OPTIMIZED CLN1 GENES AND EXPRESSION CASSETTES AND THEIR USE
This invention relates to polynucleotides comprising a nucleotide sequence encoding a PPT1 polypeptide or a fragment thereof, vectors (viral or non-viral vectors) comprising the same, and methods of using the same for delivery of the open reading frame to a cell or a subject and to treat infantile neuronal lipofuscinosis (infantile Batten disease). The polynucleotides comprise an optimized CLN1 open reading frame.
1 . A polynucleotide comprising a human CLN1 open reading frame, wherein a polynucleotide sequence of the polynucleotide or its complement is codon-optimized for expression in a human cell.
2 . The polynucleotide of claim 1 , wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least about 90% identity thereto or to its complement.
3 . An expression cassette comprising a polynucleotide comprising a human CLN1 open reading frame.
4 . The expression cassette of claim 3 , wherein the polynucleotide is the polynucleotide of claim 1 or 2 .
5 . The expression cassette of claim 3 or 4 , wherein the polynucleotide is operably linked to a promoter.
6 . The expression cassette of claim 5 , wherein the promoter is a chicken beta actin promoter.
7 . The expression cassette of any one of claims 3-6 , wherein the polynucleotide is operably linked to an enhancer.
8 . The expression cassette of claim 7 , wherein the enhancer is a cytomegalovirus enhancer.
9 . The expression cassette of any one of claims 3-8 , wherein the polynucleotide is operably linked to an intron.
10 . The expression cassette of claim 9 , wherein the intron is a hybrid/modified MVM intron.
11 . The expression cassette of any one of claims 3-10 , wherein the polynucleotide is operably linked to a polyadenylation signal.
12 . The expression cassette of claim 11 , wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal.
13 . The expression cassette of any one of claims 3-12 , further comprising at least one adeno-associated virus (AAV) inverted terminal repeat (ITR).
14 . The expression cassette of claim 13 , wherein the expression cassette comprises two AAV ITRs.
15 . The expression cassette of claim 14 , wherein the two AAV ITRs have the same nucleotide sequence.
16 . The expression cassette of claim 14 , wherein the two AAV ITRs have different nucleotide sequences.
17 . The expression cassette of any one of claims 13-16 , wherein the AAV ITRs are AAV2 ITRs.
18 . The expression cassette of any one of claims 3-17 , wherein the expression cassette is a self-complementary AAV genome.
19 . The expression cassette of any one of claims 3-18 , wherein the expression cassette comprises an enhancer, a promoter, an intron, a human CLN1 open reading frame, and a polyadenylation site.
20 . The expression cassette of claim 19 , wherein the expression cassette comprises an AAV ITR, an enhancer, a promoter, an intron, a human CLN1 open reading frame, a polyadenylation site, and an AAV ITR.
21 . The expression cassette of any one of claims 3-18 , wherein the expression cassette comprises a CMV enhancer, a chicken beta actin promoter, a hybrid/modified MVM intron, a human CLN1 open reading frame, and a bovine growth hormone polyadenylation site.
22 . The expression cassette of claim 21 , wherein the expression cassette comprises a mutant AAV ITR, a CMV enhancer, a chicken beta actin promoter, a hybrid/modified MVM intron, a human CLN1 open reading frame, a bovine growth hormone polyadenylation site, and a wild-type AAV ITR.
23 . The expression cassette of claim 22 , comprising the nucleotide sequence of SEQ ID NO: 7 or a sequence at least about 90% identical thereto.
24 . A vector comprising the polynucleotide of claim 1 or 2 or the expression cassette of any one of claims 3-16 .
25 . The vector of claim 24 , wherein the vector is a viral vector.
26 . The vector of claim 25 , wherein the vector is an AAV vector.
27 . The vector of claim 26 , wherein the AAV vector is an AAV9 vector.
28 . The vector of claim 27 , wherein the AAV vector comprises wild-type capsid proteins.
29 . The vector of claim 27 , wherein the AAV vector comprises a modified capsid protein with altered tropism compared to a wild-type capsid protein.
30 . The vector of claim 29 , wherein the modified capsid protein is liver-detargeted.
31 . A transformed cell comprising the polynucleotide of claim 1 or 2 , the expression cassette of any one of claims 3-23 , and/or the vector of any one of claims 24-30 .
32 . The transformed cell of claim 31 , wherein the polynucleotide, expression cassette, and/or vector is stably incorporated into the cell genome.
33 . A transgenic animal comprising the polynucleotide of claim 1 or 2 , the expression cassette of any one of claims 3-23 , the vector of any one of claims 24-30 , and/or the transformed cell of claim 31 or 32 .
34 . A pharmaceutical composition comprising the polynucleotide of claim 1 or 2 , the expression cassette of any one of claims 3-23 , the vector of any one of claims 24-30 , and/or the transformed cell of claim 31 or 32 in a pharmaceutically acceptable carrier.
35 . A method of expressing a CLN1 open reading frame in a cell, comprising contacting the cell with the polynucleotide of claim 1 or 2 , the expression cassette of any one of claims 3-23 , and/or the vector of any one of claims 24-30 , thereby expressing the CLN1 open reading frame in the cell.
36 . A method of expressing a CLN1 open reading frame in a subject, comprising delivering to the subject the polynucleotide of claim 1 or 2 , the expression cassette of any one of claims 3-23 , the vector of any one of claims 24-30 , and/or the transformed cell of claim 31 or 32 , thereby expressing the CLN1 open reading frame in the subject.
37 . A method of treating a disorder associated with aberrant expression of a CLN1 gene or aberrant activity of a CLN1 gene product in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of the polynucleotide of claim 1 or 2 , the expression cassette of any one of claims 3-23 , the vector of any one of claims 24-30 , and/or the transformed cell of claim 31 or 32 , thereby treating the disorder associated with aberrant expression of the CLN1 gene in the subject.
38 . The method of claim 37 , wherein the disorder associated with expression of the CLN1 gene is infantile, late-infantile, juvenile, or adult-onset neuronal ceroid lipofuscinosis.
39 . The method of any one of claims 36-38 , wherein the polynucleotide, expression cassette, vector, and/or transformed cell is delivered to the nervous system of the subject.
40 . The method of claim 39 , wherein the polynucleotide, expression cassette, vector, and/or transformed cell is delivered by intrathecal, intracerebral, intraventricular, intranasal, intra-aural, intra-ocular, or peri-ocular delivery, or any combination thereof.
41 . The method of any one of claims 36-38 , wherein the polynucleotide, expression cassette, vector, and/or transformed cell is delivered intravenously.