Compositions and methods for treating leber's hereditary optic neuropathy with nadh dehydrogenase proteins

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 recombinant nucleic acid, comprising:
 a mitochondrial targeting sequence;   a mitochondrial protein coding sequence comprising a sequence that is at least 99% identical to SEQ ID NO: 11 or 12; and   a 3′UTR nucleic acid sequence.   
     
     
         2 . The recombinant nucleic acid of  claim 1 , wherein said mitochondrial targeting sequence encodes a polypeptide comprising a peptide 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: 129-159. 
     
     
         3 . The recombinant nucleic acid of  claim 1 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2. 
     
     
         4 . The recombinant nucleic acid of  claim 1 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 3. 
     
     
         5 . The recombinant nucleic acid of  claim 1 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4. 
     
     
         6 . The recombinant nucleic acid of  claim 1 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 5. 
     
     
         7 . The recombinant nucleic acid of  claim 1 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 1. 
     
     
         8 . The recombinant nucleic acid of  claim 1 , wherein said mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 12. 
     
     
         9 . The recombinant nucleic acid of any one of  claims 1 - 8 , wherein said 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: 111-125. 
     
     
         10 . The recombinant nucleic acid of any one of  claims 1 - 8 , wherein said 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 as set forth in SEQ ID NO: 13 or SEQ ID NO: 14. 
     
     
         11 . The recombinant nucleic acid of  claim 1 , wherein said 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: 25-28, 39-42, 53-56, 67-70, and 81-84. 
     
     
         12 . A recombinant nucleic acid, comprising:
 a mitochondrial targeting sequence;   a mitochondrial protein coding sequence comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 11 or 12; and   a 3′UTR nucleic acid sequence.   
     
     
         13 . The recombinant nucleic acid of  claim 12 , wherein said mitochondrial targeting sequence comprises a sequence encodes a polypeptide selected from the group consisting of hsCOX10, hsCOX8, scRPM2, IcSirt5, 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, zmLOC100282174_hsADCK3_crATP6_hsATP5G3, 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_spilv1IcSirt5_osP06441306.24-2_hsATP5G2_ncATP9. 
     
     
         14 . The recombinant nucleic acid of  claim 12  or  13 , wherein said mitochondrial targeting sequence encodes a polypeptide comprising a peptide 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: 129-159. 
     
     
         15 . The recombinant nucleic acid of any one of  claims 12 - 14 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2 or 3. 
     
     
         16 . The recombinant nucleic acid of any one of  claims 12 - 14 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4. 
     
     
         17 . The recombinant nucleic acid of any one of  claims 12 - 14 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 5. 
     
     
         18 . The recombinant nucleic acid of any one of  claims 12 - 14 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 1. 
     
     
         19 . The recombinant nucleic acid of any one of  claims 12 - 18 , wherein said 3′UTR nucleic acid sequence is located at 3′ of said mitochondrial targeting sequence. 
     
     
         20 . The recombinant nucleic acid of any one of  claims 12 - 19 , wherein said 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. 
     
     
         21 . The recombinant nucleic acid of any one of  claims 12 - 20 , wherein said 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: 111-125. 
     
     
         22 . The recombinant nucleic acid of any one of  claims 12 - 21 , wherein said 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 as set forth in SEQ ID NO: 13 or SEQ ID NO: 14. 
     
     
         23 . The recombinant nucleic acid of any one of  claims 12 - 22 , wherein said mitochondrial targeting sequence is located at 5′ of said 3′UTR nucleic acid sequence. 
     
     
         24 . The recombinant nucleic acid of any one of  claims 12 - 22 , wherein said mitochondrial targeting sequence is located at 3′ of said mitochondrial targeting sequence. 
     
     
         25 . The recombinant nucleic acid of any one of  claims 12 - 24 , wherein said 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: 25-28, 39-42, 53-56, 67-70, and 81-84. 
     
     
         26 . The recombinant nucleic acid of any one of  claims 12 - 25 , wherein said mitochondrial protein coding sequence encodes a mitochondrial protein comprising or consisting of a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 162. 
     
     
         27 . A recombinant nucleic acid, comprising a mitochondrial protein coding sequence, wherein said mitochondrial protein coding sequence encodes a polypeptide comprising a mitochondrial protein, wherein said mitochondrial protein coding sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 11 or 12. 
     
     
         28 . The recombinant nucleic acid of  claim 27 , further comprising a mitochondrial targeting sequence. 
     
     
         29 . The recombinant nucleic acid of  claim 27  or  28 , wherein said mitochondrial targeting sequence comprises a sequence encodes a polypeptide selected from the group consisting of hsCOX10, hsCOX8, scRPM2, IcSirt5, 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, zmLOC100282174_hsADCK3_crATP6_hsATP5G3, 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_spilv1IcSirt5_osP06441306.24-2_hsATP5G2_ncATP9. 
     
     
         30 . The recombinant nucleic acid of any one of  claims 27 - 29 , wherein said mitochondrial targeting sequence encodes a polypeptide comprising a peptide 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: 129-159. 
     
     
         31 . The recombinant nucleic acid of any one of  claims 27 - 30 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 2. 
     
     
         32 . The recombinant nucleic acid of any one of  claims 27 - 31 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 3. 
     
     
         33 . The recombinant nucleic acid of any one of  claims 27 - 32 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 4. 
     
     
         34 . The recombinant nucleic acid of any one of  claims 27 - 33 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 5. 
     
     
         35 . The recombinant nucleic acid of any one of  claims 27 - 34 , wherein said mitochondrial targeting sequence comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence as set forth in SEQ ID NO: 1. 
     
     
         36 . The recombinant nucleic acid of any one of  claims 27 - 35 , further comprising a 3′UTR nucleic acid sequence. 
     
     
         37 . The recombinant nucleic acid of  claim 36 , wherein said 3′UTR nucleic acid sequence is located at 3′ of said mitochondrial targeting sequence. 
     
     
         38 . The recombinant nucleic acid of  claim 36  or  37 , wherein said 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. 
     
     
         39 . The recombinant nucleic acid of any one of  claims 36 - 38 , wherein said 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: 111-125. 
     
     
         40 . The recombinant nucleic acid of any one of  claims 36 - 39 , wherein said 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 as set forth in SEQ ID NO: 13 or SEQ ID NO: 14. 
     
     
         41 . The recombinant nucleic acid of any one of  claims 36 - 40 , wherein said mitochondrial targeting sequence is located at 5′ of said 3′UTR nucleic acid sequence. 
     
     
         42 . The recombinant nucleic acid of any one of  claims 36 - 41 , wherein said mitochondrial targeting sequence is located at 3′ of said mitochondrial targeting sequence. 
     
     
         43 . The recombinant nucleic acid of any one of  claims 36 - 42 , wherein said 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: 25-28, 39-42, 53-56, 67-70, and 81-84. 
     
     
         44 . A viral vector comprising said recombinant nucleic acid of any one of  claims 1 - 43 . 
     
     
         45 . The viral vector of  claim 44 , wherein said viral vector is an adeno-associated virus (AAV) vector. 
     
     
         46 . The viral vector of  claim 45 , wherein said 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 vectors. 
     
     
         47 . The viral vector of  claim 45  or  46 , wherein said AAV vector is a recombinant AAV (rAAV) vector. 
     
     
         48 . The viral vector of  claim 47 , wherein said rAAV vector is rAAV2 vector. 
     
     
         49 . A pharmaceutical composition, comprising an adeno-associated virus (AAV) comprising said recombinant nucleic acid of any one of  claims 1 - 43 . 
     
     
         50 . The pharmaceutical composition of  claim 49 , further comprising a pharmaceutically acceptable excipient thereof. 
     
     
         51 . A pharmaceutical composition, comprising said viral vector of any one of  claims 44 - 48 , and a pharmaceutically acceptable excipient thereof. 
     
     
         52 . The pharmaceutical composition of  claim 50  or  51 , wherein said pharmaceutically acceptable excipient comprises phosphate-buffered saline (PBS), α,α-trehalose dehydrate, L-histidine monohydrochloride monohydrate, polysorbate 20, NaCl, NaH2PO4, Na2HPO4, KH2PO4, K2HPO4, poloxamer 188, or any combination thereof. 
     
     
         53 . The pharmaceutical composition of  claim 50  or  51 , wherein said pharmaceutically acceptable excipient is selected from phosphate-buffered saline (PBS), α,α-trehalose dehydrate, L-histidine monohydrochloride monohydrate, polysorbate 20, NaCl, NaH2PO4, Na2HPO4, KH2PO4, K2HPO4, poloxamer 188, and any combination thereof. 
     
     
         54 . The pharmaceutical composition of  claim 50  or  51 , wherein said pharmaceutically acceptable excipient comprises poloxamer 188. 
     
     
         55 . The pharmaceutical composition of  claim 54 , wherein said pharmaceutically acceptable excipient comprises 0.0001%-0.01% poloxamer 188. 
     
     
         56 . The pharmaceutical composition of  claim 55 , wherein said pharmaceutically acceptable excipient comprises 0.001% poloxamer 188. 
     
     
         57 . The pharmaceutical composition of any one of  claims 50 - 56 , wherein said pharmaceutically acceptable excipient further comprises one or more salts. 
     
     
         58 . The pharmaceutical composition of  claim 57 , wherein said one or more salts comprises NaCl, NaH2PO4, Na2HPO4, and KH2PO4. 
     
     
         59 . The pharmaceutical composition of  claim 57 , wherein said one or more salts comprises 80 mM NaCl, 5 mM NaH2PO4, 40 mM Na2HPO4, and 5 mM KH2PO4. 
     
     
         60 . The pharmaceutical composition of any one of  claims 49 - 59 , wherein said pharmaceutical composition has a pH of 6-8. 
     
     
         61 . The pharmaceutical composition of  claim 60 , wherein said pharmaceutical composition has a pH of 7.2-7.4. 
     
     
         62 . The pharmaceutical composition of  claim 61 , wherein said pharmaceutical composition has a pH of 7.3. 
     
     
         63 . The pharmaceutical composition of any one of  claims 49 - 62 , wherein said pharmaceutical composition has a viral titer of at least 1.0×10 10  vg/mL. 
     
     
         64 . The pharmaceutical composition of  claim 63 , wherein said pharmaceutical composition has a viral titer of at least 5.0×10 10  vg/mL. 
     
     
         65 . The pharmaceutical composition of any one of  claims 49 - 64 , when said pharmaceutical composition is subject to five freeze/thaw cycles, said pharmaceutical composition retains at least 60%, 70%, 80%, or 90% of a viral titer as compared to the viral titer prior to the five freeze/thaw cycles. 
     
     
         66 . The pharmaceutical composition of any one of  claims 49 - 65 , wherein said pharmaceutical composition, when administered to a patient with Leber's hereditary optic neuropathy, generates a higher average recovery of vision than a comparable pharmaceutical composition without said recombinant nucleic acid. 
     
     
         67 . The pharmaceutical composition of any one of  claims 49 - 66 , wherein said pharmaceutical composition, when administered to a patient with Leber's hereditary optic neuropathy, generates a higher average recovery of vision than a comparable pharmaceutical composition comprising a recombinant nucleic acid as set forth in SEQ ID NO: 15. 
     
     
         68 . A method of treating an eye disorder, comprising administering said pharmaceutical composition of any one of  claims 49 - 67  to a patient in need thereof. 
     
     
         69 . The method of  claim 68 , wherein said eye disorder is Leber's hereditary optic neuropathy (LHON). 
     
     
         70 . The method of  claim 68  or  69 , comprising administering said pharmaceutical composition to one or both eyes of said patient. 
     
     
         71 . The method of any one of  claims 68 - 70 , wherein said pharmaceutical composition is administered via intraocular or intravitreal injection. 
     
     
         72 . The method of  claim 71 , wherein said pharmaceutical composition is administered via intravitreal injection. 
     
     
         73 . The method of  claim 72 , wherein about 0.01-0.1 mL of said pharmaceutical composition is administered via intravitreal injection. 
     
     
         74 . The method of  claim 73 , wherein about 0.05 mL of said pharmaceutical composition is administered via intravitreal injection. 
     
     
         75 . The method of any one of  claims 68 - 74 , further comprising administering methylprednisolone to said patient. 
     
     
         76 . The method of  claim 75 , wherein said methylprednisolone is administered prior to said intravitreal injection of said pharmaceutical composition. 
     
     
         77 . The method of  claim 75  or  76 , wherein said methylprednisolone is administered orally. 
     
     
         78 . The method of any one of  claims 75 - 77 , wherein said methylprednisolone is administered daily for at least 1, 2, 3, 4, 5, 6, or 7 days prior to said intravitreal injection of said pharmaceutical composition. 
     
     
         79 . The method of any one of  claims 75 - 78 , wherein said methylprednisolone is administered daily. 
     
     
         80 . The method of any one of  claims 75 - 79 , wherein a daily dosage of about 32 mg/60 kg methylprednisolone is administered. 
     
     
         81 . The method of any one of  claims 75 - 80 , wherein said methylprednisolone is administered after said intravitreal injection of said pharmaceutical composition. 
     
     
         82 . The method of any one of  claims 75 - 81 , further comprising administering creatine phosphate sodium to said patient. 
     
     
         83 . The method of  claim 82 , wherein said creatine phosphate sodium is administered intravenously. 
     
     
         84 . The method of any one of  claims 75 - 83 , wherein said methylprednisolone is administered intravenously or orally. 
     
     
         85 . The method of any one of  claims 75 - 84 , comprising administering methylprednisolone intravenously for at least one day, which is followed by administering methylprednisolone orally for at least a week. 
     
     
         86 . The method of  claim 85 , comprising administering methylprednisolone intravenously for about 3 days, which is followed by administering methylprednisolone orally for at least about 6 weeks. 
     
     
         87 . The method of any one of  claims 75 - 86 , wherein said methylprednisolone is administered intravenously at a daily dose of about 80 mg/60 kg. 
     
     
         88 . The method of any one of  claims 75 - 87 , wherein said administering said pharmaceutical composition generates a higher average recovery of vision than a comparable pharmaceutical composition without said recombinant nucleic acid. 
     
     
         89 . The method of any one of  claims 75 - 88 , wherein said administering said pharmaceutical composition generates a higher average recovery of vision than a comparable pharmaceutical composition comprising a recombinant nucleic acid as set forth in SEQ ID NO: 25.