Compositions and methods for treating Leber's hereditary optic neuropathy caused by ND4 mutations

Abstract

Provided herein is a recombinant nucleic acid comprising a mitochondrial targeting sequence, a mitochondrial protein coding sequence encoding a polypeptide comprising a mitochondrial protein, a 3'UTR nucleic acid sequence, and a polyA signal sequence. Also provided are pharmaceutical compositions comprising the recombinant nucleic acid, and methods of treating Leber's Hereditary Optic Neuropathy (LHON) using the pharmaceutical compositions.

Description

[Technical field]

[0001] The present invention relates to recombinant nucleic acids encoding mitochondrial proteins and related pharmaceutical compositions and their use for treating ocular diseases such as Leber's hereditary optic neuropathy. [Background technology]

[0002] Leber's hereditary optic neuropathy (LHON) is a mitochondrial inherited degeneration (transmitted from mother to child) of retinal ganglion cells (RGCs) and their axons, resulting in acute or subacute loss of central vision. It is primarily seen in young adult males. LHON is primarily due to mutations in the mitochondrial (not nuclear) genome and is exclusively maternally inherited, as only the egg provides mitochondria to the embryo. LHON is usually caused by one of three pathogenic mitochondrial DNA (mtDNA) point mutations. These mutations are G → A at nucleotide positions 11778 (G11778A), G → A at 3460 (G3460A), and T → C at 14484 (T14484C) in the genes for the subunits of NADH dehydrogenase subunit 4 protein (ND4), NADH dehydrogenase subunit 1 protein (ND1), and NADH dehydrogenase subunit 6 protein (ND6) of complex I within the mitochondrial oxidative phosphorylation chain. Each mutation is considered to be at significant risk of permanent vision loss. LHON usually progresses painlessly over weeks to months until binocular vision is reduced to less than 0.1, severely impacting the patient's quality of life. Two LHON mutants, G3460A and T14484C, have an 80% reduction in mitochondrial NADH dehydrogenase activity isolated from patients' platelets. Ninety percent of Chinese LHON patients carry the G11778A mutation. The G11778A mutation causes functional impairment and optic nerve damage in LHON patients by changing arginine to histidine in the ND4 protein. Therefore, there is a need to develop compositions and methods for treating LHON with higher transfection efficiency and therapeutic efficacy. Summary of the Invention

[0003] Disclosed herein is a recombinant nucleic acid comprising a mitochondrial targeting sequence and a mitochondrial protein coding sequence (in order from the 5' end to the 3' end), optionally wherein the mitochondrial protein coding sequence encodes an ND4 protein, and optionally wherein the ND4 protein comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:160.

[0004] In some embodiments, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the sequences set forth in SEQ ID NOs: 180 and 174-176. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 180. In some embodiments, the recombinant nucleic acid comprises a sequence set forth in SEQ ID NO: 180.

[0005] In some embodiments, the recombinant nucleic acid comprises a Kozak sequence located before the 5' end of the mitochondrial targeting sequence, optionally, the Kozak sequence is SEQ ID NO: 171, and optionally, there are no redundant nucleotides between the Kozak sequence and the mitochondrial targeting sequence.

[0006] In some embodiments, the recombinant nucleic acid comprises an intron sequence, optionally wherein the intron sequence is located before the 5' end of the mitochondrial targeting sequence, optionally wherein the intron sequence is located before the 5' end of the Kozak sequence, and optionally wherein the intron sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:170.

[0007] In some embodiments, the recombinant nucleic acid comprises a promoter sequence, optionally located prior to the 5' end of the mitochondrial targeting sequence, the Kozak sequence, and / or the intron sequence, and optionally comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:169.

[0008] In some embodiments, the recombinant nucleic acid comprises a 3'UTR sequence, optionally located after the 3' end of the mitochondrial protein coding sequence, and optionally comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:13.

[0009] In some embodiments, the recombinant nucleic acid comprises a polyA signal sequence, optionally wherein the polyA signal sequence is located after the 3' end of the mitochondrial protein coding sequence and / or the 3'UTR sequence, optionally wherein the polyA signal sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 172 or 173, optionally wherein the polyA signal sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 173.

[0010] In some embodiments, the recombinant nucleic acid comprises a sequence between the 3'UTR sequence and the polyA signal sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the spacer sequence set forth in SEQ ID NO:185.

[0011] In some embodiments, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:1.

[0012] In some embodiments, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:6.

[0013] In some embodiments, the recombinant nucleic acid comprises a first inverted terminal repeat (ITR) sequence and a second ITR sequence. In some embodiments, the first ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 178. In some embodiments, the second ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 179.

[0014] Disclosed herein is a recombinant nucleic acid comprising (in order from the 5' end to the 3' end) a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, and a polyA signal sequence, wherein the polyA signal sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:173.

[0015] In some embodiments, the 3'UTR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:13.

[0016] In some embodiments, the mRNA comprising a mitochondrial protein coding sequence produced by transcription of the recombinant nucleic acid has a higher expression level than the mRNA of a control recombinant nucleic acid lacking a polyA signal sequence, preferably at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 300% higher expression level than the mRNA of the control recombinant nucleic acid. In some embodiments, the control recombinant nucleic acid is substituted with a sequence comprising SEQ ID NO: 172 in place of the polyA signal sequence, and preferably the mRNA produced by transcription of the recombinant nucleic acid has an expression level at least 10%, at least 15%, or at least 20% higher than the mRNA produced by the control recombinant nucleic acid.

[0017] In some embodiments, the mitochondrial protein produced by translation of the recombinant nucleic acid has a higher expression level than the mitochondrial protein of a control recombinant nucleic acid lacking a polyA signal sequence, preferably at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 300% higher expression level than the mitochondrial protein of the control recombinant nucleic acid. In some embodiments, the control recombinant nucleic acid is substituted with a sequence comprising SEQ ID NO: 172 in place of the polyA signal sequence, preferably the mitochondrial protein produced by translation of the recombinant nucleic acid has an expression level at least 10%, at least 15%, or at least 20% higher than the mitochondrial protein produced by the control recombinant nucleic acid.

[0018] In some embodiments, the polyA signal sequence has a length of 122 base pairs or less, 125 base pairs or less, 130 base pairs or less, 140 base pairs or less, 150 base pairs or less, 160 base pairs or less, 170 base pairs or less, 180 base pairs or less, 190 base pairs or less, or 200 base pairs or less.

[0019] In some embodiments, the recombinant nucleic acid comprises a Kozak sequence located before the 5' end of the mitochondrial targeting sequence, optionally, the Kozak sequence is SEQ ID NO: 171, and optionally, there are no redundant nucleotides between the Kozak sequence and the mitochondrial targeting sequence.

[0020] In some embodiments, the recombinant nucleic acid comprises a sequence between the 3'UTR sequence and the polyA signal sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the spacer sequence set forth in SEQ ID NO:185.

[0021] Disclosed herein is a recombinant nucleic acid comprising (in order from the 5' end to the 3' end) a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, and a 3'UTR sequence, wherein the Kozak sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:13, and wherein there are no redundant nucleotides between the Kozak sequence and the mitochondrial targeting sequence.

[0022] In some embodiments, the recombinant nucleic acid further comprises a polyA signal sequence, wherein the polyA signal sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 172 or 173.

[0023] In some embodiments, the recombinant nucleic acid comprises an intron sequence, optionally wherein the intron sequence is located before the 5' end of the Kozak sequence, and optionally wherein the intron sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:170.

[0024] In some embodiments, the recombinant nucleic acid further comprises a promoter sequence, optionally comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 169. In some embodiments, the promoter sequence is located before the 5' end of the intron sequence.

[0025] Disclosed herein is a recombinant nucleic acid comprising (in order from the 5' end to the 3' end) a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, and a mitochondrial protein coding sequence, wherein the intron sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:170, optionally, the recombinant nucleic acid further comprises a 3'UTR sequence, and optionally, the recombinant nucleic acid further comprises a polyA signal sequence.

[0026] In some embodiments, the recombinant nucleic acid further comprises a first inverted terminal repeat (ITR) sequence and a second ITR sequence. In some embodiments, the first ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 178. In some embodiments, the second ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 179.

[0027] In some embodiments, there are no redundant nucleotides between the mitochondrial protein coding sequence and the 3'UTR sequence.

[0028] In some embodiments, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:1.

[0029] In some embodiments, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:6.

[0030] In some embodiments, there are no redundant nucleotides between the mitochondrial targeting sequence and the mitochondrial protein coding sequence.

[0031] In some embodiments, the recombinant nucleic acid comprises (in order from the 5' end to the 3' end) a first ITR sequence, a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, a polyA signal sequence, and a second ITR sequence, preferably the first ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:178. , the promoter sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 169, the intron sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 170, the Kozak sequence is SEQ ID NO: 171, and the mitochondrial targeting sequence is the sequence set forth in SEQ ID NO: 1. the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:6; the 3'UTR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:13; the polyA signal sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:173; and the second ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:179.

[0032] In some embodiments, the recombinant nucleic acid comprises (in order from the 5' end to the 3' end) a first ITR sequence, a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, a polyA signal sequence, and a second ITR sequence, preferably, the first ITR sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO:178, the promoter sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO:169, the intron sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO:170, and the Kozak sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO:170. No. 171, wherein the mitochondrial targeting sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO:1, the mitochondrial protein coding sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO:6, the 3'UTR sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO:13, the polyA signal sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO:173, and the second ITR sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO:179.

[0033] In some embodiments, the recombinant nucleic acid comprises (in order from the 5' end to the 3' end) a first ITR sequence, a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, a polyA signal sequence, and a second ITR sequence, preferably, the first ITR sequence comprises the sequence set forth in SEQ ID NO: 178, the promoter sequence comprises the sequence set forth in SEQ ID NO: 169, the intron sequence comprises the sequence set forth in SEQ ID NO: 170, the Kozak sequence is SEQ ID NO: 171, the mitochondrial targeting sequence comprises the sequence set forth in SEQ ID NO: 1, the mitochondrial protein coding sequence comprises the sequence set forth in SEQ ID NO: 6, the 3'UTR sequence comprises the sequence set forth in SEQ ID NO: 13, the polyA signal sequence comprises the sequence set forth in SEQ ID NO: 173, and the second ITR sequence comprises the sequence set forth in SEQ ID NO: 179.

[0034] In some embodiments, the recombinant nucleic acid comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the sequences set forth in SEQ ID NOs: 174-176 and 180. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 180. In some embodiments, the recombinant nucleic acid comprises a sequence set forth in SEQ ID NO: 180.

[0035] Disclosed herein is a viral vector comprising the recombinant nucleic acid described herein.In some embodiments, the viral vector is a recombinant adeno-associated virus (rAAV) vector.In some embodiments, the rAAV vector is a rAAV2 vector.

[0036] Disclosed herein are pharmaceutical compositions comprising the recombinant nucleic acids described herein.Disclosed herein are pharmaceutical compositions comprising the viral vectors described herein.Disclosed herein are pharmaceutical compositions comprising the recombinant adeno-associated viral (rAAV) vectors described herein.

[0037]

[0038] Disclosed herein is a pharmaceutical composition comprising the virus described herein. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, the virus comprises the recombinant nucleic acid described herein.

[0039] In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient thereof. In some embodiments, the pharma- ceutically acceptable excipient comprises phosphate buffered saline (PBS), α,α-trehalose dihydrate, L-histidine hydrochloride monohydrate, polysorbate 20, NaCl, NaH2PO4, Na2HPO4, KH2PO4, K2HPO4, poloxamer 188, or any combination thereof. In some embodiments, the pharma- ceutically acceptable excipient is selected from phosphate buffered saline (PBS), α,α-trehalose dihydrate, L-histidine monohydrochloride hydrate, polysorbate 20, NaCl, NaH2PO4, Na2HPO4, KH2PO4, K2HPO4, poloxamer 188, or any combination thereof. In some embodiments, the pharma- ceutically acceptable excipient comprises poloxamer 188. In some embodiments, the pharma- ceutically acceptable excipient comprises 0.0001% to 0.01% poloxamer 188. In some embodiments, the pharma- ceutically acceptable excipient comprises 0.001% poloxamer 188. In some embodiments, the pharma- ceutically acceptable excipient further comprises one or more salts. In some embodiments, the one or more salts comprise NaCl, Na2HPO4, and KH2PO4. In some embodiments, the pharmaceutical composition comprises a) NaCl at a concentration of 5 to 15 mg / mL, preferably NaCl at a concentration of 9 mg / mL, b) KH2PO4 at a concentration of 0.1 to 0.5 mg / mL, preferably KH2PO4 at a concentration of 0.144 mg / mL, and / or c) Na2HPO4 at a concentration of 0.5 to 1 mg / mL, preferably Na2HPO4 at a concentration of 0.795 mg / mL. In some embodiments, the pH of the pharmaceutical composition is 7.2 to 7.4. In some embodiments, the pH of the pharmaceutical composition is 7.3.

[0040] In some embodiments, the pharmaceutical composition comprises at least 1.0×10 10 vg / mL, at least 3.0 × 10 10 vg / mL, or at least 6.0 × 10 10 In some embodiments, the pharmaceutical composition has a viral titer of at least 9.0×10 vg / mL. 10It has a viral titer of 0.05 vg / mL.

[0041] In some embodiments, the pharmaceutical composition retains at least 60% of the viral titer after five freeze / thaw cycles compared to the viral titer before the freeze / thaw cycles.

[0042] Disclosed herein is the use of a pharmaceutical composition described herein in the preparation of a medicament for treating an ophthalmic disease.

[0043] Disclosed herein are methods for treating an ocular disease, comprising administering to a patient an effective amount of a pharmaceutical composition described herein.

[0044] In some embodiments, the eye disease is Leber's hereditary optic neuropathy (LHON). In some embodiments, the pharmaceutical composition is administered by intraocular or intravitreal injection. In some embodiments, the pharmaceutical composition is administered by intravitreal injection. In some embodiments, 0.01-0.1 mL of the pharmaceutical composition is administered to each eye. In some embodiments, 0.045-0.055 mL of the pharmaceutical composition is administered to each eye. In some embodiments, the adeno-associated virus is at least 1.5×10 9 vg / eye, preferably the adeno-associated virus is administered at a dose of at least 3.0×10 9 vg / eye. In some embodiments, the adeno-associated virus is administered at a dose of at least 4.5×10 9 It is administered in a dose of vg / eye.

[0045] In some embodiments, the use or method comprises administering a corticosteroid to the patient. In some embodiments, the corticosteroid comprises prednisone or methylprednisolone. In some embodiments, the corticosteroid is prednisone. In some embodiments, the corticosteroid is administered from about 2 days prior to administration of the pharmaceutical composition. In some embodiments, the corticosteroid is administered orally. In some embodiments, the corticosteroid is administered for about 28 consecutive days after initiation of administration. In some embodiments, the corticosteroid is administered daily after initiation of administration and at a decreasing dose with each week of successive administration. In some embodiments, the corticosteroid is administered at a dosage of 40 mg per day at the initiation of administration for one week, then 30 mg per day for one week, then 20 mg per day for one week, and finally 10 mg per day for one week.

[0046] In some embodiments, the use or method further comprises administering sodium creatine phosphate to the patient. In some embodiments, the sodium creatine phosphate is administered by intravenous injection. In some embodiments, administration of the pharmaceutical composition results in a higher average level of visual acuity recovery compared to administration of a pharmaceutical composition that does not include a recombinant nucleic acid.

[0047] In another aspect, disclosed herein are recombinant nucleic acids, pharmaceutical compositions, and methods for treating LHON.

[0048] In one aspect, disclosed herein is a recombinant nucleic acid comprising a mitochondrial targeting sequence, a mitochondrial protein coding sequence comprising a sequence having at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 7, 8, 10, and 12, and a 3'UTR nucleic acid sequence.

[0049] In some cases, the mitochondrial targeting sequence encodes a polypeptide comprising a peptide sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 129-159. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence set forth in SEQ ID NO: 2. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence set forth in SEQ ID NO: 3. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence set forth in SEQ ID NO: 4. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence set forth in SEQ ID NO: 5.

[0050] In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the sequence set forth in SEQ ID NO: 7 or 8. In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the sequence set forth in SEQ ID NO: 10. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the sequence set forth in SEQ ID NO: 12.

[0051] In some cases, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 111-125. In some cases, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence set forth in SEQ ID NO:13 or SEQ ID NO:14.

[0052] In some cases, the recombinant nucleic acid comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 17-20, 23-24, 27-28, 31-34, 37-38, 41-42, 45-48, 51-52, 55-56, 59-62, 65-66, 69-70, 73-76, 79-80, and 83-84.

[0053] In another aspect, disclosed herein is a recombinant nucleic acid comprising a mitochondrial targeting sequence comprising a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, and 5, a mitochondrial protein coding sequence encoding a polypeptide comprising a mitochondrial protein, and a 3'UTR nucleic acid sequence.

[0054] In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 2. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 3. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 4. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 5.

[0055] In some cases, the mitochondrial protein is selected from the group consisting of NADH dehydrogenase 4 (ND4), NADH dehydrogenase 6 (ND6), NADH dehydrogenase 1 (ND1), and variants thereof. The mitochondrial protein optionally comprises NADH dehydrogenase 4 (ND4) or a variant thereof. In some cases, the mitochondrial protein comprises a peptide sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 160. In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 6, 7, or 8. The mitochondrial protein optionally comprises NADH dehydrogenase 6 (ND6) or a variant thereof. In some cases, the mitochondrial protein comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 161. In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 9 or 10. The mitochondrial protein optionally comprises NADH dehydrogenase 1 (ND1) or a variant thereof. In some cases, the mitochondrial protein comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 162. In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 11 or 12.

[0056] In some cases, the 3'UTR nucleic acid sequence is located at the 3' end of the mitochondrial targeting sequence. In some cases, the 3'UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATP5B, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hsIRP1, hsMRPS12, hsATP5J2, rnSOD2, and hsOXA1L. In some cases, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 111-125. In some cases, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:13 or SEQ ID NO:14.

[0057] In some cases, the mitochondrial targeting sequence is located at the 5' end of the 3'UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at the 3' end of the mitochondrial targeting sequence.

[0058] In some cases, the recombinant nucleic acid comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 29-84.

[0059] In another aspect, disclosed herein is a recombinant nucleic acid comprising a mitochondrial targeting sequence, a mitochondrial protein coding sequence comprising a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 7, 8, 10, and 12, and a 3'UTR nucleic acid sequence.

[0060] In some cases, the mitochondrial targeting sequence is selected from the group consisting of hsCOX10, hsCOX8, scRPM2, lcSirt5, tbNDUS7, ncQCR2, hsATP5G2, hsLACTB, spilv1, gmCOX2, crATP6, hsOPA1, hsSDHD, hsADCK3, osP0644B06.24-2, Neurospora crassa ATP9(ncATP9), hsGHITM, hsNDUFAB1, hsATP5G3, crATP6_hsADCK3, ncATP9_ncATP9, zmLOC100282174, ncATP9_zmLOC100282174_spilv1_ncATP9, z mLOC100282174_hsADCK3_crATP6_hsATP5G3, zmLOC100282174_hsADCK3_hsATP5G3, ncATP9_zmLOC100282174, hsADCK3_zmLOC100282174_crATP6 ncATP9_zmLOC100282174_spilv1_GNFP_ncATP9, and ncATP9_zmLOC100282174_spilv1_lcSirt5_osP0644B06.24-2_hsATP5G3, crATP6_hsADCK3_zmLOC100282174_hsATP5G3, hsADCK3_zmLOC100282174, hsADCK3_zmLOC100282174_crATP6, ncATP9_zmLOC100282174_spilv1_GNFP_ncATP9, and ncATP9_zmLOC100282174_spilv1_lcSirt5_osP0644B06.24-2_hsATP5G2_ncATP9. In some cases, the mitochondrial targeting sequence encodes a polypeptide comprising a peptide sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 129-159. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 2 or 3. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 4.In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:5.

[0061] In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the sequence set forth in SEQ ID NO: 7 or 8. In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the sequence set forth in SEQ ID NO: 10. In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the sequence set forth in SEQ ID NO: 12.

[0062] In some cases, the 3'UTR nucleic acid sequence is located at the 3' end of the mitochondrial targeting sequence. In some cases, the 3'UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATP5B, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hsIRP1, hsMRPS12, hsATP5J2, rnSOD2, and hsOXA1L. In some cases, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 111-125. In some cases, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:13 or SEQ ID NO:14.

[0063] In some cases, the mitochondrial targeting sequence is located at the 5' end of the 3'UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at the 3' end of the mitochondrial targeting sequence.

[0064] In some cases, the recombinant nucleic acid comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 17-20, 23-24, 27-28, 31-34, 37-38, 41-42, 45-48, 51-52, 55-56, 59-62, 65-66, 69-70, 73-76, 79-80, and 83-84.

[0065] In another aspect, disclosed herein is a recombinant nucleic acid comprising a mitochondrial targeting sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 2, 3, and 4. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence set forth in SEQ ID NO: 2. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence set forth in SEQ ID NO: 3. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence set forth in SEQ ID NO: 4.

[0066] Optionally, the recombinant nucleic acid further comprises a mitochondrial protein coding sequence, the mitochondrial protein coding sequence encoding a polypeptide comprising a mitochondrial protein. Optionally, the mitochondrial protein is selected from the group consisting of NADH dehydrogenase 4 (ND4), NADH dehydrogenase 6 (ND6), NADH dehydrogenase 1 (ND1), and variants thereof. The mitochondrial protein optionally comprises NADH dehydrogenase 4 (ND4) or a variant thereof. Optionally, the mitochondrial protein comprises a peptide sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 160. Optionally, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 6, 7, or 8. Optionally, the mitochondrial protein comprises NADH dehydrogenase 6 (ND6) or a variant thereof. In some cases, the mitochondrial protein comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 161. In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 9 or 10. In some cases, the mitochondrial protein comprises NADH dehydrogenase 1 (ND1) or a variant thereof. In some cases, the mitochondrial protein comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 162. In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 11 or 12.

[0067] In some cases, the recombinant nucleic acid further comprises a 3'UTR nucleic acid sequence. In some cases, the 3'UTR nucleic acid sequence is located at the 3' end of the mitochondrial targeting sequence. In some cases, the 3'UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATP5B, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hsIRP1, hsMRPS12, hsATP5J2, rnSOD2, and hsOXA1L. In some cases, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 111-125. In some cases, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14. In some cases, the mitochondrial targeting sequence is located at the 5' end of the 3'UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at the 3' end of the mitochondrial targeting sequence.

[0068] In some cases, the recombinant nucleic acid comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 29-70.

[0069] In another aspect, disclosed herein is a recombinant nucleic acid comprising a mitochondrial protein coding sequence, wherein the mitochondrial protein coding sequence encodes a polypeptide comprising a mitochondrial protein, and wherein the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 7, 8, 10, and 12.

[0070] In some embodiments, the recombinant nucleic acid further comprises a mitochondrial targeting sequence. In some cases, the mitochondrial targeting sequence is selected from the group consisting of hsCOX10, hsCOX8, scRPM2, lcSirt5, tbNDUS7, ncQCR2, hsATP5G2, hsLACTB, spilv1, gmCOX2, crATP6, hsOPA1, hsSDHD, hsADCK3, osP0644B06.24-2, Neurospora crassa ATP9(ncATP9), hsGHITM, hsNDUFAB1, hsATP5G3, crATP6_hsADCK3, ncATP9_ncATP9, zmLOC100282174, ncATP9_zmLOC100282174_spilv1_ncATP9, z mLOC100282174_hsADCK3_crATP6_hsATP5G3, zmLOC100282174_hsADCK3_hsATP5G3, ncATP9_zmLOC100282174, hsADCK3_zmLOC100282174_crATP6 ncATP9_zmLOC100282174_spilv1_GNFP_ncATP9, and ncATP9_zmLOC100282174_spilv1_lcSirt5_osP0644B06.24-2_hsATP5G3, crATP6_hsADCK3_zmLOC100282174_hsATP5G3, hsADCK3_zmLOC100282174, hsADCK3_zmLOC100282174_crATP6, ncATP9_zmLOC100282174_spilv1_GNFP_ncATP9, and ncATP9_zmLOC100282174_spilv1_lcSirt5_osP0644B06.24-2_hsATP5G2_ncATP9. In some cases, the mitochondrial targeting sequence encodes a polypeptide comprising a peptide sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 129-159. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 2. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:3.In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 4. In some cases, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:5.

[0071] In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the sequence set forth in SEQ ID NO: 7 or 8. In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the sequence set forth in SEQ ID NO: 10. In some cases, the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the sequence set forth in SEQ ID NO: 12.

[0072] In some cases, the recombinant nucleic acid further comprises a 3'UTR nucleic acid sequence. In some cases, the 3'UTR nucleic acid sequence is located at the 3' end of the mitochondrial targeting sequence. In some cases, the 3'UTR nucleic acid sequence comprises a sequence selected from the group consisting of hsACO2, hsATP5B, hsAK2, hsALDH2, hsCOX10, hsUQCRFS1, hsNDUFV1, hsNDUFV2, hsSOD2, hsCOX6c, hsIRP1, hsMRPS12, hsATP5J2, rnSOD2, and hsOXA1L. In some cases, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 111-125. In some cases, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14. In some cases, the mitochondrial targeting sequence is located at the 5' end of the 3'UTR nucleic acid sequence. In some cases, the mitochondrial targeting sequence is located at the 3' end of the mitochondrial targeting sequence.

[0073] In some cases, the recombinant nucleic acid comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 17-20, 23-24, 27-28, 31-34, 37-38, 41-42, 45-48, 51-52, 55-56, 59-62, 65-66, 69-70, 73-76, 79-80, and 83-84.

[0074] In another aspect, disclosed herein is a viral vector comprising the recombinant nucleic acid disclosed herein.In some cases, the viral vector is an adeno-associated viral (AAV) vector.In some cases, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16 vector.In some cases, the AAV vector is a recombinant AAV (rAAV) vector.In some cases, the rAAV vector is a rAAV2 vector.

[0075] In another aspect, disclosed herein is a method for treating an ocular disease, comprising administering any of the pharmaceutical compositions disclosed herein to a patient in need of treatment for the ocular disease. Optionally, the ocular disease is Leber's Hereditary Optic Neuropathy (LHON). Optionally, the method comprises administering the pharmaceutical composition to one or both eyes of the patient. Optionally, the pharmaceutical composition is administered by intraocular injection or intravitreal injection. Optionally, the pharmaceutical composition is administered by intravitreal injection. Optionally, about 0.01-0.1 mL of the pharmaceutical composition is administered by intravitreal injection. Optionally, about 0.05 mL of the pharmaceutical composition is administered by intravitreal injection.

[0076] Optionally, the method further comprises administering methylprednisolone to the patient. Optionally, the methylprednisolone is administered prior to intravitreal injection of the pharmaceutical composition. Optionally, the methylprednisolone is administered orally. Optionally, the methylprednisolone is administered daily for at least 1, 2, 3, 4, 5, 6, or 7 days prior to intravitreal injection of the pharmaceutical composition. Optionally, the methylprednisolone is administered daily. Optionally, the methylprednisolone is administered at a daily dose of about 32 mg / 60 kg. Optionally, the methylprednisolone is administered after intravitreal injection of the pharmaceutical composition. Optionally, the method further comprises administering sodium creatine phosphate to the patient. Optionally, the sodium creatine phosphate is administered intravenously. Optionally, the methylprednisolone is administered intravenously or orally. In some cases, the method includes administering methylprednisolone intravenously for at least one day, followed by administering methylprednisolone orally for at least one week. In some cases, the method includes administering methylprednisolone intravenously for about three days, followed by administering methylprednisolone orally for at least about six weeks. In some cases, methylprednisolone is administered intravenously at a daily dose of about 80 mg / 60 kg. In some cases, administration of the pharmaceutical composition results in a higher average level of visual acuity recovery compared to administration of a comparable pharmaceutical composition that does not include a recombinant nucleic acid. In some cases, administration of the pharmaceutical composition results in a higher average level of visual acuity recovery than a comparable pharmaceutical composition that includes a recombinant nucleic acid as set forth in SEQ ID NO:15.

[0077] In some embodiments, the disclosure provides a method of treating an ocular disease, the method comprising administering to a patient in need of treatment for the ocular disease (a) a first pharmaceutical composition comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid, the recombinant nucleic acid comprising: (i) a nucleic acid sequence encoding a mitochondrial targeting peptide; (ii) a nucleic acid sequence encoding a mitochondrial protein, the nucleic acid sequence comprising a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 6-12; and (iii) a 3'UTR nucleic acid sequence; and (b) a second pharmaceutical composition comprising a steroid.

[0078] In some embodiments, the nucleic acid sequence encoding the mitochondrial protein encodes a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 160-162. In some embodiments, the nucleic acid sequence encoding the mitochondrial targeting peptide encodes a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 126-159. In some embodiments, the nucleic acid sequence encoding the mitochondrial targeting peptide comprises a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-5. In some embodiments, the 3'UTR nucleic acid sequence comprises a nucleic acid sequence (nucleic sequence) having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 13, 14, and 111-125.

[0079] In some embodiments, the disclosure provides a method of treating an ocular disease, the method comprising administering to a patient in need of treatment for the ocular disease (a) a first pharmaceutical composition comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid, the recombinant nucleic acid comprising (i) a nucleic acid sequence encoding a mitochondrial targeting peptide comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 126-159, (ii) a nucleic acid sequence encoding a mitochondrial protein, and (iii) a 3'UTR nucleic acid sequence; and (b) a second pharmaceutical composition comprising a steroid.

[0080] In some embodiments, the mitochondrial protein is selected from the group consisting of NADH dehydrogenase 4 (ND4), NADH dehydrogenase 6 (ND6), NADH dehydrogenase 1 (ND1), and variants thereof. In some embodiments, the nucleic acid sequence encoding the mitochondrial protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-12. In some embodiments, the nucleic acid sequence encoding the mitochondrial protein encodes a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 160-162. In some embodiments, the nucleic acid sequence encoding the mitochondrial targeting peptide comprises a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-5. In some embodiments, the 3'UTR nucleic acid sequence comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 13, 14 and 111-125.

[0081] In some embodiments, the disclosure provides a method of treating an ocular disease, comprising administering to a patient in need of treatment for the ocular disease (a) a first pharmaceutical composition comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid, the recombinant nucleic acid comprising (i) a nucleic acid sequence encoding a mitochondrial targeting peptide comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 126-159, and (ii) a sequence selected from the group consisting of SEQ ID NOs: 6-12. and (iii) a 3'UTR nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 13, 14, and 111-125; and (b) a second pharmaceutical composition comprising a steroid.

[0082] In some embodiments, the disclosure provides a method of treating an ocular disease, comprising administering to a patient in need of treatment for the ocular disease (a) a first pharmaceutical composition comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid, the recombinant nucleic acid comprising (i) a nucleic acid sequence encoding a mitochondrial targeting peptide and (ii) a nucleic acid sequence encoding a mitochondrial protein, and (b) a second pharmaceutical composition comprising a steroid. In some embodiments, the mitochondrial protein is selected from the group consisting of NADH dehydrogenase 4 (ND4), NADH dehydrogenase 6 (ND6), NADH dehydrogenase 1 (ND1), and variants thereof. In some embodiments, the 3'UTR nucleic acid sequence comprises a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 13, 14, and 111-125.

[0083] In some embodiments, the recombinant nucleic acid comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 15-84. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO:15.

[0084] In some embodiments, the first pharmaceutical composition is administered by intraocular or intravitreal injection. In some embodiments, about 0.01-0.1 mL of the first pharmaceutical composition is administered by intravitreal injection. In some embodiments, about 0.05 mL of the first pharmaceutical composition is administered by intravitreal injection. In some embodiments, the first pharmaceutical composition is administered to one or both eyes.

[0085] In some embodiments, the steroid is alclometasone dipropionate, amcinonide, beclomethasone dipropionate, beclomethasone, betamethasone benzoate, betamethasone dipropionate, betamethasone sodium phosphate, betamethasone sodium phosphate and sodium acetate, betasone valerate, clobetasol propionate, clocortolone pivalate, cortisol (hydrocortisol), cortisol acetate (hydrocortisone), cortisone butyrate (hydrocortisone), cortisol cypionate (hydrocortisone), cortisol (hydrocortisone) sodium phosphate, cortisol (hydrocortisone) sodium succinate, cortisol valerate (hydrocortisone), cortisone acetate, desoxycortisone. nido, desoximetasone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, diflorasone diacetate, fludrocortisone acetate, flunisolide, fluocinoid acetate, fluocinonide, fluorometholone, flurandrenolide, halcinonide, medrysone, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, mometasone furoate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone butylacetate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, and triamcinolone hexacetonide or synthetic analogs thereof.

[0086] In some embodiments, the steroid is a glucocorticoid, hi some embodiments, the glucocorticoid is methylprednisolone or prednisone.

[0087] In some embodiments, the methylprednisolone is formulated as a tablet or liquid for intravenous administration. In some embodiments, the steroid is administered orally or intravenously.

[0088] In some embodiments, the steroid is administered prior to administration of the first pharmaceutical composition. In some embodiments, the steroid is administered daily for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days prior to administration of the first pharmaceutical composition. In some embodiments, the steroid is methylprednisolone and is administered at a daily dose of about 30 mg / 60 kg to about 40 mg / 60 kg or about 30 mg to about 40 mg. In some embodiments, the daily dose of methylprednisolone is about 32 mg / 60 kg or 32 mg. In some embodiments, the steroid is prednisone and is administered at a daily dose of about 50 mg / 60 kg to about 70 mg / 60 kg. In some embodiments, the daily dose of prednisone is about 60 mg / 60 kg.

[0089] In some embodiments, the steroid is administered after administration of the first pharmaceutical composition. In some embodiments, the steroid is administered daily for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, or at least 15 weeks after administration of the first pharmaceutical composition.

[0090] In some embodiments, the steroid is methylprednisolone and is administered at a daily dose of about 70 mg / 60 kg to 90 mg / 60 kg or about 70 mg to 90 mg. In some embodiments, the daily dose of methylprednisolone is about 80 mg / 60 kg or 80 mg. In some embodiments, the methylprednisolone is administered for at least 2 days after administration of the first pharmaceutical composition. In some embodiments, subsequent doses of methylprednisolone are administered daily for at least 7 weeks after administration of the first pharmaceutical composition, wherein the dose of methylprednisolone is reduced week by week.

[0091] In some embodiments, the steroid is prednisone and is administered at a daily dose of about 50 mg / 60 kg to about 70 mg / 60 kg or about 50 mg to about 70 mg. In some embodiments, the daily dose of prednisone is about 60 mg / 60 kg or 60 mg. In some embodiments, the prednisone is administered for at least 7 days after administration of the first pharmaceutical composition. In some embodiments, after 7 days, the prednisone is administered at a daily dose of about 30 mg / 60 kg to about 50 mg / 60 kg or about 30 mg to 50 mg. In some embodiments, the daily dose of prednisone is about 40 mg / 60 kg or 40 mg. In some embodiments, subsequent doses of prednisone are administered daily for at least 4 days, wherein the dose of prednisone is decreased each day.

[0092] In some embodiments, the steroid is administered before or after administration of the first pharmaceutical composition.

[0093] In some embodiments, the steroid is methylprednisolone, and is administered daily for at least 7 days prior to administration of the first pharmaceutical composition, and is administered daily for at least 7 weeks after administration of the first pharmaceutical composition. In some embodiments, methylprednisolone is administered at a daily dose of about 32mg / 60kg or 32mg prior to administration of the first pharmaceutical composition. In some embodiments, methylprednisolone is administered at a daily dose of about 80mg / 60kg or 80mg for at least 2 days after administration of the first pharmaceutical composition. In some embodiments, methylprednisolone is administered at a daily dose of about 40mg / 60kg or 40mg for at least 4 days, starting 3 days after administration of the first pharmaceutical composition. In some embodiments, methylprednisolone is administered at a daily dose of about 32mg / 60kg or 32mg for at least 1 week, starting 1 week after administration of the first pharmaceutical composition. In some embodiments, methylprednisolone is administered at a daily dose of about 24 mg / 60 kg or 24 mg for at least one week starting 2 weeks after administration of the first pharmaceutical composition. In some embodiments, methylprednisolone is administered at a daily dose of about 16 mg / 60 kg or 16 mg for at least one week starting 3 weeks after administration of the first pharmaceutical composition. In some embodiments, methylprednisolone is administered at a daily dose of about 8 mg / 60 kg or 8 mg for at least one week starting 4 weeks after administration of the first pharmaceutical composition. In some embodiments, methylprednisolone is administered at a daily dose of about 6 mg / 60 kg or 6 mg for at least one week starting 5 weeks after administration of the first pharmaceutical composition. In some embodiments, methylprednisolone is administered at a daily dose of about 4 mg / 60 kg or 6 mg for at least one week starting 6 weeks after administration of the first pharmaceutical composition.

[0094] In some embodiments, the steroid is prednisone and is administered daily for at least 2 days before administration of the first pharmaceutical composition and for at least 11 weeks after administration of the first pharmaceutical composition. In some embodiments, prednisone is administered at a daily dose of about 60mg / 60kg or 60mg before administration of the first pharmaceutical composition. In some embodiments, prednisone is administered at a daily dose of about 60mg / 60kg or 60mg for at least 7 days after administration of the first pharmaceutical composition. In some embodiments, prednisone is administered at a daily dose of about 40mg / 60kg or 40mg for at least 8 days after administration of the first pharmaceutical composition. In some embodiments, prednisone is administered at a daily dose of about 20mg / 60kg or 20mg for at least 9 days after administration of the first pharmaceutical composition. In some embodiments, prednisone is administered at a daily dose of about 10 mg / 60 kg or 10 mg for at least 10 days following administration of the first pharmaceutical composition.

[0095] In some embodiments, the method further comprises administering sodium creatine phosphate to the patient, hi some embodiments, the sodium creatine phosphate is administered intravenously before and / or after administration of the first pharmaceutical composition.

[0096] In some embodiments, administration of the first pharmaceutical composition and the second pharmaceutical composition results in a higher average level of visual acuity recovery than a comparable pharmaceutical composition administered without the second pharmaceutical composition. In some embodiments, administration of the first pharmaceutical composition and the second pharmaceutical composition results in a lower incidence of adverse events than a comparable pharmaceutical composition administered without the second pharmaceutical composition. In some embodiments, the adverse events are selected from anterior chamber inflammation, vitritis, ocular hypertension, cataract removal, keratitis, vitreous hemorrhage, allergic conjunctivitis, and eye pain. In some embodiments, the higher average level of visual acuity recovery and the lower incidence of adverse events are determined in a patient population with ocular disease. In some embodiments, the patient population is ethnically matched. In some embodiments, the patient population is Chinese or Argentinian.

[0097] In some embodiments, the eye disease is Leber's hereditary optic neuropathy (LHON). In some embodiments, the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some embodiments, the AAV is AAV2.

[0098] In some embodiments, the disclosure provides a method for screening and treating patients with an ocular disease, comprising: (a) obtaining a serum sample from the patient; (b) culturing a target cell population with a composition comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid encoding a detectable marker in the presence of the serum sample; and (c) detecting an expression level of the detectable marker in the target cell population after culturing, wherein the patient is selected for treatment if the expression level of the detectable marker in the target cell population is higher than a predetermined threshold.

[0099] In some embodiments, the disclosure provides a method for screening and treating patients with an ocular disease, the method comprising: (a) culturing a target cell population with a composition comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid encoding a detectable marker in the presence of a serum sample from the patient; and (b) detecting the expression level of the detectable marker in the target cell population after culturing, wherein the patient is selected for treatment if the expression level of the detectable marker in the target cell population is higher than a predetermined threshold.

[0100] In some embodiments, the disclosure provides a method for treating an ocular disease in a patient in need of such treatment, comprising: (a) obtaining a serum sample from the patient; (b) culturing a target cell population with a composition comprising a first adeno-associated virus (AAV) comprising a first recombinant nucleic acid encoding a detectable marker in the presence of the serum sample; (c) detecting an expression level of the detectable marker in the target cell population; and (d) administering to the patient a pharmaceutical composition comprising a second AAV comprising a second recombinant nucleic acid, wherein the expression level of the detectable marker in the target cell population is greater than a predetermined threshold.

[0101] In some embodiments, the disclosure provides a method for treating an ocular disease in a patient in need of such treatment, comprising: (a) culturing a target cell population with a composition comprising a first adeno-associated virus (AAV) comprising a first recombinant nucleic acid encoding a detectable marker in the presence of a serum sample from the patient; (b) detecting an expression level of the detectable marker in the target cell population; and (c) administering to the patient a pharmaceutical composition comprising a second AAV comprising a second recombinant nucleic acid, wherein the expression level of the detectable marker in the target cell population is greater than a predetermined threshold.

[0102] In some embodiments, the detectable marker is a fluorescent protein. In some embodiments, the fluorescent protein is green fluorescent protein (GFP). In some embodiments, the detectable marker is detected by flow cytometry or qPCR. In some embodiments, the culturing step is performed for at least 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more.

[0103] In some embodiments, the predetermined threshold is about 40% of cells expressing the detectable marker when detection is performed by flow cytometry. In some embodiments, the predetermined threshold is about 0.6 of the relative expression level of the detectable marker when detection is performed by qPCR. In some embodiments, the target cell is a HEK-293T cell.

[0104] In some embodiments, the therapy is a recombinant AAV comprising a nucleic acid sequence encoding a mitochondrial protein. In some embodiments, the mitochondrial protein is selected from the group consisting of NADH dehydrogenase 4 (ND4), NADH dehydrogenase 6 (ND6), NADH dehydrogenase 1 (ND1), and variants thereof. In some embodiments, the patient comprises a mutation selected from G11778A of the ND4 gene, G3460A of the ND1 gene, and T14484C of the ND6 gene.

[0105] In some embodiments, the present disclosure provides a kit comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid encoding a detectable marker, a target cell population, and one or more reagents for detecting the detectable marker. In some embodiments, the kit further comprises a transfection reagent for transfecting the AAV into the target cell population. In some embodiments, the kit further comprises a second AAV comprising a recombinant nucleic acid encoding a mitochondrial protein. In some embodiments, the one or more reagents for detecting the detectable marker are selected from an antibody that binds to the detectable marker and one or more primer oligonucleotides specific to the recombinant nucleic acid encoding the detectable marker.

[0106] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description and the accompanying drawings which set forth by way of example only, and in which: [Brief description of the drawings]

[0107] [Figure 1] A map of the vector pAAV-CMV-ND4-3'Flag-COX10UTR-SV40 is shown. [Diagram 2] A map of the vector pAAV-CMV-ND4-3'Flag-COX10UTR-bGH is shown. [Diagram 3] A map of the vector pAAV-CMV-ND4-3'Flag-COX10UTR is shown. [Figure 4] 1 shows the mRNA expression efficiency of the plasmids in HEK293 cells. [Diagram 5] The results of experiments to test the stability of each formulation at 2-8° C. are shown. [Figure 6] The results of freeze / thaw experiments to test the stability of each formulation are shown. [Figure 7] The design of the ND4 expression cassette is shown. [Figure 8] FIG. 1 illustrates a first exemplary dosing regimen for steroids. [Figure 9] FIG. 2 illustrates a second exemplary dosing regimen for steroids. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] definition Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar or identical to those described herein can be used to carry out or test the formulations or doses of this specification, but some methods and materials are described below. Unless otherwise stated, the techniques used or contemplated herein are standard methods. These materials, methods, and examples are merely illustrative and not limiting.

[0109] As used herein and in the appended claims, the singular forms "a (an)," "and," and "the (said)" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of such substances, and reference to "the salt" refers to one or more salts (or salts) and equivalents thereof known to those skilled in the art, and so forth.

[0110] As used herein, unless otherwise stated, the term "or" may be a conjunction or a disjunction. As used herein, unless otherwise indicated, any embodiment may be combined with any other embodiment.

[0111] As used herein, unless otherwise indicated, certain inventive embodiments herein contemplate numerical ranges. Where a range is present, the range includes the endpoints of the range. Moreover, each subrange and value within the range exists as if it were expressly written.

[0112] The term "about" and its grammatical equivalents in connection with a reference numerical value, and its grammatical equivalents as used herein, can include a range of values ​​±10%, such as a range of values ​​±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the value. For example, the amount "about 10" includes the amount from 9 to 11.

[0113] The term "comprising" (and related terms such as "comprises" or "having" or "including") does not exclude that other embodiments (e.g., embodiments of any material components, compositions, methods or processes described herein) "consist of" or "essentially consist of" the recited features.

[0114] The term "subject" refers to a mammal that has been or is the object of treatment, observation, or experiment. The term "mammal" is intended to have its standard meaning and includes, for example, humans, dogs, cats, sheep, and cows. The methods described herein can be used in human treatment and veterinary applications. In some embodiments, the subject is a human.

[0115] The term "treatment" includes administration of at least one compound disclosed herein, or a pharma- ceutically acceptable salt thereof, to a mammalian subject, particularly a human subject, in need of such administration, and includes (i) inhibiting the onset of clinical symptoms of a disease, such as cancer; (ii) regressing clinical symptoms of a disease, such as cancer; and / or (iii) prophylactic treatment to prevent the onset of a disease, such as cancer.

[0116] The term "therapeutically effective amount" of a chemical entity described herein refers to an amount that, when administered to a human or non-human subject, is effective to provide a therapeutic benefit, such as ameliorating symptoms, slowing the progression of a disease, or preventing a disease.

[0117] As used herein, unless otherwise specified, the terms "nucleic acid" and "polynucleotide" are used interchangeably.

[0118] As used herein, unless otherwise indicated, a drug dose of Xmg / 60kg refers to the use of Xmg of drug per 60kg of patient weight. For example, a drug dose of 100mg / 60kg means that a patient weighing 60kg needs to take 100mg of drug, and similarly, another patient weighing 30kg needs to take 50mg of drug.

[0119] As used herein, unless otherwise indicated, the term "operably linked" refers to a functional connection between two or more sequences. For example, a functional link between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows expression of the polynucleotide of interest. In this sense, the term "operably linked" refers to an arrangement of a regulatory region and a coding sequence to be transcribed such that the regulatory region is effective in regulating the transcription or translation of the coding sequence of interest. In some embodiments disclosed herein, the term "operably linked" refers to a configuration in which a regulatory sequence is positioned relative to a sequence encoding a polypeptide or functional RNA so as to induce or regulate the expression or cellular localization of the mRNA, polypeptide, and / or functional RNA encoding the polypeptide, and a promoter is operably linked to a nucleic acid sequence when it is capable of mediating the transcription of the nucleic acid sequence. Functionally linked elements may be contiguous or non-contiguous. Basic techniques for operably linking two or more DNA sequences are well known to those of skill in the art, and these methods are described in many standard molecular biology texts (see, e.g., Maniatis et al., "Molecular Cloning: A Laboratory Manual" 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0120] Nucleic Acid and Polypeptide Sequences In one aspect of the present specification, a recombinant nucleic acid is provided that includes one or more sequences selected from a first ITR sequence, a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, a polyA signal sequence, and a second ITR sequence. In some embodiments, the recombinant nucleic acid includes all of these sequences. In some embodiments, the recombinant nucleic acid includes (in order from the 5' end to the 3' end): a first ITR sequence, a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, a polyA signal sequence, and a second ITR sequence, and in some embodiments, the different sequences are operably linked to each other. In some embodiments, the different regulatory sequences are operably linked to the mitochondrial protein coding sequence.

[0121] Table 1 is the nucleic acid and polypeptide sequences disclosed herein. The first column lists the SEQ ID NO of each sequence. The second column is a description of the nucleic acid or polypeptide construct. For example, the construct COX10-ND4-3'UTR (SEQ ID NO:15) is a nucleic acid that binds to the nucleic acid sequences of COX10 (SEQ ID NO:1), ND4 (SEQ ID NO:6) and 3'UTR (SEQ ID NO:13).

[0122] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

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[0123] Adeno-associated virus (AAV) Adeno-associated virus (AAV) is a small virus that infects humans and some other primate species. The compositions disclosed herein primarily comprise an adeno-associated virus (AAV) genome or a derivative thereof.

[0124] AAV genome is a polynucleotide sequence that encodes the functions required for the production of AAV virus particles.These functions include the AAV functions that function in the replication and packaging cycle of host cells, including the encapsidation of AAV genome into AAV virus particles.Naturally occurring AAV viruses are replication-deficient and depend on trans-acting helper functions to complete the replication and packaging cycle.Therefore, the AAV genome of the vector of the present invention is generally replication-deficient.

[0125] The AAV genome can be in a positive or negative single-stranded form, or in a double-stranded form. When the double-stranded form is used, the expression of the transgene can be promoted since it does not require a step of DNA replication in the target cell.

[0126] AAV genome can be derived from any naturally occurring serotype or isolate or clade of AAV.Therefore, AAV genome can be the complete genome of naturally occurring AAV virus.As known to those skilled in the art, naturally occurring AAV virus can be classified according to various biological systems.

[0127] Generally, AAV viruses are referred to by serotype. Serotypes correspond to AAV variant subspecies, which have unique reactivity due to the expression profile of capsid surface antigens and can be used to distinguish them from other variant subspecies. Usually, viruses with a certain AAV serotype do not cross-react efficiently with neutralizing antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, as well as recombinant serotypes such as Rec2 and Rec3, which have been recently identified from primate brain.

[0128] A preferred AAV serotype for use in the present invention is AAV2. Other serotypes of particular interest for use in the present invention include AAV4, AAV5 and AAV8, which are capable of efficiently transducing ocular tissues such as the retinal pigment epithelium. The AAV serotype used may be an AAV serotype other than AAV4. Reviews of AAV serotypes can be found in Choi et al. (Curr Gene Ther. 2005; 5(3); 299-310) and Wu et al. (Molecular Therapy. 2006; 14(3), 316-327). The sequences of the AAV genome or elements of the AAV genome (including ITR sequences, rep or cap genes) used in the present invention can be derived from the following accession numbers for the AAV whole genome sequence: Adeno-associated virus 1 NC_002077, AF063497; Adeno-associated virus 2 NC_001401; Adeno-associated virus 3 NC_001729; Adeno-associated virus 3BNC_001863; Adeno-associated virus 4 NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associated virus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC_005889, AY388617.

[0129] AAV viruses may also be referred to as clades or clones, which refers to the phylogenetic relationship of naturally occurring AAV viruses and generally refers to a phylogenetic group of AAV viruses that can be traced back to a common ancestor and includes all its descendants. Additionally, AAV viruses may also be referred to as specific isolates, i.e., genetic isolates of a particular AAV virus found in nature. The term genetic isolates refers to a group of AAV viruses that have undergone limited genetic mixing with other naturally occurring AAV viruses, thereby forming a population that is identifiable at the genetic level.

[0130] Examples of AAV clades and isolates that may be used in the present invention include: Clade A: AAV1 NC_002077, AF063497, AAV6 NC_001862, Hu.48 AY530611, Hu 43 AY530606, Hu 44 AY530607, Hu 46 AY530609; Clade B: Hu.19 AY530584, Hu.20 AY530586, Hu 23 AY530589, Hu22 AY530588, Hu24 AY530590, Hu21 AY530587, Hu27 AY530592, Hu28 AY530593, Hu 29 AY530594, Hu63 AY530595, Hu63 AY530596, Hu63 AY530597, Hu63 AY530598, Hu63 AY530599, Hu63 AY530590, Hu63 AY530591, Hu63 AY530592, Hu63 AY530593, Hu63 AY530594, Hu63 AY530595, Hu63 AY530596, Hu63 AY530597, Hu63 AY530598, Hu63 AY530599, Hu63 AY530591, Hu63 AY530592, Hu63 AY530593, Hu63 AY530594, Hu63 AY530595, Hu63 AY530596, Hu63 AY530597, Hu63 AY530598, Hu63 AY530 AY530624, Hu64 AY530625, Hu13 AY530578, Hu56 AY530618, Hu57 AY530619, Hu49 AY530612, Hu58 AY530620, Hu34 AY530598, Hu35 AY530599, AAV2 NC_001401, Hu45 AY530608, Hu47 AY530610, Hu51 AY530613, Hu52 AY530614, Hu T41 AY695378, Hu S17 AY695376, Hu T88 AY695375, Hu T71 AY695374, Hu T70 AY695373, Hu T40 AY695372, Hu T32 AY695371, Hu T17 AY695370, Hu LG15 AY695377; Clade C: Hu9 AY530629, Hu10 AY530576, Hu11 AY530577, Hu53 AY530615, Hu55 AY530617, Hu54 AY530616, Hu7 AY530628, Hu18 AY530583, Hu15 AY530580, Hu16 AY530581, Hu25 AY530591, Hu60 AY530622, Ch5 AY243021, Hu3 AY530595, Hu1 AY530575, Hu4 AY530602 Hu2, AY530585, Hu61 AY530623;<h2 style=";text-align:left;direction:ltr">RhD:Rh62 AY530573, Rh48 AY530561, Rh54 AY530567, Rh55 AY530568, Cy2 AY243020, AAV7 AF513851, Rh35 AY243000, Rh37 AY242998, Rh36 AY242999, Cy6 AY243016, Cy4 AY243018, Cy3 AY243019, Cy5 AY243017, Rh13 AY243013; RhE:Rh38 AY530558、Hu66 AY530626、Hu42 AY530605、Hu67 AY530627、Hu40 AY530603、Hu41 AY530604、Hu37 AY530600、Rh40 AY530559、Rh2 AY243007、Bb1 AY243023、Bb2 AY243022、Rh10 AY243015、Hu17 AY530582、Hu6 AY530621、Rh25 AY530557、Pi2 AY530554、Pi1 AY530553、Pi3 AY530555、Rh57 AY530569、Rh50 AY530563、Rh49 AY530562、Hu39 AY530601、Rh58 AY530570、Rh61 AY530572、Rh52 AY530565、Rh53 AY530566、Rh51 AY530564、Rh64 AY530574、Rh43 AY530560、AAV8 AF513852、Rh8 AY242997、Rh1 AY530556;クレードF:Hu14 (AAV9) AY530579,Hu31 AY530596,Hu32 AY530597,AAV 3 NC_001729,AAV 3B NC_001863、AAV4 NC_001829、Rh34 AY243001、Rh33 AY243002、Rh32 AY243003。;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0131] <h2 style=";text-align:left;direction:ltr"> Those skilled in the art can select the appropriate AAV serotype, clade, clone or isolate for use in the present invention based on general knowledge.For example, it has been shown that AAV5 capsid can efficiently transduce primate cone photoreceptors, as shown by the successful correction of inherited color blindness (Mancuso et al., Nature 2009, 461: 784-7).

[0132] However, it should be understood that the present invention also encompasses the use of AAV genomes of other serotypes that may not yet be identified or characterized. AAV serotypes determine the tissue specificity (or tropism) of AAV virus infection. Thus, the preferred AAV serotypes of AAV viruses used for administration to patients according to the present invention are those that have a natural tropism or high efficiency for infecting target cells in the eye of LHON. Thus, the AAV serotypes of AAV viruses used for administration to patients can be serotypes that infect the neurosensory retina and retinal pigment epithelium.

[0133] Usually, the AAV genome of a naturally occurring serotype or isolate or clade of AAV contains at least one inverted terminal repeat (ITR) sequence. The ITR sequence acts in cis to provide a functional origin of replication, allowing the integration and excision of the vector into the genome of the cell. The selectable ITR sequence may be derived from any of the AAV serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, as well as recombinant serotypes, such as Rec2 and Rec3, recently identified from primate brain, non-primate AAV, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV. The preferred ITR sequence is that of AAV2 and its variants.

[0134] In a preferred embodiment, one or more ITR sequences are flanked by polynucleotide sequences encoding ND4 or a variant thereof.

[0135] AAV genomes generally also contain packaging genes, such as the rep and / or cap genes, which code for packaging functions of AAV viral particles. The rep gene codes for one or more of the proteins Rep78, Rep68, Rep52, and Rep40 or variants thereof. The cap gene codes for one or more capsid proteins, such as VP1, VP2, and VP3 or variants thereof. These proteins constitute the capsid of the AAV viral particle. Capsid variants are discussed below.

[0136] A promoter is operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19, and p40 promoters (Laughlin et al., 1979, PNAS, 76:5567-5571). For example, the p5 and p19 promoters are commonly used to express the rep gene, and the p40 promoter is commonly used to express the cap gene.

[0137] Thus, as mentioned above, the AAV genome used in the vector of the present invention can be the complete genome of a naturally occurring AAV virus. For example, a vector containing a complete AAV genome can be used to prepare AAV virus in vitro. However, although such vectors can in principle be administered to patients, this is rarely done in practice. The AAV genome is preferably derivatized for the purpose of administering to patients. Such derivatization is standard in the art, and the present invention includes the use of any known derivative of the AAV genome, and derivatives that can be generated by applying techniques known in the art. Derivatization of the AAV genome and AAV capsid has been reviewed by Coura and Nardi (Virology Journal, 2007, 4:99) and Choi et al. and Wu et al.

[0138] The derivative of AAV genome includes any shortened or modified form of AAV genome that allows the in vivo expression of ND4 transgene from the vector of the present invention.Usually, it is possible to shorten AAV genome significantly so that it contains minimal viral sequence but retains the above-mentioned functions.This is preferable for safety reasons, to reduce the risk of vector recombination with wild-type virus and to avoid the induction of cellular immune response due to the presence of viral gene protein in target cells.

[0139] Generally, the derivative comprises at least one inverted terminal repeat (ITR) sequence, preferably two or more ITRs, for example, two or more ITRs. One or more ITRs may be derived from AAV genomes with different serotypes, or may be chimeric or mutated ITRs. A preferred mutated ITR is one that lacks trs (terminal resolution site). This deletion allows the genome to be replicated continuously to generate a single-stranded genome that contains both coding and complementary sequences, i.e., a self-complementary AAV genome. This allows the expression of the transgene to be promoted by eliminating the need for DNA replication in the target cell.

[0140] Either end of one or more ITRs is preferably flanked by a polynucleotide sequence encoding ND4, ND6, ND1, or variants thereof. The inclusion of one or more ITRs is preferred to aid in the formation of concatemers of the vector of the present invention in the nucleus of the host cell, for example, following the conversion of single-stranded vector DNA to double-stranded DNA by the action of the host cell DNA polymerase. The formation of such episomal concatemers protects the vector construct during the life of the host cell, thereby allowing long-term expression of the transgene in vivo.

[0141] In a preferred embodiment, the ITR elements are the only sequences that are retained from the original AAV genome in the derivative. Thus, the derivative preferably does not contain the rep and / or cap genes of the original genome, as well as any other sequences of the original genome. This is preferred for the reasons mentioned above, and also to reduce the possibility that the vector will be integrated into the host cell genome. Furthermore, the reduced size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) into the vector in addition to the transgene.

[0142] Thus, referring to the AAV2 genome, the following portions may be removed in the derivatives of the invention: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes (note: do not confuse the rep gene of the wild-type AAV genome with the human gene ND4, which is the human gene altered in LHON). However, in some embodiments, including in vitro embodiments, the derivatives may further comprise one or more rep and / or cap genes or other viral sequences of the AAV genome. Since naturally occurring AAV viruses integrate at a high frequency into a specific site on human chromosome 19, with only a negligible frequency of random integration, retention of the integrative capacity of the vector may be acceptable in therapeutic situations.

[0143] Where the derivative genome comprises genes encoding the capsid proteins, i.e., VP1, VP2 and / or VP3, the derivatives may be chimeric, shuffled or capsid-modified derivatives of one or more naturally occurring AAV viruses. In particular, the invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones or isolates of AAV within the same vector, i.e., pseudotyping.

[0144] Chimeric, shuffled or capsid modified derivatives are usually selected to confer one or more desired functions to the viral vector. Thus, these derivatives may show improved efficiency of gene delivery, reduced immunogenicity (humoral or cellular), altered tropism range, and / or improved targeting of specific cell types compared to AAV viral vectors containing naturally occurring AAV genomes (e.g., genomes of AAV2). Improved efficiency of gene delivery can be brought about by improved binding of cell surface receptors or co-receptors, improved internalization, improved intracellular and nuclear translocation, improved uncoating of viral particles, and improved conversion of single-stranded genomes to double-stranded forms. Improved efficiency may also be related to altered tropism range or targeting of specific cell populations, so that the vector dose is not diluted by administration to tissues where it is not needed.

[0145] Chimeric capsid proteins include proteins produced by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes.This can be done, for example, by marker rescue method, in which non-infectious capsid sequence of one serotype is co-transfected with capsid sequence of a different serotype, and capsid sequence with desired properties is selected using directional selection.Capsid sequence of different serotypes can be changed by homologous recombination in cells to produce novel chimeric capsid proteins.

[0146] Chimeric capsid proteins also include proteins produced by engineering a capsid protein sequence to transfer particular capsid protein domains, surface loops, or particular amino acid residues between two or more capsid proteins (e.g., between two or more capsid proteins of different serotypes).

[0147] Shuffled or chimeric capsid proteins may be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes may be generated by randomly fragmenting the sequences of related AAV genes, such as the sequences of genes encoding capsid proteins of several different serotypes, and then reassembling the fragments in a self-priming polymerase reaction, which may result in crossovers at regions of sequence homology. A library of hybrid AAV genes thus created by shuffling the capsid genes of several serotypes may be screened to identify viral clones with the desired functions. Similarly, error-prone PCR may be used to randomly mutate AAV capsid genes to generate a diverse library of variants that are then selected for desired properties.

[0148] The sequence of the capsid gene may also be genetically modified to introduce specific deletions, substitutions or insertions with respect to the native wild-type sequence. In particular, the capsid gene may be modified by inserting the sequence of an unrelated protein or peptide within the open reading frame of the capsid coding sequence or at the N-terminus and / or C-terminus of the capsid coding sequence.

[0149] The unrelated protein or peptide may advantageously act as a ligand for a particular cell type, thereby providing improved binding to target cells or improving the specificity of targeting of the vector to a particular cell population. An example is the use of RGD peptide to block uptake into the retinal pigment epithelium, thereby enhancing transduction of surrounding retinal tissue (Cronin et al., 2008 ARVO Abstract:D1048). The unrelated protein may also be an epitope or affinity tag that aids in purification of the viral particles as part of the production process. The insertion site is usually selected so as not to interfere with other functions of the viral particle, e.g., internalization, translocation of the viral particle. Those skilled in the art can identify suitable sites for insertion based on general knowledge. Particular sites are disclosed by Choi et al., referenced above.

[0150] The invention further encompasses the provision of sequences of the AAV genome in an order and organization different from that of the native AAV genome. The invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from two or more viruses. Such chimeric genes can be composed of sequences from two or more related viral proteins of different viral species.

[0151] The vectors of the present invention have the form of a polynucleotide sequence that includes the AAV genome or a derivative thereof, and sequences encoding ND4, ND6, ND1 or variants thereof.

[0152] For the avoidance of doubt, the present invention also provides AAV virus particles comprising the vector of the present invention.The AAV particles of the present invention include transcapsid forms, in which the AAV genome or derivatives with ITRs of one serotype are packaged in the capsid of a different serotype.The AAV particles of the present invention also include mosaic forms, in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral envelope.The AAV particles also include chemically modified forms that carry ligands adsorbed on the capsid surface.For example, such ligands may include antibodies that are used to target specific cell surface receptors.

[0153] The present invention further provides a host cell comprising a vector or an AAV viral particle of the invention.

[0154] Recombinant Nucleic Acid Sequences Also disclosed herein are recombinant nucleic acid sequences comprising polynucleotide sequences encoding NADH dehydrogenase subunit-4 (ND4), NADH dehydrogenase subunit-1 (ND1) and NADH dehydrogenase subunit-6 (ND6) polypeptides or variants thereof.

[0155] The polynucleotide sequence of ND4 is set forth in SEQ ID NO: 6 and encodes the protein set forth in SEQ ID NO: 160. Further nucleic acid sequences of ND4 are SEQ ID NOs: 7 and 8.

[0156] Variants of SEQ ID NO: 160 may include truncations, mutants or homologs thereof, and any transcriptional variants thereof that encode a functional ND4 polypeptide. Any homolog referred to herein generally shares at least 70% identity with the relevant region of ND4 and is capable of functionally complementing the polypeptide deficiency.

[0157] Homology / identity can be measured using known methods. For example, the UWGCG package provides the BESTFIT program that can be used to calculate homology (e.g., used with its default settings) (Devereux et al. 1984) Nucleic Acids Research 12, 387-395). PILEUP and BLAST algorithms can be used to calculate homology or lineup sequences (usually with their default settings), for example, as described in Altschul SF (1993) J Mol Evol 36: 290-300; Altschul, S, F et al. 1990) J Mol Biol 215: 403-10. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0158] In a preferred embodiment, the recombinant nucleic acid sequence can encode a polypeptide having at least 55%, 65%, 70%, 75%, 80%, 85%, 90%, more preferably at least 95%, 97%, 99%, 99.5%, or 100% identity to the relevant region of the ND4 protein (SEQ ID NO: 160) over at least 20 (preferably at least 30, e.g., at least 40, 60, 100, 200, 300, 400 or more contiguous amino acids), or even over the entire sequence of the recombinant nucleic acid. The relevant region is the region that confers the functional activity of ND4.

[0159] Alternatively and preferably, the recombinant nucleic acid sequence can encode a polypeptide having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 95%, 97%, 99%, 99.5%, or 100% identity over its entire sequence to the full-length ND4 (SEQ ID NO: 160). Typically, the recombinant nucleic acid sequence differs from the relevant region of ND4 (SEQ ID NO: 160) by at least 2, 5, 10, 20, 40, 50, or 60 mutations (each of which may be a substitution, insertion, or deletion), or by fewer mutations.

[0160] The ND4 polypeptide of the recombinant nucleic acid may have a percent identity (i.e., at least 70%, 80%, or 90%, more preferably at least 95%, 97%, 99% identity) to a particular region of SEQ ID NO:160 that is equal to any of the specified percent identity values ​​over any of the sequence lengths listed above.

[0161] Variants of ND4 (SEQ ID NO: 160) also include truncations. Any truncation can be used as long as the variant is functional. Truncation is generally performed to remove sequences that are not essential for protein activity and / or do not affect the conformation of the folded protein, especially the folding of the active site. Suitable truncations can be routinely identified by systematically truncating sequences of various lengths from the N-terminus or C-terminus. A preferred truncation is the N-terminus, and all other sequences except the catalytic domain can be removed.

[0162] Variants of ND4 (SEQ ID NO: 160) further include mutations with one or more (e.g., 2, 3, 4, 5-10, 10-20, 20-40, or more amino acid insertions, substitutions, or deletions) with respect to a particular region of ND4 (SEQ ID NO: 160). Deletions and insertions are preferably made outside the catalytic domain, as described below. Substitutions are also typically made in regions that are not essential for protease activity and / or do not affect the conformation of the folded protein.

[0163] Substitution preferably introduces one or more conservative changes, replacing an amino acid with another amino acid of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acids can have the same polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acids they replace. Alternatively, conservative changes can also introduce another aromatic or aliphatic amino acid in place of the aromatic or aliphatic amino acid originally present. Conservative amino acid changes are well known in the art and can be selected according to the properties of the amino acid.

[0164] Similarly, preferred variants of the polynucleotide sequence of ND4 (SEQ ID NO:6) include polynucleotides having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5% identity to the relevant region of ND4 (SEQ ID NO:6). Preferably, the variants exhibit these levels of identity to the full-length ND4 (SEQ ID NO:6) throughout their sequence.

[0165] Proteins or mRNAs can be targeted to mitochondria using mitochondrial targeting sequences (MTS) and 3' untranslated regions (3'UTRs). The charge, length, and structure of MTSs can be important for protein import into mitochondria. Certain 3'UTRs can induce mRNA localization to the mitochondrial surface, thereby facilitating co-translational protein import into mitochondria.

[0166] The polynucleotide sequences of the mitochondrial targeting sequences are: hsCOX10, hsCOX8, scRPM2, lcSirt5, tbNDUS7, ncQCR2, hsATP5G2, hsLACTB, spilv1, gmCOX2, crATP6, hsOPA1, hsSDHD, hsADCK3, osP0644B06.24-2, Neurospora crassa ATP9 (ncATP9), hsGHITM, hsNDUFAB1, hsATP5G3, crATP6_hsADCK3, ncATP9_zmLOC100282174 ... , zmLOC100282174_hsADCK3_hsATP5G3, ncATP9_zmLOC100282174, hsADCK3_zmLOC100282174_crATP6 _hsATP5G3, crATP6_hsADCK3_zmLOC100282174_hsATP5G3, hsADCK3_zmLOC100282174, hsADCK3_zmLOC100282174_crATP6, ncATP9_zmLOC100282174_spilv1_GNFP_ncATP9, and ncATP9_zmLOC100282174_spilv1_lcSirt5_osP0644B06.24-2_hsATP5G2_ncATP9 (see Table 1 for SEQ ID NOs). In one example, the polynucleotide sequence COX10 (SEQ ID NO: 1, 2, or 3) can encode the mitochondrial targeting sequence MTS-COX10 (SEQ ID NO: 126). In another example, the polynucleotide sequence COX8 (SEQ ID NO: 4) can encode the mitochondrial targeting sequence MTS-COX8 (SEQ ID NO: 127). In another example, the polynucleotide sequence OPA1 (SEQ ID NO: 5) can encode the mitochondrial targeting sequence MTS-OPA1 (SEQ ID NO: 128).

[0167] The 3'UTR nucleic acid sequence can be selected from the group consisting of hsACO2 (SEQ ID NO:111), hsATP5B (SEQ ID NO:112), hsAK2 (SEQ ID NO:113), hsALDH2 (SEQ ID NO:114), hsCOX10 (SEQ ID NO:115), hsUQCRFS1 (SEQ ID NO:116), hsNDUFV1 (SEQ ID NO:117), hsNDUFV2 (SEQ ID NO:118), hsSOD2 (SEQ ID NO:119), hsCOX6c (SEQ ID NO:120), hsIRP1 (SEQ ID NO:121), hsMRPS12 (SEQ ID NO:122), hsATP5J2 (SEQ ID NO:123), rnSOD2 (SEQ ID NO:124), and hsOXA1L (SEQ ID NO:125). The 3'UTR nucleic acid sequence may be a variant having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any of the 3'UTR nucleic acid sequences listed herein. For example, the 3'UTR nucleic acid sequence may be SEQ ID NO: 13 or 14.

[0168] Also disclosed herein is a recombinant nucleic acid sequence comprising a mitochondrial targeting sequence, a mitochondrial protein coding sequence, and a 3'UTR nucleic acid sequence. For example, the recombinant nucleic acid sequence can be selected from SEQ ID NOs: 15-84. The recombinant nucleic acid sequence can be a variant having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any of the 3'UTR nucleic acid sequences listed herein.

[0169] Promoters and Regulatory Sequences The vectors of the present invention also contain elements that allow for expression of the disclosed transgenes in vitro or in vivo. Thus, the vectors generally contain a promoter sequence operably linked to a polynucleotide sequence encoding the transgene or a variant thereof.

[0170] Any suitable promoter can be used. The promoter sequence can be constitutively active (i.e., functional in any host cell background), or can be active only in a specific host cell environment, thereby allowing targeted expression of the transgene in a specific cell type. The promoter can exhibit inducible expression in response to the presence of another factor, for example a factor present in the host cell. In any case, when the vector is administered for therapy, the promoter needs to be functional in the background of retinal cells.

[0171] In some embodiments, it is preferred that the promoter exhibits retinal cell-specific expression, so that the transgene is expressed only in a population of retinal cells. Thus, expression from the promoter may be retina-specific, for example restricted to only cells of the neurosensory retina and retinal pigment epithelium.

[0172] A preferred promoter for the ND4 transgene includes the chicken beta actin (CBA) promoter, optionally in combination with the cytomegalovirus (CME) enhancer element. In some cases, a preferred promoter for the ND4 transgene includes the CAG promoter. A particularly preferred promoter is a hybrid CBA / CAG promoter, such as the promoter used in the rAVE expression cassette. Examples of promoters based on human sequences that drive retina-specific gene expression include rhodospin kinase for rods and cones (Allocca et al., 2007, J Viol 81:11372-80), PR2.1 for cones only (Mancuso et al. 2009, Nature) and / or RPE65 for the retinal pigment epithelium (Bainbridge et al., 2008, N Eng J Med).

[0173] In some embodiments, the promoter comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 169. In some embodiments, the promoter comprises a sequence that differs from the sequence set forth in SEQ ID NO: 169 by at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the promoter has the sequence of SEQ ID NO: 169.

[0174] The vector of the present invention may also contain one or more additional regulatory sequences that may act pre- or post-transcriptionally. The regulatory sequence may be part of the native ND4 locus or may be a heterologous regulatory sequence. The vector of the present invention may contain some part of the 5'UTR or 3'UTR from the native ND4 locus.

[0175] Regulatory sequences are any sequences that act to promote the expression of a transgene, i.e., to increase the expression of a transcript, improve the nuclear export of mRNA, or increase its stability. Such regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites. In the context of the vector of the present invention, such regulatory sequences are cis-acting. However, the present invention also encompasses the use of trans-acting regulatory sequences located on additional gene constructs.

[0176] A preferred post-transcriptional regulatory element for use in the vectors of the invention is the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) or a variant thereof. Another regulatory sequence that can be used in the vectors of the invention is the scaffold attachment region (SAR).

[0177] Introns Introns, also known as spacer sequences, refer to segments of genes or mRNA molecules that are intervening sequences in the DNA of eukaryotic cells that have no coding function. These sequences are transcribed into precursor RNA and are removed by splicing, and therefore are not present in the final mature RNA molecule.

[0178] In some embodiments, the recombinant nucleic acid of the invention comprises an intron. In some embodiments, the intron is located between the promoter and the Kozak sequence. In some embodiments, the intron is located between the promoter and the mitochondrial targeting sequence. In some embodiments, the intron is located between the promoter and the mitochondrial protein coding sequence.

[0179] In some embodiments, the intron comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 170. In some embodiments, the intron comprises a sequence that differs by at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the sequence set forth in SEQ ID NO: 170. In some embodiments, the intron has the sequence of SEQ ID NO: 170.

[0180] Kozak sequence Kozak sequences (Kozak consensus sequences) are present in eukaryotic mRNAs, and can be recognized by ribosomes on the mRNA and used as a translation initiation site.

[0181] In some embodiments, a recombinant nucleic acid of the invention comprises a Kozak sequence. In some embodiments, the Kozak sequence is SEQ ID NO: 171. In some embodiments, the Kozak sequence differs from SEQ ID NO: 171 by at most 1, 2, or 3 nucleotides.

[0182] In some technical descriptions in the art, the Kozak sequence is as set forth in SEQ ID NO: 171, and is immediately adjacent to the 3' end with a translation initiation sequence four nucleotides in length, and the complete adjacent sequence is GCCACCATGG (SEQ ID NO: 177), with nucleotides -4 to -2 being the start codon ATG. Therefore, when the Kozak sequence is linked to a coding sequence (e.g., when the Kozak sequence is placed before a mitochondrial targeting sequence), it will be understood by those skilled in the art that the Kozak sequence in front of the coding sequence does not include this four nucleotide translation initiation sequence.

[0183] PolyA tail A polyadenylic acid (polyA) tail is usually located downstream of the coding region of a gene and functions to terminate transcription. In some embodiments, the recombinant nucleic acid comprises a polyA tail. In some embodiments, the polyA tail has 150 to 200 adenylic acid residues.

[0184] In some embodiments, the recombinant nucleic acid comprises a polyA tail of a simian vacuolating virus (SV40 polyA). In some embodiments, the polyA tail of a simian vacuolating virus comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 173. In some embodiments, the polyA tail of a simian vacuolating virus differs from the sequence set forth in SEQ ID NO: 173 by at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the polyA tail of a simian vacuolating virus has the sequence of SEQ ID NO: 173.

[0185] In some embodiments, the recombinant nucleic acid comprises a polyA tail of bovine growth factor (bGH polyA). In some embodiments, the polyA tail of bovine growth factor comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 172. In some embodiments, the polyA tail of bovine growth factor differs from the sequence set forth in SEQ ID NO: 172 by at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the polyA tail of bovine growth factor has the sequence of SEQ ID NO: 172.

[0186] In some embodiments, the polyA signal sequence has a length of 122 base pairs (bp) or less, 125 base pairs or less, 130 base pairs or less, 140 base pairs or less, 150 base pairs or less, 160 base pairs or less, 170 base pairs or less, 180 base pairs or less, 190 base pairs or less, 200 base pairs or less, 220 base pairs or less, 240 base pairs or less, 260 base pairs or less, 280 base pairs or less, or 300 base pairs or less.

[0187] Vector preparation The vectors of the present invention can be prepared by standard means well known in the art for providing vectors for gene therapy. Thus, suitable vector preparations can be prepared using well-established public domain transfection, packaging, and purification methods.

[0188] As discussed above, the vectors of the present invention may contain the entire genome of a naturally occurring AAV virus in addition to the polynucleotide sequence encoding ND4 or a variant thereof, however, typically a derivative genome is used (e.g., a derivative having at least one inverted terminal repeat (ITR) sequence but which may lack AAV genes such as rep or cap).

[0189] In such embodiments, additional genetic constructs providing AAV and / or helper virus functions are provided to the host cell along with the derived genome to allow assembly of the derived genome into an AAV viral particle. These additional constructs typically include genes encoding the structural AAV capsid proteins, i.e., cap, VP1, VP2, VP3, and genes encoding other functions required for the AAV life cycle, such as rep. The choice of structural capsid proteins provided on the additional constructs determines the serotype of the packaged viral vector.

[0190] Particularly preferred packaged viral vectors for use in the present invention contain a derivatized genome of AAV2 combined with AAV5 or AAV8 capsid proteins, and typically contain one or more ITRs of AAV2.

[0191] As noted above, because the AAV virus is replication incompetent, helper virus functions, preferably adenovirus helper functions, are also provided, usually on one or more additional constructs, to enable AAV replication.

[0192] All of the above additional constructs may be provided in the host cell as a plasmid or other episomal element, or alternatively, one or more of the constructs may be integrated into the genome of the host cell.

[0193] In these embodiments, the present invention provides a method for producing the vector of the present invention. The method comprises providing a vector comprising an adeno-associated virus (AAV) genome or a derivative thereof and a polynucleotide sequence encoding ND4 or a variant thereof in a host cell, and providing a means for the replication and assembly of the vector into an AAV viral particle. Preferably, the method comprises providing a vector comprising a derivative of the AAV genome and a polynucleotide sequence encoding ND4 or a variant thereof, together with one or more additional genetic constructs encoding AAV and / or helper virus functions. Typically, the derivative of the AAV genome comprises at least one ITR. Optionally, the method further comprises purifying the assembled viral particle. Additionally, the method may comprise formulating the viral particle for therapeutic use.

[0194] Treatment methods and medical uses As discussed above, the present inventors have unexpectedly demonstrated that the vectors of the present invention can be used to correct the cellular dysfunction underlying LHON. In particular, the present inventors have shown that the use of vectors can correct the defects associated with LHON. This provides a means by which the degenerative process of the disease can be treated, inhibited, mitigated, or prevented.

[0195] Thus, the present invention provides a method of treating or preventing LHON in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a vector encoding a mitochondrial protein as described herein by direct retinal, subretinal or intravitreal injection. In some embodiments, the method further comprises administering a steroid before, during and / or after administration of the vector encoding a mitochondrial protein, thereby treating or preventing LHON in the patient.

[0196] Vectors suitable for use in the methods of the invention include the vectors described herein, equivalent vectors encoding mitochondrial proteins, and their biosimilars. Equivalent vectors encoding mitochondrial proteins suitable for use in the methods of the invention include those described in the art, such as those described by Guy et.al in Ophthalmology 2017;124:1621-1634 and those described by Vignal et.al in Ophthalmology 2018;6:945-947, each of which is incorporated herein by reference in its entirety. A biosimilar is a biological product that is highly similar to an existing FDA approved reference product ("RP") and has no clinically significant differences. A "highly similar" product is one that has the same purity, chemical properties, and biological activity as the reference product. However, minor differences between the reference product and the clinically inactive components of the proposed biosimilar product are acceptable. For example, these differences may include slight differences in stabilizers or buffers compared to those used in the reference product, and slight differences during the manufacturing process (i.e., acceptable intra-product variability) are expected. FDA will carefully evaluate any differences between the proposed biosimilar product and the reference product to ensure that the biosimilar product meets the high FDA approval standards. "No clinically significant differences" means that the biosimilar product is not clinically significant different in safety, purity, and potency (safety and effectiveness) from the reference product, as demonstrated by human pharmacokinetic (exposure) and pharmacodynamic (response) studies, clinical immunogenicity evaluations, and, if necessary, further clinical trials.

[0197] In some embodiments, a patient in need of treatment with the methods provided herein has one or more mitochondrial DNA (mtDNA) point mutations. In some embodiments, the patient has a point mutation in a gene encoding a protein of complex I in the mitochondrial oxidative phosphorylation chain. For example, the patient may have one or more point mutations in the MT-ND4 gene (also known as ND4, NCBI gene ID: 4538), which encodes the NADH dehydrogenase subunit 4 protein (ND4). In some embodiments, the patient has a point mutation at nucleotide position 11778 of the ND4 gene. In some embodiments, the point mutation is G11778A in the ND4 gene. In some embodiments, the patient in need of treatment with the methods provided herein has a G11778A point mutation in the ND4 gene and is of Chinese and / or Argentinean descent.

[0198] In some embodiments, the patient in need of treatment with the methods provided herein has a G11778A point mutation in the ND4 gene and is of Argentinean descent. In some embodiments, the patient in need of treatment with the methods provided herein has a G11778A point mutation in the ND4 gene and is of Chinese descent.

[0199] In a related aspect, the invention provides the use of a vector of the invention in a method for treating or preventing LHON by administering the vector to a patient by direct retinal, subretinal or intravitreal injection.Further, the invention provides the use of a vector of the invention in the manufacture of a medicament for treating or preventing LHON by direct retinal, subretinal or intravitreal injection.

[0200] In all these embodiments, the vector of the present invention can be administered to prevent the onset of one or more symptoms of LHON. The patient may be asymptomatic. The subject may be predisposed to the disease. The method or use can include determining whether the subject is at risk of developing LHON or has LHON. Such a subject is administered a prophylactically effective amount of the vector. A prophylactically effective amount is an amount that prevents the onset of one or more symptoms of the disease.

[0201] Alternatively, the vector may be administered to cure existing symptoms of the disease when the symptoms of the disease appear in the subject. A prophylactically effective amount of the vector is administered to such a subject. A therapeutically effective amount is an amount that is effective in improving one or more symptoms of the disease. Such an amount can also prevent, delay, or reverse some of the loss of peripheral vision associated with LHON. Such an amount can also prevent, delay, or reverse the onset of LHON.

[0202] A typical single dose is 1010-1012 genome particles depending on the amount of remaining retinal tissue that requires transduction. A genome particle is defined herein as an AAV capsid containing a single-stranded DNA molecule that can be quantified using sequence-specific methods such as real-time PCR. The dose may be administered as a single dose, but may be repeated for both eyes or if the vector is not targeted to the correct area of ​​the retina for any reason (e.g., surgical complications). Treatment is preferably a one-time permanent treatment for each eye, but repeated injections, for example, in the future and / or with different AAV serotypes, may also be considered.

[0203] The invention also provides a method for monitoring the treatment or prevention of LHON in a patient, comprising measuring the ex vivo activity of retinal cells obtained from said patient following administration of an AAV vector of the invention by direct retinal, subretinal or intravitreal injection, such that the efficacy of the treatment can be determined.

[0204] In some embodiments, the disclosure provides a method of treating an ocular disease (e.g., LHON) comprising administering a therapeutically effective amount of a vector described herein and a steroid. Exemplary steroids include, but are not limited to, alclometasone dipropionate, amcinonide, beclomethasone dipropionate, betamethasone, betamethasone benzoate, betamethasone dipropionate, betamethasone sodium phosphate, betamethasone sodium phosphate and sodium acetate, betasone valerate, clobetasol propionate, clocortolone pivalate, cortisol (hydrocortisol), cortisol acetate (hydrocortisone), cortisone butyrate (hydrocortisone), cortisol cypionate (hydrocortisone), cortisol (hydrocortisone) sodium phosphate, cortisol (hydrocortisone) sodium succinate, cortisol valerate (hydrocortisone), cortisone acetate, desonide, squalene ... In some embodiments, the steroid is a glucocorticoid, and may be selected from the group consisting of methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, mometasone furoate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone butylacetate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, and triamcinolone hexacetonide, or synthetic analogs thereof, or combinations thereof. In some embodiments, the steroid is selected from the group consisting of prednisone, methylprednisolone, and methylprednisolone sodium succinate.

[0205] In some embodiments, the steroid is methylprednisolone. In some embodiments, the methylprednisolone is formulated as a tablet, e.g., MEDROL® tablet, for oral administration. For example, in some embodiments, the methylprednisolone is formulated as a tablet containing one or more inactive ingredients, such as calcium stearate, cornstarch, sodium erythrosine, lactose, mineral oil, sorbic acid, sucrose, or FD&C Yellow No. 6. In some embodiments, the methylprednisolone is formulated as a liquid for administration by injection, e.g., SOLU-MEDROL®. For example, in some embodiments, methylprednisolone sodium succinate is formulated as a liquid containing one or more inactive ingredients, such as sodium dihydrogen phosphate anhydrous, disodium hydrogen phosphate anhydrous, or lactose hydrate, and optionally a preservative, such as benzyl alcohol. In some embodiments, the steroid is MEDROL® or SOLU-MEDROL®, including its common forms.

[0206] In some embodiments, about 1 mg / 60 kg to about 100 mg / 60 kg, about 1 mg / 60 kg to about 80 mg / 60 kg, about 1 mg / 60 kg to about 60 mg / 60 kg, about 1 mg / 60 kg to about 40 mg / 60 kg, about 1 mg / 60 kg to about 20 mg / 60 kg, about 20 mg / 60 kg to about 100 mg / 60 kg, about 20 mg / 60 kg to about 80 mg / 60 kg, about 20 mg / 60 kg to about 60 mg / 60 kg, The patient is administered one or more doses of steroids in the range of about 20 mg / 60 kg to about 40 mg / 60 kg, about 40 mg / 60 kg to about 100 mg / 60 kg, about 40 mg / 60 kg to about 80 mg / 60 kg, about 40 mg / 60 kg to about 60 mg / 60 kg, about 60 mg / 60 kg to about 100 mg / 60 kg, about 60 mg / 60 kg to about 80 mg / 60 kg, or about 80 mg / 60 kg to about 100 mg / 60 kg. In some embodiments, one or more doses of steroids of about 4 mg / 60 kg, 6 mg / 60 kg, 8 mg / 60 kg, 10 mg / 60 kg, 16 mg / 60 kg, 20 mg / 60 kg, 24 mg / 60 kg, 32 mg / 60 kg, 40 mg / 60 kg, 48 mg / 60 kg, 60 mg / 60 kg, or 80 mg / 60 kg are administered to the patient. In some embodiments, one or more doses of steroids ranging from about 1 mg to about 96 mg are administered to the patient. In some embodiments, one or more doses of steroids of at least about 1 mg are administered to the patient. In some embodiments, one or more doses of steroids of about 96 mg or less are administered to the patient.In some embodiments, the amount of the active ingredient may be about 1 mg to about 2 mg, about 1 mg to about 4 mg, about 1 mg to about 8 mg, about 1 mg to about 16 mg, about 1 mg to about 32 mg, about 1 mg to about 64 mg, about 1 mg to about 96 mg, about 2 mg to about 4 mg, about 2 mg to about 8 mg, about 2 mg to about 16 mg, about 2 mg to about 32 mg, about 2 mg to about 64 mg, about 2 mg to about 96 mg, about 4 mg to about 8 mg, about 4 mg to about 16 mg, or about 4 mg. The patient is administered one or more doses of steroid ranging from about 1 mg, about 2 mg, about 4 mg, about 8 mg, about 16 mg, about 32 mg, about 4 mg, about 64 mg, about 4 mg, about 96 mg, about 8 mg, about 16 mg, about 8 mg, about 32 mg, about 8 mg, about 64 mg, about 8 mg, about 96 mg, about 16 mg, about 32 mg, about 16 mg, about 64 mg, about 16 mg, about 96 mg, about 32 mg, about 64 mg, about 32 mg, about 96 mg, or about 64 mg, about 96 mg. In some embodiments, the patient is administered one or more doses of steroid ranging from about 1 mg, about 2 mg, about 4 mg, about 8 mg, about 16 mg, about 32 mg, about 64 mg, or about 96 mg. In some embodiments, the steroid is methylprednisolone (e.g., MEDROL®). In some embodiments, the steroid is prednisone.

[0207] In some embodiments, one or more doses of steroid (i.e., one or more doses of preoperative steroid) are administered prior to administration of the therapeutic vector. In some embodiments, a daily dose of steroid is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 days prior to administration of the therapeutic vector. In some embodiments, a daily dose of steroid is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 days prior to administration of the therapeutic vector. In some embodiments, a daily dose of steroid is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 days prior to administration of the therapeutic vector. In some embodiments, a daily dose of steroid is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 days prior to administration of the therapeutic vector. Preoperative steroids are administered to the patient in one or more doses ranging from 20 mg / 60 kg to about 40 mg / 60 kg, about 40 mg / 60 kg to about 100 mg / 60 kg, about 40 mg / 60 kg to about 80 mg / 60 kg, about 40 mg / 60 kg to about 60 mg / 60 kg, about 60 mg / 60 kg to about 100 mg / 60 kg, about 60 mg / 60 kg to about 80 mg / 60 kg, or about 80 mg / 60 kg to about 100 mg / 60 kg. In some embodiments, the patient is administered one or more doses of preoperative steroids of about 4 mg / 60 kg, 6 mg / 60 kg, 8 mg / 60 kg, 10 mg / 60 kg, 16 mg / 60 kg, 20 mg / 60 kg, 24 mg / 60 kg, 32 mg / 60 kg, 40 mg / 60 kg, 48 mg / 60 kg, 60 mg / 60 kg, or 80 mg / 60 kg. In some embodiments, the patient is administered one or more doses of preoperative steroids ranging from about 1 mg to about 96 mg. In some embodiments, the patient is administered one or more doses of preoperative steroids of at least about 1 mg. In some embodiments, the patient is administered one or more doses of preoperative steroids of about 96 mg or less.In some embodiments, the amount of the active ingredient may be about 1 mg to about 2 mg, about 1 mg to about 4 mg, about 1 mg to about 8 mg, about 1 mg to about 16 mg, about 1 mg to about 32 mg, about 1 mg to about 64 mg, about 1 mg to about 96 mg, about 2 mg to about 4 mg, about 2 mg to about 8 mg, about 2 mg to about 16 mg, about 2 mg to about 32 mg, about 2 mg to about 64 mg, about 2 mg to about 96 mg, about 4 mg to about 8 mg, about 4 mg to about 16 mg, or about 4 mg. The patient is administered one or more doses of preoperative steroids ranging from about 1 mg, about 2 mg, about 4 mg, about 8 mg, about 16 mg, about 32 mg, about 64 mg, about 4 mg, about 96 mg, about 8 mg, about 16 mg, about 8 mg, about 32 mg, about 64 mg, about 8 mg, about 96 mg, about 16 mg, about 32 mg, about 16 mg, about 64 mg, about 16 mg, about 96 mg, about 32 mg, about 64 mg, about 32 mg, about 96 mg, or about 64 mg, about 96 mg. In some embodiments, the patient is administered one or more doses of preoperative steroids ranging from about 1 mg, about 2 mg, about 4 mg, about 8 mg, about 16 mg, about 32 mg, about 64 mg, or about 96 mg. In some embodiments, the steroid is methylprednisolone (e.g., MEDROL®). In some embodiments, the steroid is prednisone.

[0208] In some embodiments, about 20 mg / 60 kg to about 45 mg / 60 kg, about 25 mg / 60 kg to about 45 mg / 60 kg, about 30 mg / 60 kg to about 45 mg / 60 kg, about 35 mg / 60 kg to about 45 mg / 60 kg, about 40 mg / 60 kg to about 45 mg / 60 kg, about 20 mg / 60 kg to about 40 mg / 60 kg, about 25 mg / 60 kg to about 40 mg / 60 kg, about 30 mg / 60 kg to about 40 mg / 60 kg g, about 35 mg / 60 kg to about 40 mg / 60 kg, about 20 mg / 60 kg to about 35 mg / 60 kg, about 25 mg / 60 kg to about 35 mg / 60 kg, about 30 mg / 60 kg to about 35 mg / 60 kg, about 20 mg / 60 kg to about 30 mg / 60 kg, about 25 mg / 60 kg to about 30 mg / 60 kg, or about 20 mg / 60 kg to about 25 mg / 60 kg of preoperative steroids are administered to the patient. In some embodiments, the patient is administered one or more doses of preoperative steroids of about 25 mg / 60 kg, about 26 mg / 60 kg, about 27 mg / 60 kg, about 28 mg / 60 kg, about 29 mg / 60 kg, about 30 mg / 60 kg, about 31 mg / 60 kg, about 32 mg / 60 kg, about 33 mg / 60 kg, about 34 mg / 60 kg, or about 35 mg / 60 kg. In some embodiments, the steroid is methylprednisolone (e.g., MEDROL®). In some embodiments, a daily dose of methylprednisolone (e.g., MEDROL®) ranging from 25 mg / 60 kg to about 45 mg / 60 kg is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days prior to administration of the therapeutic vector. In some embodiments, methylprednisolone (e.g., MEDROL®) is administered at a daily dose of about 32 mg / 60 kg for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days prior to administration of the therapeutic vector.

[0209] In some embodiments, one or more doses of preoperative steroids ranging from about 50 mg / 60 kg to about 70 mg / 60 kg, about 55 mg / 60 kg to about 70 mg / 60 kg, about 60 mg / 60 kg to about 70 mg / 60 kg, about 65 mg / 60 kg to about 70 mg / 60 kg, about 50 mg / 60 kg to about 65 mg / 60 kg, about 55 mg / 60 kg to about 65 mg / 60 kg, about 60 mg / 60 kg to about 65 mg / 60 kg, about 50 mg / 60 kg to about 60 mg / 60 kg, about 55 mg / 60 kg to about 60 mg / 60 kg, or about 50 mg / 60 kg to about 55 mg / 60 kg. In some embodiments, the patient is administered one or more preoperative doses of about 55 mg / 60 kg, about 56 mg / 60 kg, about 57 mg / 60 kg, about 58 mg / 60 kg, about 59 mg / 60 kg, about 60 mg / kg, about 61 mg / 60 kg, about 62 mg / 60 kg, about 63 mg / 60 kg, about 64 mg / 60 kg, or about 65 mg / 60 kg. In some embodiments, the steroid is prednisone. In some embodiments, a daily dose of prednisolone ranging from about 50 mg / 60 kg to about 70 mg / 60 kg is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days prior to administration of the therapeutic vector. In some embodiments, a daily dose of about 32 mg / 60 kg of prednisone is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 days prior to administration of the therapeutic vector.

[0210] In some embodiments, one or more doses of steroids (i.e., one or more doses of postoperative steroids) are administered after administration of the therapeutic vector. In some embodiments, a daily dose of steroids is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days after administration of the therapeutic vector. In some embodiments, a daily dose of steroids is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 days, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks after administration of the therapeutic vector. In some embodiments, the amount of the amine is about 1 mg / 60 kg to about 100 mg / 60 kg, about 1 mg / 60 kg to about 80 mg / 60 kg, about 1 mg / 60 kg to about 60 mg / 60 kg, about 1 mg / 60 kg to about 40 mg / 60 kg, about 1 mg / 60 kg to about 20 mg / 60 kg, about 20 mg / 60 kg to about 100 mg / 60 kg, about 20 mg / 60 kg to about 80 mg / 60 kg, about 20 mg / 60 kg to about 60 mg / 60 kg, about The patient is administered one or more doses of postoperative steroids ranging from 20 mg / 60 kg to about 40 mg / 60 kg, about 40 mg / 60 kg to about 100 mg / 60 kg, about 40 mg / 60 kg to about 80 mg / 60 kg, about 40 mg / 60 kg to about 60 mg / 60 kg, about 60 mg / 60 kg to about 100 mg / 60 kg, about 60 mg / 60 kg to about 80 mg / 60 kg, or about 80 mg / 60 kg to about 100 mg / 60 kg. In some embodiments, the patient is administered one or more doses of postoperative steroids of about 96 mg or less. In some embodiments, the patient is administered one or more doses of postoperative steroids of at least about 1 mg. In some embodiments, the patient is administered one or more doses of postoperative steroids of about 96 mg or less.In some embodiments, the amount of the active ingredient may be about 1 mg to about 2 mg, about 1 mg to about 4 mg, about 1 mg to about 8 mg, about 1 mg to about 16 mg, about 1 mg to about 32 mg, about 1 mg to about 64 mg, about 1 mg to about 96 mg, about 2 mg to about 4 mg, about 2 mg to about 8 mg, about 2 mg to about 16 mg, about 2 mg to about 32 mg, about 2 mg to about 64 mg, about 2 mg to about 96 mg, about 4 mg to about 8 mg, about 4 mg to about 16 mg, or about 4 mg. The patient is administered one or more doses of postoperative steroids ranging from about 1 mg, about 2 mg, about 4 mg, about 8 mg, about 16 mg, about 32 mg, about 64 mg, about 4 mg, about 96 mg, about 8 mg, about 16 mg, about 8 mg, about 32 mg, about 64 mg, about 8 mg, about 96 mg, about 16 mg, about 32 mg, about 16 mg, about 64 mg, about 16 mg, about 96 mg, about 32 mg, about 64 mg, about 32 mg, about 96 mg, or about 64 mg, about 96 mg. In some embodiments, the patient is administered one or more doses of postoperative steroids ranging from about 1 mg, about 2 mg, about 4 mg, about 8 mg, about 16 mg, about 32 mg, about 64 mg, or about 96 mg. In some embodiments, the steroid is methylprednisolone (e.g., MEDROL®). In some embodiments, the steroid is prednisone.

[0211] In some embodiments, one or more doses of postoperative steroids in the range of about 70 mg / 60 kg to about 90 mg / 60 kg, 75 mg / 60 kg to about 90 mg / 60 kg, about 80 mg / 60 kg to about 90 mg / 60 kg, about 85 mg / 60 kg to about 90 mg / 60 kg, about 70 mg / 60 kg to about 85 mg / 60 kg, about 75 mg / 60 kg to about 85 mg / 60 kg, about 80 mg / 60 kg to about 85 mg / 60 kg, about 70 mg / 60 kg to about 80 mg / 60 kg, about 75 mg / 60 kg to about 80 mg / 60 kg, or about 70 mg / 60 kg to about 75 mg / 60 kg. In some embodiments, the patient is administered one or more doses of postoperative steroids of about 75 mg / 60 kg, about 76 mg / 60 kg, about 77 mg / 60 kg, about 78 mg / 60 kg, about 79 mg / 60 kg, about 80 mg / 60 kg, about 81 mg / 60 kg, about 82 mg / 60 kg, about 83 mg / 60 kg, about 84 mg / 60 kg, or about 85 mg / 60 kg. In some embodiments, the steroid is methylprednisolone sodium succinate (e.g., SOLU-MEDROL®). In some embodiments, a daily dose of methylprednisolone sodium succinate (e.g., SOLU-MEDROL®) ranging from about 70 mg to 90 mg / 60 kg is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days after administration of the therapeutic vector. In some embodiments, a daily dose of about 80 mg / 60 kg of methylprednisolone sodium succinate (e.g., SOLU-MEDROL®) is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 days following administration of the therapeutic vector.

[0212] In some embodiments, one or more doses of postoperative steroids in the range of about 30 mg / 60 kg to about 50 mg / 60 kg, 35 mg / 60 kg to about 50 mg / 60 kg, about 40 mg / 60 kg to about 50 mg / 60 kg, about 45 mg / 60 kg to about 50 mg / 60 kg, about 30 mg / 60 kg to about 45 mg / 60 kg, about 35 mg / 60 kg to about 45 mg / 60 kg, about 40 mg / 60 kg to about 45 mg / 60 kg, about 30 mg / 60 kg to about 40 mg / 60 kg, about 35 mg / 60 kg to about 40 mg / 60 kg, or about 30 mg / 60 kg to about 35 mg / 60 kg are administered to the patient. In some embodiments, the patient is administered one or more doses of postoperative steroids of about 35 mg / 60 kg, about 36 mg / 60 kg, about 37 mg / 60 kg, about 38 mg / 60 kg, about 39 mg / 60 kg, about 40 mg / 60 kg, about 41 mg / 60 kg, about 42 mg / 60 kg, about 43 mg / 60 kg, about 44 mg / 60 kg, or about 45 mg / 60 kg. In some embodiments, the steroid is methylprednisolone (e.g., MEDROL®). In some embodiments, a daily dose of methylprednisolone (e.g., MEDROL®) ranging from about 30 mg / 60 kg to about 50 mg / 60 kg is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector. In some embodiments, methylprednisolone (e.g., MEDROL®) is administered at a daily dose of about 40 mg / 60 kg for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector.

[0213] In some embodiments, about 20 mg / 60 kg to about 45 mg / 60 kg, 25 mg / 60 kg to about 45 mg / 60 kg, about 30 mg / 60 kg to about 45 mg / 60 kg, about 35 mg / 60 kg to about 45 mg / 60 kg, about 40 mg / 60 kg to about 45 mg / 60 kg, about 20 mg / 60 kg to about 40 mg / 60 kg, about 25 mg / 60 kg to about 40 mg / 60 kg, about 30 mg / 60 kg to about 40 mg / 60 kg , about 35 mg / 60 kg to about 40 mg / 60 kg, about 20 mg / 60 kg to about 35 mg / 60 kg, about 25 mg / 60 kg to about 35 mg / 60 kg, about 30 mg / 60 kg to about 35 mg / 60 kg, about 20 mg / 60 kg to about 30 mg / 60 kg, about 25 mg / 60 kg to about 30 mg / 60 kg, or about 20 mg / 60 kg to about 25 mg / 60 kg. In some embodiments, the patient is administered one or more doses of postoperative steroids of about 25 mg / 60 kg, about 26 mg / 60 kg, about 27 mg / 60 kg, about 28 mg / 60 kg, about 29 mg / 60 kg, about 30 mg / 60 kg, about 31 mg / 60 kg, about 32 mg / 60 kg, about 33 mg / 60 kg, about 34 mg / 60 kg, or about 35 mg / 60 kg. In some embodiments, the steroid is methylprednisolone (e.g., MEDROL®). In some embodiments, a daily dose of methylprednisolone (e.g., MEDROL®) ranging from about 20 mg / 60 kg to about 45 mg / 60 kg is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector. In some embodiments, methylprednisolone (e.g., MEDROL®) is administered at a daily dose of about 32 mg / 60 kg for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector.

[0214] In some embodiments, one or more doses of postoperative steroids in the range of about 15 mg / 60 kg to about 35 mg / 60 kg, 20 mg / 60 kg to about 35 mg / 60 kg, about 25 mg / 60 kg to about 35 mg / 60 kg, about 30 mg / 60 kg to about 35 mg / 60 kg, about 15 mg / 60 kg to about 30 mg / 60 kg, about 20 mg / 60 kg to about 30 mg / 60 kg, about 25 mg / 60 kg to about 30 mg / 60 kg, about 15 mg / 60 kg to about 25 mg / 60 kg, about 20 mg / 60 kg to about 25 mg / 60 kg, or about 15 mg / 60 kg to about 20 mg / 60 kg. In some embodiments, the patient is administered one or more doses of postoperative steroids of about 20 mg / 60 kg, about 21 mg / 60 kg, about 22 mg / 60 kg, about 23 mg / 60 kg, about 24 mg / 60 kg, about 25 mg / 60 kg, about 26 mg / 60 kg, about 27 mg / 60 kg, about 28 mg / 60 kg, about 29 mg / 60 kg, or about 30 mg / 60 kg. In some embodiments, the steroid is methylprednisolone (e.g., MEDROL®). In some embodiments, a daily dose of about 15 mg / 60 kg to about 35 mg / 60 kg methylprednisolone (e.g., MEDROL®) is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector. In some embodiments, methylprednisolone (e.g., MEDROL®) is administered at a daily dose of about 24 mg / 60 kg for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector.

[0215] In some embodiments, one or more doses of postoperative steroids in the range of about 5 mg / 60 kg to about 25 mg / 60 kg, 10 mg / 60 kg to about 25 mg / 60 kg, about 15 mg / 60 kg to about 25 mg / 60 kg, about 20 mg / 60 kg to about 25 mg / 60 kg, about 5 mg / 60 kg to about 20 mg / 60 kg, about 10 mg / 60 kg to about 20 mg / 60 kg, about 15 mg / 60 kg to about 20 mg / 60 kg, about 5 mg / 60 kg to about 15 mg / 60 kg, about 10 mg / 60 kg to about 15 mg / 60 kg, or about 5 mg / 60 kg to about 10 mg / 60 kg are administered to the patient. In some embodiments, the patient is administered one or more doses of postoperative steroids ranging from about 10 mg / 60 kg, about 11 mg / 60 kg, about 12 mg / 60 kg, about 13 mg / 60 kg, about 14 mg / 60 kg, about 15 mg / 60 kg, about 16 mg / 60 kg, about 17 mg / 60 kg, about 18 mg / 60 kg, about 19 mg / 60 kg, or about 20 mg / 60 kg. In some embodiments, the steroid is methylprednisolone (e.g., MEDROL®). In some embodiments, a daily dose of about 5 mg / 60 kg to about 25 mg / 60 kg of methylprednisolone (e.g., MEDROL®) is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector. In some embodiments, methylprednisolone (e.g., MEDROL®) is administered at a daily dose of about 16 mg / 60 kg for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector.

[0216] In some embodiments, one or more doses of postoperative steroids ranging from about 1 mg / 60 kg to about 20 mg / 60 kg, 5 mg / 60 kg to about 20 mg / 60 kg, about 10 mg / 60 kg to about 20 mg / 60 kg, about 15 mg / 60 kg to about 20 mg / 60 kg, about 1 mg / 60 kg to about 15 mg / 60 kg, about 5 mg / 60 kg to about 15 mg / 60 kg, about 10 mg / 60 kg to about 15 mg / 60 kg, about 1 mg / 60 kg to about 10 mg / 60 kg, about 5 mg / 60 kg to about 10 mg / 60 kg, or about 1 mg / 60 kg to about 5 mg / 60 kg are administered to the patient. In some embodiments, the patient is administered one or more doses of a postoperative steroid in the range of about 1 mg / 60 kg, about 2 mg / 60 kg, about 3 mg / 60 kg, about 4 mg / 60 kg, about 5 mg / 60 kg, about 6 mg / 60 kg, about 7 mg / 60 kg, about 8 mg / 60 kg, about 9 mg / 60 kg, about 10 mg / 60 kg, about 11 mg / 60 kg, about 12 mg / 60 kg, about 13 mg / 60 kg, about 14 mg / 60 kg, or about 15 mg / 60 kg. In some embodiments, the steroid is methylprednisolone (e.g., MEDROL®). In some embodiments, a daily dose of about 1 mg / 60 kg to about 20 mg / 60 kg of methylprednisolone (e.g., MEDROL®) is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 days after administration of the therapeutic vector. In some embodiments, a daily dose of about 8 mg / 60 kg of methylprednisolone (e.g., MEDROL®) is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 days after administration of the therapeutic vector. In some embodiments, a daily dose of about 6 mg / 60 kg of methylprednisolone (e.g., MEDROL®) is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 days after administration of the therapeutic vector.In some embodiments, methylprednisolone (e.g., MEDROL®) is administered at a daily dose of about 4 mg / 60 kg for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector.

[0217] In some embodiments, one or more doses of postoperative steroids in the range of about 30 mg / 60 kg to about 50 mg / 60 kg, 35 mg / 60 kg to about 50 mg / 60 kg, about 40 mg / 60 kg to about 50 mg / 60 kg, about 45 mg / 60 kg to about 50 mg / 60 kg, about 30 mg / 60 kg to about 45 mg / 60 kg, about 35 mg / 60 kg to about 45 mg / 60 kg, about 40 mg / 60 kg to about 45 mg / 60 kg, about 30 mg / 60 kg to about 40 mg / 60 kg, about 35 mg / 60 kg to about 40 mg / 60 kg, or about 30 mg / 60 kg to about 35 mg / 60 kg are administered to the patient. In some embodiments, a postoperative steroid is administered to the patient at one or more doses of about 35 mg / 60 kg, about 36 mg / 60 kg, about 37 mg / 60 kg, about 38 mg / 60 kg, about 39 mg / 60 kg, about 40 mg / 60 kg, about 41 mg / 60 kg, about 42 mg / 60 kg, about 43 mg / 60 kg, about 44 mg / 60 kg, or about 45 mg / 60 kg. In some embodiments, the steroid is prednisone. In some embodiments, a daily dose of about 30 mg / 60 kg to about 50 mg / 60 kg prednisone is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days after administration of the therapeutic vector. In some embodiments, a daily dose of about 40 mg / 60 kg of prednisone is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector.

[0218] In some embodiments, one or more doses of postoperative steroids in the range of about 10 mg / 60 kg to about 30 mg / 60 kg, 15 mg / 60 kg to about 30 mg / 60 kg, about 20 mg / 60 kg to about 30 mg / 60 kg, about 25 mg / 60 kg to about 30 mg / 60 kg, about 10 mg / 60 kg to about 25 mg / 60 kg, about 15 mg / 60 kg to about 25 mg / 60 kg, about 20 mg / 60 kg to about 25 mg / 60 kg, about 10 mg / 60 kg to about 20 mg / 60 kg, about 15 mg / 60 kg to about 20 mg / 60 kg, or about 10 mg / 60 kg to about 15 mg / 60 kg. In some embodiments, the patient is administered one or more doses of postoperative steroids ranging from about 15 mg / 60 kg, about 16 mg / 60 kg, about 17 mg / 60 kg, about 18 mg / 60 kg, about 19 mg / 60 kg, about 20 mg / 60 kg, about 21 mg / 60 kg, about 22 mg / 60 kg, about 23 mg / 60 kg, about 24 mg / 60 kg, or about 25 mg / 60 kg. In some embodiments, the steroid is prednisone. In some embodiments, a daily dose of about 10 mg / 60 kg to about 30 mg / 60 kg of prednisone is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days after administration of the therapeutic vector. In some embodiments, a daily dose of about 20 mg / 60 kg of prednisone is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector.

[0219] In some embodiments, one or more doses of postoperative steroids ranging from about 1 mg / 60 kg to about 20 mg / 60 kg, 5 mg / 60 kg to about 20 mg / 60 kg, about 10 mg / 60 kg to about 20 mg / 60 kg, about 15 mg / 60 kg to about 20 mg / 60 kg, about 1 mg / 60 kg to about 15 mg / 60 kg, about 5 mg / 60 kg to about 15 mg / 60 kg, about 10 mg / 60 kg to about 15 mg / 60 kg, about 1 mg / 60 kg to about 10 mg / 60 kg, about 5 mg / 60 kg to about 10 mg / 60 kg, or about 1 mg / 60 kg to about 5 mg / 60 kg are administered to the patient. In some embodiments, the patient is administered one or more doses of postoperative steroids ranging from about 1 mg / 60 kg, about 2 mg / 60 kg, about 3 mg / 60 kg, about 4 mg / 60 kg, about 5 mg / 60 kg, about 6 mg / 60 kg, about 7 mg / 60 kg, about 8 mg / 60 kg, about 9 mg / 60 kg, about 10 mg / 60 kg, about 11 mg / 60 kg, about 12 mg / 60 kg, about 13 mg / 60 kg, about 14 mg / 60 kg, or about 15 mg / 60 kg. In some embodiments, the steroid is prednisone. In some embodiments, a daily dose of about 1 mg / 60 kg to about 20 mg / 60 kg of prednisone is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days after administration of the therapeutic vector. In some embodiments, a daily dose of about 10 mg / 60 kg of prednisone is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector.

[0220] In some embodiments, the patient is administered one or more doses of post-operative steroids in the range of about 1 g / 60 kg to about 15 g / 60 kg, about 5 g / 60 kg to about 15 g / 60 kg, about 10 g / 60 kg to about 15 g / 60 kg, about 1 g / 60 kg to about 10 g / 60 kg, about 5 g / 60 kg to about 10 g / 60 kg, or about 1 g / 60 kg to about 5 g / 60 kg. In some embodiments, the patient is administered one or more doses of postoperative steroids ranging from about 1 mg / 60 kg, about 2 mg / 60 kg, about 3 mg / 60 kg, about 4 mg / 60 kg, about 5 mg / 60 kg, about 6 mg / 60 kg, about 7 mg / 60 kg, about 8 mg / 60 kg, about 9 mg / 60 kg, about 10 mg / 60 kg, about 11 mg / 60 kg, about 12 mg / 60 kg, about 13 mg / 60 kg, about 14 mg / 60 kg, or about 15 mg / 60 kg. In some embodiments, the steroid is sodium creatine phosphate. In some embodiments, a daily dose of about 1 mg / 60 kg to about 15 mg / 60 kg of sodium creatine phosphate is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days after administration of the therapeutic vector. In some embodiments, sodium creatine phosphate is administered at a daily dose of about 2 mg / 60 kg for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or at least 10 days following administration of the therapeutic vector.

[0221] In some embodiments, the patient is administered an intravenous dose of sodium creatine phosphate (2 g / 60 kg) and an intravenous dose of methylprednisolone sodium succinate (e.g., SOL-MEDROL®, 80 mg / 60 kg) on ​​the day the therapeutic AAV vector is administered, with both of the above agents administered for three consecutive days following administration of the therapeutic AAV vector. On the third day following administration of the therapeutic AAV vector, the patient is administered a dose of 40 mg / 60 kg of methylprednisolone (e.g., MEDROL®) tablets for four consecutive days. On the seventh day following administration of the therapeutic AAV vector, the patient is administered a dose of 32 mg / 60 kg of methylprednisolone (e.g., MEDROL®) tablets for seven consecutive days. On the fourteenth day following administration of the therapeutic AAV vector, the patient is administered a dose of 24 mg / 60 kg of methylprednisolone (e.g., MEDROL®) tablets for seven consecutive days. On the 21st day after administration of the therapeutic AAV vector, the patient is administered methylprednisolone (e.g., MEDROL®) tablets at a dose of 16 mg / 60 kg for 7 consecutive days. On the 28th day after administration of the therapeutic AAV vector, the patient is administered methylprednisolone (e.g., MEDROL®) tablets at a dose of 8 mg / 60 kg for 7 consecutive days. On the 35th day after administration of the therapeutic AAV vector, the patient is administered methylprednisolone (e.g., MEDROL®) tablets at a dose of 6 mg / 60 kg for 7 consecutive days. On the 42nd day after administration of the therapeutic AAV vector, the patient is administered methylprednisolone (e.g., MEDROL®) tablets at a dose of 4 mg / 60 kg for 7 consecutive days.

[0222] In some embodiments, 7 days prior to administration of the therapeutic AAV vector, the patient is administered a 32 mg / 60 kg dose of methylprednisolone (e.g., MEDROL®) tablets. On the day the therapeutic AAV vector is administered, an intravenous dose of methylprednisolone sodium succinate (e.g., SOL-MEDROL®, 80 mg / 60 kg) is administered once a day for three consecutive days. On the third day after administration of the therapeutic AAV vector, the patient is administered a 40 mg / 60 kg dose of methylprednisolone (e.g., MEDROL®) tablets for four consecutive days. On the seventh day after administration of the therapeutic AAV vector, the patient is administered a 32 mg / 60 kg dose of methylprednisolone (e.g., MEDROL®) tablets for seven consecutive days. On the 14th day after administration of the therapeutic AAV vector, the patient is administered methylprednisolone (e.g., MEDROL®) tablets at a dose of 24 mg / 60 kg for 7 consecutive days. On the 21st day after administration of the therapeutic AAV vector, the patient is administered methylprednisolone (e.g., MEDROL®) tablets at a dose of 16 mg / 60 kg for 7 consecutive days. On the 28th day after administration of the therapeutic AAV vector, the patient is administered methylprednisolone (e.g., MEDROL®) tablets at a dose of 8 mg / 60 kg for 7 consecutive days. On the 35th day after administration of the therapeutic AAV vector, the patient is administered methylprednisolone (e.g., MEDROL®) tablets at a dose of 6 mg / 60 kg for 7 consecutive days. On day 42 following administration of the therapeutic AAV vector, the patient is administered methylprednisolone (e.g., MEDROL®) tablets at a dose of 4 mg / 60 kg for 7 consecutive days. An exemplary schematic diagram of a treatment regimen for gene therapy of LHON is shown in FIG.

[0223] In some embodiments, the patient is administered a dose of 60 mg / 60 kg of prednisone tablets prior to administration of the therapeutic AAV vector, and is administered 7 consecutive days of prednisone tablets after administration of the therapeutic AAV vector. On the 8th day after administration of the therapeutic AAV vector, the patient is administered a dose of 40 mg / kg of prednisone tablets for 1 day. On the 9th day after administration of the therapeutic AAV vector, the patient is administered a dose of 20 mg / kg of prednisone tablets for 1 day. On the 10th day after administration of the therapeutic AAV vector, the patient is administered a dose of 10 mg / kg of prednisone tablets for 1 day. An exemplary schematic diagram of a treatment regimen for gene therapy of LHON is shown in FIG. 9.

[0224] Dosage and intervals can be adjusted individually to be sufficient to maintain therapeutic effect, and one of ordinary skill in the art would be able to optimize an effective local dose without undue experimentation.

[0225] In some embodiments, administration of a steroid before, during, and / or after administration of a therapeutic AAV vector described herein results in a higher mean visual acuity recovery, e.g., in a population of at least 10 patients, compared to administration of a comparable therapeutic AAV vector without a steroid. In some embodiments, administration of a steroid before, during, and / or after administration of a therapeutic AAV vector described herein results in a lower incidence of adverse events, e.g., in a population of at least 10 patients, compared to administration of a comparable therapeutic AAV vector without a steroid. In some embodiments, the adverse events are selected from anterior chamber inflammation, vitritis, ocular hypertension, cataract removal, keratitis, vitreous hemorrhage, allergic conjunctivitis, and eye pain.

[0226] In some embodiments, the increased mean visual recovery and reduced incidence of adverse events achieved by the methods of the present invention are determined in comparison to a patient population having an ocular disease treated by using a steroid-free therapeutic AAV vector before, during and / or after administration of the therapeutic AAV vector. In some embodiments, the patient population treated by the methods of the present disclosure and the patient population treated using the equivalent therapeutic AAV vector are ethnically matched. In some embodiments, the patient population is Chinese or Argentinian.

[0227] Diagnostic methods and kits In some embodiments, the present disclosure provides a method for screening a patient for the treatment of an ocular disease. In such an embodiment, the method comprises culturing a target cell population with a composition comprising an AAV comprising a recombinant nucleic acid sequence encoding a detectable marker in the presence of a serum sample obtained from the patient, and detecting the expression level of the detectable marker in the target cells after culturing, and the patient is selected for treatment if the expression level of the detectable marker in the target cells is higher than a predetermined threshold. In some embodiments, the method further comprises administering to the patient a pharmaceutical composition comprising an AAV, the AAV comprising a recombinant nucleic acid sequence encoding a mitochondrial protein.

[0228] The method of screening patients for treatment of ocular diseases described herein uses a serum sample obtained from the patient to evaluate the immune response of a particular patient to a recombinant viral vector for delivery of a therapeutic protein. Soluble factors (e.g., antibodies) present in the patient's serum can prevent viral infection of target cells, thereby reducing delivery of the therapeutic protein and / or reducing efficacy of the pharmaceutical composition. The method of the present disclosure can measure the level of infectivity of target cells by detecting the marker using AAV encoding a detectable marker. In some embodiments, the present disclosure provides a method for identifying patients who exhibit low immune reactivity to AAV compositions and selecting such patients for treatment with a therapeutic AAV vector described herein. In some embodiments, the present disclosure provides a method for identifying patients who exhibit high immune reactivity to AAV compositions and excluding such patients from future treatment with a therapeutic AAV vector described herein.

[0229] In some embodiments, the expression level of detectable marker in target cells correlates with the patient's immune response to the AAV vector.For example, serum from a patient that shows high immunoreactivity to AAV contains soluble factors that prevent AAV encoding detectable marker from infecting target cells and prevent the expression of detectable marker in target cells.In such a case, the expression level of detectable marker in target cells cultured in the presence of patient serum is reduced compared to the expression level of detectable marker in target cells cultured in the absence of patient serum.Alternatively, serum from a patient that shows low immunoreactivity to AAV contains little or no soluble factors that prevent AAV encoding detectable marker from infecting target cells.In such a case, the expression level of detectable marker in target cells cultured in the presence of patient serum is the same or not significantly reduced compared to the expression level of detectable marker in target cells cultured in the absence of patient serum.

[0230] The detectable marker may be any protein or nucleic acid molecule that is not endogenously expressed by the target cell and / or the AAV vector. Examples of detectable markers include, but are not limited to, FLAG tags, polyhistidine tags (e.g., 6xHis), SNAP tags, Halo tags, cMyc tags, glutathione-S-transferase tags, avidin, enzymes, fluorescent proteins, luminescent proteins, chemiluminescent proteins, bioluminescent proteins, and phosphorescent proteins.In some embodiments, the fluorescent protein is a blue / UV protein (e.g., BFP, TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T-Sapphire), a cyan protein (e.g., CFP, eCFP, Cerulean, SCFP3A, mTurcoise, mTurcooise2, monomeric Midorishi-Cyan, TagCfp, and mTFP1), a green protein (e.g., GFP, eGFP, meGFP (A208K mutation), Emerald, Superfolder GFP, monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, and mNeon). Green), yellow proteins (e.g., YFP, eYFP, Citrine, Venus, SYFP2, and TagYFP), orange proteins (monomers Kusabira-Orange, mKOκ, mKO2, mOrange, and mOrange2), red proteins (e.g., RFP, mRaspberry, mCherry, mTiragele, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2), far-red proteins (e.g., mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP), near-infrared proteins (e.g., TagRFP657, IFP1.4, and iRFP), long Stokes shift proteins (e.g., mKeima, Red, LSS-mKate1, LSS-mKate2 and mBeRFP), light-activated proteins (e.g., PA-GFP, PAmCherry1 and PATagRFP), photoconverting proteins (Kaede(green), Kaede(red), KikGR1(green), KikGR1(red), PS-CFP2, PS-CFP2, mEos2(green), mEos2(red), mEos3.2(green), mEos3.2(red), PSmOrange and PSmOrange), and photoswitching proteins (e.g., Dronpa).In some embodiments, the detectable marker can be selected from AmCyan, AsRed, DsRed2, DsRed Express, E2-Crimson, HcRed, ZsGreen, ZsYellow, mCherry, mStrawberry, mOrange, mBanana, mPlum, mRasberry, tdTomato, DsRed monomer, and / or AcGFP, all available from Clontech. In certain embodiments, the detectable marker is GFP.

[0231] Detectable markers can be detected by methods commonly known in the art, including but not limited to flow cytometry, qPCR, Western blot, ELISA, and immunohistochemistry. In certain embodiments, the detection method is a high-throughput detection method, such as flow cytometry or qPCR, and multiple patient samples can be analyzed simultaneously. In some embodiments, the detection method is flow cytometry. In some embodiments, the detection method is qPCR.

[0232] In some embodiments, a predetermined threshold of the expression level of detectable marker is set to screen patients for treatment of eye disorders. In some embodiments, patients who meet or exceed this threshold are selected for treatment with the therapeutic AAV vectors described herein. In some embodiments, patients who do not meet this threshold are excluded from future treatment with the therapeutic AAV vectors described herein or need to undergo an immunosuppressive regimen before starting treatment with the therapeutic AAV vectors described herein.

[0233] In some embodiments, the predetermined threshold can be expressed as an absolute expression level of the detectable marker in the test sample. Patients with expression levels higher than the absolute expression level are characterized as suitable for gene therapy, and / or patients with expression levels lower than the absolute expression level are characterized as unsuitable for gene therapy. For example, in some embodiments where the detectable marker is detected by qPCR, the predetermined threshold is an absolute expression level of 0.2 or greater. In such embodiments, if the absolute expression level of the detectable marker is 0.2 or greater, the patient is characterized as suitable for gene therapy, and if the absolute expression level of the detectable marker is less than 0.2, the patient is characterized as unsuitable for gene therapy. In some embodiments, the predetermined threshold is an absolute expression level of 0.6 or greater. In such embodiments, if the absolute expression level of the detectable marker is 0.6 or greater, the patient is characterized as suitable for gene therapy, and if the absolute expression level of the detectable marker is less than 0.6, the patient is characterized as unsuitable for gene therapy.

[0234] In some embodiments where the detectable marker is detected by flow cytometry, the predetermined threshold is an absolute expression level of 20% or more of marker-positive target cells in the test sample (e.g., the percentage of GFP-positive cells is 20% or more). In such embodiments, if the absolute expression level of the detectable marker is the absolute expression level of 20% or more of marker-positive target cells, the patient is characterized as suitable for gene therapy, and if the absolute expression level of the detectable marker is the absolute expression level of less than 20% of marker-positive target cells, the patient is characterized as unsuitable for gene therapy. In some embodiments, the predetermined threshold is an absolute expression level of 40% or more of target cells in the test sample (e.g., the percentage of GFP-positive cells is 40% or more). In such embodiments, if the absolute expression level of the detectable marker is the absolute expression level of 40% or more of marker-positive target cells, the patient is characterized as suitable for gene therapy, and if the absolute expression level of the detectable marker is the absolute expression level of less than 40% of marker-positive target cells, the patient is characterized as unsuitable for gene therapy.

[0235] In some embodiments, the predetermined threshold can be expressed as a relative expression level of the detectable marker in the test sample (i.e., by detecting the expression of the detectable marker in the test sample relative to the control sample). Patients having an expression level higher than the relative expression level are characterized as suitable for gene therapy, and / or patients having an expression level lower than the relative expression level are characterized as unsuitable for gene therapy. In some embodiments, where the detectable marker is detected by flow cytometry, the predetermined threshold is a relative expression level of 40% or more of marker-positive target cells in the test sample (e.g., the percentage of GFP-positive cells is 40% or more). In such embodiments, if the absolute expression level of the detectable marker is the absolute expression level of 40% or more of marker-positive target cells, the patient is characterized as suitable for gene therapy, and if the absolute expression level of the detectable marker is the absolute expression level of less than 40% of marker-positive target cells, the patient is characterized as unsuitable for gene therapy. In some embodiments, where the detectable marker is detected by flow cytometry, the predetermined threshold is a relative expression level of 80% or more of marker-positive target cells in the test sample (e.g., the percentage of GFP-positive cells is 80% or more). In such embodiments, a patient is characterized as suitable for gene therapy if the absolute expression level of the detectable marker is equal to or greater than the absolute expression level of 80% of marker-positive target cells, and a patient is characterized as unsuitable for gene therapy if the absolute expression level of the detectable marker is equal to or less than the absolute expression level of 80% of marker-positive target cells.

[0236] In some embodiments, the patient screened and / or selected for treatment with the methods described herein has one or more mtDNA point mutations. In some embodiments, the patient has a point mutation in a gene encoding a protein of complex I in the mitochondrial oxidative phosphorylation chain. For example, the patient may have one or more point mutations in the ND4 gene. In some embodiments, the patient has a point mutation at nucleotide position 11778 of the ND4 gene. In some embodiments, the point mutation is G11778A in the ND4 gene. In some embodiments, the patient screened and / or selected for treatment with the methods described herein has a G11778A point mutation in the ND4 gene and is of Chinese and / or Argentinean descent.

[0237] In some embodiments, patients screened and / or selected for treatment with the methods described herein have a G11778A point mutation in the ND4 gene and are of Argentinean descent. In some embodiments, patients screened and / or selected for treatment with the methods described herein have a G11778A point mutation in the ND4 gene and are of Chinese descent.

[0238] In some embodiments, the present disclosure provides a kit for screening patients for treatment of an ocular disease and / or for selecting patients for treatment of an ocular disease. In such embodiments, the kit includes an AAV comprising a recombinant nucleic acid encoding a detectable marker and one or more reagents for detecting the detectable marker. In some embodiments, the one or more reagents for detecting the detectable marker are selected from an antibody that binds to the detectable marker and one or more primer oligonucleotides specific for the recombinant nucleic acid encoding the detectable marker.

[0239] In some embodiments, the kit further comprises one or more reagents for reconstituting and / or diluting the AAV vector and / or detecting the reagent components. In some embodiments, the kit further comprises one or more additional reagents, such as buffers, wash buffers and / or cell culture media for introducing the AAV vector into cells. The components of the kit may be in separate containers or together in a single container.

[0240] In addition to the above components, in some embodiments, the kit further comprises instructions for using the components of the kit to carry out the methods of the present disclosure. The instructions for carrying out the methods of the present invention are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Thus, the instructions can be present in the kit as a package insert or on the label of the container of the kit or the components of the kit (i.e., attached to the package or subpackage). In other embodiments, the instructions are present as a data file stored electronically on a suitable computer-readable storage medium (e.g., CD-ROM, magnetic disk, and flash drive). In yet another embodiment, the actual instructions are not present in the kit, but a means for obtaining the instructions from a remote source, for example, via the Internet, is provided. An example of this embodiment is a kit that includes a website from which the instructions can be viewed and / or downloaded. As with the instructions, such a means for obtaining the instructions is recorded on a suitable substrate.

[0241] Pharmaceutical Compositions and Excipients The vector of the present invention can be formulated into pharmaceutical compositions. These compositions can contain, in addition to the vector, pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other materials can be determined by those skilled in the art depending on the route of administration (i.e., in this case, retinal, subretinal, or intravitreal injection).

[0242] Pharmaceutical compositions are usually in liquid form. Liquid pharmaceutical compositions usually contain liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, glucose or other sugar solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol, for example. In some cases, surfactants such as pluronic acid (PF68, also known as poloxamer 188) can be used.

[0243] When injected into the lesion, the active ingredient is in the form of an aqueous solution that is pyrogen-free and has appropriate pH, isotonicity, and stability. Those skilled in the art can easily prepare appropriate solutions using isotonic solvents such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be added as necessary.

[0244] For delayed release, the vectors can be contained in pharmaceutical compositions formulated for sustained release, such as microcapsules formed from biocompatible polymers or liposome carrier systems by methods well known in the art.

[0245] In some embodiments, disclosed herein is a pharmaceutical composition comprising an adeno-associated virus (AAV) comprising any of the recombinant nucleic acids disclosed herein. In some cases, the pharmaceutical composition further comprises a pharma- ceutical acceptable excipient thereof.

[0246] In some cases, the pharma- ceutically acceptable excipient comprises phosphate buffered saline (PBS), α,α-trehalose dihydrate, L-histidine monohydrochloride monohydrate, polysorbate 20, NaCl, NaH2PO4, Na2HPO4, KH2PO4, K2HPO4, poloxamer 188, or any combination thereof. In some cases, the pharma-ceutically acceptable excipient is selected from the group consisting of phosphate buffered saline (PBS), α,α-trehalose dihydrate, L-histidine monohydrochloride monohydrate, polysorbate 20, NaCl, NaH2PO4, Na2HPO4, KH2PO4, K2HPO4, poloxamer 188, or any combination thereof.

[0247] In some cases, the pharma- ceutical acceptable excipient comprises poloxamer 188. Poloxamer 188 is an ethylene oxide-polyoxypropylene glycol copolymer. As a surfactant, poloxamer 188 has dispersing, stabilizing, and emulsifying functions. In some cases, the pharma- ceutical acceptable excipient comprises 0.0001%-0.01% poloxamer 188. In some cases, the pharma- ceutical acceptable excipient comprises 0.0005%-0.005% poloxamer 188. In some cases, the pharma- ceutical acceptable excipient comprises 0.0007%-0.002% poloxamer 188. In some cases, the pharma- ceutical acceptable excipient comprises 0.0008%-0.0012% poloxamer 188. In some cases, the pharma- ceutical acceptable excipient comprises 0.0009%-0.0011% poloxamer 188. In some cases, the pharma- ceutically acceptable excipient comprises 0.001% (0.01%) poloxamer 188.

[0248] In some cases, the pharma- ceutically acceptable excipient further comprises one or more salts. In some cases, the one or more salts comprise NaCl, NaH2PO4, Na2HPO4, and KH2PO4. In some cases, the one or more salts comprise 80 mM NaCl, 5 mM NaH2PO4, 40 mM Na2HPO4, and 5 mM KH2PO4. In some cases, the one or more salts comprise NaCl, Na2HPO4, and KH2PO4. In some cases, the one or more salts comprise 154 mM NaCl, 5.6 mM Na2HPO4, and 8.4 mM KH2PO4. In some cases, the one or more salts comprise NaCl, Na2HPO4, and KH2PO4. In some cases, the one or more salts are NaCl, Na2HPO4, and KH2PO4. In some cases, the NaCl has a concentration of 5-15 mg / mL. In some cases, NaCl has a concentration of 9 mg / mL. In some cases, KH2PO4 has a concentration of 0.1 to 0.5 mg / mL. In some cases, KH2PO4 has a concentration of 0.144 mg / mL. In some cases, Na2HPO4 has a concentration of 0.5 to 1 mg / mL. In some cases, Na2HPO4 has a concentration of 0.795 mg / mL.

[0249] In some cases, the pharmaceutical composition has a pH of 6 to 8. In some cases, the pharmaceutical composition has a pH of 7.2 to 7.4. In some cases, the pharmaceutical composition has a pH of 7.3. In some cases, the pharmaceutical composition has a pH of at least 1.0×10 10 Optionally, the pharmaceutical composition has a viral titer of at least 5.0×10 vg / mL. 10 It has a viral titer of 0.05 vg / mL.

[0250] In some cases, the pharmaceutical composition retains at least 60%, 70%, 80% or 90% of the viral titer when subjected to five freeze / thaw cycles compared to the viral titer prior to the five freeze / thaw cycles. In some cases, the pharmaceutical composition, when administered to patients with Leber's hereditary optic neuropathy, provides a greater mean visual acuity recovery than a comparable pharmaceutical composition that does not include the recombinant nucleic acid.

[0251] In some cases, the pharmaceutical composition is stored in a container of a particular material. In some instances, the container is formed of a cycloolefin polymer.

[0252] sample A sample suitable for use in the methods described herein can be a nucleic acid sample from a subject. As used herein, a "nucleic acid sample" can include RNA or DNA, or a combination thereof. In another embodiment, a "polypeptide sample" (e.g., a peptide or protein or a fragment derived therefrom) can be used to verify information that an amino acid has been changed by a genetic variant. Nucleic acids and polypeptides can be extracted from one or more samples, including, but not limited to, blood, saliva, urine, oral mucosal scrapings, sputum, serum, tears, skin, tissue, or hair. Nucleic acid information analysis can be performed on the nucleic acid sample. As used herein, "nucleic acid information" includes the nucleic acid sequence itself, the presence or absence of genetic mutations in the nucleic acid sequence, physical properties that vary depending on the nucleic acid sequence (e.g., Tm), and the amount of nucleic acid (e.g., number of mRNA copies). "Nucleic acid" refers to any one of DNA, RNA, DNA containing artificial nucleotides, or RNA containing artificial nucleotides. As used herein, "purified nucleic acid" includes cDNA, fragments of genomic nucleic acid, nucleic acid generated using polymerase chain reaction (PCR), nucleic acid generated by restriction enzyme treatment of genomic nucleic acid, recombinant nucleic acid, and chemically synthesized nucleic acid molecules. "Recombinant" nucleic acid molecules include nucleic acid molecules that are made by artificially combining two otherwise separated sequence segments, e.g., nucleic acid fragments separated by chemical synthesis or by genetic engineering techniques. As used herein, "polypeptides" include proteins, protein fragments, and peptides that are isolated from natural sources, produced by recombinant techniques, or chemically synthesized. Polypeptides can have one or more modifications, such as post-translational modifications (e.g., glycosylation and phosphorylation) or any other modification (e.g., pegylation). Polypeptides can include one or more non-naturally occurring amino acids (e.g., amino acids with side chain modifications).

[0253] In some embodiments, the nucleic acid sample may include cells or tissues, such as cell lines. Exemplary cell types from which nucleic acids can be obtained using the methods described herein include, but are not limited to, blood cells, such as B lymphocytes, T lymphocytes, white blood cells, red blood cells, macrophages, or neutrophils; muscle cells, such as skeletal muscle cells, smooth muscle cells, or cardiomyocytes; germ cells, such as sperm or eggs; epithelial cells; connective tissue cells, such as adipocytes and chondrocytes; fibroblasts or osteoblasts; neurons; astrocytes; stromal cells; organ-specific cells, such as kidney cells, pancreatic cells, liver cells, or keratinocytes; stem cells; or any cells that arise from these. The cells from which nucleic acids can be obtained may be blood cells or specific types of blood cells, including, for example, hematopoietic stem cells, or cells that arise from hematopoietic stem cells, such as red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, or platelets. Generally, any type of stem cell can be used, including but not limited to embryonic stem cells, adult stem cells, or pluripotent stem cells.

[0254] In some embodiments, the nucleic acid sample can be processed for RNA or DNA isolation. In some embodiments, the RNA or DNA in a cell or tissue sample can be separated from other components of the nucleic acid sample. Standard techniques can be used. For example, cells can be recovered from the nucleic acid sample by centrifuging the cell sample and resuspending the pelleted cells, for example, in a buffer (e.g., phosphate buffered saline (PBS)). In some embodiments, the cell suspension can be centrifuged to obtain a cell pellet, after which the cells can be lysed and DNA can be extracted. In some embodiments, the nucleic acid sample can be concentrated and / or purified to isolate DNA. Any nucleic acid sample, including a nucleic acid sample derived from a subject and subjected to any further processing, is considered to be derived from a subject. In some embodiments, RNA or DNA can be extracted from a nucleic acid sample using standard techniques and kits well known in the art, including, for example, phenol extraction, QIAAMP® Tissue Kit (Qiagen, Chatsworth, Calif.), WIZARD® Genomic DNA Purification Kit (Promega), or the Qiagen Autopure method using Puregene chemistry, and highly stable DNA can be purified that is ideal for archiving.

[0255] In some embodiments, determining the identity or copy number of an allele can (but need not) include obtaining a nucleic acid sample comprising RNA and / or DNA from the subject, and / or assessing the identity, copy number, presence or absence, and chromosomal location of one or more genetic variations within the genomic DNA derived from the nucleic acid sample (i.e., the genome of the subject).

[0256] The individual or organization performing the assay need not actually physically analyze the nucleic acid sample from the subject. In some embodiments, the method may include using information obtained by a third party's analysis of the nucleic acid sample. In some embodiments, the method may include steps performed at more than one location. For example, a nucleic acid sample may be obtained from a subject at a first location, such as a medical institution, or in the case of a self-test kit, at the subject's home. The nucleic acid sample may be analyzed at the same location or at a second location (e.g., a laboratory or other testing facility).

[0257] nucleic acid The nucleic acids and polypeptides described herein may be used in the methods and kits of the present disclosure. In some embodiments, aptamers that specifically bind to the nucleic acids and polypeptides described herein may be used in the methods and kits of the present disclosure. As used herein, nucleic acid may include deoxyribonucleotides (DNA) or ribonucleotides (RNA), whether simple or polymeric, naturally occurring or non-naturally occurring, double-stranded or single-stranded, coding (e.g., a gene to be translated) or non-coding (e.g., a regulatory region), or any fragment, derivative, mimic, or complement thereof. In some embodiments, nucleic acid may include oligonucleotides, nucleotides, polynucleotides, nucleic acid sequences, genomic sequences, complementary DNA (cDNA), antisense nucleic acids, DNA regions, probes, primers, genes, regulatory regions, introns, exons, open reading frames, binding sites, target nucleic acids, and allele-specific nucleic acids.

[0258] As used herein, the term "probe" includes a nucleic acid fragment for examining a nucleic acid in a sample using a hybridization reaction based on nucleic acid complementarity.

[0259] As used herein, "hybrid" includes duplexes formed within the same type of nucleic acid, as discussed above, or between different types of nucleic acids, including DNA-DNA, DNA-RNA, RNA-RNA, etc.

[0260] As used herein, an "isolated" nucleic acid is one that has been separated from the nucleic acids that normally flank the gene or nucleotide sequence (e.g., in a genomic sequence) and / or purified completely or partially from other transcribed sequences (e.g., in an RNA library). For example, an isolated nucleic acid of the present disclosure can be substantially isolated relative to the complex cellular environment in which the naturally occurring nucleic acid resides, the medium used when the nucleic acid is produced by recombinant techniques, or the chemical precursors or other chemicals used when the nucleic acid is chemically synthesized. In some cases, the isolated material can form part of a composition, such as, for example, a crude extract, a buffer system, or a reagent mixture that contains other substances. In some embodiments, the material can be purified to substantial homogeneity using methods known in the art, for example, by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g., HPLC). With respect to genomic DNA (gDNA), the term "isolated" can also refer to a nucleic acid that is isolated from the chromosome with which the genomic DNA is naturally associated. For example, an isolated nucleic acid molecule may contain less than about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotides that are contiguous with the nucleic acid molecule in the gDNA of the cell from which the nucleic acid molecule is derived.

[0261] A nucleic acid may be fused to other coding or regulatory sequences or may be considered isolated. For example, recombinant DNA contained in a vector is included in the definition of "isolated" as used herein. In some embodiments, isolated nucleic acid may include recombinant DNA molecules in heterologous host cells or organisms, and partially or substantially purified DNA molecules in solution. Isolated nucleic acid also encompasses in vivo and in vitro RNA transcripts of the DNA molecules of the present disclosure. Isolated nucleic acid molecules or nucleotide sequences can be synthesized chemically or by recombinant synthesis. Such isolated nucleotide sequences can be used, for example, in the manufacture of the encoded polypeptide, as probes to isolate homologous sequences (e.g., from other mammalian species), for genetic mapping (e.g., by in situ hybridization to chromosomes), or to detect gene expression in tissues (e.g., human tissues), for example, by Northern blot analysis or other hybridization techniques disclosed herein. The present disclosure also relates to nucleic acid sequences that hybridize, for example, under highly stringent hybridization conditions for selective hybridization, with the nucleotide sequences described herein below. Such nucleic acid sequences can be detected and / or isolated by allele-specific or sequence-specific hybridization (e.g., under highly stringent conditions). Stringent conditions and methods of nucleic acid hybridization are well known to those skilled in the art (see, for example, Current Protocols in Molecular Biology, Ausubel, F. et al, John Wiley & Sons, (1998) and Kraus, M. and Aaronson, S., Methods Enzymol., 200:546-556 (1991), which are incorporated herein by reference in their entirety).

[0262] Calculation of "identity" or "percent identity" between two or more nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the sequence of the first sequence). The nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by each sequence (i.e., % identity = number of identical positions / total number of positions x 100). For example, if a position in the first sequence is occupied by a nucleotide at the same position as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. As used herein, the terms "identical" and "identity" are intended to be synonymous when used to describe the degree of sequence identity, unless otherwise indicated. For example, being at least 99% identical to a particular sequence means having at least 99% identity to that sequence.

[0263] In some embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the length of the reference sequence. The actual comparison between two sequences can be performed in a well-known manner, for example, using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90, 5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0), as described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (1997). When using BLAST and Gapped BLAST programs, any relevant parameters of the respective programs (e.g., NBLAST) can be used. For example, sequence comparison parameters may be set at score=100, word length=12, or may be altered (e.g., W=5 or W=20). Other examples include the algorithms of Myers and Miller, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA. In some embodiments, the percent identity between two amino acid sequences may be achieved using, for example, the GAP program of the GCG software package (Accelrys, Cambridge, UK).

[0264] A "probe" or "primer" can be an oligonucleotide that hybridizes in a base-specific manner to a complementary strand of a nucleic acid molecule. A probe can include a primer, which can be a single-stranded oligonucleotide probe that can serve as an initiation point for template-directed DNA synthesis using methods including, but not limited to, polymerase chain reaction (PCR) and ligase chain reaction (LCR) for amplification of a target sequence. The oligonucleotides described herein can include segments or fragments of nucleic acid sequences or their complements. In some embodiments, the DNA segment can be 5 to 10,000 contiguous bases and can range from 5, 10, 12, 15, 20, or 25 nucleotides to 10, 15, 20, 25, 30, 40, 50, 100, 200, 500, 1000, or 10,000 nucleotides. In addition to DNA and RNA, probes and primers may further comprise polypeptide nucleic acids (PNAs), as described in Nielsen, P. et al., Science 254: 1497-1500 (1991). A probe or primer may comprise a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and in certain embodiments, about 40, 50, 60 or 75 contiguous nucleotides of a nucleic acid molecule.

[0265] The present disclosure further provides an isolated nucleic acid, such as a probe or primer, that comprises a fragment or portion that can selectively hybridize to a nucleic acid that comprises or consists of a nucleotide sequence, where the nucleotide sequence can comprise at least one polymorphism or polymorphic allele contained in the genetic variation described herein, or a wild-type nucleotide located at the same position, or a complement thereof. In some embodiments, the probe or primer can have at least 70% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with the adjacent nucleotide sequence or with the complement of the adjacent nucleotide sequence.

[0266] In some embodiments, the nucleic acid probe can be an oligonucleotide capable of hybridizing to a complementary region of a gene associated with a condition comprising a genetic variation described herein (e.g., LHON). The nucleic acid fragments of the present disclosure can be used as probes or primers in assays such as those described herein.

[0267] Nucleic acids of the present disclosure as described above can be identified and isolated using standard molecular biology techniques well known to those of skill in the art. In some embodiments, DNA can be amplified and / or labeled (e.g., radioactively or fluorescently labeled) and used as a probe for screening, for example, a cDNA library derived from an organism. cDNA can be derived from mRNA and contained in an appropriate vector. For example, corresponding clones can be isolated, DNA obtained after in vivo excision, and the cloned inserts can be sequenced in either or both directions by art-recognized methods to identify the correct reading frame encoding a polypeptide of appropriate molecular weight. Using these or similar methods, the polypeptide and the DNA encoding this polypeptide can be isolated, sequenced, and further characterized.

[0268] In some embodiments, the nucleic acid may include one or more polymorphisms, variations, or mutations, such as single nucleotide polymorphisms (SNPs), single nucleotide variations (SNVs), copy number variations (CNVs), such as insertions, deletions, inversions, and translocations. In some embodiments, the nucleic acid may include analogs, such as phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, or modified nucleic acids, such as modified backbone residues or linkages, or nucleic acids combined with carbohydrates, lipids, polypeptides, or other materials, or peptide nucleic acids (PNAs), such as chromatin, ribosomes, and transcriptosomes. In some embodiments, the nucleic acid may include nucleic acids of various structures, such as A DNA, B DNA, Z-form DNA, siRNA, tRNA, and ribozymes. In some embodiments, the nucleic acid may be naturally occurring or unnaturally occurring polymorphic, e.g., having one or more sequence differences, e.g., additions, deletions, and / or substitutions, compared to a reference sequence. In some embodiments, the reference sequence may be based on publicly available information, such as the UC Santa Cruz Human Genome Browser Gateway (genome.ucsc.edu / cgi-bin / hgGateway) or the NCBI website (www.ncbi.nlm.nih.gov). In some embodiments, the reference sequence may be determined by a practitioner of the present disclosure using methods well known in the art, for example, by sequencing a reference nucleic acid.

[0269] In some embodiments, the probe may hybridize to an allele, SNP, SNV, or CNV, as described herein. In some embodiments, the probe may bind to another marker sequence associated with LHON, as described herein.

[0270] Those skilled in the art know how to design probes such that sequence-specific hybridization can occur only if a particular allele is present in the genomic sequence from the test nucleic acid sample. The present disclosure may also be simplified to practice using any convenient genotyping method, including commercially available techniques and methods for genotyping specific genetic mutations.

[0271] Control probes may also be used, for example, probes that bind to less variable sequences, such as repetitive DNA associated with the centromere of a chromosome, may be used as controls. In some embodiments, the probes may be obtained from commercial sources. In some embodiments, the probes may be, for example, chemically or in vitro synthesized, or made from chromosomal or genomic DNA by standard techniques. In some embodiments, sources of DNA that may be used include genomic DNA, cloned DNA sequences, and somatic cell hybrids that contain one or a part of a human chromosome together with the host's normal chromosome set, and chromosomes purified by flow cytometry or microdissection. The region of interest may be isolated by cloning or by site-specific amplification using PCR.

[0272] One or more nucleic acids, such as probes or primers, may also be labeled by direct labeling, for example, to include detectable labels.Detectable labels may include any label that can be detected by physical, chemical, or biological methods, such as radioactive labels such as 32P or 3H, fluorescent labels such as FITC, chromophore labels, affinity ligand labels, enzyme labels such as alkaline phosphatase, horseradish peroxidase, or I2 galactosidase, enzyme cofactor labels, hapten conjugate labels such as digoxigenin or dinitrophenyl, Raman signal-generating labels, magnetic labels, spin labels, epitope labels such as FLAG or HA epitopes, luminescent labels, heavy atom labels, nanoparticle labels, electrochemical labels, light scattering labels, spherical shell labels, semiconductor nanocrystal labels such as quantum dots (described in U.S. Pat. No. 6,207,392), and probes labeled with any other signal-generating labels known to those skilled in the art, where the label can allow the probe to be visualized with or without secondary detection molecules. Nucleotides may be directly incorporated into the probe by standard techniques such as nick translation, random priming, PCR labeling, etc. As used herein, "signal" includes any signal that is suitably detected and measured by suitable means, including fluorescent, radioactive, chemiluminescent, and the like.

[0273] Non-limiting examples of labeling moieties for detection include, but are not limited to, suitable enzymes, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; members of binding pairs capable of forming complexes, such as streptavidin / biotin, avidin / biotin, or antigen / antibody complexes, such as rabbit IgG and anti-rabbit IgG; fluorophores, such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, and the like. Fluorescent dyes include, but are not limited to, fluorescein, eosin, green fluorescent protein, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexes, such as those containing europium and terbium, cyanine dye family members, such as Cy3 and Cy5, molecular beacons and their fluorescent derivatives, and others known in the art as described, for example, in Principles of of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999), and others known in the art, such as those described in the 6th Edition of the Molecular Probes Handbook by Richard P. Hoagland; luminescent materials such as luminol; light scattering or plasmon resonant materials such as gold or silver particles or quantum dots; or radioactive materials including 14C, 123I, 124I, 125I, Tc99m, 32P, 33P, 35S or 3H.

[0274] Other labels, such as backbone labels, may also be used in the disclosed methods. Backbone labels include nucleic acid stains that bind to nucleic acids in a sequence-independent manner. Non-limiting examples include intercalating dyes, such as phenanthridines and acridines (e.g., ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, and ACMA); some minor groove binders, such as indoles and imidazoles (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580, and DAPI); and other nucleic acid stains, such as acridine orange (also capable of intercalating), 7-AAD, actinomycin D, LDS751, and hydroxystilbamidine. All of the aforementioned nucleic acid dyes are commercially available from suppliers such as Molecular Probes, Inc. Further examples of nucleic acid dyes include the following dyes from Molecular Probes: cyanine dyes, e.g., SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO -PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, - SYTO-16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84 and -85 (orange), SYTO-64, -17, -59, -61, -62, -60, and -63 (red).

[0275] In some embodiments, different colored fluorophores are used so that each probe, within or not within a set, can be clearly visualized, for example, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), 5-(and-6)-carboxy-X-rhodamine, Lissamine rhodamine B, 5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC), 7-diethylaminocoumarin-3-carboxylic acid, tetramethylrhodamine-5-(and-6)-isothiocyanate, 5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein-5-(and-6)-carboxamido]hexanoic acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3 -indacene)propionic acid, eosin-5-isothiocyanate, erythrosine-5-isothiocyanate, TRITC, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), and CASCADE™ Blue acetylazide may be selected. In some embodiments, fluorescently labeled probes can be viewed using a fluorescent microscope and appropriate filters for each fluorophore, or by using dual or triple bandpass filter sets to view multiple fluorophores. In some embodiments, techniques such as flow cytometry may be used to examine the hybridization pattern of the probes.

[0276] In other embodiments, the probe may be indirectly labeled, for example, with biotin or digoxigenin, or may be labeled with radioisotopes such as 32P and / or 3H. As a non-limiting example, a probe indirectly labeled with biotin may be detected by avidin conjugated to a detectable marker. For example, avidin may be conjugated to an enzymatic marker such as alkaline phosphatase or horseradish peroxidase. In some embodiments, the enzymatic marker may be detected using a colorimetric reaction using a substrate and / or catalyst for the enzyme. In some embodiments, a catalyst for alkaline phosphatase may be used, for example, 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. In some embodiments, a catalyst may be used for horseradish peroxidase, for example, diaminobenzoic acid.

[0277] Formulations, routes of administration and effective doses Yet another aspect of the present disclosure relates to formulations, routes of administration, and effective doses of pharmaceutical compositions comprising the agents or combinations of agents of the present disclosure. Such pharmaceutical compositions can be used to treat conditions such as LHON, as described above.

[0278] The compounds of the present disclosure may be administered as pharmaceutical formulations, including those suitable for oral (including buccal and sublingual), rectal, nasal, topical, transdermal patch, pulmonary, vaginal, suppository, or parenteral (including intraocular, intravitreal, intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous, and intravenous) administration, or in a form suitable for administration by aerosolization, inhalation, or insufflation. General information regarding drug delivery systems may be found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore Md. (1999)).

[0279] In various embodiments, pharmaceutical compositions include carriers and excipients (including, but not limited to, buffers, carbohydrates, mannitol, polypeptides, amino acids, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents, and / or preservatives), water, oils (e.g., oils of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, and sesame oil), saline, aqueous dextrose and glycerol solutions, flavoring agents, coloring agents, degreasing agents, and other acceptable additives, adjuvants, or binders, as well as other pharma- ceutically acceptable auxiliary substances to approximate physiological conditions, e.g., pH buffers, tonicity adjusting agents, emulsifying agents, and wetting agents. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. In some embodiments, the pharmaceutical formulation is substantially free of preservatives. In other embodiments, the pharmaceutical formulation may include at least one preservative. General methods for pharmaceutical dosage forms can be found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott, Williams, & Wilkins, Baltimore Md. (1999)). The compositions of the present disclosure can be administered using any suitable carrier known to those skilled in the art, but it can be recognized that the type of carrier may vary depending on the method of administration.

[0280] Compounds can be encapsulated in liposomes using known technology.Biodegradable microspheres can also be used as carriers for the pharmaceutical compositions of the present disclosure.Suitable biodegradable microspheres are disclosed in, for example, U.S. Patent Nos. 4,897,268, 5,075,109, 5,928,647, 5,811,128, 5,820,883, 5,853,763, 5,814,344 and 5,942,252.

[0281] Compounds may be administered in the form of liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to subjects are well known to those skilled in the art. A method for encapsulating biological materials in liposomes is described in U.S. Pat. No. 4,789,734, the contents of which are incorporated herein by reference. Essentially, the material is dissolved in an aqueous solution, appropriate phospholipids and lipids are added, along with a surfactant if necessary, and the material is dialyzed or sonicated as necessary. A review of known methods is provided by G. Gregoriadis, Chapter 14, "Liposomes," Drug Carriers in Biology and Medicine, pp.2.sup.87-341 (Academic Press, 1979).

[0282] Microspheres formed of polymers or polypeptides are well known to those skilled in the art and can be tailored to pass through the gastrointestinal tract directly into the bloodstream. Alternatively, compounds can be incorporated and the microspheres or composites of microspheres can be implanted for sustained release over a period ranging from days to months. See, for example, U.S. Patent Nos. 4,906,474, 4,925,673, and 3,625,214, the contents of which are incorporated herein by reference, and Jein, TIPS 19:155-157 (1998).

[0283] The concentration of the drug can be adjusted, the pH of the buffered solution can be adjusted, and the isotonicity can be adjusted to be compatible with intraocular or intravitreal injection.

[0284] The compound of the present disclosure can be formulated as a sterile solution or suspension in a suitable solvent. This pharmaceutical composition can be sterilized by conventional well-known sterilization techniques or can be sterile filtered. The resulting aqueous solution can be packaged for immediate use or can be lyophilized, and the lyophilized formulation can be combined with a sterile solution prior to administration. Suitable formulations and additional carriers are described in Remington "The Science and Practice of Pharmacy" (20th Ed., Lippincott Williams & Wilkins, Baltimore MD).

[0285] The agent or its pharma- ceutically acceptable salt may be provided alone or in combination with one or more other agents, or in one or more other forms. For example, a formulation may contain one or more agents in a particular ratio, depending on the relative potency and intended indication of each agent. For example, in a composition for targeting two different host targets, and where the potencies are similar, an approximately 1:1 ratio of agents may be used. The two forms may be combined together in the same dosage unit, e.g., one cream, suppository, tablet, capsule, aerosol spray, or packet of powder to be dissolved in a drink, or the two forms may be combined in separate units, e.g., two creams, two suppositories, two tablets, two capsules, a tablet and a liquid to dissolve the tablet, two aerosol sprays, or a packet of powder and a liquid to dissolve the powder.

[0286] The term "pharmaceutically acceptable" refers to salts that retain the biological effectiveness and properties of the agents used in this disclosure and which are not biologically or otherwise undesirable.

[0287] Common salts are those of inorganic ions, such as sodium, potassium, calcium, and magnesium ions. Such salts include those formed with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid, or maleic acid. Furthermore, if the drug contains a carboxyl or other acidic group, it can be converted to a pharma-ceutically acceptable addition salt with an inorganic or organic base. Examples of suitable bases include sodium hydroxide, potassium hydroxide, ammonia, cyclohexylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, and the like.

[0288] Pharmaceutically acceptable esters or amides refer to salts that retain the biological effectiveness and properties of the agents used in this disclosure and are not biologically or otherwise undesirable. Common esters include ethyl esters, methyl esters, isobutyl esters, ethylene glycol esters, etc. Common amides include unsubstituted amides, alkyl amides, dialkyl amides, etc.

[0289] In some embodiments, the agent may be administered in combination with one or more other compounds, forms, and / or agents, such as those described above. Pharmaceutical compositions containing one or more other active agents may also be formulated to include a particular molar ratio. For example, the molar ratio of the first active agent to the other active agent may be from about 99:1 to about 1:99. In some subsets of embodiments, the molar ratio of the first active agent to the other active agent may range from about 80:20 to about 20:80, about 75:25 to about 25:75, about 70:30 to about 30:70, about 66:33 to about 33:66, about 60:40 to about 40:60, about 50:50, or about 90:10 to about 10:90. The molar ratio of the first active agent to the other active agent may be about 1:9, and in some embodiments, about 1:1. The two drugs, forms and / or compounds may be combined together in the same dosage unit, e.g., one cream, suppository, tablet, capsule, or packet of powder to be dissolved in a drink, or the two drugs, forms and / or compounds may be combined in separate units, e.g., two creams, suppositories, tablets, two capsules, a tablet and a liquid to dissolve the tablet, an aerosol spray, a packet of powder and a liquid to dissolve the powder, etc.

[0290] If necessary or desired, the agents and / or agent combinations can also be administered with further agents. The choice of agents that can be co-administered with the agents and / or agent combinations of the present disclosure can depend, at least in part, on the condition being treated.

[0291] The agent (or a pharma- ceutically acceptable salt, ester, or amide thereof) can be administered alone or in the form of a pharmaceutical composition in which the active agent is combined or mixed with one or more pharma- ceutically acceptable carriers. As used herein, a pharmaceutical composition may be any composition prepared for administration to a subject. Pharmaceutical compositions for use according to the present disclosure can be formulated in a conventional manner using one or more physiologically acceptable carriers, including, for example, excipients, diluents, and / or adjuvants that facilitate processing of the active agent into an administrable formulation. The appropriate formulation may depend, at least in part, on the route of administration selected. The agent used in the present disclosure, or a pharma- ceutical acceptable salt, ester, or amide thereof, can be administered to a subject using a wide variety of routes or modes of administration, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, intraocular, intravitreal, and intramuscular administration, as well as inhalation.

[0292] In some embodiments, the agent may be in solution using oil or non-aqueous solvents, for example, due to the presence of large lipophilic moieties. Alternatively, other formulations such as emulsions, suspensions, or liposomal formulations may be used. With respect to liposomal formulations, any known method may be used to prepare liposomes for treating conditions. See, for example, Bangham et al., J. Mol. Biol. 23: 238-252 (1965) and Szoka et al., Proc. Natl Acad. Sci. USA 75: 4194-4198 (1978). Ligands may also be attached to liposomes to direct these compositions to specific sites of action. The agents of the present disclosure may also be included in foods, such as cream cheese, butter, salad dressing, or ice cream, to facilitate solubilization, administration, and / or compliance in certain subject populations.

[0293] The compound of the present disclosure can be formulated for parenteral administration (for example, by injection, intraocular or intravitreal injection), and can also be provided in unit dosage form in ampoules, pre-filled syringes, small volume injections, or in multi-dose containers with added preservative.This composition can have the form of suspension, solution, or emulsion in oily or aqueous solvent, for example, solution in aqueous polyethylene glycol solution, etc.

[0294] For injectable formulations, the solvent can be selected from those known in the art to be suitable, including aqueous or oily suspensions or emulsions, as well as sesame, corn, cottonseed, or peanut oil, as well as elixirs, mannitol, dextrose, or sterile aqueous solutions and similar pharmaceutical vehicles. The formulation may also include biocompatible, biodegradable polymeric compositions, such as poly(lactic-co-glycolic acid). These materials can be prepared into microspheres or nanospheres, loaded with drugs, and further coated or derivatized to provide excellent sustained release performance. Suitable solvents for periocular or intraocular injection include, for example, suspensions of therapeutic agents in injection grade water, liposomes, and solvents suitable for lipophilic substances. Other solvents for periocular or intraocular injection are known in the art.

[0295] In some embodiments, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to humans. Generally, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic, such as lidocaine, to ease pain at the injection site. Generally, each component is provided individually in unit dosage form or as a mixture, for example, as a lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet, indicating the amount of active agent. If the composition is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0296] For administration by injection, the active compound can be formulated in aqueous solutions, particularly physiologically compatible buffers such as Hank's solution, Ringer's solution or physiological saline buffer. The solutions may contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active compound can be in powder form and reconstituted with a suitable solvent, for example, sterile pyrogen-free water, prior to use. In some embodiments, the pharmaceutical composition does not include an adjuvant or any other substance added to enhance the immune response stimulated by the peptide. In some embodiments, the pharmaceutical composition includes a substance that inhibits the immune response to the peptide. Methods of formulation are well known in the art and are disclosed, for example, in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton P.

[0297] In some embodiments, eye diseases can be effectively treated by using ophthalmic solutions, suspensions, ointments or inserts containing the drug or drug combinations of the present disclosure. Ophthalmic solutions can be prepared by dissolving the active ingredient in a sterile aqueous solution such as saline, buffered solution, or by combining powder compositions and dissolving them before use. Other solvents known in the art can also be used, including, but not limited to, balanced salt solutions, saline solutions, water-soluble polyethers such as polyethylene glycol, polyvinyls such as polyvinyl alcohol and povidone, cellulose derivatives such as methylcellulose and hydroxypropylmethylcellulose, petroleum derivatives such as mineral oil and white petrolatum, animal fats such as lanolin, polymers of acrylic acid such as carboxypolymethylene gel, vegetable fats such as peanut oil, and polysaccharides such as dextran, and glycosaminoglycans such as sodium hyaluronate. Additives commonly used in eye drops can also be used if necessary. Such additives include isotonicity agents (e.g., sodium chloride, etc.), buffers (e.g., boric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), preservatives (e.g., benzalkonium chloride, benzethonium chloride, thiobutanol, etc.), viscosity enhancers (e.g., sugars such as lactose, mannitol, maltose, etc.; hyaluronic acid or salts thereof such as sodium hyaluronate, potassium hyaluronate, etc.; mucopolysaccharides such as chondroitin sulfate; e.g., sodium polyacrylate, carboxyvinyl polymers, crosslinked polyacrylates, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylcellulose, or other agents known to those skilled in the art).

[0298] The solubility of each component of the composition may be enhanced by the use of surfactants or other suitable co-solvents in the composition, such as polysorbates 20, 60, and 80, Pluronic® F68, F-84, and P-103, cyclodextrins, or other agents known to those skilled in the art. The concentration of such co-solvents used may be about 0.01% to 2% by weight.

[0299] The compositions of the present disclosure may be packaged in multi-dose form. A preservative may be preferred to prevent microbial contamination during use. Suitable preservatives include benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenethyl alcohol, disodium edetate, sorbic acid, Onamer M, or other agents known to those skilled in the art. In prior art ophthalmic products, the use concentration of such preservatives may be 0.004% to 0.02%. In the compositions of the present application, the preservative, preferably benzalkonium chloride, may be used at a concentration of 0.001% to less than 0.01% by weight, (e.g., 0.001% to 0.008% by weight, preferably about 0.005% by weight). It has been found that a concentration of 0.005% benzalkonium chloride may be sufficient to protect the compositions of the present disclosure from microbial attack.

[0300] In some embodiments, the agents of the present disclosure are delivered in a soluble form rather than in a suspension form, which allows for more rapid and quantitative absorption to the site of action.Generally, formulations such as gels, creams, lotions, suppositories, and ointments can provide areas that are exposed to the agents of the present disclosure for a long period of time, whereas formulations in the form of solutions, such as sprays, can provide more immediate and short-term exposure.

[0301] It is further expected that the compounds of the present disclosure can be releasably bound to biocompatible polymers for use in sustained release formulations on, in, or bound to inserts for local, intraocular, periocular, or systemic administration.Controlled release from biocompatible polymers can also be used with water-soluble polymers to form eye-droppable formulations.For example, controlled release from biocompatible polymers such as PLGA microspheres or nanospheres can be used in formulations suitable for intraocular implantation or injection for sustained release administration, and any suitable biodegradable and biocompatible polymers can be used.

[0302] Further Numbered Embodiments Further embodiments of the present invention are set forth in the following numbered paragraphs.

[0303] Item 1: A recombinant nucleic acid comprising (in order from the 5' end to the 3' end) a mitochondrial targeting sequence and a mitochondrial protein coding sequence, optionally wherein the mitochondrial protein coding sequence encodes an ND4 protein, and optionally wherein the ND4 protein comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 160.

[0304] Item 2: The recombinant nucleic acid according to Item 1, comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the sequences set forth in SEQ ID NOs: 180 and 174 to 176.

[0305] Item 3: The recombinant nucleic acid of item 1 or 2, comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 180.

[0306] Item 4: The recombinant nucleic acid according to any one of Items 1 to 3, comprising the sequence set forth in SEQ ID NO: 180.

[0307] Item 5: The recombinant nucleic acid according to any one of items 1 to 4, comprising a Kozak sequence located before the 5' end of the mitochondrial targeting sequence, optionally wherein the Kozak sequence is SEQ ID NO: 171, and optionally wherein there are no redundant nucleotides between the Kozak sequence and the mitochondrial targeting sequence.

[0308] Item 6: The recombinant nucleic acid according to any one of items 1 to 5, comprising an intron sequence, optionally wherein the intron sequence is located before the 5' end of the mitochondrial targeting sequence, optionally wherein the intron sequence is located before the 5' end of the Kozak sequence, and optionally wherein the intron sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 170.

[0309] Item 7: The recombinant nucleic acid according to any one of Items 1 to 6, comprising a promoter sequence, optionally the promoter sequence is located before the 5' end of the mitochondrial targeting sequence, the Kozak sequence, and / or the intron sequence, and optionally the promoter sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 169.

[0310] Item 8: The recombinant nucleic acid according to any one of items 1 to 7, comprising a 3'UTR sequence, optionally located after the 3' end of the mitochondrial protein coding sequence, and optionally comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 13.

[0311] Item 9: The recombinant nucleic acid according to any one of items 1 to 8, comprising a polyA signal sequence, optionally wherein the polyA signal sequence is located after the 3' end of the mitochondrial protein coding sequence and / or the 3'UTR sequence, optionally wherein the polyA signal sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence set forth in SEQ ID NO: 172 or 173, optionally wherein the polyA signal sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence set forth in SEQ ID NO: 173.

[0312] Item 10: The recombinant nucleic acid according to any one of Items 1 to 9, comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the spacer sequence set forth in SEQ ID NO: 185 between the 3'UTR sequence and the polyA signal sequence.

[0313] Item 11: the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:1; and / or 11. The recombinant nucleic acid according to any one of items 1 to 10, wherein the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:6.

[0314] Item 12: The recombinant nucleic acid according to any one of items 1 to 11, further comprising a first inverted terminal repeat (ITR) sequence and a second ITR sequence, optionally wherein the first ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 178, and the second ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 179.

[0315] Item 13: (in order from the 5' end to the 3' end) A recombinant nucleic acid comprising a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, and a polyA signal sequence, The recombinant nucleic acid, wherein the polyA signal sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:173.

[0316] Item 14: The recombinant nucleic acid of Item 13, wherein the 3'UTR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:13.

[0317] Item 15: The recombinant nucleic acid according to any one of Items 9 to 14, wherein an mRNA comprising a mitochondrial protein coding sequence produced by transcription of the recombinant nucleic acid has a higher expression level than an mRNA of a control recombinant nucleic acid lacking the polyA signal sequence, preferably at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 300% higher expression level than the mRNA of the control recombinant nucleic acid.

[0318] Item 16: The recombinant nucleic acid of item 15, wherein the control recombinant nucleic acid is substituted with a sequence comprising SEQ ID NO: 172 at the position of the polyA signal sequence, and preferably the mRNA produced by transcription of the recombinant nucleic acid has an expression level that is at least 10%, at least 15%, or at least 20% higher than the mRNA produced by the control recombinant nucleic acid.

[0319] Item 17: The recombinant nucleic acid according to any one of Items 9 to 16, wherein the mitochondrial protein produced by translation of the recombinant nucleic acid has a higher expression level than the mitochondrial protein of the control recombinant nucleic acid lacking the polyA signal sequence, preferably at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 300% higher expression level than the mitochondrial protein of the control recombinant nucleic acid.

[0320] Item 18: The recombinant nucleic acid of Item 17, wherein the control recombinant nucleic acid is substituted with a sequence comprising SEQ ID NO: 172 at the position of the polyA signal sequence, and preferably the mitochondrial protein produced by translation of the recombinant nucleic acid has an expression level that is at least 10%, at least 15%, or at least 20% higher than the mitochondrial protein produced by the control recombinant nucleic acid.

[0321] Item 19: The recombinant nucleic acid according to any one of Items 9 to 18, wherein the polyA signal sequence has a length of 122 base pairs or less, 125 base pairs or less, 130 base pairs or less, 140 base pairs or less, 150 base pairs or less, 160 base pairs or less, 170 base pairs or less, 180 base pairs or less, 190 base pairs or less, or 200 base pairs or less.

[0322] Item 20: The recombinant nucleic acid according to any one of Items 13 to 19, further comprising a Kozak sequence located before the 5' end of the mitochondrial targeting sequence, optionally wherein the Kozak sequence is SEQ ID NO: 171, and optionally wherein there are no redundant nucleotides between the Kozak sequence and the mitochondrial targeting sequence.

[0323] Item 21: The recombinant nucleic acid according to any one of Items 13 to 20, comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the spacer sequence set forth in SEQ ID NO: 185 between the 3'UTR sequence and the polyA signal sequence.

[0324] Item 22: (in order from the 5' end to the 3' end) A recombinant nucleic acid comprising a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, and a 3'UTR sequence, the Kozak sequence is SEQ ID NO: 171, the 3'UTR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 13; and The recombinant nucleic acid, wherein there are no redundant nucleotides between the Kozak sequence and the mitochondrial targeting sequence.

[0325] Item 23: The recombinant nucleic acid of Item 22, further comprising a polyA signal sequence, wherein the polyA signal sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 172 or 173.

[0326] Item 24: The recombinant nucleic acid according to any one of Items 13 to 23, further comprising an intron sequence, optionally wherein the intron sequence is located before the 5' end of the Kozak sequence, and optionally wherein the intron sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 170.

[0327] Item 25: The recombinant nucleic acid according to any one of Items 13 to 24, further comprising a promoter sequence, optionally comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 169.

[0328] Item 26: The recombinant nucleic acid according to Item 25, wherein the promoter sequence is located before the 5' end of the intron sequence.

[0329] Item 27: (In order from the 5' end to the 3' end) A recombinant nucleic acid comprising a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, and a mitochondrial protein coding sequence, the intron sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 170; Optionally, said recombinant nucleic acid further comprises a 3'UTR sequence, and optionally, said recombinant nucleic acid further comprises a polyA signal sequence.

[0330] Item 28: The recombinant nucleic acid according to any one of Items 13 to 27, further comprising a first inverted terminal repeat (ITR) sequence and a second ITR sequence, optionally wherein the first ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 178, and the second ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 179.

[0331] Item 29: The recombinant nucleic acid according to any one of Items 13 to 28, wherein there are no redundant nucleotides between the mitochondrial protein coding sequence and the 3'UTR sequence.

[0332] Item 30: the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:1; and / or 30. The recombinant nucleic acid according to any one of items 13 to 29, wherein the mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:6.

[0333] Item 31: The recombinant nucleic acid according to any one of Items 13 to 30, wherein there are no redundant nucleotides between the mitochondrial targeting sequence and the mitochondrial protein coding sequence.

[0334] Item 32: A recombinant nucleic acid comprising (in order from the 5' end to the 3' end) a first ITR sequence, a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, a polyA signal sequence, and a second ITR sequence, Preferably, the first ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 178, the promoter sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 169, the intron sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 170, the Kozak sequence is SEQ ID NO: 171, the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 1, and the mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 1 32. The recombinant nucleic acid according to any one of items 1 to 31, wherein the trichomeoprotein coding sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 6, the 3'UTR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 13, the polyA signal sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 173, and the second ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 179.

[0335] Item 33: A recombinant nucleic acid comprising (in order from the 5' end to the 3' end) a first ITR sequence, a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, a polyA signal sequence, and a second ITR sequence, Preferably, the first ITR sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO: 178, the promoter sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO: 169, the intron sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO: 170, the Kozak sequence is SEQ ID NO: 171, the mitochondrial targeting sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO: 1, 33. The recombinant nucleic acid according to any one of items 1 to 32, wherein the mitochondrial protein coding sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO: 6, the 3'UTR sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO: 13, the polyA signal sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO: 173, and the second ITR sequence comprises a sequence having at least 99%, at least 99.5%, or 100% identity to the sequence set forth in SEQ ID NO: 179.

[0336] Item 34: A recombinant nucleic acid comprising (in order from the 5' end to the 3' end) a first ITR sequence, a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, a polyA signal sequence, and a second ITR sequence, 34. The recombinant nucleic acid according to any one of items 1 to 33, wherein the first ITR sequence comprises the sequence set forth in SEQ ID NO: 178, the promoter sequence comprises the sequence set forth in SEQ ID NO: 169, the intron sequence comprises the sequence set forth in SEQ ID NO: 170, the Kozak sequence is SEQ ID NO: 171, the mitochondrial targeting sequence comprises the sequence set forth in SEQ ID NO: 1, the mitochondrial protein coding sequence comprises the sequence set forth in SEQ ID NO: 6, the 3'UTR sequence comprises the sequence set forth in SEQ ID NO: 13, the polyA signal sequence comprises the sequence set forth in SEQ ID NO: 173, and the second ITR sequence comprises the sequence set forth in SEQ ID NO: 179.

[0337] Item 35: The recombinant nucleic acid according to any one of Items 13 to 34, comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the sequences set forth in SEQ ID NOs: 174 to 176 and 180.

[0338] Item 36: The recombinant nucleic acid according to any one of Items 13 to 34, comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the sequences set forth in SEQ ID NO: 180, preferably a sequence identical to SEQ ID NO: 180.

[0339] Item 37: A viral vector comprising the recombinant nucleic acid according to any one of items 1 to 36.

[0340] Item 38: The viral vector of item 37, wherein the viral vector is a recombinant adeno-associated viral (rAAV) vector.

[0341] Item 39: The viral vector of Item 38, wherein the rAAV vector is a rAAV2 vector.

[0342] Item 40: A pharmaceutical composition comprising the recombinant nucleic acid according to any one of items 1 to 36.

[0343] Item 41: A pharmaceutical composition comprising a viral vector, wherein the viral vector comprises the recombinant nucleic acid according to any one of items 1 to 36.

[0344] Item 42: The pharmaceutical composition of item 41, wherein the viral vector is an adeno-associated viral (AAV) vector.

[0345] Item 43: The pharmaceutical composition according to any one of Items 40 to 42, further comprising a pharma- ceutically acceptable excipient thereof.

[0346] Item 44: The pharmaceutical composition of item 43, wherein the pharma- ceutical acceptable excipient comprises phosphate buffered saline (PBS), α,α-trehalose dihydrate, L-histidine hydrochloride monohydrate, polysorbate 20, NaCl, NaH2PO4, Na2HPO4, KH2PO4, K2HPO4, poloxamer 188, or any combination thereof.

[0347] Item 45: The pharmaceutical composition according to Item 43, wherein the pharma- ceutically acceptable excipient is selected from phosphate buffered saline (PBS), α,α-trehalose dihydrate, L-histidine hydrochloride monohydrate, polysorbate 20, NaCl, NaH2PO4, Na2HPO4, KH2PO4, K2HPO4, poloxamer 188, and any combination thereof.

[0348] Item 46: The pharmaceutical composition according to any one of Items 43 to 45, wherein the pharma- ceutically acceptable excipient comprises poloxamer 188.

[0349] Item 47: The pharmaceutical composition according to Item 46, wherein the pharma- ceutical acceptable excipient comprises 0.0001% to 0.01% poloxamer 188.

[0350] Item 48: The pharmaceutical composition of item 46, wherein the pharma- ceutical acceptable excipient comprises 0.001% poloxamer 188.

[0351] Item 49: The pharmaceutical composition according to any one of Items 46 to 48, wherein the pharma- ceutically acceptable excipient further comprises one or more salts.

[0352] Item 50: The pharmaceutical composition of item 49, wherein the one or more salts include NaCl, Na2HPO4, and KH2PO4.

[0353] Item 51: a) NaCl at a concentration of 5 to 15 mg / mL, preferably NaCl at a concentration of 9 mg / mL; b) KH2PO4 at a concentration of 0.1 to 0.5 mg / mL, preferably KH2PO4 at a concentration of 0.144 mg / mL, and / or c) The pharmaceutical composition according to item 49, comprising Na2HPO4 in a concentration of 0.5 to 1 mg / mL, preferably Na2HPO4 in a concentration of 0.795 mg / mL.

[0354] Item 52: The pharmaceutical composition according to any one of Items 40 to 51, having a pH of 7.2 to 7.4.

[0355] Item 53: The pharmaceutical composition according to Item 52, having a pH of 7.3.

[0356] Item 54: At least 1.0×10 10 vg / mL, at least 3.0 × 10 10 vg / mL, or at least 6.0 × 10 10 54. The pharmaceutical composition according to any one of items 40 to 53, having a viral titer of 0.01 to 0.05 vg / mL.

[0357] Item 55: At least 9.0×10 10 55. The pharmaceutical composition of item 54, having a viral titer of 10 ...

[0358] Item 56: The pharmaceutical composition according to any one of items 40 to 55, which retains at least 60% of the viral titer after five freeze / thaw cycles compared to the viral titer before the five freeze / thaw cycles.

[0359] Item 57: Use of the pharmaceutical composition according to any one of items 40 to 56 in the preparation of a medicament for treating an eye disease.

[0360] Item 58: A method for treating an eye disease, comprising administering to a patient an effective amount of the pharmaceutical composition according to any one of items 40 to 56.

[0361] Item 59: The use of item 57 or the method of item 58, wherein the eye disease is Leber's hereditary optic neuropathy (LHON).

[0362] Item 60: The use or method according to any one of items 57 to 59, wherein the pharmaceutical composition is administered by intraocular or intravitreal injection.

[0363] Item 61: The use or method of item 60, wherein the pharmaceutical composition is administered by intravitreal injection.

[0364] Item 62: The use or method according to any one of items 57 to 61, wherein 0.01 mL to 0.1 mL of the pharmaceutical composition is administered into each eye.

[0365] Item 63: The use or method according to Item 62, wherein 0.045 to 0.055 mL of the pharmaceutical composition is administered into each eye.

[0366] Item 64: The adeno-associated virus is at least 1.5 × 10 9 vg of adeno-associated virus, preferably containing at least 3.0×10 9 64. The use or method according to any one of items 57 to 63, wherein the administration is at a dose of vg.

[0367] Item 65: The adeno-associated virus is at least 4.5 × 10 9 64. The use or method of any one of claims 57 to 63, wherein the therapeutic agent is administered intraocularly at a dose of 100 mg / mL.

[0368] Item 66: The use or method according to any one of items 57 to 65, further comprising administering a corticosteroid to the patient.

[0369] Item 67: The use or method of item 66, wherein the corticosteroid comprises prednisone or methylprednisolone.

[0370] Item 68: The use or method of item 67, wherein the corticosteroid is prednisone.

[0371] Item 69: The use or method according to any one of items 66 to 68, wherein the corticosteroid is administered starting from about 2 days prior to administration of the pharmaceutical composition.

[0372] Item 70: The use or method according to any one of items 66 to 69, wherein the corticosteroid is administered orally.

[0373] Item 71: The use or method of any one of items 66 to 70, wherein the corticosteroid is administered for about 28 consecutive days after the initiation of said administration.

[0374] Item 72: The use or method of any one of items 66 to 71, wherein the corticosteroid is administered daily after initiation of said administration and at a decreasing dose with each week of continued administration.

[0375] Item 73: The use or method of any one of items 66 to 72, wherein the corticosteroid is administered at a dosage of 40 mg per day for 1 week at the start of administration, then 30 mg per day for 1 week, then 20 mg per day for 1 week, and finally 10 mg per day for 1 week.

[0376] Item 74: The use or method of any one of items 57 to 73, further comprising administering sodium creatine phosphate to the patient.

[0377] Item 75: The use or method of item 74, wherein the sodium creatine phosphate is administered by intravenous injection.

[0378] Item 76: The use or method of any one of items 57 to 75, wherein administration of the pharmaceutical composition results in a higher average level of visual acuity recovery compared to administration of a pharmaceutical composition not comprising the recombinant nucleic acid. EXAMPLES

[0379] The present invention will be further described below in conjunction with the following examples. It should be understood that these examples are not intended to limit the scope of the present invention, but are merely intended to illustrate the present invention. Unless otherwise indicated, the following examples can use the methods and conditions disclosed in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.

[0380] Example 1. Construction and optimization of ND4 plasmid I. Plasmid Construction and Preparation

[0381] The mitochondrial targeting sequence (MTS) COX10 (SEQ ID NO:1) was ligated to the human ND4 coding sequence (SEQ ID NO:6) and 3'UTR sequence (SEQ ID NO:13), a FLAG tag peptide was added before the terminator, a CMV promoter (SEQ ID NO:169) and a chimeric intron (SEQ ID NO:170) were added to the 5' end, a bGH (SEQ ID NO:172) or SV40 (SEQ ID NO:173) poly(A) tail (or none) was ligated to the 3' end, and the AAV2 plasmid backbone was inserted to obtain pAAV-CMV-ND4-3'Flag-COX10UTR Plasmid A (Figure 3, SEQ ID NO:174), pAAV-CMV-ND4-3'Flag-COX10UTR-bGH Plasmid B (Figure 2, SEQ ID NO:175), and pAAV-CMV-ND4-3'Flag-COX10UTR-SV40 Plasmid C (Figure 1, SEQ ID NO:176). Each ligation product was transformed into E. coli and a single colony was selected for enzymatic digestion verification and sequencing verification.

[0382] II. Cell transfection with plasmids

[0383] 1. One day prior to transfection, HEK293T cells were trypsinized, counted, and plated to reach 70%-80% confluence on the day of transfection.

[0384] 2. For each well of cells, 2.0 μg of plasmid DNA was diluted in 125 μL of serum-free DMEM medium and 6 μL of PEI reagent (1 μg / μL) was diluted in 125 μL of DMEM medium.

[0385] 3. The diluted DNA and PEI were mixed and incubated at room temperature for 20 minutes.

[0386] 4. The compounds obtained above were added directly to each well, and the culture plate was gently shaken to mix the wells.

[0387] 5. The cells were cultured at 37°C in 5% CO2 for 48 hours.

[0388] 6. After removing the medium, the cells were washed with PBS, trypsinized, centrifuged, and then harvested for further use.

[0389] III. Measuring mRNA content by qPCR

[0390] 1. Extraction of total RNA (using a kit)

[0391] 1) 10cm culture plate 2 The cells were lysed by adding 1 mL of lysis solution RZ per sample, and the cells were disrupted several times with a sampler until the solution became transparent. The solution was then allowed to stand at 15 to 30°C for 5 minutes to completely separate the nucleic acid-protein compounds.

[0392] 2) 200 μL of chloroform was added, covered with a tube cover, shaken vigorously for 15 seconds, left at room temperature for 3 minutes, and then centrifuged at 12,000 rpm (approximately 13,400 × g) for 10 minutes at 4 ° C to obtain a sample, which was separated into three layers including a yellow organic phase, an intermediate layer, and a colorless aqueous phase. The aqueous phase mainly contained RNA and was about 50% in volume of the cell lysis solution RZ reagent used. The aqueous phase was transferred to a new tube for operation in the next step.

[0393] 3) 0.5 volumes of absolute ethanol were gradually added and mixed thoroughly (in this case, precipitates may be present). The resulting solution and precipitate were transferred to the adsorption column CR3 and centrifuged at 12,000 rpm (approximately 13,400 x g) for 30 seconds at 4°C. If the solution and mixture could not be added to the adsorption column CR3 all at once, the solution and precipitate were transferred to the adsorption column CR3 in two portions and centrifuged at 12,000 rpm (approximately 13,400 x g) for 30 seconds at 4°C, after which the waste liquid in the collection tube was discarded.

[0394] 4) 500 μL of the deproteinized solution RD was added to the adsorption column CR3, and centrifuged at 12,000 rpm (approximately 13,400 × g) at 4°C for 30 seconds, the waste liquid was discarded, and CR3 was placed in a collection tube.

[0395] 5) 500 μL of the rinse solution RW was added to the adsorption column CR3, and the column was left to stand at room temperature for 2 minutes, and then centrifuged at 12,000 rpm (approximately 13,400×g) at 4° C. for 30 seconds. The waste liquid was discarded, and this procedure was repeated once.

[0396] 6) The adsorption column was placed in a 2 mL collection tube and centrifuged at 12,000 rpm (approximately 13,400 x g) at 4°C for 2 minutes to remove residual liquid.

[0397] 7) The adsorption column CR3 was transferred to a new 1.5 mL centrifuge tube, 30 to 100 μL of RNase-Free ddH2O was added, and the resulting mixture was left to stand at room temperature for 2 minutes and centrifuged at 12,000 rpm (approximately 13,400 × g) at 4 °C for 2 minutes.

[0398] 8) The RNA concentration was measured using an ultra-micro ultraviolet spectrophotometer.

[0399] 2. Reverse Transcription

[0400] 1) A solution containing 2 μg of RNA and 4 μL of 5× FastKing-RT SuperMix was added to a PCR tube, followed by adding ribonuclease-free deionized water up to 20 μL.

[0401] 2) The tubes were incubated in a PCR machine at 42°C for 15 minutes, then incubated at 95°C for 3 minutes to inactivate the reverse transcriptase.

[0402] 3. Quantitative PCR

[0403] 1) The following reaction system was prepared using 0.2 mL PCR tubes, and each reverse transcription product was prepared in three tubes. 10 μL of 2× qPCR Mix, 0.4 μL of Rox, 1 μL of 10 μM gene primer, 2.0 μL of reverse transcription product, and 5.6 μL of ddH2O were used.

[0404] Amplification primers for the target gene flag:

[0405] Forward primer: 5'-AGACCATGACGGTGAT-3' (SEQ ID NO: 181), and

[0406] Reverse primer: 5'-CTTGTCATCGTCATCCT-3' (SEQ ID NO: 182).

[0407] Amplification primers for the internal reference gene actin:

[0408] Forward primer: 5'-GGACTTCGAGCAAGAGATGG-3' (SEQ ID NO: 183), and

[0409] Reverse primer: 5'-AGGAAGGAAGGCTGGAAGAG-3' (SEQ ID NO: 184).

[0410] 2) PCR amplification

[0411] Pre-denaturation: 95℃, 30 seconds,

[0412] 40 cycles: 95°C, 5 sec → 55°C, 30 sec → 72°C, 30 sec,

[0413] Dissociation curve: 55℃→95℃ at a rate of 0.5℃ / 10 seconds

[0414] 3) To process the results ΔΔ The CT method is as follows: A=CT(target gene, test sample)-CT(internal control gene, test sample), B=CT(target gene, control sample)-CT(internal control gene, control sample), K=AB, fold expression=2-K.

[0415] IV. Experimental Results and Discussion

[0416] The expression of ND4 was detected at the mRNA level, and the results showed that compared with pAAV2-CMV-rND4-COX10UTR plasmid A, pAAV2-CMV-rND4-COX10UTR-bGH polyA plasmid B and pAAV2-CMV-rND4-COX10UTR-SV40 plasmid C (Figure 4) significantly improved the abundance of ND4 mRNA in HEK293 cells, with plasmid C being better than plasmid B. The above results indicate that more ND4 protein can be expressed in cells by codon optimization and the addition of various combinations of posttranscriptional regulators, and a better effect can be obtained when SV40 polyA is used as the polyA tail.

[0417] Example 2. Packaging and production of viruses 1. HEK293T cells that were over 90% confluent were passaged in culture dishes at a ratio of 1:3.

[0418] 2. Approximately 1 to 2 hours before plasmid transformation, serum was replaced with serum-free medium, and the target gene plasmid and helper plasmid were introduced into HEK293T cells using a transfection reagent.

[0419] 3. 24 hours after plasmid transformation, the serum-free medium was replaced with fresh medium.

[0420] 4. 72 hours after transfection, virus recovery was performed. The cells were pipetted with the medium and centrifuged, and the medium supernatant and cell precipitate were collected separately. The virus in the medium supernatant was precipitated with PEG8000 overnight, and the virus precipitate was collected.

[0421] 5. The virus mixture was purified by density gradient centrifugation using iodixanol and then concentrated in ultrafiltration tubes.

[0422] Example 3. Formulation Development 3.1 Safety testing of major ingredients

[0423] The injection contains the following auxiliary agents: potassium dihydrogen phosphate anhydrous, disodium hydrogen phosphate anhydrous, sodium chloride, water for injection, and poloxamer 188. Of these, potassium dihydrogen phosphate anhydrous, disodium hydrogen phosphate anhydrous, sodium chloride, and water for injection have been proven to be practical and safe as components of PBS in the formulation of conventional ophthalmic injections.

[0424] The data show that AAV2 virus particles have limited solubility and are prone to aggregation under high concentration and repeated freezing / thawing conditions. In addition, AAV virus particles have a strong adsorption effect on the container surface. Therefore, the applicant added poloxamer 188 to the injection solution to keep the injection solution in a relatively stable state.

[0425] The following samples were prepared: PBS + 0.01% poloxamer 188 + 10 mg / mL iodixanol, PBS + 0.01% poloxamer 188, PBS + 10 mg / mL iodixanol, PBS + 0.001% poloxamer 188 + 1 mg / mL iodixanol, and PBS + 0.001% poloxamer 188. Each sample was intravitreally injected into the eyes of New Zealand rabbits. The grouping results are shown in Table 2 below. [Table 2]

[0426] After the injection, clinical observations, intraocular pressure measurements, and general ophthalmologic examinations were performed on the New Zealand rabbits. On the 22nd day of the experiment, the experimental New Zealand rabbits were euthanized and subjected to gross anatomical observations and histopathological observations.

[0427] (1) Clinical Observations: During the experiment, all animals showed no abnormal clinical signs except for ocular symptoms.

[0428] (2) Intraocular pressure: On day 22 of the experiment, intraocular pressure in the left eye of female animals in the test group (PBS + 10 mg / mL iodoxanol) was significantly reduced (p≦0.05) compared to the injection group, and at other time points, intraocular pressure in the same eye of the same sex in all test sample groups was not statistically different from the injection group. The difference was random with no clear time-response relationship and therefore unrelated to the test sample.

[0429] (3) General eye examination:

[0430] conjunctiva:

[0431] Most animals developed mild bulbar hyperemia in the left eye on day 1 after treatment, which fully resolved by day 8. The injection group may have had bulbar hyperemia, but this was not dose- or sample-related. Therefore, the bulbar hyperemia was considered to be related to the injection procedure and not related to the test material.

[0432] Female animals in groups 2, 3, and 6 were observed to have mild chemosis in the left eye in one-third of eyes, with full recovery by day 4. chemosis was unrelated to the test article as the incidence was low and no clear dose-response relationship was observed.

[0433] Lens and vitreous humor:

[0434] Male animals in Group 4 were observed to have uniformly sized, gray-white punctate particles in the anterior vitreous humor of the left eye from days 8 to 22, and female animals in Group 5 were observed to have gray-white punctate particle deposits in the anterior lens capsule of the left eye and uniformly sized, gray-white punctate particles in the anterior vitreous humor from days 8 to 22, all of which were mild. The above abnormalities were incidental, without any apparent time / dose-response relationship, and therefore unrelated to the test material.

[0435] (4) Gross anatomy and histopathology

[0436] Euthanasia was performed on day 22 of the study. Several animals in the injection group and all test article groups had mild scattered inflammatory cell infiltration of the corneal limbus in the left and / or right eye. Since this lesion was also observed in the right eye of animals in the injection group and several untreated animals, this lesion may be a basal or natural lesion of the test animals and therefore is considered to be unrelated to the administered test article.

[0437] All animals were observed under a microscope to have no other significant abnormal pathological changes in the eye.

[0438] Conclusion: Following a single intravitreal injection of the test samples PBS + 0.01% poloxamer 188 + 10 mg / mL iodixanol, PBS + 0.01% poloxamer 188, PBS + 10 mg / mL iodixanol, and PBS + 0.001% poloxamer 188 (50 μL / eye) into New Zealand rabbits, the animals did not exhibit any ocular toxic reactions related to each of the test samples.

[0439] This demonstrates the safety of 0.01% poloxamer 188 as an adjuvant used in intraocular injections.

[0440] 3.2 Testing of the stability of the formulation

[0441] The specific formulation designs are shown in Table 3. [Table 3]

[0442] Each product was manufactured according to the above formulation and tested for stability. The experimental design for testing the stability of each formulation is shown in Tables 4 and 5. [Table 4]

[0443] The titers of viral genome in formulations 1 and 2 were measured separately on days 0, 1, and 5 to compare the stability of each formulation. [Table 5]

[0444] The viral genome titers in Formulations 1 and 2 were measured separately after 0, 1, 2, 3, 4 and 5 freeze / thaw cycles to compare the stability of each formulation.

[0445] The stability of each formulation was tested according to the above two schemes, and the results are shown in Figure 5 (results of the experiment to test the stability of each formulation at 2-8°C) and Figure 6 (results of the freeze / thaw experiment to test the stability of each formulation). [Table 6]

[0446] As shown in Table 6, in experiments to test the stability of the formulations stored at 2-8° C., the viral genome titer in formulation 1 decreased significantly and the viral genome titer in formulation 2 did not change significantly. Furthermore, the RSD of the viral genome titer in formulation 1 was 153.67% and the RSD of the viral genome titer in formulation 2 was 14.39%. Thus, formulation 2 is more stable than formulation 1.

[0447] [Table 7]

[0448] As shown in Table 7, in the freeze / thaw experiment, the viral genome titer in Formulation 1 decreased significantly, while the viral genome titer in Formulation 2 did not change significantly. Furthermore, the RSD of the viral genome titer in Formulation 1 was 113.25%, while the RSD of the viral genome titer in Formulation 2 was 10.53%. Therefore, Formulation 2 is more stable than Formulation 1.

[0449] In conclusion, the formulations containing poloxamer 188 are more stable than those without poloxamer 188.

[0450] Example 4. A Phase I / II / III, Single-Arm, Multicenter, Two-Stage Clinical Study to Evaluate the Safety and Efficacy of Gene Therapy for ND4 Mutation-Associated Leber's Hereditary Optic Neuropathy (LHON) This study is a Phase I / II / III, single-arm, multicenter, two-stage clinical trial to evaluate the safety and efficacy of NR082 ophthalmic injection in subjects with ND4 mutation-associated LHON. After admission, all subjects who met the inclusion criteria were administered study drug therapy and a systemic immunomodulatory regimen, i.e., a therapeutic dose of corticosteroid (prednisone) orally for 28 days to prevent or reduce ocular inflammation and possible immune reactions associated with IVT injection of NR082 ophthalmic injection. The specific dosing regimen of prednisone was 40 mg per day for 1 week starting 2 days before the subject's injection treatment, followed by 30 mg per day for 1 week, then 20 mg per day for 1 week, then 10 mg per day for 1 week, and finally discontinued.

[0451] The study period for each subject included a screening period, a 52-week (±4) follow-up period, and a 4-year long-term safety follow-up period. Screening period (day -42 to day -2), Hospitalization: Group allocation (-2 days), Hospitalization: Treatment (1st day), Hospitalization: Safety observation (days 2 to 8), Visits 1-6: Follow-up (week 2 (day 15) to week 52), and Visits 7–14: Long-term follow-up and end of study (every 6 months for 4 years).

[0452] The treatment groups and durations were as follows: The maximum study duration for each subject is 52 (± 4) weeks, including the screening period, and 4 years of long-term follow-up.

[0453] The doses used were as follows: 1.5×10 9 vg, 0.05 mL eye / dose (low dose), and 4.5×10 9 vg, 0.05 mL eye / dose (high dose).

[0454] Primary efficacy analysis The primary efficacy endpoint was the proportion of subjects with an improvement in BCVA in the injected eye of ≥0.3 LogMAR compared with baseline after 52 weeks (±4 weeks) of treatment.

[0455] Secondary Efficacy Analyses The percentage of subjects with ≥0.3 LogMAR improvement in BCVA in the injected eye compared to baseline was summarized by descriptive statistics (subject counts and associated percentages) for categorical variables according to treatment dose group and visit (if applicable). Exact Cloner-Pearson 95% confidence intervals for the above response variables were calculated according to visit (if applicable) or based on the usual approximation (if applicable). [Table 8]

[0456] As shown in Table 8, the number of subjects who showed an improvement of 0.3 LogMAR or more in best corrected visual acuity (BCVA) compared to baseline was 1 (16.7%), 1 (16.7%), and 2 (33.3%) in the low-dose group at weeks 2, 6, and 12, respectively, and 4 (66.7%), 4 (66.7%), and 4 (66.7%) in the high-dose group at weeks 2, 6, and 12, respectively.

[0457] Changes in BCVA (LogMAR) in the injected eye compared to baseline and improvements in BCVA in the injected eye compared to minimum will be summarized by descriptive statistics of continuous variables (including number of observations [n], mean, standard deviation [SD], median, minimum [min], maximum [max]) based on treatment dose group and visit (if applicable), as well as changes in visual field parameters (visual field index, mean visual field defect, pattern standard deviation value), contrast sensitivity parameters, and visual evoked potential (VEP) waveform parameters in injected and non-injected eyes compared to baseline.

[0458] [Table 9]

[0459] As shown in Table 9, mean BCVA compared to baseline increased by 0.080 LogMAR, 0.103 LogMAR, and 0.245 LogMAR in the low-dose group at weeks 2, 6, and 12, and by 0.367 LogMAR, 0.338 LogMAR, and 0.408 LogMAR in the high-dose group at weeks 2, 6, and 12, respectively. [Table 10]

[0460] As shown in Table 10, the mean visual field index compared to baseline increased by 6.2%, 4.2%, and 6.7% in the low-dose group at weeks 2, 6, and 12, respectively, and increased by 1.0%, 12.2%, and 13.5% in the high-dose group at weeks 2, 6, and 12, respectively.

[0461] [Table 11]

[0462] As shown in Table 11, the mean mean visual field loss compared to baseline increased by 2.373 dB, 1.507 dB, and 2.450 dB in the low-dose group at weeks 2, 6, and 12, respectively, and increased by 0.688 dB, 4.413 dB, and 5.130 dB in the high-dose group at weeks 2, 6, and 12, respectively.

[0463] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are presented by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the present invention, and that methods and structures encompassed within the scope of the claims and their equivalents are encompassed therein.

[0464] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an acknowledgment or any form of suggestion that they form part of the available prior art or general knowledge in any country in the world.

Claims

**Claim 1** A recombinant nucleic acid comprising a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, and a polyA signal sequence (in that order from the 5'-end to the 3'-end), wherein the polyA signal sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO:

173. **Claim 2** The recombinant nucleic acid according to claim 1, comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of the sequences set forth in SEQ ID NO: 174, 176, and 180. **Claim 3** The recombinant nucleic acid according to claim 1, comprising a Kozak sequence located upstream of the 5'-end of the mitochondrial targeting sequence, optionally wherein the Kozak sequence is SEQ ID NO: 171, and optionally wherein there are no redundant nucleotides between the Kozak sequence and the mitochondrial targeting sequence. **Claim 4** The recombinant nucleic acid according to claim 1, comprising an intron sequence, optionally wherein the intron sequence is located upstream of the 5'-end of the mitochondrial targeting sequence, optionally wherein the intron sequence is located upstream of the 5'-end of the Kozak sequence, and optionally wherein the intron sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO:

170. **Claim 5** The recombinant nucleic acid according to claim 1, comprising a promoter sequence, optionally wherein the promoter sequence is located upstream of the 5'-end of the mitochondrial targeting sequence, the Kozak sequence, and / or the intron sequence, and optionally wherein the promoter sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO:

169. **Claim 6** The recombinant nucleic acid according to claim 1, wherein the 3'UTR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO:

13. **Claim 7**: The mitochondrial protein coding sequence encodes an ND4 protein, and optionally, the ND4 protein comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

160. The recombinant nucleic acid according to claim 1. **Claim 8** The recombinant nucleic acid according to claim 1, comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with the spacer sequence set forth in SEQ ID NO: 185 between the 3'UTR sequence and the polyA signal sequence. **Claim 9** The mitochondrial targeting sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO: 1, and / or The mitochondrial protein coding sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO:

6. The recombinant nucleic acid according to claim 1. **Claim 10** Further comprising a first inverted terminal repeat (ITR) sequence and a second ITR sequence, and optionally, the first ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO: 178, and the second ITR sequence comprises a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO:

179. The recombinant nucleic acid according to claim 1. **Claim 11** The recombinant nucleic acid according to claim 1, wherein there are no redundant nucleotides between the mitochondrial protein coding sequence and the 3'UTR sequence. **Claim 12** The recombinant nucleic acid according to claim 1, wherein there are no redundant nucleotides between the mitochondrial targeting sequence and the mitochondrial protein coding sequence. **Claim 13** In order (in the 5'-end to 3'-end direction), a first ITR sequence, a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, a poly A signal sequence, and a second ITR sequence are included. Preferably, the first ITR sequence includes a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO: 178; the promoter sequence includes a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO: 169; the intron sequence includes a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO: 170; the Kozak sequence is SEQ ID NO: 171; the mitochondrial targeting sequence includes a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO: 1; the mitochondrial protein coding sequence includes a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO: 6; the 3'UTR sequence includes a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO: 13; the poly A signal sequence includes a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO: 173; and the second ITR sequence includes a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence set forth in SEQ ID NO:

179. The recombinant nucleic acid according to claim 1. [

14. ] In order (in the 5'-end to 3'-end direction), it includes a first ITR sequence, a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, a polyA signal sequence, and a second ITR sequence. Preferably, the first ITR sequence includes a sequence having at least 99%, at least 99.5%, or 100% identity with the sequence set forth in SEQ ID NO: 178; the promoter sequence includes a sequence having at least 99%, at least 99.5%, or 100% identity with the sequence set forth in SEQ ID NO: 169; the intron sequence includes a sequence having at least 99%, at least 99.5%, or 100% identity with the sequence set forth in SEQ ID NO: 170; the Kozak sequence is SEQ ID NO: 171; the mitochondrial targeting sequence includes a sequence having at least 99%, at least 99.5%, or 100% identity with the sequence set forth in SEQ ID NO: 1; the mitochondrial protein coding sequence includes a sequence having at least 99%, at least 99.5%, or 100% identity with the sequence set forth in SEQ ID NO: 6; the 3'UTR sequence includes a sequence having at least 99%, at least 99.5%, or 100% identity with the sequence set forth in SEQ ID NO: 13; the polyA signal sequence includes a sequence having at least 99%, at least 99.5%, or 100% identity with the sequence set forth in SEQ ID NO: 173; and the second ITR sequence includes a sequence having at least 99%, at least 99.5%, or 100% identity with the sequence set forth in SEQ ID NO:

179. The recombinant nucleic acid according to claim 1.

15. In order (in the 5'-end to 3'-end direction), it includes a first ITR sequence, a promoter sequence, an intron sequence, a Kozak sequence, a mitochondrial targeting sequence, a mitochondrial protein coding sequence, a 3'UTR sequence, a polyA signal sequence, and a second ITR sequence. Preferably, the first ITR sequence comprises the sequence set forth in SEQ ID NO: 178, the promoter sequence comprises the sequence set forth in SEQ ID NO: 169, the intron sequence comprises the sequence set forth in SEQ ID NO: 170, the Kozak sequence is SEQ ID NO: 171, the mitochondrial targeting sequence comprises the sequence set forth in SEQ ID NO: 1, the mitochondrial protein coding sequence comprises the sequence set forth in SEQ ID NO: 6, the 3'UTR sequence comprises the sequence set forth in SEQ ID NO: 13, the poly A signal sequence comprises the sequence set forth in SEQ ID NO: 173, and the second ITR sequence comprises the sequence set forth in SEQ ID NO: 179, the recombinant nucleic acid according to claim 1.

16. A viral vector comprising the recombinant nucleic acid according to any one of claims 1 to 15.

17. The viral vector according to claim 16, wherein the viral vector is a recombinant adeno-associated virus (rAAV) vector.

18. The viral vector according to claim 17, wherein the rAAV vector is an rAAV2 vector.

19. A pharmaceutical composition comprising an adeno-associated virus (AAV) comprising the recombinant nucleic acid according to any one of claims 1 to 15, Preferably, the pharmaceutical composition further comprises its pharmaceutically acceptable excipient, Preferably, the pharmaceutically acceptable excipient comprises 0.0001% to 0.01% of poloxamer 188, Preferably, the pharmaceutically acceptable excipient further comprises one or more salts, and the one or more salts comprise NaCl, Na2HPO4, and KH2PO4, Preferably, the pH of the pharmaceutical composition is 7.2 to 7.4, Preferably, the pharmaceutical composition has a viral titer of at least 1.0×1010 vg / mL, at least 3.0×1010 vg / mL, at least 6.0×1010 vg / mL, or at least 9.0×1010 vg / mL, a pharmaceutical composition.

20. A pharmaceutical for use in the treatment of an eye disease, comprising the pharmaceutical composition according to claim 19, Preferably, the eye disease is Leber hereditary optic neuropathy (LHON), Preferably, the pharmaceutical is administered by intravitreal injection or intravitreal injection, Preferably, the adeno-associated virus is administered at a dose of at least 1.5 × 109 vg / eye, at least 3.0 × 109 vg / eye, or at least 4.5 × 109 vg / eye, a pharmaceutical.