U.S. patent application number 16/457332 was filed with the patent office on 2020-01-09 for methods and compositions for treating brain diseases.
This patent application is currently assigned to UNIVERSITY OF IOWA RESEARCH FOUNDATION. The applicant listed for this patent is UNIVERSITY OF IOWA RESEARCH FOUNDATION. Invention is credited to Young Hong Chen, Beverly L. Davidson, Luis Tecedor.
Application Number | 20200009267 16/457332 |
Document ID | / |
Family ID | 52393945 |
Filed Date | 2020-01-09 |
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United States Patent
Application |
20200009267 |
Kind Code |
A1 |
Davidson; Beverly L. ; et
al. |
January 9, 2020 |
METHODS AND COMPOSITIONS FOR TREATING BRAIN DISEASES
Abstract
The present disclosure provides methods of treating a disease or
delivering a therapeutic agent to a mammal comprising administering
to the mammal's cisterna magna and/or ventricle an rAAV particle
containing a vector comprising a nucleic acid encoding a
therapeutic protein inserted between a pair of AAV inverted
terminal repeats in a manner such that cells with access to the
cerebrospinal fluid (CSF) express the therapeutic agent and in
certain embodiments secretes the therapeutic agent into the CSF for
distribution to the brain.
Inventors: |
Davidson; Beverly L.; (Iowa
City, IA) ; Tecedor; Luis; (Iowa City, IA) ;
Chen; Young Hong; (Iowa City, IA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF IOWA RESEARCH FOUNDATION |
Iowa City |
IA |
US |
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|
Assignee: |
UNIVERSITY OF IOWA RESEARCH
FOUNDATION
Iowa City
IA
|
Family ID: |
52393945 |
Appl. No.: |
16/457332 |
Filed: |
June 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14907776 |
Jan 26, 2016 |
10391184 |
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PCT/US2014/047338 |
Jul 20, 2014 |
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16457332 |
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61859157 |
Jul 26, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/06 20180101;
A61P 25/14 20180101; A61P 3/00 20180101; A61P 25/28 20180101; A61K
48/0058 20130101; A61P 29/00 20180101; A61P 25/16 20180101; A61P
25/00 20180101; A61P 21/02 20180101; C12N 2750/14143 20130101; A61K
48/00 20130101; A61K 48/0075 20130101; A61K 31/343 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 31/343 20060101 A61K031/343 |
Claims
1. A method of delivering a therapeutic agent to the central
nervous system of a mammal, comprising administering to the
mammal's cisterna magna an rAAV particle comprising an AAV capsid
protein and a vector comprising a nucleic acid encoding a
therapeutic agent inserted between a pair of AAV inverted terminal
repeats in a manner effective to infect cells that contact the
cerebrospinal fluid (CSF) of the mammal such that the cells express
the therapeutic agent in the mammal.
2. A method of treating a disease in a mammal comprising
administering to the mammal's cisterna magna an rAAV particle
comprising an AAV capsid protein and a vector comprising a nucleic
acid encoding a therapeutic agent inserted between a pair of AAV
inverted terminal repeats in a manner effective to infect cells
that contact the cerebrospinal fluid (CSF) of the mammal, wherein
the cell expresses the therapeutic agent so as to treat the
disease.
3. A method of delivering a therapeutic agent to the central
nervous system of a mammal, comprising administering to the
mammal's brain ventricle, subarachnoid space and/or intrathecal
space an rAAV particle comprising an AAV capsid protein and a
vector comprising a nucleic acid encoding a therapeutic agent
inserted between a pair of AAV inverted terminal repeats in a
manner effective to infect cells that contact the cerebrospinal
fluid (CSF) of the mammal such that the cells express the
therapeutic agent in the mammal.
4. A method of treating a disease in a mammal comprising
administering to the mammal's brain ventricle, subarachnoid space
and/or intrathecal space an rAAV particle comprising an AAV capsid
protein and a vector comprising a nucleic acid encoding a
therapeutic agent inserted between a pair of AAV inverted terminal
repeats in a manner effective to infect cells that contact the
cerebrospinal fluid (CSF) of the mammal, wherein the cell expresses
the therapeutic agent so as to treat the disease.
5. The method of claim 1, wherein the cell expresses the
therapeutic agent and secretes the therapeutic agent into the
CSF.
6. The method of claim 1, wherein the cell is an ependymal, pial,
endothelial, brain ventricle, and/or meningeal cell.
7. The method of claim 1, further comprising additionally
administering the rAAV to the mammal's brain ventricle,
subarachnoid space and/or intrathecal space.
8. The method of claim 1, wherein the mammal is a non-rodent
mammal.
9-10. (canceled)
11. The method of claim 8, wherein the non-rodent mammal is a
primate.
12. The method of claim 11, wherein the primate is human.
13. The method of claim 1, wherein the therapeutic agent is a
therapeutic nucleic acid.
14. The method of claim 1, wherein the therapeutic agent is a
protein.
15. The method of claim 14, wherein the nucleic acid encodes a
lysosomal hydrolase.
16. The method of claim 15, wherein the protein is TPP1.
17. The method of claim 1, wherein the disease is a lysosomal
storage disease (LSD).
18. The method of claim 17, wherein the LSD is infantile or late
infantile ceroid lipofuscinoses (LINCL), neuronopathic Gaucher,
Juvenile Batten, Fabry, MLD, Sanfilippo A, Hunter, Krabbe, Morquio,
Pompe, Niemann-Pick C, Tay-Sachs, Hurler (MPS-I H), Sanfilippo B,
Maroteaux-Lamy, Niemann-Pick A, Cystinosis, Hurler-Scheie (MPS-I
H/S), Sly Syndrome (MPS VII), Scheie (MPS-I S), Infantile Batten,
GM1 Gangliosidosis, Mucolipidosis type II/III, or Sandhoff
disease.
19. The method of claim 18, wherein the disease is LINCL.
20-25. (canceled)
26. The method of claim 1, wherein the rAAV particle is injected at
1-5 locations in the brain.
27. (canceled)
28. The method of claim 1, wherein the rAAV particle is an rAAV2,
rAAV4, rAAV5 and/or rAAV9 particle.
29. The method of claim 28, wherein the rAAV particle is an rAAV2
particle.
30. (canceled)
31. The method of claim 1, wherein the therapeutic agent is
administered in a single dose to the mammal's cisterna magna.
32. The method of claim 1, further comprising administering an
immunesuppression agent.
33. The method of claim 32, wherein the immuesuppression agent is
an anti-inflammatory agent.
34. The method of claim 33, wherein the anti-inflammatory agent is
mycophenolate.
35. The method of claim 1, wherein the rAAV is administered at a
dose of about 1-5 ml of 1.times.10.sup.5-1.times.10.sup.16
vg/ml.
36-37. (canceled)
38. The method of claim 2, further comprising additionally
administering the rAAV to the mammal's brain ventricle,
subarachnoid space and/or intrathecal space.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 14/907,776, filed Jan. 26, 2016, which
is a U.S. national stage application of International Patent
Application No. PCT/US2014/047338, filed Jul. 20, 2014, which
claims priority to U.S. Provisional Patent Application No.
61/859,157, filed Jul. 26, 2013. The entirety of the applications
is hereby incorporated by reference herein.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 13, 2014, is named 17023_139WO1_SL.txt and is 30,720 bytes
in size.
BACKGROUND
[0003] Gene transfer is now widely recognized as a powerful tool
for analysis of biological events and disease processes at both the
cellular and molecular level. More recently, the application of
gene therapy for the treatment of human diseases, either inherited
(e.g., ADA deficiency) or acquired (e.g., cancer or infectious
disease), has received considerable attention. With the advent of
improved gene transfer techniques and the identification of an ever
expanding library of defective gene-related diseases, gene therapy
has rapidly evolved from a treatment theory to a practical
reality.
[0004] Traditionally, gene therapy has been defined as a procedure
in which an exogenous gene is introduced into the cells of a
patient in order to correct an inborn genetic error. Although more
than 4500 human diseases are currently classified as genetic,
specific mutations in the human genome have been identified for
relatively few of these diseases. Until recently, these rare
genetic diseases represented the exclusive targets of gene therapy
efforts. Accordingly, most of the NIH approved gene therapy
protocols to date have been directed toward the introduction of a
functional copy of a defective gene into the somatic cells of an
individual having a known inborn genetic error. Only recently, have
researchers and clinicians begun to appreciate that most human
cancers, certain forms of cardiovascular disease, and many
degenerative diseases also have important genetic components, and
for the purposes of designing novel gene therapies, should be
considered "genetic disorders." Therefore, gene therapy has more
recently been broadly defined as the correction of a disease
phenotype through the introduction of new genetic information into
the affected organism.
[0005] In in vivo gene therapy, a transferred gene is introduced
into cells of the recipient organism in situ that is, within the
recipient. In vivo gene therapy has been examined in several animal
models. Several recent publications have reported the feasibility
of direct gene transfer in situ into organs and tissues such as
muscle, hematopoietic stem cells, the arterial wall, the nervous
system, and lung. Direct injection of DNA into skeletal muscle,
heart muscle and injection of DNA-lipid complexes into the
vasculature also has been reported to yield a detectable expression
level of the inserted gene product(s) in vivo.
[0006] Treatment of diseases of the central nervous system, e.g.,
inherited genetic diseases of the brain, remains an intractable
problem. Examples of such are the lysosomal storage diseases and
Alzheimer's disease. Collectively, the incidence of lysosomal
storage diseases (LSD) is 1 in 10,000 births world wide, and in 65%
of cases, there is significant central nervous system (CNS)
involvement. Proteins deficient in these disorders, when delivered
intravenously, do not cross the blood-brain barrier, or, when
delivered directly to the brain, are not widely distributed. Thus,
therapies for the CNS deficits need to be developed.
SUMMARY
[0007] The present invention provides a method of delivering a
therapeutic agent (e.g., protein or nucleic acid) to the central
nervous system of a mammal, comprising administering to the
mammal's cisterna magna an rAAV particle comprising an AAV capsid
protein and a vector comprising a nucleic acid encoding a
therapeutic agent inserted between a pair of AAV inverted terminal
repeats in a manner effective to infect cells that contact the
cerebrospinal fluid (CSF) of in the mammal such that the cells
express the therapeutic agent in the mammal.
[0008] The present invention provides a method of treating a
disease in a mammal comprising administering to the mammal's
cisterna magna an rAAV particle comprising an AAV capsid protein
and a vector comprising a nucleic acid encoding a therapeutic agent
(e.g., a therapeutic nucleic acid or a nucleic acid encoding a
protein) inserted between a pair of AAV inverted terminal repeats
in a manner effective to infect cells that contact the
cerebrospinal fluid (CSF) in the mammal, wherein the cell expresses
the therapeutic agent so as to treat the disease.
[0009] In certain embodiments, the AAV particle is an rAAV2
particle. As used herein, the term AAV2/1 is used to mean an AAV2
ITR and AAV1 capsid, the term AAV2/2 is an AAV2 ITR and AAV2
capsid, the term AAV2/4 is an AAV2 ITR and AAV4 capsid, etc. In
certain embodiments, the AAV particle is an rAAV8 particle. In
certain embodiments, the AAV particle is an rAAV9 particle. In
certain embodiments, the AAV particle is an rAAVrh10 particle. In
certain embodiments, the rAAV capsid has at least 80% homology to
AAV2 capsid protein VP1, VP2, and/or VP3. In certain embodiments,
the rAAV2 capsid has 100% homology to AAV2 capsid VP1, VP2, and/or
VP3. In certain embodiments, the rAAV capsid has at least 80%
homology to AAV4 capsid protein VP1, VP2, and/or VP3. In certain
embodiments, the rAAV4 capsid has 100% homology to AAV4 capsid VP1,
VP2, and/or VP3. In certain embodiments, the rAAV capsid has at
least 80% homology to AAV9 capsid protein VP1, VP2, and/or VP3. In
certain embodiments, the rAAV9 capsid has 100% homology to AAV9
capsid VP1, VP2, and/or VP3.
[0010] In certain embodiments, the rAAV particle is an rAAV2
particle that infects the non-rodent ependymal cell at an rate of
more than 20% than the infectivity rate of AAV4, such as at a rate
of more than 50% or 100%, 1000% or 2000% than the infectivity rate
of AAV4.
[0011] In certain embodiments, the cell expresses the therapeutic
agent and secretes the therapeutic agent into the CSF. In certain
embodiments, the cell is an ependymal, pial, endothelial or
meningeal cell. In certain embodiments, the method further
comprises additionally administering the rAAV to the non-human
primate's brain ventricle, subarachnoid space and/or intrathecal
space.
[0012] The present invention provides a method of delivering a
nucleic acid to a brain cell of a mammal comprising administering
to the brain cell an AAV particle containing a vector comprising
the nucleic acid inserted between a pair of AAV inverted terminal
repeats, thereby delivering the nucleic acid to the brain cell. In
certain embodiments, the rAAV is an rAAV2 particle that infects the
brain cell at an rate of more than 20% than the infectivity rate of
AAV4, such as at a rate of more than 50% or 100%, 1000% or 2000%
than the infectivity rate of AAV4.
[0013] In certain embodiments, the disease is a lysosomal storage
disease (LSD). In certain embodiments, the LSD is infantile or late
infantile ceroid lipofuscinoses, neuronopathic Gaucher, Juvenile
Batten, Fabry, MLD, Sanfilippo A, Hunter, Krabbe, Morquio, Pompe,
Niemann-Pick C, Tay-Sachs, Hurler (MPS-I H), Sanfilippo B,
Maroteaux-Lamy, Niemann-Pick A, Cystinosis, Hurler-Scheie (MPS-I
H/S), Sly Syndrome (MPS VII), Scheie (MPS-I S), Infantile Batten,
GM1 Gangliosidosis, Mucolipidosis type II/III, or Sandhoff disease.
In certain embodiments, the disease is LINCL. In certain
embodiments, the disease is a neurodegenerative disease, such as
Alzheimer's disease, Huntington's disease, ALS, hereditary spastic
hemiplegia, primary lateral sclerosis, spinal muscular atrophy,
Kennedy's disease, a polyglutamine repeat disease, or Parkinson's
disease.
[0014] In certain embodiments, the mammal is a non-rodent mammal,
such as a primate, horse, sheep, goat, pig, or dog. In certain
embodiments, the primate is a human.
[0015] In certain embodiments, the therapeutic agent is a
therapeutic nucleic acid. In certain embodiments, the therapeutic
agent is a protein.
[0016] In certain embodiments, the nucleic acid encodes a lysosomal
hydrolase. In certain embodiments, the nucleic acid encodes
TPP1.
[0017] In certain embodiments, the therapeutic protein is a
protective ApoE isoform protein. As used herein, the term
"protective ApoE isoform" is used to distinguish ApoE isoforms that
decrease the risk of Alzheimer's disease by at least 5%, such as
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more.
[0018] In certain embodiments, the protective ApoE isoform has at
least about 80% homology to ApoE 2. In certain embodiments, the
protective ApoE isoform has 100% homology to ApoE 2.
[0019] In certain embodiments, the rAAV particle is injected at 1-3
locations in the brain, such as at one, two, or three locations in
the brain.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A-1C together are an alignment of AAV2 (SEQ ID NO:1)
and AAV4 (SEQ ID NO:2) proteins and FIGS. 1D-1J together are an
alignment of AAV2 (SEQ ID NO:3) and AAV4 (SEQ ID NO:4) nucleotides
based on the sequence from AAV2 (NC_001401) and AAV4
(NC_001829).
[0021] FIG. 2 shows an illustration of "cross correction" between
cells. Sands and Davidson,
[0022] Mol Ther 13(5):839-849, 2006.
[0023] FIG. 3. Top: Immunohistochemical staining for human TPP1
after AAV2 mediated delivery into a LINCL dog model that is
deficient in canine TPP 1. Left, treated dog. Right, untreated
deficient animal. Compare the strong positive staining on the left
to the background staining in the right panel. Bottom. Western blot
for TPP1 showing the presence of human TPP1 in the treated,
deficient (LINCL) dog. Both normal and deficient dogs do not show
the presence of the band, as they do not express human TPP1.
[0024] FIG. 4A. Microphotographs showing the representative
autofluorescence depictive of the pathological accumulation of
lipofuscin in the neuronal ceroid lipofuscinoses. Left panel,
autofluorescence in an AAV2.TPP1 treated LINCL dog. Right panel,
autofluoresence in a control, untreated LINCL dog. Note the
reduction in autofluorence with therapy.
[0025] FIG. 4B. MRI scans of an untreated LINCL dog (upper left), a
untreated normal dog (upper right), and two AAV2.TPP1 treated dogs
(lower panels). The volumes of vector delivered are indicated in
the lower left of the bottom panels. The viral titer was
approximately 1e13 genomes/mL.
[0026] FIG. 4C. Volumetric reconstructions of the ventricles of the
dogs imaged in 4B (left panels). The graph in the right panel
denotes the volumes from the images in the left panels. Note the
extensive reduction in ventricular volume even with these low doses
of vector (stated in FIG. 4B legend).
[0027] FIG. 4D. Immunohistochemical staining in varying brain
regions shows extensive distribution of TPP1 protein after AAV.TPP1
gene transfer to the ventricular system of the LINCL dog. Top
panels are coronal sections from the dog brain atlas; the lower
right insets present the sagittal view of the coronal image. The
immunohistochemically stained sections below the panels from the
atlas show the extent of staining in sections from those regions.
Together the data show extensive distribution of enzyme.
[0028] FIG. 5. huTPP1 enzyme activity in CSF following AAV.TPP1
delivery declined shortly after viral gene transfer. Left panel:
TPP1 activity in CSF in treated Animals Co and S exceeds normal
activity levels very soon after AAV.TPP1 gene transfer, and then
rapidly declines to undetectable levels. Animals N, Po and Pi are
normal or heterozygous dogs and are shown for reference only of the
range of TPP1 activity levels in clinically normal dogs.
[0029] FIG. 6 shows the results of pre-treating with mycophenolate
on providing for sustained activity.
[0030] FIG. 7. Introduction of mycophenolate at the time of enzyme
activity decline, or prior to gene transfer, dramatically improves
the durability of TPP1 expression in dog after AAV.TPP1 delivery to
ependyma. Left and right upper graphs: Enzyme activity as a
function of time. Also indicated is the time at which mycophenolate
was administered. Note the high and sustained levels after recover
from loss of expression in Animals SR and B, and the extremely high
sustained levels in Animal F. Thus, mycophenolate pre-treatment in
animals null for recombinant protein helps provide for sustained
gene expression in transduced brain cells. Lower graph: Expansion
from the upper right graph to demonstrate that there is enzyme over
and above background levels, and close to normal levels or above
(0.1-0.4 pmol/mg).
[0031] FIG. 8. Sustained enzyme expression in CSF elevates
interstitial levels of enzyme. Enzyme activity in various brain
regions is above normal.
[0032] FIGS. 9A and 9B. Immunohistochemical staining in varying
brain regions shows extensive distribution of TPP1 protein after
AAV.TPP1 gene transfer to the ventricular system of the LINCL dog.
Representative panels from the dog brain atlas show the region of
the brain being evaluated, which is also depicted by the line.
Together the data show extensive distribution of enzyme.
[0033] FIG. 10 AAV.TPP1 gene therapy delays the onset of disease
phenotypes (appearance of the first red line on the left vs. the
first blue line on the left) and the progression of disease (the
spacing of the red lines vs. the spacing of the blue lines). Animal
life span was nearly doubled in some dogs, others are still under
evaluation.
[0034] FIG. 11 Animals with sustained TPP1 secretion from ependymal
show evidence of enzyme activity in peripheral organs and brain
dura. For two animals, BG and SR, there was notable enzyme activity
in brain dura and also the liver.
[0035] FIG. 12 The approach used to provide clinical benefit to the
LINCL dog is translatable to primates. Rhesus macaques were given
an intraventricular injection of AAV2.TPP1 (1.5 mL of 1e13 vector
genomes/mL) and TPP1 activity in brain stem (Medulla; left graph)
and CSF (right graph) measured 3 months after gene transfer. These
are normal monkeys with normal levels of TPP1 activity (range
noted--Control). In all but one animal, the enzyme activity exceeds
that of normal monkeys. Evidence of TPP1 activity in monkey brain 3
months after gene transfer using immunohistochemistry staining
against the recombinant human TPP1 expressed from the AAV
vector.
[0036] FIG. 13 shows the vestibular area (brainstem) in the
non-human primates.
[0037] FIG. 14 provides the Human TPP1 amino acid sequence (SEQ ID
NO: 5).
[0038] FIGS. 15A and 15B together provide the Human TPP1 nucleic
acid sequence (SEQ ID NO: 6).
[0039] FIG. 16 provides the Macaca mulatta TPP1 amino acid sequence
(SEQ ID NO: 7).
[0040] FIG. 17 provides the Macaca fascicularis TPP1 amino acid
sequence (SEQ ID NO: 8).
DETAILED DESCRIPTION
[0041] Adeno associated virus (AAV) is a small nonpathogenic virus
of the parvoviridae family. AAV is distinct from the other members
of this family by its dependence upon a helper virus for
replication. In the absence of a helper virus, AAV may integrate in
a locus specific manner into the q arm of chromosome 19. The
approximately 5 kb genome of AAV consists of one segment of single
stranded DNA of either plus or minus polarity. The ends of the
genome are short inverted terminal repeats which can fold into
hairpin structures and serve as the origin of viral DNA
replication. Physically, the parvovirus virion is non-enveloped and
its icosohedral capsid is approximately 20 nm in diameter.
[0042] To-date numerous serologically distinct AAVs have been
identified, and more than a dozen have been isolated from humans or
primates. The genome of AAV2 is 4680 nucleotides in length and
contains two open reading frames (ORFs). The left ORF encodes the
non-structural Rep proteins, Rep 40, Rep 52, Rep 68 and Rep 78,
which are involved in regulation of replication and transcription
in addition to the production of single-stranded progeny genomes.
Furthermore, two of the Rep proteins have been associated with the
preferential integration of AAV genomes into a region of the q arm
of human chromosome 19. Rep68/78 has also been shown to possess NTP
binding activity as well as DNA and RNA helicase activities. The
Rep proteins possess a nuclear localization signal as well as
several potential phosphorylation sites. Mutation of one of these
kinase sites resulted in a loss of replication activity.
[0043] The ends of the genome are short inverted terminal repeats
(ITR) which have the potential to fold into T-shaped hairpin
structures that serve as the origin of viral DNA replication.
Within the ITR region two elements have been described which are
central to the function of the ITR, a GAGC repeat motif and the
terminal resolution site (trs). The repeat motif has been shown to
bind Rep when the ITR is in either a linear or hairpin
conformation. This binding serves to position Rep68/78 for cleavage
at the trs which occurs in a site- and strand-specific manner. In
addition to their role in replication, these two elements appear to
be central to viral integration. Contained within the chromosome 19
integration locus is a Rep binding site with an adjacent trs. These
elements have been shown to be functional and necessary for locus
specific integration.
[0044] The AAV virion is a non-enveloped, icosohedral particle
approximately 25 nm in diameter, consisting of three related
proteins referred to as VP1, VP2 and VP3. The right ORF encodes the
capsid proteins VP1, VP2, and VP3. These proteins are found in a
ratio of 1:1:10 respectively and are all derived from the
right-hand ORF. The capsid proteins differ from each other by the
use of alternative splicing and an unusual start codon. Deletion
analysis has shown that removal or alteration of VP1 which is
translated from an alternatively spliced message results in a
reduced yield of infections particles. Mutations within the VP3
coding region result in the failure to produce any single-stranded
progeny DNA or infectious particles. An AAV particle is a viral
particle comprising an AAV capsid protein. An AAV capsid
polypeptide can encode the entire VP1, VP2 and VP3 polypeptide. The
particle can be a particle comprising AAV2 and other AAV capsid
proteins (i.e., a chimeric protein, such as AAV4 and AAV2).
Variations in the amino acid sequence of the AAV2 capsid protein
are contemplated herein, as long as the resulting viral particle
comprises the AAV2 capsid remains antigenically or immunologically
distinct from AAV4, as can be routinely determined by standard
methods. Specifically, for example, ELISA and Western blots can be
used to determine whether a viral particle is antigenically or
immunologically distinct from AAV4. Furthermore, the AAV2 viral
particle preferably retains tissue tropism distinct from AAV4.
[0045] An AAV2 particle is a viral particle comprising an AAV2
capsid protein. An AAV2 capsid polypeptide encoding the entire VP1,
VP2, and VP3 polypeptide can overall have at least about 63%
homology (or identity) to the polypeptide having the amino acid
sequence encoded by nucleotides set forth in SEQ ID NO:1 (AAV2
capsid protein). The capsid protein can have about 70% homology,
about 75% homology, 80% homology, 85% homology, 90% homology, 95%
homology, 98% homology, 99% homology, or even 100% homology to the
protein set forth in SEQ ID NO:1. The capsid protein can have about
70% identity, about 75% identity, 80% identity, 85% identity, 90%
identity, 95% identity, 98% identity, 99% identity, or even 100%
identity to the protein set forth in SEQ ID NO:1. The particle can
be a particle comprising another AAV and AAV2 capsid protein, i.e.,
a chimeric protein. Variations in the amino acid sequence of the
AAV2 capsid protein are contemplated herein, as long as the
resulting viral particle comprising the AAV2 capsid remains
antigenically or immunologically distinct from AAV4, as can be
routinely determined by standard methods. Specifically, for
example, ELISA and Western blots can be used to determine whether a
viral particle is antigenically or immunologically distinct from
AAV4. Furthermore, the AAV2 viral particle preferably retains
tissue tropism distinction from AAV4, such as that exemplified in
the examples herein, though an AAV2 chimeric particle comprising at
least one AAV2 coat protein may have a different tissue tropism
from that of an AAV2 particle consisting only of AAV2 coat
proteins.
[0046] As indicated in FIGS. 1A and 1B, AAV2 capsid sequence and
AAV4 capsid sequence are about 60% homologous. In certain
embodiments, the AAV2 capsid comprises (or consists of) a sequence
that is at least 65% homologous to the amino acid sequence set
forth in SEQ ID NO:1.
[0047] In certain embodiments, the invention further provides an
AAV2 particle containing, i.e., encapsidating, a vector comprising
a pair of AAV2 inverted terminal repeats. The nucleotide sequence
of AAV2 ITRs is known in the art. Furthermore, the particle can be
a particle comprising both AAV4 and AAV2 capsid protein, i.e., a
chimeric protein. Moreover, the particle can be a particle
encapsidating a vector comprising a pair of AAV inverted terminal
repeats from other AAVs (e.g., AAV1-AAV9 and AAVrh10). The vector
encapsidated in the particle can further comprise an exogenous
nucleic acid inserted between the inverted terminal repeats.
[0048] The following features of AAV have made it an attractive
vector for gene transfer. AAV vectors have been shown in vitro to
stably integrate into the cellular genome; possess a broad host
range; transduce both dividing and non dividing cells in vitro and
in vivo and maintain high levels of expression of the transduced
genes. Viral particles are heat stable, resistant to solvents,
detergents, changes in pH, temperature, and can be concentrated on
CsC1 gradients or by other means. The present invention provides
methods of administering AAV particles, recombinant AAV vectors,
and recombinant AAV virions. For example, an AAV2 particle is a
viral particle comprising an AAV2 capsid protein, or an AAV4
particle is a viral particle comprising an AAV4 capsid protein. A
recombinant AAV2 vector is a nucleic acid construct that comprises
at least one unique nucleic acid of AAV2. A recombinant AAV2 virion
is a particle containing a recombinant AAV2 vector. To be
considered within the term "AAV2 ITRs" the nucleotide sequence must
retain one or both features described herein that distinguish the
AAV2 ITR from the AAV4 ITR: (1) three (rather than four as in AAV4)
"GAGC" repeats and (2) in the AAV2 ITR Rep binding site the fourth
nucleotide in the first two "GAGC" repeats is a C rather than a
T.
[0049] The promoter to drive expression of the protein or the
sequence encoding another agent to be delivered can be any desired
promoter, selected by known considerations, such as the level of
expression of a nucleic acid functionally linked to the promoter
and the cell type in which the vector is to be used. Promoters can
be an exogenous or an endogenous promoter. Promoters can include,
for example, known strong promoters such as SV40 or the inducible
metallothionein promoter, or an AAV promoter, such as an AAV p5
promoter. Additional examples of promoters include promoters
derived from actin genes, immunoglobulin genes, cytomegalovirus
(CMV), adenovirus, bovine papilloma virus, adenoviral promoters,
such as the adenoviral major late promoter, an inducible heat shock
promoter, respiratory syncytial virus, Rous sarcomas virus (RSV),
etc.
[0050] The AAV vector can further comprise an exogenous
(heterologous) nucleic acid functionally linked to the promoter. By
"heterologous nucleic acid" is meant that any heterologous or
exogenous nucleic acid can be inserted into the vector for transfer
into a cell, tissue or organism. The nucleic acid can encode a
polypeptide or protein or an antisense RNA, for example. By
"functionally linked" is meant such that the promoter can promote
expression of the heterologous nucleic acid, as is known in the
art, such as appropriate orientation of the promoter relative to
the heterologous nucleic acid. Furthermore, the heterologous
nucleic acid preferably has all appropriate sequences for
expression of the nucleic acid, as known in the art, to
functionally encode, i.e., allow the nucleic acid to be expressed.
The nucleic acid can include, for example, expression control
sequences, such as an enhancer, and necessary information
processing sites, such as ribosome binding sites, RNA splice sites,
polyadenylation sites, and transcriptional terminator sequences.
The nucleic acid can encode more than one gene product, limited
only by the size of nucleic acid that can be packaged.
[0051] The heterologous nucleic acid can encode beneficial proteins
that replace missing or defective proteins required by the subject
into which the vector in transferred or can encode a cytotoxic
polypeptide that can be directed, e.g., to cancer cells or other
cells whose death would be beneficial to the subject. The
heterologous nucleic acid can also encode antisense RNAs that can
bind to, and thereby inactivate, mRNAs made by the subject that
encode harmful proteins. In one embodiment, antisense
polynucleotides can be produced from a heterologous expression
cassette in an AAV viral construct where the expression cassette
contains a sequence that promotes cell-type specific
expression.
[0052] Examples of heterologous nucleic acids which can be
administered to a cell or subject as part of the present AAV vector
can include, but are not limited to the nucleic acids encoding
therapeutic agents, such as lysosomal hydrolases; tumor necrosis
factors (TNF), such as TNF-alpha; interferons, such as
interferon-alpha, interferon-beta, and interferon-gamma;
interleukins, such as IL-1, IL-1beta, and ILs-2 through -14;
GM-CSF; adenosine deaminase; secreted factors such as growth
factors; ion channels; chemotherapeutics; lysosomal proteins;
anti-apoptotic gene products; proteins promoting neural survival
such as glutamate receptors and growth factors; cellular growth
factors, such as lymphokines; soluble CD4; Factor VIII; Factor IX;
T-cell receptors; LDL receptor; ApoE; ApoC; alpha-1 antitrypsin;
ornithine transcarbamylase (OTC); cystic fibrosis transmembrane
receptor (CFTR); insulin; Fc receptors for antigen binding domains
of antibodies, such as immunoglobulins; and antisense sequences
which inhibit viral replication, such as antisense sequences which
inhibit replication of hepatitis B or hepatitis non-A, non-B virus.
Furthermore, the nucleic acid can encode more than one gene
product, limited only by the size of nucleic acid that can be
packaged.
[0053] An AAV2 particle is a viral particle comprising an AAV2
capsid protein. Variations in the amino acid sequence of the AAV2
capsid protein are contemplated herein, as long as the resulting
viral particle comprising the AAV2 capsid remains antigenically or
immunologically distinct from AAV4, as can be routinely determined
by standard methods. Specifically, for example, ELISA and Western
blots can be used to determine whether a viral particle is
antigenically or immunologically distinct from other AAV
serotypes.
[0054] The term "polypeptide" as used herein refers to a polymer of
amino acids and includes full-length proteins and fragments
thereof. Thus, "protein" and "polypeptide" are often used
interchangeably herein. Substitutions can be selected by known
parameters to be neutral. As will be appreciated by those skilled
in the art, the invention also includes those polypeptides having
slight variations in amino acid sequences or other properties. Such
variations may arise naturally as allelic variations (e.g. due to
genetic polymorphism) or may be produced by human intervention
(e.g., by mutagenesis of cloned DNA sequences), such as induced
point, deletion, insertion and substitution mutants. Minor changes
in amino acid sequence are generally preferred, such as
conservative amino acid replacements, small internal deletions or
insertions, and additions or deletions at the ends of the
molecules. These modifications can result in changes in the amino
acid sequence, provide silent mutations, modify a restriction site,
or provide other specific mutations.
[0055] The present method provides a method of delivering a nucleic
acid to a cell comprising administering to the cell an AAV particle
containing a vector comprising the nucleic acid inserted between a
pair of AAV inverted terminal repeats, thereby delivering the
nucleic acid to the cell. Administration to the cell can be
accomplished by any means, including simply contacting the
particle, optionally contained in a desired liquid such as tissue
culture medium, or a buffered saline solution, with the cells. The
particle can be allowed to remain in contact with the cells for any
desired length of time, and typically the particle is administered
and allowed to remain indefinitely. For such in vitro methods, the
virus can be administered to the cell by standard viral
transduction methods, as known in the art and as exemplified
herein. Titers of virus to administer can vary, particularly
depending upon the cell type, but will be typical of that used for
AAV transduction in general. Additionally the titers used to
transduce the particular cells in the present examples can be
utilized. The cells can include any desired cell in humans as well
as other large (non-rodent) mammals, such as primates, horse,
sheep, goat, pig, and dog.
[0056] More specifically, the present invention provides a method
of delivering a nucleic acid to a cell with contact to the
circulating CSF, such as an ependymal cell, a pial cell, meningeal
cell, a brain endothelial cell, comprising administering to the
cell an AAV particle containing a vector comprising the nucleic
acid inserted between a pair of AAV inverted terminal repeats,
thereby delivering the nucleic acid to the cell.
[0057] The present invention further provides a method of
delivering a nucleic acid to a cell in a subject comprising
administering to the subject an AAV particle comprising the nucleic
acid inserted between a pair of AAV inverted terminal repeats,
thereby delivering the nucleic acid to a cell in the subject.
[0058] Also provided is a method of delivering a nucleic acid to an
ependymal, pial or other meningeal cell in a subject comprising
administering to the subject an AAV particle comprising the nucleic
acid inserted between a pair of AAV inverted terminal repeats,
thereby delivering the nucleic acid to the ependymal, pial or other
meningeal cell in the subject.
[0059] In certain embodiments, the amino acid sequence that targets
brain vascular endothelium targets brain vascular endothelium in a
subject that has a disease, e.g., a lysosomal storage disease.
[0060] In certain embodiments, the amino acid sequence that targets
brain vascular endothelium targets brain vascular endothelium in a
subject that does not have a lysosomal storage disease.
[0061] In certain embodiments, the viral vector comprises a nucleic
acid sequence encoding a therapeutic agent. In certain embodiments,
the therapeutic agent is TPP1.
[0062] Certain embodiments of the present disclosure provide a cell
comprising a viral vector as described herein.
[0063] Certain embodiments of the present disclosure provide a
method of treating a disease in a mammal comprising administering a
viral vector or the cell as described herein to the mammal.
[0064] In certain embodiments, the mammal is human.
[0065] In certain embodiments, the disease is a lysosomal storage
disease (LSD). In certain embodiments, the LSD is infantile or late
infantile ceroid lipofuscinoses, Gaucher, Juvenile Batten, Fabry,
MLD, Sanfilippo A, Late Infantile Batten, Hunter, Krabbe, Morquio,
Pompe, Niemann-Pick C, Tay-Sachs, Hurler (MPS-I H), Sanfilippo B,
Maroteaux-Lamy, Niemann-Pick A, Cystinosis, Hurler-Scheie (MPS-I
H/S), Sly Syndrome (MPS VII), Scheie (MPS-I S), Infantile Batten,
GM1 Gangliosidosis, Mucolipidosis type IUIII, or Sandhoff
disease.
[0066] In certain embodiments, the disease is a neurodegenerative
disease. In certain embodiments, the neurodegenerative disease is
Alzheimer's disease, Huntington's disease, ALS, hereditary spastic
hemiplegia, primary lateral sclerosis, spinal muscular atrophy,
Kennedy's disease, a polyglutamine repeat disease, or Parkinson's
disease.
[0067] Certain embodiments of the present disclosure provide a
method to deliver an agent to the central nervous system of a
subject, comprising administering to the CSF with a viral vector
described herein so that the transduced ependymal, pial,
endothelial and/or other meningeal cells express the therapeutic
agent and deliver the agent to the central nervous system of the
subject. In certain embodiments, the viral vector transduces
ependymal, pial, endothelial and/or other meningeal cells.
[0068] Certain embodiments of the present disclosure provide a
viral vector or cell as described herein for use in medical
treatments.
[0069] Certain embodiments of the present disclosure provide a use
of a viral vector or cell as described herein to prepare a
medicament useful for treating a disease, e.g., a lysosomal storage
disease, in a mammal.
[0070] The vector may further comprise a lysosomal enzyme (e.g., a
lysosomal hydrolase), a secreted protein, a nuclear protein, or a
cytoplasmic protein. As used herein, the term "secreted protein"
includes any secreted protein, whether naturally secreted or
modified to contain a signal sequence so that it can be
secreted.
[0071] Certain embodiments of the present disclosure provide a use
of a viral vector or cell as described herein to prepare a
medicament useful for treating a disease, e.g., Alzheimer's
disease, in a mammal.
[0072] The vector may further comprise a protective ApoE isoform
protein. As used herein, the term "secreted protein" includes any
secreted protein, whether naturally secreted or modified to contain
a signal sequence so that it can be secreted. Nucleic acid is
"operably linked" when it is placed into a functional relationship
with another nucleic acid sequence. Generally, "operably linked"
means that the DNA sequences being linked are contiguous. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice. Additionally, multiple
copies of the nucleic acid encoding enzymes may be linked together
in the expression vector. Such multiple nucleic acids may be
separated by linkers.
[0073] The present disclosure also provides a mammalian cell
containing a vector described herein. The cell may be human, and
may be from brain. The cell type may be a stem or progenitor cell
population.
[0074] The present disclosure provides a method of treating a
disease such as a genetic disease or cancer in a mammal by
administering a polynucleotide, polypeptide, expression vector, or
cell described herein. The genetic disease or cancer may be a
lysosomal storage disease (LSD) such as infantile or late infantile
ceroid lipofuscinoses, Gaucher, Juvenile Batten, Fabry, MLD,
Sanfilippo A, Late Infantile Batten, Hunter, Krabbe, Morquio,
Pompe, Niemann-Pick C, Tay-Sachs, Hurler (MPS-I H), Sanfilippo B,
Maroteaux-Lamy, Niemann-Pick A, Cystinosis, Hurler-Scheie (MPS-I
H/S), Sly Syndrome (MPS VII), Scheie (MPS-I S), Infantile Batten,
GM1 Gangliosidosis, Mucolipidosis type II/III, or Sandhoff
disease.
[0075] The genetic disease may be a neurodegenerative disease, such
as Huntington's disease, ALS, hereditary spastic hemiplegia,
primary lateral sclerosis, spinal muscular atrophy, Kennedy's
disease, Alzheimer's disease, a polyglutamine repeat disease, or
focal exposure such as Parkinson's disease.
[0076] Certain aspects of the disclosure relate to polynucleotides,
polypeptides, vectors, and genetically engineered cells (modified
in vivo), and the use of them. In particular, the disclosure
relates to a method for gene or protein therapy that is capable of
both systemic delivery of a therapeutically effective dose of the
therapeutic agent.
[0077] According to one aspect, a cell expression system for
expressing a therapeutic agent in a mammalian recipient is
provided. The expression system (also referred to herein as a
"genetically modified cell") comprises a cell and an expression
vector for expressing the therapeutic agent. Expression vectors
include, but are not limited to, viruses, plasmids, and other
vehicles for delivering heterologous genetic material to cells.
Accordingly, the term "expression vector" as used herein refers to
a vehicle for delivering heterologous genetic material to a cell.
In particular, the expression vector is a recombinant adenoviral,
adeno-associated virus, or lentivirus or retrovirus vector.
[0078] The expression vector further includes a promoter for
controlling transcription of the heterologous gene. The promoter
may be an inducible promoter (described below). The expression
system is suitable for administration to the mammalian recipient.
The expression system may comprise a plurality of non-immortalized
genetically modified cells, each cell containing at least one
recombinant gene encoding at least one therapeutic agent.
[0079] The cell expression system is formed in vivo. According to
yet another aspect, a method for treating a mammalian recipient in
vivo is provided. The method includes introducing an expression
vector for expressing a heterologous gene product into a cell of
the patient in situ, such as via intravenous administration. To
form the expression system in vivo, an expression vector for
expressing the therapeutic agent is introduced in vivo into the
mammalian recipient i.v., where the vector migrates via the
vasculature to the brain.
[0080] According to yet another aspect, a method for treating a
mammalian recipient in vivo is provided. The method includes
introducing the target protein into the patient in vivo.
[0081] The expression vector for expressing the heterologous gene
may include an inducible promoter for controlling transcription of
the heterologous gene product. Accordingly, delivery of the
therapeutic agent in situ is controlled by exposing the cell in
situ to conditions, which induce transcription of the heterologous
gene.
[0082] The mammalian recipient may have a condition that is
amenable to gene replacement therapy. As used herein, "gene
replacement therapy" refers to administration to the recipient of
exogenous genetic material encoding a therapeutic agent and
subsequent expression of the administered genetic material in situ.
Thus, the phrase "condition amenable to gene replacement therapy"
embraces conditions such as genetic diseases (i.e., a disease
condition that is attributable to one or more gene defects),
acquired pathologies (i.e., a pathological condition which is not
attributable to an inborn defect), cancers and prophylactic
processes (i.e., prevention of a disease or of an undesired medical
condition). Accordingly, as used herein, the term "therapeutic
agent" refers to any agent or material, which has a beneficial
effect on the mammalian recipient. Thus, "therapeutic agent"
embraces both therapeutic and prophylactic molecules having nucleic
acid or protein components.
[0083] According to one embodiment, the mammalian recipient has a
genetic disease and the exogenous genetic material comprises a
heterologous gene encoding a therapeutic agent for treating the
disease. In yet another embodiment, the mammalian recipient has an
acquired pathology and the exogenous genetic material comprises a
heterologous gene encoding a therapeutic agent for treating the
pathology. According to another embodiment, the patient has a
cancer and the exogenous genetic material comprises a heterologous
gene encoding an anti-neoplastic agent. In yet another embodiment
the patient has an undesired medical condition and the exogenous
genetic material comprises a heterologous gene encoding a
therapeutic agent for treating the condition.
[0084] As used herein, the terms "a protective ApoE isoform,"
"lysosomal enzyme," a "secreted protein," a "nuclear protein," or a
"cytoplasmic protein" include variants or biologically active or
inactive fragments of these polypeptides. A "variant" of one of the
polypeptides is a polypeptide that is not completely identical to a
native protein. Such variant protein can be obtained by altering
the amino acid sequence by insertion, deletion or substitution of
one or more amino acid. The amino acid sequence of the protein is
modified, for example by substitution, to create a polypeptide
having substantially the same or improved qualities as compared to
the native polypeptide. The substitution may be a conserved
substitution. A "conserved substitution" is a substitution of an
amino acid with another amino acid having a similar side chain. A
conserved substitution would be a substitution with an amino acid
that makes the smallest change possible in the charge of the amino
acid or size of the side chain of the amino acid (alternatively, in
the size, charge or kind of chemical group within the side chain)
such that the overall peptide retains its spacial conformation but
has altered biological activity. For example, common conserved
changes might be Asp to Glu, Asn or Gln; His to Lys, Arg or Phe;
Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanine is
commonly used to substitute for other amino acids. The 20 essential
amino acids can be grouped as follows: alanine, valine, leucine,
isoleucine, proline, phenylalanine, tryptophan and methionine
having nonpolar side chains; glycine, serine, threonine, cystine,
tyrosine, asparagine and glutamine having uncharged polar side
chains; aspartate and glutamate having acidic side chains; and
lysine, arginine, and histidine having basic side chains.
[0085] The amino acid changes are achieved by changing the codons
of the corresponding nucleic acid sequence. It is known that such
polypeptides can be obtained based on substituting certain amino
acids for other amino acids in the polypeptide structure in order
to modify or improve biological activity. For example, through
substitution of alternative amino acids, small conformational
changes may be conferred upon a polypeptide that results in
increased activity. Alternatively, amino acid substitutions in
certain polypeptides may be used to provide residues, which may
then be linked to other molecules to provide peptide-molecule
conjugates which, retain sufficient properties of the starting
polypeptide to be useful for other purposes.
[0086] One can use the hydropathic index of amino acids in
conferring interactive biological function on a polypeptide,
wherein it is found that certain amino acids may be substituted for
other amino acids having similar hydropathic indices and still
retain a similar biological activity. Alternatively, substitution
of like amino acids may be made on the basis of hydrophilicity,
particularly where the biological function desired in the
polypeptide to be generated in intended for use in immunological
embodiments. The greatest local average hydrophilicity of a
"protein", as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity. Accordingly, it is noted
that substitutions can be made based on the hydrophilicity assigned
to each amino acid.
[0087] In using either the hydrophilicity index or hydropathic
index, which assigns values to each amino acid, it is preferred to
conduct substitutions of amino acids where these values are .+-.2,
with .+-.1 being particularly preferred, and those with in .+-.0.5
being the most preferred substitutions.
[0088] The variant protein has at least 50%, at least about 80%, or
even at least about 90% but less than 100%, contiguous amino acid
sequence homology or identity to the amino acid sequence of a
corresponding native protein.
[0089] The amino acid sequence of the variant polypeptide
corresponds essentially to the native polypeptide's amino acid
sequence. As used herein "correspond essentially to" refers to a
polypeptide sequence that will elicit a biological response
substantially the same as the response generated by the native
protein. Such a response may be at least 60% of the level generated
by the native protein, and may even be at least 80% of the level
generated by native protein.
[0090] A variant may include amino acid residues not present in the
corresponding native protein or deletions relative to the
corresponding native protein. A variant may also be a truncated
"fragment" as compared to the corresponding native protein, i.e.,
only a portion of a full-length protein. Protein variants also
include peptides having at least one D-amino acid.
[0091] The variant protein may be expressed from an isolated DNA
sequence encoding the variant protein. "Recombinant" is defined as
a peptide or nucleic acid produced by the processes of genetic
engineering. It should be noted that it is well-known in the art
that, due to the redundancy in the genetic code, individual
nucleotides can be readily exchanged in a codon, and still result
in an identical amino acid sequence.
[0092] The present disclosure provides methods of treating a
disease in a mammal by administering an expression vector to a cell
or patient. For the gene therapy methods, a person having ordinary
skill in the art of molecular biology and gene therapy would be
able to determine, without undue experimentation, the appropriate
dosages and routes of administration of the expression vector used
in the novel methods of the present disclosure.
[0093] According to one embodiment, the cells are transformed or
otherwise genetically modified in vivo. The cells from the
mammalian recipient are transformed (i.e., transduced or
transfected) in vivo with a vector containing exogenous genetic
material for expressing a heterologous (e.g., recombinant) gene
encoding a therapeutic agent and the therapeutic agent is delivered
in situ.
[0094] As used herein, "exogenous genetic material" refers to a
nucleic acid or an oligonucleotide, either natural or synthetic,
that is not naturally found in the cells; or if it is naturally
found in the cells, it is not transcribed or expressed at
biologically significant levels by the cells. Thus, "exogenous
genetic material" includes, for example, a non-naturally occurring
nucleic acid that can be transcribed into anti-sense RNA, as well
as a "heterologous gene" (i.e., a gene encoding a protein which is
not expressed or is expressed at biologically insignificant levels
in a naturally-occurring cell of the same type).
[0095] In the certain embodiments, the mammalian recipient has a
condition that is amenable to gene replacement therapy. As used
herein, "gene replacement therapy" refers to administration to the
recipient of exogenous genetic material encoding a therapeutic
agent and subsequent expression of the administered genetic
material in situ. Thus, the phrase "condition amenable to gene
replacement therapy" embraces conditions such as genetic diseases
(i.e., a disease condition that is attributable to one or more gene
defects), acquired pathologies (i.e., a pathological condition
which is not attributable to an inborn defect), cancers and
prophylactic processes (i.e., prevention of a disease or of an
undesired medical condition). Accordingly, as used herein, the term
"therapeutic agent" refers to any agent or material, which has a
beneficial effect on the mammalian recipient. Thus, "therapeutic
agent" embraces both therapeutic and prophylactic molecules having
nucleic acid (e.g., antisense RNA) and/or protein components.
[0096] Alternatively, the condition amenable to gene replacement
therapy is a prophylactic process, i.e., a process for preventing
disease or an undesired medical condition. Thus, the instant
disclosure embraces a cell expression system for delivering a
therapeutic agent that has a prophylactic function (i.e., a
prophylactic agent) to the mammalian recipient.
[0097] In summary, the term "therapeutic agent" includes, but is
not limited to, agents associated with the conditions listed above,
as well as their functional equivalents. As used herein, the term
"functional equivalent" refers to a molecule (e.g., a peptide or
protein) that has the same or an improved beneficial effect on the
mammalian recipient as the therapeutic agent of which is it deemed
a functional equivalent.
[0098] The above-disclosed therapeutic agents and conditions
amenable to gene replacement therapy are merely illustrative and
are not intended to limit the scope of the instant disclosure. The
selection of a suitable therapeutic agent for treating a known
condition is deemed to be within the scope of one of ordinary skill
of the art without undue experimentation.
AAV Vectors
[0099] In one embodiment, a viral vector of the disclosure is an
AAV vector. An "AAV" vector refers to an adeno-associated virus,
and may be used to refer to the naturally occurring wild-type virus
itself or derivatives thereof. The term covers all subtypes,
serotypes and pseudotypes, and both naturally occurring and
recombinant forms, except where required otherwise. As used herein,
the term "serotype" refers to an AAV which is identified by and
distinguished from other AAVs based on capsid protein reactivity
with defined antisera, e.g., there are eight known serotypes of
primate AAVs, AAV-1 to AAV-9 and AAVrh10. For example, serotype
AAV2 is used to refer to an AAV which contains capsid proteins
encoded from the cap gene of AAV2 and a genome containing 5' and 3'
ITR sequences from the same AAV2 serotype. As used herein, for
example, rAAV1 may be used to refer an AAV having both capsid
proteins and 5'-3' ITRs from the same serotype or it may refer to
an AAV having capsid proteins from one serotype and 5'-3' ITRs from
a different AAV serotype, e.g., capsid from AAV serotype 2 and ITRs
from AAV serotype 5. For each example illustrated herein the
description of the vector design and production describes the
serotype of the capsid and 5'-3' ITR sequences. The abbreviation
"rAAV" refers to recombinant adeno-associated virus, also referred
to as a recombinant AAV vector (or "rAAV vector").
[0100] An "AAV virus" or "AAV viral particle" refers to a viral
particle composed of at least one AAV capsid protein (preferably by
all of the capsid proteins of a wild-type AAV) and an encapsidated
polynucleotide. If the particle comprises heterologous
polynucleotide (i.e., a polynucleotide other than a wild-type AAV
genome such as a transgene to be delivered to a mammalian cell), it
is typically referred to as "rAAV".
[0101] In one embodiment, the AAV 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
DNA of interest and a transcriptional termination region. The
control elements are selected to be functional in a mammalian cell.
The resulting construct which contains the operatively linked
components is flanked (5' and 3') with functional AAV ITR
sequences.
[0102] By "adeno-associated virus inverted terminal repeats" or
"AAV ITRs" is meant the art-recognized regions found at each end of
the AAV genome which function together in cis as origins of DNA
replication and as packaging signals for the virus. AAV ITRs,
together with the AAV rep coding region, provide for the efficient
excision and rescue from, and integration of a nucleotide sequence
interposed between two flanking ITRs into a mammalian cell
genome.
[0103] The nucleotide sequences of AAV ITR regions are known. As
used herein, an "AAV ITR" need not have the wild-type nucleotide
sequence depicted, but may be altered, e.g., by the insertion,
deletion or substitution of nucleotides. Additionally, the AAV ITR
may be derived from any of several AAV serotypes, including without
limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, etc. Furthermore,
5' and 3' ITRs which flank a selected nucleotide sequence in an AAV
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
heterologous sequence into the recipient cell genome when AAV Rep
gene products are present in the cell.
[0104] In one embodiment, AAV ITRs can be derived from any of
several AAV serotypes, including without limitation, AAV1, AAV2,
AAV3, AAV4, AAV5, AAV7, 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.
[0105] In one embodiment, AAV capsids can be derived from AAV2.
Suitable DNA molecules for use in AAV vectors will be less than
about 5 kilobases (kb), less than about 4.5 kb, less than about
4kb, less than about 3.5 kb, less than about 3 kb, less than about
2.5 kb in size and are known in the art.
[0106] In one embodiment, the selected nucleotide 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.
Examples include, but are not limited to, the SV40 early promoter,
mouse mammary tumor virus LTR promoter; adenovirus major late
promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a
cytomegalovirus (CMV) promoter such as the CMV immediate early
promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, pol
II promoters, pol III promoters, synthetic promoters, hybrid
promoters, and the like. 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.).
[0107] In one embodiment, both heterologous promoters and other
control elements, such as CNS-specific and inducible promoters,
enhancers and the like, will be of particular use. Examples of
heterologous promoters include the CMV promoter. Examples of
CNS-specific promoters include those isolated from the genes from
myelin basic protein (MBP), glial fibrillary acid protein (GFAP),
and neuron specific enolase (NSE). Examples of inducible promoters
include DNA responsive elements for ecdysone, tetracycline, hypoxia
and aufin.
[0108] In one embodiment, the AAV expression vector which harbors
the DNA 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.
[0109] 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. For example, ligations can be
accomplished in 20 mM Tris-Cl pH 7.5, 10 mM MgC.sub.2, 10 mM DTT,
33 .mu.g/ml BSA, 10 mM-50 mM NaCl, and either 40 uM 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.
[0110] Additionally, chimeric genes can be produced synthetically
to include AAV ITR sequences arranged 5' and 3' of one or more
selected nucleic acid sequences. Preferred codons for expression of
the chimeric gene sequence in mammalian CNS cells can be used. The
complete chimeric sequence is assembled from overlapping
oligonucleotides prepared by standard methods.
[0111] In order to produce rAAV virions, an AAV expression vector
is introduced into a suitable host cell using known techniques,
such as by transfection. A number of transfection techniques are
generally known in the art. See, e.g., Sambrook et al. (1989)
Molecular Cloning, a laboratory manual, Cold Spring Harbor
Laboratories, New York. Particularly suitable transfection methods
include calcium phosphate co-precipitation, direct micro-injection
into cultured cells, electroporation, liposome mediated gene
transfer, lipid-mediated transduction, and nucleic acid delivery
using high-velocity microprojectiles.
[0112] In one embodiment, suitable host cells for producing rAAV
virions include microorganisms, yeast cells, insect cells, and
mammalian cells, that can be, or have been, used as recipients of a
heterologous DNA molecule. 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) can be used
in the practice of the present disclosure. Particularly, the human
cell line 293 is a human embryonic kidney cell line that has been
transformed with adenovirus type-5 DNA fragments, and expresses the
adenoviral Ela and E1b genes. The 293 cell line is readily
transfected, and provides a particularly convenient platform in
which to produce rAAV virions.
[0113] 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. 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.
[0114] 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.
[0115] In one embodiment, 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. A number of other vectors
have been described which encode Rep and/or Cap expression
products.
[0116] Methods of delivery of viral vectors include injecting the
AAV2 into the CSF. Generally, rAAV virions may be introduced into
cells of the CNS 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 rAAV virions 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.
[0117] Suitable methods for the delivery and introduction of
transduced cells into a subject have been described. For example,
cells can be transduced in vitro by combining recombinant AAV
virions with CNS cells e.g., in appropriate media, and screening
for those cells harboring the DNA of interest can be screened using
conventional techniques such as Southern blots and/or PCR, or by
using selectable markers. Transduced cells can then be formulated
into pharmaceutical compositions, described more fully below, and
the composition introduced into the subject by various techniques,
such as by grafting, intramuscular, intravenous, subcutaneous and
intraperitoneal injection.
[0118] In one embodiment, pharmaceutical compositions will comprise
sufficient genetic material to produce a therapeutically effective
amount of the nucleic acid of interest, i.e., an amount sufficient
to reduce or ameliorate symptoms of the disease state in question
or an amount sufficient to confer the desired benefit. The
pharmaceutical 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, Tween80, 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).
[0119] It should be understood that more than one transgene could
be expressed by the delivered viral vector. Alternatively, separate
vectors, each expressing one or more different transgenes, can also
be delivered to the CNS as described herein. Furthermore, it is
also intended that the viral vectors delivered by the methods of
the present disclosure be combined with other suitable compositions
and therapies.
[0120] As is apparent to those skilled in the art in view of the
teachings of this specification, an effective amount of viral
vector which must be added can be empirically determined.
Administration can be effected in one dose, continuously or
intermittently throughout the course of treatment. Methods of
determining the most effective means and dosages of administration
are well known to those of skill in the art and will vary with the
viral vector, the composition of the therapy, the target cells, and
the subject being treated. Single and multiple administrations can
be carried out with the dose level and pattern being selected by
the treating physician.
[0121] In certain embodiments, the rAAV is administered at a dose
of about 1-5 ml of 1.times.10.sup.5 -1.times.10.sup.16 vg/ml. In
certain embodiments, the rAAV is administered at a dose of about
1-3 ml of 1.times.10.sup.7-1.times.10.sup.14 vg/ml. In certain
embodiments, the rAAV is administered at a dose of about 1-2 ml of
1.times.10.sup.8-1.times.10.sup.13 vg/ml.
[0122] Formulations containing the rAAV particles will contain an
effective amount of the rAAV particles in a vehicle, the effective
amount being readily determined by one skilled in the art. The rAAV
particles may typically range from about 1% to about 95% (w/w) of
the composition, or even higher or lower if appropriate. The
quantity to be administered depends upon factors such as the age,
weight and physical condition of the animal or the human subject
considered for treatment. Effective dosages can be established by
one of ordinary skill in the art through routine trials
establishing dose response curves. The subject is treated by
administration of the rAAV particles in one or more doses. Multiple
doses may be administered as is required to maintain adequate
enzyme activity.
[0123] Vehicles including water, aqueous saline, artificial CSF, or
other known substances can be employed with the subject invention.
To prepare a formulation, the purified composition can be isolated,
lyophilized and stabilized. The composition may then be adjusted to
an appropriate concentration, optionally combined with an
anti-inflammatory agent, and packaged for use.
TPP1 Protein
[0124] In certain embodiments, the nucleic acid being administered
encodes TPP1, a TPP1 that has substantial identity to wildtype
TPP1, and/or a variant, mutant or fragment of TPP 1. The human TPP1
amino acid sequence is provided in FIG. 14, and the nucleic acid
sequence is provided in FIGS. 15A and 15B. FIG. 16 provides the
Macaca mulatta TPP1 amino acid sequence, and FIG. 17 provides the
Macaca fascicularis TPP1 amino acid sequence. In certain
embodiments, the TPP1 protein can have about 70% homology, about
75% homology, 80% homology, 85% homology, 90% homology, 95%
homology, 98% homology, 99% homology, or even 100% homology to the
protein set forth in FIG. 14, 16 or 17. The TPP1 protein can have
about 70% identity, about 75% identity, 80% identity, 85% identity,
90% identity, 95% identity, 98% identity, 99% identity, or even
100% identity to the protein set forth in FIG. 14, 16 or 17.
[0125] A mutant protein refers to the protein encoded by a gene
having a mutation, e.g., a missense or nonsense mutation in TPP1.
The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form, composed of monomers (nucleotides) containing
a sugar, phosphate and a base that is either a purine or
pyrimidine. Unless specifically limited, the term encompasses
nucleic acids containing known analogs of natural nucleotides that
have similar binding properties as the reference nucleic acid and
are metabolized in a manner similar to naturally occurring
nucleotides. Unless otherwise indicated, a particular nucleic acid
sequence also encompasses conservatively modified variants thereof
(e.g., degenerate codon substitutions) and complementary sequences,
as well as the sequence explicitly indicated. Specifically,
degenerate codon substitutions may be achieved by generating
sequences in which the third position of one or more selected (or
all) codons is substituted with mixed-base and/or deoxyinosine
residues.
[0126] A "nucleic acid fragment" is a portion of a given nucleic
acid molecule. Deoxyribonucleic acid (DNA) in the majority of
organisms is the genetic material while ribonucleic acid (RNA) is
involved in the transfer of information contained within DNA into
proteins. Fragments and variants of the disclosed nucleotide
sequences and proteins or partial-length proteins encoded thereby
are also encompassed by the present invention. By "fragment" or
"portion" is meant a full length or less than full length of the
nucleotide sequence encoding, or the amino acid sequence of, a
polypeptide or protein. In certain embodiments, the fragment or
portion is biologically functional (i.e., retains 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 99% or 100% of enzymatic activity of the wildtype
TPP1).
[0127] A "variant" of a molecule is a sequence that is
substantially similar to the sequence of the native molecule. For
nucleotide sequences, variants include those sequences that,
because of the degeneracy of the genetic code, encode the identical
amino acid sequence of the native protein. Naturally occurring
allelic variants such as these can be identified with the use of
molecular biology techniques, as, for example, with polymerase
chain reaction (PCR) and hybridization techniques. Variant
nucleotide sequences also include synthetically derived nucleotide
sequences, such as those generated, for example, by using
site-directed mutagenesis, which encode the native protein, as well
as those that encode a polypeptide having amino acid substitutions.
Generally, nucleotide sequence variants of the invention will have
at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least
85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, to 98%, sequence identity to the native (endogenous)
nucleotide sequence. In certain embodiments, the variant is
biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
99% or 100% of enzymatic activity of the wildtype TPP1).
[0128] "Conservatively modified variations" of a particular nucleic
acid sequence refers to those nucleic acid sequences that encode
identical or essentially identical amino acid sequences. Because of
the degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given polypeptide. For instance,
the codons CGT, CGC, CGA, CGG, AGA and AGG all encode the amino
acid arginine. Thus, at every position where an arginine is
specified by a codon, the codon can be altered to any of the
corresponding codons described without altering the encoded
protein. Such nucleic acid variations are "silent variations,"
which are one species of "conservatively modified variations."
Every nucleic acid sequence described herein that encodes a
polypeptide also describes every possible silent variation, except
where otherwise noted. One of skill in the art will recognize that
each codon in a nucleic acid (except
[0129] ATG, which is ordinarily the only codon for methionine) can
be modified to yield a functionally identical molecule by standard
techniques. Accordingly, each "silent variation" of a nucleic acid
that encodes a polypeptide is implicit in each described
sequence.
[0130] The term "substantial identity" of polynucleotide sequences
means that a polynucleotide comprises a sequence that has at least
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least
90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or
99% sequence identity, compared to a reference sequence using one
of the alignment programs described using standard parameters. One
of skill in the art will recognize that these values can be
appropriately adjusted to determine corresponding identity of
proteins encoded by two nucleotide sequences by taking into account
codon degeneracy, amino acid similarity, reading frame positioning,
and the like. Substantial identity of amino acid sequences for
these purposes normally means sequence identity of at least 70%, at
least 80%, 90%, or even at least 95%.
[0131] The term "substantial identity" in the context of a peptide
indicates that a peptide comprises a sequence with at least 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%,
93%, or 94%, or even, 95%, 96%, 97%, 98% or 99%, sequence identity
to the reference sequence over a specified comparison window. An
indication that two peptide sequences are substantially identical
is that one peptide is immunologically reactive with antibodies
raised against the second peptide. Thus, a peptide is substantially
identical to a second peptide, for example, where the two peptides
differ only by a conservative substitution.
Apolipoprotein E (ApoE)
[0132] There are several different human apolipoprotein E (ApoE)
isoforms, the presence of some of these isoforms in the brain
increase the risk for Alzheimer's disease (AD), whereas the
presence of other isoforms decreases the risk for AD. The presence
of the ApoE c4 isoform is a strong genetic risk factor for
late-onset, sporadic AD. (Casellano et al., Sci Transl Med,
3(89):89ra57 (29 Jun. 2011).) The ApoE 4 allele strongly increases
AD risk and decreases age of onset. On the other hand, the presence
of the ApoE 2 allele appears to decrease AD risk. It is suggested
that human ApoE isoforms differentially affect the clearance or
synthesis of amyloid-.beta. (A.beta.) in vivo.
[0133] In certain embodiments, the nucleic acid being administered
encodes ApoE, a ApoE that has substantial identity to wildtype
ApoE, or a variant, mutant and/or or fragment of ApoE. In certain
embodiments, the nucleic acid encodes ApoE 2, an ApoE 2 that has
substantial identity to wildtype ApoE 2, and/or a variant, mutant
or fragment of ApoE 2.
Immunesuppression Agents
[0134] In certain embodiments, an immunesuppression agent is also
administered to the mammal. In certain embodiments, the
immuesuprression agent is an anti-inflammatory agent. In certain
embodiments, the anti-inflammatory agent is mycophenolate. In
certain embodiments, the anti-inflammatory agent is administered
prior to the administration of the rAAV particles. In certain
embodiments, the anti-inflammatory agent is administered
concurrently to the administration of the rAAV particles. In
certain embodiments, the anti-inflammatory agent is administered
subsequent to the administration of the rAAV particles.
[0135] In certain embodiments, the anti-inflammatory agent is
administered parenterally, such as by intramuscular or subcutaneous
injection in an appropriate vehicle. Other modes of administration,
however, such as oral, intranasal or intradermal delivery, are also
acceptable. In certain embodiments, a composition comprising the
rAAV particle and the anti-inflammatory agent is prepared and the
anti-inflammatory agent and rAAV particle are administered
simultaneously to the mammal's cisterna magna and/or to the
mammal's brain ventricle, subarachnoid space and/or intrathecal
space.
Methods for Introducing Genetic Material into Cells
[0136] The exogenous genetic material (e.g., a cDNA encoding one or
more therapeutic proteins) is introduced into the cell in vivo by
genetic transfer methods, such as transfection or transduction, to
provide a genetically modified cell. Various expression vectors
(i.e., vehicles for facilitating delivery of exogenous genetic
material into a target cell) are known to one of ordinary skill in
the art.
[0137] As used herein, "transfection of cells" refers to the
acquisition by a cell of new genetic material by incorporation of
added DNA. Thus, transfection refers to the insertion of nucleic
acid into a cell using physical or chemical methods. Several
transfection techniques are known to those of ordinary skill in the
art including: calcium phosphate DNA co-precipitation;
DEAE-dextran; electroporation; cationic liposome-mediated
transfection; and tungsten particle-faciliated microparticle
bombardment. Strontium phosphate DNA co-precipitation is another
possible transfection method.
[0138] In contrast, "transduction of cells" refers to the process
of transferring nucleic acid into a cell using a DNA or RNA virus.
A RNA virus (i.e., a retrovirus) for transferring a nucleic acid
into a cell is referred to herein as a transducing chimeric
retrovirus. Exogenous genetic material contained within the
retrovirus is incorporated into the genome of the transduced cell.
A cell that has been transduced with a chimeric DNA virus (e.g., an
adenovirus carrying a cDNA encoding a therapeutic agent), will not
have the exogenous genetic material incorporated into its genome
but will be capable of expressing the exogenous genetic material
that is retained extrachromosomally within the cell.
[0139] Typically, the exogenous genetic material includes the
heterologous gene (usually in the form of a cDNA comprising the
exons coding for the therapeutic protein) together with a promoter
to control transcription of the new gene. The promoter
characteristically has a specific nucleotide sequence necessary to
initiate transcription. Optionally, the exogenous genetic material
further includes additional sequences (i.e., enhancers) required to
obtain the desired gene transcription activity. For the purpose of
this discussion an "enhancer" is simply any non-translated DNA
sequence which works contiguous with the coding sequence (in cis)
to change the basal transcription level dictated by the promoter.
The exogenous genetic material may introduced into the cell genome
immediately downstream from the promoter so that the promoter and
coding sequence are operatively linked so as to permit
transcription of the coding sequence. A retroviral expression
vector may include an exogenous promoter element to control
transcription of the inserted exogenous gene. Such exogenous
promoters include both constitutive and inducible promoters.
[0140] Naturally-occurring constitutive promoters control the
expression of essential cell functions. As a result, a gene under
the control of a constitutive promoter is expressed under all
conditions of cell growth. Exemplary constitutive promoters include
the promoters for the following genes which encode certain
constitutive or "housekeeping" functions: hypoxanthine
phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR),
adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase,
phosphoglycerol mutase, the actin promoter, and other constitutive
promoters known to those of skill in the art. In addition, many
viral promoters function constitutively in eucaryotic cells. These
include: the early and late promoters of SV40; the long terminal
repeats (LTRs) of Moloney Leukemia Virus and other retroviruses;
and the thymidine kinase promoter of Herpes Simplex Virus, among
many others. Accordingly, any of the above-referenced constitutive
promoters can be used to control transcription of a heterologous
gene insert.
[0141] Genes that are under the control of inducible promoters are
expressed only or to a greater degree, in the presence of an
inducing agent, (e.g., transcription under control of the
metallothionein promoter is greatly increased in presence of
certain metal ions). Inducible promoters include responsive
elements (REs) which stimulate transcription when their inducing
factors are bound. For example, there are REs for serum factors,
steroid hormones, retinoic acid and cyclic AMP. Promoters
containing a particular RE can be chosen in order to obtain an
inducible response and in some cases, the RE itself may be attached
to a different promoter, thereby conferring inducibility to the
recombinant gene. Thus, by selecting the appropriate promoter
(constitutive versus inducible; strong versus weak), it is possible
to control both the existence and level of expression of a
therapeutic agent in the genetically modified cell. If the gene
encoding the therapeutic agent is under the control of an inducible
promoter, delivery of the therapeutic agent in situ is triggered by
exposing the genetically modified cell in situ to conditions for
permitting transcription of the therapeutic agent, e.g., by
intraperitoneal injection of specific inducers of the inducible
promoters which control transcription of the agent. For example, in
situ expression by genetically modified cells of a therapeutic
agent encoded by a gene under the control of the metallothionein
promoter, is enhanced by contacting the genetically modified cells
with a solution containing the appropriate (i.e., inducing) metal
ions in situ.
[0142] Accordingly, the amount of therapeutic agent that is
delivered in situ is regulated by controlling such factors as: (1)
the nature of the promoter used to direct transcription of the
inserted gene, (i.e., whether the promoter is constitutive or
inducible, strong or weak); (2) the number of copies of the
exogenous gene that are inserted into the cell; (3) the number of
transduced/transfected cells that are administered (e.g.,
implanted) to the patient; (4) the size of the implant (e.g., graft
or encapsulated expression system); (5) the number of implants; (6)
the length of time the transduced/transfected cells or implants are
left in place; and (7) the production rate of the therapeutic agent
by the genetically modified cell. Selection and optimization of
these factors for delivery of a therapeutically effective dose of a
particular therapeutic agent is deemed to be within the scope of
one of ordinary skill in the art without undue experimentation,
taking into account the above-disclosed factors and the clinical
profile of the patient.
[0143] In addition to at least one promoter and at least one
heterologous nucleic acid encoding the therapeutic agent, the
expression vector may include a selection gene, for example, a
neomycin resistance gene, for facilitating selection of cells that
have been transfected or transduced with the expression vector.
Alternatively, the cells are transfected with two or more
expression vectors, at least one vector containing the gene(s)
encoding the therapeutic agent(s), the other vector containing a
selection gene. The selection of a suitable promoter, enhancer,
selection gene and/or signal sequence (described below) is deemed
to be within the scope of one of ordinary skill in the art without
undue experimentation.
[0144] The therapeutic agent can be targeted for delivery to an
extracellular, intracellular or membrane location. If it is
desirable for the gene product to be secreted from the cells, the
expression vector is designed to include an appropriate secretion
"signal" sequence for secreting the therapeutic gene product from
the cell to the extracellular milieu. If it is desirable for the
gene product to be retained within the cell, this secretion signal
sequence is omitted. In a similar manner, the expression vector can
be constructed to include "retention" signal sequences for
anchoring the therapeutic agent within the cell plasma membrane.
For example, all membrane proteins have hydrophobic transmembrane
regions, which stop translocation of the protein in the membrane
and do not allow the protein to be secreted. The construction of an
expression vector including signal sequences for targeting a gene
product to a particular location is deemed to be within the scope
of one of ordinary skill in the art without the need for undue
experimentation.
EXAMPLE 1
Methods of Gene Transfer in Large Mammals
[0145] Lysosomal storage disorders (LSDs) constitute a large class
of inherited metabolic disorders. Most LSDs are caused by lysosomal
enzyme deficiencies which lead to organ damage and often central
nervous system (CNS) degeneration. Late infantile neuronal ceroid
lipofuscinosis (LINCL) is an autosomal recessive neurodegenerative
disease caused by mutations in a ceroid-lipofuscinosis (CLN),
neuronal 2 gene CLN2, which encodes the lysosomal protease
tripeptidyl peptidase 1 (TPP1). LINCL is characterized clinically
by normal birth and early development, onset of seizures by 18-24
months, progressive motor and cognitive decline, and premature
death. The disease is due to a deficiency in TPP1, which is a
soluble, M-6-P decorated lysosomal enzyme.
[0146] Enzyme-replacement therapy (ERT) is currently available for
lysosomal storage diseases affecting peripheral tissues, but has
not been used in patients with central nervous system (CNS)
involvement. A recent study investigated whether enzyme delivery
through the cerebrospinal fluid was a potential alternative route
to the CNS for LINCL (Chang et al., Molecular Therapy 16:649-656,
2008). Treated mice showed attenuated neuropathology, and decreased
resting tremor relative to vehicle-treated mice.
[0147] In the present work, it was investigated whether global
delivery of a vector could be effectively performed in order to
achieve steady-state levels of enzyme in the cerebrospinal fluid
(CSF) by means of injection in the brain. Studies were performed in
a dog model of LINCL.
[0148] The LINCL dogs are normal at birth, but develop neurological
signs around 7 months, testable cognitive deficits at .about.5-6
months, seizures at 10-11 months, and progressive visual loss. The
CLN2 gene mutation in the LINCL dog renders the TPP1 protein
non-functional, and TPP1 protein is undetectable. With disease
progression, brain tissues shrink, leading to enlarged ventricular
spaces in the brain. Neurological symptoms include decline in
balance and motor functions, loss of vision, tremors.
[0149] Affected LINCL pups were given gene therapy at three months
of age. For gene therapy, AAV2-CLN2 generated (see WO 2012/135857),
and was injected at a single site (lateral ventricle) or at two
sites (lateral ventricle plus cisterna magna) in the brain. Needles
were placed into the ventricle, or into the ventricle and cistern
magna, and vector infused slowly over several minutes. While much
of the TPP1 made within a cell stayed in that cell, a portion was
secreted and taken up by neighboring cells. This property of
secretion and uptake is called "cross-correction" (FIG. 2).
Cross-correction is valuable in the context of gene therapy in that
if the CLN2 gene is transferred to strategically situated cells in
the LINCL brain, then this can allow for cross-correction of many
surrounding cells.
[0150] In the present study, the problem of globally delivering the
therapeutic vector took advantage of the CSF flow in the brain by
targeting cells that line the ventricles and cells that make up the
meninges. AAV2-CLN2 was injected at a single site (lateral
ventricle) or at two sites (lateral ventricle plus cisterna magna)
in the brain. TPP1 expression was observed in Cln2.sup.-/- dogs
after AAV delivery (FIG. 3). A significant positive impact was
observed on ventricular volume. The effect of AAV.TPP1 on
autofluorescence was also evaluated (FIG. 4A). FIG. 4B shows T1-MM
images of untreated and treated dogs. FIG. 4C shows the effects of
AAV.TPP1 in LINCL dogs. FIG. 4D shows huTPP1 distribution after
AAV2/2-huCLN2 administration.
[0151] In untreated affected dogs, ventricular spaces enlarge to
ten times the size of normal dogs, whereas AAV2-CLN2 gene therapy
significantly reduced this effect. Further, a broad distribution of
enzyme was observed, as was a clinical benefit (lifespan and
clinical examination). Without treatment, affected dogs show signs
of disease in all 22 tests by 30 weeks of age. They reach end-stage
disease and must be euthanized between 45 and 48 weeks of age. In
dogs that received AAV2-CLN2 gene therapy, the onset of every one
of these signs was delayed or prevented.
[0152] An increase in TPP1 activity was observed in CSF after
combined cisterna and ventricular delivery.
[0153] Thus, in the LINCL dog, AAV2-CLN2 gene transfer resulted in
TPP1 protein replenishment to many areas of the brain, and the
results indicated that AAV2-CLN2 gene transfer provided significant
therapeutic effects, reduced or delayed symptoms and improved the
quality of life for the LINCL dogs.
[0154] The huTPP1 activity in CSF declined shortly after injection
(FIG. 5). A broad distribution of enzyme was observed, but the
levels were low at the time of sacrifice 6-8 months post-gene
therapy. It was postulated that the decline in activity was a
result of an immune response to the human enzyme in the dogs. In
order to inhibit the decline in activity, an anti-inflammatory
agent (mycophenolate) was introduced. The results indicated that
the anti-inflammatory agent did not inhibit the enzymatic activity
of the huTPP1, and was effective in extending the length of time
that the enzyme activity was present (FIG. 6), and sustained enzyme
activity levels were observed.
[0155] High caTPP1 activity in CSF was observed along the time
after AAV2caCLN2 intraventricular injection and early mycophenolate
treatment (FIG. 7).
[0156] An increase in TPP1 enzyme activity was observed in many
tissues two months post-administration (FIGS. 8, 9A and 9B).
[0157] FIG. 10 shows the onset of clinical signs in LINCL dogs.
caTPP1 activity was observed in meninges and peripheral tissue,
such as the liver (FIG. 11).
[0158] Thus, the inventors have shown the transformation of
pendymal cells by AAV2/2, that canine TPP1 enzyme was produced and
flowed with CSF, and that mycopheolate treatment pro rot caCLN2
injection could prevent immunoresponse in dogs.
EXAMPLE 2
Studies in Non-Human Primates
[0159] Using techniques similar to those described above, the
inventors observed that AAVeGFP transduced ependyma in nonhuman
primate brain. In vivo assessment of
[0160] AAV2/2.TPP1 delivery in rhesus brain was performed by
injecting AAV.TPP1 into the ventricle or cisterna magna, harvesting
the tissue 4-12 weeks later, and evaluating the TPP1 activity in
CSF or tissue lysates (FIG. 12). Activity was observed in the
vestibular area (brainstem) in the non-human primates (FIG. 13).
Thus, the ventricular lining cells provided a source of recombinant
enzyme for broad CNS distribution.
[0161] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification this invention has been described in relation to
certain preferred embodiments thereof, and many details have been
set forth for purposes of illustration, it will be apparent to
those skilled in the art that the invention is susceptible to
additional embodiments and that certain of the details described
herein may be varied considerably without departing from the basic
principles of the invention.
[0162] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to") unless otherwise noted. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0163] Embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those embodiments may become apparent to
those of ordinary skill in the art upon reading the foregoing
description. The inventors expect skilled artisans to employ such
variations as appropriate, and the inventors intend for the
invention to be practiced otherwise than as specifically described
herein. Accordingly, this invention includes all modifications and
equivalents of the subject matter recited in the claims appended
hereto as permitted by applicable law. Moreover, any combination of
the above-described elements in all possible variations thereof is
encompassed by the invention unless otherwise indicated herein or
otherwise clearly contradicted by context.
Sequence CWU 1
1
81735PRTAdeno-associated virus 1Met Ala Ala Asp Gly Tyr Leu Pro Asp
Trp Leu Glu Asp Thr Leu Ser1 5 10 15Glu Gly Ile Arg Gln Trp Trp Lys
Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30Lys Pro Ala Glu Arg His Lys
Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly
Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Glu Ala Asp
Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Arg Gln Leu
Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95Asp Ala
Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105
110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys
Lys Arg 130 135 140Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser
Ser Gly Thr Gly145 150 155 160Lys Ala Gly Gln Gln Pro Ala Arg Lys
Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Ala Asp Ser Val Pro
Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190Ala Ala Pro Ser Gly
Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 195 200 205Ala Pro Met
Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220Ser
Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile225 230
235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
Leu 245 250 255Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp
Asn His Tyr 260 265 270Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp
Phe Asn Arg Phe His 275 280 285Cys His Phe Ser Pro Arg Asp Trp Gln
Arg Leu Ile Asn Asn Asn Trp 290 295 300Gly Phe Arg Pro Lys Arg Leu
Asn Phe Lys Leu Phe Asn Ile Gln Val305 310 315 320Lys Glu Val Thr
Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335Thr Ser
Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345
350Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp
355 360 365Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn
Gly Ser 370 375 380Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu
Tyr Phe Pro Ser385 390 395 400Gln Met Leu Arg Thr Gly Asn Asn Phe
Thr Phe Ser Tyr Thr Phe Glu 405 410 415Asp Val Pro Phe His Ser Ser
Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430Leu Met Asn Pro Leu
Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445Asn Thr Pro
Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 450 455 460Ala
Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly465 470
475 480Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn
Asn 485 490 495Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His
Leu Asn Gly 500 505 510Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met
Ala Ser His Lys Asp 515 520 525Asp Glu Glu Lys Phe Phe Pro Gln Ser
Gly Val Leu Ile Phe Gly Lys 530 535 540Gln Gly Ser Glu Lys Thr Asn
Val Asp Ile Glu Lys Val Met Ile Thr545 550 555 560Asp Glu Glu Glu
Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575Gly Ser
Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr 580 585
590Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp
595 600 605Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro
His Thr 610 615 620Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly
Phe Gly Leu Lys625 630 635 640His Pro Pro Pro Gln Ile Leu Ile Lys
Asn Thr Pro Val Pro Ala Asn 645 650 655Pro Ser Thr Thr Phe Ser Ala
Ala Lys Phe Ala Ser Phe Ile Thr Gln 660 665 670Tyr Ser Thr Gly Gln
Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys 675 680 685Glu Asn Ser
Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr 690 695 700Asn
Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr705 710
715 720Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu
725 730 7352734PRTAdeno-associated virus 2Met Thr Asp Gly Tyr Leu
Pro Asp Trp Leu Glu Asp Asn Leu Ser Glu1 5 10 15Gly Val Arg Glu Trp
Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro Lys 20 25 30Ala Asn Gln Gln
His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro Gly 35 40 45Tyr Lys Tyr
Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro Val 50 55 60Asn Ala
Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp Gln65 70 75
80Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp
85 90 95Ala Glu Phe Gln Gln Arg Leu Gln Gly Asp Thr Ser Phe Gly Gly
Asn 100 105 110Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu
Glu Pro Leu 115 120 125Gly Leu Val Glu Gln Ala Gly Glu Thr Ala Pro
Gly Lys Lys Arg Pro 130 135 140Leu Ile Glu Ser Pro Gln Gln Pro Asp
Ser Ser Thr Gly Ile Gly Lys145 150 155 160Lys Gly Lys Gln Pro Ala
Lys Lys Lys Leu Val Phe Glu Asp Glu Thr 165 170 175Gly Ala Gly Asp
Gly Pro Pro Glu Gly Ser Thr Ser Gly Ala Met Ser 180 185 190Asp Asp
Ser Glu Met Arg Ala Ala Ala Gly Gly Ala Ala Val Glu Gly 195 200
205Gly Gln Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys
210 215 220Asp Ser Thr Trp Ser Glu Gly His Val Thr Thr Thr Ser Thr
Arg Thr225 230 235 240Trp Val Leu Pro Thr Tyr Asn Asn His Leu Tyr
Lys Arg Leu Gly Glu 245 250 255Ser Leu Gln Ser Asn Thr Tyr Asn Gly
Phe Ser Thr Pro Trp Gly Tyr 260 265 270Phe Asp Phe Asn Arg Phe His
Cys His Phe Ser Pro Arg Asp Trp Gln 275 280 285Arg Leu Ile Asn Asn
Asn Trp Gly Met Arg Pro Lys Ala Met Arg Val 290 295 300Lys Ile Phe
Asn Ile Gln Val Lys Glu Val Thr Thr Ser Asn Gly Glu305 310 315
320Thr Thr Val Ala Asn Asn Leu Thr Ser Thr Val Gln Ile Phe Ala Asp
325 330 335Ser Ser Tyr Glu Leu Pro Tyr Val Met Asp Ala Gly Gln Glu
Gly Ser 340 345 350Leu Pro Pro Phe Pro Asn Asp Val Phe Met Val Pro
Gln Tyr Gly Tyr 355 360 365Cys Gly Leu Val Thr Gly Asn Thr Ser Gln
Gln Gln Thr Asp Arg Asn 370 375 380Ala Phe Tyr Cys Leu Glu Tyr Phe
Pro Ser Gln Met Leu Arg Thr Gly385 390 395 400Asn Asn Phe Glu Ile
Thr Tyr Ser Phe Glu Lys Val Pro Phe His Ser 405 410 415Met Tyr Ala
His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile 420 425 430Asp
Gln Tyr Leu Trp Gly Leu Gln Ser Thr Thr Thr Gly Thr Thr Leu 435 440
445Asn Ala Gly Thr Ala Thr Thr Asn Phe Thr Lys Leu Arg Pro Thr Asn
450 455 460Phe Ser Asn Phe Lys Lys Asn Trp Leu Pro Gly Pro Ser Ile
Lys Gln465 470 475 480Gln Gly Phe Ser Lys Thr Ala Asn Gln Asn Tyr
Lys Ile Pro Ala Thr 485 490 495Gly Ser Asp Ser Leu Ile Lys Tyr Glu
Thr His Ser Thr Leu Asp Gly 500 505 510Arg Trp Ser Ala Leu Thr Pro
Gly Pro Pro Met Ala Thr Ala Gly Pro 515 520 525Ala Asp Ser Lys Phe
Ser Asn Ser Gln Leu Ile Phe Ala Gly Pro Lys 530 535 540Gln Asn Gly
Asn Thr Ala Thr Val Pro Gly Thr Leu Ile Phe Thr Ser545 550 555
560Glu Glu Glu Leu Ala Ala Thr Asn Ala Thr Asp Thr Asp Met Trp Gly
565 570 575Asn Leu Pro Gly Gly Asp Gln Ser Asn Ser Asn Leu Pro Thr
Val Asp 580 585 590Arg Leu Thr Ala Leu Gly Ala Val Pro Gly Met Val
Trp Gln Asn Arg 595 600 605Asp Ile Tyr Tyr Gln Gly Pro Ile Trp Ala
Lys Ile Pro His Thr Asp 610 615 620Gly His Phe His Pro Ser Pro Leu
Ile Gly Gly Phe Gly Leu Lys His625 630 635 640Pro Pro Pro Gln Ile
Phe Ile Lys Asn Thr Pro Val Pro Ala Asn Pro 645 650 655Ala Thr Thr
Phe Ser Ser Thr Pro Val Asn Ser Phe Ile Thr Gln Tyr 660 665 670Ser
Thr Gly Gln Val Ser Val Gln Ile Asp Trp Glu Ile Gln Lys Glu 675 680
685Arg Ser Lys Arg Trp Asn Pro Glu Val Gln Phe Thr Ser Asn Tyr Gly
690 695 700Gln Gln Asn Ser Leu Leu Trp Ala Pro Asp Ala Ala Gly Lys
Tyr Thr705 710 715 720Glu Pro Arg Ala Ile Gly Thr Arg Tyr Leu Thr
His His Leu 725 73032208DNAAdeno-associated virus 3atggctgccg
atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60cagtggtgga
agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac
120gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa
cggactcgac 180aagggagagc cggtcaacga ggcagacgcc gcggccctcg
agcacgacaa agcctacgac 240cggcagctcg acagcggaga caacccgtac
ctcaagtaca accacgccga cgcggagttt 300caggagcgcc ttaaagaaga
tacgtctttt gggggcaacc tcggacgagc agtcttccag 360gcgaaaaaga
gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg
420ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc
gggaaccgga 480aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg
gtcagactgg agacgcagac 540tcagtacctg acccccagcc tctcggacag
ccaccagcag ccccctctgg tctgggaact 600aatacgatgg ctacaggcag
tggcgcacca atggcagaca ataacgaggg cgccgacgga 660gtgggtaatt
cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc
720accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta
caaacaaatt 780tccagccaat caggagcctc gaacgacaat cactactttg
gctacagcac cccttggggg 840tattttgact tcaacagatt ccactgccac
ttttcaccac gtgactggca aagactcatc 900aacaacaact ggggattccg
acccaagaga ctcaacttca agctctttaa cattcaagtc 960aaagaggtca
cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt
1020caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc
gcatcaagga 1080tgcctcccgc cgttcccagc agacgtcttc atggtgccac
agtatggata cctcaccctg 1140aacaacggga gtcaggcagt aggacgctct
tcattttact gcctggagta ctttccttct 1200cagatgctgc gtaccggaaa
caactttacc ttcagctaca cttttgagga cgttcctttc 1260cacagcagct
acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag
1320tacctgtatt acttgagcag aacaaacact ccaagtggaa ccaccacgca
gtcaaggctt 1380cagttttctc aggccggagc gagtgacatt cgggaccagt
ctaggaactg gcttcctgga 1440ccctgttacc gccagcagcg agtatcaaag
acatctgcgg ataacaacaa cagtgaatac 1500tcgtggactg gagctaccaa
gtaccacctc aatggcagag actctctggt gaatccgggc 1560ccggccatgg
caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc
1620atctttggga agcaaggctc agagaaaaca aatgtggaca ttgaaaaggt
catgattaca 1680gacgaagagg aaatcaggac aaccaatccc gtggctacgg
agcagtatgg ttctgtatct 1740accaacctcc agagaggcaa cagacaagca
gctaccgcag atgtcaacac acaaggcgtt 1800cttccaggca tggtctggca
ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860attccacaca
cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa
1920caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc
ttcgaccacc 1980ttcagtgcgg caaagtttgc ttccttcatc acacagtact
ccacgggaca ggtcagcgtg 2040gagatcgagt gggagctgca gaaggaaaac
agcaaacgct ggaatcccga aattcagtac 2100acttccaact acaacaagtc
tgttaatgtg gactttactg tggacactaa tggcgtgtat 2160tcagagcctc
gccccattgg caccagatac ctgactcgta atctgtaa
220842205DNAAdeno-associated virus 4atgactgacg gttaccttcc
agattggcta gaggacaacc tctctgaagg cgttcgagag 60tggtgggcgc tgcaacctgg
agcccctaaa cccaaggcaa atcaacaaca tcaggacaac 120gctcggggtc
ttgtgcttcc gggttacaaa tacctcggac ccggcaacgg actcgacaag
180ggggaacccg tcaacgcagc ggacgcggca gccctcgagc acgacaaggc
ctacgaccag 240cagctcaagg ccggtgacaa cccctacctc aagtacaacc
acgccgacgc ggagttccag 300cagcggcttc agggcgacac atcgtttggg
ggcaacctcg gcagagcagt cttccaggcc 360aaaaagaggg ttcttgaacc
tcttggtctg gttgagcaag cgggtgagac ggctcctgga 420aagaagagac
cgttgattga atccccccag cagcccgact cctccacggg tatcggcaaa
480aaaggcaagc agccggctaa aaagaagctc gttttcgaag acgaaactgg
agcaggcgac 540ggaccccctg agggatcaac ttccggagcc atgtctgatg
acagtgagat gcgtgcagca 600gctggcggag ctgcagtcga gggcggacaa
ggtgccgatg gagtgggtaa tgcctcgggt 660gattggcatt gcgattccac
ctggtctgag ggccacgtca cgaccaccag caccagaacc 720tgggtcttgc
ccacctacaa caaccacctc tacaagcgac tcggagagag cctgcagtcc
780aacacctaca acggattctc caccccctgg ggatactttg acttcaaccg
cttccactgc 840cacttctcac cacgtgactg gcagcgactc atcaacaaca
actggggcat gcgacccaaa 900gccatgcggg tcaaaatctt caacatccag
gtcaaggagg tcacgacgtc gaacggcgag 960acaacggtgg ctaataacct
taccagcacg gttcagatct ttgcggactc gtcgtacgaa 1020ctgccgtacg
tgatggatgc gggtcaagag ggcagcctgc ctccttttcc caacgacgtc
1080tttatggtgc cccagtacgg ctactgtgga ctggtgaccg gcaacacttc
gcagcaacag 1140actgacagaa atgccttcta ctgcctggag tactttcctt
cgcagatgct gcggactggc 1200aacaactttg aaattacgta cagttttgag
aaggtgcctt tccactcgat gtacgcgcac 1260agccagagcc tggaccggct
gatgaaccct ctcatcgacc agtacctgtg gggactgcaa 1320tcgaccacca
ccggaaccac cctgaatgcc gggactgcca ccaccaactt taccaagctg
1380cggcctacca acttttccaa ctttaaaaag aactggctgc ccgggccttc
aatcaagcag 1440cagggcttct caaagactgc caatcaaaac tacaagatcc
ctgccaccgg gtcagacagt 1500ctcatcaaat acgagacgca cagcactctg
gacggaagat ggagtgccct gacccccgga 1560cctccaatgg ccacggctgg
acctgcggac agcaagttca gcaacagcca gctcatcttt 1620gcggggccta
aacagaacgg caacacggcc accgtacccg ggactctgat cttcacctct
1680gaggaggagc tggcagccac caacgccacc gatacggaca tgtggggcaa
cctacctggc 1740ggtgaccaga gcaacagcaa cctgccgacc gtggacagac
tgacagcctt gggagccgtg 1800cctggaatgg tctggcaaaa cagagacatt
tactaccagg gtcccatttg ggccaagatt 1860cctcataccg atggacactt
tcacccctca ccgctgattg gtgggtttgg gctgaaacac 1920ccgcctcctc
aaatttttat caagaacacc ccggtacctg cgaatcctgc aacgaccttc
1980agctctactc cggtaaactc cttcattact cagtacagca ctggccaggt
gtcggtgcag 2040attgactggg agatccagaa ggagcggtcc aaacgctgga
accccgaggt ccagtttacc 2100tccaactacg gacagcaaaa ctctctgttg
tgggctcccg atgcggctgg gaaatacact 2160gagcctaggg ctatcggtac
ccgctacctc acccaccacc tgtaa 22055563PRTHomo sapiens 5Met Gly Leu
Gln Ala Cys Leu Leu Gly Leu Phe Ala Leu Ile Leu Ser1 5 10 15Gly Lys
Cys Ser Tyr Ser Pro Glu Pro Asp Gln Arg Arg Thr Leu Pro 20 25 30Pro
Gly Trp Val Ser Leu Gly Arg Ala Asp Pro Glu Glu Glu Leu Ser 35 40
45Leu Thr Phe Ala Leu Arg Gln Gln Asn Val Glu Arg Leu Ser Glu Leu
50 55 60Val Gln Ala Val Ser Asp Pro Ser Ser Pro Gln Tyr Gly Lys Tyr
Leu65 70 75 80Thr Leu Glu Asn Val Ala Asp Leu Val Arg Pro Ser Pro
Leu Thr Leu 85 90 95His Thr Val Gln Lys Trp Leu Leu Ala Ala Gly Ala
Gln Lys Cys His 100 105 110Ser Val Ile Thr Gln Asp Phe Leu Thr Cys
Trp Leu Ser Ile Arg Gln 115 120 125Ala Glu Leu Leu Leu Pro Gly Ala
Glu Phe His His Tyr Val Gly Gly 130 135 140Pro Thr Glu Thr His Val
Val Arg Ser Pro His Pro Tyr Gln Leu Pro145 150 155 160Gln Ala Leu
Ala Pro His Val Asp Phe Val Gly Gly Leu His His Phe 165 170 175Pro
Pro Thr Ser Ser Leu Arg Gln Arg Pro Glu Pro Gln Val Thr Gly 180 185
190Thr Val Gly Leu His Leu Gly Val Thr Pro Ser Val Ile Arg Lys Arg
195 200 205Tyr Asn Leu Thr Ser Gln Asp Val Gly Ser Gly Thr Ser Asn
Asn Ser 210 215 220Gln Ala Cys Ala Gln Phe Leu Glu Gln Tyr Phe His
Asp Ser Asp Leu225 230 235 240Ala Gln Phe Met Arg Leu Phe Gly Gly
Asn Phe Ala His Gln Ala Ser 245
250 255Val Ala Arg Val Val Gly Gln Gln Gly Arg Gly Arg Ala Gly Ile
Glu 260 265 270Ala Ser Leu Asp Val Gln Tyr Leu Met Ser Ala Gly Ala
Asn Ile Ser 275 280 285Thr Trp Val Tyr Ser Ser Pro Gly Arg His Glu
Gly Gln Glu Pro Phe 290 295 300Leu Gln Trp Leu Met Leu Leu Ser Asn
Glu Ser Ala Leu Pro His Val305 310 315 320His Thr Val Ser Tyr Gly
Asp Asp Glu Asp Ser Leu Ser Ser Ala Tyr 325 330 335Ile Gln Arg Val
Asn Thr Glu Leu Met Lys Ala Ala Ala Arg Gly Leu 340 345 350Thr Leu
Leu Phe Ala Ser Gly Asp Ser Gly Ala Gly Cys Trp Ser Val 355 360
365Ser Gly Arg His Gln Phe Arg Pro Thr Phe Pro Ala Ser Ser Pro Tyr
370 375 380Val Thr Thr Val Gly Gly Thr Ser Phe Gln Glu Pro Phe Leu
Ile Thr385 390 395 400Asn Glu Ile Val Asp Tyr Ile Ser Gly Gly Gly
Phe Ser Asn Val Phe 405 410 415Pro Arg Pro Ser Tyr Gln Glu Glu Ala
Val Thr Lys Phe Leu Ser Ser 420 425 430Ser Pro His Leu Pro Pro Ser
Ser Tyr Phe Asn Ala Ser Gly Arg Ala 435 440 445Tyr Pro Asp Val Ala
Ala Leu Ser Asp Gly Tyr Trp Val Val Ser Asn 450 455 460Arg Val Pro
Ile Pro Trp Val Ser Gly Thr Ser Ala Ser Thr Pro Val465 470 475
480Phe Gly Gly Ile Leu Ser Leu Ile Asn Glu His Arg Ile Leu Ser Gly
485 490 495Arg Pro Pro Leu Gly Phe Leu Asn Pro Arg Leu Tyr Gln Gln
His Gly 500 505 510Ala Gly Leu Phe Asp Val Thr Arg Gly Cys His Glu
Ser Cys Leu Asp 515 520 525Glu Glu Val Glu Gly Gln Gly Phe Cys Ser
Gly Pro Gly Trp Asp Pro 530 535 540Val Thr Gly Trp Gly Thr Pro Asn
Phe Pro Ala Leu Leu Lys Thr Leu545 550 555 560Leu Asn
Pro63487DNAHomo sapiens 6cgcggaaggg cagaatggga ctccaagcct
gcctcctagg gctctttgcc ctcatcctct 60ctggcaaatg cagttacagc ccggagcccg
accagcggag gacgctgccc ccaggctggg 120tgtccctggg ccgtgcggac
cctgaggaag agctgagtct cacctttgcc ctgagacagc 180agaatgtgga
aagactctcg gagctggtgc aggctgtgtc ggatcccagc tctcctcaat
240acggaaaata cctgacccta gagaatgtgg ctgatctggt gaggccatcc
ccactgaccc 300tccacacggt gcaaaaatgg ctcttggcag ccggagccca
gaagtgccat tctgtgatca 360cacaggactt tctgacttgc tggctgagca
tccgacaagc agagctgctg ctccctgggg 420ctgagtttca tcactatgtg
ggaggaccta cggaaaccca tgttgtaagg tccccacatc 480cctaccagct
tccacaggcc ttggcccccc atgtggactt tgtgggggga ctgcaccatt
540ttcccccaac atcatccctg aggcaacgtc ctgagccgca ggtgacaggg
actgtaggcc 600tgcatctggg ggtaaccccc tctgtgatcc gtaagcgata
caacttgacc tcacaagacg 660tgggctctgg caccagcaat aacagccaag
cctgtgccca gttcctggag cagtatttcc 720atgactcaga cctggctcag
ttcatgcgcc tcttcggtgg caactttgca catcaggcat 780cagtagcccg
tgtggttgga caacagggcc ggggccgggc cgggattgag gccagtctag
840atgtgcagta cctgatgagt gctggtgcca acatctccac ctgggtctac
agtagccctg 900gccggcatga gggacaggag cccttcctgc agtggctcat
gctgctcagt aatgagtcag 960ccctgccaca tgtgcatact gtgagctatg
gagatgatga ggactccctc agcagcgcct 1020acatccagcg ggtcaacact
gagctcatga aggctgctgc tcggggtctc accctgctct 1080tcgcctcagg
tgacagtggg gccgggtgtt ggtctgtctc tggaagacac cagttccgcc
1140ctaccttccc tgcctccagc ccctatgtca ccacagtggg aggcacatcc
ttccaggaac 1200ctttcctcat cacaaatgaa attgttgact atatcagtgg
tggtggcttc agcaatgtgt 1260tcccacggcc ttcataccag gaggaagctg
taacgaagtt cctgagctct agcccccacc 1320tgccaccatc cagttacttc
aatgccagtg gccgtgccta cccagatgtg gctgcacttt 1380ctgatggcta
ctgggtggtc agcaacagag tgcccattcc atgggtgtcc ggaacctcgg
1440cctctactcc agtgtttggg gggatcctat ccttgatcaa tgagcacagg
atccttagtg 1500gccgcccccc tcttggcttt ctcaacccaa ggctctacca
gcagcatggg gcaggactct 1560ttgatgtaac ccgtggctgc catgagtcct
gtctggatga agaggtagag ggccagggtt 1620tctgctctgg tcctggctgg
gatcctgtaa caggctgggg aacacccaac ttcccagctt 1680tgctgaagac
tctactcaac ccctgaccct ttcctatcag gagagatggc ttgtcccctg
1740ccctgaagct ggcagttcag tcccttattc tgccctgttg gaagccctgc
tgaaccctca 1800actattgact gctgcagaca gcttatctcc ctaaccctga
aatgctgtga gcttgacttg 1860actcccaacc ctaccatgct ccatcatact
caggtctccc tactcctgcc ttagattcct 1920caataagatg ctgtaactag
cattttttga atgcctctcc ctccgcatct catctttctc 1980ttttcaatca
ggcttttcca aagggttgta tacagactct gtgcactatt tcacttgata
2040ttcattcccc aattcactgc aaggagacct ctactgtcac cgtttactct
ttcctaccct 2100gacatccaga aacaatggcc tccagtgcat acttctcaat
ctttgcttta tggcctttcc 2160atcatagttg cccactccct ctccttactt
agcttccagg tcttaacttc tctgactact 2220cttgtcttcc tctctcatca
atttctgctt cttcatggaa tgctgacctt cattgctcca 2280tttgtagatt
tttgctcttc tcagtttact cattgtcccc tggaacaaat cactgacatc
2340tacaaccatt accatctcac taaataagac tttctatcca ataatgattg
atacctcaaa 2400tgtaagatgc gtgatactca acatttcatc gtccaccttc
ccaaccccaa acaattccat 2460ctcgtttctt cttggtaaat gatgctatgc
tttttccaac caagccagaa acctgtgtca 2520tcttttcacc ccaccttcaa
tcaacaagtc ctcaatcaac aagtcctact gactgcacat 2580cttaaatata
tctttatcag tccacaagtc cttccaatta tatttcccaa gtatatctag
2640aacttatcca cttatatccc cactgctact accttagttt agggctatat
tctcttgaaa 2700aaaagtgtcc ttacttcctg ccaatcccca agtcatcttc
cagagtaaaa tgcaaatccc 2760atcaggccac ttggatgaaa acccttcaag
gattactgga tagaattcag gctttcccct 2820ccagccccca atcatagctc
acaaaccttc cttgctattt gttcttaagt aaaaaatcat 2880ttttcctcct
ccctccccaa accccaagga actctcactc ttgctcaagc tgttccgtcc
2940ccttaccacc cctgatacaa ctgccaggtt aatttccaga attcttgcaa
gactcagttc 3000agaagtcacc ttctttcgtg aatgttttga ttccctgagg
ctactttatt ttggtatggc 3060tgaaaaatcc tagattttct aaacaaaacc
tgtttgaatc ttggttctga tatggactag 3120gagagagact gggtcaagta
agcttatctc cctgaggctg tttcctcgtc tgttaagtgt 3180gaatatcaat
acctgccttt cataatcacc agggaataaa gtggaataat gttgataaca
3240gtgcttggca cctggaagta ggtggcagat gttaacgccc ttcctccctt
gcactgcgcc 3300ccctgtgcct acctctagca ttgtaacgac cacatagtat
tgaaatggcc agtttacttg 3360tctgccttcc tttccaagac cgttggtgcc
tagaggacta gaatcgtgtc ctatttaact 3420ttgtgttccc aggtcctagc
tcaggagttg gcaaataaga attaaatgtc tgctacaccg 3480aaacaaa
34877152PRTMacaca mulatta 7Gln Ala Gly Phe Ala Thr Ala Asp His Ser
Ser Gln Glu Thr Glu Thr1 5 10 15Glu Lys Ala Met Asp Arg Leu Ala Arg
Gly Ala Gln Ser Val Pro Asn 20 25 30Asp Ser Pro Ala Gln Gly Glu Gly
Thr His Ser Glu Glu Glu Gly Phe 35 40 45Ala Met Asp Glu Glu Asp Ser
Asp Gly Glu Leu Asn Thr Trp Glu Leu 50 55 60Ser Glu Gly Thr Asn Cys
Pro Pro Lys Glu Gln Pro Gly Asp Ile Phe65 70 75 80Asn Glu Asp Trp
Asp Leu Glu Leu Lys Ala Asp Gln Gly Asn Pro Tyr 85 90 95Asp Ala Asp
Asp Ile Gln Glu Ser Ile Ser Gln Glu Leu Lys Pro Trp 100 105 110Val
Cys Cys Ala Pro Gln Gly Asp Met Ile Tyr Asp Pro Ser Trp His 115 120
125His Pro Pro Pro Leu Ile Pro His Tyr Ser Lys Met Val Phe Glu Thr
130 135 140Gly Gln Phe Asp Asp Ala Glu Asp145 1508152PRTMacaca
fascicularis 8Gln Ala Gly Phe Ala Thr Ala Asp His Ser Ser Gln Glu
Arg Glu Thr1 5 10 15Glu Lys Ala Met Asp Arg Leu Ala Arg Gly Ala Gln
Ser Val Pro Asn 20 25 30Asp Ser Pro Ala Arg Gly Glu Gly Thr His Ser
Glu Glu Glu Gly Phe 35 40 45Ala Met Asp Glu Glu Asp Ser Asp Gly Glu
Leu Asn Thr Trp Glu Leu 50 55 60Ser Glu Gly Thr Asn Cys Pro Pro Lys
Glu Gln Pro Gly Asp Ile Phe65 70 75 80Asn Glu Asp Trp Asp Leu Glu
Leu Lys Ala Asp Gln Gly Asn Pro Tyr 85 90 95Asp Ala Asp Asp Ile Gln
Glu Ser Ile Ser Gln Glu Leu Lys Pro Trp 100 105 110Val Cys Cys Ala
Pro Gln Gly Asp Met Ile Tyr Asp Pro Ser Trp His 115 120 125His Pro
Pro Pro Leu Ile Pro His Tyr Ser Lys Met Val Phe Glu Thr 130 135
140Gly Gln Phe Asp Asp Ala Glu Asp145 150
* * * * *