Compositions and methods for treating leber's hereditary optic neuropathy

Abstract

Disclosed herein is a recombinant nucleic acid, comprising: a mitochondrial targeting sequence; a mitochondrial protein coding sequence, wherein said mitochondrial protein coding sequence encodes a polypeptide comprising a mitochondrial protein; and a 3′UTR nucleic acid sequence. Also disclosed is a pharmaceutical composition comprising the recombinant nucleic acid and a method of treating Leber's hereditary optic neuropathy (LHON) using the pharmaceutical composition.

What is claimed is: 
     
         1 . A method of treating an eye disorder, comprising administering to a patient in need thereof
 a) a first pharmaceutical composition comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid comprising:
 i) a nucleic acid sequence encoding a mitochondrial targeting peptide; 
 ii) a nucleic acid sequence encoding a mitochondrial protein comprising a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 6-12; and 
 iii) a 3′UTR nucleic acid sequence; and 
   b) a second pharmaceutical composition comprising a steroid.   
     
     
         2 . The method of  claim 1 , wherein the nucleic acid sequence encoding the mitochondrial protein encodes a polypeptide comprising an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO:
 160-162.   
     
     
         3 . The method of  claim 1  or  claim 2 , wherein the nucleic acid sequence encoding a mitochondrial targeting peptide encodes a polypeptide comprising an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 126-159. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the nucleic acid sequence encoding a mitochondrial targeting peptide comprises a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 1-5. 
     
     
         5 . The method of any one of  claims 1 - 4 , wherein the 3′UTR nucleic acid sequence comprises a nucleic sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 13, 14, and 111-125. 
     
     
         6 . A method of treating an eye disorder, comprising administering to a patient in need thereof
 a) a first pharmaceutical composition comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid comprising:
 i) a nucleic acid sequence encoding a mitochondrial targeting peptide comprising an amino sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 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.   
     
     
         7 . The method of  claim 6 , wherein said mitochondrial protein is selected from the group consisting of NADH dehydrogenase 4 (ND4), NADH dehydrogenase 6 (ND6), NADH dehydrogenase 1 (ND1), and variants thereof. 
     
     
         8 . The method of  claim 6  or  7 , wherein the nucleic acid sequence encoding a mitochondrial protein comprises a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6-12. 
     
     
         9 . The method of  claim 7  or  8 , wherein nucleic acid sequence encoding a mitochondrial protein encodes a polypeptide comprising an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 160-162. 
     
     
         10 . The method of any one of  claims 7 - 9 , wherein the nucleic acid sequence encoding a mitochondrial targeting peptide comprises a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 1-5. 
     
     
         11 . The method of any one of  claims 7 - 10 , wherein the 3′UTR nucleic acid sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 13, 14, and 111-125. 
     
     
         12 . A method of treating an eye disorder, comprising administering to a patient in need thereof
 a) a first pharmaceutical composition comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid comprising:
 i) a nucleic acid sequence encoding a mitochondrial targeting peptide comprising an amino sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 126-159; 
 ii) a nucleic acid sequence encoding a mitochondrial protein comprising a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 6-12; and 
 iii) a 3′UTR nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 13, 14, and 111-125; and 
   (b) a second pharmaceutical composition comprising a steroid.   
     
     
         13 . A method of treating an eye disorder, comprising administering to a patient in need thereof
 (a) a first pharmaceutical composition comprising an adeno-associated virus (AAV) comprising a 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.   
     
     
         14 . The method of  claim 13 , wherein 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. 
     
     
         15 . The method of  claim 13  or  14 , wherein the 3′UTR nucleic acid sequence comprises a nucleic sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 13, 14, and 111-125. 
     
     
         16 . The method of any one of  claims 1 - 15 , wherein the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 15-84. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the recombinant nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 15. 
     
     
         18 . The method of any one of  claims 1 - 17 , wherein the first pharmaceutical composition is administered via intraocular or intravitreal injection. 
     
     
         19 . The method of any one of  claims 1 - 18 , wherein about 0.01-0.1 mL of the first pharmaceutical composition is administered via intravitreal injection. 
     
     
         20 . The method of any one of  claims 1 - 19 , wherein about 0.05 mL of the first pharmaceutical composition is administered via intravitreal injection. 
     
     
         21 . The method of any one of  claims 1 - 20 , wherein the first pharmaceutical composition is administered to one or both eyes of the patient. 
     
     
         22 . The method of any one of  claims 1 - 21 , wherein the steroid selected from the group consisting of alclometasone diproprionate, amcinonide, beclomethasone diproprionate, betamethasone, betamethasone benzoate, betamethasone diproprionate, betamethasone sodium phosphate, betamethasone sodium phosphate and acetate, betamethasone valerate, clobetasol proprionate, clocortolone pivalate, cortisol (hydrocortisone), cortisol (hydrocortisone) acetate, cortisol (hydrocortisone) butyrate, cortisol (hydrocortisone) cypionate, cortisol (hydrocortisone) sodium phosphate, cortisol (hydrocortisone) sodium succinate, cortisol (hydrocortisone) valerate, cortisone acetate, desonide, desoximetasone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, diflorasone diacetate, fludrocortisone acetate, flunisolide, fluocinolone acetonide, fluocinonide, fluorometholone, flurandrenolide, halcinonide, medrysone, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, mometasone furoate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, and triamcinolone hexacetonide or a synthetic analog thereof. 
     
     
         23 . The method of any one of  claims 1 - 22 , wherein steroid is a glucocorticoid. 
     
     
         24 . The method of  claim 23 , wherein the glucocorticoid is methylprednisolone or prednisone. 
     
     
         25 . The method of  claim 24 , wherein the methylprednisolone is formulated as a tablet or as a liquid for intravenous administration. 
     
     
         26 . The method of any one of  claims 1 - 25 , wherein the steroid is administered orally or intravenously. 
     
     
         27 . The method of any one of  claims 1 - 26 , wherein the steroid is administered prior to administration of the first pharmaceutical composition. 
     
     
         28 . The method of  claim 27 , wherein the steroid is administered daily for at least 1, 2, 3, 4, 5, 6, or 7 days prior to the administration of the first pharmaceutical composition. 
     
     
         29 . The method of  claim 28 , wherein the steroid is methylprednisolone and is administered at a daily dosage of about 30 mg/60 kg to about 40 mg/60 kg or about 30 mg to about 40 mg. 
     
     
         30 . The method of  claim 29 , wherein the daily dosage of methylprednisolone is about 32 mg/60 kg or 32 mg. 
     
     
         31 . The method of  claim 28 , wherein the steroid is prednisone and is administered at a daily dosage of about 50 mg/60 kg to about 70 mg/60 kg. 
     
     
         32 . The method of  claim 31 , wherein the daily dosage of prednisone is about 60 mg/60 kg. 
     
     
         33 . The method of any one of  claims 1 - 26 , wherein the steroid is administered after the administration of the first pharmaceutical composition. 
     
     
         34 . The method of  claim 33 , wherein 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 the administration of the first pharmaceutical composition. 
     
     
         35 . The method of  claim 34 , wherein the steroid is methylprednisolone and is administered at a daily dosage of between about 70 mg/60 kg and 90 mg/60 kg or between about 70 mg and 90 mg. 
     
     
         36 . The method of  claim 35 , wherein the daily dosage of methylprednisolone is about 80 mg/60 kg or 80 mg. 
     
     
         37 . The method of  claim 35  or  36 , wherein the methylprednisolone is administered for at least two days after the administration of the first pharmaceutical composition. 
     
     
         38 . The method of  claim 37 , wherein subsequent doses of methylprednisolone are administered daily for at least 7 weeks after the administration of the first pharmaceutical composition and wherein the dosage of the methylprednisolone is decreased on a weekly basis. 
     
     
         39 . The method of  claim 34 , the steroid is predisone and is administered at a daily dosage of between about 50 mg/60 kg and 70 mg/60 kg or between about 50 mg and about 70 mg. 
     
     
         40 . The method of  claim 39 , wherein the daily dosage of predisone is about 60 mg/60 kg or about 60 mg. 
     
     
         41 . The method of  claim 39  or  40 , wherein the predisone is administered for at least seven days after the administration of the first pharmaceutical composition. 
     
     
         42 . The method of  claim 41 , wherein after seven days, the predisone is administered at a daily dosage of between about 30 mg/60 kg and about 50 mg/60 kg or between about 30 mg and 50 mg. 
     
     
         43 . The method of  claim 42 , wherein the daily dosage of predisone is about 40 mg/60 kg or 40 mg. 
     
     
         44 . The method of  claim 43 , wherein subsequent doses of predisone are administered daily for at least 4 days and wherein the dosage of the predisone is decreased on a daily basis. 
     
     
         45 . The method of any one of  claims 1 - 44 , wherein the steroid is administered prior to and after the administration of the first pharmaceutical compound. 
     
     
         46 . The method of  claim 45 , wherein the steroid is methylprednisolone and is administered daily for at least seven days prior to the administration of the first pharmaceutical compound and daily for at least 7 weeks after administration of the first pharmaceutical compound. 
     
     
         47 . The method of  claim 46 , wherein the methylprednisolone is administered prior to the administration of the first pharmaceutical compound at a daily dosage of about 32 mg/60 kg or 32 mg. 
     
     
         48 . The method of  claim 46  or  47 , wherein the methylprednisolone is administered at a daily dosage of about 80 mg/60 kg or 80 mg for at least 2 days after the administration of the first pharmaceutical compound. 
     
     
         49 . The method of  claim 48 , wherein beginning three days after administration of the first pharmaceutical compound, the methylprednisolone is administered at a daily dosage of about 40 mg/60 kg or 40 mg for at least 4 days. 
     
     
         50 . The method of  claim 49 , wherein beginning one week after administration of the first pharmaceutical compound, the methylprednisolone is administered at a daily dosage of about 32 mg/60 kg or 32 mg for at least one week. 
     
     
         51 . The method of  claim 50 , wherein beginning two weeks after administration of the first pharmaceutical compound, the methylprednisolone is administered at a daily dosage of about 24 mg/60 kg or 24 mg for at least one week. 
     
     
         52 . The method of  claim 51 , wherein beginning three weeks after administration of the first pharmaceutical compound, the methylprednisolone is administered at a daily dosage of about 16 mg/60 kg or 16 mg for at least one week. 
     
     
         53 . The method of  claim 52 , wherein beginning four weeks after administration of the first pharmaceutical compound, the methylprednisolone is administered at a daily dosage of about 8 mg/60 kg or 8 mg for at least one week. 
     
     
         54 . The method of  claim 53 , wherein beginning five weeks after administration of the first pharmaceutical compound, the methylprednisolone is administered at a daily dosage of about 6 mg/60 kg or 6 mg for at least one week. 
     
     
         55 . The method of  claim 54 , wherein beginning six weeks after administration of the first pharmaceutical compound, the methylprednisolone is administered at a daily dosage of about 4 mg/60 kg or 4 mg for at least one week. 
     
     
         56 . The method of  claim 45 , wherein the steroid is prednisone and is administered daily for at least two days prior to the administration of the first pharmaceutical compound and daily for at least eleven days after administration of the first pharmaceutical compound. 
     
     
         57 . The method of  claim 56 , wherein the prednisone is administered prior to the administration of the first pharmaceutical compound at a daily dosage of about 60 mg/60 kg or 60 mg. 
     
     
         58 . The method of  claim 56  or  57 , wherein the prednisone is administered at a daily dosage of about 60 mg/60 kg or 60 mg for at least seven days after the administration of the first pharmaceutical compound. 
     
     
         59 . The method of  claim 58 , wherein eight days after administration of the first pharmaceutical compound, the prednisone is administered at a daily dosage of about 40 mg/60 kg or 40 mg for at least one day. 
     
     
         60 . The method of  claim 59 , wherein nine days after administration of the first pharmaceutical compound, the prednisone is administered at a daily dosage of about 20 mg/60 kg or 20 mg for at least one day. 
     
     
         61 . The method of  claim 60 , wherein ten days after administration of the first pharmaceutical compound, the prednisone is administered at a daily dosage of about 10 mg/60 kg or 10 mg for at least one day. 
     
     
         62 . The method of any one of  claims 1 - 61 , further comprising administering sodium creatine phosphate to the patient. 
     
     
         63 . The method of  claim 62 , wherein said sodium creatine phosphate is administered intravenously prior to or after the administration of the first pharmaceutical composition. 
     
     
         64 . The method of any one of  claims 1 - 63 , wherein administration of the first and second pharmaceutical compositions generates a higher average recovery of vision than a comparable pharmaceutical composition administered without the second pharmaceutical composition. 
     
     
         65 . The method of any one of  claims 1 - 64 , wherein administration of the first and second pharmaceutical compositions generates a lower incidence of an adverse event than a comparable pharmaceutical composition administered without the second pharmaceutical composition. 
     
     
         66 . The method of  claim 65 , wherein the adverse event is selected from anterior chamber inflammation, vitritis, ocular hypertension, cataract removal, keratitis, vitreous hemorrhage, allergic conjunctivitis, and eye pain. 
     
     
         67 . The method of any one of  claims 64 - 66 , wherein the higher average recovery of vision and the lower incidence of an adverse event is determined in a population of patients with the eye disorder. 
     
     
         68 . The method of  claim 67 , wherein the population of patients are ethnically matched. 
     
     
         69 . The method of  claim 68 , wherein the population of patients are Chinese or Argentinian. 
     
     
         70 . The method of any one of  claims 1 - 69 , wherein the eye disorder is Leber's hereditary optic neuropathy (LHON). 
     
     
         71 . The method of any one of  claims 1 - 70 , wherein the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AA8, AAV9, and AAV10. 
     
     
         72 . The method of any one of  claims 1 - 71 , wherein the AAV is AAV2. 
     
     
         73 . A method of screening patients for treatment of an eye disorder, the method comprising:
 (a) obtaining a serum sample from a patient;   (b) culturing a population of target cells with a composition comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid encoding a detectable label in the presence of the serum sample; and   (c) detecting the expression level of the detectable label in the target cell population after the culturing, wherein the patient is selected for the treatment if the expression level of the detectable label in the target cell population is higher than a pre-determined threshold.   
     
     
         74 . A method of treating an eye disorder for a patient in need thereof, comprising:
 (a) obtaining a serum sample from a patient;   (b) culturing a population of target cells with a composition comprising a first adeno-associated virus (AAV) comprising a first recombinant nucleic acid encoding a detectable label in the presence of the serum sample;   (c) detecting the expression level of the detectable label 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 label in the target cell population is higher than a pre-determined threshold.   
     
     
         75 . The method of  claim 73  or  74 , wherein the detectable label is a fluorescent protein. 
     
     
         76 . The method of  claim 75 , wherein the fluorescent protein is green fluorescent protein (GFP). 
     
     
         77 . The method of any one of  claims 73 - 76 , wherein the detectable label is detected by flow cytometry or qPCR. 
     
     
         78 . The method of any one of  claims 73 - 77 , wherein the pre-determined threshold is about 40% of cells expressing the detectable label when detected by flow cytometry. 
     
     
         79 . The method of any one of  claims 73 - 77 , wherein the pre-determined threshold is a relative expression level of the detectable label of about 0.6 when detected by qPCR. 
     
     
         80 . The method of any one of  claims 73 - 79 , wherein the target cells are HEK-293 T cells. 
     
     
         81 . The method of any one of  claims 73 - 80 , wherein the treatment is a recombinant AAV comprising a nucleic acid sequence encoding a mitochondrial protein. 
     
     
         82 . The method of  claim 81 , wherein 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. 
     
     
         83 . The method of any one of  claims 73 - 82 , wherein the patient comprises a mutation selected from G11778A in the ND4 gene, G3460A in the ND1 gene, and T14484C in the ND6 gene. 
     
     
         84 . The method of any one of  claims 73 - 83 , wherein the culturing step is at least 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer. 
     
     
         85 . A kit, comprising an adeno-associated virus (AAV) comprising a recombinant nucleic acid encoding a detectable label, a population of target cells, and one or more reagents for detecting the detectable label. 
     
     
         86 . The kit of  claim 85 , further comprising a transfection reagent for transfecting the population of target cells with the AAV. 
     
     
         87 . The kit of  claim 85  or  86 , further comprising a second AAV comprising a recombinant nucleic acid encoding a mitochondrial protein. 
     
     
         88 . The kit of any one of  claims 85 - 87 , wherein the one or more reagents for detecting the detectable label are selected an antibody that binds to the detectable label and one or more primer oligonucleotides specific for the recombinant nucleic acid encoding the detectable label.