative embodiments, the
composition is administered to cells in vivo.
[0017] In further embodiments, the invention is directed to a
method of providing an SMN protein to a subject with spinal
muscular atrophy (SMA) comprising administering a recombinant AAV
virion comprising an AAV vector as described above to cells of a
subject in need thereof. In certain embodiments the composition is
administered to cells in vitro to transduce the cells and the
transduced cells are administered to the subject. In alternative
embodiments, the composition is administered to cells in vivo.
[0018] In each of the methods above, the composition can be
administered via direct spinal cord injection. In other
embodiments, the composition is administered via
intracerebroventricular injection. In additional embodiments, the
composition is administered into a cerebral lateral ventricle. In
certain embodiments, the composition is administered into both
cerebral lateral ventricles. In other embodiments, the composition
is administered via both intracerebroventricular injection and
direct spinal cord injection. In additional embodiments, the
composition is administered by intrathecal injection.
[0019] These and other embodiments of the subject invention will
readily occur to those of skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows survival of mice treated with AAVhSMN1 versus
untreated SMA mice. Treatment with AAVhSMN1 increased survival in
SMA mice. Untreated SMA mice (n=34, open circles) had a median life
span of 15 days. SMA mice treated at P0 with AAVhSMN1 (n=24, closed
circles) had a median lifespan of 50 days (p<0.0001), which was
a+233% increase in longevity.
[0021] FIGS. 2A-2C show the effect of gene therapy treatment on SMN
levels in the spinal cord. Shown are hSMN protein levels in
injected lumbar (FIG. 2A), thoracic (FIG. 2B) and cervical (FIG.
2C) segments compared to untreated SMA and wild-type mice. Western
blots were performed on the lumbar, thoracic and cervical segments
of the spinal cord at 16, 58-66 and 120-220 days after injection.
The western blots from the three segments were quantified and, to
control for protein levels, SMN was normalized to .beta.-tubulin
and plotted as a percentage of age-matched wild type. Key (and
n-values): SMA, untreated knockout (n=5 at 16 days); AAV,
AAV8-hSMN-treated SMA mice (n=7 at 16 days, n=5 at 58-66 days);
scAAV, scAAV8-hSMN-treated SMA mice (n=5 at each time point).
Values represent the mean.+-.SEM.
[0022] FIGS. 3A-3J show the sub-cellular distribution of hSMN
protein and expression in motor neurons of the spinal cord in
treated and untreated SMA mice. hSMN protein was abundantly
detected in the cytoplasm of transduced cells (FIGS. 3A and 3B).
Furthermore, hSMN protein was detected in the nucleus, as
illustrated by the pair of gem-like structures (arrowhead)
magnified in the inset (FIG. 3A). hSMN protein was also detected in
the dendrites (FIGS. 3B and 3C) and axons (FIG. 3D) of neurons.
hSMN protein was not detectable on the tissue sections from
untreated SMA mice (FIG. 3E). Co-localization of hSMN protein (FIG.
3F) with mouse ChAT (FIG. 3G) showed that a subset of transduced
cells were motor neurons (FIGS. 3H and 31). The percentage of ChAT
cells that were immuno-positive for hSMN protein was determined at
16 (white bars) and 58-66 (black bars) days (FIG. 3J). Values
represent the mean.+-.SEM.
[0023] FIG. 4 shows motor neuron cell counts in the spinal cord in
treated and untreated SMA mice. Shown are the average numbers of
ChAT immuno-positive neurons counted on 10 .mu.m tissue sections
for each group. Numbers represent counts of every tenth section
from different levels of the cervical, thoracic, lumbar and sacral
segments. Values represent the mean.+-.SEM. Key: *, p<0.05; **,
p<0.01; ***, p<0.001.
[0024] FIGS. 5A-5C show the myofiber cross-section area from muscle
groups in treated and untreated SMA mice. The myofiber
cross-section area from multiple muscle groups was increased with
AAVhSMN1 treatment. Stacked graphs of the quadriceps, gastrocnemius
and intercostal muscles from 16 (FIG. 5A) and 58-66 (FIG. 5B) days
showed that the distribution of myofiber sizes were similar between
the treated SMA and the wild type mice. The overall average at 16
days showed that the myofiber cross-section area was significantly
higher with treatment (FIG. 5C). Furthermore at 58-66 days, the
average area was statistically similar between treated SMA mice and
age-matched wild-type in the gastrocnemius and intercostal muscles
(FIG. 5C). Values represent the mean.+-.SEM. Key: WT, untreated
wild type; HET, untreated heterozygote; SMA, untreated knockout;
AAV, AAVhSMN1-treated SMA mice; *, p<0.05; **, p<0.01; ***,
p<0.001.
[0025] FIGS. 6A-6F show the structure of the NMJ in muscles in
treated and untreated SMA mice. The structure in the quadriceps,
gastrocnemius, and intercostal was improved with gene therapy.
Shown are the neuromuscular junction (NMJ) from the quadriceps of
untreated SMA (FIG. 6A), treated SMA (FIG. 6B), and untreated wild
type (FIG. 6C) mice at 16 days, and from treated SMA (FIG. 6D) and
untreated wild type (FIG. 6E) mice at 58-66 days. The pre- and
post-synaptic NMJ was labeled with a neurofilament antibody (green)
and with .alpha.-bungarotoxin staining (red), respectively. The
arrowhead in the main panel points to the NMJ that is highlighted
in the insets below. At least 100 NMJs was randomly scored in each
muscle per animal. A normal NMJ was defined as having a
pre-synaptic terminus that did not exhibit the abnormal
accumulation of neurofilament protein shown in FIG. 6A. Values in
FIG. 6F represent the mean.+-.SEM. Key: WT, untreated wild type;
HET, untreated heterozygote; SMA, untreated knockout; AAV,
AAVhSMN1-treated SMA mice; *, p<0.05; **, p<0.01; ***,
p<0.001. Scale bars: 20 .mu.m.
[0026] FIGS. 7A-7F show the results of behavioral tests in treated
and untreated SMA mice. Treated SMA mice showed significant
improvements on behavioral tests. Treated SMA (asterisk) and
untreated wild-type (WT) mice were substantially fitter than
untreated SMA mice (labeled `x`) at 16 days (FIG. 7A). Treated SMA
mice were also significantly heavier than untreated SMA controls
from day 11 and onwards (FIG. 7B). Treated SMA mice performed
significantly better than untreated SMA mice on the righting reflex
(FIG. 7C), negative geotaxis (FIG. 7D), grip strength (FIG. 7E) and
hindlimb splay (FIG. 7F) tests. Treated SMA mice were statistically
identical to wild-type and heterozygote mice on the righting reflex
and negative geotaxis tests at 12-16 days (FIGS. 7C and 7D). Values
represent the mean.+-.SEM. Key: untreated WT (open circle),
untreated heterozygote (open triangle); untreated SMA (open
square); AAVhSMN1-treated SMA mice (closed square); *, p<0.05;
**, p<0.01; ***, p<0.001.
[0027] FIG. 8 shows survival of scAAVhSMN1-treated and untreated
mice. Treatment with scAAVhSMN1 increased survival in SMA mice. SMA
mice treated at P0 with scAAVhSMN1 (n=17, closed triangles) had a
median lifespan of 157 days (p<0.0001), compared to 16 days in
untreated SMA mice (n=47, open circles).
[0028] FIGS. 9A-9B (SEQ ID NOS:1 and 2) show the coding DNA
sequence (FIG. 9A) and the corresponding amino acid sequence (FIG.
9B) of a representative human survival motor neuron (SMN1)
gene.
[0029] FIGS. 10A-10F shows that scAAV8-hSMN expression increases
motor neuron counts and improves NMJ in SMA mice. FIG. 10A shows
the percentage of mChAT immunopositive cells that co-localized with
hSMN expression in the thoracic-lumbar region at 16 days
post-injection. FIGS. 10B-10F show the average numbers of mChAT
immunopositive cells in the lumbar (FIG. 10B), thoracic (FIG. 10C),
and cervical (FIG. 10D) segments, and the average percentages of
collapsed NMJs in the quadriceps (FIG. 10E) and intercostal (FIG.
10F) muscles at 16, 58-66 and 214-269 days. As a reference for
FIGS. 10E and 10F, 75-90% of NMJ in the quadriceps and intercostal
muscles of untreated SMA mice contained an aberrant collapsed
structure at 16 days (see FIG. 6F). Key and n-values: SMA,
untreated knockout (open bars, n=8 at 16 days), AAV, AAV8-hSMN
(hatched bars, n=8 at 16 days, n=5 at 58-66 days); scAAV,
scAAV8-hSMN (closed bars, n=5 at each time point); WT, untreated WT
(checkered bars, n=8 at 16 days, n=5 each at 58-66 and 216-269
days). Values represent the mean.+-.SEM. Statistical comparisons
were performed with one-way ANOVA and Bonferroni multiple post hoc
tests at 16 days (FIGS. 100B-10F). The unpaired two-tailed student
t-test compared 1) the two vectors to each other at 16 days (FIG.
100A) and 58-66 days (FIGS. 10B-10D); 2) the relative number of
ChAT cells in the 58-66 d and 214-269 d groups with scAAV8-hSMN
treatment (FIGS. 10B-10D); 3) the relative number of abnormal NMJs
between the age-matched untreated WT and scAAV8-hSMN-treated SMA
mice at 214-269 days (E, F); *p<0.05, **p<0.01,
***p<0.001.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, recombinant DNA techniques and immunology, within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Fundamental Virology, 2nd Edition, vol. I
& II (B. N. Fields and D. M. Knipe, eds.); Handbook of
Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell
eds., Blackwell Scientific Publications); T. E. Creighton,
Proteins: Structures and Molecular Properties (W.H. Freeman and
Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers,
Inc., current addition); Sambrook, et al., Molecular Cloning: A
Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S.
Colowick and N. Kaplan eds., Academic Press, Inc.). All
publications, patents and patent applications cited herein, whether
supra or infra, are hereby incorporated by reference in their
entirety.
1. Definitions
[0031] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0032] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "an interleukin receptor" includes
a mixture of two or more such receptors, and the like.
[0033] The terms "polypeptide" and "protein," used interchangeably
herein, or a nucleotide sequence encoding the same, refer to a
protein or nucleotide sequence, respectively, that represents
either a native sequence, a variant thereof or a fragment thereof.
The full-length proteins, with or without the signal sequence, and
fragments thereof, as well as proteins with modifications, such as
deletions, additions and substitutions (either conservative or
non-conservative in nature), to the native sequence, are intended
for use herein, so long as the protein maintains the desired
activity. These modifications may be deliberate, as through
site-directed mutagenesis, or may be accidental, such as through
mutations of hosts which produce the proteins or errors due to PCR
amplification. Accordingly, active proteins substantially
homologous to the parent sequence, e.g., proteins with 70 . . . 80
. . . 85 . . . 90 . . . 95 . . . 98 . . . 99% etc. identity that
retain the desired activity of the native molecule, are
contemplated for use herein.
[0034] A "native" polypeptide, such as a survival motor neuron
(SMN) polypeptide, refers to a polypeptide having the same amino
acid sequence as the corresponding molecule derived from nature.
Such native sequences can be isolated from nature or can be
produced by recombinant or synthetic means. The term "native"
sequence specifically encompasses naturally-occurring truncated or
secreted forms of the specific molecule (e.g., an extracellular
domain sequence), naturally-occurring variant forms (e.g.,
alternatively spliced forms) and naturally-occurring allelic
variants of the polypeptide. In various embodiments of the
invention, the native molecules disclosed herein are mature or
full-length native sequences comprising the full-length amino acids
sequences shown in the accompanying figures. However, while some of
the molecules disclosed in the accompanying figures begin with
methionine residues designated as amino acid position 1 in the
figures, other methionine residues located either upstream or
downstream from amino acid position 1 in the figures may be
employed as the starting amino acid residue for the particular
molecule. Alternatively, depending on the expression system used,
the molecules described herein may lack an N-terminal
methionine.
[0035] By "variant" is meant an active polypeptide as defined
herein having at least about 80% amino acid sequence identity with
the corresponding full-length native sequence, a polypeptide
lacking the signal peptide, an extracellular domain of a
polypeptide, with or without a signal peptide, or any other
fragment of a full-length polypeptide sequence as disclosed herein.
Such polypeptide variants include, for instance, polypeptides
wherein one or more amino acid residues are added, or deleted, at
the N- and/or C-terminus of the full-length native amino acid
sequence. Ordinarily, a variant will have at least about 80% amino
acid sequence identity, alternatively at least about 81% amino acid
sequence identity, alternatively at least about 82% amino acid
sequence identity, alternatively at least about 83% amino acid
sequence identity, alternatively at least about 84% amino acid
sequence identity, alternatively at least about 85% amino acid
sequence identity, alternatively at least about 86% amino acid
sequence identity, alternatively at least about 87% amino acid
sequence identity, alternatively at least about 88% amino acid
sequence identity, alternatively at least about 89% amino acid
sequence identity, alternatively at least about 90% amino acid
sequence identity, alternatively at least about 91% amino acid
sequence identity, alternatively at least about 92% amino acid
sequence identity, alternatively at least about 93% amino acid
sequence identity, alternatively at least about 94% amino acid
sequence identity, alternatively at least about 95% amino acid
sequence identity, alternatively at least about 96% amino acid
sequence identity, alternatively at least about 97% amino acid
sequence identity, alternatively at least about 98% amino acid
sequence identity and alternatively at least about 99% amino acid
sequence identity to the corresponding full-length native sequence.
Ordinarily, variant polypeptides are at least about 10 amino acids
in length, such as at least about 20 amino acids in length, e.g.,
at least about 30 amino acids in length, alternatively at least
about 40 amino acids in length, alternatively at least about 50
amino acids in length, alternatively at least about 60 amino acids
in length, alternatively at least about 70 amino acids in length,
alternatively at least about 80 amino acids in length,
alternatively at least about 90 amino acids in length,
alternatively at least about 100 amino acids in length,
alternatively at least about 150 amino acids in length,
alternatively at least about 200 amino acids in length,
alternatively at least about 300 amino acids in length, or
more.
[0036] Particularly preferred variants include substitutions that
are conservative in nature, i.e., those substitutions that take
place within a family of amino acids that are related in their side
chains. Specifically, amino acids are generally divided into four
families: (1) acidic--aspartate and glutamate; (2) basic--lysine,
arginine, histidine; (3) non-polar--alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan; and (4)
uncharged polar--glycine, asparagine, glutamine, cysteine, serine
threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are
sometimes classified as aromatic amino acids. For example, it is
reasonably predictable that an isolated replacement of leucine with
isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar conservative replacement of an amino
acid with a structurally related amino acid, will not have a major
effect on the biological activity. For example, the polypeptide of
interest may include up to about 5-10 conservative or
non-conservative amino acid substitutions, or even up to about
15-25 or 50 conservative or non-conservative amino acid
substitutions, or any number between 5-50, so long as the desired
function of the molecule remains intact.
[0037] "Homology" refers to the percent identity between two
polynucleotide or two polypeptide moieties. Two DNA, or two
polypeptide sequences are "substantially homologous" to each other
when the sequences exhibit at least about 50%, preferably at least
about 75%, more preferably at least about 80%-85%, preferably at
least about 90%, and most preferably at least about 95%-98%
sequence identity over a defined length of the molecules. As used
herein, substantially homologous also refers to sequences showing
complete identity to the specified DNA or polypeptide sequence.
[0038] In general, "identity" refers to an exact
nucleotide-to-nucleotide or amino acid-to-amino acid correspondence
of two polynucleotides or polypeptide sequences, respectively.
Percent identity can be determined by a direct comparison of the
sequence information between two molecules by aligning the
sequences, counting the exact number of matches between the two
aligned sequences, dividing by the length of the shorter sequence,
and multiplying the result by 100. Readily available computer
programs can be used to aid in the analysis, such as ALIGN,
Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O.
Dayhoff ed., 5 Suppl. 3:353-358, National Biomedical Research
Foundation, Washington, D.C., which adapts the local homology
algorithm of Smith and Waterman Advances in Appl. Math. 2:482-489,
1981 for peptide analysis. Programs for determining nucleotide
sequence identity are available in the Wisconsin Sequence Analysis
Package, Version 8 (available from Genetics Computer Group,
Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs,
which also rely on the Smith and Waterman algorithm. These programs
are readily utilized with the default parameters recommended by the
manufacturer and described in the Wisconsin Sequence Analysis
Package referred to above. For example, percent identity of a
particular nucleotide sequence to a reference sequence can be
determined using the homology algorithm of Smith and Waterman with
a default scoring table and a gap penalty of six nucleotide
positions.
[0039] Another method of establishing percent identity in the
context of the present invention is to use the MPSRCH package of
programs copyrighted by the University of Edinburgh, developed by
John F. Collins and Shane S. Sturrok, and distributed by
IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of
packages the Smith-Waterman algorithm can be employed where default
parameters are used for the scoring table (for example, gap open
penalty of 12, gap extension penalty of one, and a gap of six).
From the data generated the "Match" value reflects "sequence
identity."Other suitable programs for calculating the percent
identity or similarity between sequences are generally known in the
art, for example, another alignment program is BLAST, used with
default parameters. For example, BLASTN and BLASTP can be used
using the following default parameters: genetic code=standard;
filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;
Descriptions=50 sequences; sort by=HIGH SCORE;
Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+Swiss protein+Spupdate+PIR. Details of these programs
are well known in the art.
[0040] Alternatively, homology can be determined by hybridization
of polynucleotides under conditions which form stable duplexes
between homologous regions, followed by digestion with
single-stranded-specific nuclease(s), and size determination of the
digested fragments. DNA sequences that are substantially homologous
can be identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular
system. Defining appropriate hybridization conditions is within the
skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning,
supra; Nucleic Acid Hybridization, supra.
[0041] By the term "degenerate variant" is intended a
polynucleotide containing changes in the nucleic acid sequence
thereof, that encodes a polypeptide having the same amino acid
sequence as the polypeptide encoded by the polynucleotide from
which the degenerate variant is derived.
[0042] A "coding sequence" or a sequence which "encodes" a selected
polypeptide, is a nucleic acid molecule which is transcribed (in
the case of DNA) and translated (in the case of mRNA) into a
polypeptide in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxy) terminus. A
transcription termination sequence may be located 3' to the coding
sequence.
[0043] By "vector" is meant any genetic element, such as a plasmid,
phage, transposon, cosmid, chromosome, virus, virion, etc., which
is capable of replication when associated with the proper control
elements and which can transfer gene sequences to cells. Thus, the
term includes cloning and expression vehicles, as well as viral
vectors.
[0044] By "recombinant vector" is meant a vector that includes a
heterologous nucleic acid sequence which is capable of expression
in vivo.
[0045] By "recombinant virus" is meant a virus that has been
genetically altered, e.g., by the addition or insertion of a
heterologous nucleic acid construct into the particle.
[0046] The term "transgene" refers to a polynucleotide that is
introduced into a cell and is capable of being transcribed into RNA
and optionally, translated and/or expressed under appropriate
conditions. In one aspect, it confers a desired property to a cell
into which it was introduced, or otherwise leads to a desired
therapeutic or diagnostic outcome.
[0047] The terms "genome particles (gp)," or "genome equivalents,"
as used in reference to a viral titer, refer to the number of
virions containing the recombinant AAV DNA genome, regardless of
infectivity or functionality. The number of genome particles in a
particular vector preparation can be measured by procedures such as
described in the Examples herein, or for example, in Clark et al.,
Hum. Gene Ther. (1999) 10:1031-1039; and Veldwijk et al., Mol.
Ther. (2002) 6:272-278.
[0048] The terms "infection unit (iu)," "infectious particle," or
"replication unit," as used in reference to a viral titer, refer to
the number of infectious recombinant AAV vector particles as
measured by the infectious center assay, also known as replication
center assay, as described, for example, in McLaughlin et al., J.
Virol. (1988) 62:1963-1973.
[0049] The term "transducing unit (tu)" as used in reference to a
viral titer, refers to the number of infectious recombinant AAV
vector particles that result in the production of a functional
transgene product as measured in functional assays such as
described in Examples herein, or for example, in Xiao et al., Exp.
Neurobiol. (1997) 144:1 13-124; or in Fisher et al., J. Virol.
(1996) 70:520-532 (LFU assay).
[0050] The term "transfection" is used to refer to the uptake of
foreign DNA by a cell, and a cell has been "transfected" when
exogenous DNA has been introduced inside the cell membrane. A
number of transfection techniques are generally known in the art.
See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al.
(1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor
Laboratories, New York, Davis et al. (1986) Basic Methods in
Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
Such techniques can be used to introduce one or more exogenous DNA
moieties into suitable host cells.
[0051] The term "heterologous" as it relates to nucleic acid
sequences such as coding sequences and control sequences, denotes
sequences that are not normally joined together, and/or are not
normally associated with a particular cell. Thus, a "heterologous"
region of a nucleic acid construct or a vector is a segment of
nucleic acid within or attached to another nucleic acid molecule
that is not found in association with the other molecule in nature.
For example, a heterologous region of a nucleic acid construct
could include a coding sequence flanked by sequences not found in
association with the coding sequence in nature. Another example of
a heterologous coding sequence is a construct where the coding
sequence itself is not found in nature (e.g., synthetic sequences
having codons different from the native gene). Similarly, a cell
transformed with a construct which is not normally present in the
cell would be considered heterologous for purposes of this
invention. Allelic variation or naturally occurring mutational
events do not give rise to heterologous DNA, as used herein.
[0052] A "nucleic acid" sequence refers to a DNA or RNA sequence.
The term captures sequences that include any of the known base
analogues of DNA and RNA such as, but not limited to
4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxyl-methyl) uracil,
5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,
1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine,
2-methyladenine, 2-methylguanine, 3-methyl-cytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0053] The term DNA "control sequences" refers collectively to
promoter sequences, polyadenylation signals, transcription
termination sequences, upstream regulatory domains, origins of
replication, internal ribosome entry sites ("IRES"), enhancers, and
the like, which collectively provide for the replication,
transcription and translation of a coding sequence in a recipient
cell. Not all of these control sequences need always be present so
long as the selected coding sequence is capable of being
replicated, transcribed and translated in an appropriate host
cell.
[0054] The term "promoter" is used herein in its ordinary sense to
refer to a nucleotide region comprising a DNA regulatory sequence,
wherein the regulatory sequence is derived from a gene which is
capable of binding RNA polymerase and initiating transcription of a
downstream (3'-direction) coding sequence. Transcription promoters
can include "inducible promoters" (where expression of a
polynucleotide sequence operably linked to the promoter is induced
by an analyte, cofactor, regulatory protein, etc.), "repressible
promoters" (where expression of a polynucleotide sequence operably
linked to the promoter is induced by an analyte, cofactor,
regulatory protein, etc.), and "constitutive promoters".
[0055] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, control sequences operably linked to a
coding sequence are capable of effecting the expression of the
coding sequence. The control sequences need not be contiguous with
the coding sequence, so long as they function to direct the
expression thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter sequence
and the coding sequence and the promoter sequence can still be
considered "operably linked" to the coding sequence.
[0056] The term "nervous system" includes both the central nervous
system and the peripheral nervous system. The term "central nervous
system" or "CNS" includes all cells and tissue of the brain and
spinal cord of a vertebrate. The term "peripheral nervous system"
refers to all cells and tissue of the portion of the nervous system
outside the brain and spinal cord. Thus, the term "nervous system"
includes, but is not limited to, neuronal cells, glial cells,
astrocytes, cells in the cerebrospinal fluid (CSF), cells in the
interstitial spaces, cells in the protective coverings of the
spinal cord, epidural cells (i.e., cells outside of the dura
mater), cells in non-neural tissues adjacent to or in contact with
or innervated by neural tissue, cells in the epineurium,
perineurium, endoneurium, funiculi, fasciculi, and the like.
[0057] "Active" or "activity" for purposes of the present invention
refers to forms of a therapeutic protein which retain a biological
activity of the corresponding native or naturally occurring
polypeptide. The activity may be greater than, equal to, or less
than that observed with the corresponding native or naturally
occurring polypeptide.
[0058] By "isolated" when referring to a nucleotide sequence, is
meant that the indicated molecule is present in the substantial
absence of other biological macromolecules of the same type. Thus,
an "isolated nucleic acid molecule which encodes a particular
polypeptide" refers to a nucleic acid molecule which is
substantially free of other nucleic acid molecules that do not
encode the subject polypeptide; however, the molecule may include
some additional bases or moieties which do not deleteriously affect
the basic characteristics of the composition.
[0059] For the purpose of describing the relative position of
nucleotide sequences in a particular nucleic acid molecule
throughout the instant application, such as when a particular
nucleotide sequence is described as being situated "upstream,"
"downstream," "3-prime (3')" or "5-prime (5')" relative to another
sequence, it is to be understood that it is the position of the
sequences in the "sense" or "coding" strand of a DNA molecule that
is being referred to as is conventional in the art.
[0060] The term "about", particularly in reference to a given
quantity, is meant to encompass deviations of plus or minus five
percent.
[0061] The terms "subject", "individual" or "patient" are used
interchangeably herein and refer to a vertebrate, preferably a
mammal. Mammals include, but are not limited to, murines, rodents,
simians, humans, farm animals, sport animals and pets.
[0062] The term "modulate" as used herein means to vary the amount
or intensity of an effect or outcome, e.g., to enhance, augment,
diminish, reduce or eliminate.
[0063] As used herein, the term "ameliorate" is synonymous with
"alleviate" and means to reduce or lighten. For example, one may
ameliorate the symptoms of a disease or disorder by making the
disease or symptoms of the disease less severe.
[0064] The terms "therapeutic," "effective amount" or
"therapeutically effective amount" of a composition or agent, as
provided herein, refer to a sufficient amount of the composition or
agent to provide the desired response, such as the prevention,
delay of onset or amelioration of symptoms in a subject or an
attainment of a desired biological outcome, such as correction of
neuropathology, e.g., cellular pathology associated with a motor
neuronal disease such as spinal muscular atrophy (SMA). The term
"therapeutic correction" refers to that degree of correction that
results in prevention or delay of onset or amelioration of symptoms
in a subject. The exact amount required will vary from subject to
subject, depending on the species, age, and general condition of
the subject, the severity of the condition being treated, and the
particular macromolecule of interest, mode of administration, and
the like. An appropriate "effective" amount in any individual case
may be determined by one of ordinary skill in the art using routine
experimentation.
[0065] "Treatment" or "treating" a particular disease includes: (1)
preventing the disease, i.e. preventing the development of the
disease or causing the disease to occur with less intensity in a
subject that may be exposed to or predisposed to the disease but
does not yet experience or display symptoms of the disease, (2)
inhibiting the disease, i.e., arresting the development or
reversing the disease state, or (3) relieving symptoms of the
disease i.e., decreasing the number of symptoms experienced by the
subject, as well as changing the cellular pathology associated with
the disease.
2. Modes of Carrying Out the Invention
[0066] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
formulations or process parameters as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
[0067] Although a number of methods and materials similar or
equivalent to those described herein can be used in the practice of
the present invention, the preferred materials and methods are
described herein.
[0068] Central to the present invention is the discovery that
delivery of rAAV virions containing the human survival motor neuron
1 (hSMN1) cDNA to the CNS of an aggressive mouse model of spinal
muscular atrophy (SMA), produced expression of SMN1 throughout the
spinal cord. Treated SMA mice contained a higher number of motor
neurons compared to untreated, age-matched mutants. In addition,
the evaluation of myofiber size demonstrated that the size of
individual myofibers from a variety of muscle groups in treated SMA
mice approximated those observed in wild-type mice. Furthermore,
the structure of the neuromuscular junction (NMJ) in treated SMA
mice was similar to wild-type mice, which was in contrast to
untreated SMA that showed abnormal accumulation of neurofilament
protein at the pre-synaptic termini. Treated SMA mice also
displayed significant improvements on a battery of behavioral tests
suggesting that the NMJ was functional. Importantly, recombinant
AAV treated mice had a significantly increased lifespan as compared
to their untreated counterparts. SMA mice treated with a
self-complementary rAAV vector also displayed a remarkable
improvement in median survival, even as compared to treatment with
conventional, non-self-complementary rAAV vectors.
[0069] These results demonstrate that CNS-directed, AAV-mediated
SMN1 gene augmentation is highly efficacious in addressing both the
neuronal and muscular pathologies of SMA and evidence the utility
of viral gene therapy as a therapeutic strategy for treating and
preventing neuronal and muscular pathologies, such as SMA, as well
as other diseases that affect motor function. The gene therapy
techniques described herein can be used alone, or in conjunction
with traditional drugs.
[0070] In order to further an understanding of the invention, a
more detailed discussion is provided below regarding motor neuron
pathologies and therapeutic molecules, as well as various gene
delivery methods for use with the present invention.
Motor Neuron Pathologies and Therapeutic Molecules
[0071] The subject invention provides compositions and methods to
modulate, correct or augment motor function in a subject afflicted
with a motor neuron disorder or with motor neuronal damage. For the
purpose of illustration only, the subject may suffer from one or
more of spinal muscular atrophy (SMA), amytrophic lateral sclerosis
(ALS), spinal bulbar muscular atrophy, spinal cerebellar ataxia,
primary lateral sclerosis (PLS), or traumatic spinal cord injury.
Without being bound by a particular theory, the pathology
associated with motor neuron damage may include motor neuron
degeneration, gliosis, neurofilament abnormalities, loss of
myelinated fibers in corticospinal tracts and ventral roots. For
example, two types of onset have been recognized--bulbar onset,
which affects the upper motor neurons (cortex and brainstem motor
neurons), affects the facial muscles, speech, and swallowing; and
limb onset, which affects the lower motor neurons (spinal cord
motor neurons), is reflected by spasticity, generalized weakness,
muscular atrophy, paralysis, and respiratory failure. In ALS,
subjects have both bulbar and limb onset. In PLS, subjects just
have bulbar onset.
[0072] Thus, in certain embodiments, the subject is provided with
rAAV constructs that encode a biologically active molecule, the
expression of which in the CNS results in at least partial
correction of neuropathology and/or stabilization of disease
progression, such as the prevention, delay of onset or amelioration
of symptoms in a subject or an attainment of a desired biological
outcome, including for example, a change in the cellular pathology
associated with a motor neuronal disease described above.
[0073] By way of example, the transgene present in the rAAV
construct may be, but is not limited to, survival motor neuron
protein (via the SMN1 gene or SMN2 gene), insulin growth factor-1
(IGF-1), calbindin D28, parvalbumin, HIFl-alpha, SIRT-2, VEGF such
as VEGF165, CNTF (Ciliary neurotrophic factor), sonic hedgehog
(shh), erythropoietin (EPO), lysyl oxidase (LOX), progranulin,
prolactin, ghrelin, neuroserpin, angiogenin, and placenta
lactogen.
[0074] The molecular basis of SMA, an autosomal recessive
neuromuscular disorder, is the homozygous loss of the survival
motor neuron gene 1 (SMN1), which may also be known as SMN
Telomeric. A nearly identical copy of the SMN1 gene, called SMN2,
which may also be known as SMN Centromeric, is found in humans and
modulates the disease severity. Expression of the normal SMN1 gene
results solely in expression of survival motor neuron (SMN)
protein. Expression of the SMN2 gene results in approximately
10-20% of the SMN protein and 80-90% of an unstable/non-functional
SMNdelta7 protein. Only 10% of SMN2 transcripts encode a functional
full-length protein identical to SMN1. This functional difference
between both genes results from a translationally silent mutation
that, however, disrupts an exonic splicing enhancer causing exon 7
skipping in most SMN2 transcripts. SMN protein plays a
well-established role in assembly of the spliceosome and may also
mediate mRNA trafficking in the axon and nerve terminus of
neurons.
[0075] The nucleotide and amino acid sequences of various SMN1
molecules and SMN proteins are known. See, for example, FIGS.
9A-9B; NCBI accession numbers NM.sub.--000344 (human),
NP.sub.--000335 (human), NM.sub.--011420 (mouse), EU 791616
(porcine), NM.sub.--001131470 (orangutan), NM.sub.--131191
(zebrafish), BC062404 (rat), NM.sub.--001009328 (cat),
NM.sub.--001003226 (dog), NM 175701 (cow). Similarly, various SMN2
sequences are known. See, e.g., NCBI accession numbers
NM.sub.--022876, NM 022877, NM.sub.--017411, NG.sub.--008728,
BC.sub.--000908, BC070242, DQ185039 (all human).
[0076] Insulin-like growth factor 1 (IGF-I) is a therapeutic
protein for the treatment of neurodegenerative disorders, including
motor neuron disorders, due to its many actions at different levels
of neuraxis (see Dore et al., Trends Neurosci (1997) 20:326-331).
For example, in the brain it is thought to reduce both neuronal and
glial apoptosis, protect neurons against toxicity induced by iron,
colchicine, calcium destabilizers, peroxides, and cytokines. It
also appears to modulate the release of neurotransmitters
acetylcholine and glutamate and induce the expression of
neurofilament, tublin, and myelin basic protein. In the spinal
cord, IGF-I is believed to modulate ChAT activity and attenuate
loss of cholinergic phenotype, enhance motor neuron sprouting,
increase myelination, inhibit demyelination, stimulate motor neuron
proliferation and differentiation from precursor cells, and promote
Schwann cell division, maturation, and growth. In the muscle, IGF-I
appears to induce acetylcholine receptor cluster formation at the
neuromuscular junction and increase neuromuscular function and
muscle strength.
[0077] The IGF-1 gene has a complex structure, which is well-known
in the art. It has at least two alternatively spliced mRNA products
arising from the gene transcript. There is a 153 amino acid
peptide, known by several names including IGF-IA or IGF-IEa, and a
195 amino acid peptide, known by several names including IGF-IB or
IGF-IEb. The Eb form is also be known as Ec in humans. The mature
form of IGF-I is a 70 amino acid polypeptide. Both IGF-IEa and
IGF-IEb contain the 70 amino acid mature peptide, but differ in the
sequence and length of their carboxyl-terminal extensions. The
IGF-1 proteins, as well as the peptide sequences of IGF-IEa and
IGF-IEb are known and described in, e.g., International Publication
No. WO 2007/146046, incorporated herein by reference in its
entirety. The genomic and functional cDNAs of human IGF-I, as well
as additional information regarding the IGF-I gene and its
products, are available at Unigene Accession No.
NM.sub.--000618.
[0078] Calbindin D28K (also referred to as calbindin D28) and
parvalbumin are calcium-binding proteins believed to be involved in
calcium buffering. Without being bound by a particular theory,
calcium homeostasis appears to be altered in subjects with motor
neuron disorders (e.g., ALS) and low levels of calbindin-D28K
and/or parvalbumin may increase the vulnerability of motor neurons
by reducing their ability to handle an increased calcium load. This
reduction may lead to cell injury and eventual motor neuron death.
Further evidence suggests that neurons rich in calcium-binding
proteins, such as calbindin D28K and parvalbumin, are resistant to
degeneration.
[0079] HIF-I is a heterodimeric protein composed of two subunits:
(i) a constitutively expressed .beta. subunit also known as aryl
hydrocarbon nuclear translocator (ARNT) (which is shared by other
related transcription factors (e.g., the dioxin/aryl hydrocarbon
receptor (DR/AhR)); and (ii) an .alpha. subunit (see, e.g.,
International publication No. WO 96/39426, describing the recent
affinity purification and molecular cloning of HIF-I.alpha.. Both
subunits are members of the basic helix-loop-helix (bHLH)--PAS
family of transcription factors. These domains regulate DNA binding
and dimerization. The transactivation domain resides in the
C-terminus of the protein. The basic region consists of
approximately 15 predominantly basic amino acids responsible for
direct DNA binding. This region is adjacent to two amphipathic a
helices, separated by a loop of variable length, which forms the
primary dimerization interface between family members (Moore, et
al., Proc. Natl. Acad. Sci. USA (2000) 97:10436-10441). The PAS
domain encompasses 200-300 amino acids containing two loosely
conserved, largely hydrophobic regions approximately 50 amino
acids, designated PAS A and PAS B. The HIF-I.alpha. subunit is
unstable during normoxic conditions, overexpression of this subunit
in cultured cells under normal oxygen levels is capable of inducing
expression of genes normally induced by hypoxia. Replacement of the
C terminal (or transactivation) region of the hypoxia-inducible
factor protein with a strong transactivation domain from a
transcriptional activator protein such as, for example, Herpes
Simplex Virus (HSV) VP16, NF.kappa.B or yeast transcription factors
GAL4 and GCN4, is designed to stabilize the protein under normoxic
conditions and provide strong, constitutive, transcriptional
activation. See, e.g., International Publication No. WO 2008/042420
for a description and sequence of a representative stabilized
hypoxia-inducible factor protein that is a hybrid/chimeric fusion
protein consisting of the DNA-binding and dimerization domains from
HIF-I.alpha. and the transactivation domain from the HSV VP16
protein, incorporated herein by reference in its entirety. See,
also, U.S. Pat. Nos. 6,432,927 and 7,053,062 for a description of a
constitutively stable hybrid HIF-I.alpha., both of which are
incorporated by reference herein in their entirety, Members of the
vascular endothelial growth factor (VEGF) family are among the most
powerful modulators of vascular biology. They regulate
vasculogenesis, angiogenesis, and vascular maintenance. Four
different molecular variants of VEGF have been described. The 165
amino acid variant is the predominant molecular form found in
normal cells and tissues. A less abundant, shorter form with a
deletion of 44 amino acids between positions 116 and 159
(VEGF.sub.121), a longer form with an insertion of 24 basic
residues in position 116 (VEGF.sub.189), and another longer form
with an insertion of 41 amino acids (VEGF.sub.206), which includes
the 24 amino acid insertion found in VEGF.sub.189, are also known.
VEGF.sub.121 and VEGF.sub.165 are soluble proteins. VEGF.sub.189
and VEGF.sub.206 appear to be mostly cell-associated. All of the
versions of VEGF are biologically active. See, e.g., Tischer et
al., J. Biol. Chem. (1991) 266:11947-11954, describing the sequence
of VEGF.sub.165 (see, also, GenBank Accession no. AB021221),
VEGF.sub.121 (see, also, GenBank Accession no. AF214570) and
VEGF.sub.189; and Houck et al., Mol. Endocrinol. (1991)
5:1806-1814, describing the sequence of VEGF.sub.206.
[0080] CNTF (Ciliary neurotrophic factor) is a neurocytokine
expressed by glial cells in peripheral nerves and the central
nervous system. CNTF is generally recognized for its function in
support and survival of non-neuronal and neuronal cell types. See
e.g., Vergara, C and Ramirez, B; Brain Res, Brain Res. Rev. (2004)
47: 161-73.
[0081] Sonic hedgehog (Shh) controls important developmental
processes, including neuronal and glial cell survival.
[0082] Erythropoietin (EPO) is a principal regulator of erythroid
progenitor cells. However, it is functionally expressed in the
nervous system and has been reported to have a neuroprotective
effects. See e.g., Bartesaghi, S., 2005. Neurotoxicology, 26:923-8.
Genes encoding human and other mammalian EPO have been cloned,
sequenced and expressed, and show a high degree of sequence
homology in the coding region across species. Wen et al.,
Blood(1993) 82:1507-1516. The sequence of the gene encoding native
human EPO, as well as methods of obtaining the same, are described
in, e.g., U.S. Pat. Nos. 4,954,437 and 4,703,008, incorporated
herein by reference in their entirety, as well as in Jacobs et al.
(1985) Nature 313:806-810; Lin et al. (1985) Proc. Natl. Acad. Sci.
USA 82:7580; International Publication Number WO 85/02610; and
European Patent Publication Number 232,034 B1. In addition, the
sequences of the genes encoding native feline, canine and porcine
EPO are known and readily available (GenBank Accession Nos.:
L10606; L13027; and L10607, respectively), and the sequence of the
gene encoding monkey (Macaca mulatta) is also known and available
(GenBank Accession No.: L10609).
[0083] Lysyl oxidase (LOX) oxidizes the side chain of peptidyl
lysine thereby converting certain lysine residues to
alpha-aminoadipic-delta-semialdehyde. This is a post-translational
change that, for example, enables the covalent cross-linking of the
component chains of collagen and elastin. It stabilizes the fibrous
deposits of these proteins in the extracellular matrix. LOX can
also oxidize lysine within a variety of cationic proteins, which
suggests that its functions are broader than stabilization or the
extracellular matrix. LOX is synthesized as a preprotein; it
emerges from the cell as proLOX and is processed proteolytically to
the active enzyme. See e.g., Lucero, HA and Kagan, HM, Cell Mol.
Life. Sci. (2006) 63:2304-2316.
[0084] Progranulin (PGRN) is a pleitropic protein. Mutations in the
gene cause frontotemporal lobar degeneration. PGRN in the CNS is
expressed by microglia and neurons and plays a role in brain
development. PGRN is also involved in multiple "tissue modeling"
processes including development, wound repair and tumorogenesis.
PGRN is converted to Granulin (GRN) by elastase enzymes. While
progranulin has trophic properties, GRNs are more akin to
inflammatory mediators. Gene expression studies from animal models
of CNS disease show a differential increase in PRGN combined with
microglial activation and inflammation. Increase in PGRN expression
may be closely related to microglial activation and
neuroinflammation. Moreover, PGRN expression is increased in
activated microglia in many neurodegenerative diseases including
motor neuron disease and Alzheimer's disease. Studies have
identified mutations in PGRN as a cause of neurodegenerative
disease and indicate the importance of PGRN function for neuronal
survival.
[0085] Oligodendrocytes, the myelinating cells of the CNS, continue
to be generated by oligodendrocyte precursor cells (OPCs)
throughout adulthood and are required for the intrinsic repair of
myelin damage in the adult CNS. The physiological events that
modulate OPC proliferation and the generation of new myelinating
oligodendrocytes in the adult CNS are largely known. Recently it
has been reported that patients with Multiple Sclerosis (MS), a
demyleinating disease, have a reduced relapse rate during the third
trimester of pregnancy suggesting that hormones influence
oligodendrocyte generation. Remission in MS patients is correlated
with a decrease in the number and size of active white matter
lesions. Pregnancy in mice results in an increase in the generation
of new oligodendrocytes and the number of myelinated axons within
the maternal CNS (Gregg et al., J. Neurosci. (2007) 27:1812-1823).
Prolactin, a hormone that plateaus during the final stage of
pregnancy, has been shown to regulate OPC proliferation during
pregnancy and promote white matter repair in virgin female mice
(Gregg et al., J. Neurosci. (2007) 27:1812-1823).
[0086] Human placenta lactogen (hPL), a hormone that also peaks
during the third trimester of pregnancy may have a similar
influence on oligodendrocyte generation. hPL has a number of
biological activities that are qualitatively similar to human
growth hormone (hGH) and prolactin and appears to be a major
regulator of IGF-I production. Both hGH and IGF-I have been shown
to be stimulators of myelination in the adult CNS (Carson et al.,
Neuron (1993) 10:729-740; Peltwon et al., Neurology (1977)
27:282-288). Therefore, the treatment of CNS diseases involving
demyelination such as MS, ALS, stroke and spinal cord injury may
benefit from PRL- or hPL-based therapies, such as by the
intraventricular injection of an rhPRL or hPL expressing viral
vector.
[0087] Ghrelin is a gastric hormone that is a mediator of growth
hormone release. See e.g. Wu, et al., Ann. Surg. (2004)
239:464.
[0088] Neuroserpin is a serpin protease inhibitor family member. In
certain CNS conditions, neuroserpin can play a neuroprotective role
potentially through the blockage of the effects of tPA. See, e.g.,
Galliciotti, G and Sonderegger, P, Front Biosci (2006) 11:33;
Simonin, et al., (2006) 26:10614; Miranda, E and Lomas, DA, Cell
MolLife Sci (2006) 63:709.
[0089] Angiogenin is a member of the RNAse superfamily. It is a
normal constituent of circulation but has also been implicated as a
risk factor in motor neuron disorders.
[0090] In certain compositions and methods of the invention, more
than one transgene encoding more than one of the therapeutic
molecules described above can be delivered, wherein each transgene
is operably linked to a promoter to enable the expression of the
trangenes from a single AAV vector. In additional methods, the
transgenes may be operably linked to the same promoter. Each
transgene encodes a biologically active molecule, expression of
which in the CNS results in at least partial correction of
neuropathology. Additionally, in cases where more than one
transgene is delivered, the transgenes may be delivered via more
than one AAV vector, wherein each AAV vector comprises a transgene
operably linked to a promoter.
[0091] The native molecules, as well as active fragments and
analogs thereof, which retain the desired biological activity, as
measured in any of the various assays and animal models including
those described further herein, are intended for use with the
present invention.
[0092] Polynucleotides encoding the desired protein for use with
the present invention can be made using standard techniques of
molecular biology. For example, polynucleotide sequences coding for
the above-described molecules can be obtained using recombinant
methods, such as by screening cDNA and genomic libraries from cells
expressing the gene, or by deriving the gene from a vector known to
include the same. The gene of interest can also be produced
synthetically, rather than cloned, based on the known sequences.
The molecules can be designed with appropriate codons for the
particular sequence. The complete sequence is then assembled from
overlapping oligonucleotides prepared by standard methods and
assembled into a complete coding sequence. See, e.g., Edge, Nature
(1981) 292:756; Nambair et al., Science (1984) 223:1299; and Jay et
al., J. Biol. Chem. (1984) 259:6311.
[0093] Thus, particular nucleotide sequences can be obtained from
vectors harboring the desired sequences or synthesized completely
or in part using various oligonucleotide synthesis techniques known
in the art, such as site-directed mutagenesis and polymerase chain
reaction (PCR) techniques where appropriate. See, e.g., Sambrook,
supra. One method of obtaining nucleotide sequences encoding the
desired sequences is by annealing complementary sets of overlapping
synthetic oligonucleotides produced in a conventional, automated
polynucleotide synthesizer, followed by ligation with an
appropriate DNA ligase and amplification of the ligated nucleotide
sequence via PCR. See, e.g., Jayaraman et al., Proc. Natl. Acad.
Sci. USA (1991) 88:4084-4088. Additionally,
oligonucleotide-directed synthesis (Jones et al., Nature (1986)
54:75-82), oligonucleotide directed mutagenesis of preexisting
nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and
Verhoeyen et al., Science (1988) 239:1534-1536), and enzymatic
filling-in of gapped oligonucleotides using T.sub.4 DNA polymerase
(Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-10033)
can be used to provide molecules for use in the subject
methods.
[0094] Once produced, the constructs are delivered using
recombinant viral vectors as described further below.
AAV Gene Delivery Techniques
[0095] The constructs described above, are delivered to the subject
in question using any of several rAAV gene delivery techniques.
Several AAV-mediated methods for gene delivery are known in the
art. As described further below, genes can be delivered either
directly to the subject or, alternatively, delivered ex vivo, to
appropriate cells, such as cells derived from the subject, and the
cells reimplanted in the subject.
[0096] Various AAV vector systems have been developed for gene
delivery. AAV vectors can be readily constructed using techniques
well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and
5,139,941; International Publication Nos. WO 92/01070 (published 23
Jan. 1992) and WO 93/03769 (published 4 Mar. 1993); Lebkowski et
al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al.,
Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter,
B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka,
N. Current Topics in Microbiol. and Immunol. (1992) 158:97-129;
Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and
Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med.
(1994) 179:1867-1875.
[0097] AAV vector systems are also described in further detail
below. The AAV genome is a linear, single-stranded DNA molecule
containing about 4681 nucleotides. The AAV genome generally
comprises an internal, nonrepeating genome flanked on each end by
inverted terminal repeats (ITRs). The ITRs are approximately 145
base pairs (bp) in length. The ITRs have multiple functions,
including providing origins of DNA replication, and packaging
signals for the viral genome. The internal nonrepeated portion of
the genome includes two large open reading frames, known as the AAV
replication (rep) and capsid (cap) genes. The rep and cap genes
code for viral proteins that allow the virus to replicate and
package into a virion. In particular, a family of at least four
viral proteins are expressed from the AAV rep region, Rep 78, Rep
68, Rep 52, and Rep 40, named according to their apparent molecular
weight. The AAV cap region encodes at least three proteins, VP1,
VP2, and VP3.
[0098] AAV has been engineered to deliver genes of interest by
deleting the internal nonrepeating portion of the AAV genome (i.e.,
the rep and cap genes) and inserting a heterologous gene between
the ITRs. The heterologous gene is typically functionally linked to
a heterologous promoter (constitutive, cell-specific, or inducible)
capable of driving gene expression in the patient's target cells
under appropriate conditions. Examples of each type of promoter are
well-known in the art. Termination signals, such as polyadenylation
sites, can also be included.
[0099] AAV is a helper-dependent virus; that is, it requires
coinfection with a helper virus (e.g., adenovirus, herpesvirus or
vaccinia), in order to form AAV virions. In the absence of
coinfection with a helper virus, AAV establishes a latent state in
which the viral genome inserts into a host cell chromosome, but
infectious virions are not produced. Subsequent infection by a
helper virus "rescues" the integrated genome, allowing it to
replicate and package its genome into an infectious AAV virion.
While AAV can infect cells from different species, the helper virus
must be of the same species as the host cell. Thus, for example,
human AAV will replicate in canine cells coinfected with a canine
adenovirus.
[0100] Recombinant AAV virions comprising the gene of interest may
be produced using a variety of art-recognized techniques described
more fully below. Wild-type AAV and helper viruses may be used to
provide the necessary replicative functions for producing rAAV
virions (see, e.g., U.S. Pat. No. 5,139,941, incorporated herein by
reference in its entirety). Alternatively, a plasmid, containing
helper function genes, in combination with infection by one of the
well-known helper viruses can be used as the source of replicative
functions (see e.g., U.S. Pat. No. 5,622,856 and U.S. Pat. No.
5,139,941, both incorporated herein by reference in their
entireties). Similarly, a plasmid, containing accessory function
genes can be used in combination with infection by wild-type AAV,
to provide the necessary replicative functions. These three
approaches, when used in combination with a rAAV vector, are each
sufficient to produce rAAV virions. Other approaches, well known in
the art, can also be employed by the skilled artisan to produce
rAAV virions.
[0101] In one embodiment of the present invention, a triple
transfection method (described in detail in U.S. Pat. No.
6,001,650, incorporated by reference herein in its entirety) is
used to produce rAAV virions because this method does not require
the use of an infectious helper virus, enabling rAAV virions to be
produced without any detectable helper virus present. This is
accomplished by use of three vectors for rAAV virion production: an
AAV helper function vector, an accessory function vector, and a
rAAV expression vector. One of skill in the art will appreciate,
however, that the nucleic acid sequences encoded by these vectors
can be provided on two or more vectors in various combinations.
[0102] As explained herein, the AAV helper function vector encodes
the "AAV helper function" sequences (i.e., rep and cap), which
function in trans for productive AAV replication and encapsidation.
Preferably, the AAV helper function vector supports efficient AAV
vector production without generating any detectable wt AAV virions
(i.e., AAV virions containing functional rep and cap genes). An
example of such a vector, pHLP19, is described in U.S. Pat. No.
6,001,650, incorporated herein by reference in its entirety. The
rep and cap genes of the AAV helper function vector can be derived
from any of the known AAV serotypes, as explained above. For
example, the AAV helper function vector may have a rep gene derived
from AAV-2 and a cap gene derived from AAV-6; one of skill in the
art will recognize that other rep and cap gene combinations are
possible, the defining feature being the ability to support rAAV
virion production.
[0103] The accessory function vector encodes nucleotide sequences
for non-AAV-derived viral and/or cellular functions upon which AAV
is dependent for replication (i.e., "accessory functions"). The
accessory functions include those functions required for AAV
replication, including, without limitation, those moieties involved
in activation of AAV gene transcription, stage specific AAV mRNA
splicing, AAV DNA replication, synthesis of cap expression
products, and AAV capsid assembly. Viral-based accessory functions
can be derived from any of the well-known helper viruses such as
adenovirus, herpesvirus, and vaccinia virus. In one embodiment, the
accessory function plasmid pLadeno5 is used (details regarding
pLadeno5 are described in U.S. Pat. No. 6,004,797, incorporated
herein by reference in its entirety). This plasmid provides a
complete set of adenovirus accessory functions for AAV vector
production, but lacks the components necessary to form
replication-competent adenovirus.
[0104] In order to further an understanding of AAV, a more detailed
discussion is provided below regarding recombinant AAV expression
vectors and AAV helper and accessory functions
[0105] Recombinant AAV Expression Vectors
[0106] Recombinant AAV (rAAV) expression vectors are constructed
using known techniques to at least provide as operatively linked
components in the direction of transcription, control elements
including a transcriptional initiation region, the polynucleotide
of interest and a transcriptional termination region. The control
elements are selected to be functional in the cell of interest,
such as in a mammalian cell. The resulting construct which contains
the operatively linked components is bounded (5' and 3') with
functional AAV ITR sequences.
[0107] The nucleotide sequences of AAV ITR regions are known. See,
e.g., Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K.
I. "Parvoviridae and their Replication" in Fundamental Virology,
2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2
sequence. AAV ITRs used in the vectors of the invention need not
have a wild-type nucleotide sequence, and may be altered, e.g., by
the insertion, deletion or substitution of nucleotides.
Additionally, AAV ITRs may be derived from any of several AAV
serotypes, including without limitation, AAV-1, AAV-2, AAV-3,
AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, etc. Furthermore, 5' and
3' ITRs which flank a selected nucleotide sequence in an AAV
expression vector need not necessarily be identical or derived from
the same AAV serotype or isolate, so long as they function as
intended, i.e., to allow for excision and rescue of the sequence of
interest from a host cell genome or vector, and to allow
integration of the DNA molecule into the recipient cell genome when
AAV Rep gene products are present in the cell.
[0108] Suitable polynucleotide molecules for use in traditional AAV
vectors will be less than or about 5 kilobases (kb) in size. The
selected polynucleotide sequence is operably linked to control
elements that direct the transcription or expression thereof in the
subject in vivo. Such control elements can comprise control
sequences normally associated with the selected gene.
Alternatively, heterologous control sequences can be employed.
Useful heterologous control sequences generally include those
derived from sequences encoding mammalian or viral genes. Non
limiting examples of promoters include, but are not limited to, the
cytomegalovirus (CMV) promoter (Kaplitt et al., Nat. Genet. (1994)
8:148-154), CMV/human 13-globin promoter (Mandel et al., J.
Neurosci. (1998) 18:4271-4284), GFAP promoter (Xu et al., Gene
Ther. (2001) 8:1323-1332), the 1.8-kb neuron-specific enolase (NSE)
promoter (Klein et al., Exp. Neurol. (1998) 150:183-194), chicken
beta actin (CBA) promoter (Miyazaki, Gene (1989) 79:269-277), the
.beta.-glucuronidase (GUSB) promoter (Shipley et al., Genetics
(1991) 10:1009-1018), and ubiquitin promoters such as those
isolated from human ubiquitin A, human ubiquitin B, and human
ubiquitin C, as described in U.S. Pat. No. 6,667,174, incorporated
herein by reference in its entirety. To prolong expression, other
regulatory elements may additionally be operably linked to the
transgene, such as, e.g., the Woodchuck Hepatitis Virus
Post-Regulatory Element (WPRE) (Donello et al., J. Virol. (1998)
72:5085-5092) or the bovine growth hormone (BGH) polyadenylation
site. In addition, sequences derived from nonviral genes, such as
the murine metallothionein gene, will also find use herein. Such
promoter sequences are commercially available from, e.g.,
Stratagene (San Diego, Calif.).
[0109] For some CNS gene therapy applications, it may be necessary
to control transcriptional activity. To this end, pharmacological
regulation of gene expression with viral vectors can been obtained
by including various regulatory elements and drug-responsive
promoters as described, for example, in Haberma et al., Gene Ther.
(1998) 5.1604-16011; and Ye et al., Science (1995) 283:88-91.
[0110] The AAV expression vector which harbors the polynucleotide
molecule of interest bounded by AAV ITRs, can be constructed by
directly inserting the selected sequence(s) into an AAV genome
which has had the major AAV open reading frames ("ORFs") excised
therefrom. Other portions of the AAV genome can also be deleted, so
long as a sufficient portion of the ITRs remain to allow for
replication and packaging functions. Such constructs can be
designed using techniques well known in the art. See, e.g., U.S.
Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos.
WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4
Mar. 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;
Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory
Press); Carter (1992) Current Opinion in Biotechnology 3:533-539;
Muzyczka (1992) Current Topics in Microbiol. and Immunol.
158:97-129; Kotin (1994) Human Gene Therapy 5:793-801; Shelling and
Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.
Med. 179:1867-1875.
[0111] Alternatively, AAV ITRs can be excised from the viral genome
or from an AAV vector containing the same and fused 5' and 3' of a
selected nucleic acid construct that is present in another vector
using standard ligation techniques, such as those described in
Sambrook et al., supra. For example, ligations can be accomplished
in 20 mM Tris-Cl pH 7.5, 10 mM MgCl.sub.2, 10 mM DTT, 33 .mu.g/ml
BSA, 10 mM-50 mM NaCl, and either 40 .mu.M ATP, 0.01-0.02 (Weiss)
units T4 DNA ligase at 0.degree. C. (for "sticky end" ligation) or
1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14.degree. C. (for
"blunt end" ligation). Intermolecular "sticky end" ligations are
usually performed at 30-100 .mu.g/ml total DNA concentrations
(5-100 nM total end concentration). AAV vectors which contain ITRs
have been described in, e.g., U.S. Pat. No. 5,139,941. In
particular, several AAV vectors are described therein which are
available from the American Type Culture Collection ("ATCC") under
Accession Numbers 53222, 53223, 53224, 53225 and 53226.
[0112] In certain embodiments, the rAAV expression vectors are
provided as self-complementary rAAV constructs. Typically, rAAV DNA
is packaged into the viral capsid as a single-stranded DNA (ssDNA)
molecule about 4600 nucleotides in length. Following infection of
the cell by the virus, the single DNA strand is converted into a
double-stranded DNA (dsDNA) form. Only the dsDNA is useful to
proteins of the cell that transcribe the contained gene or genes
into RNA. Thus, the conventional replication scheme of AAV requires
de novo synthesis of a complementary DNA strand. This step of
converting the ssDNA AAV genome into dsDNA prior to expression can
be circumvented by the use of self-complementary (sc) vectors.
[0113] Self-complementary vectors are produced by base pairing
complementary strands from two infecting viruses, which does not
require DNA synthesis (see, e.g., Nakai et al., J. Virol. (2000)
74:9451-9463). This interstrand base pairing, or strand annealing
(SA), is possible because AAV packages either the plus or minus DNA
strand with equal efficiency (Berns, K. I., Microbiol. Rev. (1990)
54:316-329).
[0114] Thus and without being limited as to theory, the need for
dsDNA conversion, either by SA or DNA synthesis, can be entirely
circumvented by packaging both strands as a single molecule. This
can be achieved by taking advantage of the tendency of AAV to
produce dimeric inverted repeat genomes during the AAV replication
cycle. If these dimers are small enough, they can be packaged in
the same manner as conventional AAV genomes, and the two halves of
the ssDNA molecule can fold and base pair to form a dsDNA molecule
of half the length. dsDNA conversion is independent of host-cell
DNA synthesis and vector concentration (McCarty et al., Gene Ther.
(2001) 8:1248-1254).
[0115] scAAV viral constructs include approximately 4.6 kb and are
able to be packaged into the normal AAV capsid. Each of the known
AAV serotypes is capable of packaging scAAV genomes with similar
efficiency (see, e.g., Sipo et al., Gene Ther. (2007)
14:1319-1329). Thus, in certain embodiments of the instant
invention, the scAAV vector comprises capsid proteins from
serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,
AAV8, or AAV9 serotypes. However, an scAAV vector may comprise
capsid proteins from any of the known serotypes or modified capsid
proteins known in the art. These scAAV vectors may also be
pseudotyped vectors comprising which contain the genome of one AAV
serotype in the capsid of a second AAV serotype. Such vectors may
comprise, for example, an AAV vector that contains the AAV2 capsid
and the AAV1 genome or an AAV vector that contains the AAV5 capsid
and the AAV 2 genome (Auricchio et al., (2001) Hum. Mol. Genet.,
10(26):3075-81).
[0116] Initially, it was believed that the transgene sequence in an
scAAV vector could only comprise approximately 2.2 kb. However, it
appears there is greater latitude in packaging capacity than
previously believed. For example, Wu et al., Human Gene Ther.
(2007) 18:171-182 successfully packaged scAAV-2 constructs
exceeding 3,300 bp and demonstrated dimeric inverted repeat genomes
that were fully DNase resistant. These vectors yielded the expected
increases in transduction efficiency over ssAAV when tested on
cultured cells.
[0117] scAAV vectors can be produced either by generating vector
plasmids that are approximately half of the conventional genome
size combined with selective purification of the infectious double
stranded form, or through the use of approximately half-genome
sized vector plasmids with a mutation in one of the terminal
resolution sequences of the AAV virus that provides for synthesis
of double-stranded virus. Both strategies generate + and - strand
viral genomes that are covalently linked at one terminal
repeat.
[0118] In particular, the generation of normal monomeric AAV
genomes relies on the efficient resolution of the two ITRs in turn,
with each round of DNA synthesis. This reaction is mediated by the
ssDNA endonuclease activity of the two larger isoforms of AAV Rep.
Nicking the ITR at the terminal resolution site is followed by DNA
elongation from the nick by host DNA polymerase. Dimeric genomes
are formed when Rep fails to nick the terminal resolution site
before it is reached by the replication complex initiated at the
other end.
[0119] The yield of dimeric genomes in a scAAV prep can be
increased dramatically by inhibiting resolution at one terminal
repeat. This is readily accomplished by deleting the terminal
resolution site sequence from one ITR, such that the Rep protein
cannot generate the essential ssDNA nick (see, e.g., McCarty et
al., Gene Ther. (2003) 10:2112-2118 and Wang et al., Gene Ther.
(2003) 10:2105-2111). The replication complex initiated at the
other ITR then copies through the hairpin and back toward the
initiating end. Replication proceeds to the end of the template
molecule, leaving a dsDNA inverted repeat with a wild-type ITR at
each end and the mutated ITR in the middle. This dimeric inverted
repeat can then undergo normal rounds of replication from the two
wild-type ITR ends. Each displaced daughter strand comprises a
ssDNA inverted repeat with a complete ITR at each end and a mutated
ITR in the middle. Packaging into the AAV capsid starts at the 3'
end of the displaced strand. Production of scAAV from constructs
with one mutated ITR typically yields more than 90% dimeric
genomes.
[0120] Production and purification of scAAV vector from mutated ITR
constructs is the same as conventional ssAAV, as described further
below. However, if dot blot or Southern blot is used, the vector
DNA is preferably applied to hybridization membranes under alkaline
conditions to prevent reannealing of the complementary strands.
Additionally, it is possible for a spurious Rep-nicking site to be
produced close enough to the mutated ITR to allow terminal
resolution and generation of monomer genomes. This can typically be
avoided by turning the transgene cassette around with respect to
the mutant and wild-type terminal repeats.
[0121] See, e.g., McCarty, D. M., Molec. Ther. (2008) 16:1648-1656;
McCarty et al., Gene Ther. (2001) 8:1248-1254; McCarty et al., Gene
Ther. (2003) 10:2112-2118; Wang et al., Gene Ther. (2003)
10:2105-2111); Wu et al., Human Gene Ther. (2007) 18:171-182; U.S.
Patent Publication Nos. 2007/0243168 and 2007/0253936, incorporated
herein by reference in their entireties; as well as the examples
herein, for methods of producing scAAV constructs.
[0122] For the purposes of the invention, suitable host cells for
producing rAAV virions from the AAV expression vectors (either
conventional or sc vectors) include microorganisms, yeast cells,
insect cells, and mammalian cells, that can be, or have been, used
as recipients of a heterologous DNA molecule and that are capable
of growth in, for example, suspension culture, a bioreactor, or the
like. The term includes the progeny of the original cell which has
been transfected. Thus, a "host cell" as used herein generally
refers to a cell which has been transfected with an exogenous DNA
sequence. Cells from the stable human cell line, 293 (readily
available through, e.g., the American Type Culture Collection under
Accession Number ATCC CRL1573) are preferred in the practice of the
present invention. Particularly, the human cell line 293 is a human
embryonic kidney cell line that has been transformed with
adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen.
Virol. 36:59), and expresses the adenoviral E1a and E1b genes
(Aiello et al. (1979) Virology 94:460). The 293 cell line is
readily transfected, and provides a particularly convenient
platform in which to produce rAAV virions.
[0123] AAV Helper Functions
[0124] Host cells containing the above-described AAV expression
vectors must be rendered capable of providing AAV helper functions
in order to replicate and encapsidate the nucleotide sequences
flanked by the AAV ITRs to produce rAAV virions. AAV helper
functions are generally AAV-derived coding sequences which can be
expressed to provide AAV gene products that, in turn, function in
trans for productive AAV replication. AAV helper functions are used
herein to complement necessary AAV functions that are missing from
the AAV expression vectors. Thus, AAV helper functions include one,
or both of the major AAV ORFs, namely the rep and cap coding
regions, or functional homologues thereof.
[0125] By "AAV rep coding region" is meant the art-recognized
region of the AAV genome which encodes the replication proteins Rep
78, Rep 68, Rep 52 and Rep 40. These Rep expression products have
been shown to possess many functions, including recognition,
binding and nicking of the AAV origin of DNA replication, DNA
helicase activity and modulation of transcription from AAV (or
other heterologous) promoters. The Rep expression products are
collectively required for replicating the AAV genome. For a
description of the AAV rep coding region, see, e.g., Muzyczka, N.
(1992) Current Topics in Microbiol. and Immunol. 158:97-129; and
Kotin, R. M. (1994) Human Gene Therapy 5:793-801. Suitable
homologues of the AAV rep coding region include the human
herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2
DNA replication (Thomson et al. (1994) Virology 204:304-311).
[0126] By "AAV cap coding region" is meant the art-recognized
region of the AAV genome which encodes the capsid proteins VP1,
VP2, and VP3, or functional homologues thereof. These Cap
expression products supply the packaging functions which are
collectively required for packaging the viral genome. For a
description of the AAV cap coding region, see, e.g., Muzyczka, N.
and Kotin, R. M. (supra).
[0127] AAV helper functions are introduced into the host cell by
transfecting the host cell with an AAV helper construct either
prior to, or concurrently with, the transfection of the AAV
expression vector. AAV helper constructs are thus used to provide
at least transient expression of AAV rep and/or cap genes to
complement missing AAV functions that are necessary for productive
AAV infection. AAV helper constructs lack AAV ITRs and can neither
replicate nor package themselves. These constructs can be in the
form of a plasmid, phage, transposon, cosmid, virus, or virion. A
number of AAV helper constructs have been described, such as the
commonly used plasmids pAAV/Ad and pIM29+45 which encode both Rep
and Cap expression products. See, e.g., Samulski et al. (1989) J.
Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol.
65:2936-2945. A number of other vectors have been described which
encode Rep and/or Cap expression products. See, e.g., U.S. Pat. No.
5,139,941.
[0128] AAV Accessory Functions
[0129] The host cell (or packaging cell) must also be rendered
capable of providing nonAAV-derived functions, or "accessory
functions," in order to produce rAAV virions. Accessory functions
are nonAAV-derived viral and/or cellular functions upon which AAV
is dependent for its replication. Thus, accessory functions include
at least those nonAAV proteins and RNAs that are required in AAV
replication, including those involved in activation of AAV gene
transcription, stage specific AAV mRNA splicing, AAV DNA
replication, synthesis of Cap expression products and AAV capsid
assembly. Viral-based accessory functions can be derived from any
of the known helper viruses.
[0130] In particular, accessory functions can be introduced into
and then expressed in host cells using methods known to those of
skill in the art. Typically, accessory functions are provided by
infection of the host cells with an unrelated helper virus. A
number of suitable helper viruses are known, including
adenoviruses; herpesviruses such as herpes simplex virus types 1
and 2; and vaccinia viruses. Nonviral accessory functions will also
find use herein, such as those provided by cell synchronization
using any of various known agents. See, e.g., Buller et al. (1981)
J. Virol. 40:241-247; McPherson et al. (1985) Virology 147:217-222;
Schlehofer et al. (1986) Virology 152:110-117.
[0131] Alternatively, accessory functions can be provided using an
accessory function vector as defined above. See, e.g., U.S. Pat.
No. 6,004,797 and International Publication No. WO 01/83797,
incorporated herein by reference in their entireties.
[0132] Nucleic acid sequences providing the accessory functions can
be obtained from natural sources, such as from the genome of an
adenovirus particle, or constructed using recombinant or synthetic
methods known in the art. As explained above, it has been
demonstrated that the full-complement of adenovirus genes are not
required for accessory helper functions. In particular, adenovirus
mutants incapable of DNA replication and late gene synthesis have
been shown to be permissive for AAV replication. Ito et al., (1970)
J. Gen. Virol. 9:243; Ishibashi et al, (1971) Virology 45:317.
Similarly, mutants within the E2B and E3 regions have been shown to
support AAV replication, indicating that the E2B and E3 regions are
probably not involved in providing accessory functions. Carter et
al., (1983) Virology 126:505. However, adenoviruses defective in
the E1 region, or having a deleted E4 region, are unable to support
AAV replication. Thus, E1A and E4 regions are likely required for
AAV replication, either directly or indirectly. Laughlin et al.,
(1982) J. Virol. 41:868; Janik et al., (1981) Proc. Natl. Acad.
Sci. USA 78:1925; Carter et al., (1983) Virology 126:505. Other
characterized Ad mutants include: E1B (Laughlin et al. (1982),
supra; Janik et al. (1981), supra; Ostrove et al., (1980) Virology
104:502); E2A (Handa et al., (1975) J. Gen. Virol. 29:239; Strauss
et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol.
35:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:2927;
Myers et al., (1981) J. Biol. Chem. 256:567); E2B (Carter,
Adeno-Associated Virus Helper Functions, in I CRC Handbook of
Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983),
supra); and E4 (Carter et al. (1983), supra; Carter (1995)).
Although studies of the accessory functions provided by
adenoviruses having mutations in the E1B coding region have
produced conflicting results, Samulski et al., (1988) J. Virol.
62:206-210, has reported that E1B55k is required for AAV virion
production, while E1B 19k is not. In addition, International
Publication WO 97/17458 and Matshushita et al., (1998) Gene Therapy
5:938-945, describe accessory function vectors encoding various Ad
genes. Particularly preferred accessory function vectors comprise
an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding
region, an adenovirus E2A 72 kD coding region, an adenovirus E1A
coding region, and an adenovirus E1B region lacking an intact
E1B55k coding region. Such vectors are described in International
Publication No. WO 01/83797.
[0133] As a consequence of the infection of the host cell with a
helper virus, or transfection of the host cell with an accessory
function vector, accessory functions are expressed which
transactivate the AAV helper construct to produce AAV Rep and/or
Cap proteins. The Rep expression products excise the recombinant
DNA (including the DNA of interest) from the AAV expression vector.
The Rep proteins also serve to duplicate the AAV genome. The
expressed Cap proteins assemble into capsids, and the recombinant
AAV genome is packaged into the capsids. Thus, productive AAV
replication ensues, and the DNA is packaged into rAAV virions. A
"recombinant AAV virion," or "rAAV virion" is defined herein as an
infectious, replication-defective virus including an AAV protein
shell, encapsidating a heterologous nucleotide sequence of interest
which is flanked on both sides by AAV ITRs.
[0134] Following recombinant AAV replication, rAAV virions can be
purified from the host cell using a variety of conventional
purification methods, such as column chromatography, CsCl
gradients, and the like. For example, a plurality of column
purification steps can be used, such as purification over an anion
exchange column, an affinity column and/or a cation exchange
column. See, for example, International Publication No. WO
02/12455. Further, if infection is employed to express the
accessory functions, residual helper virus can be inactivated,
using known methods. For example, adenovirus can be inactivated by
heating to temperatures of approximately 60.degree. C. for, e.g.,
20 minutes or more. This treatment effectively inactivates only the
helper virus since AAV is extremely heat stable while the helper
adenovirus is heat labile.
[0135] The resulting rAAV virions containing the nucleotide
sequence of interest can then be used for gene delivery using the
techniques described below.
Compositions and Delivery
[0136] A. Compositions
[0137] Once produced, the rAAV virions encoding the gene of
interest, will be formulated into compositions suitable for
delivery. Compositions will comprise sufficient genetic material to
produce a therapeutically effective amount of the gene of interest,
i.e., an amount sufficient to (1) prevent the development of the
disease or cause the disease to occur with less intensity in a
subject that may be exposed to or predisposed to the disease but
does not yet experience or display symptoms of the disease, (2)
inhibit the disease, i.e., arrest the development or reverse the
disease state, or (3) relieve symptoms of the disease i.e.,
decrease the number of symptoms experienced by the subject, as well
as change the cellular pathology associated with the disease.
[0138] Appropriate doses will also depend on the mammal being
treated (e.g., human or nonhuman primate or other mammal), age and
general condition of the subject to be treated, the severity of the
condition being treated, the mode of administration, among other
factors. An appropriate effective amount can be readily determined
by one of skill in the art and representative amounts are provided
below.
[0139] The compositions will also contain a pharmaceutically
acceptable excipient. Such excipients include any pharmaceutical
agent that does not itself induce the production of antibodies
harmful to the individual receiving the composition, and which may
be administered without undue toxicity. Pharmaceutically acceptable
excipients include, but are not limited to, sorbitol, any of the
various TWEEN compounds, and liquids such as water, saline,
glycerol and ethanol. Pharmaceutically acceptable salts can be
included therein, for example, mineral acid salts such as
hydrochlorides, hydrobromides, phosphates, sulfates, and the like;
and the salts of organic acids such as acetates, propionates,
malonates, benzoates, and the like. Additionally, auxiliary
substances, such as wetting or emulsifying agents, pH buffering
substances, and the like, may be present in such vehicles. A
thorough discussion of pharmaceutically acceptable excipients is
available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co.,
N.J. 1991).
[0140] Formulations can be liquid or solid, for example,
lyophilized. Formulations can also be administered as aerosols.
[0141] One particularly useful formulation comprises the rAAV
virion of interest in combination with one or more dihydric or
polyhydric alcohols, and, optionally, a detergent, such as a
sorbitan ester. See, for example, U.S. Pat. No. 6,764,845,
incorporated herein by reference in its entirety.
[0142] B. Delivery
[0143] Generally, the recombinant virions are introduced into the
subject using either in vivo or in vitro transduction techniques.
If transduced in vitro, the desired recipient cell will be removed
from the subject, transduced with the recombinant vector and
reintroduced into the subject. Alternatively, syngeneic or
xenogeneic cells can be used where those cells will not generate an
inappropriate immune response in the subject. Suitable cells for
delivery to mammalian host animals include mammalian cell types
from organs, tumors, or cell lines. For example, human, murine,
goat, ovine, bovine, dog, cat, and porcine cells can be used.
Suitable cell types for use include without limitation,
fibroblasts, hepatocytes, endothelial cells, keratinocytes,
hematopoietic cells, epithelial cells, myocytes, neuronal cells,
and stem cells. Additionally, neural progenitor cells can be
transduced in vitro and then delivered to the CNS.
[0144] Cells can be transduced in vitro by combining recombinant
virions with the desired cell in appropriate media, and the cells
can be screened for those cells harboring the DNA of interest using
conventional techniques such as Southern blots and/or PCR, or by
using selectable markers. Transduced cells can then be formulated
into pharmaceutical compositions, as described above, and the
composition introduced into the subject by various techniques as
described below, in one or more doses.
[0145] For in vivo delivery, the recombinant virions will be
formulated into pharmaceutical compositions and one or more dosages
may be administered directly in the indicated manner. For
identification of structures in the human brain, see, e.g., The
Human Brain Surface, Three-Dimensional Sectional Anatomy With MRI,
and Blood Supply, 2nd ed., eds. Deuteron et al., Springer Vela,
1999; Atlas of the Human Brain, eds. Mai et al., Academic Press;
1997; and Co-Planar Sterotaxic Atlas of the Human Brain:
3-Dimensional Proportional System: An Approach to Cerebral Imaging,
eds. Tamarack et al., Thyme Medical Pub., 1988. For identification
of structures in the mouse brain, see, e.g., The Mouse Brain in
Sterotaxic Coordinates, 2nd ed., Academic Press, 2000. If desired,
the human brain structure can be correlated to similar structures
in the brain of another mammal. For example, most mammals,
including humans and rodents, show a similar topographical
organization of the entorhinal-hippocampus projections, with
neurons in the lateral part of both the lateral and medial
entorhinal cortex projecting to the dorsal part or septal pole of
the hippocampus, whereas the projection to the ventral hippocampus
originates primarily from neurons in medial parts of the entorhinal
cortex (Principles of Neural Science, 4th ed., eds Kandel et al.,
McGraw-Hill, 1991; The Rat Nervous System, 2nd ed., ed. Paxinos,
Academic Press, 1995). Furthermore, layer II cells of the
entorhinal cortex project to the dentate gyrus, and they terminate
in the outer two-thirds of the molecular layer of the dentate
gyrus. The axons from layer III cells project bilaterally to the
cornu ammonis areas CA1 and CA3 of the hippocampus, terminating in
the stratum lacunose molecular layer.
[0146] To deliver the vector specifically to a particular region of
the central nervous system, especially to a particular region of
the brain, it may be administered by sterotaxic microinjection. For
example, on the day of surgery, patients will have the sterotaxic
frame base fixed in place (screwed into the skull). The brain with
sterotaxic frame base (MRI-compatible with fiduciary markings) will
be imaged using high resolution MRI. The MRI images will then be
transferred to a computer that runs stereotaxic software. A series
of coronal, sagittal and axial images will be used to determine the
target site of vector injection, and trajectory. The software
directly translates the trajectory into 3-dimensional coordinates
appropriate for the stereotaxic frame. Burr holes are drilled above
the entry site and the stereotaxic apparatus localized with the
needle implanted at the given depth. The vector in a
pharmaceutically acceptable carrier will then be injected. The
vector is then administrated by direct injection to the primary
target site and retrogradely transported to distal target sites via
axons. Additional routes of administration may be used, e.g.,
superficial cortical application under direct visualization, or
other non-stereotaxic application.
[0147] Recombinant AAV of any serotype can be used in the instant
invention, wherein the recombinant AAV may be either a
self-complementary AAV or a non-self complementary AAV. The
serotype of the viral vector used in certain embodiments of the
invention is selected from the group consisting from AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 (see, e.g., Gao et al.
(2002) PNAS, 99:11854 11859; and Viral Vectors for Gene Therapy:
Methods and Protocols, ed. Machida, Humana Press, 2003). Other
serotype besides those listed herein can be used. Furthermore,
pseudotyped AAV vectors may also be utilized in the methods
described herein. Pseudotyped AAV vectors are those which contain
the genome of one AAV serotype in the capsid of a second AAV
serotype; for example, an AAV vector that contains the AAV2 capsid
and the AAV1 genome or an AAV vector that contains the AAV5 capsid
and the AAV 2 genome (Auricchio et al., (2001) Hum. Mol. Genet.,
10(26):3075-81).
[0148] Recombinant virions or cells transduced in vitro may be
delivered directly to neural tissue such as peripheral nerves, the
retina, dorsal root ganglia, neuromuscular junction, as well as the
CNS, by injection into, e.g., the ventricular region, such as one
or both of the lateral ventricles, as well as to the striatum
(e.g., the caudate nucleus or putamen of the striatum), the
cerebellum, spinal cord, and neuromuscular junction, with a needle,
catheter or related device, using neurosurgical techniques known in
the art, such as by stereotactic injection (see, e.g., Stein et
al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS
97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993;
and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000). In an
illustrative embodiment, the delivery is accomplished by direct
injection of a high titer vector solution into the spinal cord of a
subject or patient.
[0149] In another illustrative embodiment, a method to deliver a
transgene to the spinal cord and/or the brainstem region of a
subject by administering a recombinant AAV vector containing the
transgene to at least one region of the deep cerebellar nuclei
(DCN) region of the cerebellum of the subject's brain. Deep within
the cerebellum is grey matter called the deep cerebellar nuclei
termed the medial (fastigial) nucleus, the interposed
(interpositus) nucleus and the lateral (dentate) nucleus. As used
herein, the term "deep cerebellar nuclei" collectively refers to
these three regions, wherein one or more of these three regions may
be targeted. The viral delivery is under conditions that favor
expression of the transgene in the spinal cord and/or the brainstem
region, in at least one subdivision of the spinal cord of the
subject. These subdivisions include one or more of cervical,
thoracic, lumbar or sacral.
[0150] Without being limited as to theory, one embodiment of the
invention lies in the ability to provide a therapeutic molecule
(for example, a protein or peptide) to each division of the spinal
cord. This may be accomplished by injecting an AAV vector,
including a scAAV vector into the DCN. Furthermore, it may be
important to target individual lamina within each spinal cord
division. Lamina are specific sub-regions within regions of the
brain and spinal cord. It may be desirable in certain embodiments
to target specific lamina within a certain spinal cord division.
Since motor neuron damage may occur within the upper motor neurons
as well, it may also be desirable to provide a therapeutic molecule
(for example, a protein or peptide) to the divisions of the
brainstem. In one embodiment, it may be desirable to provide the
therapeutic molecule to both the spinal cord, including some or all
subdivisions as well as to the brainstem, including some or all
subdivisions. The instant invention uses the introduction of an AAV
vector into the DCN to accomplish the above described delivery of a
therapeutic molecule to the spinal cord region(s) and/or
brainstem.
[0151] Another method for targeting spinal cord (e.g., glia) is by
intrathecal delivery, rather than into the spinal cord tissue
itself. Such delivery presents many advantages. The targeted
protein is released into the surrounding CSF and unlike viruses,
released proteins can penetrate into the spinal cord parenchyma,
just as they do after acute intrathecal injections. Indeed,
intrathecal delivery of viral vectors can keep expression local. An
additional advantage of intrathecal gene therapy is that the
intrathecal route mimics lumbar puncture administration (i.e.,
spinal tap) already in routine use in humans.
[0152] Another method for administering the recombinant vectors or
transduced cells is by delivery to dorsal root ganglia (DRG)
neurons, e.g., by injection into the epidural space with subsequent
diffusion to DRG. For example, the recombinant vectors or
transduced cells can be delivered via intrathecal cannulation under
conditions where the protein is diffused to DRG. See, e.g., Chiang
et al., ActaAnaesthesiol. Sin. (2000) 38:31-36; Jain, K. K., Expert
Opin. Investig. Drugs (2000) 9:2403-2410.
[0153] Yet another mode of administration to the CNS uses a
convection-enhanced delivery (CED) system, which is any non-manual
delivery of the vector. In one embodiment of CED, a pressure
gradient is created via the use of a non-manual delivery system. By
using CED, recombinant vectors can be delivered to many cells over
large areas of the CNS. Moreover, the delivered vectors efficiently
express transgenes in CNS cells (e.g., glial cells). Any
convection-enhanced delivery device may be appropriate for delivery
of recombinant vectors. In a preferred embodiment, the device is an
osmotic pump or an infusion pump. Both osmotic and infusion pumps
are commercially available from a variety of suppliers, for example
Alzet Corporation, Hamilton Corporation, Alza, Inc., Palo Alto,
Calif.). Typically, a recombinant vector is delivered via CED
devices as follows. A catheter, cannula or other injection device
is inserted into CNS tissue in the chosen subject. Stereotactic
maps and positioning devices are available, for example from ASI
Instruments, Warren, Mich. Positioning may also be conducted by
using anatomical maps obtained by CT and/or MRI imaging to help
guide the injection device to the chosen target. Moreover, because
the methods described herein can be practiced such that relatively
large areas of the subject take up the recombinant vectors, fewer
infusion cannula are needed. Since surgical complications are often
related to the number of penetrations, this mode of delivery serves
to reduce the side-effects seen with conventional delivery
techniques. For a detailed description regarding CED delivery, see
U.S. Pat. No. 6,309,634, incorporated herein by reference in its
entirety.
[0154] Intracerebroventricular, or intraventricular, delivery of a
recombinant AAV vector may be performed in any one or more of the
brain's ventricles, which are filled with cerebrospinal fluid
(CSF). CSF is a clear fluid that fills the ventricles, is present
in the subarachnoid space, and surrounds the brain and spinal cord.
CSF is produced by the choroid plexuses and via the weeping or
transmission of tissue fluid by the brain into the ventricles. The
choroid plexus is a structure lining the floor of the lateral
ventricle and the roof of the third and fourth ventricles. Certain
studies have indicated that these structures are capable of
producing 400-600 ccs of fluid per day consistent with an amount to
fill the central nervous system spaces four times in a day. In
adult humans, the volume of this fluid has been calculated to be
from 125 to 150 ml (4-5 oz). The CSF is in continuous formation,
circulation and absorption. Certain studies have indicated that
approximately 430 to 450 ml (nearly 2 cups) of CSF may be produced
every day. Certain calculations estimate that production equals
approximately 0.35 ml per minute in adults and 0.15 per minute in
infant humans. The choroid plexuses of the lateral ventricles
produce the majority of CSF. It flows through the foramina of Monro
into the third ventricle where it is added to by production from
the third ventricle and continues down through the aqueduct of
Sylvius to the fourth ventricle. The fourth ventricle adds more
CSF; the fluid then travels into the subarachnoid space through the
foramina of Magendie and Luschka. It then circulates throughout the
base of the brain, down around the spinal cord and upward over the
cerebral hemispheres. The CSF empties into the blood via the
arachnoid villi and intracranial vascular sinuses.
[0155] In one aspect, the disclosed methods include administering
to the CNS of an afflicted subject a rAAV virion carrying a
transgene encoding a therapeutic product and allowing the transgene
to be expressed within the CNS near the administration site at a
level sufficient to exert a therapeutic effect as the expressed
protein is transported via the CSF throughout the CNS. In some
embodiments, the methods comprise administration of a high titer
virion composition carrying a therapeutic transgene so that the
transgene product is expressed at a therapeutic level at a first
site within the CNS distal to the ultimate site of action of the
expressed product.
[0156] In experimental mice, the total volume of injected AAV
solution is for example, between 1 to 20 .mu.l. For other mammals,
including the human, volumes and delivery rates are appropriately
scaled. Treatment may consist of a single injection per target
site, or may be repeated in one or more sites. Multiple injection
sites can be used. For example, in some embodiments, in addition to
the first administration site, a composition containing a viral
vector carrying a transgene is administered to another site which
can be contralateral or ipsilateral to the first administration
site. Injections can be single or multiple, unilateral or
bilateral.
[0157] Dosage treatment may be a single dose schedule, continuously
or intermittently, or a multiple dose schedule. Moreover, the
subject may be administered as many doses as appropriate. If
multiple doses are administered, the first formulation administered
can be the same or different than the subsequent formulations.
Thus, for example, the first administration can be in the form of
an AAV vector and the second administration in the form of an
adenovirus vector, plasmid DNA, a protein composition, or the like.
Moreover, subsequent delivery can also be the same or different
than the second mode of delivery.
[0158] In addition, the subject may receive the rAAV vector of the
instant invention by a combination of the delivery methods
disclosed therein. Thus, a subject may receive injections of an AAV
vector in at least two injection sites selected from the group
consisting of intracerebroventricular injections, direct spinal
cord injections, intrathecal injections, and intraparenchymal brain
injections (e.g., the striatum, the cerebellum, including the deep
cerebellar nuclei). In one embodiment, the subject may receive rAAV
vector via 1) at least one intracerebroventricular injection, and
at least one direct spinal cord injection or 2) at least one
intracerebroventricular injection and at least one intrathecal
injection or 3) at least one intracerebroventricular injection and
at least one intraparenchymal brain injection or 4) at least one
direct spinal cord injection and at least one intrathecal injection
or 5) at least one direct spinal cord injection and at least one
intraparenchymal brain injection or 6) at least one intrathecal
injection and at least one intraparenchymal brain injection.
[0159] It should be understood that more than one transgene can be
expressed by the delivered recombinant virion. Alternatively,
separate vectors, each expressing one or more different transgenes,
can also be delivered to the subject as described herein. Thus,
multiple transgenes can be delivered concurrently or sequentially.
Furthermore, it is also intended that the vectors delivered by the
methods of the present invention be combined with other suitable
compositions and therapies. Additionally, combinations of protein
and nucleic acid treatments can be used.
[0160] Methods of determining the most effective means of
administration and therapeutically effective dosages are well known
to those of skill in the art and will vary with the vector, the
composition of the therapy, the target cells, and the subject being
treated. Therapeutically effective doses can be readily determined
using, for example, one or more animal models of the particular
disease in question. A "therapeutically effective amount" will fall
in a relatively broad range that can be determined through clinical
trials. For example, for in vivo injection of rAAV virions, a dose
will be on the order of from about 10.sup.6 to 10.sup.15 genome
particles of the recombinant virus, more preferably 10.sup.8 to
10.sup.14 genome particles recombinant virus, or any dose within
these ranges which is sufficient to provide the desired affect. In
certain embodiments, the concentration or titer of the vector in
the composition is at least: (a) .delta. 6, 7, 8, 9, 10, 15, 20,
25, or 50 (.times.10.sup.12 gp/ml); (b) .delta. 6, 7, 8, 9, 10, 15,
20, 25, or 50 (.times.10.sup.9 tu/ml); or (c) 5, 6, 7, 8, 9, 10,
15, 20, 25, or 50 (.times.10.sup.10 iu/ml).
[0161] For in vitro transduction, an effective amount of rAAV
virions to be delivered to cells will be on the order of 10.sup.8
to 10.sup.13 of the recombinant virus. The amount of transduced
cells in the pharmaceutical compositions, in turn will be from
about 10.sup.4 to 10.sup.10 cells, more preferably 10.sup.5 to
10.sup.8 cells. Other effective dosages can be readily established
by one of ordinary skill in the art through routine trials
establishing dose response curves.
[0162] Generally, from 1 .mu.l to 1 ml of composition will be
delivered, such as from 0.01 to about 0.5 ml, for example about
0.05 to about 0.3 ml, such as 0.08, 0.09, 0.1, 0.2, etc. and any
number within these ranges, of composition will be delivered.
Animal Models
[0163] Therapeutic effectiveness and safety using the AAV virions
including transgenes as described above can be tested in an
appropriate animal model. For example, animal models which appear
most similar to human disease include animal species which either
spontaneously develop a high incidence of the particular disease or
those that have been induced to do so.
[0164] In particular, several animal models for SMA are known and
have been generated. See, e.g., Sumner C. J., NeuroRx (2006)
3:235-245; Schmid et al., J. Child Neurol. (2007) 22:1004-1012. As
explained above, the molecular basis of SMA, an autosomal recessive
neuromuscular disorder, is the homozygous loss of the survival
motor neuron gene 1 (SMN1). A nearly identical copy of the SMN1
gene, called SMN2 is found in humans and modulates the disease
severity. In contrast to humans, mice have a single gene (SMN) that
is equivalent to SMN1. Homozygous loss of this gene is lethal to
embryos and results in massive cell death, which indicates that the
SMN gene product is necessary for cellular survival and function.
The introduction of 2 copies of SMN2 into mice lacking SMN rescues
the embryonic lethality, resulting in mice with the SMA phenotype
(Monani et al., Hum. Mol. Genet. (2000) 9:333-339. A high copy
number of SMN2 rescues the mice because sufficient SMN protein is
produced in motor neurons. See, also, Hsieh-Li, et al., Nat. Genet.
(2000) 24:66-70, reporting the production of transgenic mouse lines
that expressed human SMN2. In particular, transgenic mice harboring
SMN2 in the SMN-/- background show pathological changes in the
spinal cord and skeletal muscles similar to those of SMA patients.
The severity of the pathological changes in these mice correlates
with the amount of SMN protein that contained the region encoded by
exon 7. Phenotypes in this mouse model include motor neuron cell
loss, skeletal muscle atrophy, aberrant neuromuscular junctions
(NMJ), behavioral deficits, paralysis, and a shortened life span of
about two weeks. Le et al., Hum. Mol. Genet. (2005) 14:845-857.
[0165] Similarly, animal models for ALS are known. ALS is a fatal
neurodegenerative disease that is characterized by a selective loss
of motor neurons in the cortex, brain stem and spinal cord.
Progression of the disease can lead to atrophy of limb, axial and
respiratory muscles. Motor neuron cell death is accompanied by
reactive gliosis, neurofilament abnormalities, and a significant
loss of large myelinated fibers in the corticospinal tracts and
ventral roots. Although the etiology of ALS is poorly understood,
accumulating evidence indicates that sporadic (SALS) and familial
(FALS) ALS share many similar pathological features; thus,
providing a hope that the study of either form will lead to a
common treatment. FALS accounts for approximately 10% of diagnosed
cases, of which 20% are associated with dominantly inherited
mutations in Cu/Zn superoxide dismutase (SODI). Transgenic mice
that express the mutant human SODI protein (e.g., SODIG93A mice)
recapitulate many pathological features of ALS and are an available
anima model to study ALS. For SALS, a myriad of pathological
mechanisms have been implicated as the underlying cause, including
glutamate induced excitotoxicity, toxin exposure, proteasome
dysfunction, mitochondrial damage, neurofilament disorganization
and loss of neurotrophic support.
[0166] Experimental Autoimmune Encephalomyelitis (EAE), also called
Experimental Allergic Encephalomyelitis, provides an animal model
for MS. EAE resembles the various forms and stages of MS very
closely. EAE is an acute or chronic-relapsing, acquired,
inflammatory and demyelinating autoimmune disease. In order to
create the disease, animals are injected with proteins that make up
myelin, the insulating sheath that surrounds neurons. These
proteins induce an autoimmune response in the injected animals
which develop a disease process that closely resembles MS in
humans. EAE has been induced in a number of different animal
species including mice, rats, guinea pigs, rabbits, macaques,
rhesus monkeys and marmosets.
[0167] Spinal and bulbar muscular atrophy (SBMA) is an adult-onset
motor neuron disease, caused by the expansion of a trinucleotide
repeat (TNR) in exon 1 of the androgen receptor (AR) gene. This
disorder is characterized by degeneration of motor and sensory
neurons, proximal muscular atrophy, and endocrine abnormalities,
such as gynecomastia and reduced fertility. Only males develop
symptoms, while female carriers usually are asymptomatic. The
molecular basis of SBMA is the expansion of a trinucleotide CAG
repeat, which encodes the polyglutamine (polyQ) tract, in the first
exon of the androgen receptor (AR) gene. The pathologic hallmark is
nuclear inclusions (NIs) containing the mutant and truncated AR
with expanded polyQ in the residual motor neurons in the brainstem
and spinal cord as well as in some other visceral organs. Several
transgenic mouse models have been created for studying the
pathogenesis of SBMA. See, e.g., Katsuno et al., Cytogen. and
Genome Res. (2003) 100:243-251. For example, a transgenic mouse
model carrying pure 239 CAGs under human AR promoter and another
model carrying truncated AR with expanded CAGs show motor
impairment and nuclear NIs in spinal motor neurons. Transgenic mice
carrying full-length human AR with expanded polyQ demonstrate
progressive motor impairment and neurogenic pathology as well as
sexual difference of phenotypes. These models recapitulate the
phenotypic expression observed in SBMA.
[0168] Machado-Joseph disease (MJD), also called spinocerebellar
ataxia type 3, is caused by mutant ataxin-3 with a polyglutamine
expansion. Mouse models of MJD, as well as other polyglutamine
spinocerebellar ataxias have been generated. For a review of these
models, see e.g., Gould, V. F. C. NeuroRx (2005) 2:480-483.
[0169] Accordingly, animal models standard in the art are available
for the screening and/or assessment for activity and/or
effectiveness of the methods and compositions of the invention for
the treatment of motor neuron disorders.
Kits of the Invention
[0170] The invention also provides kits. In certain embodiments,
the kits of the invention comprise one or more containers
comprising recombinant vectors encoding the protein of interest.
The kits may further comprise a suitable set of instructions,
generally written instructions, relating to the use of the vectors
for any of the methods described herein.
[0171] The kits may comprise the components in any convenient,
appropriate packaging. For example, if the recombinant vectors are
provided as a dry formulation (e.g., freeze dried or a dry powder),
a vial with a resilient stopper is normally used, so that the
vectors may be easily resuspended by injecting fluid through the
resilient stopper. Ampules with non-resilient, removable closures
(e.g., sealed glass) or resilient stoppers are most conveniently
used for liquid formulations. Also contemplated are packages for
use in combination with a specific device, such as a syringe or an
infusion device such as a minipump.
[0172] The instructions relating to the use or the recombinant
vectors generally include information as to dosage, dosing
schedule, and route of administration for the intended method of
use. The containers may be unit doses, bulk packages (e.g.,
multi-dose packages) or sub-unit doses. Instructions supplied in
the kits of the invention are typically written instructions on a
label or package insert (e.g., a paper sheet included in the kit),
but machine-readable instructions (e.g., instructions carried on a
magnetic or optical storage disk) are also acceptable.
2. Experimental
[0173] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
[0174] Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
Materials and Methods
[0175] AAV Vectors.
[0176] The open reading frame of a exemplary human SMN1 gene (the
open reading frame sequence is shown in FIG. 9A; the corresponding
amino acid sequence is shown in FIG. 9B; the complete nucleotide
sequence is found at GenBank accession number NM.sub.--000344)) was
cloned into a shuttle plasmid containing either the AAV2 inverted
terminal repeats (ITR) and the 1.6 kb cytomegalovirus
enhancer/chicken 3-actin (CBA) promoter or the scAAV2 ITR and the
0.4 kb human 3-glucuronidase (GUSB) promoter. The size constraint
of the recombinant genome in the scAAV packaging reaction required
the use of a small promoter (McCarty, D. M. Molec. Ther. (2008)
16:1648-1656). Thus, the 0.4 kb GUSB promoter was chosen because it
is ubiquitously expressed throughout the CNS including motor
neurons of the spinal cord (Passini et al., J. Virol. (2001)
75:12382-12392). The recombinant plasmids were each packaged into
AAV serotype-8 capsid by triple-plasmid cotransfection of human 293
cells (see, e.g., U.S. Pat. No. 6,001,650, incorporated by
reference herein in its entirety) and virions were column-purified
as reported previously (O'Riordan et al., J.
[0177] Gene Med. (2000) 2:444-454.). The resulting vectors
AAV2/8-CBA-hSMN1 (AAV-hSMN1) and scAAV2/8-GUSB-hSMN1 (scAAV-hSMN1)
possessed titers of 8.3 e12 and 2.8 e12 genome copies per ml,
respectively.
[0178] Animals and procedures. Heterozygote (SMN.sup.+/-,
hSMN2.sup.+/+, SMNA7.sup.+/+) breeding pairs were mated and, on the
day of birth (P0), newborn pups received 3 total injections of 2
.mu.l each into the cerebral lateral ventricles of both hemispheres
and the upper lumbar spinal cord. The total doses of viral vectors
were 5.0 e10 and 1.7 e10 genome copies for AAV-hSMN1 and
scAAV-hSMN1, respectively. All the injections were performed with a
finely drawn glass micropipette needle as described (Passini et al,
J. Virol. (2001) 75:12382-12392). Following the injections, the
pups were toe-clipped and genotyped (Le et al., Hum. Mol. Genet.
(2005) 14:845-857) to identify SMA (SMN.sup.-/-, hSMN2.sup.+/+,
SMNA7.sup.+/+), heterozygote, and wild type (SMN.sup.+/+,
hSMN2.sup.+/+, SMNA7.sup.+/+) mice. All the litters were culled to
7 pups to control for litter size on survival. Some of the litters
were not injected in order to generate untreated control
groups.
[0179] Western Blots.
[0180] For biochemical analysis, treated and untreated mice were
killed at 16 and 58-66 days were perfused with phosphate-buffered
saline (PBS), the spinal cords were dissected and separated into
the lumbar, thoracic and cervical segments, and snap-frozen in
liquid nitrogen. Tissues were homogenized at a concentration of 50
mg/mL using T-Per lysis buffer and protease inhibitor cocktail
(Pierce, Rockford, Ill.). The homogenates were cleared by
centrifugation at 10,000 RCF for 6 minutes and the protein
concentration was measured by BCA assay (Pierce, Rockford, Ill.).
10-20 .mu.g of homogenate protein were resolved on a 4-12%
SDS-PAGE, transferred to nitrocellulose membrane, and probed with a
mouse monoclonal anti-SMN (1:5,000 BD Biosciences, San Jose,
Calif.) and a rabbit polyclonal anti-(3-tubulin (1:750, Santa Cruz
Biotechnology, Santa Cruz, Calif.) antibodies. The membranes were
incubated with infrared secondary antibodies (1:20,000, LI-COR
Biosciences, Lincoln NB), and protein bands were visualized by
quantitative fluorescence using Odyssey (LI-COR Biosciences).
Molecular weight markers confirmed the sizes of the bands.
[0181] Immunohistochemistry. For histological analysis, treated and
untreated mice were killed at 16 and 58-66 days were perfused with
4% paraformaldehyde (pH 7.4), the spinal cords were removed and
placed in 30% sucrose for 48-72 hours, embedded in OCT and cut into
10 .mu.m frozen sections by a cryostat. Spinal cord sections were
blocked for 1 h at room temperature (RT) and then incubated with
either a mouse monoclonal anti-SMN antibody (1:200 dilution) to
identify AAV-derived hSMN expression, or a goat polyclonal
anti-choline acetyl transferase (ChAT) antibody (Millipore;
Burlington, Mass.; 1:100 dilution) to identify motor neurons of
laminae 8 and 9 (ventral horn) of the spinal cord or a rabbit
polyclonal anti-glial fibrillary acidic protein (GFAP) antibody
(Sigma-Aldrich, 1:2,500 dilution) to detect astrocytes. Primary
antibodies were incubated for 1 h at RT followed by an overnight
incubation at 4.degree. C. in a humidified chamber. Spinal cord
sections were then incubated for 1 h at RT with either a
FITC-conjugated anti-rabbit secondary antibody or a Cy3-conjugated
anti-goat secondary antibody (Jackson ImmunoResearch; West Grove,
Pa.; 1:250 dilution). To increase the SMN and ChAT immuno-positive
signal, a TSA signal amplification kit (Perkin Elmer; Waltham,
Mass.) or a citric acid antigen retrieval protocol (Vector Labs;
Burlingame, Calif.) were performed according to the manufacturer's
instruction, respectively. Sections were cover-slipped with
Vectashield mounting media (Vector Labs; Burlingame, Calif.).
[0182] Motor Neuron Counting.
[0183] The number of ChAT immuno-positive cells was counted in the
cervical, thoracic, and lumbar segments. Bilateral counts were
performed at 100.times. magnification in the ventral horns along
the rostrocaudal axis of the three spinal cord segments. Adjacent
sections were at least 100 microns apart to prevent double counting
of the same cell. Special care was taken to compare anatomically
matched sections between different animals, and all cell counts
were assessed blind by a single observer. Cells located in laminae
8 and 9 of the spinal cord exhibiting a fluorescent ChAT signal
markedly above background were considered motor neurons.
[0184] Myofiber Size.
[0185] For histological analysis of the periphery, the fixed
quadriceps, gastrocnemius and intercostal muscles from the right
side of each mouse were processed by paraffin and stained for
hematoxylin-eosin to determine myofiber size as reported (Avila et
al., J. Clin. Invest. (2007) 117:659-671). Approximately 500
non-overlapping myofibers from each muscle per animal were randomly
selected and photographed at 60.times. magnification. The
cross-section areas of each myofiber were measured using a
Metamorph software (Molecular Devices, Sunnyvale, Calif.).
[0186] Neuromuscular Junction Staining (NMJ).
[0187] The fixed muscle groups from the left side of each mouse
were stored in PBS for NMJ analysis. In toto staining on teased
muscle fibers from the quadriceps, gastrocnemius and intercostals
muscles was performed as reported (Lesbordes et al., Hum. Mol.
Genet. (2003) 12:1233-1239). Pre-synaptic nerve terminals were
labeled by overnight incubation at 4.degree. C. with a rabbit
polyclonal antibody against the 150 kD neurofilament isoform (NF-M,
Millipore, Billerica, Mass., 1:200 dilution), followed by a
biotinylated anti-rabbit secondary antibody (Jackson
ImmunoResearch, 1:200 dilution). Acetylcholine receptors on the
muscle endplates were labeled with Alexa 555-conjugated
.alpha.-bungarotoxin (Molecular Probes, Eugene, Oreg.) at 1:5000
for 3 h at RT. Stained muscle fibers were mounted onto slides,
cover-slipped with Vectashield, and viewed under epifluorescence.
For NMJ quantification, a minimum of 100 NMJs from each muscle per
animal were randomly selected and assessed under the microscope.
Confocal images were captured using a Zeiss LSM 510-META
microscope.
[0188] Behavior Tests.
[0189] In the righting reflex, each mouse was placed on a supine
position and the time taken for the mouse to reposition itself onto
all four paws was measured. The procedure was repeated three times
for each animal, and the average of the three scores was designated
the righting score. If the mouse did not respond within 60 seconds,
the test was terminated. In the negative geotaxis, each mouse was
placed on a 45.degree.-platform facing downward. The test was
deemed a success if the mouse turned 180.degree. to the "head up"
position. Each mouse was given three attempts to complete the task
in 180 seconds or less. In the grip strength, the forelimbs and
hindlimbs were placed together on a wire grid and gently dragging
horizontally along the mesh. Resistance was recorded in grams by a
force transducer. In the hindlimb splay test, each mouse was
suspended by its tail for 5 seconds and the resulting splay was
scored based on an arbitrary system. A healthy splay of both
hindlimbs similar to that observed in wild-type mice was given a
score of 4. An acute splay angle or "weak splay" of both hindlimbs
was a score of 3. A single leg splay was assigned a score of 2. A
mouse that exhibited no splay was given a score of 1. Finally, a
score of 0 occurred when the pup pulled both hindlimbs together,
effectively crossing them over the other.
[0190] Statistics.
[0191] The behavioral tests, the number of motor neurons, the
cross-section myofiber areas, and the NMJ were analyzed with
one-way ANOVA and Bonferroni multiple post hoc comparisons and with
unpaired two-tailed student t-tests. The Kaplan-Meier survival
curve was analyzed with the log-rank test equivalent to the
Mantel-Haenszel test. All statistical analyses were performed with
GraphPad Prism v4.0 (GraphPad Software, San Diego, Calif.). Values
with p<0.05 were considered significant.
Example 1
Significant Increase in Survival with Treatment Using AAV-Mediated
SMN1 Delivery
[0192] SMA mice on postnatal day 0 (P0) were injected
intracerebroventricularly with AAV-hSMN1 into both cerebral lateral
ventricles and by direct spinal cord injection into the upper
lumbar spinal cord for a total dose of 5.0 e10 genome copies per
mouse. Treated and untreated SMA mice were randomly separated into
either a survival cohort in which all the mice were left
undisturbed and sacrificed at a humane end point, or into an
age-matched cohort in which all the mice were sacrificed at 16 days
for age-matched comparisons with end-stage untreated SMA mice.
[0193] In the survival cohort, SMA mice treated with AAV-hSMN1
showed a significant increase in median lifespan to 50 days
(p<0.0001), compared to 15 days in untreated SMA controls (FIG.
1). All of the treated SMA mice were alive at 15 days, and 87.5% of
the treated SMA mice were alive at 19 days compared to 0% in
untreated SMA. The Kaplan-Meier curve showed a bimodal survival
distribution with treatment, in which the first group died at 17-27
days and the second group at 58-66 days (FIG. 1). In the first
group, the majority of the treated SMA mice showed ambulation, but
the mice were stunted in growth and were ultimately found dead in
the cage. The second group of treated SMA mice at 58-66 days showed
ambulation and weight gain, but eventually developed severe
hindlimb necrosis that resulted in euthanasia of the animal. As
such, the 58-66 days mice were analyzed in parallel with the
16-days age-matched cohort.
Example 2
AAV-Mediated Expression of SMN in the Spinal Cord and Motor Neuron
Counts
[0194] Levels of hSMN protein increased throughout the spinal cord
following CNS administration of AAV-hSMN1. In AAV-treated SMA mice
at 16 days, there was an approximate 34.0- and 3.6-fold increase in
hSMN protein levels in the injected lumbar segment compared to
untreated SMA and wild-type mice, respectively (FIG. 2A). The
increase in hSMN protein expression extended into the other
segments, which included a >2.0-fold increase above wild type
levels in the thoracic and cervical spinal cord at 16 days (FIGS.
2B and 2C). In the second group, hSMN protein expression was
sustained in AAV-treated SMA mice at 58-66 days. The injected
lumbar and neighboring thoracic and cervical regions was
approximately 2.5-, 2.2- and 1.2-fold higher than age-matched WT
controls, respectively.
[0195] Immunostaining of tissue sections showed hSMN protein in the
dorsal and ventral horns of the spinal cord in treated SMA mice at
16 and 58-66 days (FIG. 3). Upon closer examination of the
transduced cells, vector-derived hSMN expression was detected in a
punctate pattern throughout the cytosol, and in gem-like structures
in the nucleus (FIG. 3A). Furthermore, hSMN protein was localized
to neurites in distinct granule-like structures that could be seen
spanning the length of dendrites and axons (FIGS. 3B-3D). The very
low level of endogenous hSMN protein in SMA mice was below the
threshold of immunodetection in cells (FIG. 3E).
[0196] Co-localization with ChAT and hSMN confirmed that a subset
of the transduced cells were indeed motor neurons (FIGS. 3F-31). At
16 days, approximately 18-42% of ChAT-positive cells in the lumbar,
thoracic and cervical segments of the spinal cord were transduced
by AAV-hSMN1 (FIG. 3J). This percentage was higher at 58-66 days,
in which 60-70% of motor neurons expressed exogenous hSMN in the
three spinal cord segments (FIG. 3J). There was an overall increase
in the number of motor neurons in treated SMA mice compared to
untreated mutants (FIG. 4). However, there were significantly less
motor neurons in treated SMA mice compared to wild type mice at 16
and 58-66 days (FIG. 4).
[0197] hSMN immunostaining of cervical tissue sections from
untreated, Intracerebroventricular (ICV)-only injected, and
lumbar-only injected heterozygote mice was performed. ICV
injections alone did not contribute to appreciable AAV transduction
patterns in the brain but nevertheless generated substantial
targeting of the cervical spinal cord that was not achievable with
lumbar-only injections. In particular, the ICV-only injections
resulted in the cervical spinal cord expression of hSMN. This was
in contrast to intraparenchymal injection of the lumbar segment
that showed very little transduction of the cervical spinal cord,
presumably due to the distal proximity from the injection site. On
occasion, SMN immunopositive signal that possessed a gem-like
appearance was observed in the nucleus of untreated heterozygote
and wild-type mice. However, this immunostaining pattern was not
observed in the nucleus of untreated SMA mice.
[0198] Thus, the combination of ICV and lumbar injections in P0
mice provided broad, widespread transduction of the spinal cord.
ICV injections of AAV8-hSMN targeted the cervical spinal cord for
transduction.
Example 3
Effects of AAV Treatment on Myofiber Size, the NMJ, and
Behavior
[0199] The quadriceps (proximal), gastrocnemius (distal) and
intercostal (respiratory) muscles were chosen for analysis because
they show marked degeneration. In untreated SMA mice at 16 days,
myofibers were small and the majority of individual cells contained
a cross-section area of <100 um.sup.2 (FIG. 5A). Less than 10%
of the myofibers from the untreated SMA mice contained a
cross-section area of more than 200 um.sup.2. In contrast, the
distribution of myofiber sizes in AAV-hSMN1 treated SMA mice was
similar to wild type, and many cells possessed a cross-section area
of more than 200 and more than 400 .mu.m.sup.2 at 16 and 58-66
days, respectively (FIGS. 5A and 5B). The overall average at 16
days showed that the myofibers from treated SMA mice were more than
2-fold larger than those from untreated SMA mice (FIG. 5C).
Furthermore, the average myofiber cross-section area in treated SMA
mice at 58-66 days was 67%, 76%, and 82% that of wild type mice in
the quadriceps, gastrocnemius, and intercostal, respectively (FIG.
5C).
[0200] Analysis of the neuromuscular junction (NMJ) from untreated
SMA mice at 16 days showed abnormal accumulation of neurofilament
protein at the pre-synaptic termini (FIG. 6A). Approximately 75-90%
of the pre-synaptic termini from the quadriceps, gastrocnemius, and
intercostal showed this hallmark pathology in untreated SMA mice
(FIG. 6F). In contrast, the majority of the pre-synaptic termini
from AAV-hSMN1 treated SMA mice did not contain this collapsed
structure (FIGS. 6B, 6D). Only 10-25% and 5% of the pre-synaptic
termini from treated SMA mice showed this hallmark pathology at 16
and 58-66 days, respectively (FIG. 6F). However, treatment resulted
in more branching at the pre-synaptic termini compared to wild type
(FIG. 6B-6E). On the post-synaptic NMJ from treated SMA and wild
type mice, .alpha.-bungarotoxin staining produced a `pretzel-like`
structure that was indicative of a functional network of
acetylcholine receptors (FIG. 6B-6E).
[0201] Treated and untreated mice were subjected to periodic
behavioral tests that have been validated for this animal model
(Butchbach et al., Neurobiol. Dis. (2007) 27:207-219; El-Khodar et
al., Exp. Neurol. (2008) 212:29-43). Treated SMA mice had good body
scores and ambulatory skills, whereas untreated SMA mice were
emancipated and paralyzed (FIG. 7A). Treated SMA mice were
significantly heavier than untreated SMA controls, although they
never reached wild-type size (FIG. 7B). Treated SMA mice showed a
significant improvement in righting latency (FIG. 7C). There also
was a significant improvement in the treated SMA mice to complete
the negative geotaxis test, which measures spatial locomotive
behavior (FIG. 7D). Furthermore, treated SMA mice showed
significant improvements in grip strength and in the ability to
splay their hindlimbs (FIGS. 7E, 7F).
Example 4
Effects on Longevity Using Self-Complementary AAV
[0202] Without being limited as to theory, self-complementary AAV
(scAAV) vectors are predicted to have faster expression kinetics
due to the double-stranded recombinant genome (reviewed in McCarty,
D. M. Molec. Ther. (2008) 16:1648-1656). This rapid increase in
expression may be beneficial in highly aggressive diseases or
conditions where the temporal window of intervention is small.
Thus, to determine whether earlier expression could improve
efficacy, a scAAV vector (scAAV-hSMN1) was engineered and tested.
Using the same site of injections as performed in Example 1, a dose
of 1.7 e10 genome copies of scAAV-hSMN1 was administered into P0
SMA mice.
[0203] Treatment with scAAV-hSMN1 resulted in a striking and
remarkable improvement in median survival of 157 days
(p<0.0001), which was a+214% and +881% increase compared to
AAV-hSMN1-treated and untreated SMA mice, respectively (FIG. 8).
Approximately 42% of the scAAV-treated mice possessed a more than
1000% increase (log-fold increase) in median survival. Furthermore,
scAAV2/8-GUSB-hSMN1 treatment resulted in 88% of the SMA mice
living beyond 66 days, in contrast to 0% with AAV2/8-CBA-hSMN1. The
scAAV-treated SMA mice possessed healthy body scores, were well
groomed, gained weight, and maintained ambulation throughout their
life. Interestingly, scAAV-treated SMA mice developed only mild
hindlimb necrosis that never progressed into a severe phenotype.
The majority of scAAV-treated SMA mice were sacrificed due to an
unforeseen and sudden appearance of respiratory distress, which
included audible clicking gasps when breathing and a decreased rate
of respiration.
[0204] To better understand the basis for the observed increase in
survival with scAAV8-hSMN, additional SMA mice were treated at P0
and sacrificed at 16 or 64 (58-66 d) days post-injection, and
analyzed with the long-lived scAAV8-treated mice from the survival
curve (FIG. 8). At 16 days, SMN expression levels from the
scAAV8-hSMN group were approximately 60-90% to those observed in WT
animals. These levels were substantially less than that achieved
with AAV8-hSMN treatment at this time point. In the
scAAV8-hSMN-treated SMA mice, SMN levels in both the lumbar and
thoracic segments were above or at WT levels at 58-66 and 120-220
days, respectively (FIGS. 2A and 2B). In contrast, SMN levels in
the cervical spinal cord remained relatively low at all time
points.
[0205] Comparison of AAV vector tropism in the lumbar spinal cord
was examined using hSMN immunostaining on frozen tissue sections
from untreated SMA, AAV8-hSMN-treated SMA, and scAAV8-hSMN-treated
SMA mice at 16 days and 157 days post-injection. A diffuse hSMN
immunostaining pattern consistent with glial cell morphology was
observed at 16 days with AAV8-hSMN. Doubling immunolabeling of hSMN
and mGFAP confirmed that a subset of the AAV8-hSMN-transduced cells
were astrocytes. In contrast, scAAV8 treatment resulted in hSMN
expression only in distinct cell bodies with neuronal morphology,
which did not co-localize with GFAP. Double immunolabeling of hSMN
and the motor neuron marker mChAT confirmed that a subset of cells
transduced by scAAV8-hSMN and AAV8-hSMN were motor neurons. hSMN
expression was also observed in the interneuronal cell layers of
the spinal cord with both viral vectors, as exemplified by
scAAV8-hSMN at 157 days. Thus, in contrast to AAV8-hSMN,
histological analysis of scAAV8-hSMN-treated SMA mice showed hSMN
expression was largely restricted to neurons.
[0206] Furthermore, double immunostaining with hSMN and mChAT
showed a significant increase in the percentage of motor neurons
transduced with scAAV8-hSMN compared to AAV8-hSMN (FIG. 10A). The
more efficient targeting of motor neurons with scAAV correlated
with a significant increase in the number of ChAT-positive cells
(FIGS. 10B-10D). Analysis of the NMJ in the quadriceps and
intercostal muscles at 16 days also showed a significant decrease
in the number of collapsed structures with scAAV-hSMN compared to
AAV8-hSMN (FIGS. 10E and 10F). However, there was an increase in
the number of aberrant NMJs at 216-269 days that was concomitant
with the decline of motor neuron cell counts in the scAAV-hSMN
group (FIGS. 10B-10F).
[0207] To summarize, injection of AAV8-hSMN at birth into the CNS
of a mouse model of SMA resulted in widespread expression of SMN
throughout the spinal cord that translated to a robust improvement
in skeletal muscle physiology. Treated SMA animals also displayed
significant improvements on behavioral tests indicating that the
neuromuscular junction was functional. Importantly, treatment with
AAV8-hSMN increased the median lifespan of SMA mice to 50 days
compared to 15 days for untreated controls. Moreover, SMA mice
injected with a self-complementary AAV vector resulted in improved
efficacy including a significant extension in median survival to
157 days. These data evidence that CNS-directed, AAV-mediated SMN
augmentation is highly efficacious in addressing both the neuronal
and muscular pathologies of a severe mouse model of SMA.
[0208] Thus, compositions and methods for treating spinal cord
disorders are disclosed. Although preferred embodiments of the
subject invention have been described in some detail, it is
understood that obvious variations can be made without departing
from the spirit and the scope of the invention as defined herein.
Sequence CWU 1
1
21885DNAHomo sapiensmisc_featurecDNA sequence of human survival
motor neuron (SMN1) gene 1atggcgatga gcagcggcgg cagtggtggc
ggcgtcccgg agcaggagga ttccgtgctg 60ttccggcgcg gcacaggcca gagcgatgat
tctgacattt gggatgatac agcactgata 120aaagcatatg ataaagctgt
ggcttcattt aagcatgctc taaagaatgg tgacatttgt 180gaaacttcgg
gtaaaccaaa aaccacacct aaaagaaaac ctgctaagaa gaataaaagc
240caaaagaaga atactgcagc ttccttacaa cagtggaaag ttggggacaa
atgttctgcc 300atttggtcag aagacggttg catttaccca gctaccattg
cttcaattga ttttaagaga 360gaaacctgtg ttgtggttta cactggatat
ggaaatagag aggagcaaaa tctgtccgat 420ctactttccc caatctgtga
agtagctaat aatatagaac aaaatgctca agagaatgaa 480aatgaaagcc
aagtttcaac agatgaaagt gagaactcca ggtctcctgg aaataaatca
540gataacatca agcccaaatc tgctccatgg aactcttttc tccctccacc
accccccatg 600ccagggccaa gactgggacc aggaaagcca ggtctaaaat
tcaatggccc accaccgcca 660ccgccaccac caccacccca cttactatca
tgctggctgc ctccatttcc ttctggacca 720ccaataattc ccccaccacc
tcccatatgt ccagattctc ttgatgatgc tgatgctttg 780ggaagtatgt
taatttcatg gtacatgagt ggctatcata ctggctatta tatgggtttc
840agacaaaatc aaaaagaagg aaggtgctca cattccttaa attaa 8852294PRTHomo
sapiensmisc_featureamino acid sequence of human survival motor
neuron (SMN1) gene 2Met Ala Met Ser Ser Gly Gly Ser Gly Gly Gly Val
Pro Glu Gln Glu 1 5 10 15 Asp Ser Val Leu Phe Arg Arg Gly Thr Gly
Gln Ser Asp Asp Ser Asp 20 25 30 Ile Trp Asp Asp Thr Ala Leu Ile
Lys Ala Tyr Asp Lys Ala Val Ala 35 40 45 Ser Phe Lys His Ala Leu
Lys Asn Gly Asp Ile Cys Glu Thr Ser Gly 50 55 60 Lys Pro Lys Thr
Thr Pro Lys Arg Lys Pro Ala Lys Lys Asn Lys Ser 65 70 75 80 Gln Lys
Lys Asn Thr Ala Ala Ser Leu Gln Gln Trp Lys Val Gly Asp 85 90 95
Lys Cys Ser Ala Ile Trp Ser Glu Asp Gly Cys Ile Tyr Pro Ala Thr 100
105 110 Ile Ala Ser Ile Asp Phe Lys Arg Glu Thr Cys Val Val Val Tyr
Thr 115 120 125 Gly Tyr Gly Asn Arg Glu Glu Gln Asn Leu Ser Asp Leu
Leu Ser Pro 130 135 140 Ile Cys Glu Val Ala Asn Asn Ile Glu Gln Asn
Ala Gln Glu Asn Glu 145 150 155 160 Asn Glu Ser Gln Val Ser Thr Asp
Glu Ser Glu Asn Ser Arg Ser Pro 165 170 175 Gly Asn Lys Ser Asp Asn
Ile Lys Pro Lys Ser Ala Pro Trp Asn Ser 180 185 190 Phe Leu Pro Pro
Pro Pro Pro Met Pro Gly Pro Arg Leu Gly Pro Gly 195 200 205 Lys Pro
Gly Leu Lys Phe Asn Gly Pro Pro Pro Pro Pro Pro Pro Pro 210 215 220
Pro Pro His Leu Leu Ser Cys Trp Leu Pro Pro Phe Pro Ser Gly Pro 225
230 235 240 Pro Ile Ile Pro Pro Pro Pro Pro Ile Cys Pro Asp Ser Leu
Asp Asp 245 250 255 Ala Asp Ala Leu Gly Ser Met Leu Ile Ser Trp Tyr
Met Ser Gly Tyr 260 265 270 His Thr Gly Tyr Tyr Met Gly Phe Arg Gln
Asn Gln Lys Glu Gly Arg 275 280 285 Cys Ser His Ser Leu Asn 290
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