Note: Descriptions are shown in the official language in which they were submitted.
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GENE THERAPIES FOR LYSOSOMAL DISORDERS
RELATED APPLICATIONS
This Application claims the benefit under 35 U.S.C. 119(e) of the filing date
of U.S.
Provisional Application Serial Numbers 62/832,223, filed April 10, 2019,
entitled "AAV
VECTORS ENCODING TREM2 AND USES THEREOF", 62/831,840, filed April 10, 2019,
entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", 62/831,846, filed April 10,
2019, entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", 62/831,856, filed
April 10, 2019, entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", 62/934,450,
filed November 12, 2019, entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS",
62/954,089, filed December 27, 2019, entitled "GENE THERAPIES FOR LYSOSOMAL
DISORDERS", 62/960,471, filed January 13, 2020, entitled "GENE THERAPIES FOR
LYSOSOMAL DISORDERS", 62/998,665, filed March 12, 2020, entitled "GENE
THERAPIES FOR LYSOSOMAL DISORDERS", and 62/990,246, filed March 16, 2020,
entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", the entire contents of each
of which are incorporated herein by reference.
BACKGROUND
Gaucher disease is a rare inborn error of glycosphingolipid metabolism due to
deficiency of lysosomal acid P-glucocerebrosidase (Gcase, "GBA"). Patients
suffer from non-
CNS symptoms and findings including hepatosplenomegly, bone marrow
insufficiency leading
to pancytopenia, lung disorders and fibrosis, and bone defects. In addition, a
significant number
of patients suffer from neurological manifestations, including defective
saccadic eye movements
and gaze, seizures, cognitive deficits, developmental delay, and movement
disorders including
Parkinson's disease.
Several therapeutics exist that address the peripheral disease and the
principal clinical
manifestations in hematopoietic bone marrow and viscera, including enzyme
replacement
therapies as described below, chaperone-like small molecule drugs that bind to
defective Gcase
and improve stability, and substrate reduction therapy that block the
production of substrate that
accumulate in Gaucher disease leading to symptoms and findings. However, other
aspects of
Gaucher disease (particularly those affecting the skeleton and brain) appear
refractory to
treatment.
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SUMMARY
The present disclosure relates, in part, to compositions and methods for
treating certain
central nervous system (CNS) diseases, for example neurodegenerative diseases
(e.g.,
neurodegenerative diseases listed in Table 2), synucleinopathies (e.g.,
synucleinopathies listed in
Table 3), tauopathies (tauopathies listed in Table 4), or lysosomal storage
diseases (e.g.,
lysosomal storage diseases listed in Table 5).
In addition to Gaucher disease patients (who possess mutations in both
chromosomal
alleles of GBA1 gene), patients with mutations in only one allele of GBA1 are
at highly
increased risk of Parkinson's disease (PD). The severity of PD symptoms- which
include gait
difficulty, a tremor at rest, rigidity, and often depression, sleep
difficulties, and cognitive decline
- correlate with the degree of enzyme activity reduction. Thus, Gaucher
disease patients have
the most severe course, whereas patients with a single mild mutation in GBA1
typically have a
more benign course. Mutation carriers are also at high risk of other PD-
related disorders,
including Lewy Body Dementia, characterized by executive dysfunction,
psychosis, and a PD-
like movement disorder, and multi-system atrophy, with characteristic motor
and cognitive
impairments. No therapies exist that alter the inexorable course of these
disorders.
Deficits in enzymes such as Gcase (e.g., the gene product of GBA1 gene), as
well as
common variants in many genes implicated in lysosome function or trafficking
of
macromolecules to the lysosome (e.g., Lysosomal Membrane Protein 1 (LIMP),
also referred to
as SCARB2), have been associated with increased PD risk and/or risk of Gaucher
disease (e.g.,
neuronopathic Gaucher disease, such as Type 2 Gaucher disease or Type 3
Gaucher disease).
The disclosure is based, in part, on expression constructs (e.g., vectors)
encoding one or more
genes, for example Gcase, GBA2, prosaposin, progranulin, LIMP2, GALC, CTSB,
SMPD1,
GCH1, RAB7, VPS35, IL-34, TREM2, TMEM106B, or a combination of any of the
foregoing
(or portions thereof), associated with central nervous system (CNS) diseases,
for example
Gaucher disease, PD, etc. In some embodiments, combinations of gene products
described
herein act together (e.g., synergistically) to reduce one or more signs and
symptoms of a CNS
disease when expressed in a subject.
Accordingly, in some aspects, the disclosure provides an isolated nucleic acid
comprising an expression construct encoding a Gcase (e.g., the gene product of
GBA1 gene). In
some embodiments, the isolated nucleic acid comprises a Gcase-encoding
sequence that has
been codon optimized (e.g., codon optimized for expression in mammalian cells,
for example
human cells). In some embodiments, the nucleic acid sequence encoding the
Gcase encodes a
protein comprising an amino acid sequence as set forth in SEQ ID NO: 14 (e.g.,
as set forth in
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NCBI Reference Sequence NP 000148.2). In some embodiments, the isolated
nucleic acid
comprises the sequence set forth in SEQ ID NO: 15. In some embodiments the
expression
construct comprises adeno-associated virus (AAV) inverted terminal repeats
(ITRs), for example
AAV ITRs flanking the nucleic acid sequence encoding the Gcase protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding Prosaposin (e.g., the gene product of PSAP
gene). In some
embodiments, the isolated nucleic acid comprises a prosaposin-encoding
sequence that has been
codon optimized (e.g., codon optimized for expression in mammalian cells, for
example human
cells). In some embodiments, the nucleic acid sequence encoding the prosaposin
encodes a
protein comprising an amino acid sequence as set forth in SEQ ID NO: 16 (e.g.,
as set forth in
NCBI Reference Sequence NP 002769.1). In some embodiments, the isolated
nucleic acid
comprises the sequence set forth in SEQ ID NO: 17. In some embodiments the
expression
construct comprises adeno-associated virus (AAV) inverted terminal repeats
(ITRs), for example
AAV ITRs flanking the nucleic acid sequence encoding the prosaposin protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding LIMP2/SCARB2 (e.g., the gene product of SCARB2
gene). In
some embodiments, the isolated nucleic acid comprises a SCARB2-encoding
sequence that has
been codon optimized (e.g., codon optimized for expression in mammalian cells,
for example
human cells). In some embodiments, the nucleic acid sequence encoding the
LIMP2/SCARB2
encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO:
18 (e.g., as set
forth in NCBI Reference Sequence NP 005497.1). In some embodiments, the
isolated nucleic
acid comprises the sequence set forth in SEQ ID NO: 19. In some embodiments
the expression
construct comprises adeno-associated virus (AAV) inverted terminal repeats
(ITRs), for example
AAV ITRs flanking the nucleic acid sequence encoding the SCARB2 protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding GBA2 protein (e.g., the gene product of GBA2
gene). In some
embodiments, the isolated nucleic acid comprises a GBA2-encoding sequence that
has been
codon optimized (e.g., codon optimized for expression in mammalian cells, for
example human
cells). In some embodiments, the nucleic acid sequence encoding the GBA2
encodes a protein
comprising an amino acid sequence as set forth in SEQ ID NO: 30 (e.g., as set
forth in NCBI
Reference Sequence NP 065995.1). In some embodiments, the isolated nucleic
acid comprises
the sequence set forth in SEQ ID NO: 31. In some embodiments the expression
construct
comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for
example AAV
ITRs flanking the nucleic acid sequence encoding the GBA2 protein.
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In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding GALC protein (e.g., the gene product of GALC
gene). In some
embodiments, the isolated nucleic acid comprises a GALC-encoding sequence that
has been
codon optimized (e.g., codon optimized for expression in mammalian cells, for
example human
cells). In some embodiments, the nucleic acid sequence encoding the GALC
encodes a protein
comprising an amino acid sequence as set forth in SEQ ID NO: 33 (e.g., as set
forth in NCBI
Reference Sequence NP 000144.2). In some embodiments, the isolated nucleic
acid comprises
the sequence set forth in SEQ ID NO: 34. In some embodiments the expression
construct
comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for
example AAV
ITRs flanking the nucleic acid sequence encoding the GALC protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding CTSB protein (e.g., the gene product of CTSB
gene). In some
embodiments, the isolated nucleic acid comprises a CTSB-encoding sequence that
has been
codon optimized (e.g., codon optimized for expression in mammalian cells, for
example human
cells). In some embodiments, the nucleic acid sequence encoding the CTSB
encodes a protein
comprising an amino acid sequence as set forth in SEQ ID NO: 35 (e.g., as set
forth in NCBI
Reference Sequence NP 001899.1). In some embodiments, the isolated nucleic
acid comprises
the sequence set forth in SEQ ID NO: 36. In some embodiments the expression
construct
comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for
example AAV
ITRs flanking the nucleic acid sequence encoding the CTSB protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding SMPD1 protein (e.g., the gene product of SMPD1
gene). In some
embodiments, the isolated nucleic acid comprises a SMPD1-encoding sequence
that has been
codon optimized (e.g., codon optimized for expression in mammalian cells, for
example human
cells). In some embodiments, the nucleic acid sequence encoding the SMPD1
encodes a protein
comprising an amino acid sequence as set forth in SEQ ID NO: 37 (e.g., as set
forth in NCBI
Reference Sequence NP 000534.3). In some embodiments, the isolated nucleic
acid comprises
the sequence set forth in SEQ ID NO: 38. In some embodiments the expression
construct
comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for
example AAV
ITRs flanking the nucleic acid sequence encoding the SMPD1 protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding GCH1 protein (e.g., the gene product of GCH1
gene). In some
embodiments, the isolated nucleic acid comprises a GCH1-encoding sequence that
has been
codon optimized (e.g., codon optimized for expression in mammalian cells, for
example human
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cells). In some embodiments, the nucleic acid sequence encoding the GCH1
encodes a protein
comprising an amino acid sequence as set forth in SEQ ID NO: 45 (e.g., as set
forth in NCBI
Reference Sequence NP 000534.3). In some embodiments, the isolated nucleic
acid comprises
the sequence set forth in SEQ ID NO: 46. In some embodiments the expression
construct
comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for
example AAV
ITRs flanking the nucleic acid sequence encoding the GCH1 protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding RAB7L protein (e.g., the gene product of RAB7L
gene). In some
embodiments, the isolated nucleic acid comprises a RAB7L-encoding sequence
that has been
codon optimized (e.g., codon optimized for expression in mammalian cells, for
example human
cells). In some embodiments, the nucleic acid sequence encoding the RAB7L
encodes a protein
comprising an amino acid sequence as set forth in SEQ ID NO: 47 (e.g., as set
forth in NCBI
Reference Sequence NP 003920.1). In some embodiments, the isolated nucleic
acid comprises
the sequence set forth in SEQ ID NO: 48. In some embodiments the expression
construct
comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for
example AAV
ITRs flanking the nucleic acid sequence encoding the RAB7L protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding VP535 protein (e.g., the gene product of VPS35
gene). In some
embodiments, the isolated nucleic acid comprises a VP535-encoding sequence
that has been
codon optimized (e.g., codon optimized for expression in mammalian cells, for
example human
cells). In some embodiments, the nucleic acid sequence encoding the VP535
encodes a protein
comprising an amino acid sequence as set forth in SEQ ID NO: 49 (e.g., as set
forth in NCBI
Reference Sequence NP 060676.2). In some embodiments, the isolated nucleic
acid comprises
the sequence set forth in SEQ ID NO: 50. In some embodiments the expression
construct
comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for
example AAV
ITRs flanking the nucleic acid sequence encoding the VP535 protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding IL-34 protein (e.g., the gene product of IL34
gene). In some
embodiments, the isolated nucleic acid comprises a IL-34-encoding sequence
that has been
codon optimized (e.g., codon optimized for expression in mammalian cells, for
example human
cells). In some embodiments, the nucleic acid sequence encoding the IL-34
encodes a protein
comprising an amino acid sequence as set forth in SEQ ID NO: 55 (e.g., as set
forth in NCBI
Reference Sequence NP 689669.2). In some embodiments, the isolated nucleic
acid comprises
the sequence set forth in SEQ ID NO: 56. In some embodiments the expression
construct
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comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for
example AAV
ITRs flanking the nucleic acid sequence encoding the IL-34 protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding TREM2 protein (e.g., the gene product of TREM
gene). In some
embodiments, the isolated nucleic acid comprises a TREM2-encoding sequence
that has been
codon optimized (e.g., codon optimized for expression in mammalian cells, for
example human
cells). In some embodiments, the nucleic acid sequence encoding the TREM2
encodes a protein
comprising an amino acid sequence as set forth in SEQ ID NO: 57 (e.g., as set
forth in NCBI
Reference Sequence NP 061838.1). In some embodiments, the isolated nucleic
acid comprises
the sequence set forth in SEQ ID NO: 58. In some embodiments the expression
construct
comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for
example AAV
ITRs flanking the nucleic acid sequence encoding the TREM2 protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding TMEM106B protein (e.g., the gene product of
TMEM106B
gene). In some embodiments, the isolated nucleic acid comprises a TMEM106B-
encoding
sequence that has been codon optimized (e.g., codon optimized for expression
in mammalian
cells, for example human cells). In some embodiments, the nucleic acid
sequence encoding the
TMEM106B encodes a protein comprising an amino acid sequence as set forth in
SEQ ID NO:
63 (e.g., as set forth in NCBI Reference Sequence NP 060844.2). In some
embodiments, the
isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 64. In
some embodiments
the expression construct comprises adeno-associated virus (AAV) inverted
terminal repeats
(ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the
TMEM106B
protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding progranulin (e.g., the gene product of PGRN
gene, also referred
to as GRN gene). In some embodiments, the isolated nucleic acid comprises a
prosaposin-
encoding sequence that has been codon optimized (e.g., codon optimized for
expression in
mammalian cells, for example human cells). In some embodiments, the nucleic
acid sequence
encoding the progranulin (PRGN also referred to as GRN) encodes a protein
comprising an
amino acid sequence as set forth in SEQ ID NO: 67 (e.g., as set forth in NCBI
Reference
Sequence NP 002078.1). In some embodiments, the isolated nucleic acid
comprises the
sequence set forth in SEQ ID NO: 68. In some embodiments the expression
construct comprises
adeno-associated virus (AAV) inverted terminal repeats (ITRs), for example AAV
ITRs flanking
the nucleic acid sequence encoding the prosaposin protein.
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Aspects of the disclosure relate to isolated nucleic acids and expression
constructs (e.g.,
rAAV vectors) encoding one or more inhibitory nucleic acids. In some
embodiments, one or
more inhibitory nucleic acids target a gene associated with certain central
nervous system (CNS)
diseases (e,g, SNCA, TMEM106B, RPS2 or MAPT). In some embodiments, the
inhibitory
nucleic acids are expressed alone, or in combination with one or more gene
products described
herein (e.g., GBA1, PSAP, PRGN, etc.). In some embodiments, an isolated
nucleic acid
encodes 1) an inhibitory nucleic acid targeting SNCA, and 2) GBA1 protein. In
some
embodiments, an isolated nucleic acid encodes 1) an inhibitory nucleic acid
targeting SNCA, and
2) PSAP protein. In some embodiments, an isolated nucleic acid encodes 1) an
inhibitory
nucleic acid targeting SNCA, and 2) PGRN protein (e.g., GRN protein). In some
embodiments,
an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting MAPT,
and 2) GBA1
protein. In some embodiments, an isolated nucleic acid encodes 1) an
inhibitory nucleic acid
targeting MAPT, and 2) PSAP protein. In some embodiments, an isolated nucleic
acid encodes
1) an inhibitory nucleic acid targeting MAPT, and 2) PGRN protein (e.g., GRN
protein). In
some embodiments, an isolated nucleic acid encodes 1) an inhibitory nucleic
acid targeting
TMEM106B, and 2) GBA1 protein. In some embodiments, an isolated nucleic acid
encodes 1)
an inhibitory nucleic acid targeting TMEM106B, and 2) PSAP protein. In some
embodiments, an
isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting
TMEM106B, and 2) PGRN
protein (e.g., GRN protein). In some embodiments, an isolated nucleic acid
encodes 1) an
inhibitory nucleic acid targeting RPS25, and 2) GBA1 protein. In some
embodiments, an
isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting RPS25,
and 2) PSAP
protein. In some embodiments, an isolated nucleic acid encodes 1) an
inhibitory nucleic acid
targeting RPS25, and 2) PGRN protein (e.g., GRN protein).
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding an inhibitory nucleic acid that inhibits
expression or activity of a-
Syn flanked by AAV inverted terminal repeats (ITRs). In some embodiments, the
inhibitory
nucleic acid is complementary to at least six contiguous nucleotides of the
sequence set forth in
SEQ ID NO: 90. In some embodiments, the inhibitory nucleic acid is an
inhibitory RNA
comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 20-
25. In some
embodiments, the inhibitory nucleic acid comprises the sequence set forth in
any one of SEQ ID
NOs: 94-99.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding an inhibitory nucleic acid that inhibits
expression or activity of
TMEM106B flanked by AAV inverted terminal repeats (ITRs). In some embodiments,
the
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inhibitory nucleic acid is complementary to at least six contiguous
nucleotides of the sequence
set forth in SEQ ID NO: 91. In some embodiments, the inhibitory nucleic acid
is an inhibitory
RNA comprising the nucleic acid sequence set forth in SEQ ID NO: 92 or 93. In
some
embodiments, the inhibitory nucleic acid comprises the sequence set forth in
SEQ ID NO: 65 or
66.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding an inhibitory nucleic acid that inhibits
expression or activity of
MAPT flanked by AAV inverted terminal repeats (ITRs). In some embodiments, the
inhibitory
nucleic acid is complementary to at least six contiguous nucleotides of the
sequence set forth in
SEQ ID NO: 114. In some embodiments, the inhibitory nucleic acid is an
inhibitory RNA
comprising the nucleic acid sequence set forth in SEQ ID NO: 123, 124, 127,
128, 131, 132, 135
or 136). In some embodiments, the inhibitory nucleic acid comprises the
sequence set forth in
SEQ ID NO: 125, 126, 129, 130, 133, 134, 137 or 138.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding a first gene product and a second gene product,
wherein each
gene product independently is selected from the gene products, or portions
thereof, set forth in
Table 1 or an inhibitory nucleic acid targeting a gene or gene product set
forth in Table 1. In
some embodiments, the first gene product is a protein, and the second gene
product is a protein.
In some embodiments, the first gene product is an inhibitory nucleic acid and
the second gene
product is a protein. In some embodiments, the first gene product is an
inhibitory nucleic acid
and the second gene product is an inhibitory nucleic acid.
In some embodiments, the first gene product is a Gcase protein, or a portion
thereof. In
some embodiments, the second gene product is an inhibitory nucleic acid that
targets SNCA. In
some embodiments, the interfering nucleic acid is a siRNA, shRNA, miRNA, or
dsRNA,
optionally wherein the interfering nucleic acid inhibits expression of a-Syn
protein. In some
embodiments, the isolated nucleic acid further comprises one or more
promoters, optionally
wherein each of the one or more promoters is independently a chicken-beta
actin (CBA)
promoter, a CAG promoter, a CD68 promoter, or a JeT promoter. In some
embodiments, the
isolated nucleic acid further comprising an internal ribosomal entry site
(IRES), optionally
wherein the IRES is located between the first gene product and the second gene
product.
In some embodiments, the isolated nucleic acid further comprising a self-
cleaving peptide
coding sequence, optionally wherein the self-cleaving peptide is T2A. In some
embodiments, the
expression construct comprises two adeno-associated virus (AAV) inverted
terminal repeat
(ITR) sequences flanking the first gene product and the second gene product.
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In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding a first gene product and a second gene product,
wherein each
gene product independently is selected from the gene products, or portions
thereof, set forth in
Table 1 or an inhibitory nucleic acid targeting a gene or gene product set
forth in Table 1.
In some embodiments, a first gene product or a second gene product is a Gcase
protein,
or a portion thereof. In some embodiments, a first gene product is a Gcase
protein and a second
gene product is selected from GBA2, prosaposin, progranulin, LIMP2, GALC,
CTSB, SMPD1,
GCH1, RAB7, VPS35, IL-34, TREM2, and TMEM106B.
In some embodiments, an expression construct encodes (e.g., alone or in
addition to
another gene product) an interfering nucleic acid (e.g., shRNA, miRNA, dsRNA,
etc.). In some
embodiments, an interfering nucleic acid inhibits expression of a-Synuclein (a-
Synuclein). In
some embodiments, an expression construct encodes an inhibitory nucleic acid
targeting SNCA,
and encodes one or more gene product selected from GBA1, GBA2, PSAP, PRGN,
LIMP2,
GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, and TMEM106B. In some
embodiments, an interfering nucleic acid that targets a-Synuclein comprises a
sequence set forth
in any one of SEQ ID NOs: 20-25. In some embodiments, an interfering nucleic
acid that
targets a-Synuclein binds to (e.g., hybridizes with) a sequence set forth in
any one of SEQ ID
NO: 20-25.
In some embodiments, an interfering nucleic acid inhibits expression of
TMEM106B. In
some embodiments, an expression construct encodes an inhibitory nucleic acid
targeting
TMEM106B, and encodes one or more gene product selected from GBA1, GBA2, PSAP,
PRGN, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VP535, IL-34, and TREM2. In some
embodiments, an interfering nucleic acid that targets TMEM106B comprises a
sequence set
forth in SEQ ID NO: 65 or 66. In some embodiments, an interfering nucleic acid
that targets
TMEM106B binds to (e.g., hybridizes with) a sequence set forth in SEQ ID NO:
65 or 66.
In some embodiments, an interfering nucleic acid inhibits expression of MAPT.
In some
embodiments, an expression construct encodes an inhibitory nucleic acid
targeting MAPT, and
encodes one or more gene product selected from GBA1, GBA2, PSAP, PRGN, LIMP2,
GALC,
CTSB, SMPD1, GCH1, RAB7, VP535, IL-34, TREM2, and TMEM106B. In some
embodiments, an interfering nucleic acid that targets MAPT comprises a
sequence set forth in
any one of SEQ ID NOs: 123-138. In some embodiments, an interfering nucleic
acid that
targets MAPT binds to (e.g., hybridizes with) a sequence set forth in any one
of SEQ ID NO:
123-138.
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In some embodiments, an interfering nucleic acid inhibits expression of RPS25.
In some
embodiments, an expression construct encodes an inhibitory nucleic acid
targeting RPS25, and
encodes one or more gene product selected from GBA1, GBA2, PSAP, PRGN, LIMP2,
GALC,
CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, and TMEM106B. In some
embodiments, an interfering nucleic acid that targets RPS25 comprises a
sequence set forth in
any one of SEQ ID NOs: 115-122. In some embodiments, an interfering nucleic
acid that
targets RP525 binds to (e.g., hybridizes with) a sequence set forth in any one
of SEQ ID NO:
115-122. In some embodiments, an expression construct further comprises one or
more
promoters. In some embodiments, a promoter is a chicken-beta actin (CBA)
promoter, a CAG
promoter, a CD68 promoter, or a JeT promoter. In some embodiments, a promoter
is a RNA pol
II promoter (e.g., or an RNA pol III promoter (e.g., U6, etc.).
In some embodiments, an expression construct further comprises an internal
ribosomal
entry site (IRES). In some embodiments, an IRES is located between a first
gene product and a
second gene product.
In some embodiments, an expression construct further comprises a self-cleaving
peptide
coding sequence. In some embodiments, a self-cleaving peptide is a T2A
peptide.
In some embodiments, an expression construct comprises two adeno-associated
virus
(AAV) inverted terminal repeat (ITR) sequences. In some embodiments, ITR
sequences flank a
first gene product and a second gene product (e.g., are arranged as follows
from 5'-end to 3'-
end: ITR-first gene product-second gene product-ITR). In some embodiments, one
of the ITR
sequences of an isolated nucleic acid lacks a functional terminal resolution
site (trs). For
example, in some embodiments, one of the ITRs is a AITR.
The disclosure relates, in some aspects, to rAAV vectors comprising an ITR
having a
modified "D" region (e.g., a D sequence that is modified relative to wild-type
AAV2 ITR, SEQ
ID NO: 29). In some embodiments, the ITR having the modified D region is the
5' ITR of the
rAAV vector. In some embodiments, a modified "D" region comprises an "S"
sequence, for
example as set forth in SEQ ID NO: 26. In some embodiments, the ITR having the
modified
"D" region is the 3' ITR of the rAAV vector. In some embodiments, a modified
"D" region
comprises a 3' ITR in which the "D" region is positioned at the 3' end of the
ITR (e.g., on the
outside or terminal end of the ITR relative to the transgene insert of the
vector). In some
embodiments, a modified "D" region comprises a sequence as set forth in SEQ ID
NO: 26 or 27.
In some embodiments, an isolated nucleic acid (e.g., an rAAV vector) comprises
a TRY
region. In some embodiments, a TRY region comprises the sequence set forth in
SEQ ID NO:
28.
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In some embodiments, an isolated nucleic acid described by the disclosure
comprises or
consists of, or encodes a peptide having, the sequence set forth in any one of
SEQ ID NOs: 1-
149.
In some aspects, the disclosure provides a vector comprising an isolated
nucleic acid as
described by the disclosure. In some embodiments, a vector is a plasmid, or a
viral vector. In
some embodiments, a viral vector is a recombinant AAV (rAAV) vector or a
Baculovirus
vector. In some embodiments, an rAAV vector is single-stranded (e.g., single-
stranded DNA).
In some aspects, the disclosure provides a host cell comprising an isolated
nucleic acid as
described by the disclosure or a vector as described by the disclosure.
In some aspects, the disclosure provides a recombinant adeno-associated virus
(rAAV)
comprising a capsid protein and an isolated nucleic acid or a vector as
described by the
disclosure.
In some embodiments, a capsid protein is capable of crossing the blood-brain
barrier, for
example an AAV9 capsid protein or an AAVrh.10 capsid protein. In some
embodiments, an
rAAV transduces neuronal cells and non-neuronal cells of the central nervous
system (CNS).
In some aspects, the disclosure provides a method for treating a subject
having or
suspected of having or suspected of having a central nervous system (CNS)
disease, the method
comprising administering to the subject a composition (e.g., a composition
comprising an
isolated nucleic acid or a vector or a rAAV) as described by the disclosure.
In some
embodiments, the CNS disease is a neurodegenerative disease, such as a
neurodegenerative
disease listed in Table 2. In some embodiments, the CNS disease is a
synucleinopathy, such as a
synucleinopathy listed in Table 3. In some embodiments, the CNS disease is a
tauopathy, such
as a tauopathy listed in Table 4. In some embodiments, the CNS disease is a
lysosomal storage
disease, such as a lysosomal storage disease listed in Table 5. In some
embodiments, the
lysosomal storage disease is neuronopathic Gaucher disease, such as Type 2
Gaucher disease or
Type 3 Gaucher disease.
In some embodiments, the disclosure relates to methods of treating a disease
selected
from Parkinson's Disease (e.g., Parkinson's Disease with GBA1 mutation (PD-
GBA), sporadic
Parkinson's Disease (sPD)), Gaucher Disease (e.g., neuronopathic Gaucher
disease (nGD), Type
I Gaucher Disease (T1GD), Type II Gaucher Disease (T2GD), and Type III Gaucher
Disease
(T3GD)), Dementia with Lewy Bodies (DLB), Amyotrophic lateral sclerosis (ALS),
and
Niemann-Pick Type C disease (NPC) by administering to a subject in need
thereof an isolated
nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic
acid) that encodes
GBAl.
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In some embodiments, the disclosure relates to methods of treating
Frontotemporal
Dementia (e.g., Frontotemporal Dementia with GRN mutation (FTD-GRN),
Frontotemporal
Dementia with MAPT mutation (FTD-tau), and Frontotemporal Dementia with
C90RF72
mutation (FTD-C9orf72)), Parkinson's Disease (PD), Alzheimer's Disease (AD),
Neuronal
Ceroid Lipofuscinosis (NCL), Corticobasal Degeneration (CBD), Motor Neuron
Disease
(MND), or Gaucher Disease (GD) by administering to a subject in need thereof
an isolated
nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic
acid) that encodes
PGRN (e.g. GRN).
In some embodiments, the disclosure relates to methods of treating
Synucleinopathies
(e.g., multiple system atrophy (MSA), Parkinson's Disease (PD), Parkinson's
disease with GBA1
mutation (PD-GBA), Dementia with Lewy Bodies (DLB), Dementia with Lewy Bodies
with
GBA1 mutation, and Lewy Body Disease) by administering to a subject in need
thereof an
isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated
nucleic acid) that
encodes GBA1 gene product, and an inhibitory nucleic acid targeting SNCA.
In some embodiments, the disclosure relates to methods of treating a disease
selected
from Parkinson's Disease (PD), Frontotemporal Dementia (e.g., Frontotemporal
Dementia with
GRN mutation (FTD-GRN)), Lysosomal Storage Diseases (LSDs), or Gaucher Disease
(GD) by
administering to a subject in need thereof an isolated nucleic acid (e.g., an
rAAV vector or
rAAV comprising an isolated nucleic acid) that encodes PSAP.
In some embodiments, the disclosure relates to methods of treating Alzheimer's
Disease
(AD), Nasu-Hakola Disease (NHD) or Parkinson's Disease (PD), by administering
to a subject
in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV
comprising an isolated
nucleic acid) that encodes TREM2.
In some embodiments, the disclosure relates to methods of treating Alzheimer's
disease
(AD) or Frontotemporal Dementia (Frontotemporal Dementia with MAPT mutation
(FTD-Tau),
Progressive supranuclear palsy (PSP), neurodegenerative disease, Lewy Body
Disease (LBD) or
Parkinson's Disease by administering to a subject in need thereof an isolated
nucleic acid (e.g.,
an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes
inhibitory nucleic
acids targeting MAPT.
In some aspects, the disclosure provides a method for treating a subject
having or
suspected of having Parkinson's disease, the method comprising administering
to the subject a
composition (e.g., a composition comprising an isolated nucleic acid or a
vector or a rAAV) as
described by the disclosure.
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In some embodiments, a composition comprises a nucleic acid (e.g., an rAAV
genome,
for example encapsidated by AAV capsid proteins) that encodes two or more gene
products
(e.g., CNS disease-associated gene products), for example 2, 3, 4, 5, or more
gene products
described in this application. In some embodiments, a composition comprises
two or more (e.g.,
2, 3, 4, 5, or more) different nucleic acids (e.g., two or more rAAV genomes,
for example
separately encapsidated by AAV capsid proteins), each encoding one or more
different gene
products. In some embodiments, two or more different compositions are
administered to a
subject, each composition comprising one or more nucleic acids encoding
different gene
products. In some embodiments, different gene products are operably linked to
the same
promoter type (e.g., the same promoter). In some embodiments, different gene
products are
operably linked to different promoters.
In some embodiments, administration comprises direct injection to the CNS of a
subject.
In some embodiments, direct injection is intracerebral injection,
intraparenchymal injection,
intrathecal injection, intra-cisterna manga injection, or any combination
thereof. In some
embodiments, direct injection to the CNS of a subject comprises convection
enhanced delivery
(CED).
In some embodiments, administration comprises peripheral injection. In some
embodiments, peripheral injection is intravenous injection.
In some aspects, the present disclosure provides a method for treating a
subject having or
suspected of having a central nervous system (CNS) disease, the method
comprising
administering to the subject an isolated nucleic acid comprising: (i) an
expression construct
comprising a transgene encoding one or more gene products listed in Table 1 or
an inhibitory
nucleic acid targeting a gene or gene product set forth in Table 1; and (ii)
two adeno-associated
virus (AAV) inverted terminal repeats (ITRs) flanking the expression
construct. In some
aspects, the present disclosure provides a method for treating a subject
having or suspected of
having a central nervous system (CNS) disease, the method comprising
administering to the
subject two or more types of isolated nucleic acids encoding different gene
products, where each
type of isolated nucleic acid comprises: (i) an expression construct
comprising a transgene
encoding one or more gene products listed in Table 1 or an inhibitory nucleic
acid targeting a
gene or gene product set forth in Table 1; and (ii) two adeno-associated virus
(AAV) inverted
terminal repeats (ITRs) flanking the expression construct.
In some embodiments, the transgene encodes one or more proteins selected from:
GBA1,
GBA2, PGRN (e.g., GRN), TREM2, PSAP, SCARB2, GALC, SMPD1, CTSB, RAB7L,
VPS35, GCH1, and IL34. In some embodiments, the transgene encoding one or more
gene
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products comprises a codon-optimized protein coding sequence. In some
embodiments, the
transgene encodes one or more inhibitory nucleic acids targeting SNCA, MAPT,
RPS25, and/or
TMEM106B.
In some embodiments, the AAV ITRs are AAV2 ITRs.
In some embodiments, the isolated nucleic acid is packaged into a recombinant
adeno-
associated virus (rAAV). In some embodiments, the rAAV comprises an AAV9
capsid protein.
In some embodiments, the subject is a mammal. In some embodiments, the subject
is a
human. In some embodiments, the CNS disease is a neurodegenerative disease,
synucleinopathy,
tauopathy, and/or lysosomal storage disease (LSD). In some embodiments, the
CNS disease is
listed in Table 2, Table 3, Table 4, or Table 5.
In some embodiments, the administration comprises direct injection to the CNS
of the
subject, optionally wherein the direct injection is intracerebral injection,
intraparenchymal
injection, intrathecal injection, intra-cisterna magna injection or any
combination thereof. In
some embodiments, the intra-cisterna magna injection is suboccipital injection
into the cisterna
magna. In some embodiments, the direct injection to the CNS of the subject
comprises
convection enhanced delivery (CED). In some embodiments, the administration
comprises
peripheral injection, optionally wherein the peripheral injection is
intravenous injection. In some
embodiments, the subject is administered about 1 x 1010 vg to about 1 x 1016
vg of the rAAV.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof).
FIG. 2 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and LIMP2 (SCARB2)
or a portion
thereof. The coding sequences of Gcase and LIMP2 are separated by an internal
ribosomal entry
site (IRES).
FIG. 3 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and LIMP2 (SCARB2)
or a portion
thereof. Expression of the coding sequences of Gcase and LIMP2 are each driven
by a separate
promoter.
FIG. 4 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof), LIMP2 (SCARB2) or
a portion
thereof, and an interfering RNA for a-Syn.
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FIG. 5 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof), Prosaposin (e.g.,
PSAP or a portion
thereof), and an interfering RNA for a-Syn.
FIG. 6 is a schematic depicting one embodiment of a vector comprising an
expression
.. construct encoding Gcase (e.g., GBA1 or a portion thereof) and Prosaposin
(e.g., PSAP or a
portion thereof). The coding sequences of Gcase and Prosaposin are separated
by an internal
ribosomal entry site (IRES).
FIG. 7 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding a Gcase (e.g., GBA1 or a portion thereof). In this
embodiment, the vector
comprises a CBA promoter element (CBA), consisting of four parts: the CMV
enhancer
(CMVe), CBA promoter (CBAp), Exon 1, and intron (int) to constitutively
express the codon
optimized coding sequence of human GBA1 . The 3' region also contains a WPRE
regulatory
element followed by a bGH polyA tail. Three transcriptional regulatory
activation sites are
included at the 5' end of the promoter region: TATA, RBS, and YY1. The
flanking ITRs allow
for the correct packaging of the intervening sequences. Two variants of the 5'
ITR sequence
(inset box) were evaluated; these have several nucleotide differences within
the 20-nucleotide
"D" region of wild-type AAV2 ITR. In some embodiments, an rAAV vector contains
the "D"
domain nucleotide sequence shown on the top line. In some embodiments, a rAAV
vector
comprises a mutant "D" domain (e.g., an "S" domain, with the nucleotide
changes shown on the
bottom line).
FIG. 8 is a schematic depicting one embodiment of the vector described in FIG.
6
FIG. 9 shows representative data for delivery of an rAAV comprising a
transgene
encoding a Gcase (e.g., GBA1 or a portion thereof) in a CBE mouse model of
Parkinson's
disease. Daily IP delivery of PBS vehicle, 25 mg/kg CBE, 37.5 mg/kg CBE, or 50
mg/kg CBE
(left to right) initiated at P8. Survival (top left) was checked two times a
day and weight (top
right) was checked daily. All groups started with n = 8. Behavior was assessed
by total distance
traveled in Open Field (bottom left) at P23 and latency to fall on Rotarod
(bottom middle) at
P24. Levels of the GCase substrates were analyzed in the cortex of mice in the
PBS and 25
mg/kg CBE treatment groups both with (Day 3) and without (Day 1) CBE
withdrawal.
Aggregate GluSph and GalSph levels (bottom right) are shown as pmol per mg wet
weight of
the tissue. Means are presented. Error bars are SEM. *p<0.05; **p<0.01;
***p<0.001, nominal
p-values for treatment groups by linear regression.
FIG. 10 is a schematic depicting one embodiment of a study design for maximal
rAAV
dose in a CBE mouse model. Briefly, rAAV was delivered by ICV injection at P3,
and daily
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CBE treatment was initiated at P8. Behavior was assessed in the Open Field and
Rotarod assays
at P24-25 and substrate levels were measured at P36 and P38.
FIG. 11 shows representative data for in-life assessment of maximal rAAV dose
in a
CBE mouse model. At P3, mice were treated with either excipient or 8.8e9 vg
rAAV-GBA1 via
ICV delivery. Daily IP delivery of either PBS or 25 mg/kg CBE was initiated at
P8. At the end
of the study, half the mice were sacrificed one day after their last CBE dose
at P36 (Day 1) while
the remaining half went through 3 days of CBE withdrawal before sacrifice at
P38 (Day3). All
treatment groups (excipient + PBS n = 8, rAAV-GBA1+ PBS n = 7, excipient + CBE
n = 8, and
variant + CBE n = 9) were weighed daily (top left), and the weight at P36 was
analyzed (top
right). Behavior was assessed by total distance traveled in Open Field at P23
(bottom left) and
latency to fall on Rotarod at P24 (bottom right), evaluated for each animal as
the median across
3 trials. Due to lethality, n = 7 for the excipient + CBE group for the
behavioral assays, while
n=8 for all other groups. Means across animals are presented. Error bars are
SEM. *p<0.05;
***p<0.001, nominal p-values for treatment groups by linear regression in the
CBE-treated
animals.
FIG. 12 shows representative data for biochemical assessment of maximal rAAV
dose in
a CBE mouse model. The cortex of all treatment groups (excipient + PBS n = 8,
variant + PBS
n = 7, excipient + CBE n = 7, and variant + CBE n = 9) was used to measure
GCase activity (top
left), GluSph levels (top right), GluCer levels (bottom left), and vector
genomes (bottom right)
in the groups before (Day 1) or after (Day 3) CBE withdrawal. Biodistribution
is shown as
vector genomes per 1 i.t.g of genomic DNA. Means are presented. Error bars are
SEM.
(*)p<0.1; **p<0.01; ***p<0.001, nominal p-values for treatment groups by
linear regression in
the CBE-treated animals, with collection days and gender corrected for as
covariates.
FIG. 13 shows representative data for behavioral and biochemical correlations
in a CBE
mouse model after administration of excipient + PBS, excipient + CBE, and
variant + CBE
treatment groups. Across treatment groups, performance on Rotarod was
negatively correlated
with GluCer accumulation (A, p=0.0012 by linear regression), and GluSph
accumulation was
negatively correlated with increased GCase activity (B, p=0.0086 by linear
regression).
FIG. 14 shows representative data for biodistribution of variant in a CBE
mouse model.
Presence of vector genomes was assessed in the liver, spleen, kidney, and
gonads for all
treatment groups (excipient + PBS n = 8, variant+ PBS n = 7, excipient + CBE n
= 7, and
variant+ CBE n = 9). Biodistribution is shown as vector genomes per 1 i.t.g of
genomic DNA.
Vector genome presence was quantified by quantitative PCR using a vector
reference standard
curve; genomic DNA concentration was evaluated by A260 optical density
measurement.
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Means are presented. Error bars are SEM. *p<0.05; **p<0.01; ***p<0.001,
nominal p-values
for treatment groups by linear regression in the CBE-treated animals, with
collection days and
gender corrected for as covariates.
FIG. 15 shows representative data for in-life assessment of rAAV dose ranging
in a CBE
mouse model. Mice received excipient or one of three different doses of rAAV-
GBA1 by ICV
delivery at P3: 3.2e9 vg, 1.0e10vg, or 3.2e10 vg. At P8, daily IP treatment of
25 mg/kg CBE
was initiated. Mice that received excipient and CBE or excipient and PBS
served as controls.
All treatment groups started with n = 10 (5M/5F) per group. All mice were
sacrificed one day
after their final CBE dose (P38-P40). All treatment groups were weighed daily,
and their weight
was analyzed at P36. Motor performance was assessed by latency to fall on
Rotarod at P24 and
latency to traverse the Tapered Beam at P30. Due to early lethality, the
number of mice
participating in the behavioral assays was: excipient + PBS n = 10, excipient
+ CBE n = 9, and
3.2e9 vg rAAV-GBA1+ CBE n =6, 1.0e10 vg rAAV-GBA1+ CBE n = 10, 3.2e10 vg rAAV-
GBA1+ CBE n =7. Means are presented. Error bars are SEM; * p<0.05; **p<0.01
for nominal
p-values by linear regression in the CBE-treated groups, with gender corrected
for as a covariate.
FIG. 16 shows representative data for biochemical assessment of rAAV dose
ranging in
a CBE mouse model. The cortex of all treatment groups (excipient + PBS n = 10,
excipient +
CBE n =9, and 3.2e9 vg rAAV-GBA1+ CBE n =6, 1.0e10 vg rAAV-GBA1+ CBE n = 10,
3.2e10 vg rAAV-GBA1+ CBE n = 7) was used to measure GCase activity, GluSph
levels,
GluCer levels, and vector genomes. GCase activity is shown as ng of GCase per
mg of total
protein. GluSph and GluCer levels are shown as pmol per mg wet weight of the
tissue.
Biodistribution is shown as vector genomes per 1 i.t.g of genomic DNA. Vector
genome
presence was quantified by quantitative PCR using a vector reference standard
curve; genomic
DNA concentration was evaluated by A260 optical density measurement. Vector
genome
.. presence was also measured in the liver (E). Means are presented. Error
bars are SEM.
**p<0.01; ***p<0.001 for nominal p-values by linear regression in the CBE-
treated groups,
with gender corrected for as a covariate.
FIG. 17 shows representative data for tapered beam analysis in maximal dose
rAAV-
GBA1 in a genetic mouse model. Motor performance of the treatment groups (WT +
excipient,
n = 5), 4L/PS-NA + excipient (n = 6), and 4L/PS-NA + rAAV-GBA1 (n = 5)) was
assayed by
Beam Walk 4 weeks post rAAV-GBA1 administration. The total slips and active
time are
shown as total over 5 trials on different beams. Speed and slips per speed are
shown as the
average over 5 trials on different beams. Means are presented. Error bars are
SEM.
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FIG. 18 shows representative data for in vitro expression of rAAV constructs
encoding
progranulin (PGRN) protein (also referred to as GRN protein). The left panel
shows a standard
curve of progranulin (PGRN) ELISA assay. The bottom panel shows a dose-
response of PGRN
expression measured by ELISA assay in cell lysates of HEK293T cells transduced
with rAAV.
MOI = multiplicity of infection (vector genomes per cell).
FIG. 19 shows representative data for in vitro expression of rAAV constructs
encoding
GBA1 in combination with Prosaposin (PSAP), SCARB2, and/or one or more
inhibitory nucleic
acids. Data indicate transfection of HEK293 cells with each construct resulted
in overexpression
of the transgenes of interest relative to mock transfected cells.
FIG. 20 is a schematic depicting an rAAV vectors comprising a "D" region
located on
the "outside" of the ITR (e.g., proximal to the terminus of the ITR relative
to the transgene insert
or expression construct) (top) and a wild-type rAAV vectors having ITRs on the
"inside" of the
vector (e.g., proximal to the transgene insert of the vector).
FIG. 21 a schematic depicting one embodiment of a vector comprising an
expression
construct encoding GBA2 or a portion thereof, and an interfering RNA for a-
Syn.
FIG. 22 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and
Galactosylceramidase (e.g.,
GALC or a portion thereof). Expression of the coding sequences of Gcase and
Galactosylceramidase are separated by a T2A self-cleaving peptide sequence.
FIG. 23 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and
Galactosylceramidase (e.g.,
GALC or a portion thereof). Expression of the coding sequences of Gcase and
Galactosylceramidase are separated by a T2A self-cleaving peptide sequence.
FIG. 24 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof), Cathepsin B (e.g.,
CTSB or a portion
thereof), and an interfering RNA for a-Syn. Expression of the coding sequences
of Gcase and
Cathepsin B are separated by a T2A self-cleaving peptide sequence.
FIG. 25 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof), Sphingomyelin
phosphodiesterase 1
(e.g., SMPD1 a portion thereof, and an interfering RNA for a-Syn.
FIG. 26 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and
Galactosylceramidase (e.g.,
GALC or a portion thereof). The coding sequences of Gcase and
Galactosylceramidase are
separated by an internal ribosomal entry site (IRES).
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FIG. 27 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and Cathepsin B
(e.g., CTSB or a
portion thereof). Expression of the coding sequences of Gcase and Cathepsin B
are each driven
by a separate promoter.
FIG. 28 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof), GCH1 (e.g., GCH1
or a portion
thereof), and an interfering RNA for a-Syn. The coding sequences of Gcase and
GCH1 are
separated by an T2A self-cleaving peptide sequence
FIG. 29 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof), RAB7L1 (e.g.,
RAB7L1 or a portion
thereof), and an interfering RNA for a-Syn . The coding sequences of Gcase and
RAB7L1 are
separated by an T2A self-cleaving peptide sequence.
FIG. 30 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof), GCH1 (e.g., GCH1
or a portion
thereof), and an interfering RNA for a-Syn. Expression of the coding sequences
of Gcase and
GCH1 are an internal ribosomal entry site (IRES).
FIG. 31 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding VPS35 (e.g., VPS35 or a portion thereof) and interfering
RNAs for a-Syn
and TMEM106B.
FIG. 32 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof), IL-34 (e.g., IL34
or a portion
thereof), and an interfering RNA for a-Syn. The coding sequences of Gcase and
IL-34 are
separated by T2A self-cleaving peptide sequence.
FIG. 33 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and IL-34 (e.g.,
IL34 or a portion
thereof). The coding sequences of Gcase and IL-34 are separated by an internal
ribosomal entry
site (RES).
FIG. 34 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and TREM2 (e.g.,
TREM2 or a
portion thereof). Expression of the coding sequences of Gcase and TREM2 are
each driven by a
separate promoter.
FIG. 35 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and IL-34 (e.g.,
IL34 or a portion
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thereof). Expression of the coding sequences of Gcase and IL-34 are each
driven by a separate
promoter.
FIGs. 36A-36B show representative data for overexpression of TREM2 and GBA1 in
HEK293 cells relative to control transduced cells, as measured by qPCR and
ELISA. FIG. 36A
shows data for overexpression of TREM2. FIG. 36B shows data for overexpression
of GBA1
from the same construct.
FIG. 37 shows representative data indicating successful silencing of SNCA in
vitro by
GFP reporter assay (top) and a-Syn assay (bottom).
FIG. 38 shows representative data indicating successful silencing of TMEM106B
in vitro
by GFP reporter assay (top) and a-Syn assay (bottom).
FIG. 39 is a schematic depicting one embodiments of a vector comprising an
expression
construct encoding PGRN (also referred to as GRN).
FIG. 40 shows data for transduction of HEK293 cells using rAAVs having ITRs
with
wild-type (circles) or alternative (e.g., "outside"; squares) placement of the
"D" sequence. The
rAAVs having ITRs placed on the "outside" were able to transduce cells as
efficiently as rAAVs
having wild-type ITRs.
FIG. 41 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof).
FIG. 42 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof).
FIG. 43 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and an interfering
RNA for a-Syn.
FIG. 44 is a schematic depicting one embodiments of a vector comprising an
expression
construct encoding PGRN (also referred to as GRN).
FIG. 45 is a schematic depicting one embodiments of a vector comprising an
expression
construct encoding PGRN (also referred to as GRN).
FIG. 46 is a schematic depicting one embodiments of a vector comprising an
expression
construct encoding PGRN (also referred to as GRN) and an interfering RNA for
microtubule-
associated protein tau (MAPT). The nucleic acid sequence of this vector is set
forth in SEQ ID
.. NO: 142.
FIG. 47 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and an interfering
RNA for a-Syn.
FIG. 48 is a schematic depicting one embodiments of a vector comprising an
expression
construct encoding PSAP.
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FIG. 49 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof).
FIG. 50 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and
Galactosylceramidase (e.g.,
GALC or a portion thereof).
FIG. 51 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding Gcase (e.g., GBA1 or a
portion thereof),
Prosaposin (e.g., PSAP or a portion thereof), and an interfering RNA for a-
Syn.
FIG. 52 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and an inhibitory
RNA targeting
SNCA.
FIG. 53 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding SNCA.
FIG. 54 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding an inhibitory RNA
targeting SNCA. The
inhibitory RNA is positioned within an intron between the promoter sequence
and the Gcase
encoding sequence.
FIG. 55 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding progranulin (PGRN, also
referred to as
GRN) and an inhibitory RNA targeting SNCA. The inhibitory RNA is positioned
within an
intron between the promoter sequence and the Gcase encoding sequence.
FIG. 56 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding Gcase (GBA1) and an
inhibitory RNA
targeting SNCA. The inhibitory RNA is positioned within an intron between the
promoter
sequence and the Gcase encoding sequence.
FIG. 57 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding Gcase (GBA1) and an
inhibitory RNA
targeting SNCA. The inhibitory RNA is positioned within an intron between the
promoter
sequence and the Gcase encoding sequence.
FIG. 58 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding Gcase (GBA1) and an
inhibitory RNA
targeting SNCA. The "D" sequence of the 3'ITR is positioned on the "outside"
of the vector.
FIG. 59 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding Gcase (GBA1) and an
inhibitory RNA
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targeting SNCA. The inhibitory RNA is positioned within an intron between the
promoter
sequence and the Gcase encoding sequence.
FIG. 60 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding Gcase (GBA1) and an
inhibitory RNA
targeting SNCA. The inhibitory RNA is positioned within an intron between the
promoter
sequence and the Gcase encoding sequence.
FIG. 61 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding Gcase (GBA1) and an
inhibitory RNA
targeting SNCA.
FIG. 62 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding Gcase (GBA1) and an
inhibitory RNA
targeting SNCA. The inhibitory RNA is positioned within an intron between the
promoter
sequence and the Gcase encoding sequence.
FIG. 63 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding Gcase (GBA1) and an
inhibitory RNA
targeting SNCA. The inhibitory RNA is positioned within an intron between the
promoter
sequence and the Gcase encoding sequence.
FIG. 64 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding Gcase (GBA1) and
progranulin (PGRN,
also referred to as GRN), and an inhibitory RNA targeting TMEM106B. The
inhibitory RNA is
positioned within an intron between the promoter sequence and the Gcase
encoding sequence.
FIG. 65 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding an inhibitory RNA
targeting RPS25.
FIG. 66 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding an inhibitory RNA
targeting RPS25.
FIG. 67 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding an inhibitory RNA
targeting MAPT.
FIG. 68 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding an inhibitory RNA
targeting MAPT.
FIG. 69 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding progranulin (PGRN, also
referred to as
GRN) and an inhibitory RNA targeting MAPT. The inhibitory RNA is positioned
within an
intron between the promoter sequence and the PGRN (also referred to as GRN)
encoding
sequence.
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FIG. 70 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding an inhibitory RNA
targeting MAPT.
FIG. 71 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding progranulin (PGRN, also
referred to as
GRN) and an inhibitory RNA targeting MAPT. The inhibitory RNA is positioned
within an
intron between the promoter sequence and the PGRN (also referred to as GRN)
encoding
sequence.
FIG. 72 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and an inhibitory
RNA targeting
SNCA. Nucleic acid sequence of this vector is set forth in SEQ ID NO: 141.
FIG. 73 is a schematic depicting one embodiment of a vector comprising an
expression
construct encoding Gcase (e.g., GBA1 or a portion thereof) and an inhibitory
RNA targeting
SNCA. Nucleic acid sequence of this vector is set forth in SEQ ID NO: 143.
FIG. 74 is a schematic depicting one embodiment of a plasmid comprising an
rAAV
vector that includes an expression construct encoding Gcase (GBA1) and
prosaposin (PSAP),
and an inhibitory RNA targeting SNCA. Nucleic acid sequence of this vector is
set forth in SEQ
ID NO: 144.
FIG. 75A-75C are charts showing MAPT knockdown in SY5Y Cells by RNA
interference. FIG. 75A shows that immunofluorescent stationing of the AAV
vectors using a
probe directed to BGHpA. FIG. 75B shows RT-PCR results of MAPT expression 3
and 7 days
post transduction. FIG. 75C shows the general information of the rAAV virus
stocks used for
transduction.
DETAILED DESCRIPTION
The disclosure is based, in part, on compositions and methods for expression
of
combinations of certain gene products (e.g., gene products associated with CNS
disease) in a
subject. A gene product can be a protein, a fragment (e.g., portion) of a
protein, an interfering
nucleic acid that inhibits a CNS disease-associated gene, etc. In some
embodiments, a gene
product is a protein or a protein fragment encoded by a CNS disease-associated
gene. In some
embodiments, a gene product is an interfering nucleic acid (e.g., shRNA,
siRNA, miRNA,
amiRNA, etc.) that inhibits a CNS disease-associated gene.
A CNS disease-associated gene refers to a gene encoding a gene product that is
genetically, biochemically or functionally associated with a CNS disease, such
as PD. For
example, individuals having mutations in the GBA1 gene (which encodes the
protein Gcase),
Li
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have been observed to be have an increased risk of developing PD compared to
individuals that
do not have a mutation in GBAL In another example, synucleinopathies (e.g.,
PD, etc.) are
associated with accumulation of protein aggregates comprising a-Synuclein (a-
Syn) protein;
accordingly, SNCA (which encodes a-Syn) is a PD-associated gene. In some
embodiments, an
expression cassette described herein encodes a wild-type or non-mutant form of
a CNS disease-
associated gene, for example a PD-associated gene (or coding sequence
thereof). Examples of
CNS diseases-associated genes (e.g., PD-associated genes, AD-associated genes,
FTD-
associated genes, etc.) are listed in Table 1.
Table 1: Examples of CNS disease-associated genes and gene products
Name Gene Function NCBI
Accession
No.
Lysosome membrane protein 2 SCARB21LIMP2 lysosomal receptor for
NP_005497.1
glucosylceramidase (Isoform
1),
(GBA targeting) NP
001191184.1
(Isoform 2)
Prosaposin PSAP precursor for saposins
AAH01503.1,
A, B, C, and D, which AAH07612.1,
localize to the lysosomal AAH04275.1,
compartment and AAA60303.1
facilitate the catabolism
of glycosphingolipids
with short
oligosaccharide groups
beta-Glucocerebrosidase GBA1 cleaves the beta-
NP_001005742.1
glucosidic linkage of (Isoform
1),
glucocerebroside NP
001165282.1
(Isoform 2),
NP_001165283.1
(Isoform 3)
Non-lysosomal GBA2 catalyzes the conversion
NP_065995.1
Glucosylceramidase of glucosylceramide to
(Isoform 1),
free glucose and NP
001317589.1
ceramide (Isoform 2)
Galactosylceramidase GALC removes galactose from
EAW81359.1
ceramide derivatives (Isoform
CRA_a),
EAW81360.1
(Isoform CRA_b),
EAW81362.1
(Isoform CRA_c)
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Sphingomyelin SMPD1 converts sphingomyelin EAW68726.1
phosphodiesterase 1 to ceramide (Isoform
CRA_a),
EAW68727.1
(Isoform CRA_b),
EAW68728.1
(Isoform CRA_c),
EAW68729.1
(Isoform CRA_d)
Cathepsin B CTSB thiol protease believed
AAC37547.1,
to participate in AAH95408.1,
intracellular degradation AAH10240.1
and turnover of proteins;
also implicated in tumor
invasion and metastasis
RAB7, member RAS oncogene RAB7L1 regulates vesicular AAH02585.1
family-like 1 transport
Vacuolar protein sorting- VPS35 component of
retromer NP_060676.2
associated protein 35 cargo-selective complex
GTP cyclohydrolase 1 GCH1 responsible for AAH25415.1
hydrolysis of guanosine
triphosphate to form
7. 8-dihydroneopterin
triphosphate
Interleukin 34 IL34 increases growth or AAH29804.1
survival of monocytes;
elicits activity by
binding the Colony
stimulating factor 1
receptor
Triggering receptor expressed on TREM2 forms a receptor AAF69824.1
myeloid cells 2 signaling complex with
the TYRO protein
tyrosine kinase binding
protein; functions in
immune response and
may be involved in
chronic inflammation
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Progranulin PGRN (also referred to as plays a
role in NP_002087.1
GRN) development,
inflammation, cell
proliferation and protein
homeostasis
alpha-Synuclein SNCA plays a role in
NP_001139527.1
maintaining a supply of
synaptic vesicles in
presynaptic terminals by
clustering synaptic
vesicles, and may help
regulate the release of
dopamine
Transmembrane protein 106B TMEM106B plays a role in dendrite
NP_060844.2
morphogenesis and
regulation of lysosomal
trafficking
Microtubule associated protein MAPT plays a
role in NP_005901.2
tau maintaining stability of
microtubules in axons
Isolated nucleic acids and vectors
An isolated nucleic acid may be DNA or RNA. As used herein, the term
"isolated"
means artificially produced. An "isolated nucleic acid", as used herein,
refers to nucleic acids (i)
amplified in vitro by, for example, polymerase chain reaction (PCR); (ii)
recombinantly
produced by cloning; (iii) purified, as by cleavage and gel separation; or
(iv) synthesized by, for
example, chemical synthesis. An isolated nucleic acid is one which is readily
manipulable by
recombinant DNA techniques well known in the art.
The disclosure provides, in some aspects, an isolated nucleic acids (e.g.,
rAAV vectors)
comprising an expression construct encoding one or more CNS disease-associated
genes (e.g.,
PD-associated genes), for example a Gcase, a Prosaposin, a LIMP2/SCARB2, a
GBA2, GALC
protein, a CTSB protein, a SMPD1, a GCH1 protein, a RAB7L protein, a VPS35
protein, a IL-
34 protein, a TREM2 protein, or a TMEM106B protein. The disclosure also
provides, in some
aspects, isolated nucleic acids (e.g., rAAV vectors) encoding one or more
inhibitory nucleic
acids that target one or more CNS disease-associated gene, for example SNCA,
TMEM106B,
RPS25, and MAPT. In some embodiments, the isolated nucleic acid encoding the
CNS disease-
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associated genes may further comprises coding sequences for inhibitory nucleic
acids targeting
one or more CNS disease-associated genes. In some embodiments, the CNS disease-
associated
genes and the inhibitory nucleic acids targeting CNS disease-associated genes
are encoded on
different nucleic acids.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding Gcase (e.g., the gene product of GBA1 gene).
Gcase, also referred
to as P-glucocerebrosidase or GBA, refers to a lysosomal protein that cleaves
the beta-glucosidic
linkage of the chemical glucocerebroside, an intermediate in glycolipid
metabolism. In humans,
Gcase is encoded by the GBA1 gene, located on chromosome 1. In some
embodiments, GBA1
encodes a peptide that is represented by NCBI Reference Sequence NCBI
Reference Sequence
NP 000148.2 (SEQ ID NO: 14). In some embodiments, an isolated nucleic acid
comprises a
Gcase-encoding sequence that has been codon optimized (e.g., codon optimized
for expression
in mammalian cells, for example human cells), such as the sequence set forth
in SEQ ID NO:
15.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding Prosaposin (e.g., the gene product of PSAP
gene). Prosaposin is a
precursor glycoprotein for sphingolipid activator proteins (saposins) A, B, C,
and D, which
facilitate the catabolism of glycosphingolipids with short oligosaccharide
groups. In humans,
the PSAP gene is located on chromosome 10. In some embodiments, PSAP encodes a
peptide
that is represented by NCBI Reference Sequence NP 002769.1 (e.g., SEQ ID NO:
16). In some
embodiments, an isolated nucleic acid comprises a prosaposin-encoding sequence
that has been
codon optimized (e.g., codon optimized for expression in mammalian cells, for
example human
cells), such as the sequence set forth in SEQ ID NO: 17.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding LIMP2/SCARB2 (e.g., the gene product of SCARB2 gene).
SCARB2 refers
to a membrane protein that regulates lysosomal and endosomal transport within
a cell. In
humans, SCARB2 gene is located on chromosome 4. In some embodiments, the
SCARB2 gene
encodes a peptide that is represented by NCBI Reference Sequence NP 005497.1
(SEQ ID NO:
18). In some embodiments, an isolated nucleic acid comprises the sequence set
forth in SEQ ID
NO: 19. In some embodiments the isolated nucleic acid comprises a SCARB2-
encoding
sequence that has been codon optimized.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding GBA2 protein (e.g., the gene product of GBA2 gene). GBA2
protein refers
to non-lysosomal glucosylceramidase. In humans, GBA2 gene is located on
chromosome 9. In
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some embodiments, the GBA2 gene encodes a peptide that is represented by NCBI
Reference
Sequence NP 065995.1 (SEQ ID NO: 30). In some embodiments, an isolated nucleic
acid
comprises the sequence set forth in SEQ ID NO: 31. In some embodiments the
isolated nucleic
acid comprises a GBA2-encoding sequence that has been codon optimized.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding GALC protein (e.g., the gene product of GALC gene). GALC
protein refers
to galactosylceramidase (or galactocerebrosidase), which is an enzyme that
hydrolyzes galactose
ester bonds of galactocerebroside, galactosylsphingosine, lactosylceramide,
and
monogalactosyldiglyceride. In humans, GALC gene is located on chromosome 14.
In some
.. embodiments, the GALC gene encodes a peptide that is represented by NCBI
Reference
Sequence NP 000144.2 (SEQ ID NO: 33). In some embodiments, an isolated nucleic
acid
comprises the sequence set forth in SEQ ID NO: 34. In some embodiments the
isolated nucleic
acid comprises a GALC-encoding sequence that has been codon optimized.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding CTSB protein (e.g., the gene product of CTSB gene). CTSB
protein refers to
cathepsin B, which is a lysosomal cysteine protease that plays an important
role in intracellular
proteolysis. In humans, CTSB gene is located on chromosome 8. In some
embodiments, the
CTSB gene encodes a peptide that is represented by NCBI Reference Sequence NP
001899.1
(SEQ ID NO: 35). In some embodiments, an isolated nucleic acid comprises the
sequence set
forth in SEQ ID NO: 36. In some embodiments the isolated nucleic acid
comprises a CTSB-
encoding sequence that has been codon optimized.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding SMPD1 protein (e.g., the gene product of SMPD1 gene). SMPD1
protein
refers to sphingomyelin phosphodiesterase 1, which is a hydrolase enzyme that
is involved in
sphingolipid metabolism. In humans, SMPD1 gene is located on chromosome 11. In
some
embodiments, the SMPD1 gene encodes a peptide that is represented by NCBI
Reference
Sequence NP 000534.3 (SEQ ID NO: 37). In some embodiments, an isolated nucleic
acid
comprises the sequence set forth in SEQ ID NO: 38. In some embodiments the
isolated nucleic
acid comprises a SMPD1-encoding sequence that has been codon optimized.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding GCH1 protein (e.g., the gene product of GCH1 gene). GCH1
protein refers
to GTP cyclohydrolase I, which is a hydrolase enzyme that is part of the
folate and biopterin
biosynthesis pathways. In humans, GCH1 gene is located on chromosome 14. In
some
embodiments, the GCH1 gene encodes a peptide that is represented by NCBI
Reference
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Sequence NP 000152.1 (SEQ ID NO: 45). In some embodiments, an isolated nucleic
acid
comprises the sequence set forth in SEQ ID NO: 46. In some embodiments the
isolated nucleic
acid comprises a GCH1-encoding sequence that has been codon optimized.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding RAB7L protein (e.g., the gene product of RAB7L gene). RAB7L
protein
refers to RAB7, member RAS oncogene family-like 1, which is a GTP binding
protein. In
humans, RAB7L gene is located on chromosome 1. In some embodiments, the RAB7L
gene
encodes a peptide that is represented by NCBI Reference Sequence NP 003920.1
(SEQ ID NO:
47). In some embodiments, an isolated nucleic acid comprises the sequence set
forth in SEQ ID
NO: 48. In some embodiments the isolated nucleic acid comprises a RAB7L-
encoding sequence
that has been codon optimized.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding VP535 protein (e.g., the gene product of VPS35 gene). VP535
protein refers
to vacuolar protein sorting-associated protein 35, which is part of a protein
complex involved in
retrograde transport of proteins from endosomes to the trans-Golgi network. In
humans, VPS35
gene is located on chromosome 16. In some embodiments, the VPS35 gene encodes
a peptide
that is represented by NCBI Reference Sequence NP 060676.2 (SEQ ID NO: 49). In
some
embodiments, an isolated nucleic acid comprises the sequence set forth in SEQ
ID NO: 50. In
some embodiments the isolated nucleic acid comprises a VP535-encoding sequence
that has
been codon optimized.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding IL-34 protein (e.g., the gene product of IL34 gene). IL-34
protein refers to
interleukin 34, which is a cytokine that increases growth and survival of
monocytes. In humans,
IL34 gene is located on chromosome 16. In some embodiments, the IL34 gene
encodes a
peptide that is represented by NCBI Reference Sequence NP 689669.2 (SEQ ID NO:
55). In
some embodiments, an isolated nucleic acid comprises the sequence set forth in
SEQ ID NO: 56.
In some embodiments the isolated nucleic acid comprises a IL-34-encoding
sequence that has
been codon optimized.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding TREM2 protein (e.g., the gene product of TREM2 gene). TREM2
protein
refers to triggering receptor expressed on myeloid cells 2, which is an
immunoglobulin
superfamily receptor found on myeloid cells. In humans, TREM2 gene is located
on
chromosome 6. In some embodiments, the TREM2 gene encodes a peptide that is
represented
by NCBI Reference Sequence NP 061838.1 (SEQ ID NO: 57). In some embodiments,
the
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isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 58. In
some embodiments
an isolated nucleic acid comprises a TREM2-encoding sequence that has been
codon optimized.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding TMEM106B protein (e.g., the gene product of TMEM106B gene).
TMEM106B protein refers to transmembrane protein 106B, which is a protein
involved in
dendrite morphogenesis and regulation of lysosomal trafficking. In humans,
TMEM106B gene
is located on chromosome 7. In some embodiments, the TMEM106B gene encodes a
peptide
that is represented by NCBI Reference Sequence NP 060844.2 (SEQ ID NO: 63). In
some
embodiments, an isolated nucleic acid comprises the sequence set forth in SEQ
ID NO: 64. In
some embodiments the isolated nucleic acid comprises a TMEM106B-encoding
sequence that
has been codon optimized.
Aspects of the disclosure relate to an isolated nucleic acid comprising an
expression
construct encoding progranulin protein (e.g., the gene product of GRN gene).
PGRN protein
refers to progranulin, which is a protein involved in development,
inflammation, cell
proliferation and protein homeostasis. In humans, PGRN (also referred to as
GRN) gene is
located on chromosome 17. In some embodiments, the GRN gene encodes a peptide
that is
represented by NCBI Reference Sequence NP 002078.1 (SEQ ID NO: 67). In some
embodiments, an isolated nucleic acid comprises the sequence set forth in SEQ
ID NO: 68. In
some embodiments the isolated nucleic acid comprises a PGRN-encoding sequence
(GRN-
encoding sequence) that has been codon optimized.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding a first gene product and a second gene product,
wherein each
gene product independently is selected from the gene products, or portions
thereof, set forth in
Table 1 or an inhibitory nucleic acid targeting a gene or gene product set
forth in Table 1.
In some embodiments, a gene product is encoded by a coding portion (e.g., a
cDNA) of a
naturally occurring gene. In some embodiments, a first gene product is a
protein (or a fragment
thereof) encoded by the GBA1 gene. In some embodiments, a gene product is a
protein (or a
fragment thereof) encoded by another gene listed in Table 1, for example the
SCARB2ILIMP2
gene or the PSAP gene. However, the skilled artisan recognizes that the order
of expression of a
first gene product (e.g., Gcase) and a second gene product (e.g., LIMP2, etc.)
can generally be
reversed (e.g., LIMP2 is the first gene product and Gcase is the second gene
product). In some
embodiments, a gene product is a fragment (e.g., portion) of a gene listed in
Table 1. A protein
fragment may comprise about 50%, about 60%, about 70%, about 80% about 90% or
about 99%
of a protein encoded by the genes listed in Table 1. In some embodiments, a
protein fragment
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comprises between 50% and 99.9% (e.g., any value between 50% and 99.9%) of a
protein
encoded by a gene listed in Table 1.
Pathologically, disorders such as PD and Gaucher disease are associated with
accumulation of protein aggregates composed largely of a-Synuclein (a-Syn)
protein.
Accordingly, in some embodiments, isolated nucleic acids described herein
comprise an
inhibitory nucleic acid that reduces or prevents expression of a-Syn protein.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding one or more interfering nucleic acids (e.g.,
dsRNA, siRNA,
miRNA, amiRNA, etc.) that target an microtubule-associated protein tau, MAPT
(e.g., the gene
product of MAPT gene), which is involved in Alzheimer's disease and FTD-tau.
Generally, an isolated nucleic acid as described herein may encode 1, 2, 3, 4,
5, 6, 7, 8, 9,
10, or more inhibitory nucleic acids (e.g., dsRNA, siRNA, shRNA, miRNA,
amiRNA, etc.). In
some embodiments, an isolated nucleic acid encodes more than 10 inhibitory
nucleic acids. In
some embodiments, each of the one or more inhibitory nucleic acids targets a
different gene or a
portion of a gene (e.g., a first miRNA targets a first target sequence of a
gene and a second
miRNA targets a second target sequence of the gene that is different than the
first target
sequence). In some embodiments, each of the one or more inhibitory nucleic
acids targets the
same target sequence of the same gene (e.g., an isolated nucleic acid encodes
multiple copies of
the same miRNA).
In some aspects, the disclosure provides relate to an isolated nucleic acid
comprising an
expression construct encoding one or more interfering nucleic acids (e.g.,
dsRNA, siRNA,
miRNA, amiRNA, etc.) that target an a-Synuclein protein (e.g., the gene
product of SNCA
gene). a-Synuclein protein refers to a protein found in brain tissue, which is
plays a role in
maintaining a supply of synaptic vesicles in presynaptic terminals by
clustering synaptic vesicles
and regulating the release of dopamine. In humans, SNCA gene is located on
chromosome 4. In
some embodiments, the SNCA gene encodes a peptide that is represented by NCBI
Reference
Sequence NP 001139527.1. In some embodiments, a SNCA gene comprises the
sequence set
forth in SEQ ID NO: 90.
An inhibitory nucleic acid targeting SNCA may comprise a region of
complementarity
(e.g., a region of the inhibitory nucleic acid that hybridizes to the target
gene, such as SNCA)
that is between 6 and 50 nucleotides in length. In some embodiments, an
inhibitory nucleic acid
comprises a region of complementarity with SNCA that is between about 6 and
30, about 8 and
20, or about 10 and 19 nucleotides in length. In some embodiments, an
inhibitory nucleic acid is
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complementary with at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22,
23, 24, or 25 contiguous nucleotides of a SNCA sequence.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding one or more interfering nucleic acids (e.g.,
dsRNA, siRNA,
miRNA, amiRNA, etc.) that target an TMEM106B protein (e.g., the gene product
of
TMEM106B gene). TMEM106B protein refers to transmembrane protein 106B, which
is a
protein involved in dendrite morphogenesis and regulation of lysosomal
trafficking. In humans,
TMEM106B gene is located on chromosome 7. In some embodiments, the TMEM106B
gene
encodes a peptide that is represented by NCBI Reference Sequence NP 060844.2.
In some
embodiments, a TMEM106B gene comprises the sequence set forth in SEQ ID NO:
91.
An inhibitory nucleic acid targeting TMEM106B may comprise a region of
complementarity (e.g., a region of the inhibitory nucleic acid that hybridizes
to the target gene,
such as TMEM106B) that is between 6 and 50 nucleotides in length. In some
embodiments, an
inhibitory nucleic acid comprises a region of complementarity with TMEM106B
that is between
about 6 and 30, about 8 and 20, or about 10 and 19 nucleotides in length. In
some embodiments,
an inhibitory nucleic acid is complementary with at least 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a TMEM106B
sequence.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding one or more interfering nucleic acids (e.g.,
dsRNA, siRNA,
miRNA, amiRNA, etc.) that target an ribosomal protein s25 (RP525) (e.g., the
gene product of
RPS25). RP525 protein refers to a ribosomal protein which is a subunit of the
s40 ribosome, a
protein complex involved in protein synthesis. In humans, RPS25 gene is
located on
chromosome 11. In some embodiments, the RPS25 gene encodes a peptide that is
represented
by NCBI Reference Sequence NP 001019.1. In some embodiments, a RPS25 gene
comprises
the sequence set forth in SEQ ID NO: 113.
An inhibitory nucleic acid targeting RPS25 may comprise a region of
complementarity
(e.g., a region of the inhibitory nucleic acid that hybridizes to the target
gene, such as RPS25)
that is between 6 and 50 nucleotides in length. In some embodiments, an
inhibitory nucleic acid
comprises a region of complementarity with RPS25 that is between about 6 and
30, about 8 and
20, or about 10 and 19 nucleotides in length. In some embodiments, an
inhibitory nucleic acid is
complementary with at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22,
23, 24, or 25 contiguous nucleotides of a RPS25 sequence.
In some aspects, the disclosure provides an isolated nucleic acid comprising
an
expression construct encoding one or more interfering nucleic acids (e.g.,
dsRNA, siRNA,
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miRNA, amiRNA, etc.) that target an microtubule-associated protein tau, MAPT
(e.g., the gene
product of MAPT gene). MAPT protein refers to microtubule-associated protein
tau, which is a
protein involved in microtubule stabilization. In humans, MAPT gene is located
on chromosome
17. In some embodiments, the MAPT gene encodes a peptide that is represented
by NCBI
Reference Sequence NP 005901.2. In some embodiments, a MAPT gene comprises the
sequence set forth in SEQ ID NO: 114.
An inhibitory nucleic acid targeting MAPT may comprise a region of
complementarity
(e.g., a region of the inhibitory nucleic acid that hybridizes to the target
gene, such as MAPT)
that is between 6 and 50 nucleotides in length. In some embodiments, an
inhibitory nucleic acid
comprises a region of complementarity with MAPT that is between about 6 and
30, about 8 and
20, or about 10 and 19 nucleotides in length. In some embodiments, an
inhibitory nucleic acid is
complementary with at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22,
23, 24, or 25 contiguous nucleotides of a MAPT sequence.
Aspects of the disclosure relate to isolated nucleic acids encoding one or
more gene
products (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more gene products). In some
embodiments, the one
or more gene products are two or more proteins. In some embodiments, the one
or more gene
products are two or more inhibitory nucleic acids. In some embodiments, the
one or more gene
products are one or more protein and one or more inhibitory nucleic acid. In
some aspects, the
disclosure provides an isolated nucleic acid comprising an expression
construct encoding a first
gene product and a second gene product, wherein each gene product
independently is selected
from the gene products, or portions thereof, set forth in Table 1 or an
inhibitory nucleic acid
targeting a gene or gene product set forth in Table 1. A sequence encoding an
inhibitory nucleic
acid may be placed in an untranslated region (e.g., intron, 5'UTR, 3'UTR,
etc.) of the expression
vector.
In some embodiments, a gene product is encoded by a coding portion (e.g., a
cDNA) of a
naturally occurring gene. In some embodiments, a first gene product is a
protein (or a fragment
thereof) encoded by the GBA1 gene. In some embodiments, a gene product is an
inhibitory
nucleic acid that targets (e.g., hybridizes to, or comprises a region of
complementarity with) a
PD-associated gene (e.g., SNCA). A skilled artisan recognizes that the order
of expression of a
first gene product (e.g., Gcase) and a second gene product (e.g., inhibitory
RNA targeting
SNCA) can generally be reversed (e.g., the inhibitory RNA is the first gene
product and Gcase is
the second gene product). In some embodiments, a gene product is a fragment
(e.g., portion) of
a gene listed in Table 1. A protein fragment may comprise about 50%, about
60%, about 70%,
about 80% about 90% or about 99% of a protein encoded by the genes listed in
Table 1. In
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some embodiments, a protein fragment comprises between 50% and 99.9% (e.g.,
any value
between 50% and 99.9%) of a protein encoded by a gene listed in Table 1. In
some
embodiments, a gene product (e.g., an inhibitory RNA) hybridizes to portion of
a target gene
(e.g., is complementary to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, or more
contiguous nucleotides of a target gene, for example SNCA),In some
embodiments, an
expression construct is monocistronic (e.g., the expression construct encodes
a single fusion
protein comprising a first gene product and a second gene product). In some
embodiments, an
expression construct is polycistronic (e.g., the expression construct encodes
two distinct gene
products, for example two different proteins or protein fragments).
A polycistronic expression vector may comprise a one or more (e.g., 1, 2, 3,
4, 5, or
more) promoters. Any suitable promoter can be used, for example, a
constitutive promoter, an
inducible promoter, an endogenous promoter, a tissue-specific promoter (e.g.,
a CNS- specific
promoter), etc. In some embodiments, a promoter is a chicken beta-actin
promoter (CBA
promoter), a CAG promoter (for example as described by Alexopoulou et al.
(2008) BMC Cell
Biol. 9:2; doi: 10.1186/1471-2121-9-2), a CD68 promoter, or a JeT promoter
(for example as
described by Tornoe et al. (2002) Gene 297(1-2):21-32). In some embodiments, a
promoter is
operably-linked to a nucleic acid sequence encoding a first gene product, a
second gene product,
or a first gene product and a second gene product. In some embodiments, an
expression cassette
comprises one or more additional regulatory sequences, including but not
limited to transcription
factor binding sequences, intron splice sites, poly(A) addition sites,
enhancer sequences,
repressor binding sites, or any combination of the foregoing.
In some embodiments, a nucleic acid sequence encoding a first gene product and
a
nucleic acid sequence encoding a second gene product are separated by a
nucleic acid sequence
encoding an internal ribosomal entry site (IRES). Examples of IRES sites are
described, for
example, by Mokrejs et al. (2006) Nucleic Acids Res. 34(Database issue):D125-
30. In some
embodiments, a nucleic acid sequence encoding a first gene product and a
nucleic acid sequence
encoding a second gene product are separated by a nucleic acid sequence
encoding a self-
cleaving peptide. Examples of self-cleaving peptides include but are not
limited to T2A, P2A,
E2A, F2A, BmCPV 2A, and BmIFV 2A, and those described by Liu et al. (2017) Sci
Rep. 7:
2193. In some embodiments, the self-cleaving peptide is a T2A peptide.
In some embodiments, an inhibitory nucleic acid is positioned in an intron of
an
expression construct, for example in an intron upstream of the sequence
encoding a first gene
product. An inhibitory nucleic acid can be a double stranded RNA (dsRNA),
siRNA, micro
RNA (miRNA), artificial miRNA (amiRNA), or an RNA aptamer. Generally, an
inhibitory
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nucleic acid binds to (e.g., hybridizes with) between about 6 and about 30
(e.g., any integer
between 6 and 30, inclusive) contiguous nucleotides of a target RNA (e.g.,
mRNA). In some
embodiments, the inhibitory nucleic acid molecule is an miRNA or an amiRNA,
for example an
miRNA that targets SNCA (the gene encoding a-Syn protein) or TMEM106B (e.g..
the gene
encoding TMEM106B protein). In some embodiments, the miRNA does not comprise
any
mismatches with the region of SNCA mRNA to which it hybridizes (e.g., the
miRNA is
"perfected"). In some embodiments, the inhibitory nucleic acid is an shRNA
(e.g., an shRNA
targeting SNCA or TMEM106B). In some embodiments, an inhibitory nucleic acid
is an
artificial miRNA (amiRNA) that includes a miR-155 scaffold and a SNCA or
TMEM106B
targeting sequence.
In some embodiments, an inhibitory nucleic acid is an artificial microRNA
(amiRNA).
A microRNA (miRNA) typically refers to a small, non-coding RNA found in plants
and animals
and functions in transcriptional and post-translational regulation of gene
expression. MiRNAs
are transcribed by RNA polymerase to form a hairpin-loop structure referred to
as a pri-miRNAs
which are subsequently processed by enzymes (e.g., Drosha, Pasha, spliceosome,
etc.) to for a
pre-miRNA hairpin structure which is then processed by Dicer to form a
miRNA/miRNA*
duplex (where * indicates the passenger strand of the miRNA duplex), one
strand of which is
then incorporated into an RNA-induced silencing complex (RISC). In some
embodiments, an
inhibitory RNA as described herein is a miRNA targeting SNCA or TMEM106B.
In some embodiments, an inhibitory nucleic acid targeting SNCA comprises a
miRNA/miRNA* duplex. In some embodiments, the miRNA strand of a miRNA/miRNA*
duplex comprises or consists of the sequence set forth in any one of SEQ ID
NOs: 20-25. In
some embodiments, the miRNA* strand of a miRNA/miRNA* duplex comprises or
consists of
the sequence set forth in any one of SEQ ID NOs: 20-25.
In some embodiments, an inhibitory nucleic acid targeting TMEM106B comprises a
miRNA/miRNA* duplex. In some embodiments, the miRNA strand of a miRNA/miRNA*
duplex comprises or consists of the sequence set forth in SEQ ID NO: 92 or 93.
In some
embodiments, the miRNA* strand of a miRNA/miRNA* duplex comprises or consists
of the
sequence set forth in SEQ ID NOs: 92 or 93.
An artificial microRNA (amiRNA) is derived by modifying native miRNA to
replace
natural targeting regions of pre-mRNA with a targeting region of interest. For
example, a
naturally occurring, expressed miRNA can be used as a scaffold or backbone
(e.g., a pri-miRNA
scaffold), with the stem sequence replaced by that of an miRNA targeting a
gene of interest. An
artificial precursor microRNA (pre-amiRNA) is normally processed such that one
single stable
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small RNA is preferentially generated. In some embodiments, scAAV vectors and
scAAVs
described herein comprise a nucleic acid encoding an amiRNA. In some
embodiments, the pri-
miRNA scaffold of the amiRNA is derived from a pri-miRNA selected from the
group
consisting of pri-MIR-21, pri-MIR-22, pri-MIR-26a, pri-MIR-30a, pri-MIR-33,
pri-MIR-122,
pri-MIR-375, pri-MIR-199, pri-MIR-99, pri-MIR-194, pri-MIR-155, and pri-MIR-
451. In some
embodiments, an amiRNA comprises a nucleic acid sequence targeting SNCA or
TMEM106B
and an eSIBR amiRNA scaffold, for example as described in Fowler et al.
Nucleic Acids Res.
2016 Mar 18; 44(5): e48.
In some embodiments, an amiRNA targeting SNCA comprises or consists of the
sequence set forth in any one of SEQ ID NOs: 94-99. In some embodiments, an
amiRNA
targeting TMEM106B comprises or consists of the sequence set forth in SEQ ID
NOs: 65-66. In
some embodiments, an amiRNA targeting RP525 comprises or consists of the
sequence set forth
in SEQ ID NOs: 115 to 122. In some embodiments, an amiRNA targeting MAPT
comprises or
consists of the sequence set forth in SEQ ID NOs: 123-138.
In some embodiments, an isolated nucleic acid or vector (e.g., rAAV vector)
described
by the disclosure comprises or consists of a sequence set forth in any one of
SEQ ID NOs: 1-13,
15, 17, 19-29, 31, 32, 34, 36, 38-44, 46, 48, 50-54, 56, 58-62, 64-66, and 68-
145. In some
embodiments, an isolated nucleic acid or vector (e.g., rAAV vector) described
by the disclosure
comprises or consists of a sequence that is complementary (e.g., the
complement of) a sequence
set forth in any one of SEQ ID NOs: 1-13, 15, 17, 19-29, 31, 32, 34, 36, 38-
44, 46, 48, 50-54,
56, 58-62, 64-66, and 68-145. In some embodiments, an isolated nucleic acid or
vector (e.g.,
rAAV vector) described by the disclosure comprises or consists of a sequence
that is a reverse
complement of a sequence set forth in any one of SEQ ID NOs: 1-13, 15, 17, 19-
29, 31, 32, 34,
36, 38-44, 46, 48, 50-54, 56, 58-62, 64-66, and 68-145. In some embodiments,
an isolated
nucleic acid or vector (e.g., rAAV vector) described by the disclosure
comprises or consists of a
portion of a sequence set forth in any one of SEQ ID NOs: 1-13, 15, 17, 19-29,
31, 32, 34, 36,
38-44, 46, 48, 50-54, 56, 58-62, 64-66, and 68-145. A portion may comprise at
least 25%, 50%,
60%, 70%, 80%, 90%, 95%, or 99% of a sequence set forth in any one of SEQ ID
NOs: 1-13,
15, 17, 19-29, 31, 32, 34, 36, 38-44, 46, 48, 50-54, 56, 58-62, 64-66, and 68-
145. In some
embodiments, a nucleic acid sequence described by the disclosure is a nucleic
acid sense strand
(e.g., 5' to 3' strand), or in the context of a viral sequences a plus (+)
strand. In some
embodiments, a nucleic acid sequence described by the disclosure is a nucleic
acid antisense
strand (e.g., 3' to 5' strand), or in the context of viral sequences a minus (-
) strand.
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The skilled artisan recognizes that when referring to nucleic acid sequences
comprising
or encoding inhibitory nucleic acids (e.g., dsRNA, siRNA, miRNA, amiRNA, etc.)
any one or
more thymidine (T) nucleotides or uridine (U) nucleotides in a sequence
provided herein may be
replaced with any other nucleotide suitable for base pairing (e.g., via a
Watson-Crick base pair)
with an adenosine nucleotide. For example, T may be replaced with U, and U may
be replaced
with T.
An isolated nucleic acid as described herein may exist on its own, or as part
of a vector.
Generally, a vector can be a plasmid, cosmid, phagemid, bacterial artificial
chromosome (BAC),
or a viral vector (e.g., adenoviral vector, adeno-associated virus (AAV)
vector, retroviral vector,
baculoviral vector, etc.). In some embodiments, the vector is a plasmid (e.g.,
a plasmid
comprising an isolated nucleic acid as described herein). In some embodiments,
an rAAV
vector is single-stranded (e.g., single-stranded DNA). In some embodiments,
the vector is a
recombinant AAV (rAAV) vector. In some embodiments, a vector is a Baculovirus
vector (e.g.,
an Autographa califomica nuclear polyhedrosis (AcNPV) vector).
Typically an rAAV vector (e.g., rAAV genome) comprises a transgene (e.g., an
expression construct comprising one or more of each of the following:
promoter, intron,
enhancer sequence, protein coding sequence, inhibitory RNA coding sequence,
polyA tail
sequence, etc.) flanked by two AAV inverted terminal repeat (ITR) sequences.
In some
embodiments the transgene of an rAAV vector comprises an isolated nucleic acid
as described
by the disclosure. In some embodiments, each of the two ITR sequences of an
rAAV vector is a
full-length ITR (e.g., approximately 145 bp in length, and containing
functional Rep binding site
(RBS) and terminal resolution site (trs)). In some embodiments, one of the
ITRs of an rAAV
vector is truncated (e.g., shortened or not full-length). In some embodiments,
a truncated ITR
lacks a functional terminal resolution site (trs) and is used for production
of self-complementary
AAV vectors (scAAV vectors). In some embodiments, a truncated ITR is a AITR,
for example
as described by McCarty et al. (2003) Gene Ther. 10(26):2112-8.
Aspects of the disclosure relate to isolated nucleic acids (e.g., rAAV
vectors) comprising
an ITR having one or more modifications (e.g., nucleic acid additions,
deletions, substitutions,
etc.) relative to a wild-type AAV ITR, for example relative to wild-type AAV2
ITR (e.g., SEQ
ID NO: 29). The structure of wild-type AAV2 ITR is shown in FIG. 20.
Generally, a wild-type
ITR comprises a 125 nucleotide region that self-anneals to form a palindromic
double-stranded
T-shaped, hairpin structure consisting of two cross arms (formed by sequences
referred to as
B/B' and C/C', respectively), a longer stem region (formed by sequences A/A'),
and a single-
stranded terminal region referred to as the "D" region (FIG. 20). Generally,
the "D" region of an
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ITR is positioned between the stem region formed by the A/A' sequences and the
insert
containing the transgene of the rAAV vector (e.g., positioned on the "inside"
of the ITR relative
to the terminus of the ITR or proximal to the transgene insert or expression
construct of the
rAAV vector). In some embodiments, a "D" region comprises the sequence set
forth in SEQ ID
NO: 27. The "D" region has been observed to play an important role in
encapsidation of rAAV
vectors by capsid proteins, for example as disclosed by Ling et al. (2015) J
Mol Genet Med 9(3).
The disclosure is based, in part, on that rAAV vectors comprising a "D" region
located
on the "outside" of the ITR (e.g., proximal to the terminus of the ITR
relative to the transgene
insert or expression construct) are efficiently encapsidated by AAV capsid
proteins than rAAV
vectors having ITRs with unmodified (e.g., wild-type) ITRs In some
embodiments, rAAV
vectors having a modified "D" sequence (e.g., a "D" sequence in the "outside"
position) have
reduced toxicity relative to rAAV vectors having wild-type ITR sequences.
In some embodiments, a modified "D" sequence comprises at least one nucleotide
substitution relative to a wild-type "D" sequence (e.g., SEQ ID NO: 27). A
modified "D"
sequence may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10
nucleotide substitutions
relative to a wild-type "D" sequence (e.g., SEQ ID NO: 27). In some
embodiments, a modified
"D" sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19
nucleic acid
substitutions relative to a wild-type "D" sequence (e.g., SEQ ID NO: 27). In
some
embodiments, a modified "D" sequence is between about 10% and about 99% (e.g.,
10%, 15%,
20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
99%)
identical to a wild-type "D" sequence (e.g., SEQ ID NO: 27). In some
embodiments, a modified
"D" sequence comprises the sequence set forth in SEQ ID NO: 26, also referred
to as an "S"
sequence as described in Wang et al. (1995) J Mol Biol 250(5):573-80.
An isolated nucleic acid or rAAV vector as described by the disclosure may
further
comprise a "TRY" sequence, for example as set forth in SEQ ID NO: 28 or as
described by
Francois, et al. 2005. The Cellular TATA Binding Protein Is Required for Rep-
Dependent
Replication of a Minimal Adeno-Associated Virus Type 2 p5 Element. J Virol. In
some
embodiments, a TRY sequence is positioned between an ITR (e.g. a 5' ITR) and
an expression
construct (e.g. a transgene-encoding insert) of an isolated nucleic acid or
rAAV vector.
Aspects of the disclosure relate to constructs which are configured to express
one or
more transgenes in myeloid cells (e.g., CNS myeloid cells, such as microglia)
of a subject.
Thus, in some embodiments, a construct (e.g., gene expression vector)
comprises a protein
coding sequence that is operably linked to a myeloid cell-specific promoter.
Examples of
myeloid cell-specific promoters include CD68 promoter, lysM promoter, csflr
promoter, CD11c
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promoter, c-fes promoter, and F4/80 promoter, for example as described in Lin
et al. Adv Exp
Med Biol. 2010;706:149-56. In some embodiments, a myeloid cell-specific
promoter is a CD68
promoter or a F4/80 promoter.
In some aspects, the disclosure relates to Baculovirus vectors comprising an
isolated
nucleic acid or rAAV vector as described by the disclosure. In some
embodiments, the
Baculovirus vector is an Auto grapha californica nuclear polyhedrosis (AcNPV)
vector, for
example as described by Urabe et al. (2002) Hum Gene Ther 13(16):1935-43 and
Smith et al.
(2009) Mol Ther 17(11):1888-1896.
In some aspects, the disclosure provides a host cell comprising an isolated
nucleic acid or
vector as described herein. A host cell can be a prokaryotic cell or a
eukaryotic cell. For
example, a host cell can be a mammalian cell, bacterial cell, yeast cell,
insect cell, etc. In some
embodiments, a host cell is a mammalian cell, for example a HEK293T cell. In
some
embodiments, a host cell is a bacterial cell, for example an E. coli cell.
rAAVs
In some aspects, the disclosure relates to recombinant AAVs (rAAVs) comprising
a
transgene that encodes one or more isolated nucleic acids as described herein
(e.g., an rAAV
vector encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more gene products described
herein and/or
inhibitory nucleic acids targeting gene products described herein). The term
"rAAVs" generally
refers to viral particles comprising an rAAV vector encapsidated by one or
more AAV capsid
proteins. An rAAV described by the disclosure may comprise a capsid protein
having a
serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
and AAV10, or a variant thereof. In some embodiments, a capsid protein is an
AAV9 capsid
protein or a variant thereof. In some embodiments, an AAV9 capsid protein
variant comprises a
mutation at one or more positions corresponding to T492, Y705, and Y731 of SEQ
ID NO: 147
(e.g., corresponding to those positions of AAV6). In some embodiments, the one
or more
mutations are selected from T492V, Y705F, Y73 1F, or a combination thereof. In
some
embodiments, an AAV9 capsid protein variant comprises the amino acid sequence
set forth in
SEQ ID NO: 149.
In some embodiments, an rAAV comprises a capsid protein from a non-human host,
for
example a rhesus AAV capsid protein such as AAVrh.10, AAVrh.39, etc. In some
embodiments, an rAAV described by the disclosure comprises a capsid protein
that is a variant
of a wild-type capsid protein, such as a capsid protein variant that includes
at least 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, or more than 10 (e.g., 15, 20 25, 50, 100, etc.) amino acid
substitutions (e.g.,
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mutations) relative to the wild-type AAV capsid protein from which it is
derived. In some
embodiments, an AAV capsid protein variant is an AAV1RX capsid protein, for
example as
described by Albright et al. Mol Ther. 2018 Feb 7;26(2):510-523. In some
embodiments, a
capsid protein is AAV1RX and comprises the amino acid sequence set forth in
SEQ ID NO: 146
(or is encoded by the nucleic acid sequence set forth in SEQ ID NO: 145). In
some
embodiments, a capsid protein variant is an AAV TM6 capsid protein, for
example as described
by Rosario et al. Mol Ther Methods Chu Dev. 2016; 3: 16026. In some
embodiments, an AAV6
capsid protein variant is AAV-TM6 capsid protein and comprises the amino acid
sequence set
forth in SEQ ID NO: 148.
In some embodiments, rAAVs described by the disclosure readily spread through
the
CNS, particularly when introduced into the CSF space or directly into the
brain parenchyma.
Accordingly, in some embodiments, rAAVs described by the disclosure comprise a
capsid
protein that is capable of crossing the blood-brain barrier (BBB). For
example, in some
embodiments, an rAAV comprises a capsid protein having an AAV9 or AAVrh.10
serotype.
Production of rAAVs is described, for example, by Samulski et al. (1989) J
Viral. 63(9):3822-8
and Wright (2009) Hum Gene Ther. 20(7): 698-706. In some embodiments, an rAAV
comprises a capsid protein that specifically or preferentially targets myeloid
cells, for example
microglial cells. In some embodiments, an rAAV transduces microglial cells.
In some embodiments, an rAAV as described by the disclosure (e.g., comprising
a
recombinant rAAV genome encapsidated by AAV capsid proteins to form an rAAV
capsid
particle) is produced in a Baculovirus vector expression system (BEVS).
Production of rAAVs
using BEVS are described, for example by Urabe et al. (2002) Hum Gene Ther
13(16):1935-43,
Smith et al. (2009) Mol Ther 17(11):1888-1896, U.S. Patent No. 8,945,918, U.S.
Patent No.
9,879,282, and International PCT Publication WO 2017/184879. However, an rAAV
can be
produced using any suitable method (e.g., using recombinant rep and cap
genes). In some
embodiments, an rAAV as disclosed herein is produced in HEK293 (human
embryonic kidney)
cells.
Pharmaceutical Compositions
In some aspects, the disclosure provides pharmaceutical compositions
comprising an
isolated nucleic acid or rAAV as described herein and a pharmaceutically
acceptable carrier. As
used herein, the term "pharmaceutically acceptable" refers to a material, such
as a carrier or
diluent, which does not abrogate the biological activity or properties of the
compound, and is
relatively non-toxic, e.g., the material may be administered to an individual
without causing
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undesirable biological effects or interacting in a deleterious manner with any
of the components
of the composition in which it is contained.
As used herein, the term "pharmaceutically acceptable carrier" means a
pharmaceutically
acceptable material, composition or carrier, such as a liquid or solid filler,
stabilizer, dispersing
agent, suspending agent, diluent, excipient, thickening agent, solvent or
encapsulating material,
involved in carrying or transporting a compound useful within the invention
within or to the
patient such that it may perform its intended function. Additional ingredients
that may be
included in the pharmaceutical compositions used in the practice of the
invention are known in
the art and described, for example in Remington's Pharmaceutical Sciences
(Genaro, Ed., Mack
Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
Compositions (e.g., pharmaceutical compositions) provided herein can be
administered
by any route, including enteral (e.g., oral), parenteral, intravenous,
intramuscular, intra-arterial,
intramedullary, intrathecal, subcutaneous, intraventricular, transdermal,
interdermal, rectal,
intravaginal, intraperitoneal, topical (as by powders, ointments, creams,
and/or drops), mucosal,
nasal, bucal, sublingual; by intratracheal instillation, bronchial
instillation, and/or inhalation;
and/or as an oral spray, nasal spray, and/or aerosol. Specifically
contemplated routes are oral
administration, intravenous administration (e.g., systemic intravenous
injection), regional
administration via blood and/or lymph supply, and/or direct administration to
an affected site.
In general, the most appropriate route of administration will depend upon a
variety of factors
including the nature of the agent (e.g., its stability in the environment of
the gastrointestinal
tract), and/or the condition of the subject (e.g., whether the subject is able
to tolerate oral
administration). In certain embodiments, the compound or pharmaceutical
composition
described herein is suitable for topical administration to the eye of a
subject.
In some embodiments, a composition comprises one or more (e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9,
or 10) different rAAVs, each rAAV comprising an isolated nucleic acid that
encodes a different
gene product (e.g., a different protein or inhibitory nucleic acid). The
different rAAVs may
comprise a capsid protein of the same serotype or different serotypes.
Methods
Aspects of the disclosure relate to compositions for expression of one or more
CNS
disease-associated gene products in a subject to treat CNS-associated
diseases. The one or more
CNS disease-associated gene products may be encoded by one or more isolated
nucleic acids or
rAAV vectors. In some embodiments, a subject is administered a single vector
(e.g., isolated
nucleic acid, rAAV, etc.) encoding one or more (1, 2, 3, 4, 5, or more) gene
products. In some
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embodiments, a subject is administered a plurality (e.g., 2, 3, 4, 5, or more)
vectors (e.g.,
isolated nucleic acids, rAAVs, etc.), where each vector encodes a different
CNS disease-
associated gene product.
A CNS-associated disease may be a neurodegenerative disease, synucleinopathy,
tauopathy, or a lysosomal storage disease. Examples of neurodegenerative
diseases and their
associated genes are listed in Table 2.
A "synucleinopathy" refers to a disease or disorder characterized by
accumulation,
overexpression or activity of alpha-Synuclein (the gene product of SNCA) in a
subject (e.g.,
relative to a healthy subject, for example a subject not having a
synucleinopathy). Examples of
synucleinopathies and their associated genes are listed in Table 3.
A "tauopathy" refers to a disease or disorder characterized by accumulation,
overexpression or activity of Tau protein in a subject (e.g., a healthy
subject not having a
tauopathy). Examples of tauopathies and their associated genes are listed in
Table 4.
A "lysosomal storage disease" refers to a disease characterized by abnormal
build-up of
toxic cellular products in lysosomes of a subject. Examples of lysosomal
storage diseases and
their associated genes are listed in Table 5.
In some embodiments, the disclosure relates to methods of treating a disease
selected
from Parkinson's Disease (e.g., Parkinson's Disease with GBA1 mutation (PD-
GBA), sporadic
Parkinson's Disease (sPD)), Gaucher Disease (e.g., neuronopathic Gaucher
disease (nGD), Type
I Gaucher Disease (T1GD), Type II Gaucher Disease (T2GD), and Type III Gaucher
Disease
(T3GD)), Dementia with Lewy Bodies (DLB), Amyotrophic lateral sclerosis (ALS),
and
Niemann-Pick Type C disease (NPC) by administering to a subject in need
thereof an isolated
nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic
acid) that encodes
GBAL
In some embodiments, the disclosure relates to methods of treating
Frontotemporal
Dementia (e.g., Frontotemporal Dementia with GRN mutation (FTD-GRN),
Frontotemporal
Dementia with MAPT mutation (FTD-tau), and Frontotemporal Dementia with
C90RF72
mutation (FTD-C9orf72)), Parkinson's Disease (PD), Alzheimer's Disease (AD),
Neuronal
Ceroid Lipofuscinosis (NCL), Corticobasal Degeneration (CBD), Motor Neuron
Disease
(MND), or Gaucher Disease (GD) by administering to a subject in need thereof
an isolated
nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic
acid) that encodes
PGRN (also referred to as GRN).
In some embodiments, the disclosure relates to methods of treating
Synucleinopathies
(e.g., multiple system atrophy (MSA), Parkinson's Disease (PD), Parkinson's
disease with GBA1
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mutation (PD-GBA), Dementia with Lewy Bodies (DLB), Dementia with Lewy Bodies
with
GBA1 mutation, and Lewy Body Disease) by administering to a subject in need
thereof an
isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated
nucleic acid) that
encodes GBA1 gene product, and an inhibitory nucleic acid targeting SNCA.
In some embodiments, the disclosure relates to methods of treating a disease
selected
from Parkinson's Disease (PD), Frontotemporal Dementia (e.g., Frontotemporal
Dementia with
GRN mutation (FTD-GRN)), Lysosomal Storage Diseases (LSDs), or Gaucher Disease
(GD) by
administering to a subject in need thereof an isolated nucleic acid (e.g., an
rAAV vector or
rAAV comprising an isolated nucleic acid) that encodes PSAP.
In some embodiments, the disclosure relates to methods of treating Alzheimer's
Disease
(AD), Nasu-Hakola Disease (NHD) Frontotemporal Dementia with MAPT mutation
(FTD-Tau),
or Parkinson's Disease (PD), by administering to a subject in need thereof an
isolated nucleic
acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that
encodes TREM2.
In some embodiments, the disclosure relates to methods of treating Alzheimer's
disease
(AD) or Frontotemporal Dementia (Frontotemporal Dementia with MAPT mutation
(FTD-Tau),
a tauopathy, Progressive supranuclear palsy (PSP), neurodegenerative disease,
Lewy Body
Disease (LBD) or Parkinson's Disease by administering to a subject in need
thereof an isolated
nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic
acid) that encodes
inhibitory nucleic acids targeting MAPT.
As used herein "treat" or "treating" refers to (a) preventing or delaying
onset of a CNS
disease; (b) reducing severity of a CNS disease; (c) reducing or preventing
development of
symptoms characteristic of a CNS disease; (d) and/or preventing worsening of
symptoms
characteristic of a CNS disease. Symptoms of CNS disease may include, for
example, motor
dysfunction (e.g., shaking, rigidity, slowness of movement, difficulty with
walking, paralysis),
cognitive dysfunction (e.g., dementia, depression, anxiety, psychosis),
difficulty with memory,
emotional and behavioral dysfunction.
The disclosure is based, in part, on compositions for expression of
combinations of CNS
diseases-associated genes (e.g., PD-associated gene products) in a subject
that act together (e.g.,
synergistically) to treat the disease.
Accordingly, in some aspects, the disclosure provides a method for treating a
subject
having or suspected of having CNS-associated diseases (e.g., Parkinson's
disease, AD, FTD,
etc.), the method comprising administering to the subject a composition (e.g.,
a composition
comprising an isolated nucleic acid or a vector or a rAAV) as described by the
disclosure.
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In some embodiments, a subject has one or more signs or symptoms, or has a
genetic
predisposition (e.g., a mutation in a gene listed in Table 1) to a
neurodegenerative disease listed
in Table 2. In some embodiments, a subject has one or more signs or symptoms,
or has a genetic
predisposition (e.g., a mutation in a gene listed in Table 1) to a
synucleinopathy listed in Table
3. In some embodiments, a subject has one or more signs or symptoms, or has a
genetic
predisposition (e.g., a mutation in a gene listed in Table 1) to a tauopathy
listed in Table 4. In
some embodiments, a subject has one or more signs or symptoms, or has a
genetic
predisposition (e.g., a mutation in a gene listed in Table 1) to a lysosomal
storage disease listed
in Table 5.
The disclosure is based, in part, on compositions for expression of one or
more CNS-
disease associated gene products in a subject to treat Gaucher disease. In
some embodiments,
the Gaucher disease is a neuronopathic Gaucher disease, for example Type 2
Gaucher disease or
Type 3 Gaucher disease. In some embodiments, a subject does not have PD or PD
symptoms.
Accordingly, in some aspects, the disclosure provides a method for treating a
subject
having or suspected of having neuronopathic Gaucher disease, the method
comprising
administering to the subject a composition (e.g., a composition comprising an
isolated nucleic
acid or a vector or a rAAV) as described by the disclosure.
The disclosure is based, in part, on compositions for expression of one or
more CNS-
disease associated gene products in a subject to treat Alzheimer's disease or
fronto-temporal
dementia (FTD). In some embodiments, the subject does not have Alzheimer's
disease.
Accordingly, in some aspects, the disclosure provides a method for treating a
subject
having or suspected of having FTD, the method comprising administering to the
subject a
composition (e.g., a composition comprising an isolated nucleic acid or a
vector or a rAAV) as
described by the disclosure. In some embodiments, a subject having Alzheimer's
disease or
fronto-temporal dementia (FTD) is administered an rAAV encoding Progranulin
(PGRN, also
referred to as GRN) or a portion thereof.
In some aspects, the disclosure provides a method for delivering a transgene
to
microglial cells, the method comprising administering an rAAV as described
herein to a subject.
In some embodiments, a rAAV encoding a Gcase protein for treating Type 2 or
Type 3
Gaucher disease or Parkinson's disease with a GBA1 mutation is administered to
a subject as a
single dose, and the rAAV is not administered to the subject subsequently.
In some embodiments, a rAAV encoding a Gcase protein is administered via a
single
suboccipital injection into the cisterna magna. In some embodiments, the
injection into the
cisterna magna is performed under radiographic guidance.
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A subject is typically a mammal, preferably a human. In some embodiments, a
subject is
between the ages of 1 month old and 10 years old (e.g., 1 month, 2 months, 3
months, 4, months,
months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12
months, 13
months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20
months, 21
5 months, 22 months, 23 months, 24 months, 3, years, 4 years, 5 years, 6
years, 7 years, 8 years, 9
years, 10 years, or any age therebetween). In some embodiments, a subject is
between 2 years
old and 20 years old. In some embodiments, a subject is between 30 years old
and 100 years
old. In some embodiments, a subject is older than 55 years old.
In some embodiments, a composition is administered directly to the CNS of the
subject,
for example by direct injection into the brain and/or spinal cord of the
subject. Examples of
CNS-direct administration modalities include but are not limited to
intracerebral injection,
intraventricular injection, intracisternal injection, intraparenchymal
injection, intrathecal
injection, and any combination of the foregoing. In some embodiments, a
composition is
administered to a subject by intra-cisterna magna (ICM) injection. In some
embodiments, direct
injection into the CNS of a subject results in transgene expression (e.g.,
expression of the first
gene product, second gene product, and if applicable, third gene product) in
the midbrain,
striatum and/or cerebral cortex of the subject. In some embodiments, direct
injection into the
CNS results in transgene expression (e.g., expression of the first gene
product, second gene
product, and if applicable, third gene product) in the spinal cord and/or CSF
of the subject.
In some embodiments, direct injection to the CNS of a subject comprises
convection
enhanced delivery (CED). Convection enhanced delivery is a therapeutic
strategy that involves
surgical exposure of the brain and placement of a small-diameter catheter
directly into a target
area of the brain, followed by infusion of a therapeutic agent (e.g., a
composition or rAAV as
described herein) directly to the brain of the subject. CED is described, for
example by
Debinski et al. (2009) Expert Rev Neurother. 9(10):1519-27.
In some embodiments, a composition is administered peripherally to a subject,
for
example by peripheral injection. Examples of peripheral injection include
subcutaneous
injection, intravenous injection, intra-arterial injection, intraperitoneal
injection, or any
combination of the foregoing. In some embodiments, the peripheral injection is
intra-arterial
injection, for example injection into the carotid artery of a subject.
In some embodiments, a composition (e.g., a composition comprising an isolated
nucleic
acid or a vector or a rAAV) as described by the disclosure is administered
both peripherally and
directly to the CNS of a subject. For example, in some embodiments, a subject
is administered a
composition by intra-arterial injection (e.g., injection into the carotid
artery) and by
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intraparenchymal injection (e.g., intraparenchymal injection by CED). In some
embodiments,
the direct injection to the CNS and the peripheral injection are simultaneous
(e.g., happen at the
same time). In some embodiments, the direct injection occurs prior (e.g.,
between 1 minute and
1 week, or more before) to the peripheral injection. In some embodiments, the
direct injection
occurs after (e.g., between 1 minute and 1 week, or more after) the peripheral
injection.
In some embodiments, a subject is administered an immunosuppressant prior to
(e.g.,
between 1 month and 1 minute prior to) or at the same time as a composition as
described
herein. In some embodiments, the immunosuppressant is a corticosteroid (e.g.,
prednisone,
budesonide, etc.), an mTOR inhibitor (e.g., sirolimus, everolimus, etc.), an
antibody (e.g.,
.. adalimumab, etanercept, natalizumab, etc.), or methotrexate.
The amount of composition (e.g., a composition comprising an isolated nucleic
acid or a
vector or a rAAV) as described by the disclosure administered to a subject
will vary depending
on the administration method. For example, in some embodiments, a rAAV as
described herein
is administered to a subject at a titer between about 109 Genome copies
(GC)/kg and about 1014
GC/kg (e.g., about 109 GC/kg, about 1010 GC/kg, about 1011 GC/kg, about 1012
GC/kg, about
1012 GC/kg, or about 1014 GC/kg). In some embodiments, a subject is
administered a high titer
(e.g., >1012 Genome Copies GC/kg of an rAAV) by injection to the CSF space, or
by
intraparenchymal injection. In some embodiments, a rAAV as described herein is
administered
to a subject at a dose ranging from about 1 x 1010 vector genomes (vg) to
about 1 x 1017 vg by
intravenous injection. In some embodiments, a rAAV as described herein is
administered to a
subject at a dose ranging from about 1 x 1010 vg to about 1 x 1016 vg by
injection into the
cisterna magna.
A composition (e.g., a composition comprising an isolated nucleic acid or a
vector or a
rAAV) as described by the disclosure can be administered to a subject once or
multiple times
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) times. In some embodiments, a
composition is
administered to a subject continuously (e.g., chronically), for example via an
infusion pump.
Table 2: Examples of neurodegenerative diseases
Disease Associated genes
Alzheimer's disease APP, PSEN1, PSEN2, APOE
Parkinson's disease LRRK2, PARK7, PINK], PRKN, SNCA,
GBA,
UCHL1, ATP13A2, VPS35
Huntington's disease HTT
Amyotrophic lateral sclerosis ALS2, ANG, ATXN2, C9orf72,
CHCHD10,
CHMP2B, DCTN1, ERBB4, FIG4, FUS,
HNRNPA1, MATR3, NEFH, OPTN, PFN1,
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PRPH, SETX, SIGMAR1, SMN1, SOD],
SPG11, SQSTM1, TARDBP, TBK1, TRPM7,
TUBA4A, UBQLN2, VAPB, VCP
Batten disease (Neuronal ceroid PPT1, TPP1, CLN3, CLN5, CLN6,
MFSD8,
lipofunscinosis) CLN8, CTSD, DNAJC5, CTSF, ATP13A2,
GRN, KCTD7
Friedreich's ataxia FXN
Lewy body disease APOE, GBA, SNCA, SNCB
Spinal muscular atrophy SMN1, SMN2
Multiple sclerosis CYP27B1, HLA-DRB1, IL2RA, IL7R,
TNFRSF1A
Prion disease (Creutzfeldt-Jakob disease, Fatal PRNP
familial insomnia, Gertsmann-Straussler-
Scheinker syndrome, Variably protease-
sensitive prionopathy)
Table 3: Examples of synucleinopathies
Disease Associated genes
Parkinson's disease LRRK2, PARK7, PINK], PRKN, SNCA,
GBA,
UCHL1, ATP13A2, VPS35
Dementia with Lewy bodies APOE, GBA, SNCA, SNCB
Multiple system atrophy COQ2, SNCA
Table 4: Examples of tauopathies
Disease Associated genes
Alzheimer's disease APP, PSEN1, PSEN2, APOE
Primary age-related tauopathy MAPT
Progressive supranuclear palsy MAPT
Corticobasal degeneration MAPT, GRN, C9orf72, VCP, CHMP2B,
TARDBP, FUS
Frontotemporal dementia with parkinsonism- MAPT
17
Subacute sclerosing panencephalitis SCN1A
Lytico-Bodig disease
Gangioglioma, gangliocytoma
Meningioangiomatosis
Postencephalitic parkinsonism
Chronic traumatic encephalopathy
Table 5: Examples of lysosomal storage diseases
Disease Associated genes
Niemann-Pick disease NPC1, NPC2, SMPD1
Fabry disease GLA
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Krabbe disease GALC
Gaucher disease GBA
Tach-Sachs disease HEXA
Metachromatic leukodystrophy ARSA, PSAP
Farber disease ASAH1
Galactosialidosis CTSA
Schindler disease NAGA
GM1 gangliosidosis GLB 1
GM2 gangliosidosis GM2A
Sandhoff disease HEXB
Lysosomal acid lipase deficiency LIPA
Multiple sulfatase deficiency SUMF1
Mucopolysaccharidosis Type I IDUA
Mucopolysaccharidosis Type II IDS
Mucopolysaccharidosis Type III GNS, HGSNAT, NAGLU, SGSH
Mucopolysaccharidosis Type IV GALNS, GLB1
Mucopolysaccharidosis Type VI ARSB
Mucopolysaccharidosis Type VII GUSB
Mucopolysaccharidosis Type IX HYAL1
Mucolipidosis Type II GNPTAB
Mucolipidosis Type III alpha/beta GNPTAB
Mucolipidosis Type III gamma GNPTG
Mucolipidosis Type IV MCOLN1
Neuronal ceroid lipofuscinosis PPT1, TPP1, CLN3, CLN5, CLN6,
MFSD8,
CLN8, CTSD, DNAJC5, CTSF, ATP13A2,
GRN, KCTD7
Alpha-mannosidosis MAN2B1
Beta-mannosidosis MANBA
Aspartylglucosaminuria AGA
Fucosidosis FUCA]
EXAMPLES
Example 1: rAAV vectors
AAV vectors are generated using cells, such as HEK293 cells for triple-plasmid
transfection. The ITR sequences flank an expression construct comprising a
promoter/enhancer
element for each transgene of interest, a 3' polyA signal, and
posttranslational signals such as
the WPRE element. Multiple gene products can be expressed simultaneously such
as GBA1 and
LIMP2 and/or Prosaposin, by fusion of the protein sequences; or using a 2A
peptide linker, such
as T2A or P2A, which leads 2 peptide fragments with added amino acids due to
prevention of
the creation of a peptide bond; or using an IRES element; or by expression
with 2 separate
expression cassettes. The presence of a short intronic sequence that is
efficiently spliced,
upstream of the expressed gene, can improve expression levels. shRNAs and
other regulatory
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RNAs can potentially be included within these sequences. Examples of
expression constructs
described by the disclosure are shown in FIGs. 1-8, 21-35, 39 and 41-51, and
in Table 6 below.
Table 6
Name Promoter shRNA CDS1 PolyA Bicistronic Promoter CDS2
PolyA2 Length
1 1 element 2
between
ITRs
CMVe_CBAp_GBAl_ CBA GBA1 WPRE
3741
WPRE_bGH -bGH
LT1 s_JetLong_mRNAi JetLong aSyn S CARB 2 bGH T2A GBA1
4215
aSYn_SCARB2-T2A-
GBA 1 _bGH
LIl_JetLong_S CARB 2 JetLong SCARB2 bGH IRES GBA1
4399
-IRES -GB Al_bGH
FPl_JetLong_GB Al_b JetLong GBA1 bGH JetLong S CARB 2 S
V4OL 4464
GH_JetLong_S CARB 2
_S V4OL
PrevailVector_LT2s_Je JetLong aSyn P S AP bGH T2A -
- GBA1 4353
tLong_mRNAiaSYn_P
SAP-T2A-
GBA 1 _bGH_4353nt
PrevailVector_LI2 _JetL JetLong - P S AP S ynthe IRES -
- GBA1 4337
ong_PSAP_IRES_GBA tic pA
1_SymtheticpolyA_433
7nt
PrevailVector_10s_JetL JetLong aSyn GB A2 WPRE - -
- - 4308
ong_mRNAiaS y_GB A _bGH
2_WPRE_bGH_4308nt
PrevailVector_FT4 _Jet JetLong - GBA1 S ynthe T2A - -
GALC 4373
Long_GB Al_T2A_GA tic pA
LC_SyntheticpolyA_43
73nt
PrevailVector_LT4 _Jet JetLong - GALC S ynthe T2A - -
GBA1 4373
Long_GALC_T2A_GB tic pA
A 1 _S yntheticpolyA_43
73nt
PrevailVector_LT5s_Je JetLong aSyn CTSB WPRE T2A - -
GBA1 4392
tLong_mRNAiaS yn_C _bGH
TSB-T2A-
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GBA1_WPRE_bGH_4
392nt
PrevailVector_FT110 JetLong aSyn GBA1 S ynthe T2A - -
SMPD1 4477
etLong_mRNAiaSyn_ tic pA
GBA 1 _T2S_SMPD l_S
yntheticpolyA_4477nt
PrevailVector_LI4 _JetL JetLong - GALC S ynthe IRES -
- GBA1 4820
ong_GALC_IRES_GB tic pA
A 1 _S ymtheticpolyA_4
820nt
PrevailVector_FP5_Jet JetLong - GBA1 bGH - JetLong CTSB
SV4OL 4108
Long_GB Al_bGH_Jet
Long_CTSB_SV401_41
08nt
PrevailVector_FT6s_Je JetLong aSyn GBA1 WPRE T2A - -
GCH1 4125
tLong_mRNAiaS yn_G _bGH
BA1 -T2A-
GCHl_WPRE_bGH_4
125nt
PrevailVector_LT7s_Je JetLong aSyn RAB7L1 WPRE T2A - -
GBA1 3984
tLong_mRNAiaS yn_R _bGH
AB7L1 -T2A-
GBAl_WPRE_bGH_3
984nt
PrevailVector_FI6s_Jet JetLong aSyn GBA1 bGH IRES - -
GCH1 3978
Long_mRNAiaSYn_G
B Al -TRES -
GCH 1 _bGH_3978nt
PrevailVector_9st_JetL JetLong aSyn VP S35 WPRE - -
- - 4182
ong_mRNAiaS yn_mR & _bGH
NAiTMEM106B_VPS TMEM
35_WPRE_bGH_4182 106B
nt
PrevailVector_FT12s_J JetLong aSyn GBA1 WPRE T2A - -
IL34 4104
etLong_mRNAiaSyn_ _bGH
GBA1 -T2A-
IL34_WPRE_bGH_410
4nt
PrevailVector_FI 1 2s_Je JetLong aSyn GBA1 bGH IRES -
- IL34 3957
tLong_mRNAiaSYn_G
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B Al-IRES -
IL34_bGH_3957nt
PrevailVector_FP8_Jet JetLong - GBA1 bGH - CD68 TREM2 S
V4OL 4253
Long_GB A l_bGH_CD
68_TREM2_S V401_42
53nt
PrevailVector_FP12_C CB A GBA1 bGH JetLong IL34
SV4OL 4503
MVe_CBA_GB A l_bG
H_JetLong_IL34_S V40
1_4503nt
PrevailVector_O_CMV CB A aSyn GBA1 WPRE - - - -
4004
e_CBAp_mRNAiaSyn_ _bGH
GBA1_WPRE_bGH_4
004nt
PrevailVector_Xl_SN CMVe + - SNCA WPRE - - - -
-
CA CB A _bGH
Example 2: Cell based assays of viral transduction into GBA -deficient cells
Cells deficient in GBA1 are obtained, for example as fibroblasts from GD
patients,
monocytes, or hES cells, or patient-derived induced pluripotent stem cells
(iPSCs). These cells
accumulate substrates such as glucosylceramide and glucosylsphingosine (GlcCer
and GlcSph).
Treatment of wild-type or mutant cultured cell lines with Gcase inhibitors,
such as CBE, is also
be used to obtain GBA deficient cells.
Using such cell models, lysosomal defects are quantified in terms of
accumulation of
protein aggregates, such as of a-Synuclein with an antibody for this protein
or phospho-aSyn,
followed by imaging using fluorescent microscopy. Imaging for lysosomal
abnormalities by
ICC for protein markers such as LAMP1, LAMP2, LIMP1, LIMP2, or using dyes such
as
Lysotracker, or by uptake through the endocytic compartment of fluorescent
dextran or other
markers is also performed. Imaging for autophagy marker accumulation due to
defective fusion
with the lysosome, such as for LC3, can also be performed. Western blotting
and/or ELISA is
used to quantify abnormal accumulation of these markers. Also, the
accumulation of glycolipid
substrates and products of GBA1 is measured using standard approaches.
Therapeutic endpoints (e.g., reduction of PD-associated pathology) are
measured in the
context of expression of transduction of the AAV vectors, to confirm and
quantify activity and
function. Gcase can is also quantified using protein ELISA measures, or by
standard Gcase
activity assays.
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Example 3: In vivo assays using mutant mice
This example describes in vivo assays of AAV vectors using mutant mice. In
vivo
studies of AAV vectors as above in mutant mice are performed using assays
described, for
example, by Liou et al. (2006) J. Biol. Chem. 281(7): 4242-4253, Sun et al.
(2005) J. Lipid Res.
46:2102-2113, and Farfel-Becker et al. (2011) Dis. Model Mech. 4(6):746-752.
The intrathecal or intraventricular delivery of vehicle control and AAV
vectors (e.g., at a
dose of 2x1011 vg/mouse) are performed using concentrated AAV stocks, for
example at an
injection volume between 5-10 [IL. Intraparenchymal delivery by convection
enhanced delivery
is performed.
Treatment is initiated either before onset of symptoms, or subsequent to
onset.
Endpoints measured are the accumulation of substrate in the CNS and CSF,
accumulation of
Gcase enzyme by ELISA and of enzyme activity, motor and cognitive endpoints,
lysosomal
dysfunction, and accumulation of a-Synuclein monomers, protofibrils or
fibrils.
Example 4: Chemical models of disease
This example describes in vivo assays of AAV vectors using a chemically-
induced
mouse model of Gaucher disease (e.g., the CBE mouse model). In vivo studies of
these AAV
vectors are performed in a chemically-induced mouse model of Gaucher disease,
for example as
described by Vardi et al. (2016) J Pathol. 239(4):496-509.
Intrathecal or intraventricular delivery of vehicle control and AAV vectors
(e.g., at a
dose of 2x1011 vg/mouse) are performed using concentrated AAV stocks, for
example with
injection volume between 5-10 [IL. Intraparenchymal delivery by convection
enhanced delivery
is performed. Peripheral delivery is achieved by tail vein injection.
Treatment is initiated either before onset of symptoms, or subsequent to
onset.
Endpoints measured are the accumulation of substrate in the CNS and CSF,
accumulation of
Gcase enzyme by ELISA and of enzyme activity, motor and cognitive endpoints,
lysosomal
dysfunction, and accumulation of a-Synuclein monomers, protofibrils or
fibrils.
Example 5: Clinical trials in PD, LBD, Gaucher disease patients
In some embodiments, patients having certain forms of Gaucher disease (e.g.,
GD1) have
an increased risk of developing Parkinson's disease (PD) or Lewy body dementia
(LBD). This
Example describes clinical trials to assess the safety and efficacy of rAAVs
as described by the
disclosure, in patients having Gaucher disease, PD and/or LBD.
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Clinical trials of such vectors for treatment of Gaucher disease, PD and/or
LBD are
performed using a study design similar to that described in Grabowski et al.
(1995) Ann. Intern.
Med. 122(1):33-39.
Example 6: Treatment of peripheral disease
In some embodiments, patients having certain forms of Gaucher disease exhibit
symptoms of peripheral neuropathy, for example as described in Biegstraaten et
al. (2010) Brain
133(10):2909-2919.
This example describes in vivo assays of AAV vectors as described herein for
treatment
of peripheral neuropathy associated with Gaucher disease (e.g., Type 1 Gaucher
disease).
Briefly, Type 1 Gaucher disease patients identified as having signs or
symptoms of peripheral
neuropathy are administered a rAAV as described by the disclosure. In some
embodiments, the
peripheral neuropathic signs and symptoms of the subject are monitored, for
example using
methods described in Biegstraaten et al., after administration of the rAAV.
Levels of transduced gene products as described by the disclosure present in
patients
(e.g., in serum of a patient, in peripheral tissue (e.g., liver tissue, spleen
tissue, etc.)) of a patient
are assayed, for example by Western blot analysis, enzymatic functional
assays, or imaging
studies.
Example 7: Treatment of CNS forms
This example describes in vivo assays of rAAVs as described herein for
treatment of
CNS forms of Gaucher disease. Briefly, Gaucher disease patients identified as
having a CNS
form of Gaucher disease (e.g., Type 2 or Type 3 Gaucher disease) are
administered a rAAV as
described by the disclosure. Levels of transduced gene products as described
by the disclosure
present in the CNS of patients (e.g., in serum of the CNS of a patient, in
cerebrospinal fluid
(CSF) of a patient, or in CNS tissue of a patient) are assayed, for example by
Western blot
analysis, enzymatic functional assays, or imaging studies.
Example 8: Gene therapy of Parkinson's Disease in subjects having mutations in
GBA1
This example describes administration of a recombinant adeno-associated virus
(rAAV)
encoding GBA1 to a subject having Parkinson's disease characterized by a
mutation in
GBAlgene.
The rAAV-GBA1 vector insert contains the CBA promoter element (CBA),
consisting of
four parts: the CMV enhancer (CMVe), CBA promoter (CBAp), Exon 1, and intron
(int) to
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constitutively express the codon optimized coding sequence (CDS) of human GBA1
(maroon).
The 3' region also contains a Woodchuck hepatitis virus Posttranscriptional
Regulatory Element
(WPRE) posttranscriptional regulatory element followed by a bovine Growth
Hormone polyA
signal (bGH polyA) tail. The flanking ITRs allow for the correct packaging of
the intervening
sequences. Two variants of the 5' ITR sequence (FIG. 7, inset box, bottom
sequence) were
evaluated; these variants have several nucleotide differences within the 20-
nucleotide "D"
region of the ITR, which is believed to impact the efficiency of packaging and
expression. The
rAAV-GBA1 vector product contains the "D" domain nucleotide sequence shown in
FIG. 7
(inset box, top sequence). A variant vector harbors a mutant "D" domain
(termed an "S" domain
herein, with the nucleotide changes shown by shading), performed similarly in
preclinical
studies. The backbone contains the gene to confer resistance to kanamycin as
well as a stuffer
sequence to prevent reverse packaging. A schematic depicting a rAAV-GBA1
vector is shown
in FIG. 8. The rAAV-GBA1 vector is packaged into an rAAV using AAV9 serotype
capsid
proteins.
rAAV-GBA1 is administered to a subject as a single dose via a fluoroscopy
guided sub-
occipital injection into the cisterna magna (intracisternal magna; ICM). One
embodiment of a
rAAV-GBA1 dosing regimen study is as follows:
A single dose of rAAV-GBA lis administered to patients (N=12) at one of two
dose
levels (3e13 vg (low dose); 1e14 vg (high dose), etc.) which are determined
based on the results
of nonclinical pharmacology and toxicology studies.
Initial studies were conducted in a chemical mouse model involving daily
delivery of
conduritol-b-epoxide (CBE), an inhibitor of GCase to assess the efficacy and
safety of the
rAAV-GBA1 vector and a rAAV-GBA1 S-variant construct (as described further
below).
Additionally, initial studies were performed in a genetic mouse model, which
carries a
homozygous GBA1 mutation and is partially deficient in saposins (4L/PS-NA).
Additional
dose-ranging studies in mice and nonhuman primates (NHPs) are conducted to
further evaluate
vector safety and efficacy.
Two slightly different versions of the 5' inverted terminal repeat (ITR) in
the AAV
backbone were tested to assess manufacturability and transgene expression
(FIG. 7). The 20 bp
"D" domain within the 145 bp 5' ITR is thought to be necessary for optimal
viral vector
production, but mutations within the "D" domain have also been reported to
increase transgene
expression in some cases. Thus, in addition to the viral vector rAAV-GBA1,
which harbors an
intact "D" domain, a second vector form with a mutant D domain (termed an "S"
domain herein)
was also evaluated. Both rAAV-GBA1 and the variant express the same transgene.
While both
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vectors produced virus that was efficacious in vivo as detailed below, rAAV-
GBA1, which
contains a wild-type "D" domain, was selected for further development.
To establish the CBE model of GCase deficiency, juvenile mice were dosed with
CBE, a
specific inhibitor of GCase. Mice were given CBE by IP injection daily,
starting at postnatal
day 8 (P8). Three different CBE doses (25 mg/kg, 37.5 mg/kg, 50 mg/kg) and PBS
were tested
to establish a model that exhibits a behavioral phenotype (FIG. 9). Higher
doses of CBE led to
lethality in a dose-dependent manner. All mice treated with 50 mg/kg CBE died
by P23, and 5
of the 8 mice treated with 37.5 mg/kg CBE died by P27. There was no lethality
in mice treated
with 25 mg/kg CBE. Whereas CBE-injected mice showed no general motor deficits
in the open
field assay (traveling the same distance and at the same velocity as mice
given PBS), CBE-
treated mice exhibited a motor coordination and balance deficit as measured by
the rotarod
assay.
Mice surviving to the end of the study were sacrificed on the day after their
last CBE
dose (P27, "Day 1") or after three days of CBE withdrawal (P29, "Day 3").
Lipid analysis was
performed on the cortex of mice given 25 mg/kg CBE to evaluate the
accumulation of GCase
substrates in both the Day 1 and Day 3 cohorts. GluSph and GalSph levels
(measured in
aggregate in this example) were significantly accumulated in the CBE-treated
mice compared to
PBS-treated controls, consistent with GCase insufficiency.
Based on the study described above, the 25 mg/kg CBE dose was selected since
it
produced behavioral deficits without impacting survival. To achieve widespread
GBA1
distribution throughout the brain and transgene expression during CBE
treatment, rAAV-GBA1
or excipient was delivered by intracerebroventricular (ICV) injection at
postnatal day 3 (P3)
followed by daily IP CBE or PBS treatment initiated at P8 (FIG. 10).
CBE-treated mice that received rAAV-GBA1 performed statistically significantly
better
on the rotarod than those that received excipient (FIG. 11). Mice in the
variant treatment group
did not differ from excipient treated mice in terms of other behavioral
measures, such as the total
distance traveled during testing (FIG. 11).
At the completion of the in-life study, half of the mice were sacrificed the
day after the
last CBE dose (P36, "Day 1") or after three days of CBE withdrawal (P38, "Day
3") for
biochemical analysis (FIG. 12). Using a fluorometric enzyme assay performed in
biological
triplicate, GCase activity was assessed in the cortex. GCase activity was
increased in mice that
were treated with rAAV-GBA1, while CBE treatment reduced GCase activity.
Additionally,
mice that received both CBE and rAAV-GBA1 had GCase activity levels that were
similar to
the PBS-treated group, indicating that delivery of rAAV-GBA1 is able to
overcome the
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inhibition of GCase activity induced by CBE treatment. Lipid analysis was
performed on the
motor cortex of the mice to examine levels of the substrates GluCer and
GluSph. Both lipids
accumulated in the brains of mice given CBE, and rAAV-GBA1 treatment
significantly reduced
substrate accumulation.
Lipid levels were negatively correlated with both GCase activity and
performance on the
Rotarod across treatment groups. The increased GCase activity after rAAV-GBA1
administration was associated with substrate reduction and enhanced motor
function (FIG. 13).
As shown in FIG. 14, preliminary biodistribution was assessed by vector genome
presence, as
measured by qPCR (with >100 vector genomes per 1 i.t.g genomic DNA defined as
positive).
Mice that received rAAV-GBA1, both with and without CBE, were positive for
rAAV-GBA1
vector genomes in the cortex, indicating that ICV delivery results in rAAV-
GBA1 delivery to
the cortex. Additionally, vector genomes were detected in the liver, few in
spleen, and none in
the heart, kidney or gonads. For all measures, there was no statistically
significant difference
between the Day 1 and Day 3 groups.
A larger study in the CBE model further explored efficacious doses of rAAV-
GBA1 in
the CBE model. Using the 25 mg/kg CBE dose model, excipient or rAAV-GBA1 was
delivered
via ICV at P3, and daily IP PBS or CBE treatment initiated at P8. Given the
similarity between
the groups with and without CBE withdrawal observed in the previous studies,
all mice were
sacrificed one day after the final CBE dose (P38-40). The effect of three
different rAAV-GBA1
doses was assessed, resulting in the following five groups, with 10 mice
(5M/5F) per group:
Excipient ICV + PBS IP
Excipient ICV +25 mg/kg CBE IP
3.2e9 vg (2.13e10 vg/g brain) rAAV-GBA1 ICV +25 mg/kg CBE IP
1.0e10 vg (6.67e10 vg/g brain) rAAV-GBA1 ICV +25 mg/kg CBE IP
3.2e10 vg (2.13e11 vg/g brain) rAAV-GBA1 ICV + 25 mg/kg CBE IP.
The highest dose of rAAV-GBA1 rescued the CBE treatment-related failure to
gain
weight at P37. Additionally, this dose resulted in a statistically significant
increase in
performance on the rotarod and tapered beam compared to the Excipient + CBE
treated group
(FIG. 15). Lethality was observed in several groups, including both excipient-
treated and
rAAV-GBAl-treated groups (Excipient + PBS: 0; Excipient + 25 mg/kg CBE: 1;
3.2e9 vg
rAAV-GBA1+ 25 mg/kg CBE: 4; 1.0e10 vg rAAV-GBA1+ 25 mg/kg CBE: 0; 3.2e10 vg
rAAV-GBA1+ 25 mg/kg CBE: 3).
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At the completion of the in-life study, mice were sacrificed for biochemical
analysis
(FIG. 16). GCase activity in the cortex was assessed in biological triplicates
by a fluorometric
assay. CBE-treated mice showed reduced GCase activity whereas mice that
received a high
rAAV-GBA1 dose showed a statistically significant increase in GCase activity
compared to
CBE treatment. CBE-treated mice also had accumulation of GluCer and GluSph,
both of which
were rescued by administering a high dose of rAAV-GBA1.
In addition to the established chemical CBE model, rAAV-GBAlis also evaluated
in the
4L/PS-NA genetic model, which is homozygous for the V394L GD mutation in Gbal
and is also
partially deficient in saposins, which affect GCase localization and activity.
These mice exhibit
motor strength, coordination, and balance deficits, as evidenced by their
performance in the
beam walk, rotarod, and wire hang assays. Typically the lifespan of these mice
is less than 22
weeks. In an initial study, 3 ill of maximal titer virus was delivered by ICV
at P23, with a final
dose of 2.4e10 vg (6.0e10 vg/g brain). With 6 mice per group, the treatment
groups were:
WT + Excipient ICV
4L/PS-NA + Excipient ICV
4L/PS-NA + 2.4e10 vg (6.0e10 vg/g brain) rAAV-GBA1 ICV
Motor performance by the beam walk test was assessed 4 weeks post- rAAV-GBA1
delivery. The group of mutant mice that received rAAV-GBA1 showed a trend
towards fewer
total slips and fewer slips per speed when compared to mutant mice treated
with excipient,
restoring motor function to near WT levels (FIG. 17). Since the motor
phenotypes become more
severe as these mice age, their performance on this and other behavioral tests
is assessed at later
time points. At the completion of the in-life study, lipid levels, GCase
activity, and
biodistribution are assessed in these mice.
Additional lower doses of rAAV-GBA1 are currently being tested using the CBE
model,
corresponding to 0.03x, 0.1x, and lx the proposed phase 1 high clinical dose.
Each group
includes 10 mice (5M/5F) per group:
Excipient ICV
Excipient ICV +25 mg/kg CBE IP
3.2e8 vg (2.13e9 vg/g brain) rAAV-GBA1 ICV + 25 mg/kg CBE IP
1.0e9 vg (6.67e9 vg/g brain) rAAV-GBA1 ICV + 25 mg/kg CBE IP
1.0e10 vg (6.67e10 vg/g brain) rAAV-GBA1 ICV +25 mg/kg CBE IP.
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In addition to motor phenotypes, lipid levels and GCase activity are assessed
in the
cortex. Time course of treatments and analyses are also performed.
A larger dose ranging study was initiated to evaluate efficacy and safety
data. 10 4L/PS-
NA mice (5M/5F per group) were injected with 10 ill of rAAV-GBA1. Using an
allometric
brain weight calculation, the doses correlate to 0.15x, 1.5x, 4.4x, and 14.5x
the proposed phase 1
high clinical dose. The injection groups consist of:
WT + Excipient ICV
4L/PS-NA + Excipient ICV
4L/PS-NA + 4.3e9 vg (1.1e10 vg/g brain) rAAV-GBA1 ICV
4L/PS-NA + 4.3e10 vg (1.1ell vg/g/ brain) rAAV-GBA1 ICV
4L/PS-NA + 1.3e11 vg (3.2e11 vg/g brain) rAAV-GBA1 ICV
4L/PS-NA + 4.3e11 vg (1.1e12 vg/g brain) rAAV-GBA1 ICV.
A summary of nonclinical studies in the CBE model are shown in Table 7 below.
Table 7: Summary of Results in CBE Mouse Model
Test Study Dose Cohort Behavioral Changes Lipids Enzyme BD
Material Number
o 0
t")
(24 H
rAAV- PRV-2018- 3.2e9 vg NS NS NS NS NS + -
GBA1 005 Dose- (2.13e10
ranging vg/g brain)
rAAV- 1.10e10 vg T NS NS T/S NS +
+
GBA1 in (6.67e10
CBE Model vg/g brain)
2.3e10vg S S NS S S + +
(2.13e11
vg/g brain)
Variant PRV-2018- 8.8e9 vg S N/A NS S S +
+
005 Dose- (5.9e10 vg/g
ranging brain)
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Variant in
CBE Model
Note that positive biodistribution is defined as >100 vg/1 i.t.g genomic DNA.
Abbreviations: BD = biodistribution; NS = nonsignificant; T = trend; S =
significant; N/A =
not applicable; + = positive; - = negative.
Example 9: In vitro analysis of rAAV vectors
rAAV constructs were tested in vitro and in vivo. FIG. 18 shows representative
data for
in vitro expression of rAAV constructs encoding progranulin (PGRN, also
referred to as GRN)
protein. The left panel shows a standard curve of progranulin (PGRN) ELISA
assay. The
bottom panel shows a dose-response of PGRN expression measured by ELISA assay
in cell
lysates of HEK293T cells transduced with rAAV. MOI = multiplicity of infection
(vector
genomes per cell).
A pilot study was performed to assess in vitro activity of rAAV vectors
encoding
Prosaposin (PSAP) and SCARB2, alone or in combination with GBA1 and/or one or
more
inhibitory RNAs. One construct encoding PSAP and progranulin (PGRN, also
referred to as
GRN) was also tested. Vectors tested include those shown in Table 4. "Opt"
refers to a nucleic
acid sequence codon optimized for expression in mammalian cells (e.g., human
cells). FIG. 19
shows representative data indicating that transfection of HEK293 cells with
each of the
constructs resulted in overexpression of the corresponding gene product
compared to mock
transfected cells.
A pilot study was performed to assess in vitro activity of rAAV vectors
encoding
TREM2, alone or in combination with one or more inhibitory RNAs. Vectors
tested include
those shown in Table 8. "Opt" refers to a nucleic acid sequence codon
optimized for expression
in mammalian cells (e.g., human cells). FIGs. 36A-36B show representative data
indicating that
transfection of HEK293 cells with each of the constructs resulted in
overexpression of the
corresponding gene product compared to mock transfected cells.
Table 8
ID Promoter Inhibitory RNA Promoter Transgene
100015 JL intronic SNCA JetLong Opt-
PSAP GBA1
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100039 - - JetLong Opt-PSAP-
GRN
100046 - - CBA Opt-PSAP
100014 JetLong SNCA JetLong Opt-
SCARB2 GBA1
100040 JL, CD68 opt-GB Al,
TREM2
Example 10: Testing of SNCA and TMEM106B shRNA constructs
HEK293 cells
Human embryonic kidney 293 cell line (HEK293) were used in this study
(#85120602,
Sigma-Aldrich). HEK293 cells were maintained in culture media (D-MEM
[#11995065,
Thermo Fisher Scientific] supplemented with 10% fetal bovine serum [FBS]
[#10082147,
Thermo Fisher Scientific]) containing 100 units/ml penicillin and 100 [ig/m1
streptomycin
(#15140122, Thermo Fisher Scientific).
Plasmid transfection
Plasmid transfection was performed using Lipofectamine 2000 transfection
reagent
(#11668019, Thermo Fisher Scientific) according to the manufacture's
instruction. Briefly,
HEK293 cells (#12022001, Sigma-Aldrich) were plated at the density of 3x105
cells/ml in
culture media without antibiotics. On the following day, the plasmid and
Lipofectamine 2000
reagent were combined in Opti-MEM solution (#31985062, Thermo Fisher
Scientific). After 5
minutes, the mixtures were added into the HEK293 culture. After 72 hours, the
cells were
harvested for RNA or protein extraction, or subjected to the imaging analyses.
For imaging
analyses, the plates were pre-coated with 0.01% poly-L-Lysine solution (P8920,
Sigma-Aldrich)
before the plating of cells.
Gene expression analysis by quantitative real-time PCR (qRT-PCR)
Relative gene expression levels were determined by quantitative real-time PCR
(qRT-
PCR) using Power SYBR Green Cells-to-CT Kit (#4402955, Thermo Fisher
Scientific)
according to the manufacturer's instruction. The candidate plasmids were
transiently transfected
into HEK293 cells plated on 48-well plates (7.5 x104 cells/well) using
Lipofectamine 2000
transfection reagent (0.5 p.g plasmid and 1.5 pi reagent in 50 pi Opti-MEM
solution). After 72
hours, RNA was extracted from the cells and used for reverse transcription to
synthesize cDNA
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according to the manufacturer's instruction. For quantitative PCR analysis, 2-
5 Ill of cDNA
products were amplified in duplicates using gene specific primer pairs (250 nM
final
concentration) with Power SYBR Green PCR Master Mix (#4367659, Thermo Fisher
Scientific). The primer sequences for SNCA, TMEM106B, and GAPDH genes were: 5'-
AAG
AGG GTG TTC TCT ATG TAG GC -3' (SEQ ID NO: 71), 5'- GCT CCT CCA ACA TTT
GTC ACT T -3' (SEQ ID NO: 72) for SNCA, 5'-ACA CAG TAC CTA CCG TTA TAG CA-3'
(SEQ ID NO: 73), 5'-TGT TGT CAC AGT AAC TTG CAT CA-3' (SEQ ID NO: 74) for
TMEM106B, and 5'- CTG GGC TAC ACT GAG CAC C -3' (SEQ ID NO: 75), 5'- AAG TGG
TCG TTG AGG GCA ATG -3' (SEQ ID NO: 76) for GAPDH. Quantitative PCR was
performed in a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific).
Expression
levels were normalized by the housekeeping gene GAPDH and calculated using the
comparative
CT method.
Fluorescence Imaging Analysis
EGFP reporter plasmids, which contain 3'-UTR of human SNCA gene at downstream
of
EGFP coding region, were used for the validation of SNCA and TMEM106B
knockdown
plasmids. EGFP reporter plasmids and candidate knockdown plasmids were
simultaneously
transfected into HEK293 cells plated on poly-L-Lysine coated 96-well plates
(3.0 x104
cells/well) using Lipofectamine 2000 transfection reagent (0.04 1.tg reporter
plasmid, 0.06m
knockdown plasmid and 0.3 Ill reagent in 10 Ill Opti-MEM solution). After 72
hours, the
fluorescent intensities of EGFP signal were measured at excitation 488
nm/emission 512 nm
using Varioskan LUX multimode reader (Thermo Fisher Scientific). Cells were
fixed with 4%
PFA at RT for 10 minutes, and incubated with D-PBS containing 40m/m1 7-
aminoactinomycin
D (7-AAD) for 30 min at RT. After washing with D-PBS, the fluorescent
intensities of 7-AAD
signal were measured at excitation 546 nm/emission 647 nm using Varioskan
reader to quantify
cell number. Normalized EGFP signal per 7-AAD signal levels were compared with
the control
knockdown samples.
Enzyme-linked Immunosorbent Assay (ELISA)
a-Synuclein reporter plasmids, which contain 3'-UTR of human SNCA gene or
TMEM106B
gene downstream of SNCA coding region, were used for the validation of
knockdown plasmids
at the protein level. Levels of a-synuclein protein were determined by ELISA
(#KHB0061,
Thermo Fisher Scientific) using the lysates extracted from HEK293 cells. The
candidate
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plasmids were transiently transfected into HEK293 cells plated on 48-well
plates (7.5 x104
cells/well) using Lipofectamine 2000 transfection reagent (0.1m reporter
plasmid, 0.15 1.tg
knockdown plasmid and 0.75 Ill reagent in 25 Ill Opti-MEM solution). After 72
hours, cells were
lysed in radioimmunoprecipitation assay (RIPA) buffer (#89900, Thermo Fisher
Scientific)
supplemented with protease inhibitor cocktail (#P8340, Sigma-Aldrich), and
sonicated for a few
seconds. After incubation on ice for 30 min, the lysates were centrifuged at
20,000 xg at 4 C for
min, and the supernatant was collected. Protein levels were quantified. Plates
were read in a
Varioskan plate reader at 450 nm, and concentrations were calculated using
SoftMax Pro 5
software. Measured protein concentrations were normalized to total protein
concentration
10 determined with a bicinchoninic acid assay (#23225, Thermo Fisher
Scientific).
FIG. 37 and Table 9 show representative data indicating successful silencing
of SNCA in
vitro by GFP reporter assay (top) and a-Syn assay (bottom). FIG. 38 and Table
10 show
representative data indicating successful silencing of TMEM106B in vitro by
GFP reporter assay
(top) and a-Syn assay (bottom).
Table 9
ID Promoter Knockdown Promoter
Overexpress
100007 CMV intronic SNCA mi CMV opt-GB
Al
100008 H1 SNCA sh CMV opt-GB
Al
100009 H1 SNCA Pubsh4 CMV opt-GB
Al
100014 JL intronic SNCA mi JetLong
opt-
SCARB2 GBA
100015 JL intronic SNCA mi JetLong
opt-
PSAP GBA
100016 JL intronic SNCA mi JetLong
opt-
CTSB GBA
100019 JL intronic SNCA TMEM mi JetLong opt-
VPS35
100023 JL intronic SNCA mi JetLong
opt-
GB Al IL34
100024 JL intronic SNCA mi JetLong opt-GBA2
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100028 intronic SNCA Broadsh CMV
opt-GB Al
100029 intronic SNCA Pubsh4 CMV
opt-GB Al
Table 10
ID Promoter Knockdown Promoter
Overexpress
100010 HI TMEM Pub sh CMV
opt-GRN
100011 JL intronic TMEM mi JetLong
opt-
GB Al GRN
100012 H1 TMEM sh CMV
opt-GRN
100019 JL intronic SNCA TMEM mi JetLong
opt-VPS35
Example 11: ITR "D" sequence placement and cell transduction
The effect of placement of ITR "D" sequence on cell transduction of rAAV
vectors was
investigated. HEK293 cells were transduced with Gcase-encoding rAAVs having 1)
wild-type
ITRs (e.g., "D" sequences proximal to the transgene insert and distal to the
terminus of the ITR)
or 2) ITRs with the "D" sequence located on the "outside" of the vector (e.g.,
"D" sequence
located proximal to the terminus of the ITR and distal to the transgene
insert), as shown in FIG.
20. Data indicate that rAAVs having the "D" sequence located in the "outside"
position retain
the ability to be packaged and transduce cells efficiently (FIG. 40).
Example 12: In vitro testing of Pro granulin rAAVs
FIG. 39 is a schematic depicting one embodiments of a vector comprising an
expression
construct encoding PGRN (also referred to as GRN). Progranulin is
overexpressed in the CNS
of rodents deficient in GRN, either heterozygous or homozygous for GRN
deletion, by injection
of an rAAV vector encoding PGRN (e.g., codon-optimized PGRN, also referred to
as codon-
optimized GRN), either by intraparenchymal or intrathecal injection such as
into the cisterna
magna.
Mice are injected at 2 months or 6 months of age, and aged to 6 months or 12
months
and analyzed for one or more of the following: expression level of GRN at the
RNA and protein
levels, behavioral assays (e.g., improved movement), survival assays (e.g.,
improved survival),
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microglia and inflammatory markers, gliosis, neuronal loss, Lipofuscinosis,
and/or Lysosomal
marker accumulation rescue, such as LAMPl. Assays on GRN-deficient mice are
described, for
example by Arrant et al. (2017) Brain 140: 1477-1465; Arrant et al. (2018) J.
Neuroscience
38(9):2341-2358; and Amado et al. (2018) doi:https://doi.org/10.1101/30869;
the entire
contents of which are incorporated herein by reference.
Example 13: In vitro testing of MAPT rAAVs
SY5Y cells were plated at 4x104 cells per well in a 96-well plate. The
following day,
cells were transduced with two virus stocks (Intronic eSIBR MAPT MiR615
Conserved
vector) encoding inhibitory RNA targeting MAPT (J00130 produced in a mammalian
cell-based
system, and J00122 produced in a Baculovirus-based system; shown in FIG. 75C)
in triplicates
at MOI of 2x 105 in media containing luM Hoechst. Excipient alone was used as
negative
control. The cells were harvested 72 hours later, and stained with a probe to
detect AAV vectors
expressing inhibitory RNA for MAPT. The probe targets BGHpA. FIG. 75A shows
that both
virus stocks successfully transduced SY5Y cells.
SY5Y cells were plated at 4x104 cells per well in a 96-well plate. The
following day,
cells were transduced with two virus stocks ((Intronic eSIBR MAPT MiR615
Conserved
vector) encoding inhibitory RNA targeting MAPT (J00130 and J00122; shown in
FIG. 75C) in
triplicates at MOI 2x106 in media containing luM Hoechst. Excipient alone was
used as
negative control. SY5Y cells were lysed for RNA extraction 72 hours or 7 days
after
transduction. cDNA was made from the extracted RNA using Invitrogen Power SYBR
Green
Cells-to-Ct Kit. qRT-PCR was conducted on cDNA samples and run in triplicates
using primers
for both human MAPT and GAPDH. FIG.75B shows data for knockdown of MAPT
expression
by J00130 and J00122.
EQUIVALENTS
This Application incorporates by reference the contents of the following
documents in
their entirety: the International PCT Application PCT/U52018/054225, filed
October 3 2018;
International PCT Application PCT/U52018/054223, filed October 3, 2018;
Provisional
Application Serial Numbers 62/567,296, filed October 3, 2017, entitled "GENE
THERAPIES
FOR LYSOSOMAL DISORDERS"; 62/567,311, filed October 3, 2017, entitled "GENE
THERAPIES FOR LYSOSOMAL DISORDERS"; 62/567,319, filed October 3, 2017,
entitled
"GENE THERAPIES FOR LYSOSOMAL DISORDERS"; 62/567,301, filed October 3, 2018,
entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS"; 62/567,310, filed October
3,
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2017, entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS"; 62/567,303, filed
October 3, 2017, entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS"; and
62/567,305, filed October 3, 2017, entitled "GENE THERAPIES FOR LYSOSOMAL
DISORDERS".
Having thus described several aspects of at least one embodiment of this
invention, it is
to be appreciated that various alterations, modifications, and improvements
will readily occur to
those skilled in the art. Such alterations, modifications, and improvements
are intended to be
part of this disclosure, and are intended to be within the spirit and scope of
the invention.
Accordingly, the foregoing description and drawings are by way of example
only.
While several embodiments of the present invention have been described and
illustrated
herein, those of ordinary skill in the art will readily envision a variety of
other means and/or
structures for performing the functions and/or obtaining the results and/or
one or more of the
advantages described herein, and each of such variations and/or modifications
is deemed to be
within the scope of the present invention. More generally, those skilled in
the art will readily
appreciate that all parameters, dimensions, materials, and configurations
described herein are
meant to be exemplary and that the actual parameters, dimensions, materials,
and/or
configurations will depend upon the specific application or applications for
which the teachings
of the present invention is/are used. Those skilled in the art will recognize,
or be able to
ascertain using no more than routine experimentation, many equivalents to the
specific
embodiments of the invention described herein. It is, therefore, to be
understood that the
foregoing embodiments are presented by way of example only and that, within
the scope of the
appended claims and equivalents thereto, the invention may be practiced
otherwise than as
specifically described and claimed. The present invention is directed to each
individual feature,
system, article, material, and/or method described herein. In addition, any
combination of two
or more such features, systems, articles, materials, and/or methods, if such
features, systems,
articles, materials, and/or methods are not mutually inconsistent, is included
within the scope of
the present invention.
The indefinite articles "a" and "an," as used herein in the specification and
in the claims,
unless clearly indicated to the contrary, should be understood to mean "at
least one."
The phrase "and/or," as used herein in the specification and in the claims,
should be
understood to mean "either or both" of the elements so conjoined, i.e.,
elements that are
conjunctively present in some cases and disjunctively present in other cases.
Other elements
may optionally be present other than the elements specifically identified by
the "and/or" clause,
whether related or unrelated to those elements specifically identified unless
clearly indicated to
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the contrary. Thus, as a non-limiting example, a reference to "A and/or B,"
when used in
conjunction with open-ended language such as "comprising" can refer, in one
embodiment, to A
without B (optionally including elements other than B); in another embodiment,
to B without A
(optionally including elements other than A); in yet another embodiment, to
both A and B
(optionally including other elements); etc.
As used herein in the specification and in the claims, "or" should be
understood to have
the same meaning as "and/or" as defined above. For example, when separating
items in a list,
"or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion
of at least one, but also
including more than one, of a number or list of elements, and, optionally,
additional unlisted
items. Only terms clearly indicated to the contrary, such as "only one of' or
"exactly one of,"
or, when used in the claims, "consisting of," will refer to the inclusion of
exactly one element of
a number or list of elements. In general, the term "or" as used herein shall
only be interpreted as
indicating exclusive alternatives (i.e. "one or the other but not both") when
preceded by terms of
exclusivity, such as "either," "one of," "only one of," or "exactly one of."
"Consisting
essentially of," when used in the claims, shall have its ordinary meaning as
used in the field of
patent law.
As used herein in the specification and in the claims, the phrase "at least
one," in
reference to a list of one or more elements, should be understood to mean at
least one element
selected from any one or more of the elements in the list of elements, but not
necessarily
including at least one of each and every element specifically listed within
the list of elements
and not excluding any combinations of elements in the list of elements. This
definition also
allows that elements may optionally be present other than the elements
specifically identified
within the list of elements to which the phrase "at least one" refers, whether
related or unrelated
to those elements specifically identified. Thus, as a non-limiting example,
"at least one of A and
B" (or, equivalently, "at least one of A or B," or, equivalently "at least one
of A and/or B") can
refer, in one embodiment, to at least one, optionally including more than one,
A, with no B
present (and optionally including elements other than B); in another
embodiment, to at least one,
optionally including more than one, B, with no A present (and optionally
including elements
other than A); in yet another embodiment, to at least one, optionally
including more than one, A,
and at least one, optionally including more than one, B (and optionally
including other
elements); etc.
In the claims, as well as in the specification above, all transitional phrases
such as
"comprising," "including," "carrying," "having," "containing," "involving,"
"holding," and the
like are to be understood to be open-ended, i.e., to mean including but not
limited to. Only the
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transitional phrases "consisting of' and "consisting essentially of' shall be
closed or semi-closed
transitional phrases, respectively, as set forth in the United States Patent
Office Manual of Patent
Examining Procedures, Section 2111.03.
Use of ordinal terms such as "first," "second," "third," etc., in the claims
to modify a
claim element does not by itself connote any priority, precedence, or order of
one claim element
over another or the temporal order in which acts of a method are performed,
but are used merely
as labels to distinguish one claim element having a certain name from another
element having a
same name (but for use of the ordinal term) to distinguish the claim elements.
It should also be understood that, unless clearly indicated to the contrary,
in any methods
claimed herein that include more than one step or act, the order of the steps
or acts of the method
is not necessarily limited to the order in which the steps or acts of the
method are recited.
SEQUENCES
In some embodiments, an expression cassette encoding one or more gene products
(e.g.,
a first, second and/or third gene product) comprises or consists of (or
encodes a peptide having)
a sequence set forth in any one of SEQ ID NOs: 1-149. In some embodiments, a
gene product is
encoded by a portion (e.g., fragment) of any one of SEQ ID NOs: 1-149.
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