WO2023280175A1 - Methods for treating complex i deficiencies or cancers by modulating gro3p biosynthesis - Google Patents
Methods for treating complex i deficiencies or cancers by modulating gro3p biosynthesis Download PDFInfo
- Publication number
- WO2023280175A1 WO2023280175A1 PCT/CN2022/103982 CN2022103982W WO2023280175A1 WO 2023280175 A1 WO2023280175 A1 WO 2023280175A1 CN 2022103982 W CN2022103982 W CN 2022103982W WO 2023280175 A1 WO2023280175 A1 WO 2023280175A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- complex
- gro3p
- cgpds
- cells
- biosynthesis
- Prior art date
Links
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 76
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 51
- 230000007812 deficiency Effects 0.000 title claims description 23
- 101000900194 Homo sapiens Glycerol-3-phosphate dehydrogenase 1-like protein Proteins 0.000 claims description 39
- 239000013598 vector Substances 0.000 claims description 39
- 102100022556 Glycerol-3-phosphate dehydrogenase 1-like protein Human genes 0.000 claims description 37
- 239000003112 inhibitor Substances 0.000 claims description 37
- 239000003795 chemical substances by application Substances 0.000 claims description 34
- 230000002401 inhibitory effect Effects 0.000 claims description 29
- 210000004556 brain Anatomy 0.000 claims description 23
- 230000000694 effects Effects 0.000 claims description 22
- 230000002708 enhancing effect Effects 0.000 claims description 22
- 239000002773 nucleotide Substances 0.000 claims description 19
- 125000003729 nucleotide group Chemical group 0.000 claims description 19
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 claims description 18
- 229960003105 metformin Drugs 0.000 claims description 18
- 241000702423 Adeno-associated virus - 2 Species 0.000 claims description 17
- 230000035772 mutation Effects 0.000 claims description 17
- 102100021244 Integral membrane protein GPR180 Human genes 0.000 claims description 16
- 230000014509 gene expression Effects 0.000 claims description 15
- 241000580270 Adeno-associated virus - 4 Species 0.000 claims description 13
- 241001634120 Adeno-associated virus - 5 Species 0.000 claims description 13
- 241001164825 Adeno-associated virus - 8 Species 0.000 claims description 13
- 108020005196 Mitochondrial DNA Proteins 0.000 claims description 13
- 150000001875 compounds Chemical class 0.000 claims description 12
- 150000007523 nucleic acids Chemical group 0.000 claims description 12
- 230000002255 enzymatic effect Effects 0.000 claims description 11
- 102000039446 nucleic acids Human genes 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 11
- 241000972680 Adeno-associated virus - 6 Species 0.000 claims description 10
- 101100283544 Homo sapiens GPD1 gene Proteins 0.000 claims description 10
- 241000702421 Dependoparvovirus Species 0.000 claims description 9
- 108090000565 Capsid Proteins Proteins 0.000 claims description 8
- 102100023321 Ceruloplasmin Human genes 0.000 claims description 8
- 239000013608 rAAV vector Substances 0.000 claims description 8
- 208000006136 Leigh Disease Diseases 0.000 claims description 7
- 208000017507 Leigh syndrome Diseases 0.000 claims description 7
- 208000009564 MELAS Syndrome Diseases 0.000 claims description 7
- 206010058799 Mitochondrial encephalomyopathy Diseases 0.000 claims description 7
- 230000001086 cytosolic effect Effects 0.000 claims description 7
- 241001655883 Adeno-associated virus - 1 Species 0.000 claims description 6
- 241000202702 Adeno-associated virus - 3 Species 0.000 claims description 6
- 241001164823 Adeno-associated virus - 7 Species 0.000 claims description 6
- 241000649046 Adeno-associated virus 11 Species 0.000 claims description 6
- 241000649047 Adeno-associated virus 12 Species 0.000 claims description 6
- -1 BAY84-2243 Chemical compound 0.000 claims description 6
- 208000032087 Hereditary Leber Optic Atrophy Diseases 0.000 claims description 6
- 208000001043 mitochondrial complex I deficiency Diseases 0.000 claims description 6
- 108091033319 polynucleotide Proteins 0.000 claims description 6
- 102000040430 polynucleotide Human genes 0.000 claims description 6
- 239000002157 polynucleotide Substances 0.000 claims description 6
- 230000002195 synergetic effect Effects 0.000 claims description 6
- 108700019255 Mitochondrial complex I deficiency Proteins 0.000 claims description 5
- YMTINGFKWWXKFG-UHFFFAOYSA-N fenofibrate Chemical compound C1=CC(OC(C)(C)C(=O)OC(C)C)=CC=C1C(=O)C1=CC=C(Cl)C=C1 YMTINGFKWWXKFG-UHFFFAOYSA-N 0.000 claims description 5
- 229960002297 fenofibrate Drugs 0.000 claims description 5
- 229960003243 phenformin Drugs 0.000 claims description 5
- ICFJFFQQTFMIBG-UHFFFAOYSA-N phenformin Chemical compound NC(=N)NC(=N)NCCC1=CC=CC=C1 ICFJFFQQTFMIBG-UHFFFAOYSA-N 0.000 claims description 5
- 108020005199 Dehydrogenases Proteins 0.000 claims description 4
- 241000124008 Mammalia Species 0.000 claims description 4
- 239000000556 agonist Substances 0.000 claims description 4
- 230000000259 anti-tumor effect Effects 0.000 claims description 4
- 230000004770 neurodegeneration Effects 0.000 claims description 4
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 4
- 208000011580 syndromic disease Diseases 0.000 claims description 4
- 210000004881 tumor cell Anatomy 0.000 claims description 4
- 208000028742 Leber hereditary optic neuropathy and dystonia Diseases 0.000 claims description 3
- 208000013926 Leber optic atrophy and dystonia Diseases 0.000 claims description 3
- 208000036546 leukodystrophy Diseases 0.000 claims description 3
- 208000015122 neurodegenerative disease Diseases 0.000 claims description 3
- 201000001119 neuropathy Diseases 0.000 claims description 3
- 230000007823 neuropathy Effects 0.000 claims description 3
- 230000003287 optical effect Effects 0.000 claims description 3
- 208000033808 peripheral neuropathy Diseases 0.000 claims description 3
- 102000049481 human GPD1L Human genes 0.000 claims description 2
- 208000005332 optic atrophy with demyelinating disease of CNS Diseases 0.000 claims description 2
- 230000008488 polyadenylation Effects 0.000 claims description 2
- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical compound OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 abstract description 88
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 26
- 230000002438 mitochondrial effect Effects 0.000 abstract description 19
- 201000010099 disease Diseases 0.000 abstract description 16
- 230000001225 therapeutic effect Effects 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 135
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 50
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 50
- 102100036669 Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic Human genes 0.000 description 44
- 101001072574 Homo sapiens Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic Proteins 0.000 description 44
- 230000027721 electron transport chain Effects 0.000 description 39
- 230000005764 inhibitory process Effects 0.000 description 39
- 241000699670 Mus sp. Species 0.000 description 37
- 238000003786 synthesis reaction Methods 0.000 description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 27
- 108090000623 proteins and genes Proteins 0.000 description 25
- 241000699666 Mus <mouse, genus> Species 0.000 description 23
- 238000004458 analytical method Methods 0.000 description 23
- 101150050451 ndufs4 gene Proteins 0.000 description 23
- GNGACRATGGDKBX-UHFFFAOYSA-N dihydroxyacetone phosphate Chemical compound OCC(=O)COP(O)(O)=O GNGACRATGGDKBX-UHFFFAOYSA-N 0.000 description 22
- 210000002569 neuron Anatomy 0.000 description 22
- 210000001519 tissue Anatomy 0.000 description 21
- 238000011282 treatment Methods 0.000 description 21
- 230000003247 decreasing effect Effects 0.000 description 20
- 230000001965 increasing effect Effects 0.000 description 20
- 201000011510 cancer Diseases 0.000 description 19
- 239000002207 metabolite Substances 0.000 description 17
- 230000008929 regeneration Effects 0.000 description 17
- 238000011069 regeneration method Methods 0.000 description 17
- 206010021143 Hypoxia Diseases 0.000 description 16
- 230000002829 reductive effect Effects 0.000 description 15
- 208000002267 Anti-neutrophil cytoplasmic antibody-associated vasculitis Diseases 0.000 description 14
- 230000037361 pathway Effects 0.000 description 14
- 101000604411 Homo sapiens NADH-ubiquinone oxidoreductase chain 1 Proteins 0.000 description 13
- 101000632623 Homo sapiens NADH-ubiquinone oxidoreductase chain 6 Proteins 0.000 description 13
- 102100038625 NADH-ubiquinone oxidoreductase chain 1 Human genes 0.000 description 13
- 102100028386 NADH-ubiquinone oxidoreductase chain 6 Human genes 0.000 description 13
- 238000003119 immunoblot Methods 0.000 description 13
- 238000012762 unpaired Student’s t-test Methods 0.000 description 13
- 108700019146 Transgenes Proteins 0.000 description 12
- 241000700605 Viruses Species 0.000 description 12
- 230000007954 hypoxia Effects 0.000 description 12
- 238000005259 measurement Methods 0.000 description 12
- 230000002503 metabolic effect Effects 0.000 description 12
- 230000004614 tumor growth Effects 0.000 description 12
- 239000013607 AAV vector Substances 0.000 description 11
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 description 11
- 229960004359 iodixanol Drugs 0.000 description 11
- 101000598279 Homo sapiens NADH-ubiquinone oxidoreductase chain 5 Proteins 0.000 description 10
- 102100036971 NADH-ubiquinone oxidoreductase chain 5 Human genes 0.000 description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 10
- 208000035475 disorder Diseases 0.000 description 10
- 230000004064 dysfunction Effects 0.000 description 10
- 230000034659 glycolysis Effects 0.000 description 10
- BBLGCDSLCDDALX-LKGBESRRSA-N piericidin A Chemical compound COC=1NC(C\C=C(/C)C\C=C\C(\C)=C\[C@@H](C)[C@@H](O)C(\C)=C\C)=C(C)C(=O)C=1OC BBLGCDSLCDDALX-LKGBESRRSA-N 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108091064702 1 family Proteins 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 102000015782 Electron Transport Complex III Human genes 0.000 description 8
- 108010024882 Electron Transport Complex III Proteins 0.000 description 8
- 230000036760 body temperature Effects 0.000 description 8
- 230000001419 dependent effect Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 239000012091 fetal bovine serum Substances 0.000 description 8
- 210000005260 human cell Anatomy 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 230000002018 overexpression Effects 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 7
- 210000000234 capsid Anatomy 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 230000003959 neuroinflammation Effects 0.000 description 7
- UIFFUZWRFRDZJC-UHFFFAOYSA-N Antimycin A1 Natural products CC1OC(=O)C(CCCCCC)C(OC(=O)CC(C)C)C(C)OC(=O)C1NC(=O)C1=CC=CC(NC=O)=C1O UIFFUZWRFRDZJC-UHFFFAOYSA-N 0.000 description 6
- NQWZLRAORXLWDN-UHFFFAOYSA-N Antimycin-A Natural products CCCCCCC(=O)OC1C(C)OC(=O)C(NC(=O)c2ccc(NC=O)cc2O)C(C)OC(=O)C1CCCC NQWZLRAORXLWDN-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 108091026890 Coding region Proteins 0.000 description 6
- 101000970214 Homo sapiens NADH-ubiquinone oxidoreductase chain 3 Proteins 0.000 description 6
- 101001109052 Homo sapiens NADH-ubiquinone oxidoreductase chain 4 Proteins 0.000 description 6
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 102100021668 NADH-ubiquinone oxidoreductase chain 3 Human genes 0.000 description 6
- 102100021506 NADH-ubiquinone oxidoreductase chain 4 Human genes 0.000 description 6
- ZQLBCAUKNYXILZ-UGKGYDQZSA-N Piericidin A Natural products COc1nc(CC=C(/C)C=CCC(=C[C@H](C)[C@@H](O)C(=CC)C)C)c(C)c(O)c1OC ZQLBCAUKNYXILZ-UGKGYDQZSA-N 0.000 description 6
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 6
- UIFFUZWRFRDZJC-SBOOETFBSA-N antimycin A Chemical compound C[C@H]1OC(=O)[C@H](CCCCCC)[C@@H](OC(=O)CC(C)C)[C@H](C)OC(=O)[C@H]1NC(=O)C1=CC=CC(NC=O)=C1O UIFFUZWRFRDZJC-SBOOETFBSA-N 0.000 description 6
- PVEVXUMVNWSNIG-UHFFFAOYSA-N antimycin A3 Natural products CC1OC(=O)C(CCCC)C(OC(=O)CC(C)C)C(C)OC(=O)C1NC(=O)C1=CC=CC(NC=O)=C1O PVEVXUMVNWSNIG-UHFFFAOYSA-N 0.000 description 6
- 229940009098 aspartate Drugs 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 239000007789 gas Substances 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 230000036284 oxygen consumption Effects 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 229940076788 pyruvate Drugs 0.000 description 6
- 230000003938 response to stress Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 101000632748 Homo sapiens NADH-ubiquinone oxidoreductase chain 2 Proteins 0.000 description 5
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 5
- 102100028488 NADH-ubiquinone oxidoreductase chain 2 Human genes 0.000 description 5
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 5
- 210000000133 brain stem Anatomy 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 235000014113 dietary fatty acids Nutrition 0.000 description 5
- 229930195729 fatty acid Natural products 0.000 description 5
- 239000000194 fatty acid Substances 0.000 description 5
- 150000004665 fatty acids Chemical class 0.000 description 5
- 230000009395 genetic defect Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 230000004060 metabolic process Effects 0.000 description 5
- 208000012268 mitochondrial disease Diseases 0.000 description 5
- 238000001543 one-way ANOVA Methods 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 239000008194 pharmaceutical composition Substances 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 101150053137 AIF1 gene Proteins 0.000 description 4
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 4
- 208000005623 Carcinogenesis Diseases 0.000 description 4
- 102100030395 Glycerol-3-phosphate dehydrogenase, mitochondrial Human genes 0.000 description 4
- 108020005004 Guide RNA Proteins 0.000 description 4
- 101001009678 Homo sapiens Glycerol-3-phosphate dehydrogenase, mitochondrial Proteins 0.000 description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 4
- 229930182661 Piericidin Natural products 0.000 description 4
- 102100024276 Transcription factor SOX-3 Human genes 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- 229960001230 asparagine Drugs 0.000 description 4
- 235000009582 asparagine Nutrition 0.000 description 4
- 230000036952 cancer formation Effects 0.000 description 4
- 231100000504 carcinogenesis Toxicity 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 210000003618 cortical neuron Anatomy 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000012139 lysis buffer Substances 0.000 description 4
- 235000021073 macronutrients Nutrition 0.000 description 4
- 230000007659 motor function Effects 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 4
- 210000005253 yeast cell Anatomy 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101001109060 Homo sapiens NADH-ubiquinone oxidoreductase chain 4L Proteins 0.000 description 3
- 201000000639 Leber hereditary optic neuropathy Diseases 0.000 description 3
- 102100021452 NADH-ubiquinone oxidoreductase chain 4L Human genes 0.000 description 3
- 108091093105 Nuclear DNA Proteins 0.000 description 3
- 229920002873 Polyethylenimine Polymers 0.000 description 3
- 241000288906 Primates Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 3
- 235000011089 carbon dioxide Nutrition 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000000543 intermediate Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 230000037353 metabolic pathway Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 3
- 229940045145 uridine Drugs 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- 239000013603 viral vector Substances 0.000 description 3
- UFBJCMHMOXMLKC-UHFFFAOYSA-N 2,4-dinitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O UFBJCMHMOXMLKC-UHFFFAOYSA-N 0.000 description 2
- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical compound CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 description 2
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- 206010002660 Anoxia Diseases 0.000 description 2
- 241000976983 Anoxia Species 0.000 description 2
- 229930182536 Antimycin Natural products 0.000 description 2
- 238000011729 BALB/c nude mouse Methods 0.000 description 2
- 238000009010 Bradford assay Methods 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- ACTIUHUUMQJHFO-UHFFFAOYSA-N Coenzym Q10 Natural products COC1=C(OC)C(=O)C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UHFFFAOYSA-N 0.000 description 2
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 108010089760 Electron Transport Complex I Proteins 0.000 description 2
- 102000008013 Electron Transport Complex I Human genes 0.000 description 2
- WMBWREPUVVBILR-UHFFFAOYSA-N GCG Natural products C=1C(O)=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- DHCLVCXQIBBOPH-UHFFFAOYSA-N Glycerol 2-phosphate Chemical compound OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 2
- 108010041921 Glycerolphosphate Dehydrogenase Proteins 0.000 description 2
- 102000000587 Glycerolphosphate Dehydrogenase Human genes 0.000 description 2
- 101000633984 Homo sapiens Influenza virus NS1A-binding protein Proteins 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 2
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- 102220514259 NADH-ubiquinone oxidoreductase chain 6_M64I_mutation Human genes 0.000 description 2
- 102000002023 NADH:ubiquinone oxidoreductases Human genes 0.000 description 2
- 108050009313 NADH:ubiquinone oxidoreductases Proteins 0.000 description 2
- 206010061309 Neoplasm progression Diseases 0.000 description 2
- 206010060860 Neurological symptom Diseases 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 238000010162 Tukey test Methods 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- WDJHALXBUFZDSR-UHFFFAOYSA-M acetoacetate Chemical compound CC(=O)CC([O-])=O WDJHALXBUFZDSR-UHFFFAOYSA-M 0.000 description 2
- CUJRVFIICFDLGR-UHFFFAOYSA-N acetylacetonate Chemical compound CC(=O)[CH-]C(C)=O CUJRVFIICFDLGR-UHFFFAOYSA-N 0.000 description 2
- 230000006154 adenylylation Effects 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- 230000007953 anoxia Effects 0.000 description 2
- CQIUKKVOEOPUDV-IYSWYEEDSA-N antimycin Chemical compound OC1=C(C(O)=O)C(=O)C(C)=C2[C@H](C)[C@@H](C)OC=C21 CQIUKKVOEOPUDV-IYSWYEEDSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000013060 biological fluid Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000012054 celltiter-glo Methods 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 210000001638 cerebellum Anatomy 0.000 description 2
- 235000017471 coenzyme Q10 Nutrition 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- XJWSAJYUBXQQDR-UHFFFAOYSA-M dodecyltrimethylammonium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)C XJWSAJYUBXQQDR-UHFFFAOYSA-M 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000001146 hypoxic effect Effects 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 230000003050 macronutrient Effects 0.000 description 2
- 230000006540 mitochondrial respiration Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004723 neuronal vulnerability Effects 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 210000000956 olfactory bulb Anatomy 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- NPCOQXAVBJJZBQ-UHFFFAOYSA-N reduced coenzyme Q9 Natural products COC1=C(O)C(C)=C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)C(O)=C1OC NPCOQXAVBJJZBQ-UHFFFAOYSA-N 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 102220328180 rs1386396228 Human genes 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 229940054269 sodium pyruvate Drugs 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000009469 supplementation Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 230000005751 tumor progression Effects 0.000 description 2
- 238000007492 two-way ANOVA Methods 0.000 description 2
- 229940035936 ubiquinone Drugs 0.000 description 2
- 210000004440 vestibular nuclei Anatomy 0.000 description 2
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-3',4',5,7-Tetrahydroxy-2,3-trans-flavan-3-ol Natural products C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 description 1
- WMBWREPUVVBILR-WIYYLYMNSA-N (-)-Epigallocatechin-3-o-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-WIYYLYMNSA-N 0.000 description 1
- PFTAWBLQPZVEMU-UKRRQHHQSA-N (-)-epicatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-UKRRQHHQSA-N 0.000 description 1
- 229930013783 (-)-epicatechin Natural products 0.000 description 1
- 235000007355 (-)-epicatechin Nutrition 0.000 description 1
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 description 1
- 229930014124 (-)-epigallocatechin gallate Natural products 0.000 description 1
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- HWXBTNAVRSUOJR-VKHMYHEASA-N (S)-2-hydroxyglutaric acid Chemical compound OC(=O)[C@@H](O)CCC(O)=O HWXBTNAVRSUOJR-VKHMYHEASA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 1
- 230000002407 ATP formation Effects 0.000 description 1
- 102100021921 ATP synthase subunit a Human genes 0.000 description 1
- 239000012109 Alexa Fluor 568 Substances 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 101100079514 Arabidopsis thaliana NDA1 gene Proteins 0.000 description 1
- 206010003591 Ataxia Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 108091028075 Circular RNA Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 102100023580 Cyclic AMP-dependent transcription factor ATF-4 Human genes 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 229940124186 Dehydrogenase inhibitor Drugs 0.000 description 1
- LSHVYAFMTMFKBA-UHFFFAOYSA-N ECG Natural products C=1C=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 102100039844 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-T2 Human genes 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000753741 Homo sapiens ATP synthase subunit a Proteins 0.000 description 1
- 101000905743 Homo sapiens Cyclic AMP-dependent transcription factor ATF-4 Proteins 0.000 description 1
- 101000887521 Homo sapiens Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-T2 Proteins 0.000 description 1
- 101001032837 Homo sapiens Metabotropic glutamate receptor 6 Proteins 0.000 description 1
- 101000573300 Homo sapiens NADH dehydrogenase [ubiquinone] iron-sulfur protein 2, mitochondrial Proteins 0.000 description 1
- 101000601581 Homo sapiens NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial Proteins 0.000 description 1
- 101000633503 Homo sapiens Nuclear receptor subfamily 2 group E member 1 Proteins 0.000 description 1
- 101000595674 Homo sapiens Pituitary homeobox 3 Proteins 0.000 description 1
- 101001098880 Homo sapiens Purkinje cell protein 2 homolog Proteins 0.000 description 1
- 101000609335 Homo sapiens Pyrroline-5-carboxylate reductase 1, mitochondrial Proteins 0.000 description 1
- 101000710852 Homo sapiens Retinal cone rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 102100038300 Metabotropic glutamate receptor 6 Human genes 0.000 description 1
- 101100516303 Mus musculus Ndufs4 gene Proteins 0.000 description 1
- 102100026360 NADH dehydrogenase [ubiquinone] iron-sulfur protein 2, mitochondrial Human genes 0.000 description 1
- 102100037519 NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial Human genes 0.000 description 1
- 102220483353 NADH-ubiquinone oxidoreductase chain 1_A52T_mutation Human genes 0.000 description 1
- 102220558682 NADH-ubiquinone oxidoreductase chain 3_A47T_mutation Human genes 0.000 description 1
- 102220525889 NADH-ubiquinone oxidoreductase chain 4L_V65A_mutation Human genes 0.000 description 1
- 102220505278 NADH-ubiquinone oxidoreductase chain 5_F56L_mutation Human genes 0.000 description 1
- 102220513441 NADH-ubiquinone oxidoreductase chain 6_A72V_mutation Human genes 0.000 description 1
- 102220514242 NADH-ubiquinone oxidoreductase chain 6_G36S_mutation Human genes 0.000 description 1
- 102220514243 NADH-ubiquinone oxidoreductase chain 6_I26M_mutation Human genes 0.000 description 1
- 102220514239 NADH-ubiquinone oxidoreductase chain 6_I58V_mutation Human genes 0.000 description 1
- 102220514255 NADH-ubiquinone oxidoreductase chain 6_L60S_mutation Human genes 0.000 description 1
- 102220513445 NADH-ubiquinone oxidoreductase chain 6_M64V_mutation Human genes 0.000 description 1
- 102220514240 NADH-ubiquinone oxidoreductase chain 6_Y59C_mutation Human genes 0.000 description 1
- 102100029534 Nuclear receptor subfamily 2 group E member 1 Human genes 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- 208000001715 Osteoblastoma Diseases 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 1
- 102100036088 Pituitary homeobox 3 Human genes 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 102100038998 Purkinje cell protein 2 homolog Human genes 0.000 description 1
- 102000004518 Pyrroline-5-carboxylate reductase Human genes 0.000 description 1
- 102100039407 Pyrroline-5-carboxylate reductase 1, mitochondrial Human genes 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 108091007187 Reductases Proteins 0.000 description 1
- 102100033844 Retinal cone rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma Human genes 0.000 description 1
- 101100459997 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) NDI1 gene Proteins 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 108010034546 Serratia marcescens nuclease Proteins 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 102000001435 Synapsin Human genes 0.000 description 1
- 108050009621 Synapsin Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- OHVGNSMTLSKTGN-BTVCFUMJSA-N [C].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O Chemical compound [C].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O OHVGNSMTLSKTGN-BTVCFUMJSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000001668 ameliorated effect Effects 0.000 description 1
- 230000001028 anti-proliverative effect Effects 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000004227 basal ganglia Anatomy 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000001593 brown adipocyte Anatomy 0.000 description 1
- 102220352755 c.171C>G Human genes 0.000 description 1
- 230000006860 carbon metabolism Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013000 chemical inhibitor Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 210000003477 cochlea Anatomy 0.000 description 1
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 208000010934 demyelinating disease of central nervous system Diseases 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- XMOCLSLCDHWDHP-IUODEOHRSA-N epi-Gallocatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-IUODEOHRSA-N 0.000 description 1
- VFSWRBJYBQXUTE-UHFFFAOYSA-N epi-Gallocatechin 3-O-gallate Natural products Oc1ccc2C(=O)C(OC(=O)c3cc(O)c(O)c(O)c3)C(Oc2c1)c4cc(O)c(O)c(O)c4 VFSWRBJYBQXUTE-UHFFFAOYSA-N 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000002546 full scan Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 230000004153 glucose metabolism Effects 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 150000002313 glycerolipids Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 238000002013 hydrophilic interaction chromatography Methods 0.000 description 1
- 230000001969 hypertrophic effect Effects 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000000185 intracerebroventricular administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 208000006443 lactic acidosis Diseases 0.000 description 1
- 210000005240 left ventricle Anatomy 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000003137 locomotive effect Effects 0.000 description 1
- 230000006742 locomotor activity Effects 0.000 description 1
- 238000001325 log-rank test Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000005565 malate-aspartate shuttle Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004066 metabolic change Effects 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 210000000274 microglia Anatomy 0.000 description 1
- 230000002025 microglial effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 208000001749 optic atrophy Diseases 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000003305 oral gavage Methods 0.000 description 1
- SOWBFZRMHSNYGE-UHFFFAOYSA-N oxamic acid Chemical compound NC(=O)C(O)=O SOWBFZRMHSNYGE-UHFFFAOYSA-N 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 210000002243 primary neuron Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000009684 proliferation defect Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 230000006824 pyrimidine synthesis Effects 0.000 description 1
- 108020001898 pyrroline-5-carboxylate reductase Proteins 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035806 respiratory chain Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 102220065894 rs112180170 Human genes 0.000 description 1
- 102220005384 rs33939620 Human genes 0.000 description 1
- 102220074551 rs759993423 Human genes 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 239000002924 silencing RNA Substances 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000001931 thermography Methods 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 230000005100 tissue tropism Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000012250 transgenic expression Methods 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000013414 tumor xenograft model Methods 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229940054967 vanquish Drugs 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01008—Glycerol-3-phosphate dehydrogenase (NAD+) (1.1.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01094—Glycerol-3-phosphate dehydrogenase (NAD(P)+)(1.1.1.94)
Definitions
- the present application is directed to methods for treating mitochondrial complex I diseases or cancers by modulating glycerol-3-phosphate (Gro3P) biosynthesis. Also provided are agents and pharmaceutical compositions for use in modulating Gro3P biosynthesis.
- Gro3P glycerol-3-phosphate
- NADH reductive stress also regulates tumorigenesis under hypoxia.
- Hypoxia deprives the electron acceptor oxygen to inhibit the ETC and to cause NADH reductive stress.
- Hypoxic NADH reductive stress restricts the synthesis of aspartate and serine to limit tumor growth (Diehl et al., 2019; Garcia-Bermudez et al., 2018; Sullivan et al., 2018) , and also promotes the synthesis of L-2-hydroxyglutarate from ⁇ -ketoglutarate to regulate histone methylation levels and to inhibit glycolysis and electron transport (Intlekofer et al., 2015; Oldham et al., 2015) .
- Mitochondrial disease is a group of genetic disorders characterized by defective oxidative phosphorylation, a coupled process of electron transport and ATP synthesis (Gorman et al., 2016; Russell et al., 2020; Thompson et al., 2020) .
- mitochondrial diseases especially the severe pediatric forms, are often characterized by metabolic derangements indicative of NADH reductive stress, such as lactic acidosis (Katsyuba et al., 2020; Russell et al., 2020; Thompson Legault et al., 2015) .
- NDI1 the yeast NADH: ubiquinone oxidoreductase regenerates NAD + to extend the lifespan of brain NDUFS4-knockout mice (McElroy et al., 2020) , a model of mitochondrial complex I disorder (Kruse et al., 2008; Quintana et al., 2010) .
- NADH/NAD + redox homeostasis in mitochondrial disease and tumorigenesis
- our understanding of endogenous NAD + regeneration pathways remains limited. Identification of therapeutic targets involved in NAD + regeneration would be highly desirable. Development of agents or methods for modulating such therapeutic targets would also be needed for treating pathological conditions related to ETC dysfunctions.
- the present inventors revealed Gro3P biosynthesis as an NAD + regeneration pathway evolutionarily conserved in yeast, C. elegans, mouse, and human. It has been established that enhancing Gro3P synthesis could rescue mitochondrial complex I deficiency in cultured cells. Also, the inventors revealed lacking Gro3P biosynthesis as a metabolic characteristic that sensitizes neurons to complex I deficiency and demonstrated enhancing Gro3P biosynthesis alleviates neuroinflammation and extends lifespan in the Ndufs4 -/- mice (an animal model of complex I deficiency) .
- the present disclosure relates to a method for treating complex I deficiency in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent enhancing Gro3P biosynthesis.
- the agent enhancing Gro3P biosynthesis increases the enzymatic activity, e.g. specific activity of one or more of cytosolic Gro3P dehydrogenases (cGPDs) .
- the agent is an agonist of one or more of cGPDs.
- the agent enhancing Gro3P biosynthesis increases the level, e.g. mRNA level or protein level, of one or more cGPDs.
- the one or more cGPDs is human GPD1 and/or human GPD1L.
- the agent enhancing Gro3P biosynthesis comprises a nucleic acid molecule comprising a nucleotide sequence encoding the one or more cGPDs, encoding a variant of cGPD having the same or enhanced enzymatic activity or a nucleotide sequence enhancing the expression of one or more cGPDs.
- the agent enhancing Gro3P biosynthesis is a vector comprising the nucleic acid molecule of the above paragraph.
- the vector further comprises one or more regulatory sequences selected from a promoter, an enhancer, a polyadenylation sequence, and origin of replication.
- the promoter is operatively linked to the nucleic acid of the above paragraph.
- the promoter is a constitutive promoter.
- the promoter is a tissue-specific promoter, e.g. brain-specific promoter or ocular tissue-specific promoter, or cell-type-specific promoter e.g. neuron-specific promoter.
- the vector is a viral vector, such as an adenoviral vector, a retroviral vector or a recombinant adeno-associated virus (rAAV) vector.
- the rAAV vector comprises the nucleotide sequence encoding one or more cGPDs, encoding a variant of cGPD having the same or enhanced enzymatic activity or a nucleotide sequence enhancing the expression of one or more cGPDs, as well as at least one inverted terminal repeat.
- the rAAV vector comprises two ITRs.
- the AAV ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV. 7m8, AAV2tYF, AAV11, or AAV12 serotype ITR, preferably AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV. 7m8, or AAV2tYF serotype ITR, more preferably AAV2 ITR.
- the AAV vector comprises a nucleotide sequence as shown in SEQ ID NO: 31, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%or 99%sequence identity to SEQ ID NO: 31.
- the AAV vector is encapsidated in an AAV particle.
- the AAV particle comprises capsid proteins.
- at least one, preferably two or three of the capsid proteins (VP1, VP2 or VP3) is of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV. 7m8, AAV2tYF, AAV11, or AAV12 serotype.
- the serotype of capsid protein is suitable for brain delivery, e.g.
- the AAV vector comprises a capsid of AAV2, AAV4, AAV5, AAV8, AAV9, or AAVrh. 10 serotype.
- the complex I deficiency is selected from a group consisting of Leigh syndrome, Leber’s optical hereditary neuropathy (LHON) (also known as Leber optic atrophy) , Leber optic atrophy and dystonia, mitochondrial complex I deficiency (OMIM entry 252010) , leukodystrophy, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome, optic atrophy with demyelinating disease of central nervous system (CNS) and other neurodegenerative disorders.
- LHON optical hereditary neuropathy
- MELAS mitochondrial complex I deficiency
- CNS central nervous system
- the subject is mammal, e.g. rodent, or primate, preferably human.
- the subject has at least one mutation in mtDNA encoding a complex I subunit, e.g. ND1, ND2, ND3, ND4, ND5, ND6 or ND4L.
- a complex I subunit e.g. ND1, ND2, ND3, ND4, ND5, ND6 or ND4L.
- the subject has LHON and has at least one mutation selected from Table 2 or Table 3.
- the method of the first aspect has one or more of the following effects: increased C-I-independent OCR (complex I independent oxygen consumption rate) , decreased NADH/NAD + ratio, increased ATP level, decreased mitochondrial integrative stress response, reduced cell death, deceased neuroinflammation, decreased neurodegeneration, enhanced motor function, alleviated metabolic derangements, decreased glycolysis blockade, decreased ⁇ HB level, decreased lactate level, decreased alanine level, decreased ⁇ -hydroxybutyrate/acetoacetate ( ⁇ HB/AcAc) ratio, decreased lactate/pyruvate ratio, increased aspartate level, and increased asparagine level in tissue or plasm.
- C-I-independent OCR complex I independent oxygen consumption rate
- NADH/NAD + ratio decreased NADH/NAD + ratio
- increased ATP level decreased mitochondrial integrative stress response
- reduced cell death deceased neuroinflammation
- decreased neurodegeneration decreased motor function
- alleviated metabolic derangements decreased glycolysis blockade, decreased ⁇ HB level, decreased lactate level,
- the present disclosure provides a method for treating tumor in a subject by inhibiting Gro3P biosynthesis of tumor cells.
- the method comprises administering to the subject a therapeutically effective amount of an agent inhibiting Gro3P biosynthesis of tumor cells.
- the agent inhibiting Gro3P biosynthesis inhibits the activity of one or more cGPDs, e.g. human GPD1 and/or GPD1L.
- the agent inhibiting the activity of one or more cGPDs is a chemical compound, a polypeptide or a polynucleotide.
- the method further comprises inhibiting complex I, e.g. by administering to the subject an inhibitor of complex I.
- the inhibitor of complex I is selected from a group consisted of metformin, phenformin, BAY84-2243, CAI, ME-344, fenofibrate, and mIBG.
- the agent inhibiting Gro3P biosynthesis is delivered simultaneously with the inhibitor of complex I. In a preferred embodiment, the agent inhibiting Gro3P biosynthesis is delivered after the inhibitor of complex I.
- the inhibition of Gro3P biosynthesis and the inhibition of complex I achieves synergistic anti-tumor effect.
- the present disclosure provides a pharmaceutical combination of an agent inhibiting Gro3P biosynthesis and an inhibitor of complex I.
- the agent inhibiting Gro3P biosynthesis is an agent inhibiting or eliminating the activity of one or more cGPDs.
- the cGPD is human GPD1 and/or GPD1L.
- the inhibitor of complex I is selected from a group consisted of metformin, phenformin, BAY84-2243, CAI, ME-344, fenofibrate, and mIBG.
- the pharmaceutical combination is used for treating tumor.
- the pharmaceutical combination exhibits synergistic anti-tumor effect.
- FIG. 1 is a schematic of Gro3P metabolism and chemical inhibitors of the ETC complexes. Human ETC complexes are shown. Complex-II is absent for simplicity. MIM: mitochondrial inner membrane.
- FIG. 2 shows the results of immunoblot analysis of WT and cGPD-knockout (KO) 143B, HeLa, SNB-19, and A549 cells. GPD1 is not expressed in 143B cells.
- FIG. 3 shows the results of metabolite analysis of dihydroxyacetone phosphate (DHAP) and Gro3P of WT and cGPD-KO 143B cells treated with ETC inhibitors for 2 hours.
- Pier piericidin, complex I inhibitor; Anti: antimycin, complex III inhibitor.
- Data are mean ⁇ SD from three biological replicates.
- Statistics two-tailed unpaired Student’s t-test, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- FIG. 4 shows the results of metabolite analysis of the NADH/NAD + ratio of WT and cGPD-KO 143B, HeLa, SNB-19, and A549 cells treated with ETC inhibitors for 2 hours. Data are mean ⁇ SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- FIG. 5 shows the cell number and results of immunoblot analysis of WT and cGPD-KO 143B and HeLa cells complemented with GPD1L (1L) or vector (V) .
- ETC inhibitors were treated for 4-5 days.
- Met metformin, complex I inhibitor.
- Data are mean ⁇ SD from three biological replicates.
- Statistics two-tailed unpaired Student’s t-test, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- FIG. 6 shows the metabolite analysis of Gro3P and DHAP of WT and cGPD-KO 143B cells following 0.5%oxygen treatment for 6 hours. Data are mean ⁇ SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- FIG. 7 shows cell number of WT and cGPD-KO 143B cells treated with 0.2%oxygen for 5 days in the presence or absence of 3 mM pyruvate supplementation. Data are mean ⁇ SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- FIGS. 8A-B show the result of metabolite analysis of Gro3P and DHAP (A) and the NADH/NAD + ratio (B) of WT and cGPD-KO HeLa cells following 0.5%oxygen treatment for 6 hours. Data are mean and mean ⁇ SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test; **P ⁇ 0.01, ***P ⁇ 0.001.
- FIG. 9 shows the tumor growth curve of WT and cGPD-KO 143B-and HeLa-derived xenograft tumors daily treated with vehicle (water) or 1g/kg metformin. Data are mean ⁇ SEM. Statistics: one-way ANOVA with the Tukey-Kramer test, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- FIG. 10 shows the tumor sizes at the end point of WT and cGPD-KO 143B-and HeLa-derived xenograft tumors daily treated with vehicle (water) or 1g/kg metformin. Data are mean ⁇ SEM. Statistics: one-way ANOVA with the Tukey-Kramer test, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- FIG. 11 shows the photo images of WT and cGPD-KO HeLa-derived tumors daily treated with vehicle (water) or 1g/kg metformin.
- FIG. 12A-B show tumor growth curves (A) and tumor sizes at the end point (B) of the xenograft tumors derived from WT and cGPD-KO143B cells complemented with GPD1L (1L) or vector (V) which were daily treated with vehicle (water) or 1g/kg metformin. Data are mean ⁇ SEM. Statistics: one-way ANOVA with the Tukey-Kramer test, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- FIG. 13 shows the results of immunoblot analysis of 143B cells overexpressing WT cGPDs, enzymatically-inactive cGPDs (GPD1L G14A, GPD1L K206A and GPD1 G12A , GPD1 K204A ) , or the vector control.
- FIG. 14 shows the metabolite analysis of Gro3P and DHAP of the 143B cells as in FIG. 13.
- Cells were treated with ETC inhibitors for 2 hours. Data are mean ⁇ SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. ***P ⁇ 0.001. ns: not significant.
- FIG. 15 shows the complex I-dependent and complex I-independent oxygen consumption rate (OCRs) of the 143B cells as in FIG. 13. Data are mean ⁇ SD from six biological replicates. Statistics: two-tailed unpaired Student’s t-test. ***P ⁇ 0.001. ns: not significant.
- FIG. 16 shows the NADH/NAD + ratio of WT and GPD2-KO 143B cells overexpressing vector (V) or GPD1L (1L) .
- Cells were treated with ETC inhibitors for 2 hours. Data are mean ⁇ SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. ***P ⁇ 0.001. ns: not significant.
- FIGS. 17A-D show that enhancing Gro3P synthesis promotes Gro3P shuttle activity to alleviate metabolic and proliferative defects and to repress mitochondrial integrative stress response in Complex-I impaired Cells.
- FIG. 18 shows cell number of WT and GPD2-KO 143B cells treated with ETC inhibitors (Pier: 300 nM; Met: 2.5 mM; Anti: 100 nM) for 4-5 days. Representative images are shown. Data are mean ⁇ SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. ***P ⁇ 0.001. ns: not significant.
- FIG. 19 shows the results of immunoblot analysis of WT and NDUFS2-KO 143B cells overexpressing WT or enzymatically-inactive (GPD1L K206A and GPD1 K204A ) cGPDs or the vector control.
- FIG. 20 shows the cell number of the cells as in FIG. 19 after growing for 5 days. Data are mean ⁇ SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. ***P ⁇ 0.001. ns: not significant.
- FIG. 21 shows the schematic of the beneficial effects of NAD + regeneration by the Gro3P shuttle under complex I dysfunction.
- FIGS. 22A-D (A) Cell number of 143B cells overexpressing WT cGPDs, enzymatically-inactive cGPDs (GPD1 LG14A , GPD1L K206A and GPD1 G12A , GPD1 K204A ) , or the vector control. Cells were treated with ETC inhibitors (Pier: 300 nM; Anti: 100 nM) for 5 days. (B) Immunoblot analysis and cell number of HeLa, U2OS and MEF cells overexpressing GPD1L or the vector (V) control. Cells were treated with ETC inhibitors (Pier: 300 nM; Anti: 100 nM) for 5 days.
- C The NADH/NAD + ratio of 143B cells overexpressing GPD1L or vector.
- Cells were treated individually or in combination with Piericidin (300 nM) and oxamate (lactate dehydrogenase inhibitor, 40 mM) for 25 minutes.
- D Cell survival of 143B cells treated as in (C) for 10 hours. Representative images are shown. Cell viability was measured by trypan blue staining. Live cell number relative to total cell number in DMSO-treated cells were normalized as 100%. Data are mean ⁇ SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test, ***P ⁇ 0.001.
- FIG. 23 shows the results of immunoblot analysis of Gro3P metabolic enzymes in adult C57BL/6J mouse organs. 6 ⁇ g proteins of each sample were loaded for immunoblots.
- FIG. 24 shows the immunoblot analysis of cultured cortical neurons infected with AAV-PHP. eB encoding GFP or GPD1 for 10 days.
- FIGS. 25A-B show the NADH/NAD + ratio (A) , and ATP level (B) of neurons as in FIG. 24 treated with ETC inhibitors for 2 hours. Ratio (A) and ATP level (B) in DMSO-treated non-infected neurons were normalized as 1.
- FIG. 26 shows the measurement of complex I-dependent and complex I-independent OCRs of neurons as in FIG. 24.
- DNP 2, 4-dinitrophenol, 200 ⁇ M
- FIG. 26 shows the measurement of complex I-dependent and complex I-independent OCRs of neurons as in FIG. 24.
- DNP 2, 4-dinitrophenol, 200 ⁇ M
- FIG. 27 Left: Schematic of delivering AAV-PHP. eB expressing GFP or GPD1 to the Ndufs4 -/- mice via the retro-orbital injection to increase GPD1 level in the brain; Right: Immunoblot analysis of GPD1 in mouse organs one month after AAV delivery.
- FIG. 28 shows the immunoblot analysis of Gro3P metabolic enzymes in brain regions collected 3 weeks after AAV transduction to express GPD1 or GPD1 K204A . Samples from two mouse brains for each treatment are shown.
- FIG. 29 shows the body weight of the indicated mice.
- KO + X Ndufs4 -/- mice injected with AAV-PHP.
- eB expressing gene X.
- Data are mean ⁇ SD.
- FIG. 30 shows the lifespan of the indicated mice.
- KO + X Ndufs4 -/- mice injected with AAV-PHP.
- eB expressing gene X.
- FIGS. 31A-B show the metabolite analysis of brainstem glycolysis intermediates (A) and brainstem and plasma metabolites sensitive to the NADH/NAD + ratio (B) of the WT, Ndufs4 -/- , and Ndufs4 -/- mice transduced with AAV expressing GFP or GPD1.
- KO Ndufs4 -/- .
- FIG. 32 shows the representative images of Iba1 immunofluorescence staining at postnatal day 47-50 (P47-50) of the indicated mice.
- OB olfactory bulb
- CB cerebellum
- IO inferior olive
- VN vestibular nuclei.
- Scale bar 1 mm.
- FIG. 33 shows the quantification results of Iba1 fluorescence intensity in different brain areas of the indicated mice.
- FIG. 35 shows the body temperature of the indicated mice. Data are mean ⁇ SEM. Statistics: two-way ANOVA with the Tukey-Kramer test; **P ⁇ 0.01, ***P ⁇ 0.001.
- the wording “comprise” and variations thereof such as “comprises” and “comprising” will be understood to imply the inclusion of a stated element, e.g. a component, a property, a step or a group thereof, but not the exclusion of any other elements, e.g. components, properties and steps.
- the term “comprise” or any variation thereof can be substituted with the term “contain” , “include” or sometimes “have” or equivalent variation thereof.
- the wording “comprise” also includes the scenario of “consisting of” .
- treat includes cure or at least alleviate the symptoms.
- terapéuticaally effective amount refers to the amount of an agent that, when administered to a subject for treating a disease, or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or symptom.
- the “therapeutically effective amount” can vary with the agent, the disease, disorder, and/or symptoms of the disease or disorder, severity of the disease, disorder, and/or symptoms of the disease or disorder, the age of the subject to be treated, and/or the weight of the subject to be treated.
- the “therapeutically effective amount” refers to the total amount of the combination objects for the effective treatment of a disease, a disorder or a condition.
- the term “pharmaceutical composition” refers to a composition suitable for delivering to a subject.
- the pharmaceutical composition of the present disclosure comprises an agent modulating Gro3P biosynthesis and a pharmaceutically acceptable excipient.
- the pharmaceutical composition comprises a nucleic acid or a vector encoding cGPD.
- the pharmaceutical composition comprises an inhibitor of Gro3P biosynthesis.
- Conventional pharmaceutically acceptable excipients are known in the art.
- subject it refers to a eukaryote, such as yeast, C. elegans, or an animal, preferably a mammal, e.g., a rodent, such as a mouse or a rat, a primate, preferably a higher primate, such as a human.
- a eukaryote such as yeast, C. elegans
- an animal preferably a mammal, e.g., a rodent, such as a mouse or a rat
- a primate preferably a higher primate, such as a human.
- administration when applied to a subject, e.g. an animal, including human, or to cells, tissue, organ, or biological fluid, mean contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the subject, cell, tissue, organ, or biological fluid.
- Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell.
- administration and treatment also include in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell.
- isolated nucleic acid or “isolated polynucleotide” , it means a DNA or RNA which is removed from all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature.
- An isolated nucleic acid molecule "comprising" a specific nucleotide sequence may include, in addition to the specified sequence, operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences. Due to the codon degeneracy, one skilled in the art can understand that a specific amino acid sequence can be coded by different nucleotide sequences.
- promoter is well known in the art and refers to a regulatory sequence directing the transcription of a gene. Promoters can be categorized into constitutive promoters and inducible promoters. A constitutive promoter is active at all circumstances in vivo. An inducible promoter is only active under certain condition, e.g. in the presence of stimulus, within a certain type of tissue (tissue-specific promoter) or cell (cell-type-specific promoter) , or at a certain developmental stage (developmental stage specific promoter) .
- operatively linked refers to the association of two components in a single nucleic acid stretch so that one can affect the function of the other.
- transgene refers to a nucleic acid to be transferred or introduced into a host, for example, a cell or organism.
- a transgene can be a coding sequence of a functional protein or fragment thereof, e.g. a coding sequence of GPD1 or GPD1L.
- the cellular NADH/NAD + redox balance is fundamental to metabolism (Hosios and Vander Heiden, 2018; Katsyuba et al., 2020; Xiao and Loscalzo, 2020) .
- the oxidized form NAD + functions as an electron acceptor in diverse metabolic pathways such as glycolysis, fatty acid oxidation, amino acid degradation, citric acid cycle, serine synthesis, and one carbon metabolism (Ducker and Rabinowitz, 2017; Hosios and Vander Heiden, 2018; Katsyuba et al., 2020; Lunt and Vander Heiden, 2011; Xiao and Loscalzo, 2020) .
- the continuous operation of these metabolic pathways requires NAD + regeneration by mitochondrial respiration.
- the ETC oxidizes NADH and transfers electrons to oxygen, the ultimate electron acceptor.
- the matrix arm of ADH: ubiquinone oxidoreductase or complex I is the entry point for electrons from matrix NADH into the ETC. Because mitochondrial inner membrane is impermeable to NADH, electrons from cytosolic NADH are shuttled to mitochondrial matrix through the malate-aspartate shuttle or directly transferred to the ETC through the Gro3P shuttle.
- ETC inhibition resulting from either hypoxia or genetic/pharmacological disruption of ETC function, impairs NADH oxidation and elevates the mitochondrial and cytosolic NADH/NAD + ratios, a condition termed NADH reductive stress.
- Gro3P biosynthesis is a side pathway of glycolysis. It oxidizes NADH by converting glycolysis intermediate dihydroxyacetone phosphate (DHAP) to Gro3P, a reversible reaction catalyzed by NAD + -dependent cytosolic Gro3P dehydrogenases (cGPDs) (Ansell et al., 1997; Kelly et al., 2011) . Gro3P is subsequently metabolized in three ways: 1. Condensation with acyl-CoA to generate glycerolipids (Yu et al., 2018) ; 2. dephosphorylation to generate glycerol (Mugabo et al., 2016; Nevoigt and Stahl, 1997) ; 3.
- Gro3P biosynthesis supports yeast growth under anoxia (Ansell et al., 1997) .
- cGPDs are heterogeneously expressed in mouse tissues, with highest expression in brown adipocytes and lowest expression in cerebral cortex (Ratner et al., 1981) .
- a BALB/c subline of mice lacking cGPD activity exhibited elevated lactate/pyruvate ratio in skeletal muscle (MacDonald and Marshall, 2000) .
- Gro3P synthesis was identified as a major NAD + regeneration pathway in yeast and human cells for the first time by the present inventors, which further explains the findings in previous studies as mentioned above.
- GPD glycerol 3-phosphate dehydrogenase
- cGPD cytoplasmic GPD
- mitochondrial GPD located in mitochondrial inner membrane.
- cGPD is encoded by two genes, GPD1 and GPD1L, which share around 72%sequence identity with each other.
- cGPDs catalyze a reversible redox reaction of dihydroxyacetone phosphate to glycerol 3-phosphate.
- mGPD is encoded by GPD2 and catalyzes an irreversible reaction of glycerol 3-phosphate to dihydroxyacetone phosphate.
- one or more cGPDs means one or more cytoplasmic GPD, e.g. GPD1 and/or GPD1L of human.
- complex I diseases are treated by increasing the level or activity of cGPD.
- tumors are treated by inhibiting cGPD.
- the present inventors have surprisingly discovered that inhibition of cGPD produces an enhanced inhibitory effect on tumor growth. Further, when cGPD inhibition is combined with inhibition of complex I, a synergistic inhibitory effect on tumor growth can be observed.
- the present disclosure provides a method of treating, alleviating or preventing tumor in a subject, comprising administering to the subject a pharmaceutically effective amount of an inhibitor of GPD1 and/or GPD1L.
- complex I refers to respiratory complex I, or NADH: ubiquinone oxidoreductase, which couples electron transfer from NADH to ubiquinone with transmembrane proton pumping. Being the largest respiratory complex, complex I is consisted of 44 subunits coded by both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) .
- mtDNA mitochondrial DNA
- nDNA nuclear DNA
- complex I deficiencies which is also known as mitochondrial complex I deficiencies.
- complex I deficiency and “complex I disease” is interchangeable and should be understood in a broad way to include any disorder caused by any defects of complex I, i.e. any mitochondrial complex I linked disease.
- the present disclosure provides a method for treating complex I deficiency by enhancing Gro3P biosynthesis, specifically by delivering an agent increasing the enzymatic activity or level of one or more cGPDs, e.g. human GPD1 and/or GPD1L.
- the increase of enzymatic activity can be indicated by increase of specific activity of cGPD.
- the activity of cGPDs can be enhanced by an agonist of cGPD.
- cGPD level can be achieved by multiple ways known to those skilled in the art, such as increasing gene copy number or improving protein expression.
- the complex I deficiency which can be treated by the present method has one or more diseases selected from a group consisting of Leigh syndrome, Leber’s optical hereditary neuropathy (LHON) (also known as Leber optic atrophy) , Leber optic atrophy and dystonia, mitochondrial complex I deficiency (OMIM entry 252010) , leukodystrophy, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome, optic atrophy with demyelinating disease of CNS and other neurodegenerative disorders.
- LHON optical hereditary neuropathy
- MELAS mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes
- optic atrophy with demyelinating disease of CNS and other neurodegenerative disorders One skilled in the art would understand that additional complex I deficiencies may be discovered in the future, which can also be treated by the method of the present disclosure.
- Complex I deficiency caused by nDNA can be treated by gene replacement therapy.
- mtDNA mutations cannot be readily remedied in the same way by delivering a transgene encoding complex I component to make compensation. Therefore, the method of the present disclosure is especially suitable for treating complex I deficiencies mainly caused by mtDNA mutations.
- the subject to be treated by the present method has genetic defects in mtDNA encoded complex I subunits, such as ND1, ND2, ND3, ND4, ND4L, ND5, and/or ND6.
- the subject has one or more of Leigh syndrome, LHON, or MELAS.
- Table 1 below provides an overview of genetic defects in mtDNA encoded complex I subunits and resulting phenotypes (R. J. Rodenburg, 2016, supra) .
- mutations in mtDNA encoded complex I subunits MTND1, MTND2, MTND3, MTND4, MTND5, MTND6 can also cause Leigh Syndrome or Leigh- like Syndrome. Mutations MTND3 and MTND5 are the most frequent mtDNA causes of Leigh Syndrome.
- Table 2 19 primary LHON mutations, the first 3 mutations listed (in boldface) represent approximately 95%of all cases. The remaining mutations are listed in nucleotide order.
- the subject to be treated by the present method has one or more mtDNA mutations selected from those listed in Table 2 or Table 3.
- the therapeutic effect in treating complex I deficiency achieved by the method of the present disclosure may be shown as or determined by one or more of the followings: an increased C-I-independent OCR (complex I independent oxygen consumption rate) , a decreased NADH/NAD + ratio, an increased ATP level, decreased mitochondrial integrative stress response, reduced cell death, deceased neuroinflammation, decreased neurodegeneration, enhanced motor function, alleviated metabolic derangements, including decreased glycolysis blockade, decreased ⁇ HB level, decreased lactate level, decreased alanine level, decreased ⁇ -hydroxybutyrate/acetoacetate ( ⁇ HB/AcAc) ratio, decreased lactate/pyruvate ratio, increased aspartate level, and increased asparagine level in tissue or plasma. Measurement of therapeutic effects may vary depending on the phenotype of complex I disease and can be determined by one skilled in the art.
- the complex I deficiency is treated by enhancing Gro3P biosynthesis, specifically by increasing the level of one or more cGPDs, namely GPD1 and GPD1L, or/and by increasing the enzymatic activity of cGPDs.
- the increase of cGPD level can be achieved by delivering a transgene encoding GPD1 and/or a transgene encoding GPD1L.
- the coding sequence of the cGPDs can be a wild-type nucleotide sequence or can be subjected to codon optimization so as to enhance the expression efficiency in a certain subject, e.g. human.
- the increase of enzymatic activity of cGPD can be achieved by enabling the expression of a mutated version of cGPD with enhanced enzymatic activity or by an agonist of one or more cGPDs.
- the transgene encoding GPD1 or the transgene encoding GPD1L is comprised in a viral vector.
- the viral vector is a lentiviral vector, an adenoviral vector or an adeno-associated virus (AAV) vector.
- the lentiviral vector includes envelope proteins.
- the rAAV vector of the present disclosure can comprise elements of any serotype known in the art or discovered in the future, or variant thereof.
- the vector genome of the rAAV is AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV. 7m8, AAV2tYF serotype.
- the rAAV vector comprises one or more ITRs of AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV. 7m8, AAV2tYF serotype.
- the AAV vector is self-complementary AAV (scAAV) or single stranded AAV (ssAAV) .
- the AAV vector comprises one or more ITRs from any AAV serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AV10, AAVrh10, AAV. 7m8, AAV2tYF, AAV-DJ, AAV-DJ/8, AAV11, AAV12, or variant thereof.
- the AAV vector comprises two AAV2 ITRs flanking the coding sequence of GPD1 or GPD1L.
- the transgene encoding GPD1 or GPD1L is under the control of a constitutive promoter.
- the promoter can be EF1-alpha promoter, CAG promoter, PGK promoter or CMV promoter.
- the transgene encoding GPD1 or GPD1L is under the control of a tissue-specific or cell-type-specific promoter.
- the promoter can be a neuron-specific promoter, e.g. synapsin promoter; a brain-specific promoter; or an ocular tissue-specific promoter, e.g. GRM6 promoter, PCP2 promoter, GNGT2 promoter, PDE6H promoter, PITX3 promoter, or NR2E1 promoter.
- the AAV vector comprises an adenylation signal sequence.
- the adenylation signal sequence can be bGH polyA, SV40 polyA or WPRE-SV40 poly A, preferably bGH polyA.
- the vector genome is packaged by one or more capsid.
- the capsid proteins of the present disclosure can be capsid of any AAV serotype known in the art or discovered in the future.
- the capsid protein can be selected from a group consisting of but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV. 7m8, AAV2tYF, AAV11, or AAV12 serotype.
- the capsid can be derived from the same or different AAV serotype as the ITR.
- the capsid proteins show tissue tropism for cells of the CNS (e.g. brain) or eye.
- the AAV vector comprises a capsid from of AAV2, AAV4, AAV5, AAV8, AAV9, or AAVrh. 10.
- a vector encoding the AAV replication and/or encapsidation protein and a vector encoding helper functions, such as p. Helper vectors are used along with the vector comprising the nucleotide encoding target sequence flanked by ITR to infect host cells.
- the transgene encoding GPD1 or GPD1L is constructed into closed-ended DNA.
- the transgene encoding GPD1 or GPD1L is made into a circular RNA.
- the transgene encoding GPD1 or GPD1L is comprised in a lipid nano particle (LNP) .
- LNP lipid nano particle
- the present disclosure provides a method for treating tumor or prevent tumor progression by inhibiting Gro3P biosynthesis of tumor cells.
- the method includes inhibition of both Gro3P biosynthesis and Complex I.
- cancer or “tumor” herein mean or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
- the cancer can be solid tumors, such as sarcomas, carcinomas or lymphomas, or cancers in the blood.
- the cancer expresses one or more of cGPDs.
- the method of the present disclosure is not limited to the use in treating solid tumor, especially when the inhibition of Gro3P biosynthesis is combined with the inhibition of Complex I.
- Inhibition of Complex I creates an NADH reductive stress in cancer microenvironment.
- Inhibition of Gro3P biosynthesis in the cells under such NADH reductive stress retards the cell growth and in turn slows down the progression of tumor.
- inhibition of Gro3P biosynthesis When inhibition of Gro3P biosynthesis is used in combination with inhibition of Complex I, they can be conducted simultaneously or sequentially. In a preferred embodiment, the inhibition of Gro3P can be conducted after the inhibition of Complex I.
- the inhibition of Gro3P biosynthesis is achieved by delivering of an agent, e.g. a compound, a polypeptide, or an isolated nucleotide inhibiting the one or more of cGPDs, e.g. human GPD1 or GPD1L.
- an agent e.g. a compound, a polypeptide, or an isolated nucleotide inhibiting the one or more of cGPDs, e.g. human GPD1 or GPD1L.
- the tumor or cancer to be treated expresses one or more of cGPDs.
- the agent inhibiting one or more of cGPDs is a chemical compound, including but not limited to (-) -epicatechin, (-) -epicatechin-3-gallate, (-) -epigallocatechin, or (-) -epigallocatechin-3-gallate.
- the agent inhibiting one or more of cGPDs is a polypeptide, e.g. an antibody or a fragment thereof specifically binding to one or more of cGPDs.
- the agent inhibiting one or more of cGPDs is a nucleotide-based inhibitor, including but not limited to siRNA, shRNA, miRNA, gRNA, or antisense oligonucleotides.
- the inhibition of Gro3P biosynthesis is used in combination with inhibition of complex I to achieve a synergistic anti-tumor effect.
- the inhibition of complex I is achieved by administering to the subject a therapeutically effective amount of complex I inhibitor.
- a complex I inhibitor can be a compound known to have inhibitory effect on complex I.
- the complex I inhibitor is a compound suitable for pharmaceutical use, e.g. a compound which has been proven to be safe in a clinical trial or pre-clinical studies.
- the complex I inhibitor can be selected from a group consisting of metformin, phenformin, BAY84-2243, CAI, ME-344, fenofibrate, and mIBG.
- This example describes the process of identifying Gro3P synthesis as candidate NAD + regeneration pathways under ETC dysfunction.
- a total number of 175 genes encoding NAD + -dependent and NAD (P) + dual-specific dehydrogenases/reductases in the human genome were retrieved from the KEGG database.
- 81 genes encode enzymes catalyzing 39 reactions of macronutrient metabolism (glucose, amino acids, fatty acids, nucleic acids, ketone bodies, alcohols and aldehydes) .
- macronutrient metabolism glucose, amino acids, fatty acids, nucleic acids, ketone bodies, alcohols and aldehydes
- the inventors thus focused on macronutrient metabolism because macronutrients are the most abundant substrates available in the cell, culture media, and circulating blood.
- eight candidate NAD + regeneration pathways including Gro3P synthesis (glucose metabolism) , four amino acid metabolic pathways, transhydrogenation reaction, ketone body metabolism and fatty acids desaturation were identified.
- yeast NAD + -dependent and NAD (P) + dual-specific enzymes were retrieved to examine the conservation of the eight human candidate pathways. It turned out three of them are conserved: Gro3P synthesis, proline synthesis, and fatty acid desaturation.
- cGPDs GPD1L and GPD1 were knocked out in four human cancer cell lines, 143B, Hela, SNB-19 and A549 (Fig. 2) . It was found that cGPD knockout abolished Gro3P synthesis (Fig. 3) , and significantly elevated the NADH/NAD + ratio under chemical ETC inhibition in all the four cell lines (Fig. 4) .
- Gro3P synthesis provides an indispensable NAD + regeneration activity to maintain viability and proliferation of ETC-deficient cells.
- This example investigates the correlation between Gro3P synthesis and tumor growth.
- hypoxia is a common feature of most malignant tumors. It was found that hypoxia promoted Gro3P synthesis in 143B cells (Fig. 6) . cGPD-KO 143B cells proliferated significantly slower than WT cells under hypoxia treatment, which was rescued by pyruvate supplementation (Fig. 7) . Hypoxia also promoted Gro3P synthesis in HeLa cells (Fig. 8A) . cGPD-KO HeLa cells exhibited higher NADH/NAD + ratio as compared to WT cells under hypoxia (Fig. 8B) .
- Mouse brain has very low protein levels of cGPDs (Fig. 23) .
- Examination of the Genotype-Tissue Expression (GTEx) database also supported the low transcript levels of cGPDs in human brain, especially in brain areas sensitive to complex I deficiency, such as basal ganglia (Fassone and Rahman, 2012) .
- cGPDs are almost undetectable in cultured mouse cortical neurons (Fig. 24) .
- ETC inhibition these neurons had both a high NADH/NAD + ratio (> threefold increase) (Fig. 25A) and a low ATP level ( ⁇ 30%of WT level) (Fig. 25B) , similar to what was previously observed in cGPD-KO cancer cells (Fig. 4) .
- This example determines whether enhancing cGPD expression is applicable to rescuing complex I deficiency in vivo.
- the Ndufs4 -/- mice was used, which progressively develop fatal neuroinflammation and neurodegeneration, recapitulating the characteristics of human Leigh syndrome (Jain et al., 2016; Johnson et al., 2013; Kruse et al., 2008; Quintana et al., 2010) .
- the inventors employed the recently described AAV-PHP. eB vector (Chan et al., 2017) that can extensively transduce both neurons and glia to express target genes in the brain.
- AAV-mediated delivery of NDUFS4 to the Ndufs4 -/- mice rescued the lifespan of these mice (Fig. 30) , confirming the validity of our approach.
- AAVs expressing GPD1, GFP, or the GPD1 K204A inactive-mutant were constructed and delivered to the Ndufs4 -/- mice by retro-orbital injection so that the AAV particles can diffuse in the whole mouse body.
- the enrichment of AAV virus in mouse brain was achieved by the brain high affinity capsid vector PHP. eB.
- mice delivered with AAV-GPD1 and mice delivered with AAV-GPD1 K204A brain GPD1 was increased to a comparable level as hepatic GPD1 (Fig. 27 and Fig. 28) .
- GPD1, GFP, and GPD1 K204A did not rescue body weight loss of the Ndufs4 -/- mice (Fig. 29) .
- GPD1 not GFP or GPD1 K204A significantly extended the lifespan of the Ndufs4 -/- mice by 44%, from 58.3 ⁇ 1.92 to 84.0 ⁇ 3.65 days (Fig. 30) .
- Ndufs4 -/- mice exhibited increased accumulation of glycolysis intermediates upstream of the NADH-producing step (F6P, G3P, and DHAP) (Fig. 31A) , indicating glycolysis blockade.
- the Ndufs4 -/- mice also exhibited increased ⁇ HB and lactate levels and decreased aspartate levels in the brainstem, as well as increased plasma ⁇ HB levels (Fig. 31B) , indicating elevated brainstem NADH/NAD + ratio.
- Overexpression of GPD1 but not GFP significantly alleviated these metabolic derangements (Figs. 31 A-B) .
- Ndufs4 -/- mice exhibited strong neuroinflammation manifested by the greatly-increased number and hypertrophic morphology of Iba1-positive microglia within multiple brain regions (Fig. 32 and Fig. 33) .
- Ndufs4 -/- mice progressively developed ataxia and locomotor defects (Fig. 34) and suffered from reduced body temperature (Fig. 35) .
- Expression of GPD1 but not GFP ameliorated most of the neuroinflammation and partially prevented the decline of motor function and the reduction in body temperature (Figs. 32-35) .
- 143B, HeLa, U2OS, SNB-19, A549 and MEF cells were cultured in DMEM without pyruvate supplemented with 10%fetal bovine serum (FBS) , 1%penicillin-streptomycin and 2 mM L-glutamine. All cells were incubated at 37 °Cwith 5%CO2.
- FBS fetal bovine serum
- Cortex from anesthetized P0 mouse was cut into small pieces and digested with 0.25%trypsin at 37 °C for 8 minutes and stopped by 3 volumes of DMEM supplemented with 10%FBS. Digested cortical pieces were dissociated by gentle pipetting for 5 times, rested for 3 minutes to precipitate undigested large pieces. Supernatants were transferred through a 40 ⁇ m cell strainer to remove cell aggregates. Dissociated neurons were cultured at 0.5-1 ⁇ 10 6 cells per well (24 well plate) in dish or coverslips pre-coated with poly-D-lysine in DMEM supplemented with 10%FBS. Medium was switched to Neurobasal medium supplemented with 1 mM sodium pyruvate, 2%B27 and 1%GlutaMAX 12 hours after attachment. Culture was maintained by changing medium every 3 days.
- Ndufs4 -/- mice were purchased from The Jackson Laboratory.
- BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology. Mice were maintained under a 12-hours light/dark cycle and on a standard chow diet at the specific pathogen-free (SPF) facility at the National Institute of Biological Sciences, Beijing. All mouse experiments were carried out following the national guidelines for housing and care of laboratory animals (Ministry of Health, China) and the protocol was in compliance with institutional regulations after review and approval by the Institutional Animal Care and Use Committee at National Institute of Biological Sciences, Beijing.
- SPF pathogen-free
- Lentiviruses were produced by co-transfection of HEK293T with cDNA containing lentiviral vectors FUIPW, lentiviral packaging vectors psPAX2 and pMD2. G at a ratio of 5: 3: 2.
- Virus containing supernatants were collected and filtered through 0.45 ⁇ m filters 48 hours post-transfection and stored at -80 °C.
- targeted cells were infected with virus-containing supernatants for 48 hours in the presence of 8 ⁇ g/ml polybrene. Infected cells were selected by 1-3 ⁇ g/ml puromycin. cDNA expression was validated by immunoblotting.
- gRNAs Two guide RNAs
- PX458 was then transfected to targeted cells using the Polyethylenimine (PEI) transfection reagent.
- PEI Polyethylenimine
- the seeding densities used allowed exponential proliferation for 4-5 days and final cell counts were measured 4-5 days after treatment.
- Cells were counted using TC20 automated cell counter. Relative cell number was determined by cell number of inhibitor treatment divided by cell number of vehicle treatment.
- Lysis buffer contains 20 mM Tris–HCl (pH 7.5) , 1 mM EDTA, 1%Triton X-100, 150 mM NaCl, 0.1%SDS, 2.5 mM sodium pyrophosphate, 1 mM ⁇ -glycerophosphate, a protease inhibitor cocktail and a phosphatase inhibitor. Human cells were scraped and pellets were collected. Tissues from anesthetized mice were dissected out and snap-frozen in liquid nitrogen. Tissues were weighted and cryohomogenized on dry ice.
- the NADH/NAD + ratio was measured by modification of manufacturer instructions for NAD + /NADH Glo Assay kit.
- the lysis buffer is 1%Dodecyltrimethylammonium bromide (DTAB) in 0.2 N NaOH.
- NADH/NAD + ratio in human cell lines, cells were plated in 96-well plates at 16,000 cells per well. The following day cells were incubated with DMSO or ETC inhibitors (piericidin A: 10 ⁇ M; antimycin A, 10 ⁇ M) in growth medium for 2 hours. Media were aspirated and cells were extracted by adding 125 ⁇ l ice cold lysis buffer (diluted 1: 1 with PBS) into the plate and incubated 3 minutes on an orbital shaker to ensure homogenous cell lysis. 50 ⁇ l supernatants were used to measure NADH and NAD + respectively.
- NADH NADH
- 50 ⁇ l of samples were moved to empty wells of 96 well plate and incubated at 60 °C for 15 minutes to degrade NAD + .
- NAD + 50 ⁇ l of the samples were moved to empty wells of 96 well plate, mixed with 25 ⁇ l 0.4 N HCl and incubated at 60 °C for 15 minutes to degrade NADH. Following incubations, samples were allowed to equilibrate for 10 minutes at room temperature and quenched by neutralizing with 25 ⁇ l 0.5 M Tris (NAD + ) or 50 ⁇ l of HCl/Tris (NADH) . 20 ⁇ l samples were mixed with 20 ⁇ l detection reagents in a 384 well plate. Luminescence signals were recorded after 30 minutes incubation at room temperature.
- 143B cells (2 million per sample) cultured in 6-cm dish were incubated with DMSO, 10 ⁇ M Piericidin A or 10 ⁇ M Antimycin A for 2 hours. After washing with PBS twice, cells were extracted by -80 °C pre-chilled 80%methanol for the cellular steady metabolite measurements. The remaining cell pellets were dissolved in 0.1 M KOH by shaking at 4 °C overnight. Protein concentration was measured by Bradford assay kit. The metabolic data was normalized to protein concentration.
- mice were anesthetized during tissue collection. Tissues were dissected out and snap-frozen in liquid nitrogen. Tissues were weighed and cryohomogenized on dry ice and the homogenized frozen powders were extracted by ice-cold 4/4/2 acetonitrile/methanol/water according to the weight (100 ul/10 mg) . Samples were incubated on ice for 30 minutes. During incubation, samples were mixed for 1 minute every 10 minutes. After centrifugation at 15,000 rpm for 15 minutes at 4 °C, 500 ⁇ l supernatants from each sample were transferred to a new tube and were lyophilized by Speedvac to dry pellet at 30 °C. Dried samples were stored at -80 °C.
- LC-MS analysis was performed by a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer.
- Authentic reference standard compounds were purchased from Sigma-Aldrich to confirm the retention time of each targeted metabolites. Dried samples were resuspended in 100 ⁇ l 50%methanol and filtered through 0.45 ⁇ m filters. 6 ⁇ l of each sample was injected for analysis.
- Chromatographic separation was performed on a Merck ZIC-HILIC column (2.1 ⁇ 100 mm, 3.5 ⁇ m) at a flow rate of 0.5 ml/min and maintained at 40 °C, with the mobile phases of 10 mM ammonium acetate in ACN/water 5/95 (A) and 10 mM ammonium acetate in ACN/water 95/5 (B) .
- the following gradient was applied: 0-5 min, 99%B; 5-20 min, 99-20%B; 20-21 min, 20-99%B; 21-25 min, 99%B.
- Full-scan mass spectra were acquired in the range of m/z 66.7 to 1,000 with the following ESI source settings: spray voltage: 3.5 kV, auxiliary gas heater temperature: 380 °C, capillary temperature: 320 °C, sheath gas flow rate: 35 units, auxiliary gas flow: 10 units in the positive mode; Spray voltage: 2.5 kV, auxiliary gas heater temperature: 380 °C, capillary temperature: 320 °C, sheath gas flow rate: 30 units, auxiliary gas flow: 10 units in the negative mode.
- MS1 scan parameters included resolution 60,000, AGC target 3e6, and maximum injection time 200 ms. Data processing was performed with Thermo Xcalibur software (4.2) . Results were manually validated and compounds with relative standard deviation (RSD) of quality control peak areas greater than 20 %were excluded.
- Oxygen consumption rates were determined using an XF96 Extracellular Flux Analyzer. Cell lines were plated at 10,000 cells per well in 80 ⁇ l DMEM supplemented with 10%FBS and 1%P/Sthe day before OCR measurement.
- Neurons were cultured at 50,000 cells per well (pre-coated with poly-D-lysine) with 150 ⁇ l Neurobasal medium supplemented with 1 mM sodium pyruvate, 2%B27 and 1%GlutaMAX. Neurons were infected with AAV-PHP. eB virus expressing GFP or GPD1 three days after attachment (virus packaging and infection protocol are in section below) . OCRs of neurons were measured 10 days after viral infection.
- DNP Neuronal Complex-I dependent and Complex-I independent OCRs were determined by injection of compounds: DNP (2, 4-dinitrophenol, 200 ⁇ M) , piericidin A (2 ⁇ M) followed by antimycin A (2 ⁇ M) .
- DNP is an uncoupler to stimulate maximum OCR.
- ATP level To measure ATP level, cell lines were plated in triplicates in 96-well plates at 10,000 cells per well 16 hours before measurement. Neurons were cultured and infected as the OCR experiment above. ATP levels of neurons were measured 10 days after viral infection.
- Neurons and Cell lines were incubated with DMSO, 10 ⁇ M piericidin A or 10 ⁇ M antimycin A in DMEM supplemented with 10%FBS and 1%FBS for 2 hours.
- ATP level was measured by Cell Titer-Glo Luminescent assay kit following the manufacturer instructions. Briefly, Cells were lysed by adding 20 ⁇ l CellTiter-Glo reagent into the media and mixed on an orbital shaker for 5 minutes to lyse the cells. Luminescence signals were recorded after 10 minutes equilibration at room temperature.
- RNAs were extracted using a Direct-zol RNA Miniprep Kit.
- cDNA generated by reverse transcription by 5 ⁇ All-In-One RT MasterMix, together with primers were combined with the TB Green Premix Ex Taq and assayed by quantitative PCR (qPCR) . All CT values for genes of interest were normalized against the ACTINB gene (probe/control) . The ratio (probe/control) was set to 1 for DMSO-treated cells.
- the oligonucleotides for qPCR analysis were listed below.
- V ( ⁇ /6) (L ⁇ W 2 ) .
- tumors were permitted to grow to 50 mm 3 , after which animals were randomly assigned to the metformin treatment or the vehicle group. Vehicle (water) or 1 g/kg metformin (in water) was dosed via oral gavage daily for indicated time. Tumor volume was measured every two days. Mice were killed at end points that were consistent with our mouse protocol. Primary investigator was not blinded during or after experiments, although results were qualitatively verified by blinded investigators.
- AAV Adeno-associated Virus
- AAV vectors expressing mouse Ndufs4, human GPD1, GPD1 K204A , or GFP-3 ⁇ HA were driven by the EF1 ⁇ promoter, added with bGH poly (A) signal at 3’ end of the coding sequence and flanked by AAV2 ITRs at both ends.
- nucleotide sequence of the vector construct expressing human GPD1 is shown as SEQ ID NO. 31.
- the GPD1 cDNA accession number is NM_005276.4.
- Virus package was conducted as described above.
- AAV-pro cells (80%density) were transfected with AAV vectors along with AAV packaging vectors PHP. eB or PHP. B and p. Helper at a ratio of 4: 6: 5 by the PEI transfection reagent. 60 hours later, cell pellets were scraped and collected by centrifuging into tubes containing GB buffer (10 mM Tris (PH 7.6) , 150 mM NaCl and10 mM MgCl 2 ) and glass beads. Cells were lysed by freezing in liquid nitrogen for 8 minutes followed by thawing at 37 °C for 3 minutes and then shaking for 2 minutes.
- benzonase nuclease was used to eliminate cellular DNA after incubation at 37 °C for 30 minutes.
- Virus-containing supernatants were obtained by centrifuging at 15,000 rpm for 45 minutes.
- Optiprep iodixanol solution was used to prepare different iodixanol gradient: 15%iodixanol fraction (15%iodixanol and 1 M NaCl in GB buffer) , 25%iodixanol fraction (25%iodixanol in GB buffer) , 40%iodixanol fraction (40%iodixanol in GB buffer) and 58%iodixanol fraction (58%iodixanol in GB buffer) .
- Virus was purified by iodixanol gradient ultracentrifugation at 41,000 rpm for 4 hours (Zolotukhin et al., 1999) .
- Virus titer was determined by qPCR. All centrifugations were performed at 4 °C.
- AAV Adeno-associated Virus
- Ndufs4 -/- mice and control littermates were randomly assigned to receive an intravenous injection of 150 ⁇ l of AAV-PHP.
- eB virus (4 ⁇ 10 10 genome copies/ ⁇ l, diluted in PBS) through the retro-orbital injection (Yardeni et al., 2011) .
- Neurons plated in 24 well plate after 3 days of culture were infected with 1 ⁇ l of AAV-PHP.
- eB virus (4 ⁇ 10 10 genome copies/ ⁇ l, diluted in PBS) per well. Culture was maintained by changing virus containing medium every 3 days. Experiments with neurons were performed 10 days after viral infection.
- mice body temperatures were measured three consecutive days from P29-P31 to confirm the exact body temperature. Pictures were analyzed by Acrobat Reader DC. Temperatures are shown as mean ⁇ SEM.
- mice were anesthetized during tissue collection. The chest cavity was opened and a catheter was placed in the left ventricle. The whole body was perfused with PBS and then with 4%PFA. The brain was dissected out, stored overnight in 4%paraformaldehyde (PFA) and then placed in 30%sucrose (in PBS) for two days. PFA-perfused whole brains were sectioned parasagittally or coronally by 50 ⁇ m. Immunohistochemistry was performed using an antibody recognizing the microglial marker Iba-1 or antibody recognizing HA according to previous described methods (Quintana et al., 2010) .
- the mean fluorescence intensities of Alexa Fluor 568 in different brain areas were measured with the ImageJ software. Background fluorescence was subtracted from the measured fluorescence. Quantification results are represented as the mean ⁇ SEM.
- Sample size (n) indicates biological replicates from a single representative experiment. For analyzing C. elegans lifespan data, statistical significance was determined by the log-rank test. For mouse study, Sample size (n) indicates data from a distinct mouse. The results of all experiments were validated by independent repetitions. For comparing of two groups, statistical significance was determined using a two-tailed unpaired Student’s t-test; For comparing multiple groups, statistical significance was determined by one-way ANOVA using Tukey’s test or two-way ANOVA using Tukey’s test. P values are denoted in figures as: not significant [ns] , *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
- the sequence information is shown in the following table.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Diabetes (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A method for modulating Gro3P (glycerol-3-phosphate) biosynthesis, and uses thereof, e.g. therapeutic uses thereof in treating mitochondrial complex I diseases or cancers.
Description
This application claims the benefit of PCT Application No. PCT/CN2021/104699, filed on July 06, 2021, which is hereby incorporated by reference in its entirety
The present application is directed to methods for treating mitochondrial complex I diseases or cancers by modulating glycerol-3-phosphate (Gro3P) biosynthesis. Also provided are agents and pharmaceutical compositions for use in modulating Gro3P biosynthesis.
Mitochondrial respiration oxidizes NADH and transfers electrons to oxygen. ETC (electron transport chain) dysfunction or hypoxia results in toxic NADH reductive stress. Researches in the past decades reveal that NADH reductive stress underlies proliferation defects of ETC-defective human cells. However, how cells manage NADH reductive stress remains elusive.
NADH reductive stress also regulates tumorigenesis under hypoxia. Hypoxia deprives the electron acceptor oxygen to inhibit the ETC and to cause NADH reductive stress. Hypoxic NADH reductive stress restricts the synthesis of aspartate and serine to limit tumor growth (Diehl et al., 2019; Garcia-Bermudez et al., 2018; Sullivan et al., 2018) , and also promotes the synthesis of L-2-hydroxyglutarate from α-ketoglutarate to regulate histone methylation levels and to inhibit glycolysis and electron transport (Intlekofer et al., 2015; Oldham et al., 2015) .
Mitochondrial disease is a group of genetic disorders characterized by defective oxidative phosphorylation, a coupled process of electron transport and ATP synthesis (Gorman et al., 2016; Russell et al., 2020; Thompson et al., 2020) . Given the essential role of ETC in NAD
+ regeneration, mitochondrial diseases, especially the severe pediatric forms, are often characterized by metabolic derangements indicative of NADH reductive stress, such as lactic acidosis (Katsyuba et al., 2020; Russell et al., 2020; Thompson Legault et al., 2015) . Transgenic expression of NDI1, the yeast NADH: ubiquinone oxidoreductase, regenerates NAD
+ to extend the lifespan of brain NDUFS4-knockout mice (McElroy et al., 2020) , a model of mitochondrial complex I disorder (Kruse et al., 2008; Quintana et al., 2010) . Despite the importance of NADH/NAD
+ redox homeostasis in mitochondrial disease and tumorigenesis, our understanding of endogenous NAD
+ regeneration pathways remains limited. Identification of therapeutic targets involved in NAD
+ regeneration would be highly desirable. Development of agents or methods for modulating such therapeutic targets would also be needed for treating pathological conditions related to ETC dysfunctions.
SUMMARY OF INVENTION
The present inventors revealed Gro3P biosynthesis as an NAD
+ regeneration pathway evolutionarily conserved in yeast, C. elegans, mouse, and human. It has been established that enhancing Gro3P synthesis could rescue mitochondrial complex I deficiency in cultured cells. Also, the inventors revealed lacking Gro3P biosynthesis as a metabolic characteristic that sensitizes neurons to complex I deficiency and demonstrated enhancing Gro3P biosynthesis alleviates neuroinflammation and extends lifespan in the Ndufs4
-/- mice (an animal model of complex I deficiency) .
Accordingly, in a first aspect, the present disclosure relates to a method for treating complex I deficiency in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent enhancing Gro3P biosynthesis.
In one embodiment, the agent enhancing Gro3P biosynthesis increases the enzymatic activity, e.g. specific activity of one or more of cytosolic Gro3P dehydrogenases (cGPDs) . In one embodiment, the agent is an agonist of one or more of cGPDs.
In one embodiment, the agent enhancing Gro3P biosynthesis increases the level, e.g. mRNA level or protein level, of one or more cGPDs.
In one embodiment, the one or more cGPDs is human GPD1 and/or human GPD1L.
In one embodiment, the agent enhancing Gro3P biosynthesis comprises a nucleic acid molecule comprising a nucleotide sequence encoding the one or more cGPDs, encoding a variant of cGPD having the same or enhanced enzymatic activity or a nucleotide sequence enhancing the expression of one or more cGPDs.
In one embodiment, the agent enhancing Gro3P biosynthesis is a vector comprising the nucleic acid molecule of the above paragraph. In a specific embodiment, the vector further comprises one or more regulatory sequences selected from a promoter, an enhancer, a polyadenylation sequence, and origin of replication. In one embodiment, the promoter is operatively linked to the nucleic acid of the above paragraph. In one embodiment, the promoter is a constitutive promoter. In one embodiment, the promoter is a tissue-specific promoter, e.g. brain-specific promoter or ocular tissue-specific promoter, or cell-type-specific promoter e.g. neuron-specific promoter.
In a specific embodiment, the vector is a viral vector, such as an adenoviral vector, a retroviral vector or a recombinant adeno-associated virus (rAAV) vector. In one embodiment, the rAAV vector comprises the nucleotide sequence encoding one or more cGPDs, encoding a variant of cGPD having the same or enhanced enzymatic activity or a nucleotide sequence enhancing the expression of one or more cGPDs, as well as at least one inverted terminal repeat. In one embodiment, the rAAV vector comprises two ITRs. In a preferred embodiment, the AAV ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV. 7m8, AAV2tYF, AAV11, or AAV12 serotype ITR, preferably AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV. 7m8, or AAV2tYF serotype ITR, more preferably AAV2 ITR. In a specific embodiment, the AAV vector comprises a nucleotide sequence as shown in SEQ ID NO: 31, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%or 99%sequence identity to SEQ ID NO: 31.
In one embodiment, the AAV vector is encapsidated in an AAV particle. In one embodiment, the AAV particle comprises capsid proteins. In a preferred embodiment, at least one, preferably two or three of the capsid proteins (VP1, VP2 or VP3) is of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV. 7m8, AAV2tYF, AAV11, or AAV12 serotype. In a preferred embodiment, the serotype of capsid protein is suitable for brain delivery, e.g. intraparenchymal, intrathecal, intracerebroventricular, subpial and intravenous administration delivery, or eye delivery, e.g. subretinal, intravitreal or intraocular delivery. In a specific embodiment, the AAV vector comprises a capsid of AAV2, AAV4, AAV5, AAV8, AAV9, or AAVrh. 10 serotype.
In one embodiment, the complex I deficiency is selected from a group consisting of Leigh syndrome, Leber’s optical hereditary neuropathy (LHON) (also known as Leber optic atrophy) , Leber optic atrophy and dystonia, mitochondrial complex I deficiency (OMIM entry 252010) , leukodystrophy, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome, optic atrophy with demyelinating disease of central nervous system (CNS) and other neurodegenerative disorders.
In one embodiment, the subject is mammal, e.g. rodent, or primate, preferably human.
In one embodiment, the subject has at least one mutation in mtDNA encoding a complex I subunit, e.g. ND1, ND2, ND3, ND4, ND5, ND6 or ND4L. For example, the subject has LHON and has at least one mutation selected from Table 2 or Table 3.
In one embodiment, the method of the first aspect has one or more of the following effects: increased C-I-independent OCR (complex I independent oxygen consumption rate) , decreased NADH/NAD
+ ratio, increased ATP level, decreased mitochondrial integrative stress response, reduced cell death, deceased neuroinflammation, decreased neurodegeneration, enhanced motor function, alleviated metabolic derangements, decreased glycolysis blockade, decreased αHB level, decreased lactate level, decreased alanine level, decreased β-hydroxybutyrate/acetoacetate (βHB/AcAc) ratio, decreased lactate/pyruvate ratio, increased aspartate level, and increased asparagine level in tissue or plasm.
It has also been found that human cancer cells exploit Gro3P biosynthesis to antagonize ETC inhibition and hypoxia in culture and in xenografts. In xenograft tumors, abolishing Gro3P synthesis significantly suppressed tumor growth, and synergistic effect in inhibiting tumor progression was observed when used with metformin, an inhibitor of complex I.
Accordingly, in second aspect, the present disclosure provides a method for treating tumor in a subject by inhibiting Gro3P biosynthesis of tumor cells. In a specific embodiment, the method comprises administering to the subject a therapeutically effective amount of an agent inhibiting Gro3P biosynthesis of tumor cells.
In one embodiment, the agent inhibiting Gro3P biosynthesis inhibits the activity of one or more cGPDs, e.g. human GPD1 and/or GPD1L.
In one embodiment, the agent inhibiting the activity of one or more cGPDs is a chemical compound, a polypeptide or a polynucleotide.
In a preferred embodiment, the method further comprises inhibiting complex I, e.g. by administering to the subject an inhibitor of complex I.
In specific embodiments, the inhibitor of complex I is selected from a group consisted of metformin, phenformin, BAY84-2243, CAI, ME-344, fenofibrate, and mIBG.
In one embodiment, the agent inhibiting Gro3P biosynthesis is delivered simultaneously with the inhibitor of complex I. In a preferred embodiment, the agent inhibiting Gro3P biosynthesis is delivered after the inhibitor of complex I.
In one embodiment, the inhibition of Gro3P biosynthesis and the inhibition of complex I achieves synergistic anti-tumor effect.
In third aspect, the present disclosure provides a pharmaceutical combination of an agent inhibiting Gro3P biosynthesis and an inhibitor of complex I.
In one embodiment, the agent inhibiting Gro3P biosynthesis is an agent inhibiting or eliminating the activity of one or more cGPDs. In one embodiment, the cGPD is human GPD1 and/or GPD1L.
In one embodiment, the inhibitor of complex I is selected from a group consisted of metformin, phenformin, BAY84-2243, CAI, ME-344, fenofibrate, and mIBG.
In one embodiment, the pharmaceutical combination is used for treating tumor.
In one embodiment, the pharmaceutical combination exhibits synergistic anti-tumor effect.
BRIEF DESCRIPTION OF DRAWINGS
To facilitate a better understanding of the features and advantages of the present invention, the following detailed description and accompanying drawings thereof are provided. However, one skilled in the art will understand that they are provided with a purpose of illustration rather than limitation. The scope of the invention is controlled by the appended claims.
FIG. 1 is a schematic of Gro3P metabolism and chemical inhibitors of the ETC complexes. Human ETC complexes are shown. Complex-II is absent for simplicity. MIM: mitochondrial inner membrane.
FIG. 2 shows the results of immunoblot analysis of WT and cGPD-knockout (KO) 143B, HeLa, SNB-19, and A549 cells. GPD1 is not expressed in 143B cells.
FIG. 3 shows the results of metabolite analysis of dihydroxyacetone phosphate (DHAP) and Gro3P of WT and cGPD-KO 143B cells treated with ETC inhibitors for 2 hours. Pier: piericidin, complex I inhibitor; Anti: antimycin, complex III inhibitor. Data are mean±SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test, *P<0.05, **P<0.01, ***P<0.001.
FIG. 4 shows the results of metabolite analysis of the NADH/NAD
+ ratio of WT and cGPD-KO 143B, HeLa, SNB-19, and A549 cells treated with ETC inhibitors for 2 hours. Data are mean±SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test, *P<0.05, **P<0.01, ***P<0.001.
FIG. 5 shows the cell number and results of immunoblot analysis of WT and cGPD-KO 143B and HeLa cells complemented with GPD1L (1L) or vector (V) . ETC inhibitors were treated for 4-5 days. Met: metformin, complex I inhibitor. Data are mean±SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test, *P<0.05, **P<0.01, ***P<0.001.
FIG. 6 shows the metabolite analysis of Gro3P and DHAP of WT and cGPD-KO 143B cells following 0.5%oxygen treatment for 6 hours. Data are mean±SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. *P<0.05, **P<0.01, ***P<0.001.
FIG. 7 shows cell number of WT and cGPD-KO 143B cells treated with 0.2%oxygen for 5 days in the presence or absence of 3 mM pyruvate supplementation. Data are mean±SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. *P<0.05, **P<0.01, ***P<0.001.
FIGS. 8A-B show the result of metabolite analysis of Gro3P and DHAP (A) and the NADH/NAD
+ ratio (B) of WT and cGPD-KO HeLa cells following 0.5%oxygen treatment for 6 hours. Data are mean and mean±SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test; **P<0.01, ***P<0.001.
FIG. 9 shows the tumor growth curve of WT and cGPD-KO 143B-and HeLa-derived xenograft tumors daily treated with vehicle (water) or 1g/kg metformin. Data are mean±SEM. Statistics: one-way ANOVA with the Tukey-Kramer test, *P<0.05, **P<0.01, ***P<0.001.
FIG. 10 shows the tumor sizes at the end point of WT and cGPD-KO 143B-and HeLa-derived xenograft tumors daily treated with vehicle (water) or 1g/kg metformin. Data are mean±SEM. Statistics: one-way ANOVA with the Tukey-Kramer test, *P<0.05, **P<0.01, ***P<0.001.
FIG. 11 shows the photo images of WT and cGPD-KO HeLa-derived tumors daily treated with vehicle (water) or 1g/kg metformin.
FIG. 12A-B show tumor growth curves (A) and tumor sizes at the end point (B) of the xenograft tumors derived from WT and cGPD-KO143B cells complemented with GPD1L (1L) or vector (V) which were daily treated with vehicle (water) or 1g/kg metformin. Data are mean±SEM. Statistics: one-way ANOVA with the Tukey-Kramer test, *P<0.05, **P<0.01, ***P<0.001.
FIG. 13 shows the results of immunoblot analysis of 143B cells overexpressing WT cGPDs, enzymatically-inactive cGPDs (GPD1L
G14A, GPD1L
K206A and GPD1
G12A, GPD1
K204A) , or the vector control.
FIG. 14 shows the metabolite analysis of Gro3P and DHAP of the 143B cells as in FIG. 13. Cells were treated with ETC inhibitors for 2 hours. Data are mean±SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. ***P<0.001. ns: not significant.
FIG. 15 shows the complex I-dependent and complex I-independent oxygen consumption rate (OCRs) of the 143B cells as in FIG. 13. Data are mean±SD from six biological replicates. Statistics: two-tailed unpaired Student’s t-test. ***P<0.001. ns: not significant.
FIG. 16 shows the NADH/NAD
+ ratio of WT and GPD2-KO 143B cells overexpressing vector (V) or GPD1L (1L) . Cells were treated with ETC inhibitors for 2 hours. Data are mean±SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. ***P<0.001. ns: not significant.
FIGS. 17A-D show that enhancing Gro3P synthesis promotes Gro3P shuttle activity to alleviate metabolic and proliferative defects and to repress mitochondrial integrative stress response in Complex-I impaired Cells. A, Left: heat map representing metabolite levels of WT and GPD2-KO 143B cells overexpressing vector or GPD1L. Cells were treated with DMSO or ETC inhibitors for 2 hours. For each metabolite, the mean log2-transformed fold change is relative to DMSO-treated cells (n = 3) . Arrows indicate major metabolic changes induced by GPD1L-overexpression under piericidin treatment. Right: schematic of GPD1L-overexpression (OE) effects on cells under complex I inhibition. Arrows indicate the direction of glucose carbon flux. B-D, ATP level after ETC inhibition for 2 hours (B) , metabolite analysis of aspartate and asparagine after ETC inhibition for 2 hours (C) , and qPCR analysis of representative ATF4 target genes upregulated by mitochondrial integrative stress response (Quiros et al., 2017) after ETC inhibition for 16 hours (D) in WT and GPD2-KO 143B cells overexpressing vector (V) or GPD1L (1L) .
FIG. 18 shows cell number of WT and GPD2-KO 143B cells treated with ETC inhibitors (Pier: 300 nM; Met: 2.5 mM; Anti: 100 nM) for 4-5 days. Representative images are shown. Data are mean±SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. ***P<0.001. ns: not significant.
FIG. 19 shows the results of immunoblot analysis of WT and NDUFS2-KO 143B cells overexpressing WT or enzymatically-inactive (GPD1L
K206A and GPD1
K204A) cGPDs or the vector control.
FIG. 20 shows the cell number of the cells as in FIG. 19 after growing for 5 days. Data are mean±SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test. ***P<0.001. ns: not significant.
FIG. 21 shows the schematic of the beneficial effects of NAD
+ regeneration by the Gro3P shuttle under complex I dysfunction.
FIGS. 22A-D (A) Cell number of 143B cells overexpressing WT cGPDs, enzymatically-inactive cGPDs (GPD1
LG14A, GPD1L
K206A and GPD1
G12A, GPD1
K204A) , or the vector control. Cells were treated with ETC inhibitors (Pier: 300 nM; Anti: 100 nM) for 5 days. (B) Immunoblot analysis and cell number of HeLa, U2OS and MEF cells overexpressing GPD1L or the vector (V) control. Cells were treated with ETC inhibitors (Pier: 300 nM; Anti: 100 nM) for 5 days. (C) The NADH/NAD
+ ratio of 143B cells overexpressing GPD1L or vector. Cells were treated individually or in combination with Piericidin (300 nM) and oxamate (lactate dehydrogenase inhibitor, 40 mM) for 25 minutes. (D) Cell survival of 143B cells treated as in (C) for 10 hours. Representative images are shown. Cell viability was measured by trypan blue staining. Live cell number relative to total cell number in DMSO-treated cells were normalized as 100%. Data are mean±SD from three biological replicates. Statistics: two-tailed unpaired Student’s t-test, ***P<0.001.
FIG. 23 shows the results of immunoblot analysis of Gro3P metabolic enzymes in adult C57BL/6J mouse organs. 6 μg proteins of each sample were loaded for immunoblots.
FIG. 24 shows the immunoblot analysis of cultured cortical neurons infected with AAV-PHP. eB encoding GFP or GPD1 for 10 days.
FIGS. 25A-B show the NADH/NAD
+ ratio (A) , and ATP level (B) of neurons as in FIG. 24 treated with ETC inhibitors for 2 hours. Ratio (A) and ATP level (B) in DMSO-treated non-infected neurons were normalized as 1.
FIG. 26 shows the measurement of complex I-dependent and complex I-independent OCRs of neurons as in FIG. 24. DNP (2, 4-dinitrophenol, 200 μM) is an uncoupler to stimulate maximum OCR.
FIG. 27 Left: Schematic of delivering AAV-PHP. eB expressing GFP or GPD1 to the Ndufs4
-/- mice via the retro-orbital injection to increase GPD1 level in the brain; Right: Immunoblot analysis of GPD1 in mouse organs one month after AAV delivery.
FIG. 28 shows the immunoblot analysis of Gro3P metabolic enzymes in brain regions collected 3 weeks after AAV transduction to express GPD1 or GPD1
K204A. Samples from two mouse brains for each treatment are shown.
FIG. 29 shows the body weight of the indicated mice. KO + X: Ndufs4
-/- mice injected with AAV-PHP. eB expressing gene X. Data are mean ± SD.
FIG. 30 shows the lifespan of the indicated mice. KO + X: Ndufs4
-/- mice injected with AAV-PHP. eB expressing gene X. Statistics: one-way ANOVA with the tukey-Kramer test. **P < 0.01, ***P < 0.001.
FIGS. 31A-B show the metabolite analysis of brainstem glycolysis intermediates (A) and brainstem and plasma metabolites sensitive to the NADH/NAD
+ ratio (B) of the WT, Ndufs4
-/-, and Ndufs4
-/- mice transduced with AAV expressing GFP or GPD1. KO: Ndufs4
-/-.
FIG. 32 shows the representative images of Iba1 immunofluorescence staining at postnatal day 47-50 (P47-50) of the indicated mice. OB: olfactory bulb; CB: cerebellum; IO: inferior olive; VN: vestibular nuclei. Scale bar: 1 mm.
FIG. 33 shows the quantification results of Iba1 fluorescence intensity in different brain areas of the indicated mice.
FIG. 34 shows the representative 30-min locomotor activity traces at P50 (WT: n=6; Ndufs4
-/-: n=5; Ndufs4
-/- + GFP: n=6; Ndufs4
-/- + GPD1: n=7) of the indicated mice.
FIG. 35 shows the body temperature of the indicated mice. Data are mean ± SEM. Statistics: two-way ANOVA with the Tukey-Kramer test; **P < 0.01, ***P < 0.001.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
Unless specifically defined elsewhere in the present disclosure, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, including in the appended claims, the singular forms of words such as “a” , “an” , and “the” , include their corresponding plural references unless the context clearly dictates otherwise.
The term “or” is used to mean, and is used interchangeably with, the term “and/or” unless the context clearly dictates otherwise.
In the context of the present disclosure, unless being otherwise indicated, the wording "comprise" , and variations thereof such as "comprises" and "comprising" will be understood to imply the inclusion of a stated element, e.g. a component, a property, a step or a group thereof, but not the exclusion of any other elements, e.g. components, properties and steps. When used herein the term "comprise" or any variation thereof can be substituted with the term "contain" , “include” or sometimes "have" or equivalent variation thereof. In certain embodiments, the wording “comprise” also includes the scenario of “consisting of” .
The term "treat" , “treating” or “treatment” includes cure or at least alleviate the symptoms.
The term “therapeutically effective amount” as used herein, refers to the amount of an agent that, when administered to a subject for treating a disease, or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or symptom. The “therapeutically effective amount” can vary with the agent, the disease, disorder, and/or symptoms of the disease or disorder, severity of the disease, disorder, and/or symptoms of the disease or disorder, the age of the subject to be treated, and/or the weight of the subject to be treated. In the case of combination therapy, the “therapeutically effective amount” refers to the total amount of the combination objects for the effective treatment of a disease, a disorder or a condition.
The term “pharmaceutical composition” refers to a composition suitable for delivering to a subject. The pharmaceutical composition of the present disclosure comprises an agent modulating Gro3P biosynthesis and a pharmaceutically acceptable excipient. For example, in some embodiments of treating complex I deficiency, the pharmaceutical composition comprises a nucleic acid or a vector encoding cGPD. In some embodiments of treating cancer, the pharmaceutical composition comprises an inhibitor of Gro3P biosynthesis. Conventional pharmaceutically acceptable excipients are known in the art.
In the present disclosure, by “subject” it refers to a eukaryote, such as yeast, C. elegans, or an animal, preferably a mammal, e.g., a rodent, such as a mouse or a rat, a primate, preferably a higher primate, such as a human.
The terms “administration” , “administering” , “treating” and “treatment” as used herein, when applied to a subject, e.g. an animal, including human, or to cells, tissue, organ, or biological fluid, mean contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. The term “administration” and “treatment” also include in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell.
By "isolated nucleic acid" or “isolated polynucleotide” , it means a DNA or RNA which is removed from all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. An isolated nucleic acid molecule "comprising" a specific nucleotide sequence may include, in addition to the specified sequence, operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences. Due to the codon degeneracy, one skilled in the art can understand that a specific amino acid sequence can be coded by different nucleotide sequences.
The term “promoter” is well known in the art and refers to a regulatory sequence directing the transcription of a gene. Promoters can be categorized into constitutive promoters and inducible promoters. A constitutive promoter is active at all circumstances in vivo. An inducible promoter is only active under certain condition, e.g. in the presence of stimulus, within a certain type of tissue (tissue-specific promoter) or cell (cell-type-specific promoter) , or at a certain developmental stage (developmental stage specific promoter) .
The term “operatively linked” refers to the association of two components in a single nucleic acid stretch so that one can affect the function of the other.
The term “transgene” refers to a nucleic acid to be transferred or introduced into a host, for example, a cell or organism. A transgene can be a coding sequence of a functional protein or fragment thereof, e.g. a coding sequence of GPD1 or GPD1L.
NADH/NAD
+ REDOX
The cellular NADH/NAD
+ redox balance is fundamental to metabolism (Hosios and Vander Heiden, 2018; Katsyuba et al., 2020; Xiao and Loscalzo, 2020) . The oxidized form NAD
+ functions as an electron acceptor in diverse metabolic pathways such as glycolysis, fatty acid oxidation, amino acid degradation, citric acid cycle, serine synthesis, and one carbon metabolism (Ducker and Rabinowitz, 2017; Hosios and Vander Heiden, 2018; Katsyuba et al., 2020; Lunt and Vander Heiden, 2011; Xiao and Loscalzo, 2020) . The continuous operation of these metabolic pathways requires NAD
+ regeneration by mitochondrial respiration. The ETC oxidizes NADH and transfers electrons to oxygen, the ultimate electron acceptor. The matrix arm of ADH: ubiquinone oxidoreductase or complex I is the entry point for electrons from matrix NADH into the ETC. Because mitochondrial inner membrane is impermeable to NADH, electrons from cytosolic NADH are shuttled to mitochondrial matrix through the malate-aspartate shuttle or directly transferred to the ETC through the Gro3P shuttle. ETC inhibition, resulting from either hypoxia or genetic/pharmacological disruption of ETC function, impairs NADH oxidation and elevates the mitochondrial and cytosolic NADH/NAD
+ ratios, a condition termed NADH reductive stress.
Gro3P biosynthesis and cGPDs
Gro3P biosynthesis is a side pathway of glycolysis. It oxidizes NADH by converting glycolysis intermediate dihydroxyacetone phosphate (DHAP) to Gro3P, a reversible reaction catalyzed by NAD
+-dependent cytosolic Gro3P dehydrogenases (cGPDs) (Ansell et al., 1997; Kelly et al., 2011) . Gro3P is subsequently metabolized in three ways: 1. Condensation with acyl-CoA to generate glycerolipids (Yu et al., 2018) ; 2. dephosphorylation to generate glycerol (Mugabo et al., 2016; Nevoigt and Stahl, 1997) ; 3. oxidization by FAD
2+-dependent mitochondrial Gro3P dehydrogenase (mGPD) to regenerate DHAP; this third metabolic fate for Gro3P feeds electrons to the ETC, forming the Gro3P shuttle (Mracek et al., 2013) (Fig. 1) . Notably, Gro3P biosynthesis supports yeast growth under anoxia (Ansell et al., 1997) . cGPDs are heterogeneously expressed in mouse tissues, with highest expression in brown adipocytes and lowest expression in cerebral cortex (Ratner et al., 1981) . A BALB/c subline of mice lacking cGPD activity exhibited elevated lactate/pyruvate ratio in skeletal muscle (MacDonald and Marshall, 2000) .
Gro3P synthesis was identified as a major NAD
+ regeneration pathway in yeast and human cells for the first time by the present inventors, which further explains the findings in previous studies as mentioned above.
The abbreviation “GPD” herein refers to glycerol 3-phosphate dehydrogenase (GPDH) . There are two isoforms of GPD, cytoplasmic GPD (cGPD) located in cytosol and mitochondrial GPD located in mitochondrial inner membrane. cGPD is encoded by two genes, GPD1 and GPD1L, which share around 72%sequence identity with each other. cGPDs catalyze a reversible redox reaction of dihydroxyacetone phosphate to glycerol 3-phosphate. mGPD is encoded by GPD2 and catalyzes an irreversible reaction of glycerol 3-phosphate to dihydroxyacetone phosphate.
In the context of the present disclosure, the phrase “one or more cGPDs” means one or more cytoplasmic GPD, e.g. GPD1 and/or GPD1L of human.
In one aspect of the present disclosure, complex I diseases are treated by increasing the level or activity of cGPD.
In another aspect of the present disclosure, tumors are treated by inhibiting cGPD. The present inventors have surprisingly discovered that inhibition of cGPD produces an enhanced inhibitory effect on tumor growth. Further, when cGPD inhibition is combined with inhibition of complex I, a synergistic inhibitory effect on tumor growth can be observed.
In some embodiment, the present disclosure provides a method of treating, alleviating or preventing tumor in a subject, comprising administering to the subject a pharmaceutically effective amount of an inhibitor of GPD1 and/or GPD1L.
Treatment of Complex I deficiencies
The term “complex I” refers to respiratory complex I, or NADH: ubiquinone oxidoreductase, which couples electron transfer from NADH to ubiquinone with transmembrane proton pumping. Being the largest respiratory complex, complex I is consisted of 44 subunits coded by both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) .
Genetic defects in the subunits of complex I may cause complex I deficiencies, which is also known as mitochondrial complex I deficiencies. In the context of the present disclosure, the term “complex I deficiency” and “complex I disease” is interchangeable and should be understood in a broad way to include any disorder caused by any defects of complex I, i.e. any mitochondrial complex I linked disease.
Complex I deficiency is the most common cause of pediatric mitochondrial disease (~30%) that typically presents neurological symptoms, and also causes multiple adult-onset diseases, such as Leber’s hereditary optic neuropathy (Fassone and Rahman, 2012) . Table 1 of a review by R. J. Rodenburg (Biochimica et Biophysica Acta 1857 (2016) 938–945) provides a list showing the genetic defects in the subunits of complex I and related mitochondrial diseases.
The present disclosure provides a method for treating complex I deficiency by enhancing Gro3P biosynthesis, specifically by delivering an agent increasing the enzymatic activity or level of one or more cGPDs, e.g. human GPD1 and/or GPD1L.
The increase of enzymatic activity can be indicated by increase of specific activity of cGPD. For example, the activity of cGPDs can be enhanced by an agonist of cGPD.
The increase of cGPD level can be achieved by multiple ways known to those skilled in the art, such as increasing gene copy number or improving protein expression.
In some embodiments, the complex I deficiency which can be treated by the present method has one or more diseases selected from a group consisting of Leigh syndrome, Leber’s optical hereditary neuropathy (LHON) (also known as Leber optic atrophy) , Leber optic atrophy and dystonia, mitochondrial complex I deficiency (OMIM entry 252010) , leukodystrophy, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome, optic atrophy with demyelinating disease of CNS and other neurodegenerative disorders. One skilled in the art would understand that additional complex I deficiencies may be discovered in the future, which can also be treated by the method of the present disclosure.
Complex I deficiency caused by nDNA can be treated by gene replacement therapy. However, mtDNA mutations cannot be readily remedied in the same way by delivering a transgene encoding complex I component to make compensation. Therefore, the method of the present disclosure is especially suitable for treating complex I deficiencies mainly caused by mtDNA mutations.
In some embodiments, the subject to be treated by the present method has genetic defects in mtDNA encoded complex I subunits, such as ND1, ND2, ND3, ND4, ND4L, ND5, and/or ND6. In some embodiments, the subject has one or more of Leigh syndrome, LHON, or MELAS.
Table 1 below provides an overview of genetic defects in mtDNA encoded complex I subunits and resulting phenotypes (R. J. Rodenburg, 2016, supra) .
Table 1. Overview of genetic defects in mtDNA encoded complex I subunits
It should be noted that mutations in mtDNA encoded complex I subunits MTND1, MTND2, MTND3, MTND4, MTND5, MTND6 can also cause Leigh Syndrome or Leigh- like Syndrome. Mutations MTND3 and MTND5 are the most frequent mtDNA causes of Leigh Syndrome.
For example, mutations leading to LHON are listed in Table 2 and Table 3 below. Data obtained from MITOMAP: www. mitomap. org.
Table 2: 19 primary LHON mutations, the first 3 mutations listed (in boldface) represent approximately 95%of all cases. The remaining mutations are listed in nucleotide order.
No. | Mutation | Subunit | NT Δ | AA | %Patients | |
1 | m. 11778G>A | ND4 | G-A | R340H | 69 | |
2 | m. 3460G>A | | G-A | A52T | 13 | |
3 | m. 14484T>C | ND6 | T-C | |
14 | |
4 | m. 3376G>A | ND1 | G-A | E24K | Rare | |
5 | m. 3635G>A | ND1 | G-A | S110N | Rare | |
6 | m. 3697G>A | ND1 | G-A | G131S | Rare | |
7 | m. 3700G>A | ND1 | G-A | A112T | Rare | |
8 | m. 3733G>A | ND1 | G-A | E143K | Rare | |
9 | m. 4171C>A | ND1 | C-A | L289M | Rare | |
10 | m. 10197G>A | ND3 | G-A | A47T | Rare | |
11 | m. 10663T>C | ND4L | T-C | V65A | Rare | |
12 | m. 13051G>A | ND5 | G-A | G239S | Rare | |
13 | m. 13094T>C | ND5 | T-C | V253A | Rare | |
14 | m. 14459G>A | ND6 | G-A | A72V | Rare | |
15 | m. 14482C>A | ND6 | C-A | M64I | Rare | |
16 | m. 14482C>G | ND6 | C-G | M64I | Rare | |
17 | m. 14495A>G | ND6 | A-G | L60S | Rare | |
18 | m. 14502T>C | ND6 | T-C | I58V | Rare | |
19 | m. 14568C>T | ND6 | C-T | G36S | Rare |
Table 3: Other candidate LHON mutations found as single family or singleton cases
Patient | Mutation | Subunit | NT Δ | AA | #Patients | |
1 | m. 3472T>C | ND1 | T-C | |
1 |
|
2 | m. 4025C>T | ND1 | C-T | T240M | 1 family; 3 |
|
3 | m. 4160T>C | ND1 | T-C | L285P | 1 family; 9 cases |
4 | m. 4640C>A | | C-A | I57M | 1 family; 4 |
|
5 | m. 5244G>A | | G-A | G259S | 1 |
|
6 | m. 9101T>C | ATP6 | T-C | |
1 |
|
7 | m. 9804G>A | CO3 | G-A | A200T | Multiple unrelated | |
8 | m. 10237T>C | ND3 | T-C | |
1 family; 2 |
|
9 | m. 11253T>C | ND4 | T-C | |
1 |
|
10 | m. 11696G>A | | G-A | V312I | 1 family; 11 |
|
11 | m. 14596A>T | ND6 | A-T | |
1 family; 11 |
|
12 | m. 12811T>C | ND5 | T-C | |
1 family; 2 |
|
13 | m. 12848C>T | ND5 | C-T | |
1 |
|
14 | m. 13637A>G | | A-G | Q434R | 1 family; 3 |
|
15 | m. 13730G>A | | G-A | G465E | 1 |
|
16 | m. 14279G>A | | G-A | S132L | 1 family; 2 |
|
17 | m. 14325T>C | ND6 | T-C | |
1 |
|
18 | m. 14498T>C | ND6 | T-C | |
1 case |
Accordingly, for example, the subject to be treated by the present method has one or more mtDNA mutations selected from those listed in Table 2 or Table 3.
One skilled in the art would understand that other mutations in mtDNA encoded complex I subunit leading to LHON or other disorders may be revealed in the future. The present disclosure also contemplates application of the present method in a subject with any of these mutations.
The therapeutic effect in treating complex I deficiency achieved by the method of the present disclosure may be shown as or determined by one or more of the followings: an increased C-I-independent OCR (complex I independent oxygen consumption rate) , a decreased NADH/NAD
+ ratio, an increased ATP level, decreased mitochondrial integrative stress response, reduced cell death, deceased neuroinflammation, decreased neurodegeneration, enhanced motor function, alleviated metabolic derangements, including decreased glycolysis blockade, decreased αHB level, decreased lactate level, decreased alanine level, decreased β-hydroxybutyrate/acetoacetate (βHB/AcAc) ratio, decreased lactate/pyruvate ratio, increased aspartate level, and increased asparagine level in tissue or plasma. Measurement of therapeutic effects may vary depending on the phenotype of complex I disease and can be determined by one skilled in the art.
Enhancing Gro3P by Increasing cGPDs
In the method of the present disclosure, the complex I deficiency is treated by enhancing Gro3P biosynthesis, specifically by increasing the level of one or more cGPDs, namely GPD1 and GPD1L, or/and by increasing the enzymatic activity of cGPDs.
In some embodiments, the increase of cGPD level can be achieved by delivering a transgene encoding GPD1 and/or a transgene encoding GPD1L.
In some embodiments, the coding sequence of the cGPDs, specifically GPD1 or GPD1L, can be a wild-type nucleotide sequence or can be subjected to codon optimization so as to enhance the expression efficiency in a certain subject, e.g. human.
In some embodiments, the increase of enzymatic activity of cGPD can be achieved by enabling the expression of a mutated version of cGPD with enhanced enzymatic activity or by an agonist of one or more cGPDs.
In some embodiments, the transgene encoding GPD1 or the transgene encoding GPD1L is comprised in a viral vector. For example, the viral vector is a lentiviral vector, an adenoviral vector or an adeno-associated virus (AAV) vector.
In some embodiments, the lentiviral vector includes envelope proteins.
The rAAV vector of the present disclosure can comprise elements of any serotype known in the art or discovered in the future, or variant thereof. Preferably, the vector genome of the rAAV is AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV. 7m8, AAV2tYF serotype. In one embodiment, the rAAV vector comprises one or more ITRs of AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV. 7m8, AAV2tYF serotype.
In some embodiments, the AAV vector is self-complementary AAV (scAAV) or single stranded AAV (ssAAV) .
In some embodiments, the AAV vector comprises one or more ITRs from any AAV serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AV10, AAVrh10, AAV. 7m8, AAV2tYF, AAV-DJ, AAV-DJ/8, AAV11, AAV12, or variant thereof. For example, the AAV vector comprises two AAV2 ITRs flanking the coding sequence of GPD1 or GPD1L.
In some embodiments, the transgene encoding GPD1 or GPD1L is under the control of a constitutive promoter. For example, the promoter can be EF1-alpha promoter, CAG promoter, PGK promoter or CMV promoter. In some other embodiments, the transgene encoding GPD1 or GPD1L is under the control of a tissue-specific or cell-type-specific promoter. For example, the promoter can be a neuron-specific promoter, e.g. synapsin promoter; a brain-specific promoter; or an ocular tissue-specific promoter, e.g. GRM6 promoter, PCP2 promoter, GNGT2 promoter, PDE6H promoter, PITX3 promoter, or NR2E1 promoter.
In some embodiments, the AAV vector comprises an adenylation signal sequence. For example, the adenylation signal sequence can be bGH polyA, SV40 polyA or WPRE-SV40 poly A, preferably bGH polyA.
In some embodiments, the vector genome is packaged by one or more capsid. The capsid proteins of the present disclosure can be capsid of any AAV serotype known in the art or discovered in the future. Specifically, the capsid protein can be selected from a group consisting of but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV. 7m8, AAV2tYF, AAV11, or AAV12 serotype. The capsid can be derived from the same or different AAV serotype as the ITR. Preferably, the capsid proteins show tissue tropism for cells of the CNS (e.g. brain) or eye. Specifically, the AAV vector comprises a capsid from of AAV2, AAV4, AAV5, AAV8, AAV9, or AAVrh. 10.
In one embodiment, for the assembly of rAAV viral particle, a vector encoding the AAV replication and/or encapsidation protein and a vector encoding helper functions, such as p. Helper vectors, are used along with the vector comprising the nucleotide encoding target sequence flanked by ITR to infect host cells.
In some embodiment, the transgene encoding GPD1 or GPD1L is constructed into closed-ended DNA.
In some embodiment, the transgene encoding GPD1 or GPD1L is made into a circular RNA.
In some embodiments, the transgene encoding GPD1 or GPD1L is comprised in a lipid nano particle (LNP) .
Inhibition of tumor growth
The present disclosure provides a method for treating tumor or prevent tumor progression by inhibiting Gro3P biosynthesis of tumor cells. In a preferred embodiment, the method includes inhibition of both Gro3P biosynthesis and Complex I.
The terms “cancer” or “tumor” herein mean or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. The cancer can be solid tumors, such as sarcomas, carcinomas or lymphomas, or cancers in the blood. Preferably, the cancer expresses one or more of cGPDs.
Since solid tumors often grow in hypoxic microenvironment (Hockel and Vaupel, 2001) , they may produce a more significant response to a treatment by inhibiting Gro3P biosynthesis alone as compared to leukemias. However, the method of the present disclosure is not limited to the use in treating solid tumor, especially when the inhibition of Gro3P biosynthesis is combined with the inhibition of Complex I. Inhibition of Complex I creates an NADH reductive stress in cancer microenvironment. Inhibition of Gro3P biosynthesis in the cells under such NADH reductive stress retards the cell growth and in turn slows down the progression of tumor.
When inhibition of Gro3P biosynthesis is used in combination with inhibition of Complex I, they can be conducted simultaneously or sequentially. In a preferred embodiment, the inhibition of Gro3P can be conducted after the inhibition of Complex I.
In one embodiment, the inhibition of Gro3P biosynthesis is achieved by delivering of an agent, e.g. a compound, a polypeptide, or an isolated nucleotide inhibiting the one or more of cGPDs, e.g. human GPD1 or GPD1L. In one embodiment, the tumor or cancer to be treated expresses one or more of cGPDs.
For example, the agent inhibiting one or more of cGPDs is a chemical compound, including but not limited to (-) -epicatechin, (-) -epicatechin-3-gallate, (-) -epigallocatechin, or (-) -epigallocatechin-3-gallate.
For example, the agent inhibiting one or more of cGPDs is a polypeptide, e.g. an antibody or a fragment thereof specifically binding to one or more of cGPDs.
For example, the agent inhibiting one or more of cGPDs is a nucleotide-based inhibitor, including but not limited to siRNA, shRNA, miRNA, gRNA, or antisense oligonucleotides.
In one embodiment, the inhibition of Gro3P biosynthesis is used in combination with inhibition of complex I to achieve a synergistic anti-tumor effect.
In one embodiment, the inhibition of complex I is achieved by administering to the subject a therapeutically effective amount of complex I inhibitor.
For example, a complex I inhibitor can be a compound known to have inhibitory effect on complex I. Preferably, the complex I inhibitor is a compound suitable for pharmaceutical use, e.g. a compound which has been proven to be safe in a clinical trial or pre-clinical studies. The complex I inhibitor can be selected from a group consisting of metformin, phenformin, BAY84-2243, CAI, ME-344, fenofibrate, and mIBG.
EXAMPLES
Example 1. Identification of Gro3P as NAD
+ regeneration pathway
This example describes the process of identifying Gro3P synthesis as candidate NAD
+ regeneration pathways under ETC dysfunction.
A total number of 175 genes encoding NAD
+-dependent and NAD (P)
+ dual-specific dehydrogenases/reductases in the human genome were retrieved from the KEGG database. Among the retrieved genes, 81 genes encode enzymes catalyzing 39 reactions of macronutrient metabolism (glucose, amino acids, fatty acids, nucleic acids, ketone bodies, alcohols and aldehydes) . Under ETC dysfunction, only pathways that are NAD
+-regenerating and have continuous substrate supply, as well as continuous product disposal, can substantially contribute to NAD
+ regeneration. The inventors thus focused on macronutrient metabolism because macronutrients are the most abundant substrates available in the cell, culture media, and circulating blood. By following metabolic flow starting from macronutrients, eight candidate NAD
+ regeneration pathways, including Gro3P synthesis (glucose metabolism) , four amino acid metabolic pathways, transhydrogenation reaction, ketone body metabolism and fatty acids desaturation were identified.
Next, because ETC deficiency and hypoxia are universal stresses for aerobic organisms, the inventors strongly weighted for evolutionary conservation to prioritize amongst candidate pathways. Yeast is an organism well known for its strong resistance to ETC-dysfunction and anoxia when cultured in fermentable medium (Lasserre et al., 2015) , suggesting the presence of effective NAD
+ regeneration pathways. Thus, yeast NAD
+-dependent and NAD (P)
+ dual-specific enzymes were retrieved to examine the conservation of the eight human candidate pathways. It turned out three of them are conserved: Gro3P synthesis, proline synthesis, and fatty acid desaturation. It was recently reported that fatty acid desaturation contributes to NAD
+ regeneration under complex I inhibition (Kim et al., 2019) , corroborating the fidelity of the inventors’ analysis. The two conserved and uncharacterized candidate pathways, proline synthesis and Gro3P synthesis, were further investigated.
Experimental results obtained with yeast and human cells do not support proline synthesis as a major NAD
+ regeneration pathway. The gene encoding for pyrroline-5-carboxylate reductase (Pro3 in yeast and PYCR1&2 in human) , an enzyme catalyzing the final step of proline synthesis, was deleted in yeast cells with disrupted ETC and in human osteoblastoma cell line 143B treated with ETC inhibitors, respectively, while no effects on the NADH/NAD
+ ratio or on the proliferation rates was observed, suggesting the proline synthesis is unlikely a major NAD
+ regeneration pathway in yeast and human cells. As a result, the study was continued with Gro3P synthesis as the candidate pathway.
Example 2. ETC Inhibition Promotes Gro3P Synthesis to Alleviate NADH Reductive Stress in Human Cancer Cells
To determine the contribution of Gro3P synthesis to NAD
+ regeneration, cGPDs (GPD1L and GPD1) were knocked out in four human cancer cell lines, 143B, Hela, SNB-19 and A549 (Fig. 2) . It was found that cGPD knockout abolished Gro3P synthesis (Fig. 3) , and significantly elevated the NADH/NAD
+ ratio under chemical ETC inhibition in all the four cell lines (Fig. 4) .
cGPD knockout sensitized 143B and HeLa cells to the anti-proliferative effects of ETC inhibitors (Fig. 5) . Reconstitution of GPD1L in cGPD-KO cells rescued sensitivity to complex III inhibition by antimycin but surprisingly caused strong resistance to complex I inhibition by piericidin or metformin (Fig. 5) . This is an unexpected result.
Thus, Gro3P synthesis provides an indispensable NAD
+ regeneration activity to maintain viability and proliferation of ETC-deficient cells.
Example 3. Gro3P Synthesis Antagonizes Hypoxia and Supports Growth of Xenograft Tumors
This example investigates the correlation between Gro3P synthesis and tumor growth.
Hypoxia is a common feature of most malignant tumors. It was found that hypoxia promoted Gro3P synthesis in 143B cells (Fig. 6) . cGPD-KO 143B cells proliferated significantly slower than WT cells under hypoxia treatment, which was rescued by pyruvate supplementation (Fig. 7) . Hypoxia also promoted Gro3P synthesis in HeLa cells (Fig. 8A) . cGPD-KO HeLa cells exhibited higher NADH/NAD
+ ratio as compared to WT cells under hypoxia (Fig. 8B) .
To further examine the function of Gro3P synthesis in tumorigenesis with a physiologically-relevant method, the tumor xenograft model was employed. It was found that cGPD knockout significantly suppressed the growth of 143B-and HeLa-derived xenograft tumors. Metformin treatment, which inhibits tumorigenesis through inhibiting complex I (Wheaton et al., 2014) , synergized with cGPD knockout to further suppress tumor growth (Figs. 9-11) . GPD1L reconstitution in cGPD-KO 143B-derived tumors rescued tumor growth and caused metformin resistance (Fig. 12) , similar to what was previously observed in cell culture (Fig. 5) . Therefore, Gro3P synthesis supports tumor growth in vivo and regulates sensitivity to metformin.
Example 4. Enhancing Gro3P Synthesis Promotes Gro3P Shuttle Activity to Rescue Complex I Dysfunction in Human Cancer Cell Lines
This example investigates whether overexpression of cGPDs rescues complex I dysfunction through the Gro3P shuttle. WT and enzymatically-inactive (Kelly et al., 2011) cGPDs were overexpressed in 143B cells (Fig. 13) . Overexpression of WT but not mutant cGPDs caused twenty-fold increase of Gro3P level (Fig. 14) and fourfold increase of the C-I-independent OCR (complex I independent oxygen consumption rate) (Fig. 15) . GPD2 knockout completely blocked the enhanced C-I-independent OCR (data not shown) . Therefore, enhancing Gro3P synthesis by overexpressing cGPDs can promote Gro3P shuttle activity.
In 143B cells, overexpression of GPD1L offset the elevated NADH/NAD
+ ratio under complex I but not complex III inhibition (Fig. 16) . GPD1L-overexpression in complex I inhibited cells relieved glycolysis blockage (Fig. 17A) , rescued the levels of ATP (Fig. 17B) , aspartate, and asparagine (Fig. 17C) , and repressed mitochondrial integrative stress responses (Fig. 17D) , which is also induced by NADH reductive stress (Mick et al., 2020) . All these rescue effects were blocked by GPD2 knockout and by complex III inhibition (Figs. 16 and 17A-D) .
Overexpression of cGPDs rescued the proliferation of 143B cells under complex I but not complex III inhibition; such rescue required the enzymatic activity of cGPDs (Fig. 22A) and was blocked by GPD2 knockout (Fig. 18) . Overexpression of GPD1L also rescued the proliferation of multiple cell lines under complex I inhibition (Figure 22B) . Moreover, beyond chemical inhibition, complex I was also genetically inactivated by deleting the NDUFS2 subunit in 143B cells. NDUFS2-KO 143B cells showed greatly impaired proliferation and were rescued by WT but not by enzymatically-inactive cGPDs (Fig. 19 and Fig. 20) . We further challenged 143B cells by simultaneously inhibiting complex I and lactate dehydrogenase, which led to an extremely high NADH/NAD
+ ratio (Fig. 22C) and complete cell death (Fig. 22D) . GPD1L overexpression protected cells from this metabolic crisis (Figs. 22C-D) , demonstrating the capacity of enhanced Gro3P shuttle to regenerate NAD
+. Taken together, these results establish proof-of-concept for enhancing Gro3P synthesis as a strategy to rescue complex I dysfunction through the Gro3P shuttle (Fig. 21) .
Example 5. Enhancing Gro3P Synthesis Compensates for Complex I Inhibition in Cultured Neurons
Mouse brain has very low protein levels of cGPDs (Fig. 23) . Examination of the Genotype-Tissue Expression (GTEx) database (Consortium, 2015) also supported the low transcript levels of cGPDs in human brain, especially in brain areas sensitive to complex I deficiency, such as basal ganglia (Fassone and Rahman, 2012) . A question was raised to ask whether low expression level of cGPDs contributes to neuronal vulnerability to ETC dysfunction. This example tries to address this question in cultured neurons.
Consistent with the mouse brain data, cGPDs are almost undetectable in cultured mouse cortical neurons (Fig. 24) . Upon ETC inhibition, these neurons had both a high NADH/NAD
+ ratio (> threefold increase) (Fig. 25A) and a low ATP level (< 30%of WT level) (Fig. 25B) , similar to what was previously observed in cGPD-KO cancer cells (Fig. 4) .
Overexpression of GPD1 in cultured neurons (Fig. 24) increased C-I-independent OCR (Fig. 26) , significantly decreased the NADH/NAD
+ ratio (from 3.8 to 2, Fig. 25A) , and significantly increased ATP level (from 20%to 60%, Fig. 25B) under chemical inhibition of complex I. GPD1-overexpression had no rescue effects on complex III-inhibited neurons (Figs. 25A-B) . Therefore, weak activity of Gro3P synthesis contributes to neuronal vulnerability to mitochondrial complex I dysfunction.
Example 6. Enhancing Gro3P Synthesis Suppresses Neuroinflammation, Alleviates Neurological Symptoms, and Extends the Lifespan of the Ndufs4
-/- Mice
This example determines whether enhancing cGPD expression is applicable to rescuing complex I deficiency in vivo.
The Ndufs4
-/- mice was used, which progressively develop fatal neuroinflammation and neurodegeneration, recapitulating the characteristics of human Leigh syndrome (Jain et al., 2016; Johnson et al., 2013; Kruse et al., 2008; Quintana et al., 2010) . The inventors employed the recently described AAV-PHP. eB vector (Chan et al., 2017) that can extensively transduce both neurons and glia to express target genes in the brain. AAV-mediated delivery of NDUFS4 to the Ndufs4
-/- mice rescued the lifespan of these mice (Fig. 30) , confirming the validity of our approach. On such basis, AAVs expressing GPD1, GFP, or the GPD1
K204A inactive-mutant were constructed and delivered to the Ndufs4
-/- mice by retro-orbital injection so that the AAV particles can diffuse in the whole mouse body. The enrichment of AAV virus in mouse brain was achieved by the brain high affinity capsid vector PHP. eB.
In mice delivered with AAV-GPD1 and mice delivered with AAV-GPD1
K204A, brain GPD1 was increased to a comparable level as hepatic GPD1 (Fig. 27 and Fig. 28) . GPD1, GFP, and GPD1
K204A did not rescue body weight loss of the Ndufs4
-/- mice (Fig. 29) . But strikingly, GPD1 not GFP or GPD1
K204A significantly extended the lifespan of the Ndufs4
-/- mice by 44%, from 58.3± 1.92 to 84.0± 3.65 days (Fig. 30) .
To characterize the rescue effect in detail, the brainstem metabolite levels were examined first. It was found that Ndufs4
-/- mice exhibited increased accumulation of glycolysis intermediates upstream of the NADH-producing step (F6P, G3P, and DHAP) (Fig. 31A) , indicating glycolysis blockade. The Ndufs4
-/- mice also exhibited increased αHB and lactate levels and decreased aspartate levels in the brainstem, as well as increased plasma αHB levels (Fig. 31B) , indicating elevated brainstem NADH/NAD
+ ratio. Overexpression of GPD1 but not GFP significantly alleviated these metabolic derangements (Figs. 31 A-B) .
To characterize the rescue effect in detail, brain pathology and motor function of age-matched mice were examined. Consistent with previous reports (Kruse et al., 2008; Quintana et al., 2010) , Ndufs4
-/- mice exhibited strong neuroinflammation manifested by the greatly-increased number and hypertrophic morphology of Iba1-positive microglia within multiple brain regions (Fig. 32 and Fig. 33) . Ndufs4
-/- mice progressively developed ataxia and locomotor defects (Fig. 34) and suffered from reduced body temperature (Fig. 35) . Expression of GPD1 but not GFP ameliorated most of the neuroinflammation and partially prevented the decline of motor function and the reduction in body temperature (Figs. 32-35) .
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell Lines
143B, HeLa, U2OS, SNB-19, A549 and MEF cells were cultured in DMEM without pyruvate supplemented with 10%fetal bovine serum (FBS) , 1%penicillin-streptomycin and 2 mM L-glutamine. All cells were incubated at 37 ℃with 5%CO2.
Primary Neuron
Cortex from anesthetized P0 mouse was cut into small pieces and digested with 0.25%trypsin at 37 ℃ for 8 minutes and stopped by 3 volumes of DMEM supplemented with 10%FBS. Digested cortical pieces were dissociated by gentle pipetting for 5 times, rested for 3 minutes to precipitate undigested large pieces. Supernatants were transferred through a 40 μm cell strainer to remove cell aggregates. Dissociated neurons were cultured at 0.5-1×10
6 cells per well (24 well plate) in dish or coverslips pre-coated with poly-D-lysine in DMEM supplemented with 10%FBS. Medium was switched to Neurobasal medium supplemented with 1 mM sodium pyruvate, 2%B27 and 1%GlutaMAX 12 hours after attachment. Culture was maintained by changing medium every 3 days.
Mice
Ndufs4
-/- mice were purchased from The Jackson Laboratory. BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology. Mice were maintained under a 12-hours light/dark cycle and on a standard chow diet at the specific pathogen-free (SPF) facility at the National Institute of Biological Sciences, Beijing. All mouse experiments were carried out following the national guidelines for housing and care of laboratory animals (Ministry of Health, China) and the protocol was in compliance with institutional regulations after review and approval by the Institutional Animal Care and Use Committee at National Institute of Biological Sciences, Beijing.
METHOD DETAILS
Lentivirus Production and Generation of Cells Stably Expressing cDNAs
Lentiviruses were produced by co-transfection of HEK293T with cDNA containing lentiviral vectors FUIPW, lentiviral packaging vectors psPAX2 and pMD2. G at a ratio of 5: 3: 2. Virus containing supernatants were collected and filtered through 0.45 μm filters 48 hours post-transfection and stored at -80 ℃. For infection, targeted cells were infected with virus-containing supernatants for 48 hours in the presence of 8 μg/ml polybrene. Infected cells were selected by 1-3 μg/ml puromycin. cDNA expression was validated by immunoblotting.
Generation of Knockout Cell Lines
Generation of knockout cell lines by the CRISPR–Cas9 technology was performed as described (Ran et al., 2013) . Two guide RNAs (gRNAs) were designed for targeting each gene. The oligonucleotides were listed below. Annealed gRNAs were cloned into the gRNA-expression plasmid PX458. PX458 was then transfected to targeted cells using the Polyethylenimine (PEI) transfection reagent. Two days after transfection, GFP-positive cells were sorted into single clones and were grown for 2 weeks or longer. The resultant colonies were trypsinized and expanded. Knockout clones were selected after immunoblotting examination.
Proliferation Assay of Human Cancer Cells
All the cell proliferation analyses were performed in growth medium: DMEM supplemented with 10%FBS, 1%P/Sand 50 μg/ml uridine with media changes every 48 hours. Uridine compensates for the pyrimidine synthesis function of mitochondrial Complex-III, which is blocked under Complex-III inhibition and is not related to the NADH/NAD
+ ratio (Bajzikova et al., 2019; Martinez-Reyes et al., 2020) . Cell lines growing in log phase were trypsinized, counted, and plated onto 6 well dishes in 2 ml growth medium with respiratory chain inhibitors at indicated concentrations. Initial seeding density was 25,000 cells per well. For all conditions the seeding densities used allowed exponential proliferation for 4-5 days and final cell counts were measured 4-5 days after treatment. Cells were counted using TC20 automated cell counter. Relative cell number was determined by cell number of inhibitor treatment divided by cell number of vehicle treatment.
Immunoblotting of Human Cancer Cells and Mouse Tissue Samples
Lysis buffer contains 20 mM Tris–HCl (pH 7.5) , 1 mM EDTA, 1%Triton X-100, 150 mM NaCl, 0.1%SDS, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, a protease inhibitor cocktail and a phosphatase inhibitor. Human cells were scraped and pellets were collected. Tissues from anesthetized mice were dissected out and snap-frozen in liquid nitrogen. Tissues were weighted and cryohomogenized on dry ice.
Human cell pellets and homogenized frozen mouse tissue powders were extracted by sonication in the lysis buffer. Cell lysates were clarified by centrifuging at 15,000 rpm for 10 min at 4 ℃. Protein concentrations were determined by Bradford assay kit following the manufacturer instructions. Lysates were resolved by SDS–PAGE at 120 V. Proteins were transferred for 1.5 hours at 400 mA to PVDF membranes. Membranes were incubated with primary antibodies in 5%nonfat milk in PBST overnight. Membranes were washed three times in PBST before incubation in 1: 10,000 species-specific HRP-conjugated antibody. Membranes were washed again three times in PBST before visualization with ECL substrate.
Measurement of the NADH/NAD
+ ratio in Human Cancer Cells
The NADH/NAD
+ ratio was measured by modification of manufacturer instructions for NAD
+/NADH Glo Assay kit. The lysis buffer is 1%Dodecyltrimethylammonium bromide (DTAB) in 0.2 N NaOH.
To measure the NADH/NAD
+ ratio in human cell lines, cells were plated in 96-well plates at 16,000 cells per well. The following day cells were incubated with DMSO or ETC inhibitors (piericidin A: 10 μM; antimycin A, 10 μM) in growth medium for 2 hours. Media were aspirated and cells were extracted by adding 125 μl ice cold lysis buffer (diluted 1: 1 with PBS) into the plate and incubated 3 minutes on an orbital shaker to ensure homogenous cell lysis. 50 μl supernatants were used to measure NADH and NAD
+respectively.
To measure NADH, 50 μl of samples were moved to empty wells of 96 well plate and incubated at 60 ℃ for 15 minutes to degrade NAD
+. To measure NAD
+, 50 μl of the samples were moved to empty wells of 96 well plate, mixed with 25 μl 0.4 N HCl and incubated at 60 ℃ for 15 minutes to degrade NADH. Following incubations, samples were allowed to equilibrate for 10 minutes at room temperature and quenched by neutralizing with 25 μl 0.5 M Tris (NAD
+) or 50 μl of HCl/Tris (NADH) . 20 μl samples were mixed with 20 μl detection reagents in a 384 well plate. Luminescence signals were recorded after 30 minutes incubation at room temperature.
Metabolite Extraction from Human Cancer Cells and Mouse Tissues
For metabolite extraction from human cells, 143B cells (2 million per sample) cultured in 6-cm dish were incubated with DMSO, 10 μM Piericidin A or 10 μM Antimycin A for 2 hours. After washing with PBS twice, cells were extracted by -80 ℃ pre-chilled 80%methanol for the cellular steady metabolite measurements. The remaining cell pellets were dissolved in 0.1 M KOH by shaking at 4 ℃ overnight. Protein concentration was measured by Bradford assay kit. The metabolic data was normalized to protein concentration.
For metabolite extraction from mouse tissues, mice were anesthetized during tissue collection. Tissues were dissected out and snap-frozen in liquid nitrogen. Tissues were weighed and cryohomogenized on dry ice and the homogenized frozen powders were extracted by ice-cold 4/4/2 acetonitrile/methanol/water according to the weight (100 ul/10 mg) . Samples were incubated on ice for 30 minutes. During incubation, samples were mixed for 1 minute every 10 minutes. After centrifugation at 15,000 rpm for 15 minutes at 4 ℃, 500 μl supernatants from each sample were transferred to a new tube and were lyophilized by Speedvac to dry pellet at 30 ℃. Dried samples were stored at -80 ℃.
LC-MS Analysis of Metabolites
LC-MS analysis was performed by a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer. Authentic reference standard compounds were purchased from Sigma-Aldrich to confirm the retention time of each targeted metabolites. Dried samples were resuspended in 100 μl 50%methanol and filtered through 0.45 μm filters. 6 μl of each sample was injected for analysis. Chromatographic separation was performed on a Merck ZIC-HILIC column (2.1×100 mm, 3.5 μm) at a flow rate of 0.5 ml/min and maintained at 40 ℃, with the mobile phases of 10 mM ammonium acetate in ACN/water 5/95 (A) and 10 mM ammonium acetate in ACN/water 95/5 (B) . The following gradient was applied: 0-5 min, 99%B; 5-20 min, 99-20%B; 20-21 min, 20-99%B; 21-25 min, 99%B. Full-scan mass spectra were acquired in the range of m/z 66.7 to 1,000 with the following ESI source settings: spray voltage: 3.5 kV, auxiliary gas heater temperature: 380 ℃, capillary temperature: 320 ℃, sheath gas flow rate: 35 units, auxiliary gas flow: 10 units in the positive mode; Spray voltage: 2.5 kV, auxiliary gas heater temperature: 380 ℃, capillary temperature: 320 ℃, sheath gas flow rate: 30 units, auxiliary gas flow: 10 units in the negative mode. MS1 scan parameters included resolution 60,000, AGC target 3e6, and maximum injection time 200 ms. Data processing was performed with Thermo Xcalibur software (4.2) . Results were manually validated and compounds with relative standard deviation (RSD) of quality control peak areas greater than 20 %were excluded.
Measurement of Complex-I-Dependent and Complex-I-Independent Oxygen Consumption Rates in Human Cancer Cells and Cultured Mouse Cortical Neurons
Oxygen consumption rates (OCRs) were determined using an XF96 Extracellular Flux Analyzer. Cell lines were plated at 10,000 cells per well in 80 μl DMEM supplemented with 10%FBS and 1%P/Sthe day before OCR measurement.
Neurons were cultured at 50,000 cells per well (pre-coated with poly-D-lysine) with 150 μl Neurobasal medium supplemented with 1 mM sodium pyruvate, 2%B27 and 1%GlutaMAX. Neurons were infected with AAV-PHP. eB virus expressing GFP or GPD1 three days after attachment (virus packaging and infection protocol are in section below) . OCRs of neurons were measured 10 days after viral infection.
Before measurements, cells were washed 2 times in assay media: XF Base Medium supplemented with 25 mM glucose and 4 mM glutamine and adjusted to pH 7.4. Cells were then incubated in 180 μl assay media at 37℃ without CO
2 for 1 hour. Measurements were performed at consecutive intervals of mixing (3 minutes) and measurement (3 minutes) . Complex-I dependent and Complex-I independent OCRs of cell lines were determined by injection of compounds: piericidin A (2 μM) followed by antimycin A (2 μM) . Neuronal Complex-I dependent and Complex-I independent OCRs were determined by injection of compounds: DNP (2, 4-dinitrophenol, 200 μM) , piericidin A (2 μM) followed by antimycin A (2 μM) . DNP is an uncoupler to stimulate maximum OCR.
Measurement of Cellular ATP Level in Human Cancer Cells and Cultured Mouse Cortical Neurons
To measure ATP level, cell lines were plated in triplicates in 96-well plates at 10,000 cells per well 16 hours before measurement. Neurons were cultured and infected as the OCR experiment above. ATP levels of neurons were measured 10 days after viral infection.
Neurons and Cell lines were incubated with DMSO, 10 μM piericidin A or 10 μM antimycin A in DMEM supplemented with 10%FBS and 1%FBS for 2 hours. ATP level was measured by Cell Titer-Glo Luminescent assay kit following the manufacturer instructions. Briefly, Cells were lysed by adding 20 μl CellTiter-Glo reagent into the media and mixed on an orbital shaker for 5 minutes to lyse the cells. Luminescence signals were recorded after 10 minutes equilibration at room temperature.
Quantitive PCR Analysis of Mitochondrial Integrative Stress Response in 143B Cells
350,000 143B cells were seeded in 6-cm dishes in 4 ml DMEM 24 hours prior to inhibitor treatment. Cells were incubated with DMSO, 10 μM piericidin A or 10 μM antimycin A for 12 hours in the presence of 50 μg/ml uridine. Total RNAs were extracted using a Direct-zol RNA Miniprep Kit. cDNA, generated by reverse transcription by 5×All-In-One RT MasterMix, together with primers were combined with the TB Green Premix Ex Taq and assayed by quantitative PCR (qPCR) . All CT values for genes of interest were normalized against the ACTINB gene (probe/control) . The ratio (probe/control) was set to 1 for DMSO-treated cells. The oligonucleotides for qPCR analysis were listed below.
Xenograft Tumor Growth
Cancer cells were suspended in 125 μl PBS and injected into the flanks of 5 week-old, female BALB/c nude mice at 1×10
6 per injection for 143B cells and 5×10
6 cells per injection for HeLa cells. When tumors became palpable, tumor volume was measured by calipers in two dimensions and the volumes were estimated using the equation V = (π /6) (L×W
2) . For experiments determining the efficacy of metformin treatment in tumors, tumors were permitted to grow to 50 mm
3, after which animals were randomly assigned to the metformin treatment or the vehicle group. Vehicle (water) or 1 g/kg metformin (in water) was dosed via oral gavage daily for indicated time. Tumor volume was measured every two days. Mice were killed at end points that were consistent with our mouse protocol. Primary investigator was not blinded during or after experiments, although results were qualitatively verified by blinded investigators.
Adeno-associated Virus (AAV) Package
To produce AAV particles, three plasmids were required: the AAV vector, the capsid vector PHP. eB, and the p. Helper vector. AAV vectors expressing mouse Ndufs4, human GPD1, GPD1
K204A, or GFP-3×HA were driven by the EF1α promoter, added with bGH poly (A) signal at 3’ end of the coding sequence and flanked by AAV2 ITRs at both ends.
Specifically, the nucleotide sequence of the vector construct expressing human GPD1 is shown as SEQ ID NO. 31. The GPD1 cDNA accession number is NM_005276.4. Virus package was conducted as described above.
AAV-pro cells (80%density) were transfected with AAV vectors along with AAV packaging vectors PHP. eB or PHP. B and p. Helper at a ratio of 4: 6: 5 by the PEI transfection reagent. 60 hours later, cell pellets were scraped and collected by centrifuging into tubes containing GB buffer (10 mM Tris (PH 7.6) , 150 mM NaCl and10 mM MgCl
2) and glass beads. Cells were lysed by freezing in liquid nitrogen for 8 minutes followed by thawing at 37 ℃ for 3 minutes and then shaking for 2 minutes. After repeating this freezing-thawing-shaking procedure for 5 times, benzonase nuclease was used to eliminate cellular DNA after incubation at 37 ℃ for 30 minutes. Virus-containing supernatants were obtained by centrifuging at 15,000 rpm for 45 minutes. Optiprep iodixanol solution was used to prepare different iodixanol gradient: 15%iodixanol fraction (15%iodixanol and 1 M NaCl in GB buffer) , 25%iodixanol fraction (25%iodixanol in GB buffer) , 40%iodixanol fraction (40%iodixanol in GB buffer) and 58%iodixanol fraction (58%iodixanol in GB buffer) . Virus was purified by iodixanol gradient ultracentrifugation at 41,000 rpm for 4 hours (Zolotukhin et al., 1999) . Virus titer was determined by qPCR. All centrifugations were performed at 4 ℃.
Adeno-associated Virus (AAV) Delivery to Ndufs4
-/- Mice and Cultured Neurons
At P16-18, Ndufs4
-/- mice and control littermates were randomly assigned to receive an intravenous injection of 150 μl of AAV-PHP. eB virus (4×10
10 genome copies/μl, diluted in PBS) through the retro-orbital injection (Yardeni et al., 2011) .
Neurons plated in 24 well plate after 3 days of culture were infected with 1 μl of AAV-PHP. eB virus (4×10
10 genome copies/μl, diluted in PBS) per well. Culture was maintained by changing virus containing medium every 3 days. Experiments with neurons were performed 10 days after viral infection.
Measurements of Body Temperature
A thermal imaging camera was used to measure mouse body temperature. The pictures were taken at cochlea site of free moved mice at ~P30, P40, and P50 days. Six pictures were taken for one mouse. For a single time point, such as P30 body temperature, mice body temperatures were measured three consecutive days from P29-P31 to confirm the exact body temperature. Pictures were analyzed by Acrobat Reader DC. Temperatures are shown as mean ± SEM.
Histology
Mice were anesthetized during tissue collection. The chest cavity was opened and a catheter was placed in the left ventricle. The whole body was perfused with PBS and then with 4%PFA. The brain was dissected out, stored overnight in 4%paraformaldehyde (PFA) and then placed in 30%sucrose (in PBS) for two days. PFA-perfused whole brains were sectioned parasagittally or coronally by 50 μm. Immunohistochemistry was performed using an antibody recognizing the microglial marker Iba-1 or antibody recognizing HA according to previous described methods (Quintana et al., 2010) .
QUANTIFICATION AND STATISTICAL ANALYSIS
Quantification of Iba1 Fluorescence Intensity
The mean fluorescence intensities of Alexa Fluor 568 in different brain areas were measured with the ImageJ software. Background fluorescence was subtracted from the measured fluorescence. Quantification results are represented as the mean ± SEM.
Statistical Analyses
Data were presented as the mean ± standard deviation (SD) unless otherwise indicated. Sample size (n) indicates biological replicates from a single representative experiment. For analyzing C. elegans lifespan data, statistical significance was determined by the log-rank test. For mouse study, Sample size (n) indicates data from a distinct mouse. The results of all experiments were validated by independent repetitions. For comparing of two groups, statistical significance was determined using a two-tailed unpaired Student’s t-test; For comparing multiple groups, statistical significance was determined by one-way ANOVA using Tukey’s test or two-way ANOVA using Tukey’s test. P values are denoted in figures as: not significant [ns] , *P < 0.05, **P < 0.01, ***P < 0.001.
The materials used in the experiments as described above can be obtained from following sources.
The sequence information is shown in the following table.
Claims (28)
- A method for treating mitochondrial complex I deficiency in a subject, comprising administering to the subject a therapeutically effective amount of an agent enhancing Gro3P biosynthesis.
- The method of claim 1, wherein the agent enhancing Gro3P biosynthesis increases the level or enzymatic activity of one or more cytosolic Gro3P dehydrogenases (cGPDs) .
- The method of claim 2, wherein the one or more cGPDs is human GPD1 and/or human GPD1L.
- The method of claim 2 or claim 3, wherein the agent is a nucleic acid molecule comprising a nucleotide sequence encoding the one or more of cGPDs, a nucleotide sequence encoding a variant of cGPD having the same or enhanced enzymatic activity as compared to wild-type cGPD, or a nucleotide sequence enhancing the expression of one or more cGPDs.
- The method of claim 2 or claim 3, wherein the agent is a vector comprising the nucleic acid molecule of claim 4.
- The method of claim 5, wherein the vector further comprises a promoter which controls the expression of the nucleotide sequence, preferably the promoter is selected from a constitutive promoter, a tissue-specific promoter or a cell-type-specific promoter, more preferably a neuro-specific, a brain-specific promoter or an ocular tissue-specific promoter.
- The method of claim 5 or claim 6, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.
- The method of claim 7, wherein the rAAV vector comprises at least one, preferably two ITRs.
- The method of claim 8, wherein the ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV. 7m8, or AAV2tYF, AAV11, AAV12 serotype ITR, preferably AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV. 7m8, or AAV2tYF serotype ITR, more preferably AAV2 ITR.
- The method of any one of claims 7 to 9, wherein the rAAV vector comprises a polyadenylation sequence, preferably bGH polyA, SV40 polyA or WPRE-SV40 poly A, more preferably bGH polyA.
- The method of any one of claims 7 to 10, wherein the rAAV vector comprises a nucleotide sequence as shown in SEQ ID NO: 31 or a nucleotide sequence having at least 80%homology to SEQ ID NO: 31.
- The method of any one of claims 7 to 11, wherein the rAAV vector is encapsidated in capsid proteins, preferably of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV. 7m8, AAV2tYF, AAV11, or AAV12 serotype.
- The method of claim 12, wherein at least one of the capsid proteins is of AAV2, AAV4, AAV5, AAV8, AAV9, or AAVrh. 10 serotype.
- The method of claim 2 or claim 3, wherein the agent is an agonist of one or more of cGPDs.
- The method of any one of claims 1-14, wherein the complex I deficiency is selected from a group consisting of Leigh syndrome, Leber’s optical hereditary neuropathy (LHON) , Leber optic atrophy and dystonia, mitochondrial complex I deficiency (OMIM entry 252010) , leukodystrophy, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome, optic atrophy with demyelinating disease of CNS and other neurodegenerative diseases caused by complex I deficiency.
- The method of any one of claims 1-15, wherein the subject is a mammal, preferably human.
- The method of any one of claims 1-16, wherein the subject has at least one mutation in the mtDNA encoding complex I subunit.
- A method for treating tumor in a subject, comprising administering to the subject a therapeutically effective amount of an agent inhibiting Gro3P biosynthesis of tumor cells.
- The method of claim 18, wherein the agent inhibiting Gro3P biosynthesis is a chemical compound, a polypeptide, or a polynucleotide inhibiting the activity of one or more cGPDs.
- The method of claim 19, wherein the cGPD is human GPD1 and/or GPD1L.
- The method of any one of claims 18 to 20, further comprising administering to the subject a therapeutically effective amount of an inhibitor of complex I.
- The method of claim 21, wherein the inhibitor of complex I is selected from a group consisted of metformin, phenformin, BAY84-2243, CAI, ME-344, fenofibrate, and mIBG.
- A pharmaceutical combination of an agent inhibiting Gro3P biosynthesis and an inhibitor of complex I.
- The combination of claim 23, wherein the agent inhibiting Gro3P biosynthesis is an agent inhibiting or eliminating the activity of one or more cGPDs.
- The combination of claim 24, wherein the cGPD is human GPD1 and/or GPD1L.
- The combination of claim 23, wherein the inhibitor of complex I is selected from a group consisted of metformin, phenformin, BAY84-2243, CAI, ME-344, fenofibrate, and mIBG.
- The combination of any one of claims 23-26, which is used for treating tumor.
- The combination of any one of claims 23-27, which exhibits synergistic anti-tumor effect.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2021104699 | 2021-07-06 | ||
CNPCT/CN2021/104699 | 2021-07-06 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2023280175A1 true WO2023280175A1 (en) | 2023-01-12 |
WO2023280175A9 WO2023280175A9 (en) | 2023-02-09 |
Family
ID=84801295
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2022/103982 WO2023280175A1 (en) | 2021-07-06 | 2022-07-05 | Methods for treating complex i deficiencies or cancers by modulating gro3p biosynthesis |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2023280175A1 (en) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001021820A1 (en) * | 1999-09-22 | 2001-03-29 | National Research Council Of Canada | Transgenic manipulation of sn-glycerol-3-phosphate and glycerol production with a feedback defective glycerol-3-phosphate dehydrogenase gene |
CN101319221A (en) * | 2008-07-01 | 2008-12-10 | 上海大学 | Glycerol-3- phosphoric desaturase gene relating with glycerol synthesis and uses thereof |
CN104520428A (en) * | 2012-02-17 | 2015-04-15 | 费城儿童医院 | Aav vector compositions and methods for gene transfer to cells, organs and tissues |
US20180221457A1 (en) * | 2015-07-31 | 2018-08-09 | Val-Chum, Limited Partnership | Glycerol-3-phosphate phosphatase activators |
US20190211103A1 (en) * | 2018-01-05 | 2019-07-11 | Gnt Biotech & Medicals Corporation | Pharmaceutical combination and method for regulation of tumor microenvironment and immunotherapy |
CN110699451A (en) * | 2019-07-19 | 2020-01-17 | 广州市第一人民医院(广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院) | GPD1 expression quantity detection kit and application of metformin in assisting cancer treatment |
CN112689515A (en) * | 2018-07-17 | 2021-04-20 | 国家医疗保健研究所 | Compositions and methods for increasing or enhancing transformation of gene therapy vectors and for removing or reducing immunoglobulins |
-
2022
- 2022-07-05 WO PCT/CN2022/103982 patent/WO2023280175A1/en unknown
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001021820A1 (en) * | 1999-09-22 | 2001-03-29 | National Research Council Of Canada | Transgenic manipulation of sn-glycerol-3-phosphate and glycerol production with a feedback defective glycerol-3-phosphate dehydrogenase gene |
CN101319221A (en) * | 2008-07-01 | 2008-12-10 | 上海大学 | Glycerol-3- phosphoric desaturase gene relating with glycerol synthesis and uses thereof |
CN104520428A (en) * | 2012-02-17 | 2015-04-15 | 费城儿童医院 | Aav vector compositions and methods for gene transfer to cells, organs and tissues |
US20180221457A1 (en) * | 2015-07-31 | 2018-08-09 | Val-Chum, Limited Partnership | Glycerol-3-phosphate phosphatase activators |
US20190211103A1 (en) * | 2018-01-05 | 2019-07-11 | Gnt Biotech & Medicals Corporation | Pharmaceutical combination and method for regulation of tumor microenvironment and immunotherapy |
CN112689515A (en) * | 2018-07-17 | 2021-04-20 | 国家医疗保健研究所 | Compositions and methods for increasing or enhancing transformation of gene therapy vectors and for removing or reducing immunoglobulins |
CN110699451A (en) * | 2019-07-19 | 2020-01-17 | 广州市第一人民医院(广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院) | GPD1 expression quantity detection kit and application of metformin in assisting cancer treatment |
Also Published As
Publication number | Publication date |
---|---|
WO2023280175A9 (en) | 2023-02-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7094236B2 (en) | Compositions, methods and uses for mutant AAV and gene transfer into cells, organs and tissues | |
EP3288594B1 (en) | Dual aav vector system for crispr/cas9 mediated correction of human disease | |
CN105408486B (en) | Capsid-modified RAAV3 vector compositions and uses in gene therapy of human liver cancer | |
US20190300903A1 (en) | Recombinant virus products and methods for inhibition of expression of dux4 | |
BR112020001940A2 (en) | cell models of and therapies for eye diseases | |
KR102398039B1 (en) | Selective gene therapy expression system | |
CA3091806A1 (en) | Novel adeno-associated virus (aav) vectors, aav vectors having reduced capsid deamidation and uses therefor | |
US20180169269A1 (en) | Methods of delivery of transgenes for treating brain diseases | |
KR20210006327A (en) | Novel adeno-associated virus (AAV) vectors with reduced capsid deamidation and uses thereof | |
US20040142416A1 (en) | Treatment for phenylketonuria | |
US11717560B2 (en) | Compositions comprising nucleic acid molecules and methods of treating ATPase-mediated diseases | |
KR20220004696A (en) | Compositions useful for the treatment of Pompe disease | |
JP2022046792A (en) | Modified u6 promoter systems for tissue-specific expression | |
TW202022116A (en) | Variant rnai against alpha-synuclein | |
CN116134134A (en) | Trifunctional adeno-associated virus (AAV) vectors for the treatment of C9ORF 72-related diseases | |
Fröhlich et al. | Dual-function AAV gene therapy reverses late-stage Canavan disease pathology in mice | |
US20210324417A1 (en) | Products and methods for inhibition of expression of mutant gars protein | |
TW201837173A (en) | shRNA expression cassette, polynucleotide sequence carrying same and application thereof sequentially containing a DNA sequence for expressing shRNA and a filling sequence according to a sequence 5'-3' | |
BR112020015798A2 (en) | COMPOSITIONS OF ADEN-ASSOCIATED VIRUSES TO RESTORE PAH GENE FUNCTION AND METHODS OF USING THE SAME | |
Ojano-Dirain et al. | An animal model of PDH deficiency using AAV8-siRNA vector-mediated knockdown of pyruvate dehydrogenase E1α | |
WO2023280175A1 (en) | Methods for treating complex i deficiencies or cancers by modulating gro3p biosynthesis | |
US20230090989A1 (en) | AAV-Mediated Targeting of MIRNA in the Treatment of X-Linked Disorders | |
US20230323390A1 (en) | Methods and compositions for expressing phenylalanine hydroxylase | |
US20220098614A1 (en) | Compositions and Methods for Treating Oculopharyngeal Muscular Dystrophy (OPMD) | |
JP2023515442A (en) | Muscle-specific nucleic acid regulatory elements and methods and uses thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22836924 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |