RECOMBINANT AAV VECTORS AND METHODS OF USING THE SAME
20210189424 · 2021-06-24
Assignee
Inventors
Cpc classification
C12N2750/14143
CHEMISTRY; METALLURGY
C12N2750/14141
CHEMISTRY; METALLURGY
A61K48/005
HUMAN NECESSITIES
C12N15/86
CHEMISTRY; METALLURGY
International classification
C12N15/86
CHEMISTRY; METALLURGY
Abstract
The present disclosure relates to recombinant vectors expressing the human ND4 gene, methods of preparing recombinant vectors expressing the human ND4 gene, and uses thereof. Recombinant AAV2 vectors as disclosed herein are useful in treating Leber Hereditary Optic Neuroretinopathy (LHON), including ND4-related LHON.
Claims
1. A recombinant AAV2 vector comprising: a 3′UTR Cox10 sequence comprising SEQ ID No: 1, a nucleic acid sequence encoding an NADH dehydrogenase 4 (ND4) polypeptide comprising SEQ ID No: 13, and a nucleic acid sequence encoding an MTS Cox10 polypeptide comprising SEQ ID No: 11 or SEQ ID No: 12.
2. (canceled)
3. A recombinant AAV2 vector comprising: a 3′UTR Cox10 sequence comprising SEQ ID No: 14, a nucleic acid sequence encoding an NADH dehydrogenase 4 (ND4) polypeptide comprising SEQ ID No: 13, and a nucleic acid sequence encoding an MTS Cox10 polypeptide comprising SEQ ID No: 11 or SEQ ID No: 12.
4. (canceled)
5. A recombinant AAV2 vector comprising: a 3′UTR Cox10 sequence comprising SEQ ID No: 1, a nucleic acid sequence encoding ND4 comprising SEQ ID No: 2 or SEQ ID No: 17, and an MTS Cox10 sequence comprising SEQ ID No: 3.
6. (canceled)
7. (canceled)
8. (canceled)
9. A recombinant AAV2 vector comprising: a 3′UTR Cox10 sequence comprising SEQ ID No: 14, a nucleic acid sequence encoding ND4 comprising SEQ ID No: 15 or SEQ ID No: 18, and an MTS Cox10 sequence comprising SEQ ID No: 16.
10. (canceled)
11. (canceled)
12. (canceled)
13. The recombinant AAV2 vector of claim 1, further comprising: an HBB2 intron sequence comprising SEQ ID No: 4, a CMV promoter sequence comprising SEQ ID No: 5, a first ITR sequence comprising SEQ ID No: 6, and a second ITR sequence comprising SEQ ID No: 7.
14. (canceled)
15. The recombinant AAV2 vector of claim 3, further comprising: an HBB2 intron sequence comprising SEQ ID No: 24, a CMV promoter sequence comprising SEQ ID No: 25, a first ITR sequence comprising SEQ ID No: 26, and a second ITR sequence comprising SEQ ID No: 27.
16. (canceled)
17. A recombinant AAV2 vector of claim 5, further comprising: an HBB2 intron sequence consisting of SEQ ID No: 4, a CMV promoter sequence consisting of SEQ ID No: 5, a first ITR sequence consisting of SEQ ID No: 6, and a second ITR sequence consisting of SEQ ID No: 7.
18. A recombinant AAV2 vector of claim 9 further comprising: an HBB2 intron sequence consisting of SEQ ID No: 24, a CMV promoter sequence consisting of SEQ ID No: 25, a first ITR sequence consisting of SEQ ID No: 26, and a second ITR sequence consisting of SEQ ID No: 27.
19. A method of treating Leber Hereditary Optic Neuroretinopathy in a patient in need thereof, comprising administering to the patient an effective amount of the recombinant AAV2 vector according to claim 1.
20. A method of treating Leber Hereditary Optic Neuroretinopathy in a patient in need thereof, comprising administering to the patient an effective amount of the recombinant vector according to claim 1, wherein the patient has experienced a disease duration of less than nine months.
21. A method of treating Leber Hereditary Optic Neuroretinopathy in a patient in need thereof, comprising administering to the patient an effective amount of the recombinant AAV2 vector according to claim 1, wherein the patient has experienced a disease duration of six to nine months.
22. A method of treating Leber Hereditary Optic Neuroretinopathy in a patient in need thereof, comprising administering to the patient an effective amount of the recombinant AAV2 vector according to claim 1, wherein the patient has a baseline visual acuity of <about 1.6 LogMAR.
23. A method of treating Leber Hereditary Optic Neuroretinopathy in a patient in need thereof, comprising administering to the patient an effective amount of the recombinant AAV2 vector according to claim 1, wherein the patient has experienced a disease duration of less than nine months and the patient has a baseline visual acuity of <about 1.6 LogMAR.
24. A method of treating Leber Hereditary Optic Neuroretinopathy in a patient in need thereof, comprising administering to the patient an effective amount of the recombinant AAV2 vector according to claim 1, wherein the patient has experienced a disease duration of six to nine months and the patient has a baseline visual acuity of <about 1.6 LogMAR.
25. The method according to claim 19, wherein the Leber Hereditary Optic Neuroretinopathy is ND4-related Leber Hereditary Optic Neuroretinopathy.
26. The method according to claim 19, wherein the recombinant AAV2 vector is administered intravitreally.
27. The method according to claim 19, wherein the recombinant AAV2 vector is administered intravitreally in an amount of about 109 to 1011 viral genomes per eye.
28. The method according to claim 19, wherein the recombinant AAV2 vector is administered intravitreally in an amount of about 10.sup.10 to 10.sup.11 viral genomes per eye.
29. The method according to claim 19, wherein the recombinant AAV2 vector is administered intravitreally in an amount of about 5.0×10.sup.10 to 1.0×10.sup.11 viral genomes per eye.
30. The method according to claim 19, wherein the recombinant AAV2 vector is administered intravitreally in an amount of about 9.0×10.sup.10 viral genomes per eye.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0153] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several non-limiting embodiments of the present disclosure and together with the description, serve to explain the principles of the disclosure.
[0154]
[0155]
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DETAILED DESCRIPTION OF THE DISCLOSURE
[0161] Disclosed herein in some embodiments are recombinant vectors expressing a gene encoding the human NADH dehydrogenase type 4 (ND4) protein ND4 (SEQ ID NO: 13). Also disclosed herein are methods of treating LHON by administration of recombinant AAV2 vectors expressing the human ND4 protein.
[0162] The term “a,” “an,” or “the” refers to one or to more than one of the grammatical object of the article. The term may mean “one,” “one or more,” “at least one,” or “one or more than one.” By way of example, “an element” means one element or more than one element. The term “or” means “and/or” unless otherwise stated. The term “including” or “containing” is not limiting.
[0163] The term “codon” is meant to refer to a sequence of three nucleotides, e.g., deoxyribonucleotides or ribonucleotides, which together form a unit of a genetic code that encodes an amino acid. The term “genetic code” is meant to refer to the full set of relationships between codons and amino acids used by living cells. The genetic code is highly similar among all organisms, and a person of ordinary skill in the art would understand that the terms “universal genetic code” or “standard genetic code” is meant to refer to the most common genetic code, used by most organisms including humans. In some embodiments, the universal genetic code is the genetic code used in eukaryotic cells. In some embodiments, the universal genetic code is the genetic code for nuclear genes. The term “mitochondria genetic code” is meant to refer to the code used in mitochondria, that sets out the codes for mitochondria nucleic acids and proteins. In some embodiments, the mitochondria genetic code is the vertebrate mitochondria code. In some embodiments, the mitochondria genetic code is the human mitochondria code. Codon usage in the mitochondria vs. the universal genetic code is described in Lewin, Genes V, Oxford University Press; New York 1994, the content of which is incorporated by reference.
[0164] The human NADH dehydrogenase type 4 (ND4) protein is a subunit of NADH dehydrogenase (ubiquinone), which is targeted to the mitochondrial inner membrane, and is the largest of the five complexes of the electron transport chain. The ND4 gene, also known as mitochondrially encoded NADH dehydrogenase 4 (MT-ND4), is located in the human mitochondria DNA. Exemplary nucleic acid sequences encoding the ND4 protein include but are not limited to NCBI NC_012920.1. In some embodiments, the nucleic acid sequence encoding an ND4 polypeptide may be a mitochondrial nucleic acid, or a nuclear nucleic acid encoding for the human ND4 polypeptide. In some embodiments, the nucleic acid sequence encoding an ND4 polypeptide may be any nucleic acid sequence encoding a human ND4 polypeptide. In some embodiments, the nucleic acid sequence encoding a human ND4 protein comprises SEQ ID NO: 2, 15, 17 or 18. Exemplary amino acid sequences for the human ND4 polypeptide include but are not limited to Genbank ACF70814.1. In some embodiments, the amino acid sequence of the human ND4 polypeptide comprises SEQ ID NO: 13.
TABLE-US-00001 TABLE 1 Sequences of various embodiments of the disclosure SEQ ID NO: 2 ND4 1374 ATGAGAAACCAGTGATAATGTCTGGGTTCAGGGACAGCAGCA (3′ to 5′) nt GGATGGGAGACAGATGCATAAACATCAGGGTGTTCTCTCTGGT GAATGAGGGCTTCATGTTATTAATGTGGTGAGTCAGTGAGCCC CACTGGGTAGTGGTAAACATGTACAGGCTGTAGAGGGCTGTG ACCAGCATGTTCAGTCCTGTCAGGAGCAGGGTGATGTTGCTC CAGGAGAATGTTGTCACCAGCACTGACAGCTCTCCCAGCAGG TTAATTGTAGGGGGCAGAGCCAGGTTGGCCAGACTAGCCAGG AGCCACCAGAAAGCCATCAGTGGGAGCAGGGTCTGGAGCCCC TGACTCAGGATCATAATTCTTGAGTGAGTTCTTTCATAGTTGCT ATTTGCCAGGCAGAACAGGAGGCTGCTGGTCAGCCCATGAGC AATCATGAGGATCACTGCCCCAGTAAAGGACCAGGGTGTCTG AATCAGAATGGCAGTCACCACCAGTGCCATGTGGCTGATGGA GCTGTAAGCAATCAGGCTCTTGAGGTCAGTCTGCCTGAGACA GATGGAGCTGGTCATGATCATACCCCAGAGGCTCAGAACCAG GAAAGGGTAAGCCATGTGCTTTGTCAGAGGGTTCAGGATCAG GGTGAGCCTCATCATACCATAACCACCCAGCTTCAGGAGGACA GCAGCCAGGACCATGGAGCCAGCTATTGGAGCTTCCACATGA GCCTTGGGGAGCCAGAGGTGCAGGCCATAGAGGGGCATCTTC ACCATAAAGGCCATTGTATAAGCCAGCCACATCAGGTTGTTTG CCCAGGAGTTACTCAGCTCCTGGGCAGTCAGAGTCAGCAGGA GGATGTTCAGGCTACCCAGTGTGTTGTGGGTATAGATCAGTGC AATCAGCAGGGGCAGTGAGCCCACCAGTGTATAAAAGAGAAA GTAGGTGCCTGCATTCAGCCTCTCAGGCTGATTTCCCCACCTA GTGATGATGGCCAGGGTTGGGATGAGAGTGGTCTCAAAGAAG ATATAGAACATGATCAGCTCAGTGGCTGTGAAGGTCATAATCA GGCTGATTTGCAGGCTAATGAGCATGGACAGGTACAGCTTTTT CCTTGACAGAGGCTCTGAGCTGAGGTGCCTCTGACTGGCCAT GATAGTCAGAGGCAGCAGCCATGTGGTCAGCATGAGCAGGGG GGTTGTCAGGGGATCAGAGGAAAAGGTAGGGGAGCATGAAAA GAGGTTATTATTAATCTGGTTGAAAAACAGCAGTGGGATGATG CTGATAATCAGGCTGTGGGTTGTGGTGTTAATCCAAATCATGT GCTTTTTGCTCAGCCATGTGAGAGGCAGCAGCATGATGGTTG GCACAATCAGCTTCAGCA SEQ ID NO: 17 ND4 1380 TCAGGATGAGAAACCAGTGATAATGTCTGGGTTCAGGGACAG (3′ to 5′) nt CAGCAGGATGGGAGACAGATGCATAAACATCAGGGTGTTCTCT CTGGTGAATGAGGGCTTCATGTTATTAATGTGGTGAGTCAGTG AGCCCCACTGGGTAGTGGTAAACATGTACAGGCTGTAGAGGG CTGTGACCAGCATGTTCAGTCCTGTCAGGAGCAGGGTGATGTT GCTCCAGGAGAATGTTGTCACCAGCACTGACAGCTCTCCCAG CAGGTTAATTGTAGGGGGCAGAGCCAGGTTGGCCAGACTAGC CAGGAGCCACCAGAAAGCCATCAGTGGGAGCAGGGTCTGGA GCCCCTGACTCAGGATCATAATTCTTGAGTGAGTTCTTTCATAG TTGCTATTTGCCAGGCAGAACAGGAGGCTGCTGGTCAGCCCA TGAGCAATCATGAGGATCACTGCCCCAGTAAAGGACCAGGGT GTCTGAATCAGAATGGCAGTCACCACCAGTGCCATGTGGCTG ATGGAGCTGTAAGCAATCAGGCTCTTGAGGTCAGTCTGCCTGA GACAGATGGAGCTGGTCATGATCATACCCCAGAGGCTCAGAA CCAGGAAAGGGTAAGCCATGTGCTTTGTCAGAGGGTTCAGGA TCAGGGTGAGCCTCATCATACCATAACCACCCAGCTTCAGGAG GACAGCAGCCAGGACCATGGAGCCAGCTATTGGAGCTTCCAC ATGAGCCTTGGGGAGCCAGAGGTGCAGGCCATAGAGGGGCA TCTTCACCATAAAGGCCATTGTATAAGCCAGCCACATCAGGTT GTTTGCCCAGGAGTTACTCAGCTCCTGGGCAGTCAGAGTCAG CAGGAGGATGTTCAGGCTACCCAGTGTGTTGTGGGTATAGATC AGTGCAATCAGCAGGGGCAGTGAGCCCACCAGTGTATAAAAG AGAAAGTAGGTGCCTGCATTCAGCCTCTCAGGCTGATTTCCCC ACCTAGTGATGATGGCCAGGGTTGGGATGAGAGTGGTCTCAA AGAAGATATAGAACATGATCAGCTCAGTGGCTGTGAAGGTCAT AATCAGGCTGATTTGCAGGCTAATGAGCATGGACAGGTACAGC TTTTTCCTTGACAGAGGCTCTGAGCTGAGGTGCCTCTGACTGG CCATGATAGTCAGAGGCAGCAGCCATGTGGTCAGCATGAGCA GGGGGGTTGTCAGGGGATCAGAGGAAAAGGTAGGGGAGCAT GAAAAGAGGTTATTATTAATCTGGTTGAAAAACAGCAGTGGGA TGATGCTGATAATCAGGCTGTGGGTTGTGGTGTTAATCCAAAT CATGTGCTTTTTGCTCAGCCATGTGAGAGGCAGCAGCATGATG GTTGGCACAATCAGCTTCAGCAT SEQ ID NO: 15 ND4 1374 TGCTGAAGCTGATTGTGCCAACCATCATGCTGCTGCCTCTCAC (5′ to 3′) nt ATGGCTGAGCAAAAAGCACATGATTTGGATTAACACCACAACC CACAGCCTGATTATCAGCATCATCCCACTGCTGTTTTTCAACCA GATTAATAATAACCTCTTTTCATGCTCCCCTACCTTTTCCTCTG ATCCCCTGACAACCCCCCTGCTCATGCTGACCACATGGCTGCT GCCTCTGACTATCATGGCCAGTCAGAGGCACCTCAGCTCAGA GCCTCTGTCAAGGAAAAAGCTGTACCTGTCCATGCTCATTAGC CTGCAAATCAGCCTGATTATGACCTTCACAGCCACTGAGCTGA TCATGTTCTATATCTTCTTTGAGACCACTCTCATCCCAACCCTG GCCATCATCACTAGGTGGGGAAATCAGCCTGAGAGGCTGAAT GCAGGCACCTACTTTCTCTTTTATACACTGGTGGGCTCACTGC CCCTGCTGATTGCACTGATCTATACCCACAACACACTGGGTAG CCTGAACATCCTCCTGCTGACTCTGACTGCCCAGGAGCTGAGT AACTCCTGGGCAAACAACCTGATGTGGCTGGCTTATACAATGG CCTTTATGGTGAAGATGCCCCTCTATGGCCTGCACCTCTGGCT CCCCAAGGCTCATGTGGAAGCTCCAATAGCTGGCTCCATGGT CCTGGCTGCTGTCCTCCTGAAGCTGGGTGGTTATGGTATGATG AGGCTCACCCTGATCCTGAACCCTCTGACAAAGCACATGGCTT ACCCTTTCCTGGTTCTGAGCCTCTGGGGTATGATCATGACCAG CTCCATCTGTCTCAGGCAGACTGACCTCAAGAGCCTGATTGCT TACAGCTCCATCAGCCACATGGCACTGGTGGTGACTGCCATTC TGATTCAGACACCCTGGTCCTTTACTGGGGCAGTGATCCTCAT GATTGCTCATGGGCTGACCAGCAGCCTCCTGTTCTGCCTGGC AAATAGCAACTATGAAAGAACTCACTCAAGAATTATGATCCTGA GTCAGGGGCTCCAGACCCTGCTCCCACTGATGGCTTTCTGGT GGCTCCTGGCTAGTCTGGCCAACCTGGCTCTGCCCCCTACAA TTAACCTGCTGGGAGAGCTGTCAGTGCTGGTGACAACATTCTC CTGGAGCAACATCACCCTGCTCCTGACAGGACTGAACATGCT GGTCACAGCCCTCTACAGCCTGTACATGTTTACCACTACCCAG TGGGGCTCACTGACTCACCACATTAATAACATGAAGCCCTCAT TCACCAGAGAGAACACCCTGATGTTTATGCATCTGTCTCCCAT CCTGCTGCTGTCCCTGAACCCAGACATTATCACTGGTTTCTCA T SEQ ID NO: 18 ND4 1380 ATGCTGAAGCTGATTGTGCCAACCATCATGCTGCTGCCTCTCA (5′ to 3′) nt CATGGCTGAGCAAAAAGCACATGATTTGGATTAACACCACAAC CCACAGCCTGATTATCAGCATCATCCCACTGCTGTTTTTCAACC AGATTAATAATAACCTCTTTTCATGCTCCCCTACCTTTTCCTCT GATCCCCTGACAACCCCCCTGCTCATGCTGACCACATGGCTG CTGCCTCTGACTATCATGGCCAGTCAGAGGCACCTCAGCTCA GAGCCTCTGTCAAGGAAAAAGCTGTACCTGTCCATGCTCATTA GCCTGCAAATCAGCCTGATTATGACCTTCACAGCCACTGAGCT GATCATGTTCTATATCTTCTTTGAGACCACTCTCATCCCAACCC TGGCCATCATCACTAGGTGGGGAAATCAGCCTGAGAGGCTGA ATGCAGGCACCTACTTTCTCTTTTATACACTGGTGGGCTCACT GCCCCTGCTGATTGCACTGATCTATACCCACAACACACTGGGT AGCCTGAACATCCTCCTGCTGACTCTGACTGCCCAGGAGCTG AGTAACTCCTGGGCAAACAACCTGATGTGGCTGGCTTATACAA TGGCCTTTATGGTGAAGATGCCCCTCTATGGCCTGCACCTCTG GCTCCCCAAGGCTCATGTGGAAGCTCCAATAGCTGGCTCCAT GGTCCTGGCTGCTGTCCTCCTGAAGCTGGGTGGTTATGGTAT GATGAGGCTCACCCTGATCCTGAACCCTCTGACAAAGCACATG GCTTACCCTTTCCTGGTTCTGAGCCTCTGGGGTATGATCATGA CCAGCTCCATCTGTCTCAGGCAGACTGACCTCAAGAGCCTGAT TGCTTACAGCTCCATCAGCCACATGGCACTGGTGGTGACTGC CATTCTGATTCAGACACCCTGGTCCTTTACTGGGGCAGTGATC CTCATGATTGCTCATGGGCTGACCAGCAGCCTCCTGTTCTGCC TGGCAAATAGCAACTATGAAAGAACTCACTCAAGAATTATGATC CTGAGTCAGGGGCTCCAGACCCTGCTCCCACTGATGGCTTTC TGGTGGCTCCTGGCTAGTCTGGCCAACCTGGCTCTGCCCCCT ACAATTAACCTGCTGGGAGAGCTGTCAGTGCTGGTGACAACAT TCTCCTGGAGCAACATCACCCTGCTCCTGACAGGACTGAACAT GCTGGTCACAGCCCTCTACAGCCTGTACATGTTTACCACTACC CAGTGGGGCTCACTGACTCACCACATTAATAACATGAAGCCCT CATTCACCAGAGAGAACACCCTGATGTTTATGCATCTGTCTCC CATCCTGCTGCTGTCCCTGAACCCAGACATTATCACTGGTTTC TCATCCTGA SEQ ID NO: 13 ND4 459 MLKLIVPTIMLLPLTWLSKKHMIWINTTTHSLIISIIPLLFFNQINNNLF AA SCSPTFSSDPLTTPLLMLTTWLLPLTIMASQRHLSSEPLSRKKLYL SMLISLQISLIMTFTATELIMFYIFFETTLIPTLAIITRWGNQPERLNA GTYFLFYTLVGSLPLLIALIYTHNTLGSLNILLLTLTAQELSNSWANN LMWLAYTMAFMVKMPLYGLHLWLPKAHVEAPIAGSMVLAAVLLKL GGYGMMRLTLILNPLTKHMAYPFLVLSLWGMIMTSSICLRQTDLK SLIAYSSISHMALVVTAILIQTPWSFTGAVILMIAHGLTSSLLFCLAN SNYERTHSRIMILSQGLQTLLPLMAFWWLLASLANLALPPTINLLG ELSVLVTTFSWSNITLLLTGLNMLVTALYSLYMFTTTQWGSLTHHI NNMKPSFTRENTLMFMHLSPILLLSLNPDIITGFSS
[0165] Without being bound by theory, mitochondrial genes may use a mitochondrial genetic code which is different from the universal genetic code used by nuclear genes. When a mitochondrial gene is inserted in a recombinant vector to be expressed in the nucleus, the mitochondrial nucleic acid sequence may be recoded in accordance with the universal genetic code, in order to be correctly expressed and/or translated outside the mitochondria. In some embodiments, a mitochondria-encoded gene may be recoded to form a nuclear-encoded version of the same gene. In some embodiments, the nuclear-encoded version is produced by codon substitution of the mitochondrial nucleic acid. In some embodiments, the nuclear-encoded version is produced by codon substitution to replace the codons of the mitochondrial genetic code with codons of the universal genetic code. Codon usage in the mitochondria vs. the universal genetic code is described in Lewin, Genes V, Oxford University Press; New York 1994, the content of which is incorporated by reference. Exemplary codon substitutions include but are not limited to UGA to UGG, AGA to UAA, UAG or UGA, AGG to UAA, UAG or UGA, AUA to AUG, CUG or GUG, AUU to AUG, CUG or GUG. In some embodiments, the nucleic acid encoding a human ND4 polypeptide is the sequence of a naturally occurring mitochondrial nucleic acid, recoded in accordance with the universal genetic code.
[0166] Due to the degeneracy of the genetic code, most amino acids can be encoded by multiple synonymous codons (Grantham et al., Nucleic Acids Res., 8(1):r49-r62 (1980). Without being bound by theory, synonymous codons naturally occur with different frequencies in different organisms. The choice of codons may affect protein expression, structure, and function. When expressing a recombinant protein, one may select specific codons to optimize for expression in a chosen host system, thus recoding by taking into account the preferred codon usage. In some embodiments, recoding is done taking into account the preferred usage codon of mammalian cells. In some embodiments, recoding is done taking into account the preferred codon usage in humans.
[0167] In some embodiments, the nucleic acid sequence encoding a human ND4 protein, recoded in accordance with the universal genetic code, and taking into account the human preferred usage codon comprises the nucleic acid sequence SEQ ID NO: 2 (3′ to 5′ sequence) or its reverse complement SEQ ID NO: 15 (5′ to 3′ sequence).
[0168] In some embodiments, the nucleic acid sequence encoding human ND4 protein, recoded in accordance with the universal genetic code, and taking into account the human preferred usage codon comprises the nucleic acid sequence SEQ ID NO: 17 (3′ to 5′ sequence) or its reverse complement SEQ ID NO: 18 (5′ to 3′ sequence).
[0169] The term “vector” refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, the vector is a DNA vector. In some embodiments, the vector is a circular vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is double-stranded. In some embodiments, the vector is single-stranded.
[0170] In some embodiment, the recombinant vector disclosed herein is a recombinant viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector, chimeric AAV vector, adenoviral vector, retroviral vector, lentiviral vector, DNA viral vector, herpes simplex viral vector, baculoviral vector, or any mutant or derivative thereof. In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (AAV) vector. In some embodiments, by an “AAV vector” is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8 and AAV-9. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, e.g., the rep and/or cap genes, while retaining functional flanking inverted terminal repeat (ITR) sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences that in cis provide for replication and packaging (e.g., functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. An “AAV vector” may also refer to the protein shell or capsid, which provides an efficient vehicle for delivery of vector nucleic acid to the nucleus of target cells. In some embodiments, the recombinant viral vector is a recombinant AAV2 vector. In some embodiments, a recombinant vector of the disclosure is a recombinant AAV vector, of serotype 2 (rAAV2/2).
[0171] In some embodiments, a recombinant AAV vector disclosed herein comprises a nucleic acid sequence encoding the ND4 protein, and operatively linked gene regulatory control sequences, including but not limited to promoters, enhancers, termination signals. Without being bound by theory, a cytomegalovirus (CMV) immediate early promoter may provide high and sustained expression levels of an operatively linked nucleic acid sequence in a cell. In some embodiments, the recombinant AAV vector of the disclosure comprises a cytomegalovirus (CMV) immediate early promoter. Without being bound by theory, intronic sequences incorporated into recombinant nucleic acid sequences or transgenes may stabilize mRNA levels and increase expression of an operatively linked nucleic acid sequence. In some embodiments, the recombinant AAV2 vector of the disclosure comprises a beta-globin (HBB2) derived intronic sequence.
[0172] In some embodiments, a recombinant AAV2 vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the human NADH dehydrogenase 4 (ND4) under the control of the cytomegalovirus immediate early promoter (CMV) in an intron-containing expression cassette (beta globin intron, HBB2), further comprising viral inverted terminal repeats (ITRs) from AAV2/2 (
TABLE-US-00002 TABLE 2 Sequences of various embodiments of the disclosure SEQ ID NO: 1 3′UTR 595 aacatgtggaatccccagtcccagtgcacagcagccgggtcctagctattggtattgt Cox10 nt tagaaggactatggttgagaatgtgtggggtggggaaaaccaaaaatgcaggccctgg (3′ to ctcagtcaccaggaagggggtgctccgaggcctttgagggacctgagctcacagaact 5′) acagacagcagatcatgaggctcacactttctggccaccaagtgccactctgctgggc atgtggagtgtgcgtgtggtgtggtggtaaggatggaaccaaaagaggaagctgtgta tgtgtacccccatgtgaggaggaagaaacagaatagagggtggggttggaggagagat gtataaagaccctcaaagggaaaaataattccttttttgtattcactgactgagctga tgcatttcttatttggggagcattttgggtaatatttaaaaaaaaaaaaaactgtcaa gtgatcactgggcaccgaattcgtttataatcttgttctaaacccagcaatttctctt cttgtgttccagaattaccacaacatgctcgcctggcagcggagggaaaggggcggtg ggcgtcccagtgctc SEQ ID NO: 14 3′UTR 595 gagcactgggacgcccaccgcccctttccctccgctgccaggcgagcatgttgtggta Cox10 nt attctggaacacaagaagagaaattgctgggtttagaacaagattataaacgaattcg (5′ to gtgcccagtgatcacttgacagttttttttttttttaaatattacccaaaatgctccc 3′) caaataagaaatgcatcagctcagtcagtgaatacaaaaaaggaattatttttccctt tgagggtctttatacatctctcctccaaccccaccctctattctgtttcttcctcctc acatgggggtacacatacacagcttcctcttttggttccatccttaccaccacaccac acgcacactccacatgcccagcagagtggcacttggtggccagaaagtgtgagcctca tgatctgctgtctgtagttctgtgagctcaggtccctcaaaggcctcggagcaccccc ttcctggtgactgagccagggcctgcatttttggttttccccaccccacacattctca accatagtccttctaacaataccaatagctaggacccggctgctgtgcactgggactg gggattccacatgtt SEQ ID NO: 3 MTS 84 nt agtcctcctttccaggtaccacacactcccccccacacagccagtcaggagcctggat Cox10 gacagtgtatgggggctggcagccat (3′ to 5′) SEQ ID NO: 16 MTS 84 nt atggctgccagcccccatacactgtcatccaggctcctgactggctgtgtggggggga Cox10 gtgtgtggtacctggaaaggaggact (5′ to 3′) SEQ ID NO: 4 HBB2 398 aattctttgccaaagtgatgggccagcacacagaccagcacgttgcccaggagctgtg intron nt ggaggaagataagaggtatgaacatgattagcaaaagggcctagcttggactcagaat (3′ to aatccagccttatcccaaccataaaataaaagcagaatggtagctggattgtagctgc 5′) tattagcaatatgaaacctcttacatcagttacaatttatatgcagaaatatttatat gcagaaatattgctattgccttaacccagaaattatcactgttattctttagaatggt gcaaagaggcatgatacattgtatcattattgccctgaaagaaagagattagggaaag tattagaaataagataaacaaaaaagtatattaaaagaagaaagcatttt SEQ ID NO: 24 HBB2 398 aaaatgctttcttcttttaatatacttttttgtttatcttatttctaatactttccct intron nt aatctctttctttcagggcaataatgatacaatgtatcatgcctctttgcaccattct (5′ to aaagaataacagtgataatttctgggttaaggcaatagcaatatttctgcatataaat 3′) atttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagctac aatccagctaccattctgcttttattttatggttgggataaggctggattattctgag tccaagctaggcccttttgctaatcatgttcatacctcttatcttcctcccacagctc ctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaatt SEQ ID NO: 5 CMV 654 ggaggctggatcggtcccggtgtcttctatggaggtcaaaacagcgtggatggcgtct promoter nt ccaggcgatctgacggttcactaaacgagctctgcttatatagacctcccaccgtaca (3′ cgcctaccgcccatttgcgtcaatggggcggagttgttacgacattttggaaagtccc to 5′) gttgattttggtgccaaaacaaactcccattgacgtcaatggggtggagacttggaaa tccccgtgagtcaaaccgctatccacgcccattgatgtactgccaaaaccgcatcacc atggtaatagcgatgactaatacgtagatgtactgccaagtaggaaagtcccataagg tcatgtactgggcataatgccaggcgggccatttaccgtcattgacgtcaataggggg cgtacttggcatatgatacacttgatgtactgccaagtgggcagtttaccgtaaatac tccacccattgacgtcaatggaaagtccctattggcgttactatgggaacatacgtca ttattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggccatttaccgt aagttatgtaacgcggaactccatatatgggctatgaactaatgaccccgtaattgat tactattaataactag SEQ ID NO: 25 CMV 654 ctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagtt promoter nt ccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgc (5′ ccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccatt to 3′) gacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgta tcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcat tatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattag tcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcg gtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttt tggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgc aaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaa ccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgg gaccgatccagcctcc SEQ ID NO: 6 ITR (3′ 128 aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactga to 5′) nt ggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagc gagcgagcgcgc SEQ ID NO: 26 ITR (5′ 128 gcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgaccttt to 3′) nt ggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatca ctaggggttcct SEQ ID NO: 7 ITR (3′ 130 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacct to 5′) nt ttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccat cactaggggttcct SEQ ID NO: 27 ITR (5′ 130 aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactga to 3′) nt ggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagc gagcgagcgcgcag
[0173] In some embodiments, the recombinant AAV2 vector of the disclosure comprises a coding sequence of human ND4 that is codon-optimized for improved expression in human cells.
[0174] In some embodiments, a recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0175] (i) a 3′UTR Cox/0 sequence comprising SEQ ID NO: 1; [0176] (ii) a coding sequence ND4 comprising SEQ ID NO: 2; and [0177] (iii) an MTS Cox/0 sequence comprising SEQ ID NO: 3.
[0178] In some embodiments, a recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0179] (i) a 3′UTR Cox/0 sequence consisting of SEQ ID NO: 1; [0180] (ii) a coding sequence ND4 consisting of SEQ ID NO: 2; and [0181] (iii) an MTS Cox/0 sequence consisting of SEQ ID NO: 3.
[0182] In some embodiments, a recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0183] (i) a 3′UTR Cox/0 sequence comprising SEQ ID NO: 1; [0184] (ii) a coding sequence ND4 comprising SEQ ID NO: 17; and [0185] (iii) an MTS Cox/0 sequence comprising SEQ ID NO: 3.
[0186] In some embodiments, a recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0187] (i) a 3′UTR Cox/0 sequence consisting of SEQ ID NO: 1; [0188] (ii) a coding sequence ND4 consisting of SEQ ID NO: 17; and [0189] (iii) an MTS Cox/0 sequence consisting of SEQ ID NO: 3.
[0190] In some embodiments, a recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0191] (i) a 3′UTR Cox/0 sequence comprising SEQ ID NO: 14; [0192] (ii) a coding sequence ND4 comprising SEQ ID NO: 15; and [0193] (iii) an MTS Cox/0 sequence comprising SEQ ID NO: 16
[0194] In some embodiments, a recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0195] (i) a 3′UTR Cox/0 sequence consisting of SEQ ID NO: 14; [0196] (ii) a coding sequence ND4 consisting of SEQ ID NO: 15; and [0197] (iii) an MTS Cox/0 sequence consisting of SEQ ID NO: 16.
[0198] In some embodiments, a recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0199] (i) a 3′UTR Cox/0 sequence comprising SEQ ID NO: 14; [0200] (ii) a coding sequence ND4 comprising SEQ ID NO: 18; and [0201] (iii) an MTS Cox/0 sequence comprising SEQ ID NO: 16.
[0202] In some embodiments, a recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0203] (i) a 3′UTR Cox/0 sequence consisting of SEQ ID NO: 14; [0204] (ii) a coding sequence ND4 consisting of SEQ ID NO: 18; and [0205] (iii) an MTS Cox/0 sequence consisting of SEQ ID NO: 16.
[0206] In some embodiments, a recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0207] (i) a 3′UTR Cox/0 sequence of:
TABLE-US-00003 (SEQ ID NO: 1) aacatgtggaatccccagtcccagtgcacagcagccgggtcctagctatt ggtattgttagaaggactatggttgagaatgtgtggggtggggaaaacca aaaatgcaggccctggctcagtcaccaggaagggggtgctccgaggcctt tgagggacctgagctcacagaactacagacagcagatcatgaggctcaca ctttctggccaccaagtgccactctgctgggcatgtggagtgtgcgtgtg gtgtggtggtaaggatggaaccaaaagaggaagctgtgtatgtgtacccc catgtgaggaggaagaaacagaatagagggtggggttggaggagagatgt ataaagaccctcaaagggaaaaataattccttttttgtattcactgactg agctgatgcatttcttatttggggagcattttgggtaatatttaaaaaaa aaaaaaactgtcaagtgatcactgggcaccgaattcgtttataatcttgt tctaaacccagcaatttctcttcttgtgttccagaattaccacaacatgc tcgcctggcagcggagggaaaggggcggtgggcgtcccagtgctc [0208] (ii) a coding sequence ND4 of:
TABLE-US-00004 (SEQ ID NO: 2) atgagaaaccagtgataatgtctgggttcagggacagcagcaggatggga gacagatgcataaacatcagggtgttctctctggtgaatgagggcttcat gttattaatgtggtgagtcagtgagccccactgggtagtggtaaacatgt acaggctgtagagggctgtgaccagcatgttcagtcctgtcaggagcagg gtgatgttgctccaggagaatgttgtcaccagcactgacagctctcccag caggttaattgtagggggcagagccaggttggccagactagccaggagcc accagaaagccatcagtgggagcagggtctggagcccctgactcaggatc ataattcttgagtgagttctttcatagttgctatttgccaggcagaacag gaggctgctggtcagcccatgagcaatcatgaggatcactgccccagtaa aggaccagggtgtctgaatcagaatggcagtcaccaccagtgccatgtgg ctgatggagctgtaagcaatcaggctcttgaggtcagtctgcctgagaca gatggagctggtcatgatcataccccagaggctcagaaccaggaaagggt aagccatgtgctttgtcagagggttcaggatcagggtgagcctcatcata ccataaccacccagcttcaggaggacagcagccaggaccatggagccagc tattggagcttccacatgagccttggggagccagaggtgcaggccataga ggggcatcttcaccataaaggccattgtataagccagccacatcaggttg tttgcccaggagttactcagctcctgggcagtcagagtcagcaggaggat gttcaggctacccagtgtgttgtgggtatagatcagtgcaatcagcaggg gcagtgagcccaccagtgtataaaagagaaagtaggtgcctgcattcagc ctctcaggctgatttccccacctagtgatgatggccagggttgggatgag agtggtctcaaagaagatatagaacatgatcagctcagtggctgtgaagg tcataatcaggctgatttgcaggctaatgagcatggacaggtacagcttt ttccttgacagaggctctgagctgaggtgcctctgactggccatgatagt cagaggcagcagccatgtggtcagcatgagcaggggggttgtcaggggat cagaggaaaaggtaggggagcatgaaaagaggttattattaatctggttg aaaaacagcagtgggatgatgctgataatcaggctgtgggttgtggtgtt aatccaaatcatgtgctttttgctcagccatgtgagaggcagcagcatga tggttggcacaatcagcttcagca [0209] (iii) an MTS Cox/0 sequence of:
TABLE-US-00005 (SEQ ID NO: 3) agtcctcctttccaggtaccacacactcccccccacacagccagtcagga gcctggatgacagtgtatgggggctggcagccat [0210] (iv) an HBB2 intron sequence of:
TABLE-US-00006 (SEQ ID NO: 4) aattctttgccaaagtgatgggccagcacacagaccagcacgttgcccag gagctgtgggaggaagataagaggtatgaacatgattagcaaaagggcct agcttggactcagaataatccagccttatcccaaccataaaataaaagca gaatggtagctggattgtagctgctattagcaatatgaaacctcttacat cagttacaatttatatgcagaaatatttatatgcagaaatattgctattg ccttaacccagaaattatcactgttattctttagaatggtgcaaagaggc atgatacattgtatcattattgccctgaaagaaagagattagggaaagta ttagaaataagataaacaaaaaagtatattaaaagaagaaagcatttt [0211] (v) a CMV promoter sequence of:
TABLE-US-00007 (SEQ ID NO: 5) ggaggctggatcggtcccggtgtcttctatggaggtcaaaacagcgtgga tggcgtctccaggcgatctgacggttcactaaacgagctctgcttatata gacctcccaccgtacacgcctaccgcccatttgcgtcaatggggcggagt tgttacgacattttggaaagtcccgttgattttggtgccaaaacaaactc ccattgacgtcaatggggtggagacttggaaatccccgtgagtcaaaccg ctatccacgcccattgatgtactgccaaaaccgcatcaccatggtaatag cgatgactaatacgtagatgtactgccaagtaggaaagtcccataaggtc atgtactgggcataatgccaggcgggccatttaccgtcattgacgtcaat agggggcgtacttggcatatgatacacttgatgtactgccaagtgggcag tttaccgtaaatactccacccattgacgtcaatggaaagtccctattggc gttactatgggaacatacgtcattattgacgtcaatgggcgggggtcgtt gggcggtcagccaggcgggccatttaccgtaagttatgtaacgcggaact ccatatatgggctatgaactaatgaccccgtaattgattactattaataa ctag [0212] (vi) an ITR sequence of:
TABLE-US-00008 (SEQ ID NO: 6) aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcg ctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccg ggcggcctcagtgagcgagcgagcgcgc,
and [0213] (vii) an ITR sequence of:
TABLE-US-00009 (SEQ ID NO: 7) ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcg ggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagg gagtggccaactccatcactaggggttcct.
[0214] In some embodiments, a recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0215] (i) a 3′UTR Cox/0 sequence comprising SEQ ID NO: 1, [0216] (ii) a coding sequence ND4 comprising SEQ ID NO: 17, [0217] (iii) an MTS Cox/0 sequence comprising SEQ ID NO: 3, [0218] (iv) an HBB2 intron sequence comprising SEQ ID NO: 4, [0219] (v) a CMV promoter sequence comprising SEQ ID NO: 5, [0220] (vi) an ITR sequence comprising SEQ ID NO: 6, and [0221] (vii) an ITR sequence comprising SEQ ID NO: 7.
[0222] In some embodiments, the recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0223] (i) a 3′UTR Cox/0 sequence comprising SEQ ID NO: 14, [0224] (ii) a coding sequence ND4 comprising SEQ ID NO: 15, [0225] (iii) an MTS Cox/0 sequence comprising SEQ ID NO: 16, [0226] (iv) an HBB2 intron sequence comprising SEQ ID NO: 24, [0227] (v) a CMV promoter sequence comprising SEQ ID NO: 25, [0228] (vi) an ITR sequence comprising SEQ ID NO: 26, and [0229] (vii) an ITR sequence comprising SEQ ID NO: 27.
[0230] In some embodiments, the recombinant AAV vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding the gene of the human NADH dehydrogenase 4 (ND4), and comprises: [0231] (i) a 3′UTR Cox/0 sequence comprising SEQ ID NO: 14, [0232] (ii) a coding sequence ND4 comprising SEQ ID NO: 18, [0233] (iii) an MTS Cox/0 sequence comprising SEQ ID NO: 16, [0234] (iv) an HBB2 intron sequence comprising SEQ ID NO: 24, [0235] (v) a CMV promoter sequence comprising SEQ ID NO: 25, [0236] (vi) an ITR sequence comprising SEQ ID NO: 26, and [0237] (vii) an ITR sequence comprising SEQ ID NO: 27.
[0238] In some embodiment, the recombinant vector of the disclosure further comprises: [0239] (i) an HBB2 intron sequence comprising SEQ ID NO: 4, [0240] (ii) a CMV promoter sequence comprising SEQ ID NO: 5, [0241] (iii) an ITR sequence comprising SEQ ID NO: 6, and [0242] (iv) an ITR sequence comprising SEQ ID No: 7.
[0243] In some embodiment, the recombinant vector of the disclosure further comprises: [0244] (i) an HBB2 intron sequence consisting of SEQ ID NO: 4, [0245] (ii) a CMV promoter sequence consisting of SEQ ID NO: 5, [0246] (iii) an ITR sequence consisting of SEQ ID NO: 6, and [0247] (iv) an ITR sequence consisting of SEQ ID No: 7.
[0248] In some embodiment, the recombinant vector of the disclosure further comprises: [0249] (i) an HBB2 intron sequence comprising SEQ ID NO: 24, [0250] (ii) a CMV promoter sequence comprising SEQ ID NO: 25, [0251] (iii) an ITR sequence comprising SEQ ID NO: 26, and [0252] (iv) an ITR sequence comprising SEQ ID No: 27.
[0253] In some embodiment, the recombinant vector of the disclosure further comprises: [0254] (i) an HBB2 intron sequence consisting of SEQ ID NO: 24, [0255] (ii) a CMV promoter sequence consisting of SEQ ID NO: 25, [0256] (iii) an ITR sequence consisting of SEQ ID NO: 26, and [0257] (iv) an ITR sequence consisting of SEQ ID No: 27.
[0258] In some embodiments, a recombinant vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding a ND4 protein, and comprises: [0259] a 3′UTR Cox/0 sequence comprising SEQ ID NO: 14, [0260] a nucleic acid sequence encoding an ND4 polypeptide comprising SEQ ID NO: 13, and [0261] a nucleic acid sequence encoding an MTS Cox/0 polypeptide sequence comprising SEQ ID NO: 11.
[0262] In some embodiments, a recombinant vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding a ND4 protein, and comprises: [0263] a 3′UTR Cox/0 sequence comprising SEQ ID NO: 14, [0264] a nucleic acid sequence encoding an ND4 polypeptide SEQ ID NO: 13, and [0265] a nucleic acid sequence encoding MTS Cox/0 polypeptide sequence comprising SEQ ID NO: 12.
[0266] In some embodiments, a recombinant vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding a ND4 protein, and comprises: [0267] a 3′UTR Cox/0 sequence comprising SEQ ID NO: 1, [0268] a nucleic acid sequence encoding an ND4 polypeptide comprising SEQ ID NO: 13, and [0269] a nucleic acid sequence encoding an MTS Cox/0 polypeptide sequence comprising SEQ ID NO: 11.
[0270] In some embodiments, a recombinant vector of the disclosure is a recombinant adeno-associated virus (AAV), serotype 2, (rAAV2/2) encoding a ND4 protein, and comprises: [0271] a 3′UTR Cox/0 sequence comprising SEQ ID NO: 1, [0272] a nucleic acid sequence encoding an ND4 polypeptide SEQ ID NO: 13, and [0273] a nucleic acid sequence encoding MTS Cox/0 polypeptide sequence comprising SEQ ID NO: 12.
TABLE-US-00010 TABLE 3 Sequences of various embodiments of the disclosure SEQ ID Cox10 MTS 20 MAASPHTLSSRLLTGCVGGS NO: 11 (cleaved) AA SEQ ID Cox10 MTS 28 MAASPHTLSSRLLTGCVGGSVWYLERRT NO: 12 (uncleaved) AA
[0274] Sequences such as promoters, introns or ITR are well known to person of ordinary skill in the art who can easily interchange each of them with other elements known in the art.
[0275] Recombinant vectors of the disclosure are useful in treating Leber Hereditary Optic Neuroretinopathy (LHON), including ND4-related LHON.
[0276] In some embodiments, a recombinant vector of the disclosure is administered to a patient in need thereof via intravitreal injection.
[0277] In some embodiments, a recombinant vector of the disclosure is administered to a patient in need thereof via a single intravitreal injection.
[0278] In some embodiments, a recombinant viral vector of the disclosure is administered to patients in need thereof in one or more doses of about 10.sup.9 to 10.sup.11 vg (viral genomes) per eye. In some embodiments, a recombinant AAV2 vector of the disclosure is administered to patients in need thereof in one or more doses of about 10.sup.10 vg per eye, for example 9×10.sup.10 vg per eye.
[0279] One aspect of the disclosure pertains to a pAAV-ND4 transfer plasmid that, in some embodiments, may be used in the preparation of a recombinant AAV2 vector of the disclosure.
[0280] In some embodiments, a pAAV-ND4 transfer plasmid of the disclosure comprises the following functional elements and sequences:
3′UTR Cox10: nt 11-605=595 bp
[0281] Coding sequence ND4: nt 618-1997=1380 bp
MTS Cox10: nt 1998-2081=84 bp
[0282] HBB2 intron: nt 2124-2616=493 bp
CMV promoter: nt 2624-3283=660 bp
ITR: nt 3327-3454=128 bp
[0283] F1 origin: nt 3872-4327=456 bp
Kana R gene: nt 4482-5273=792 bp
COLE1 origin: nt 5488-6102=615 bp
ITR: nt 6324-6453=130 bp.
[0284]
TABLE-US-00011 TABLE 4 Annotated regions within one embodiment of the pAAV-ND4 plasmid Name Start Stop Sequence 3′UTR 11 605 aacatgtggaatccccagtcccagtgcacagcagccgggtcctagctattggtattgttagaa COX10 ggactatggttgagaatgtgtggggtggggaaaaccaaaaatgcaggccctggctcagtca (polyA ccaggaagggggtgctccgaggcctttgagggacctgagctcacagaactacagacagca sequence gatcatgaggctcacactttctggccaccaagtgccactctgctgggcatgtggagtgtgcgtg underlined) tggtgtggtggtaaggatggaaccaaaagaggaagctgtgtatgtgtacccccatgtgagga ggaagaaacagaatagagggtggggttggaggagagatgtataaagaccctcaaaggga aaaataattccttttttgtattcactgactgagctgatgcatttcttatttggggagcattttgggtaat atttaaaaaaaaaaaaaactgtcaagtgatcactgggcaccgaattcgtttataatcttgttcta aacccagcaatttctcttcttgtgttccagaattaccacaacatgctcgcctggcagcggaggg aaaggggcggtgggcgtcccagtgctc ND4 618 1997 tcaggatgagaaaccagtgataatgtctgggttcagggacagcagcaggatgggagacag atgcataaacatcagggtgttctctctggtgaatgagggcttcatgttattaatgtggtgagtcagt gagccccactgggtagtggtaaacatgtacaggctgtagagggctgtgaccagcatgttcag tcctgtcaggagcagggtgatgttgctccaggagaatgttgtcaccagcactgacagctctcc cagcaggttaattgtagggggcagagccaggttggccagactagccaggagccaccagaa agccatcagtgggagcagggtctggagcccctgactcaggatcataattcttgagtgagttcttt catagttgctatttgccaggcagaacaggaggctgctggtcagcccatgagcaatcatgagg atcactgccccagtaaaggaccagggtgtctgaatcagaatggcagtcaccaccagtgcca tgtggctgatggagctgtaagcaatcaggctcttgaggtcagtctgcctgagacagatggagc tggtcatgatcataccccagaggctcagaaccaggaaagggtaagccatgtgctttgtcaga gggttcaggatcagggtgagcctcatcataccataaccacccagcttcaggaggacagcag ccaggaccatggagccagctattggagcttccacatgagccttggggagccagaggtgcag gccatagaggggcatcttcaccataaaggccattgtataagccagccacatcaggttgtttgc ccaggagttactcagctcctgggcagtcagagtcagcaggaggatgttcaggctacccagtg tgttgtgggtatagatcagtgcaatcagcaggggcagtgagcccaccagtgtataaaagaga aagtaggtgcctgcattcagcctctcaggctgatttccccacctagtgatgatggccagggttg ggatgagagtggtctcaaagaagatatagaacatgatcagctcagtggctgtgaaggtcata atcaggctgatttgcaggctaatgagcatggacaggtacagctttttccttgacagaggctctg agctgaggtgcctctgactggccatgatagtcagaggcagcagccatgtggtcagcatgagc aggggggttgtcaggggatcagaggaaaaggtaggggagcatgaaaagaggttattatta atctggttgaaaaacagcagtgggatgatgctgataatcaggctgtgggttgtggtgttaatcc aaatcatgtgctttttgctcagccatgtgagaggcagcagcatgatggttggcacaatcagcttc agcat MTS Cox10 1998 2081 agtcctcctttccaggtaccacacactcccccccacacagccagtcaggagcctggatgaca gtgtatgggggctggcagccat betaglobin 2124 2616 atcccaattctttgccaaagtgatgggccagcacacagaccagcacgttgcccaggagctgt intron gggaggaagataagaggtatgaacatgattagcaaaagggcctagcttggactcagaata atccagccttatcccaaccataaaataaaagcagaatggtagctggattgtagctgctattagc aatatgaaacctcttacatcagttacaatttatatgcagaaatatttatatgcagaaatattgctatt gccttaacccagaaattatcactgttattctttagaatggtgcaaagaggcatgatacattgtatc attattgccctgaaagaaagagattagggaaagtattagaaataagataaacaaaaaagtat attaaaagaagaaagcattttttgtgggcctatagactctataggcggtacttacgtcactcttgg cacggggaatccgcgttccaatgcaccgttcccggccgggattcg CMV 2624 3283 ggaggctggatcggtcccggtgtcttctatggaggtcaaaacagcgtggatggcgtctccagg promoter cgatctgacggttcactaaacgagctctgcttatatagacctcccaccgtacacgcctaccgcc catttgcgtcaatggggcggagttgttacgacattttggaaagtcccgttgattttggtgccaaaa caaactcccattgacgtcaatggggtggagacttggaaatccccgtgagtcaaaccgctatc cacgcccattgatgtactgccaaaaccgcatcaccatggtaatagcgatgactaatacgtag atgtactgccaagtaggaaagtcccataaggtcatgtactgggcataatgccaggcgggcca tttaccgtcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtactgccaagt gggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccctattggcgttactatg ggaacatacgtcattattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggcc atttaccgtaagttatgtaacgcggaactccatatatgggctatgaactaatgaccccgtaattg attactattaataactagacgcgt 5′ ITR 3327 3454 aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccg ggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcga gcgcgc F1 origin 3872 4327 acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgc tacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccg gctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacct cgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggt ttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaaca ctcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaa atgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaattt Kana R gene 4482 5273 attgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctat gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggg gcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggc agcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcact gaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctca ccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgat ccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgg atggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccag ccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgaccca tggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtgg ccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaaga gcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcag cgcatcgccttctatcgccttcttgacgagttcttctga ColE1 origin 5488 6102 aaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccg ctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttc agcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaag aactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtgg cgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtc gggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaact gagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgg acaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagg gggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgt gatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcg 3′ ITR 6324 6453 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttg gtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcacta ggggttcct
[0285] In some embodiments, a pAAV-ND4 transfer plasmid of the disclosure comprises: [0286] (i) a 3′UTR Cox/0 sequence of SEQ ID NO: 1, [0287] (ii) a coding sequence ND4 of SEQ ID NO: 2, [0288] (iii) an MTS Cox/0 sequence of SEQ ID NO: 3, [0289] (iv) an HBB2 intron sequence of SEQ ID NO: 4, [0290] (v) a CMV promoter sequence of SEQ ID NO: 5, [0291] (vi) an ITR sequence of SEQ ID NO: 6, and [0292] (vii) an ITR sequence of SEQ ID NO: 7.
[0293] In some embodiments, a pAAV-ND4 transfer plasmid of the disclosure comprises: [0294] (i) a 3′UTR Cox/0 sequence of SEQ ID NO: 1, [0295] (ii) a coding sequence ND4 of SEQ ID NO: 17, [0296] (iii) an MTS Cox/0 sequence of SEQ ID NO: 3, [0297] (iv) an HBB2 intron sequence of SEQ ID NO: 4, [0298] (v) a CMV promoter sequence of SEQ ID NO: 5, [0299] (vi) an ITR sequence of SEQ ID NO: 6, and [0300] (vii) an ITR sequence of SEQ ID NO: 7.
[0301] In some embodiments, a pAAV-ND4 transfer plasmid of the disclosure comprises: [0302] (i) a 3′UTR Cox/0 sequence of SEQ ID NO: 14, [0303] (ii) a coding sequence ND4 of SEQ ID NO: 15, [0304] (iii) an MTS Cox/0 sequence of SEQ ID NO: 16, [0305] (iv) an HBB2 intron sequence of SEQ ID NO: 24, [0306] (v) a CMV promoter sequence of SEQ ID NO: 25, [0307] (vi) an ITR sequence of SEQ ID NO: 26, and [0308] (vii) an ITR sequence of SEQ ID NO: 27.
[0309] In some embodiments, a pAAV-ND4 transfer plasmid of the disclosure comprises: [0310] (i) a 3′UTR Cox/0 sequence of SEQ ID NO: 14, [0311] (ii) a coding sequence ND4 of SEQ ID NO: 18, [0312] (iii) an MTS Cox/0 sequence of SEQ ID NO: 16, [0313] (iv) an HBB2 intron sequence of SEQ ID NO: 24, [0314] (v) a CMV promoter sequence of SEQ ID NO: 25, [0315] (vi) an ITR sequence of SEQ ID NO: 26, and [0316] (vii) an ITR sequence of SEQ ID NO: 27.
[0317] In some embodiments, a pAAV-ND4 transfer plasmid of the disclosure comprises:
TABLE-US-00012 (SEQ ID NO: 22) gctcggtccgaacatgtggaatccccagtcccagtgcacagcagccgggtcctagctattggtattgttaga aggactatggttgagaatgtgtggggtggggaaaaccaaaaatgcaggccctggctcagtcaccaggaa gggggtgctccgaggcctttgagggacctgagctcacagaactacagacagcagatcatgaggctcaca ctttctggccaccaagtgccactctgctgggcatgtggagtgtgcgtgtggtgtggtggtaaggatggaacca aaagaggaagctgtgtatgtgtacccccatgtgaggaggaagaaacagaatagagggtggggttggag gagagatgtataaagaccctcaaagggaaaaataattccttttttgtattcactgactgagctgatgcatttctt atttggggagcattttgggtaatatttaaaaaaaaaaaaaactgtcaagtgatcactgggcaccgaattcgttt ataatcttgttctaaacccagcaatttctcttcttgtgttccagaattaccacaacatgctcgcctggcagcgga gggaaaggggcggtgggcgtcccagtgctcagatctctcgagtcaggatgagaaaccagtgataatgtct gggttcagggacagcagcaggatgggagacagatgcataaacatcagggtgttctctctggtgaatgagg gcttcatgttattaatgtggtgagtcagtgagccccactgggtagtggtaaacatgtacaggctgtagagggc tgtgaccagcatgttcagtcctgtcaggagcagggtgatgttgctccaggagaatgttgtcaccagcactga cagctctcccagcaggttaattgtagggggcagagccaggttggccagactagccaggagccaccagaa agccatcagtgggagcagggtctggagcccctgactcaggatcataattcttgagtgagttctttcatagttgc tatttgccaggcagaacaggaggctgctggtcagcccatgagcaatcatgaggatcactgccccagtaaa ggaccagggtgtctgaatcagaatggcagtcaccaccagtgccatgtggctgatggagctgtaagcaatc aggctcttgaggtcagtctgcctgagacagatggagctggtcatgatcataccccagaggctcagaaccag gaaagggtaagccatgtgctttgtcagagggttcaggatcagggtgagcctcatcataccataaccaccca gcttcaggaggacagcagccaggaccatggagccagctattggagcttccacatgagccttggggagcc agaggtgcaggccatagaggggcatcttcaccataaaggccattgtataagccagccacatcaggttgttt gcccaggagttactcagctcctgggcagtcagagtcagcaggaggatgttcaggctacccagtgtgttgtg ggtatagatcagtgcaatcagcaggggcagtgagcccaccagtgtataaaagagaaagtaggtgcctgc attcagcctctcaggctgatttccccacctagtgatgatggccagggttgggatgagagtggtctcaaagaa gatatagaacatgatcagctcagtggctgtgaaggtcataatcaggctgatttgcaggctaatgagcatgga caggtacagctttttccttgacagaggctctgagctgaggtgcctctgactggccatgatagtcagaggcagc agccatgtggtcagcatgagcaggggggttgtcaggggatcagaggaaaaggtaggggagcatgaaaa gaggttattattaatctggttgaaaaacagcagtgggatgatgctgataatcaggctgtgggttgtggtgttaat ccaaatcatgtgctttttgctcagccatgtgagaggcagcagcatgatggttggcacaatcagcttcagcata gtcctcctttccaggtaccacacactcccccccacacagccagtcaggagcctggatgacagtgtatgggg gctggcagccatgtcgactctagaggatccccggggaattcaatcgatgttcgaatcccaattctttgccaaa gtgatgggccagcacacagaccagcacgttgcccaggagctgtgggaggaagataagaggtatgaaca tgattagcaaaagggcctagcttggactcagaataatccagccttatcccaaccataaaataaaagcaga atggtagctggattgtagctgctattagcaatatgaaacctcttacatcagttacaatttatatgcagaaatattt atatgcagaaatattgctattgccttaacccagaaattatcactgttattctttagaatggtgcaaagaggcatg atacattgtatcattattgccctgaaagaaagagattagggaaagtattagaaataagataaacaaaaaag tatattaaaagaagaaagcattttttgtgggcctatagactctataggcggtacttacgtcactcttggcacggg gaatccgcgttccaatgcaccgttcccggccgggattcgaatccgcggaggctggatcggtcccggtgtctt ctatggaggtcaaaacagcgtggatggcgtctccaggcgatctgacggttcactaaacgagctctgcttata tagacctcccaccgtacacgcctaccgcccatttgcgtcaatggggcggagttgttacgacattttggaaagt cccgttgattttggtgccaaaacaaactcccattgacgtcaatggggtggagacttggaaatccccgtgagtc aaaccgctatccacgcccattgatgtactgccaaaaccgcatcaccatggtaatagcgatgactaatacgt agatgtactgccaagtaggaaagtcccataaggtcatgtactgggcataatgccaggcgggccatttaccg tcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtactgccaagtgggcagtttaccgtaa atactccacccattgacgtcaatggaaagtccctattggcgttactatgggaacatacgtcattattgacgtca atgggcgggggtcgttgggcggtcagccaggcgggccatttaccgtaagttatgtaacgcggaactccata tatgggctatgaactaatgaccccgtaattgattactattaataactagacgcgtgcggccgtagataagtag catggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgct cgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagc gagcgagcgcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgca gcctgaatggcgaatggattccagacgattgagcgtcaaaatgtaggtatttccatgagcgtttttccgttgca atggctggcggtaatattgttctggatattaccagcaaggccgatagtttgagttcttctactcaggcaagtgat gttattactaatcaaagaagtattgcgacaacggttaatttgcgtgatggacagactcttttactcggtggcctc actgattataaaaacacttctcaggattctggcgtaccgttcctgtctaaaatccctttaatcggcctcctgtttag ctcccgctctgattctaacgaggaaagcacgttatacgtgctcgtcaaagcaaccatagtacgcgccctgta gcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagc gcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggc tccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgta gtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttc caaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattg gttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtgg cacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatga gacaataaccctgataaatgcttcaataatagcacctagatcaagagacaggatgaggatcgtttcgcatg attgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggca caacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtca agaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacga cgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaa gtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgc ggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagc acgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgat gcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggc ggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgacc gcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttct gaattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatcaggt ggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcat gaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttct tgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgc cggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtcc ttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatc ctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccgg ataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctac accgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgg acaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacg cctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagggggg cggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacat gttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgca gccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccg cctctccccgcgcgttggccgattcattaatgcagctgcgcgctcgctcgctcactgaggccgcccgggcaa agcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtg gccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatcta
[0318] In some other embodiments, a pAAV-ND4 transfer plasmid of the disclosure comprises the following functional elements and sequences:
3′UTR Cox10: nt 11-605=595 bp
[0319] Coding sequence ND4: nt 618-1997=1380 bp
MTS Cox10: nt 1998-2081=84 bp
[0320] HBB2 intron: nt 2124-2616=493 bp
CMV promoter: nt 2624-3283=660 bp
ITR: nt 3327-3454=128 bp
[0321] F1 origin: nt 3827-4282=456 bp
Kana R gene: nt 4437-5228=792 bp
COLE1 origin: nt 5443-6057=615 bp
ITR: nt 6279-6408=130 bp.
[0322]
TABLE-US-00013 TABLE 5 Annotated regions within one embodiment of the pAAV-ND4 plasmid Name Start Stop Sequence 3′UTR 11 605 aacatgtggaatccccagtcccagtgcacagcagccgggtcctagctattggtattgttagaa COX10 ggactatggttgagaatgtgtggggtggggaaaaccaaaaatgcaggccctggctcagtca (polyA ccaggaagggggtgctccgaggcctttgagggacctgagctcacagaactacagacagca sequence gatcatgaggctcacactttctggccaccaagtgccactctgctgggcatgtggagtgtgcgtg underlined) tggtgtggtggtaaggatggaaccaaaagaggaagctgtgtatgtgtacccccatgtgagga ggaagaaacagaatagagggtggggttggaggagagatgtataaagaccctcaaaggga aaaataattccttttttgtattcactgactgagctgatgcatttcttatttggggagcattttgggtaat atttaaaaaaaaaaaaaactgtcaagtgatcactgggcaccgaattcgtttataatcttgttcta aacccagcaatttctcttcttgtgttccagaattaccacaacatgctcgcctggcagcggaggg aaaggggcggtgggcgtcccagtgctc ND4 618 1997 tcaggatgagaaaccagtgataatgtctgggttcagggacagcagcaggatgggagacag atgcataaacatcagggtgttctctctggtgaatgagggcttcatgttattaatgtggtgagtcagt gagccccactgggtagtggtaaacatgtacaggctgtagagggctgtgaccagcatgttcag tcctgtcaggagcagggtgatgttgctccaggagaatgttgtcaccagcactgacagctctcc cagcaggttaattgtagggggcagagccaggttggccagactagccaggagccaccagaa agccatcagtgggagcagggtctggagcccctgactcaggatcataattcttgagtgagttcttt catagttgctatttgccaggcagaacaggaggctgctggtcagcccatgagcaatcatgagg atcactgccccagtaaaggaccagggtgtctgaatcagaatggcagtcaccaccagtgcca tgtggctgatggagctgtaagcaatcaggctcttgaggtcagtctgcctgagacagatggagc tggtcatgatcataccccagaggctcagaaccaggaaagggtaagccatgtgctttgtcaga gggttcaggatcagggtgagcctcatcataccataaccacccagcttcaggaggacagcag ccaggaccatggagccagctattggagcttccacatgagccttggggagccagaggtgcag gccatagaggggcatcttcaccataaaggccattgtataagccagccacatcaggttgtttgc ccaggagttactcagctcctgggcagtcagagtcagcaggaggatgttcaggctacccagtg tgttgtgggtatagatcagtgcaatcagcaggggcagtgagcccaccagtgtataaaagaga aagtaggtgcctgcattcagcctctcaggctgatttccccacctagtgatgatggccagggttg ggatgagagtggtctcaaagaagatatagaacatgatcagctcagtggctgtgaaggtcata atcaggctgatttgcaggctaatgagcatggacaggtacagctttttccttgacagaggctctg agctgaggtgcctctgactggccatgatagtcagaggcagcagccatgtggtcagcatgagc aggggggttgtcaggggatcagaggaaaaggtaggggagcatgaaaagaggttattatta atctggttgaaaaacagcagtgggatgatgctgataatcaggctgtgggttgtggtgttaatcc aaatcatgtgctttttgctcagccatgtgagaggcagcagcatgatggttggcacaatcagcttc agcat MTS Cox10 1998 2081 agtcctcctttccaggtaccacacactcccccccacacagccagtcaggagcctggatgaca gtgtatgggggctggcagccat betaglobin 2124 2616 atcccaattctttgccaaagtgatgggccagcacacagaccagcacgttgcccaggagctgt intron gggaggaagataagaggtatgaacatgattagcaaaagggcctagcttggactcagaata atccagccttatcccaaccataaaataaaagcagaatggtagctggattgtagctgctattagc aatatgaaacctcttacatcagttacaatttatatgcagaaatatttatatgcagaaatattgctatt gccttaacccagaaattatcactgttattctttagaatggtgcaaagaggcatgatacattgtatc attattgccctgaaagaaagagattagggaaagtattagaaataagataaacaaaaaagtat attaaaagaagaaagcattttttgtgggcctatagactctataggcggtacttacgtcactcttgg cacggggaatccgcgttccaatgcaccgttcccggccgggattcg CMV 2624 3283 ggaggctggatcggtcccggtgtcttctatggaggtcaaaacagcgtggatggcgtctccagg promoter cgatctgacggttcactaaacgagctctgcttatatagacctcccaccgtacacgcctaccgcc catttgcgtcaatggggcggagttgttacgacattttggaaagtcccgttgattttggtgccaaaa caaactcccattgacgtcaatggggtggagacttggaaatccccgtgagtcaaaccgctatc cacgcccattgatgtactgccaaaaccgcatcaccatggtaatagcgatgactaatacgtag atgtactgccaagtaggaaagtcccataaggtcatgtactgggcataatgccaggcgggcca tttaccgtcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtactgccaagt gggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccctattggcgttactatg ggaacatacgtcattattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggcc atttaccgtaagttatgtaacgcggaactccatatatgggctatgaactaatgaccccgtaattg attactattaataactagacgcgt 5′ ITR 3327 3454 aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccg ggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcga gcgcgc F1 origin 3827 4282 acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgc tacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccg gctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacct cgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggt ttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaaca ctcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaa atgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaattt Kana R gene 4437 5228 attgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctat gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggg gcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggc agcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcact gaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctca ccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgat ccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgg atggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccag ccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgaccca tggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtgg ccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaaga gcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcag cgcatcgccttctatcgccttcttgacgagttcttctga ColE1 origin 5443 6057 aaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccg ctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttc agcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaag aactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtgg cgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtc gggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaact gagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgg acaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagg gggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgt gatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcg 3′ ITR 6279 6408 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttg gtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcacta ggggttcct
[0323] In some embodiments, a pAAV-ND4 transfer plasmid of the disclosure comprises:
TABLE-US-00014 (SEQ ID NO: 23) gctcggtccgaacatgtggaatccccagtcccagtgcacagcagccgggtcctagctattggtattgttaga aggactatggttgagaatgtgtggggtggggaaaaccaaaaatgcaggccctggctcagtcaccaggaa gggggtgctccgaggcctttgagggacctgagctcacagaactacagacagcagatcatgaggctcaca ctttctggccaccaagtgccactctgctgggcatgtggagtgtgcgtgtggtgtggtggtaaggatggaacca aaagaggaagctgtgtatgtgtacccccatgtgaggaggaagaaacagaatagagggtggggttggag gagagatgtataaagaccctcaaagggaaaaataattccttttttgtattcactgactgagctgatgcatttctt atttggggagcattttgggtaatatttaaaaaaaaaaaaaactgtcaagtgatcactgggcaccgaattcgttt ataatcttgttctaaacccagcaatttctcttcttgtgttccagaattaccacaacatgctcgcctggcagcgga gggaaaggggcggtgggcgtcccagtgctcagatctctcgagtcaggatgagaaaccagtgataatgtct gggttcagggacagcagcaggatgggagacagatgcataaacatcagggtgttctctctggtgaatgagg gcttcatgttattaatgtggtgagtcagtgagccccactgggtagtggtaaacatgtacaggctgtagagggc tgtgaccagcatgttcagtcctgtcaggagcagggtgatgttgctccaggagaatgttgtcaccagcactga cagctctcccagcaggttaattgtagggggcagagccaggttggccagactagccaggagccaccagaa agccatcagtgggagcagggtctggagcccctgactcaggatcataattcttgagtgagttctttcatagttgc tatttgccaggcagaacaggaggctgctggtcagcccatgagcaatcatgaggatcactgccccagtaaa ggaccagggtgtctgaatcagaatggcagtcaccaccagtgccatgtggctgatggagctgtaagcaatc aggctcttgaggtcagtctgcctgagacagatggagctggtcatgatcataccccagaggctcagaaccag gaaagggtaagccatgtgctttgtcagagggttcaggatcagggtgagcctcatcataccataaccaccca gcttcaggaggacagcagccaggaccatggagccagctattggagcttccacatgagccttggggagcc agaggtgcaggccatagaggggcatcttcaccataaaggccattgtataagccagccacatcaggttgttt gcccaggagttactcagctcctgggcagtcagagtcagcaggaggatgttcaggctacccagtgtgttgtg ggtatagatcagtgcaatcagcaggggcagtgagcccaccagtgtataaaagagaaagtaggtgcctgc attcagcctctcaggctgatttccccacctagtgatgatggccagggttgggatgagagtggtctcaaagaa gatatagaacatgatcagctcagtggctgtgaaggtcataatcaggctgatttgcaggctaatgagcatgga caggtacagctttttccttgacagaggctctgagctgaggtgcctctgactggccatgatagtcagaggcagc agccatgtggtcagcatgagcaggggggttgtcaggggatcagaggaaaaggtaggggagcatgaaaa gaggttattattaatctggttgaaaaacagcagtgggatgatgctgataatcaggctgtgggttgtggtgttaat ccaaatcatgtgctttttgctcagccatgtgagaggcagcagcatgatggttggcacaatcagcttcagcata gtcctcctttccaggtaccacacactcccccccacacagccagtcaggagcctggatgacagtgtatgggg gctggcagccatgtcgactctagaggatccccggggaattcaatcgatgttcgaatcccaattctttgccaaa gtgatgggccagcacacagaccagcacgttgcccaggagctgtgggaggaagataagaggtatgaaca tgattagcaaaagggcctagcttggactcagaataatccagccttatcccaaccataaaataaaagcaga atggtagctggattgtagctgctattagcaatatgaaacctcttacatcagttacaatttatatgcagaaatattt atatgcagaaatattgctattgccttaacccagaaattatcactgttattctttagaatggtgcaaagaggcatg atacattgtatcattattgccctgaaagaaagagattagggaaagtattagaaataagataaacaaaaaag tatattaaaagaagaaagcattttttgtgggcctatagactctataggcggtacttacgtcactcttggcacggg gaatccgcgttccaatgcaccgttcccggccgggattcgaatccgcggaggctggatcggtcccggtgtctt ctatggaggtcaaaacagcgtggatggcgtctccaggcgatctgacggttcactaaacgagctctgcttata tagacctcccaccgtacacgcctaccgcccatttgcgtcaatggggcggagttgttacgacattttggaaagt cccgttgattttggtgccaaaacaaactcccattgacgtcaatggggtggagacttggaaatccccgtgagtc aaaccgctatccacgcccattgatgtactgccaaaaccgcatcaccatggtaatagcgatgactaatacgt agatgtactgccaagtaggaaagtcccataaggtcatgtactgggcataatgccaggcgggccatttaccg tcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtactgccaagtgggcagtttaccgtaa atactccacccattgacgtcaatggaaagtccctattggcgttactatgggaacatacgtcattattgacgtca atgggcgggggtcgttgggcggtcagccaggcgggccatttaccgtaagttatgtaacgcggaactccata tatgggctatgaactaatgaccccgtaattgattactattaataactagacgcgtgcggccgtagataagtag catggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgct cgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagc gagcgagcgcgcagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcag cctgaatggcgaatggcgattccgttgcaatggctggcggtaatattgttctggatattaccagcaaggccga tagtttgagttcttctactcaggcaagtgatgttattactaatcaaagaagtattgcgacaacggttaatttgcgt gatggacagactcttttactcggtggcctcactgattataaaaacacttctcaggattctggcgtaccgttcctgt ctaaaatccctttaatcggcctcctgtttagctcccgctctgattctaacgaggaaagcacgttatacgtgctcg tcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcg tgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccgg ctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaa aaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttgga gtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgattt ataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaa caaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttcta aatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacctagatcaa gagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtg gagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtca gcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggc agcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgg gaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgaga aagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccacc aagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctgga cgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcg aggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattc atcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaa gagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcg ccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcat ctgtgcggtatttcacaccgcatcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttct aaatacattcaaatatgtatccgctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagac cccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaa accaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttca gcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagc accgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccg ggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacgggggggtcgtgcacac agcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgcc acgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgc acgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagc gtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggtt cctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcct ttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcg gaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctgcgcgctcg ctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgc catgctacttatctac
[0324] In some embodiments, a pAAV-ND4 transfer plasmid of the disclosure comprises a Kanamycin resistance gene to allow for antibiotic selection. In some embodiments, a pAAV-ND4 transfer plasmid of the disclosure comprises an f1 origin of replication sequence to allow for replication of the plasmid. In some embodiments, a pAAV-ND4 transfer plasmid of the disclosure comprises a ColE1 origin of replication sequence to allow for replication of plasmid.
[0325] Thus, in some embodiments, a pAAV-ND4 transfer plasmid of the disclosure further comprises: [0326] (i) an f1 origin of replication sequence comprising:
TABLE-US-00015 (SEQ ID No: 8) acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgc agcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgcttt cttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaa atcgggggctccctttagggttccgatttagtgctttacggcacctcgac cccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctg atagacggtttttcgccctttgacgttggagtccacgttctttaatagtg gactcttgttccaaactggaacaacactcaaccctatctcggtctattct tttgatttataagggattttgccgatttcggcctattggttaaaaaatga gctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgctta caattt, [0327] (ii) a Kanamycin resistance gene sequence comprising:
TABLE-US-00016 (SEQ ID No: 9) attgaacaagatggattgcacgcaggttctccggccgcttgggtggagag gctattcggctatgactgggcacaacagacaatcggctgctctgatgccg ccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagacc gacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatc gtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtca ctgaagcgggaagggactggctgctattgggcgaagtgccggggcaggat ctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctga tgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgacc accaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggt cttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagc cgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcg tcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggc cgcttttctggattcatcgactgtggccggctgggtgtggcggaccgcta tcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcg aatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcg cagcgcatcgccttctatcgccttcttgacgagttcttctga,
and [0328] (iii) a ColE1 origin of replication sequence comprising:
TABLE-US-00017 (SEQ ID No: 10) aaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgca aacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagc taccaactctttttccgaaggtaactggcttcagcagagcgcagatacca aatactgtccttctagtgtagccgtagttaggccaccacttcaagaactc tgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctg ctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatag ttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacaca gcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtg agctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtat ccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagg gggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgac ttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaa aacgccagcaacgcg.
[0329] Generation of a pAAV-ND4 transfer plasmid of the disclosure can be accomplished using a suitable genetic engineering technique known in the art (see, e.g., Green, et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Press, (2012)).
[0330] In some embodiments, a recombinant AAV vector of the disclosure is produced by tri-transfection in a transitory packaging cell line with (i) a pAAV-ND4 transfer plasmid of the disclosure (e.g., that shown in
[0331] In some embodiments, the packaging cell line comprises the human embryonic kidney 293 (HEK 293) cell line.
[0332] In some embodiments, the rep/cap plasmid is pRep2Cap2 plasmid. In some embodiments, the rep/cap plasmid is pRep2Cap2 plasmid comprising the following elements (
[0337] In some embodiments, the adenovirus helper plasmid is pXX6 plasmid. In some embodiments, the adenovirus helper plasmid is pXX6 plasmid comprising the following elements (
[0342] Patients suffering from LHON and treated with the recombinant vectors disclosed herein may receive therapeutic benefit, e.g., by an improvement in visual acuity. The term “treatment” as used herein, is defined as the application or administration of a therapeutic agent to a subject, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, one or more symptoms of the disease, or the predisposition toward the disease. As long as the compositions of the disclosure either alone or in combination with another therapeutic agent cure, heal, alleviate, relive, alter, remedy, ameliorate, improve or affect at least one symptom of LHON being treated, as compared to that symptom in the absence of treatment, the result is considered a treatment of the underlying disorder regardless of whether all the symptoms of the disorder are cured, healed, alleviated, relieved, altered, remedied, ameliorated, improved or affected or not. Treatment may be achieved using an “effective amount” of a therapeutic agent, which shall be understood to embrace partial and complete treatment, e.g., partial or complete curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease, one or more symptoms of the disease, or the predisposition toward the disease. An “effective amount” of may be determined empirically. Likewise, a “therapeutically effective amount” is a concentration or which is effective for achieving a stated therapeutic effect.
[0343] In one embodiment, the term “treating” comprises the step of administering an effective dose, or effective multiple doses, of a composition comprising a nucleic acid, a vector, a recombinant virus, or a pharmaceutical composition as disclosed herein, to an animal (including a human being) in need thereof. If the dose is administered prior to development of a disorder/disease, the administration is prophylactic. If the dose is administered after the development of a disorder/disease, the administration is therapeutic. In embodiments, an effective dose is a dose that detectably alleviates (either eliminates or reduces) at least one symptom associated with the disorder/disease state being treated, that slows or prevents progression to a disorder/disease state, that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival. The term encompasses but does not require complete treatment (i.e., curing) and/or prevention.
[0344] In some embodiments, the titer of recombinant vector administered is measured in viral genomes (vg). In some embodiments, the titer of recombinant vector administered is measured by quantitative polymerase chain reaction (qPCR). In some embodiments, the titer of recombinant vector administered is measured by digital droplet PCR (ddPCR). In some embodiments, recombinant AAV vector is administered intravitreally at an amount of about 1.0×10.sup.9 to 1.0×10.sup.12 vg per eye. In some embodiments, recombinant AAV vector is administered intravitreally at an amount of about 5.0×10.sup.9 to 5×10.sup.11 vg per eye. In some embodiments, recombinant AAV vector is administered intravitreally at an amount of about 1.0×10.sup.10 to 1×10.sup.11 vg per eye. In some embodiments, recombinant AAV vector is administered intravitreally at an amount of about 9×10.sup.11 vg per eye. The titer of recombinant vector may be measured by PCR from primers that hybridize within the recombinant vector. Examples of primers include but are not limited to: CTCCATCACTAGGGGTTCCTTG AAV22mers.F (SEQ ID NO: 19) GTAGATAAGTAGCATGGC AAV18mers.R (SEQ ID NO: 20) TAGTTAATGATTAACCC AAV_MGB.P (SEQ ID NO: 21)
[0345] In some embodiments, the recombinant vector of the disclosure, e.g. an AAV, serotype 2, (rAAV) encoding the gene of the human NADH dehydrogenase 4 (ND4), comprises: [0346] a 3′UTR Cox/0 sequence comprising SEQ ID NO:1, [0347] a nucleic acid sequence encoding an ND4 polypeptide comprising SEQ ID NO: 13, and [0348] a nucleic acid sequence encoding an MTS Cox/0 polypeptide comprising SEQ ID NO: 11,
[0349] is administered at an effective dose into a patient in need thereof. In some embodiments, the patient suffers from LHON.
[0350] In some embodiments, the recombinant vector of the disclosure, e.g. an AAV, serotype 2, (rAAV) encoding the gene of the human NADH dehydrogenase 4 (ND4), comprises: [0351] a 3′UTR Cox/0 sequence comprising SEQ ID NO:1, [0352] a nucleic acid sequence encoding an ND4 polypeptide comprising SEQ ID NO: 13, and [0353] a nucleic acid sequence encoding an MTS Cox/0 polypeptide comprising SEQ ID NO: 12,
[0354] is administered at an effective dose into a patient in need thereof. In some embodiments, the patient suffers from LHON.
[0355] In some embodiments, the recombinant vector of the disclosure, e.g. an AAV, serotype 2, (rAAV) encoding the gene of the human NADH dehydrogenase 4 (ND4), comprises: [0356] a 3′UTR Cox/0 sequence comprising SEQ ID NO: 14, [0357] a nucleic acid sequence encoding an ND4 polypeptide comprising SEQ ID NO: 13, and [0358] a nucleic acid sequence encoding an MTS Cox/0 polypeptide comprising SEQ ID NO: 11,
[0359] is administered at an effective dose into a patient in need thereof. In some embodiments, the patient suffers from LHON.
[0360] In some embodiments, the recombinant vector of the disclosure, e.g. an AAV, serotype 2, (rAAV) encoding the gene of the human NADH dehydrogenase 4 (ND4), comprises: [0361] a 3′UTR Cox/0 sequence comprising SEQ ID NO: 14, [0362] a nucleic acid sequence encoding an ND4 polypeptide comprising SEQ ID NO: 13, and [0363] a nucleic acid sequence encoding an MTS Cox/0 polypeptide comprising SEQ ID NO: 12,
[0364] is administered at an effective dose into a patient in need thereof. In some embodiments, the patient suffers from LHON.
[0365] Onset of LHON may be determined by the presence of symptoms. In some embodiments, the recombinant vectors are administered to patients with disease onset of less than 9 months, e.g., 6 to 9 months, 3 to 6 months, or 1 to 3 months. In some embodiments, the recombinant vectors are administered to patients with disease onset of more than 9 months, e.g., for 12 months, for 2 years, or for 3 years. In some embodiments, the patient shows one or more symptoms of LHON, e.g., loss in visual acuity.
[0366] A scale to measure visual acuity in a patient may be expressed as the (decadic) logarithm of the minimum angle of resolution (MAR) (Bailey I L, Lovie J E. I, Am. J. Optom. Physiol. Opt., 53 (11): 740-745 (1976)). The LogMAR scale converts the geometric sequence of a traditional chart to a linear scale. It measures visual acuity loss: positive values indicate vision loss, while negative values denote normal or better visual acuity. In some embodiments, visual acuity of a patient suffering from LHON is measured by the LogMar Scale. In some embodiments, visual acuity of a patient suffering from LHON is measured by the Snellen Scale.
[0367] Another commonly used measure of visual acuity is the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity charts, which is capable of quantifying visual acuity to very low vision levels (Ferris et al., Am. J. Opthalmol., 94:91-96 (1982)). In some embodiments, visual acuity of a patient suffering from LHON is measured by the ETDRS charts.
[0368] Contrast is determined by the difference in the color and brightness of an object and other objects within the same field of view. Patients suffering from LHON may have reduced sensitivity for contrast. Another scale that measures visual acuity may be the Pelli-Robson contrast sensitivity chart (Pelli et al., Clin. Vision Sci., 2(3):187-199 (1988). In some embodiments, visual acuity of a patient suffering from LHON is measured by a Pelli Robson chart.
[0369] In some embodiments, treatment is administered in patients with visual acuity at before treatment e.g., at baseline, of <2.0 LogMAR, e.g., <1.8, <1.6, <1.4, <1.2, <1.0, or <0.8 LogMAR. In some embodiments, treatment is administered in patients with visual acuity at before treatment e.g., at baseline, of at least 3 letters, e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 letters.
[0370] Efficacy or response to treatment may be measured by reversal or amelioration of disease symptoms. In some embodiments, a baseline visual acuity is measured before administration of treatment. In some embodiments, efficacy or response to treatment is measured by an increase in visual acuity. In some embodiments, efficacy or response to treatment is measured by an increase in visual acuity after treatment compared to the baseline before treatment. In some embodiments, efficacy or response to the treatment is measured by the difference between ETDRS scores before and after treatment. In some embodiments, efficacy or response to the treatment is measured by a difference of at least +5.0 ETDRS score, e.g., at least +6.0, +7.0, +8.0, +9.0, +10.0, +11.0, +12.0, +13.0, +14.0, +15.0, or +16.0 after treatment compared to baseline. In some embodiments, efficacy or response to the treatment is measured by a difference of at least 0.05 LogMAR, e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 after treatment compared to baseline.
[0371] As disclosed herein, and without being bound by theory, patients who respond to treatment with a recombinant vector of the disclosure (e.g. patients for which an increase in visual acuity was observed) may include those patients with a disease duration (e.g., vision loss) at baseline of less than 9 months, for example, of 6 to 9 months, and/or with visual acuity at baseline of <1.6 LogMAR. In some embodiments, a criterion (e.g., a disease duration as measured by vision loss at baseline of less than 9 months, or of 6 to 9 months, and/or visual acuity at baseline of <1.6 LogMAR) may be used to identify a patient sub-population that is expected to respond better to treatment with recombinant vector of the disclosure (e.g., a patient population for which an increase in visual acuity may be expected).
[0372] The present disclosure further describes the use of recombinant vector encoding a human NADH dehydrogenase 4 (ND4) polypeptide and comprising (i) a nucleic acid sequence encoding a MTS Cox10 sequence comprising SEQ ID NO: 11, (ii) a nucleic acid sequence encoding a NADH dehydrogenase 4 (ND4) polypeptide comprising SEQ ID No: 13, and (iii) a 3′UTR Cox10 sequence comprising SEQ ID NO: 14 (or its reverse complement SEQ ID NO: 1), in the treatment of Leber Hereditary Optic Neuroretinopathy (LHON) for a group of patients with (i) disease duration at baseline of less than 9 months (e.g. 6 to 9 months) and/or (ii) visual acuity at baseline of less than 1.6 LogMAR.
[0373] The present disclosure also describes a method of treating patients suffering from LHON, with (i) disease duration at baseline of less than 9 months (e.g. 6 to 9 months) and/or (ii) visual acuity at baseline of less than 1.6 LogMAR, comprising administering an effective amount of a recombinant vector encoding a human NADH dehydrogenase 4 (ND4) polypeptide and comprising (i) a nucleic acid sequence encoding a MTS Cox10 sequence comprising SEQ ID NO: 11, (ii) a nucleic acid sequence encoding NADH dehydrogenase 4 (ND4) polypeptide comprising SEQ ID No: 13, and (iii) a 3′UTR Cox10 sequence comprising SEQ ID NO: 14 (or its reverse complement SEQ ID NO: 1).
[0374] The present disclosure is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.
Example 1
[0375] The safety and efficacy of a vector, as disclosed herein, comprising a recombinant adeno-associated virus (AAV) vector, serotype 2, containing the human mitochondrial ND4 gene (rAAV2/2-ND4) (“Vector A”) in patients having Leber Hereditary Optic Neuroretinopathy was investigated.
[0376] Patients participating in the study suffered vision loss for a duration of greater than six months up to one year. Enrolled subjects had a confirmed G11778A mutation in the ND4 gene. Enrolled subjects also had baseline vision greater than or equal to Count Fingers.
[0377] Each patient had one eye randomly selected to receive a single injection of Vector A, while the other eye received a sham injection. In a first patient group, the right eye (OD) was treated with Vector A, while the left eye (OS) was sham-treated. In a second patient group, the right eye (OD) was sham-treated, while the left eye (OS) was treated with Vector A.
[0378] Treatment with Vector A was by means of intravitreal injection containing 9×10.sup.10 viral genomes in 90 μL balanced salt solution plus 0.001% Pluronic F68®. Sham-treatment comprised intravitreal injection that was performed by applying pressure to the eye at the location of a typical intravitreal injection procedure, using the blunt end of a syringe without a needle.
[0379] Comparisons were made between vector A-treated eyes versus sham-treated eyes in changes from baseline (pre-study) point and week 48 of LogMAR acuity derived from the number of letters patients read on the ETDRS chart at week 48 post-treatment. In a separate mode of comparison, the better-seeing eye of each patient was determined at visit 1, prior to randomization, based on vision testing results. Better-seeing eyes that received Vector A were compared to better-seeing eyes that received the sham injection. A similar analysis was performed for the worse-seeing eyes. As would be understood by a person having ordinary skill in the art, ETDRS (Early Treatment Diabetic Retinopathy Study) is a measurement of visual acuity that is capable of quantifying visual acuity to very low vision levels.
[0380] As would be understood by a person having ordinary skill in the art, MAR refers to minimum angle of resolution (in minutes of arc) of the stroke width of the smallest letter recognized. The logarithm of MAR (LogMAR) and, by way of a non-limiting example, LogMAR charts, are used to determine visual acuity. (Johnston, A., Association of Contact Lens Manufacturers Year Book 2011-2016, pp. 38-39 (2016)).
[0381] Patient demographics at baseline are presented in Table 6.
TABLE-US-00018 TABLE 6 Statistic Study N subjects 37 Demographics N males (%) 29 (78.4) Mean age - years (SD) 34.2 (15.2) .sup. Median age - years (range) 30 (15, 67) Vision loss duration (VLD) Eyes with available data 74 Mean VLD - days (SD) 270.9 (59.4) Median VLD - days (range) .sup. 262 (181, 364) Simultaneous bilateral onset N subjects (%) 7 (19%) SD: standard deviation, VLD: vision loss duration
[0382] Data pertaining to patient visual acuity at baseline are presented in Table 7.
TABLE-US-00019 TABLE 7 Statistic Study Treated eyes Sham eyes N subjects 37 N eyes with available data 74 All eyes Mean LogMAR (SD) 1.6 1.66 1.55 (0.4) (0.5) (0.42) Median LogMAR (range) 1.6 1.6 1.5 (0.7, 3.2) (0.8, 3.17) (0.7, 2.81) Best-seeing eyes Mean LogMAR (SD) 1.5 1.44 1.50 (0.4) (0.33) (0.38) Median LogMAR (range) 1.5 1.45 1.50 (0.7, 2.2) (0.8, 2.09) (0.7, 2.22) Worst-seeing eyes Mean LogMAR (SD) 1.7 1.84 1.61 (0.5) (0.55) (0.48) Median LogMAR (range) 1.6 1.6 1.55 (0.8, 3.2) (0.8, 3.17) (0.9, 2.81)
[0383] At 48 weeks, a favorable safety profile of Vector A was reported. 75% of adverse events (AEs) were ocular. 50% of ocular AEs were related to Vector A, and 48% of ocular AEs were related to the procedure. The most common ocular AEs comprised anterior chamber inflammation (15%), vitritis (9%), punctate keratitis (9%), and IOP elevation (8%).
[0384] At 48 weeks, tRNFL (temporal retinal nerve fiber layer)/PM (papillomacular) bundle thickness was significantly preserved in treated eyes and decreased in untreated eyes. Data pertaining to the change of RNFL temporal quadrant from baseline to week 48 are provided in Table 8.
TABLE-US-00020 TABLE 8 Secondary Efficacy Analysis Change of RNFL Temporal Quadrant from Baseline to Week 48 RNFL Quadrant Temporal LS Mean 95% (μm) n (SE) .sup.[a] CI .sup.[a] p-value Change from Baseline to 37 −0.6 −2.6, 1.4 Week 48 All-Treated Eyes (1.0) Change from Baseline to 35 −3.4 −5.4, −1.3 Week 48 All-Sham Eyes (1.0) Difference between All- 35 2.8 0.0359 .sup.[a] Treated Eyes and All- (0.2, Sham Eyes Treatment 5.4) Effect (95% CI) Wilcoxon Signed-Rank 0.0416 Test .sup.[a] A mixed model of analysis of covariance (ANCOVA) was used with change from baseline at week 48 as the response, and subject, eyes of the subject as random factor, treatment and the baseline GCL Thickness/Volume value as covariates in the model. LS mean refers to least-squares mean.
[0385] At 48 weeks, GCL (Ganglion cell layer) volume was significantly preserved in treated eyes and decreased in untreated eyes. At least these results suggested that the biological targets of Vector A were successfully engaged. Data pertaining to the change of GCL volume and topographical map from baseline to week 48 are presented in Table 9.
TABLE-US-00021 TABLE 9 Secondary Efficacy Analysis Change of GCL Volume and Topographical Map from Baseline to Week 48 GCL Macular Volume LS Mean 95% (mm.sup.3) n (SE) .sup.[a] CI .sup.[a] p-value Change from Baseline to 36 −0.003 −0.028, 0.022 Week 48 All-Treated Eyes (0.012) Change from Baseline to 36 −0.038 −0.062, −0.013 Week 48 All-Sham Eyes (0.012) Difference between All- 36 0.035 0.0189 [a] Treated Eyes and All- (0.006, Sham Eyes Treatment 0.063) Effect (95% CI) Wilcoxon Signed-Rank 0.0448 .sup. Test .sup.[a] A mixed model of analysis of covariance (ANCOVA) was used with change from baseline at week 48 as the response, and subject, eyes of the subject as random factor, treatment and the baseline GCL Thickness/Volume value as covariates in the model.
[0386] At 48 weeks, visual acuity improvement was observed in both eyes (−0.21 LogMAR on average). Data pertaining to the change of LogMAR from baseline to week 48 are presented in Table 10. No statistically significant difference between treated and untreated eyes was observed.
TABLE-US-00022 TABLE 10 Primary Efficacy Analysis Change of LogMAR from Baseline to Week 48 LS Mean 95% Visual acuity (LogMAR) n (SE) .sup.[a] CI .sup.[a] p-value Change from Baseline to 37 −0.218 −0.328, −0.108 Week 48 All-Treated Eyes (0.055) Change from Baseline to 37 −0.211 −0.320, −0.101 Week 48 All-Sham Eyes (0.055) Difference between All- 37 −0.007 0.8942 .sup.[a] Treated Eyes and All- (−0.118, Sham Eyes Treatment 0.103) Effect (95% CI) Wilcoxon Signed-Rank 0.3875 Test .sup.[a] A mixed model of analysis of covariance (ANCOVA) was used with change from baseline at week 48 as the response, and subject, eyes of the subject as random factor, treatment and the baseline LogMAR value as covariates in the model.
[0387] At 48 weeks, visual field testing was performed using Humphrey® Visual Field analysis (mean deviation and foveal threshold). Data pertaining to the visual field testing are presented in Table 11 and Table 12. No difference between treated and untreated eyes was observed.
TABLE-US-00023 TABLE 11 Mean (SD) VF Foveal Threshold (dB) n Treated eyes n Sham eyes Baseline 13 19.6 14 21.9 (16.9) (15.0) Week 48 25 14.9 25 13.6 (11.4) (11.6) Change from baseline 9 0.7 11 −0.5 (8.9) (11.9)
TABLE-US-00024 TABLE 12 Mean (SD) VF Mean Deviation (dB) n Treated eyes n Sham eyes Baseline 37 −25.99 37 −24.94 (8.37) (9.70) Week 48 37 −22.83 37 −22.94 (9.43) (9.80) Change from baseline 37 3.15 37 2.00 (6.96) (5.04)
[0388] At 48 weeks, contrast sensitivity was assessed using the Pelli-Robson chart (see also
TABLE-US-00025 TABLE 13 Log of Contrast Mean (SD) Sensitivity (LogCS) n Treated eyes n Sham eyes Baseline 37 0.25 37 0.35 (0.40) (0.46) Week 48 37 0.45 37 0.43 (0.50) (0.49) Change from baseline 37 0.20 37 0.08 (0.36) (0.28)
[0389] Color vision was tested using the Farnsworth-Munsell 100-hue color test. At baseline, extremely poor scores for color discrimination were observed. At week 48, no difference between treated and untreated eyes was observed. Data pertaining to color vision tested are presented in Table 14.
TABLE-US-00026 TABLE 14 Mean (SD) Total Error Score n Treated eyes n Sham eyes Baseline 28 649.3 28 661.9 (420.5) (433.7) Week 48 28 623.6 28 622.3 (400.4) (430.3) Change from baseline 28 −25.7 28 −39.6 (235.6) (172.3)
[0390] Quality of life was assessed at week 48 using the Visual Functional Questionnaire-25 (VFQ-25). Data from selected sub-scales are presented in Table 15. Although the difference in scores between scores is small, treatment of the patient's worse-seeing eye appeared to lead to improved quality of life metrics. Such a trend was observed across all sections of the questionnaire.
TABLE-US-00027 TABLE 15 Mean scores at Week 48 (SD) Best-seeing Best-seeing Selected sub-scales n Treated eyes n Sham eyes General Vision 16 38.8 21 42.9 (13.6) (20.3) Near Activities 16 32.3 21 35.3 (19.7) (19.7) Distant Activities 16 42.4 21 47.0 (22.3) (24.8)
[0391] Study data were further analyzed to identify patient populations that were especially responsive to treatment with Vector A (e.g., patients for which an increase in visual acuity was observed).
[0392] Data pertaining to the change in visual acuity from baseline for on-chart best-seeing eyes treated with Vector A and for on-chart best-seeing eyes that were sham-treated are presented in Table 16. “On-chart” refers to subjects who can read at least three letters on an ETDRS chart and/or having visual acuity below 1.6 LogMAR.
TABLE-US-00028 TABLE 16 Change from baseline in 95% ETDRS visual acuity LS Mean Confidence letters (LogMAR) n (SE) Interval equivalent Best-seeing 12 −0.236 −0.405, −0.067 +12 treated eyes (0.082) Best-seeing 17 −0.075 −0.216, 0.067 +4* Sham eyes (0.069) p-value .sup.a 0.1466 .sup.a Significance of the difference between All-treated and All-Sham with respect to change of LogMAR from baseline. *Does not statistically differ from 0.
[0393] The difference in the change in ETDRS score relative to baseline was measured. As shown in Table 17, this difference was greater for the set of on-chart best-seeing eyes relative to the set of all on-chart eyes (+6.1 versus+4.5).
TABLE-US-00029 TABLE 17 Change from Difference baseline in Treated Sham Treated - ETDRS score eyes eyes Untreated (n = 24) (n = 28) All eyes +8.8 +4.3 +4.5 (n = 13) (n = 17) Best-seeing eyes +9.8 +3.7 +6.1
[0394] Among the set of on-chart eyes treated with Vector A and for which an increase in visual acuity was measured at week 48, 75% ( 12/16) had a disease duration at baseline of 6 to 9 months, while 25% ( 4/16) had a disease duration at baseline of 9 to 12 months (Cf. Table 18). Among the set of on-chart sham-treated eyes for which an increase in visual acuity was measured at week 48, 50% ( 8/16) had a disease duration at baseline of 6 to 9 months, while 50% ( 8/16) had a disease duration at baseline of 9 to 12 months.
TABLE-US-00030 TABLE 18 Disease duration at baseline 6 to 9 months 9 to 12 months Total Treated eyes 12 4 16 (75%) (25%) 8 8 Sham eyes (50%) (50%) 16 Total 20 12 32
[0395] In a further analysis, “responder” referred to improvement in visual acuity in on-chart patients of at least 0.25 LogMAR (+12.5 ETDRS equivalent). As shown in Table 19, 24.0% of all on-chart eyes treated with Vector A and 14.3% of all on-chart sham-treated eyes were characterized as “Responder Eyes.”
TABLE-US-00031 TABLE 19 All On-chart All On-chart Treated eyes Sham eyes Responder eye 6 4 (24.0%) (14.3%) Non-Responder eye 19 24 McNemar p-value 0.5637
[0396] In a further analysis, “responder” referred to improvement in visual acuity in best-seeing eyes of on-chart patients of at least 0.25 LogMAR (+12.5 ETDRS equivalent). As shown in Table 20, 25.0% of on-chart best-seeing eyes treated with Vector A and 5.6% of best-seeing on-chart sham-treated eyes were characterized as “Responder Eyes.”
TABLE-US-00032 TABLE 20 On-chart eyes Best-seeing Best-seeing Treated eyes Sham eyes Responder eye 3 1 (25.0%) (5.6%) Non-Responder eye 9 16 McNemar p-value 0.2785
[0397] Study data were further analyzed using a generalized estimating equation (GEE) model to assess the effect of treatment with Vector A on achievement of a 20/200 visual acuity endpoint. Results showed that eyes treated with Vector A were significantly more likely to achieve the 20/200 visual acuity endpoint than were sham-treated eyes (p=0.0005). The odds ratio was 18.45 (lower 95% boundary=3.60).
[0398] Data pertaining to the number of eyes legally blind at baseline and, of those eyes blind at baseline, the number of eyes rescued from legal blindness are presented in Table 21. In this context, a legally-blind eye is defined as having visual acuity worse than 20/200.
TABLE-US-00033 TABLE 21 Treated eyes Sham eyes Number of eyes legally blind at 24 25 baseline Of those eyes blind at baseline, 2 1 number of eyes rescued from legal (8.3%) (4.0%) blindness
[0399] Analysis of the study data indicated that the set of patients who responded better to treatment with Vector A (e.g. patients for which an increase in visual acuity was observed) included those patients having a disease duration (e.g., vision loss) at baseline of less than 9 months, for example, of 6 to 9 months, and/or with visual acuity at baseline of <1.6 LogMAR. Thus, in some embodiments, these criteria (a disease duration (e.g., vision loss) at baseline of less than 9 months, for example, of 6 to 9 months and/or visual acuity at baseline of <1.6 LogMAR) may be used to identify a patient sub-population expected to better respond to treatment with Vector A (e.g., a patient population for which an increase in visual acuity may be expected).
Example 2
[0400] The trial evaluated the safety and efficacy of a single intravitreal injection of Vector A (rAAV2/2-ND4) in 37 subjects whose visual loss due to 11778-ND4 Leber Hereditary Optic Neuropathy (LHON) commenced between 6 and 12 months prior to study treatment. Week 96 is the last of the scheduled readouts for the trial and marks the time when the data are unmasked, providing access to individual patient profiles.
[0401] At Week 96, Vector A-treated eyes showed a mean improvement of −0.308 LogMAR compared to baseline, equivalent to +15.4 ETDRS letters or 3 lines on the ETDRS vision chart (
[0402] Consistent with natural history, subjects experienced an initial point of low visual acuity, or nadir. The nadir is defined as the lowest post-treatment BCVA as measured by LogMAR up to the week of measurement. Eyes of trial subjects recovered significantly. By week 96, Vector A-treated eyes had gained+28 more letters relative to their nadir.
TABLE-US-00034 TABLE 22 Recovery of BCVA from nadir as measured by difference from nadir in ETDRS letters equivalent, mean and standard deviation n Week 48 Week 72 Week 96 Vector A- 37 +24.1 +27.4 +28.1 treated eyes (21.1) (21.8) (22.0) Sham-treated 37 +20.3 +22.6 +23.2 eyes (23.4) (25.5) (24.5)
[0403] At Week 96, low-contrast visual acuity, as measured on the Pelli-Robson chart showed a similar trend of improvement for both Vector A-treated eyes and sham-treated eyes. The trajectories of sham- and Vector A-treated eyes did not track each other as closely as BCVA. Mean contrast sensitivity showed a more robust improvement versus baseline over the course of the trial (
[0404] The proportion of Vector A-treated eyes that achieved at least a −0.2 LogMAR or +10 ETDRS letters equivalent improvement versus baseline at Week 96 is statistically significantly higher than the corresponding proportion of sham-treated eyes (65% vs. 46%, p-value=0.0348). Vector A-treated eyes were also significantly more likely than sham-treated eyes to achieve another measure of treatment success improving by at least 15 ETDRS letters at Week 96 from on-chart acuity at baseline, or avoiding the US legal blindness threshold of 20/200 at Week 96 (32% vs. 16%, p=0.0196).
[0405] Based on a generalized estimating equations (GEE) model, Vector A-treated eyes were 2.8 times more likely to be at or above 20/200 than sham-treated eyes (p=0.0094). When only eyes that were strictly above the threshold were considered, the odds ratio rose to 3.6 (p=0.0032).
[0406] Additionally, 68% of trial subjects achieved a spontaneous “clinically relevant recovery (CRR)” in at least one eye at Week 96, defined by an improvement of (a) at least 10 ETDRS letters from on-chart visual acuity, or (b) an improvement from off-chart visual acuity to being able to read at least 5 ETDRS letters. Vector A-treated eyes were significantly more likely to achieve this than sham-treated eyes (62% vs. 43%, p=0.0348). In comparison, in a previous natural history study, only 15% of patients with the same 11778A mutation achieved CRR.
[0407] In terms of quality of life, improvement in visual function were reflected in scores on the National Eye Institute Visual Function Questionnaire-25 (NEI VFQ-25) survey, a validated, vision-specific quality-of-life instrument completed by trial subjects. As shown in Table 23, mean composite score and means of relevant sub-scale scores continued to improve over baseline, particularly for the ability to carry out near and distance activities. The increase over baseline of the mean sub-scale scores exceeded those that have been associated with a 15-letter improvement in BCVA in other ocular diseases.
TABLE-US-00035 TABLE 23 Meaningful Improvements in Quality of Life Scores Reported by Patients (NEI VFQ-25) - Mean change from baseline (absolute score and percentage) Composite Near Distance Role General Mental Score** Activities Activities Dependency Difficulties Vision Health Week 48 +7.2 +10.4 +9.6 +12.4 +14.5 +10.3 +11.2 +23.2% +65.1% +49.8% +100.6% + 65.0% +50.9% +81.9% Week 72 +8.1 +9.5 +8.2 +18.9 +15.2 +11.9 +15.2 +25.2% +58.1% +42.5% +130.2% +70.9% +54.1% +105.6% Week 96 +9.5 +13.3 +10.7 +18.5 +15.9 +6.5 +16.1 +28.8% +78.1% +47.4% +130.2% +78.9% +32.4% +108.2% Clinically +3.90 to +4.67 to +5.15 to +4.72 to +3.31 to +4.38 to +4.70 to Relevant +4.34 +6.06 +5.38 +4.98 +4.70 +4.82 +4.88 Difference **The composite score is an average of the vision-targeted sub-scale scores, excluding the general health rating question.
[0408] Structural metrics indicate that GS010-treated eyes maintained the stability achieved in previous readouts in ganglion cell volume. The differential effect of therapy was, however, more prominent in previous readouts.