Synthetic mmab genes and aav vectors to treat cobalamin b deficiency

Abstract

Synthetic polynucleotides encoding human MMAB (Metabolism Of Cobalamin Associated B) enzyme (synMMAB) and exhibiting augmented expression in cell culture and / or in a mammal are described herein. Related recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the synMMAB polynucleotides are also described. Methods of treating a disease or condition mediated by MMAB enzyme are also described.

Description

SYNTHETIC MMAB GENES AND AAV VECTORS TO TREAT COBALAMIN B DEFICIENCYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 499,013, filed April 28, 2023, which is incorporated by reference.STATEMENT REGARDINGFEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under project number 1ZIAHG200318-19 by the National Institutes of Health, National Human Genome Research Institute. The Government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 96,899 Byte XML file named “770632.XML," dated April 5, 2024.BACKGROUND OF THE INVENTION

[0004] Isolated methylmalonic acidemia (MMA) comprises a relatively common and heterogeneous group of inborn errors of metabolism. There are four major distinct genetic etiologies of isolated MMA. Mutations in the methylmalonyl-CoA mutase (MMUT) apoenzyme, the mitochondrial 5’-deoxyadenosylco(I)balamin transferase (MMAB) (Metabolism Of Cobalamin Associated B), or in the MMAA (Metabolism Of Cobalamin Associated A) enzyme define the mut, cblB, and cblA complementation groups, respectively. Cells from patients with absent MMUT activity are designated muf\ cells with detectable but abnormal activity are designated mut. Children with MMAA mutations have a greater chance of responding clinically to supplemental vitamin Bn, and usually are not as severely affected as children with mut forms of MMA. Most of those with MMAB mutations (cblB class MMA) clinically resemble patients with muf MMA for the reason that they have completely absent MMUT activity because they cannot synthesize 5’-deoxyadenosylcobalamin, thecofactor for the hMUT enzyme. Patients with cblD-variant 2 MMA, an ultra-rare inborn error of metabolism (IEM) with only several patients in the world recognized to date, resemble muf and non-B12 responsive cblB patients in terms of biochemical and clinical severity.

[0005] Most affected individuals with vitamin B 12 non-responsive isolated MMA display severe multisystemic disease characterized by metabolic instability, chronic renal disease, and neurological complications. The treatment entails adherence to a low protein diet, carnitine supplementation and vigilant clinical monitoring. However, despite meticulous medical management, patients with MMUT and MMAB deficiency suffer from substantial mortality and morbidity related to the disease. Most commonly, premature death occurs in the setting of an acute metabolic crisis and has led to the use of elective liver or liver / kidney transplantation as a treatment some patients. While these experimental surgical procedures do not completely cure the biochemical phenotype, metabolic stability is restored after liver transplantation and the propensity for acute decompensation is eliminated. However, the patients must adhere to a life-long regimen of anti -rejection and immune suppression medicines and suffer the sequelae experienced by all transplant recipients, including the risk for graft failure, rejection, and have an increased incidence of malignancy. There are currently no gene therapies that have been developed for this severe pediatric metabolic disorder. Accordingly, there exists a need for improved compositions and methods for treating diseases and conditions mediated by MMAB enzyme.BRIEF SUMMARY OF THE INVENTION

[0006] An aspect of the invention provides a synthetic MMAB (Metabolism Of Cobalamin Associated B) polynucleotide (syriMMAB) selected from the group consisting of:(a) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-6;(b) a polynucleotide having a nucleic acid sequence with at least about 80% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-6 and encoding a polypeptide having an amino acid sequence at least about 90% identical to SEQ ID NO: 21 and having equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 7 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 7; and (c) a polynucleotide having a nucleic acid sequence with at least about 80% identity to the nucleic acid sequence of nucleotides 97-753 of any one of SEQ ID NOs: 1-6 and encoding a polypeptide having an amino acid sequence at least about 90% identical toSEQ ID NO: 23, and having equivalent or increased expression in a mammalian host cell relative to expression of nucleotides 97-753 of SEQ ID NO: 7 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 97-753 of SEQ ID NO: 7.

[0007] Further aspects of the invention provide recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the synMMAB polynucleotides of the invention.

[0008] Still further aspects of the invention provide methods of treating a disease or condition mediated by MMAB enzyme.

[0009] Another aspect of the invention provides a method of detecting the presence of a synthetic MMAB polynucleotide (synMMAB) in a biological sample from a mammal, the method comprising: (a) obtaining at least one test sample comprising isolated nucleic acid from a biological sample from a mammal; (b) contacting any of the inventive synthetic polynucleotide described herein with the at least one test sample under conditions allowing for a complex to form between the synthetic polynucleotide and the isolated nucleic acid of the test sample; (c) detecting the complex; and (d) comparing a presence of the complex in the at least one test sample with an absence of complex from a negative sample that lacks the synthetic polynucleotide, wherein detection of the complex is indicative of the presence of the synthetic polynucleotide in the biological sample from the mammal.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0010] Figure 1 A is a map of the electrophoresis PCR products of MMAB Tl-4, Tl-5, and Tl-8 that used PCR primers flanking the site for the guide RNA that was used to engineer mutations in the MMAB gene.

[0011] Figure IB is an alignment of the sequences of MMAB Tl-NC, MMAB Tl-4, MMAB Tl-5, and MMAB Tl-8. The gRNA sites are shaded in black and INDEL mutations are marked by arrows.

[0012] Figure 1C is an image of a western blot showing the level of MMAB expression in HEK 293 MMAB KO Tl-4 cells (lane 1), HEK 293 MMAB KO Tl-8 cells (lane 2), HEK 293 MMAB Tl-5 cells (lane 3), and parental control HEK 293 cells (lane 4).

[0013] Figures 2A-2E show an alignment of MMAB polynucleotides.

[0014] Figure 2F is a phylogenetic tree of MMAB polynucleotides.

[0015] Figures 3 A-3B are a map of the single-stranded AAV cassette (Figure 3 A) and the self-complementary AAV cassette (Figure 3B).

[0016] Figures 4A-4C are images of western blots for expression of MMAB and bar graphs showing the results of western blot analysis in HEK 293 MMAB KO Tl-8 cells transfected with no plasmid (Tl-8-0), pAAV EF1L MMAB WT plasmid (Tl-8-1), pAAV EF1L synMMABl (Tl-8-2), pAAV EF1L synMMAB2 plasmid (Tl-8-3), pscAAV EFIs MMAB WT BGH plasmid (T 1-8-4), pscAAV EFIs MMAB WT RGB plasmid (T 1-8-5), pscAAV EFIs synMMABl plasmid (T 1-8-6), pscAAV EFIs synMMAB2 plasmid (T 1-8-7), pscAAV EFIs synMMAB3 plasmid (T 1-8-8), pscAAV EFIs synMMAB4 plasmid (T 1-8-9), pscAAV EFIs synMMAB5 plasmid (Tl-8- 10), and pscAAV EFIs synMMAB6 plasmid (Tl- 8-11), compared to HEK293 MMAB WT cells when using 2.5 pg of plasmid (Figure 4A), 1 pg of plasmid (Figure 4B), and 0.25 pg of plasmid (Figure 4C).

[0017] Figures 5A-5C are images of western blots for expression of MMAB and bar graphs showing the results of western blot analysis in HEK 293 MMAB KO Tl-4 cells transfected with no plasmid (Tl-4-0), pAAV EF1L MMAB WT plasmid (Tl-4-1), pAAV EF1L synMMABl (Tl-4-2), pAAV EF1L synMMAB2 plasmid (Tl-4-3), pscAAV EFIs MMAB WT BGH plasmid (T 1-4-4), pscAAV EFIs MMAB WT RGB plasmid (T 1-4-5), pscAAV EFIs synMMABl plasmid (T 1-4-6), pscAAV EFIs synMMAB2 plasmid (T 1-4-7), pscAAV EFIs synMMAB3 plasmid (T 1-4-8), pscAAV EFIs synMMAB4 plasmid (T 1-4-9), pscAAV EFIs synMMAB5 plasmid (Tl-4- 10), and pscAAV EFIs synMMAB6 plasmid (Tl- 4-11), compared to HEK293 MMAB WT when using 2.5 pg of plasmid (Figure 5A), 1 pg of plasmid (Figure 5B), and 0.25 pg of plasmid (Figure 5C).

[0018] Figures 6A-6B are an image of a western blot for expression of MMAB (Figure 6A) and a bar graph showing the results of western blot analysis in HEK 293 MMAB KO Tl- 8 cells given no treatment, or infected with AAV8 EF1L synMMAB2(ss) (striped bars) at moiety of infection (moi) doses of 5E+4, 1E+5, 5E+5, and 1E+6 or AAV8 EFIs synMMAB5(y>c) (solid bars) at moiety of infection (moi) doses of 2.5E+4, 5E+4, 2.5E+5, and 5E+6, compared to HEK293 MMAB WT cells (Figure 6B).

[0019] Figures 7A-7B are an image of a western blot for expression of MMAB (Figure 7A) and a bar graph showing the results of western blot analysis in HEK 293 MMAB KO Tl- 8 cells given no treatment, or infected with AAV9 EF1L synMMAB2(ss) (striped bars) at moiety of infection (moi) doses of 5E+4, 1E+5, 5E+5, and 1E+6 or AAV9 EFIssynMMAB5(sc) (solid bars) at moiety of infection (moi) doses of 2.5E+4, 5E+4, 2.5E+5, and 5E+6, compared to HEK293 MMAB WT cells (Figure 7B).

[0020] Figures 8A-8B are an image of a western blot for expression of MMAB (Figure 10A) and a bar graph showing the results of western blot analysis in HEK 293 MMAB KO Tl-4 cells given no treatment, or infected with AAV8 EF1L synMMAB2(ss) (striped bars) at moiety of infection (moi) doses of 5E+4, 1E+5, 5E+5, and 1E+6 or AAV8 EFIs synMMAB5(y>c) (solid bars) at moiety of infection (moi) doses of 2.5E+4, 5E+4, 2.5E+5, and 5E+6, compared to HEK293 MMAB WT cells (Figure 8B).

[0021] Figures 9A-9B are an image of a western blot for expression of MMAB (Figure 9A) and a bar graph showing the results of western blot analysis in HEK 293 MMAB KO Tl- 4 cells (striped bars) and HEK 293 MMAB KO Tl-8 cells (solid bars) given no treatment, or infected with AAV9 EF1L synMMAB2(ss) at moiety of infection (moi) doses of 5E+4, 1E+5, 5E+5, and 1E+6, compared to HEK293 MMAB WT cells (Figure 9B).

[0022] Figure 10 presents a line graph showing the probability of survival for WT mice, heterozygous MmabWT / A3'7mice, homozygous MmabA3'7 / A3'7mice, and homozygous MmabA3-7 A3-7mice systemically administered scAAV9 EFIs synMMAB5 on DOL1 (Mt+ scAAV9 EFIs syriMMAB5). Figure 10 presents data collected from mice treated with approximately 5el0 GC / pup.

[0023] Figure 11 is a bar graph showing circulating plasma methylmalonic acid (MMA) levels in micromoles (uM) in MMABWT / WT;TgROSA26'MCK'Mmafemice, MMABWT / R180W'TgROSA26'MCK'Mmafemice MMABR180W / R180W'TgROSA26'MCK'Mma6mice and MMABR180W / R180W;TgROSA26'MCK'Mmafemice treated with the ssAAV9 EF1L synMMAB2 or scAAV9 EFIs synMMAB5 vectors at a time point in young adulthood (circle) and at a time point two weeks later or two weeks after infection.

[0024] Figure 12 is a line graph showing results of a 1-13C propionate oxidation in MMAR ‘'3',t3 ,TpRosA26-MCK-A / mafe mice (circle) MM ABR180W / R180W,TgROSA26~MCK~A7mafemice (square), and MMABR180W / R180W;TgROSA26'MCK'Mmafemice treated with the ssAAV9 EF1L synMMAB2 (upward triangle) or scAAV9 EFIs synMMAB5 (downward triangle) vectors two weeks after infection.

[0025] Figures 13A-13B is an image of a western blot (WB) for expression of MMAB and a bar graph showing the results of western blot analysis in the liver of threeMM ABWT / WT' TpRoSA26-MCK-A / mafe mice three MMABWT / R380W,TgROSA26~MCK~A7mafemice three MM ABR38ow / Ri SOW . TgRos A26-MCK-A / mafe mice three MM / \ RRISOW / RI8OW. T^IWS A 26- \ IC K-A / >»a / > micetreated with the ssAAV9 EF1L synMMAB2, and three MMABR180W / R180W;TgROSA26'MCK'Mmafemice treated with the scAAV9 EFIs synMMAB5 vectors (Figure 13 A), and a bar graph showing the same western blot analysis with the data averaged for three mice in each group (Figure 13B).

[0026] Figure 13C is a bar graph showing the results of RT-qPCR analysis of MMAB mRNA expression levels in the liver of MMABWT / WT;TgROSA26'MCK'Mmafemice(circle),and MMABR180W / R180W;TgROSA26'MCK'Mmafemice treated with the ssAAV9 EF1L synMMAB2 or scAAV9 EFIs synMMAB5 vectors (square) compared to endogenous levels of mouse Mmab mRNA (circles).

[0027] Figures 14A-14B is an image of a western blot for expression of MMAB and a bar graph showing the results of western blot analysis in the kidney of threeMM ABWT / WT' TpRoSA26-MCK-A / mafe mice three MMABWT / R180W,TgROSA26~MCK~A^mafemice three MM A BR 180 W / R 18ow •TgRQs A26-MCK-Mmafemice three MM A BR' 8Q W / R ' so w . T^ROS \ 26- \ ic K-A / >»a / > mice treated with the ssAAV9 EF1L synMMAB2, and three MMABR180W / R180W;TgROSA26'MCK'Mmafemice treated with the scAAV9 EFIs synMMAB5 vectors (Figure 14A), and a bar graph showing the same western blot analysis with the data averaged for three mice in each group (Figure 14B).

[0028] Figure 14C is a bar graph showing the results of RT-qPCR analysis of MMAB mRNA expression levels in the kidney of MMABWT / WT;TgROSA26'MCK'Mmafemice(circle),mice(circle) MM A BR' so W / R ' so w . T^RCAS \ 26- \ ic K-A / >»a / > mice(circle), and MMABR180W / R180W;TgROSA26'MCK'Mmafemice treated with the ssAAV9 EF1L synMMAB2 or scAAV9 EFIs synMMAB5 vectors (square) compared to endogenous levels of mouse Mmab mRNA (circles).

[0029] Figure 15 is an illustration of the process of ss MMAB AAV vector optimization for humans showing the changes in plasmid maps to develop the Final ss AAV cassette (pAAV EF IL MMAB Kan).

[0030] Figures 16A-16B are images of western blots bar graphs showing the results of western blot analysis for expression of MMAB in HEK 293 MMAB KO Tl-4 cells (Figure 16A) and HEK 293 MMAB KO Tl-8 cells (Figure 16B) for cells given no treatment, or transfected with pAAV EF1L synMMAB2, pAAV EF1L synMMAB2 KC0002, pAAV EF1L MMAB KAN, compared to HEK 293 MMAB WT cells.

[0031] Figure 17 is an illustration of the process of sc MMAB AAV vector optimization for humans showing the changes in plasmid maps to develop the Final sc AAV cassette (pscAAV EF 1 s MMAB Kan).

[0032] Figure 18 is an image of a western blot showing the results of expression of MMAB in HEK 293 MMAB KO Tl-4 cells given no treatment, or transfected with pAAV EF IL synMMAB2, pAAV EF IL MMAB Kan, pscAAV EF 1 s synMMAB5, pscAAV EF 1 s MMAB Kan, compared to HEK 293 MMAB WT cells.

[0033] Figure 19 is a bar graph showing expression of MMAB in HEK 293 MMAB KO Tl-4 cells given no treatment, or transfected with pAAV EF1L synMMAB2, pAAV EF1L MMAB Kan, pscAAV EFIs synMMAB5, pscAAV EFIs MMAB Kan, compared to HEK 293 MMAB WT cells.

[0034] Figure 20A is a plot of data representing the survival of MmabA3'7 / Q228Xmice challenged with a diet consisting of a 10-fold increase in Isoleucine and Valine with (®) and without (•) treatment with the AAV9.EF1LMMAB Kan vector. Figure 20B is a plot of data representing the pMMA levels measured from Mmctb ''3'1 / Q228Xmice, Mmab Q228X / Q228Xmice, and ATmaZ>A3'7 / A3'7;TgMCK'Mmab(C57BL / 6) mice challenged with the high Isoleucine / Valine diet with and without treatment the AAV9.EF1 LMMAB Kan vector.

[0035] Figures 21A-21C present data concerning the phenotype of the Mmab^’11^’7(C57BL / 6 x FVB / N) mouse strain. Figure 21 A presents a survivability probability plot comparing MmaZ>A3'7 / A3'7(C57BL / 6 x FVB / N) homozygous mice with heterozygous mice and wild-type mice. Figure 2 IB presents data concerning the average mass of Mmab^3'1^3'7(C57BL / 6 x FVB / N) homozygous mice, heterozygous mice, and wild-type mice measured between 22 and 26 days old; data for males are presented in the left column, those for females are presented in the right column. Figure 21C presents data concerning the pMMA levels for MmaZ>A3'7 / A3'7(C57BL / 6 x FVB / N) homozygous mice, heterozygous mice, and wild-type mice.

[0036] Figure 22 presents graphs presenting data concerning the metabolic response in Mmab^3'11^3'1mice as compared to wild-type mice and after scAAV9 treatment. Data concerning pMMA are presented in the left panel, while data 1-13C propionate oxidation are presented in the right panel. The vectors employed were those depicted in Figure 3, which were delivered at approximately 5E+10 viral genomes (VG) / pup. Data were obtained six months for wild-type mice and post-neonatal treatment of A7 / wAA3_7 / A3'7mice with scAAV9, except those for untreated Mmab^3'11^3'1mice, which were collected at 25 days of age.

[0037] Figure 23 presents images demonstrating the histopathology in Mmab^3'11^3'1mouse liver (middle panel), as compared to wild-type mice (left panel) and after neonatal scAAV9 MMAB gene therapy administered to A / maZ>A3'7 / A3'7mice (right panel). Data for the untreated Mmab^3'11^3'1mice and wild-type mice were collected at 45 days of age, while those for the treated Mmab^3'11^3'1mice were collected at 51 days of age.

[0038] Figure 24 presents data concerning the levels of MMAB expression seen in the liver, heart, kidney, and muscle tissue from untreated Mmab^3'11^3'1mice, as compared to wild-type mice and after neonatal scAAV9 MMAB gene therapy administered to MmabK3~7 / A3'7mice. Data were obtained six months for wild-type mice and post-neonatal treatment of MmaZ>A3'7 / A3'7mice with scAAV9, while those for untreated MmaZ>A3'7 / A3'7mice, which were collected at 25 days of age.

[0039] Figures 25A-25D present images of tissue obtained from MmabA3'7 / A3'7(C57BL / 6 x FVB / N) mice following neonatal treatment with scAAV9 EFIs synMMAB5 (5E10 GC / pup) and probed with synMMAB5 RNA in an RNA in situ hybridization study (RNAscope). Figure 25A depicts brain, liver, heart, kidney, muscle, spleen. Figure 25B depicts liver tissue; Figure 25C depicts heart tissue; and Figure 25D depicts kidney tissue.

[0040] Figures 26A-26F present images of Western blots for expression of MMAB (Figures 26A, 26C, and 26E, top panel: AAV9 EF1L MMAB Kan (ss); bottom panel: AAV9 EFIs MMAB Kan (sc)) and bar graphs (Figures 26B, 26D, and 26F) showing the results of western blot analysis in HEK 293 MMAB KO Tl-8 cells (Figures 26A-26D) or HEK 293 MMAB KO Tl-4 cells (Figures 26E and 26F) given no treatment, or infected with AAV9 EF1L MMAB Kan (ss) or AAV9 EFIs MMAB Kan (sc) at moiety of infection (moi) doses of 5E+4, 1E+5, 5E+5, and 1E+6 (Figure 26A and 26B), or AAV9 EF1L MMAB Kan (ss) at moiety of infection (moi) doses of 5E+4, 1E+5, 5E+5 and 1E+6 , and AAV9 EFIs MMAB Kan (sc) at moiety of infection (moi) doses of 1E+5, 2E+5, 1E+6, and 2E6 (Figures 26C, 26D, 26E, 26F), compared to HEK293 MMAB WT cells. For the Western blotting, total protein was loaded at 12.5 pg / lane (Figures 26A and 26B) or 25pg / lane (Figures 26C, 26D, 26E and 26F). The vectors employed in the experiments leading to Figures 26A and 26B were constructed independently of those employed for Figures 26C-26F.

[0041] Figures 27A and 27B are line graphs showing the probability of survival for WT mice (+ / +), heterozygous MmabWT / A3'7((C57BL / 6 x FVB / N)) mice (+ / -), homozygous MmabA3-7 A3-7((C57BL / 6 x FVB / N)) mice (- / -), and homozygous MmabA3'7 / A3'7mice systemically administered AAV9.EF1L MMAB Kan (ss) or scAAV9.EFls MMAB Kan (sc)on DOL1 or at wean. The vectors employed in the experiment leading to Figure 27 A were constructed independently of that employed for Figure 27B.

[0042] Figures 28A and 28B present data concerning the metabolic response in Mmab '3'7 / A3'7(C57BL / 6 x FVB / N) mice as compared to heterozygotes and wild-type mice and to the homozygotes after treatment with AAV9.EF1L MMAB Kan (ssAAV9) or scAAV9.EFls MMAB Kan (scAAV9). Data concerning pMMA are presented in Figure 28A; data concerning FGF21 levels are presented in Figure 28B.

[0043] Figures 29A and 29B present data concerning the expression of MMAB in the liver (Figure 29A) and kidney (Figure 29B) tissue of Mmab^3'1l^3'1(C57BL / 6 x FVB / N) mice as compared to heterozygotes and wild-type mice and to the homozygotes after treatment with AAV9.EF1L MMAB Kan (ss) or scAAV9.EFls MMAB Kan (sc).

[0044] Figure 30 is a graph presenting the survival probability of mouse strains including the ATmaZ>A3'7 / A3'7;TgMCK'Mmab(C57BL / 6) strain.

[0045] Figure 31 presents data concerning the pMMA levels for mouse strains containing the TgMCK-Mmaballele, including mice also homozygous for the Mmab '3'1trait, heterozygous Mmab3, 1''3'1mice, and wild-type with respect to the Mmab '3'1trait.

[0046] Figure 32 presents data concerning the levels of MMAB expression seen in the liver, heart, kidney, and muscle tissue from untreated ATmaZ>A3'7 / A3'7;TgMCK'Mmab(C57BL / 6) mice and in TgMCK-Mmabmice,which were either wild-type or heterozygous with respect to Mmab.

[0047] Figures 33A and 33B present data concerning plasma MMAB levels measured in untreated (pretreated) neonatal ATmaZ>A3'7 / A3'7;TgMCK'Mmab(C57BL / 6) mice and for those mice 14days, 30 days, 60 days and 90 days (as indicated in each figure, respectively) posttreatment with either AAV9.EF1L MMAB Kan (ss) or scAAV9.EFls MMAB Kan (sc). The vectors employed in the experiment leading to Figure 33A were constructed independently of that employed for Figure 33B.DETAILED DESCRIPTION OF THE INVENTION

[0048] Aspects of the invention provide synthetic MMAB polynucleotides (synMMAB polynucleotides) that may provide any one or more of a variety of advantages. For example, the inventive synMMAB polynucleotides may provide enhanced expression in mammalian cells. Compared to wild-type (WT) human polynucleotides encoding MMAB enzyme, the inventive synMMAB polynucleotides are codon-optimized to enhance expression uponadministration. The inventive synMMAB polynucleotides may provide any one or more of improved survival, reduction in metabolites, increased metabolic capacity to C13 propionic acid oxidation, and robust MMAB expression in the liver and kidney, e.g., in a mouse model of model of cblB MMA.

[0049] The inventive synMMAB polynucleotides may, advantageously, be useful as a therapeutic, via viral or non-viral mediated gene delivery, to restore MMAB enzyme function in MMA patients. The inventive synMMAB polynucleotides may reduce or prevent metabolic instability and may ameliorate disease progression. The inventive synMMAB polynucleotides may also be useful for the in vitro production of MMAB enzyme for use in enzyme replacement therapy for MMA. Because the MMAB enzyme may also mediate other disorders of branched chain amino acid oxidation, the inventive synMMAB polynucleotides may be useful for treating conditions other than cblB MMA.

[0050] The inventive synMMAB polynucleotides may provide increased expression of the MMAB gene relative to naturally occurring human AZAMB sequences. In aspects, the inventive synMMAB polynucleotides were designed to not alter the naturally occurring human MMAB enzyme amino acid sequence. They were also designed to have any one or more of increased transcriptional, translational, and protein refolding efficacy. This design was accomplished by, e.g., any one or more of the evaluation of human codon biases, evaluation of GC, CpG, and negative GpC content, evaluation of the interaction between the codon and anti-codon, reducing or eliminating cryptic splicing sites and RNA instability motifs, and compatibility with the vector backbones.

[0051] As used herein, “MMAB” refers to human Metabolism Of Cobalamin Associated B enzyme (also referred to as “MMAB enzyme,” “cob(I)alamin adenosyltransferase,” or “5’- deoxyadenosylco(I)balamin transferase”). This protein catalyzes the final step in the conversion of vitamin B(12) into 5 ’-adenosylcobalamin (AdoCbl), a vitamin Bl 2-containing coenzyme for methylmalonyl-CoA mutase. The amino acid sequence of human WT MMAB enzyme is set forth in SEQ ID NO: 21. The gene encoding naturally occurring human MMAB enzyme is referred to as MMAB (also referred to as “cblB”). The WT human MMAB nucleic acid sequence is set forth in SEQ ID NO: 7. The polynucleotides encoding synthetic MMAB enzyme are referred to as synMMAB. Naturally occurring human AZAMB is referred to as AZAMB, while synthetic AZAMB is designated as synMMAB, even though the two are identical at the amino acid level.

[0052] The MMAB enzyme is processed after transcription, translation, and translocation into the mitochondrial inner space. During this importation and maturation process, the mitochondrial transit peptide, which corresponds to amino acids 1-32 of SEQ ID NO: 21, are removed to produce the mature MMAB enzyme, which is comprised of residues 33-250. The amino acid sequence of the wild type MMAB mitochondrial transit peptide is MAVCGLGSRLGLGSRLGLRGCFGAARLLYPRF (SEQ ID NO: 22). The amino acid sequence of the mature MMAB enzyme is SEQ ID NO: 23.

[0053] An aspect of the invention provides a synthetic MMAB polynucleotide (syriMMAB) selected from the group consisting of: (a) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-6; (b) a polynucleotide having a nucleic acid sequence with at least about 80% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-6 and encoding a polypeptide having an amino acid sequence at least about 90% identical to SEQ ID NO: 21, and having equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 7 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 7; and (c) a polynucleotide having a nucleic acid sequence with at least about 80% identity to the nucleic acid sequence of nucleotides 97-753 of any one of SEQ ID NOs: 1-6 and encoding a polypeptide having an amino acid sequence at least about 90% identical to SEQ ID NO: 23, and having equivalent or increased expression in a mammalian host cell relative to expression of nucleotides 97-753 of SEQ ID NO: 7 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 97-753 of SEQ ID NO: 7.

[0054] In one aspect of the invention, codon optimization was employed to create a highly active and synthetic MMAB polynucleotide. Accordingly, in an aspect, the synthetic polynucleotide is codon-optimized. “Codon optimization” refers to the process of altering a naturally occurring polynucleotide sequence to enhance expression in the target organism, e.g., humans. Codon optimization involves determining the relative frequency of a codon in the protein-encoding genes in the human genome. For example, isoleucine can be encoded by AUU, AUC, or AU A, but in the human genome, AUC (47%), AUU (36%), and AUA (17%) are variably used to encode isoleucine in proteins. Therefore, in the proper sequence context, AUA would be changed to AUC to allow this codon to be more efficiently translated in human cells.

[0055] In the subject application, the human MMAB gene has been altered to replace codons that occur less frequently in human genes with those that occur more frequentlyand / or with codons that are frequently found in highly expressed human genes. A series of synthetic codon optimized and adjusted MMAB genes were developed (Table 2). An alignment (Figures 2A-2E) and phylogenetic analysis prepared with CLUSTAL W (Figure 2F) reveals that the synMMAB polynucleotides are highly divergent at the nucleotide level while maintaining WT MMAB amino acid sequence identity.

[0056] Thus, as aspect of the invention comprises MMAB enzyme-encoding, synthetic polynucleotides comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-6.

[0057] Another aspect of the invention provides a polynucleotide having a nucleic acid sequence with at least about 80% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-6 and encoding a polypeptide having an amino acid sequence at least about 90% identical to SEQ ID NO: 21 and having equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 7 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 7. Additional aspects of the invention provide a polynucleotide having a nucleic acid sequence with at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-6 and encoding a polypeptide having an amino acid sequence at least about 90% identical to SEQ ID NO: 21, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 7.

[0058] Another aspect of the invention provides a polynucleotide having a nucleic acid sequence with at least about 80% identity to the nucleic acid sequence of nucleotides 97-753 of any one of SEQ ID NOs: 1-6 and encoding a polypeptide having an amino acid sequence at least about 90% identical to SEQ ID NO: 23, and having equivalent or increased expression in a mammalian host cell relative to expression of nucleotides 97-753 of SEQ ID NO: 7 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 97-753 of SEQ ID NO: 7. Additional aspects of the invention provide a polynucleotide having a nucleic acid sequence with at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the nucleic acidsequence of nucleotides 97-753 of any one of SEQ ID NOs: 1-6 and encoding a polypeptide having an amino acid sequence at least about 90% identical to SEQ ID NO: 23, wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 97-753 of SEQ ID NO: 7.

[0059] In one aspect, the inventive synthetic polynucleotide encodes a polypeptide with 100% sequence identity to the naturally occurring, human MMAB enzyme (SEQ ID NO: 21) or the mature WT MMAB enzyme of SEQ ID NO: 23.

[0060] In another aspect, inventive synthetic polynucleotide encodes a polypeptide with at least about 90% identity to SEQ ID NO: 21 or the mature WT MMAB enzyme of SEQ ID NO: 23 and retaining the naturally occurring human MMAB protein function i.e., the capacity to catalyze the final step in the conversion of vitamin B(12) into adenosylcobalamin (AdoCbl). Additional aspects provide a synthetic polynucleotide that encodes a polypeptide with at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 21 or the mature WT MMAB enzyme of SEQ ID NO: 23 and retaining the naturally occurring human MMAB protein function.

[0061] In one aspect, the MMAB enzyme encoded by the inventive polynucleotides retains at least about 90% of the naturally occurring human MMAB protein function, i.e., the capacity to catalyze the final step in the conversion of vitamin B(12) into adenosylcobalamin (AdoCbl). In another aspect, the encoded MMAB enzyme retains at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the naturally occurring human MMAB enzyme function. This enzyme function can be measured, for example, by measuring the ability to provide any one or more of improved survival, reduction in metabolites, and increased metabolic capacity to C13 propionic acid oxidation e.g., in a mouse model of model of cblB MMA, for example, as described in the Examples below.

[0062] In some aspects, the synthetic polynucleotide exhibits improved expression in a mammalian host cell relative to the expression of naturally occurring human MMAB polynucleotide sequence. The improved expression is due to the polynucleotide comprising codons that have been optimized relative to the naturally occurring human MMAB polynucleotide sequence of SEQ ID NO: 7 (or nucleotides 97-753 of SEQ ID NO: 7). In one aspect, the synthetic polynucleotide has at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of less commonly used codons replaced with more commonly used codons. In additional aspects, the polynucleotide has at least about 85%, at least about 90%, or at least about 95% of less commonly used codons replaced with more commonly used codons, and demonstrate equivalent or enhanced expression of MMAB in a mammalian cell as compared to the expression of SEQ ID NO: 7 (or nucleotides 97-753 of SEQ ID NO: 7) in the same mammalian host cell.

[0063] In some aspects, the synthetic polynucleotide sequences of the invention provide equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 7 (or nucleotides 97-753 of SEQ ID NO: 7) (as demonstrated by expression of the polynucleotide of SEQ ID NO: 7 (or nucleotides 97-753 of SEQ ID NO: 7) in the same mammalian host cell). In some aspects, the inventive polynucleotide exhibits an increase in expression in a mammalian host cell relative to the expression SEQ ID NO: 7 (or nucleotides 97-753 of SEQ ID NO: 7) in the same mammalian host cell. In some aspects, the inventive polynucleotide preferably encodes a polypeptide that retains at least about 80% of the enhanced MMAB expression (as demonstrated by expression of the polynucleotide of any one of SEQ ID NOs: 1-6 (or nucleotides 97-753 of any one of SEQ ID NOs: 1-6) in a mammalian host cell). In additional aspects, the polypeptide retains at least 85%, at least about 90%, at least about 95% or about 100% of the enhanced expression observed with the polynucleotide of any one of SEQ ID NOs: 1-6 (or nucleotides 97-753 of any one of SEQ ID NOs: 1-6) in a mammalian host cell. In aspects, the mammalian host cell is a human host cell or a mouse host cell. In some aspects, the mammalian host cell is a kidney cell or a liver cell from human or mouse.

[0064] In designing the synMMAB of the present invention, the following considerations were balanced. For example, the fewer changes that are made to the nucleotide sequence of SEQ ID NO: 7 (or nucleotides 97-753 of SEQ ID NO: 7), the less potential there is to alter the secondary structure of the sequence, which can have a significant impact on gene expression. The introduction of undesirable restriction sites was also reduced, facilitating the subcloning of synMMAB into the recombinant expression vector. However, a greater number of changes to the nucleotide sequence of SEQ ID NO: 7 (or nucleotides 97-753 of SEQ ID NO: 7) allows for more convenient identification of the translated and expressed message, e.g. mRNA, in vivo. Additionally, a greater number of changes to the nucleotide sequence of SEQ ID NO: 7 (or nucleotides 97-753 of SEQ ID NO: 7) provides for increased likelihood ofgreater expression. These considerations were balanced when arriving at the nucleic acid sequences of SEQ ID NO: 1-6 (or nucleotides 97-753 of any one of SEQ ID NOs: 1-6). The polynucleotide sequences encoding synMMAB allow for increased expression of the synMMAB polynucleotide relative to naturally occurring human MMAB polynucleotides. Because the sequences are novel, they may facilitate detection using nucleic acid-based assays.

[0065] MMAB has a total of 250 amino acids and synMMAB contains approximately 250 codons corresponding to said amino acids. In SEQ ID NOs: 1-6, codons were changed from that of the natural human MMAB. However, despite changes from SEQ ID NO: 7, SEQ ID NOs: 1-6 encode the amino acid sequence SEQ ID NO: 21 of WT, human MMAB. Codons for SEQ ID NOs: 1-6 were changed in accordance with the equivalent amino acid positions of SEQ ID NO: 21. In this aspect, the amino acid sequence for natural human MMAB has been retained.

[0066] Similarly, in nucleotides 97-753 of SEQ ID NOs: 1-6, codons were changed from that of the natural human MMAB. However, despite changes from nucleotides 97-753 of SEQ ID NO: 7, nucleotides 97-753 of SEQ ID NOs: 1-6 encode the amino acid sequence SEQ ID NO: 23 of WT, mature human MMAB. Codons for nucleotides 97-753 of SEQ ID NOs: 1-6 were changed in accordance with the equivalent amino acid positions of SEQ ID NO: 23. In this aspect, the amino acid sequence for natural human mature MMAB has been retained.

[0067] It can be appreciated that partial reversion of the designed synMMAB to codons that are found in MMAB can be expected to result in nucleic acid sequences that, when incorporated into appropriate vectors, can also exhibit the desirable properties of any one or more of SEQ ID NOs: 1-6 (or nucleotides 97-753 of any one of SEQ ID NOs: 1-6). For example, such partial reversion or hybrid variants can have MMAB expression from a vector inserted into an appropriate host cell that is equivalent to that of any one or more of SEQ ID NOs: 1-6 (or nucleotides 97-753 of any one of SEQ ID NOs: 1-6). For example, aspects of the invention include nucleic acids in which at least about 1 altered codon, at least about 2 altered codons, at least about 3, altered codons, at least about 4 altered codons, at least about 5 altered codons, at least about 6 altered codons, at least about 7 altered codons, at least about 8 altered codons, at least about 9 altered codons, at least about 10 altered codons, at least about 11 altered codons, at least about 12 altered codons, at least about 13 altered codons, at least about 14 altered codons, at least about 15 altered codons, at least about 16 alteredcodons, at least about 17 altered codons, at least about 18 altered codons, at least about 20 altered codons, at least about 25 altered codons, at least about 30 altered codons, at least about 35 altered codons, at least about 40 altered codons, at least about 50 altered codons, at least about 55 altered codons, at least about 60 altered codons, at least about 65 altered codons, at least about 70 altered codons, at least about 75 altered codons, at least about 80 altered codons, at least about 85 altered codons, at least about 90 altered codons, at least about 95 altered codons, at least about 100 altered codons, at least about 110 altered codons, at least about 120 altered codons, at least about 130 altered codons, at least about 130 altered codons, at least about 140 altered codons, at least about 150 altered codons, at least about 160 altered codons, at least about 170 altered codons, or at least about 180 altered codons, in any one of SEQ ID NOs: 1-6 (or nucleotides 97-753 of any one of SEQ ID NOs: 1-6) are reverted to native codons according to SEQ ID NO: 7 (or nucleotides 97-753 of SEQ ID NO: 7), and having equivalent or increased expression in a mammalian host cell as compared to expression of any one of SEQ ID NOs: 1-6 (or nucleotides 97-753 of any one of SEQ ID NOs: 1-6) in the same mammalian host cell. Alternately, at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the altered codon positions in any one of SEQ ID NOs: 1-6 (or nucleotides 97-753 of any one of SEQ ID NOs: 1-6) are reverted to the native sequence according to SEQ ID NO: 7 (or nucleotides 97-753 of SEQ ID NO: 7), and having equivalent or increased expression in a mammalian host cell as compared to expression of any one of SEQ ID NOs: 1-6 (or nucleotides 97-753 of any one of SEQ ID NOs: 1-6) in the same mammalian host cell.

[0068] The exact differences between SEQ ID NOs: 1-6 and SEQ ID NO: 7 demonstrate that the synMMAB polynucleotides are distinct from wild type human MMAB. Table 3 depicts the pairwise nucleotide identity between SEQ ID NOs: 1-6 and SEQ ID NO: 7. The values range between 73.1% and 77.8%. The exact positions of variation are depicted in a base-by- base comparison in Figures 2A-2E vs Table 3, and a phylogenetic analysis presented in FIG 2F further demonstrates that SEQ ID NOs: 1-6 are distinct.

[0069] In some aspects, polynucleotides of the present invention do not share 100% identity with SEQ ID NO: 7 (or nucleotides 97-753 of SEQ ID NO: 7). In other words, in some aspects, polynucleotides having 100% identity with SEQ ID NO: 7 (or nucleotides 97- 753 of SEQ ID NO: 7) are excluded from the aspects of the present invention.

[0070] In one aspect of a synthetic polynucleotide according to the invention, the nucleic acid sequence is a DNA sequence (e.g., a cDNA sequence). In another aspect, the nucleic acid sequence is a RNA sequence or peptide-modified nucleic acid sequence.

[0071] In another aspect, the invention is directed to a recombinant expression vector comprising any of the synMMAB polynucleotides described herein. In aspects of the invention, the recombinant expression vector may be single-stranded or self-complementary.

[0072] In an aspect of the invention, the recombinant expression vector may be a viral vector, such as a lentiviral vector, a retroviral vector, an alphaviral vector, a vaccinial viral vector, an adenoviral vector, a herpes viral vector, a fowl pox viral vector, or an adeno- associated viral (AAV) vector. While, in the Examples presented herein, studied vectors are AAV vectors derived from strains or serotypes 8 or 9 (AAV8 or AAV9 vectors), when the inventive vector is an AAV vector, such need not be limited to these exemplary strains or serotypes but can instead be from any suitable strain or serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV44.9, AAAV, BAAV, and the like). In other aspects of the invention, the recombinant expression vector may be a non-viral vector, such as plasmid DNA, liposome-DNA complex (lipoplexes), or polymer-DNA complex (polyplexes).

[0073] In certain aspects, the recombinant expression vector comprises an expression cassette. The expression cassette may, for example, include any one or more of a 5’ inverted terminal repeat (ITR), a promoter, one or more introns, a 5’ untranslated region (Kozak sequence), any of the inventive synMMAB polynucleotides described herein, an mRNA stability element, such as the WPRE (woodchuck post-transcriptional regulatory element) or HPRE (hepatitis B derived post-translational response element), a polyadenylation signal (e.g., bovine growth hormone polyadenylation signal (BGH A) or the rabbit beta-globin polyadenylation signal (rBGA)), CpG nucleotides, and a 3’ ITR.

[0074] In another aspect of the recombinant expression vector according to the invention, the synthetic polynucleotide is operably linked to an expression control sequence (e.g., a promoter). The promoter may vary with the type of viral recombinant expression vector used. For example, the promoter may be a viral promoter. Viral promoters include, for example, the ubiquitous cytomegalovirus immediate early (CMV-IE) promoter, the chicken beta-actin (CBA) promoter, the simian virus 40 (SV40) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, the Moloney murine leukemia virus (MoMLV) LTR promoter, and other retroviral LTR promoters. In a preferred aspect, the promoter is theEFIS (elongation factor 1 promoter, short) promoter or the EF1L (elongation factor 1 promoter, long) promoter.

[0075] In one specific aspect, the inventive synMMAB polynucleotide could be placed under the transcriptional control of a ubiquitous or tissue-specific promoter. The use of a tissue-specific promoter can restrict unwanted MMAB expression, as well as facilitate persistent MMAB expression. The inventive synMMAB polynucleotide could then be delivered into the systemic circulation, portal vein, or directly injected into a tissue or organ, such as the liver or kidney. In an aspect of the invention, the recombinant expression vector is configured for expression of the synMMAB polynucleotide in one or both of hepatic tissue and renal tissue. In addition to the liver or kidney, the brain, pancreas, eye, heart, lungs, bone marrow, and muscle may constitute targets for therapy. Other tissues or organs may be additionally contemplated as targets for therapy.

[0076] Tissue-specific promoters include, without limitation, Apo A-I, ApoE, hAAT, transthyretin, liver-enriched activator, albumin, PEPCK, and RNAPu promoters (liver), PAI- 1, ICAM-2 (endothelium), MCK, SMC a-actin, myosin heavy-chain, and myosin light-chain promoters (muscle), cytokeratin 18, CFTR (epithelium), GFAP, NSE, Synapsin I, Preproenkephalin, dpH, prolactin, and myelin basic protein promoters (neuronal), and ankyrin, a-spectrin, globin, HLA-DRa, CD4, glucose 6-phosphatase, and dectin-2 promoters (erythroid).

[0077] Regulatable promoters (for example, ligand-inducible or stimulus-inducible promoters) are also contemplated for expression constructs according to the invention. The inventive recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression.

[0078] In yet another aspect, the synMMAB polynucleotide could be used in ex vivo applications via packaging into a recombinant expression vector to create an integrating recombinant expression vector that could be used to permanently correct any cell type from a patient with MMAB deficiency. The synMMA / / -transduced and corrected cells could then be used as a cellular therapy. Examples might include CD34+ stem cells, primary hepatocytes, or fibroblasts derived from patients with MMAB deficiency. Fibroblasts could be reprogrammed to other cell types using iPS methods well known to practitioners of the art. In yet another aspect, the synMMAB polynucleotide could be recombined using genomic engineering and editing techniques that are well known to practitioners of the art, such asZFNs, TALENS and Cas / CRISPR, into the MMAB locus, a genomic safe harbor site, such as AAVS1, or into another advantageous location, such as into rDNA, the albumin locus, SERPINA 5 or 7, PCSK9, GAPDH, or a suitable expressed pseudogene.

[0079] In an aspect of the invention, the recombinant expression vector comprises the nucleic acid sequence of any one of SEQ ID NOs: 9-10 and 13-20.

[0080] Another aspect of the invention further provides a host cell comprising any of the polynucleotides or any of the recombinant expression vectors described herein. As used herein, the term “host cell” refers to any type of cell that can contain the inventive recombinant expression vector. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Examples of host cells are known in the art and include, for instance, DH5a E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., a DH5a cell. For purposes of producing an MMAB enzyme, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. The host cell may be, for example, a kidney cell or a liver cell from human or mouse.

[0081] Also provided by an aspect of the invention is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described herein, in addition to at least one other cell, e.g., a host cell (e.g., a kidney or liver cell), which does not comprise any of the recombinant expression vectors. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) comprising the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one aspect of the invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0082] The inventive synthetic polynucleotides, recombinant expression vectors, host cells (including populations thereof), all of which are collectively referred to as “inventivesynMMAB material(s)” hereinafter, can be formulated into a composition, such as a pharmaceutical composition. In this regard, an aspect of the invention provides a pharmaceutical composition comprising any of the inventive synthetic polynucleotides, recombinant expression vectors, or host cells (including populations thereof), described herein, and a pharmaceutically acceptable carrier.

[0083] A pharmaceutical composition for treating an mammal by gene therapy may comprise a therapeutically effective amount of a recombinant expression vector comprising the synMMAB polynucleotide or a viral particle produced by or obtained from same. The pharmaceutical composition may be for human or non-human mammal usage. Typically, a physician will determine the actual dosage which will be most suitable for an individual mammal, and it will vary with the age, weight, and response of the particular individual mammal.

[0084] The composition may, in specific aspects, comprise a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. Such materials should be non-toxic and should not interfere with the efficacy of the synMMAB polynucleotide. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in Remington: The Science and Practice of Pharmacy, 23rdEd., Academic Press (2020).

[0085] As used herein, a "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In certain aspects, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0086] The choice of pharmaceutical carrier, excipient, or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient, ordiluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system). For oral administration, excipients such as starch or lactose may be used. For parenteral administration, a sterile aqueous solution may be used, optionally containing other substances, such as salts or monosaccharides to make the solution isotonic with blood.

[0087] A pharmaceutical composition according to the invention may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The pharmaceutical compositions may be administered to a patient alone, or in combination with other agents, modulators, or drugs (e.g., antibiotics).

[0088] The pharmaceutical composition may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. Additional dosage forms contemplated include: in the form of a suppository or pessary; in the form of a lotion, solution, cream, ointment or dusting powder; by use of a skin patch; in capsules or ovules; in the form of elixirs, solutions, or suspensions; in the form of tablets or lozenges.

[0089] Routes of delivery of a synMMAB polynucleotide according to the invention may include, without limitation, injection (systemic or at target site), for example, intradermal, subcutaneous, intravenous, intraperitoneal, intraocular, subretinal, renal artery, hepatic vein, intramuscular injection; physical, including ultrasound(-mediated transfection), electric field- induced molecular vibration, electroporation, transfection using laser irradiation, photochemical transfection, gene gun (particle bombardment); parenteral and oral (including inhalation aerosols and the like).

[0090] Vehicles for delivery of a synthetic MMAB polynucleotide (synMMAB) according to the invention may include, without limitation, viral vehicles (for example, AAV, adenovirus, baculovirus, retrovirus, lentivirus, foamy virus, herpes virus, Moloney murine leukemia virus, Vaccinia virus, and hepatitis virus) and non-viral vehicles (for example, naked DNA, mini-circles, CELID, doggy-bone DNA, liposomes, ligand-polylysine-DNA complexes, nanoparticles, cationic polymers, including polycationic polymers such as dendrimers, synthetic peptide complexes, artificial chromosomes, and polydispersedpolymers). Thus, dosage forms contemplated include injectables, aerosolized particles, capsules, and other oral dosage forms.

[0091] In another aspect, the invention comprises a method of treating a disease or condition mediated by MMAB enzyme. The disease or condition can, in one aspect, be MMA or an MMAB deficiency. In an aspect of the invention, the disease or condition is cblB type MMA. This method may comprise administering to a mammal in need thereof a therapeutic amount of any of the inventive synthetic polynucleotides, recombinant expression vectors, host cells, populations of cells, or pharmaceutical compositions described herein. The MMAB enzyme is processed after transcription, translation, and translocation into the mitochondrial inner space.

[0092] The MMAB enzyme contains a mitochondrial transit peptide, SEQ ID: 22, that is removed to produce the mature MMAB enzyme SEQ ID: 23 in the mitochondrial matrix. In another aspect of this invention, the respective nucleic acids within nucleotides 1-96 of SEQ IDs: 1-6 may be used to generate heterologous transit peptides. Nucleotides 97-753 of SEQ ID NOs: 1-6 may be used to generate the mature MMAB enzyme that could be used for enzyme replacement therapy or as a source of processed MMAB that might be suitable for use in enzymology, to discover vitamin B 12 (adosylcobalamin) analogues and inhibitors, and as the active component of enzyme replacement therapy.

[0093] Enzyme replacement therapy involves the administration of the functional enzyme (MMAB) to a mammal in a manner so that the enzyme administered will catalyze the reactions in the body that the mammal’s own defective or deleted enzyme cannot. In enzyme replacement therapy, the defective enzyme can be replaced in vivo or repaired in vitro using the synthetic polynucleotide according to the invention. The functional enzyme molecule can be isolated or produced in vitro, for example. Enzyme replacement therapy may be accomplished by administration of the synthetic MMAB protein orally, sub-cutaneously, intra-muscularly, intravenously, or by other therapeutic delivery routes.

[0094] Accordingly, an aspect of the invention provides a method of treating a disease or condition mediated by MMAB enzyme, comprising: producing the MMAB enzyme, with or without the transit peptide, by expressing any of the inventive synthetic polynucleotides or recombinant expression vectors described herein by a host cell, and purifying the enzyme from the host cell; and administering to a mammal in need thereof the purified MMAB enzyme. The host cells may be as described herein with respect to other aspects of the invention. Methods of purifying enzymes are known in the art.

[0095] In another aspect, the invention includes the mature MMAB enzyme, e.g., of SEQ ID NO: 23, attached to a carrier, synthetic or heterologous mitochondrial leader sequence, charged or lipophilic small molecule to direct toward the mitochondria; conjugated or covalently modified to a peptide that targets the mitochondrial matrix; or encapsulated to deliver the mature MMAB to a subcellular organelle, cell type or tissue.

[0096] Methods for producing recombinant enzymes in vitro are known in the art. In vitro enzyme expression systems include, without limitation, cell-based systems (bacterial (for example, Escherichia coli. Corynebacterium, Pseudomonas fhiorescens). yeast (for example, Saccharomyces cerevisiae. Pichia Pasloris). insect cell (for example, Baculovirus- infected insect cells, non-lytic insect cell expression), and eukaryotic systems (for example, Leishmania)) and cell-free systems (using purified RNA polymerase, ribosomes, tRNA, ribonucleotides). Viral in vitro expression systems are likewise known in the art. The enzyme isolated or produced according to the above-iterated methods exhibits, in specific aspects, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or about 100% homology to the naturally occurring human MMAB enzyme (with or without the transit peptide).

[0097] Gene therapy can involve in vivo gene therapy (direct introduction of the genetic material into the cell or body) or ex vivo gene transfer, which usually involves genetically altering cells prior to administration of the genetically altered cells. In one aspect, genome editing, or genome editing with engineered nucleases (GEEN) may be performed with the synMMAB polynucleotides of the present invention allowing synMMAB DNA to be inserted, replaced, or removed from a genome using artificially engineered nucleases. Any known engineered nuclease may be used such as Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR / Cas system, and engineered meganuclease re-engineered homing endonucleases. Alternately, the synMMAB polynucleotides of the present invention, in combination with a CASP / CRISPR, ZFN, or TALEN, can be used to engineer correction at the locus in a mammal’s cell either in vivo or ex vivo, then, in one aspect, use that corrected cell, such as a fibroblast or lymphoblast, to create an iPS or other stem cell for use in cellular therapy.

[0098] Accordingly, an aspect of the invention provides a method of treating a disease or condition mediated by MMAB enzyme, comprising administering to a cell of a mammal in need thereof any of the inventive polynucleotides described herein, wherein thepolynucleotide is inserted into the cell of the mammal via genome editing on the cell of the mammal using a nuclease selected from the group of ZFNs, TALENs, the clustered regularly interspaced short palindromic repeats (CRISPR / cas system) and meganuclease re-engineered homing endonucleases on a cell from the mammal. In an aspect of the invention, the method comprises administering the polynucleotide to an isolated cell of the mammal, and the method further comprises administering the cell to the mammal. In another aspect of the invention, the method comprises administering the polynucleotide to the cell of the mammal in vivo.

[0099] The inventive methods of treating a disease or condition mediated by MMAB enzyme may comprise administering a therapeutically effective amount of the synMMAB material (or the genetically altered cell) to the mammal. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the synMMAB material may vary according to factors such as the disease state, age, sex, and weight of the mammal, and the ability of the synMMAB material to elicit a desired response in the mammal. A therapeutically effective amount is also one in which any toxic or detrimental effects of the synMMAB material are outweighed by the therapeutically beneficial effects.

[0100] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of the synMMAB material calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.

[0101] The term “treat,” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment of the disease or condition in a mammal. Furthermore, the treatment provided by the inventive method can include treatment of one or more conditions or symptoms of the disease or condition being treated. In another aspect, the invention is directed to thepreclinical amelioration or rescue from the condition or disease state, for example, MMA, that the afflicted mammal exhibits. This may include symptoms, such as lethargy, lethality, metabolic acidosis, and biochemical perturbations, such as increased levels of methylmalonic acid in blood, urine, and body fluids.

[0102] The mammal referred to in the inventive methods can be any mammal. As used herein, the term “mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0103] Another aspect of the invention provides a method of detecting the presence of a synthetic MMAB polynucleotide (synMMAB) in a biological sample from a mammal. Such methods may be useful for any of a variety of applications, for example, detecting the presence of the synMMAB polynucleotide in a biological sample from a mammal that has been administered any of the synMMAB materials as described herein.

[0104] The method may comprise obtaining at least one test sample comprising nucleic acid from a biological sample from a mammal. The biological sample may be a sample of any tissue from the mammal, for example, blood, kidney, liver, or any of the other tissues and organs described herein with respect to other aspects of the invention. In an aspect, the nucleic acid is isolated or purified from the biological sample to form the test sample. In another aspect, the test sample comprises the biological sample, and the nucleic acid in the biological sample is tested in situ.

[0105] The method may further comprise contacting any of the inventive synMMAB polynucleotides described herein with the at least one test sample under conditions allowing for a complex to form between the synMMAB polynucleotide and the nucleic acid of the test sample. In this regard, the method comprises contacting the nucleic acid of the test sample with the inventive synMMAB polynucleotide under conditions which allow the inventive synMMAB polynucleotide to specifically hybridize with the nucleic acid of the test sample as is known in the art. The method may comprise amplifying the inventive synMMABpolynucleotide and the nucleic acid of the test sample nucleic acid using any suitable type of polymerase chain reaction (PCR) as is known in the art.

[0106] In an aspect, the inventive synMMAB polynucleotide further comprises a detectable label. The label may be any label suitable for detecting hybridization, e.g., a complex, of the inventive synMMAB polynucleotide with the nucleic acid of the test sample. Exemplary detectable labels may include any one or more of radioactive labels, nonradioactive labels, fluorescent labels, and chemiluminescent labels.

[0107] The method may further comprise detecting the complex. The complex may be detected using, for example, a radioactive label or a dye as is known in the art. In a preferred embodiment, the method comprises measuring light emitted from a fluorescent dye using, e.g., a laser. Detecting the complex may, optionally, further comprise measuring the amount of complex formed.

[0108] The method may further comprise comparing a presence of the complex in the at least one test sample with an absence of complex from a negative sample that lacks the inventive synMMAB polynucleotide. The presence of complex from the at least one test sample is indicative of the presence of the inventive synMMAB polynucleotide in the test sample and the absence of complex from the at least one test sample is indicative of the absence of the inventive synMMAB polynucleotide in the test sample. In an aspect of the invention, the method comprises determining a background level of signal generated by the label in the negative sample that lacks the inventive synMMAB polynucleotide and comparing the background level of signal with the level of signal detected in the test sample. A level of signal that is higher or lower in the test sample as compared to that measured in the negative sample may be indicative of the presence of the inventive synMMAB polynucleotide in the test sample.

[0109] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.EXAMPLE 1

[0110] This example demonstrates the development of HEK293 mutant MMAB knockout cell lines using CRISPR CAS9.

[0111] Using CRISPR gene editing, HEK293 cells were transfected with Cas9 and CRISPR guide RNAs obtained from GenScript USA Inc. and subjected to single cell cloning. After single cell cloning was completed, a wild type clone, Tl-5, and two knockout clonesTl-4 and Tl-8 were further pursued and analyzed (Table 1). PCR was performed on Tl-4, Tl-5, and Tl-8 followed by gel electrophoresis (Figure 1A). Sequencing was also performed on Tl-4, Tl-5, and Tl-8 and analyzed using CRISPR analysis tool (CAT) (Figure IB).Clone Tl-5 had no mutations, clone Tl-4 possessed a homozygous mutation MMAB c.566dupG p.(Cysl89TrpfsTer30), and Clone Tl-8 possessed the compound genotype mutation MMAB c.566dupG p.(Cysl89TrpfsTer30) and c.567_569del p.(Argl91del). Western blotting was performed for MMAB levels in HEK293T cells and HEK293 MMAB clone Tl-4 cells, HEK293 MMAB clone Tl-5 cells, and HEK293 MMAB clone Tl-8 cells using 50 micrograms of cell lysate and Proteintech Ab (#11137-1-AP) at a dilution of 1 : 1000 (Figure 1C). The HEK293 MMAB clone Tl-4 cells and HEK293 MMAB clone Tl-8 cells both showed a lack of immunoreactive MMAB. These results suggest that the HEK293 MMAB clone Tl-4 and HEK293 MMAB clone Tl-8 cell lines harbor pathogenic mutations, with no immunoreactive enzyme MMAB.TABLE 1

[0112] In summary, these engineered cell lines lack MMAB expression and can be used to assess plasmid and AAV vectors designed to express MMAB. These knockout (KO) cell lines were employed in the experiments described below. A potency assay is presented for AAV8 and AAV9 vectors that follows the general approach: in a 6 well plate, variable doses of AAV8 or AAV9 are used to infect the MMAB Tl-4 and / or Tl-8 cells; the cells are incubated for 48 hours and then harvested; and the lysate is subjected to western analysis to detect MMAB.EXAMPLE 2

[0113] This example demonstrates the development of synthetic codon optimized and adjusted MMAB genes.

[0114] A series of synthetic codon optimized and adjusted MMAB genes were developed (Table 2). A pairwise comparison of wild type MMAB with synMMAB polynucleotides was carried out. The percentage of nucleotide identity is listed in Table 3. A sequence alignment of wild type MMAB with synMMAB polynucleotides is shown in Figures 2A-2E. Analyses of wild type MMAB and synthetic MMAB polynucleotides (synMMAB) were carried out withCLUSTAL W for phylogenetic analysis (Figure 2F). These analyses revealed that the synMMAB polynucleotides are highly divergent at the nucleotide level while maintaining wild-type MMAB amino acid sequence identity.TABLE 2TABLE 3EXAMPLE 3

[0115] This example demonstrates the development of AAV 8 and AAV 9 vectors to express the synthetic MMAB polynucleotides.

[0116] To examine function of the synMMAB polynucleotides, each gene was cloned into an AAV expression plasmid for the testing of MMAB protein after transfection into the MMAB knockout cell line described in Example 1. The plasmid contained an AAV backbone designed to express an AAV transgene either in the single-strand configuration (Figure 3 A) or self-complementary configuration (Figure 3B). The plasmid sequences are listed in Table 4. Table 5 summarizes the expression vectors studied.TABLE 4TTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTGAATTCATGGCTGTGTGCGGCCTGGGGAGCCGTCTTGGCCTGGGGAGCCGTCTTGGCCTGCGCGGGTGCTTCGGCGCCGCCAGGCTCCTGTATCCCCGTTTCCAGAGCCGCGGCCCTCAGGGCGTGGAAGACGGGGACAGGCCACAGCCTTCCTCGAAGACACCCAGGATCCCCAAGATTTACACCAAAACGGGAGACAAAGGGTTTTCTAGTACCTTCACAGGAGAAAGGAGACCCAAAGATGACCAAGTGTTTGAAGCCGTGGGAACTACAGATGAATTAAGTTCAGCTATTGGGTTTGCTCTGGAATTAGTCACAGAAAAGGGCCATACATTTGCCGAAGAGCTTCAGAAAATCCAGTGCACATTGCAGGACGTCGGCTCGGCCCTGGCGACACCATGCTCCTCGGCCCGGGAGGCTCACTTAAAGTATACCACGTTCAAGGCGGGGCCCATCCTGGAGCTGGAGCAGTGGATCGACAAGTACACCAGCCAGCTCCCACCACTCACGGCCTTCATCCTGCCTTCGGGAGGCAAGATCAGCTCGGCGCTGCATTTCTGCCGGGCCGTGTGCCGCCGGGCCGAGAGACGTGTGGTGCCTCTTGTCCAGATGGGAGAGACCGATGCGAACGTGGCCAAGTTCTTAAACAGACTCAGTGACTATCTCTTCACGCTAGCCAGATATGCAGCCATGAAGGAGGGGAATCAAGAGAAAATATACATGAAAAATGACCCATCGGCCGAGTCTGAGGGACTCTGAGGCCTCGAGATAACAGGCCTATTGATTGGAAAGTTTGTCAACGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTCAGTAAACAGTATATGACCCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACCTCATCGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTATACATCGTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAATGTCATCGATATCGTCGACTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACTAGACTAGTCCTGCAGGTACGTAAGCGGCCGCGGCCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCATATGACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATTTGCCATCCAGCTGATATCCCCTATAGTGAGTCGTATTACATGGTCATAGCTGTTTCCTGGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGAAAAGCTGGGTCATATGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAA AGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGC GCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAGGCTCCG GTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGG GAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGG AAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTA TATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGA ACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATG GCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCC GAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCC CCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGC GAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATT TAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAA TGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGAC GGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGG CCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCT GGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCG GCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGC TCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACA AAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTAC CGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTT TAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGG AGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCT TTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTT TTCTTCCATTTCAGGTGTCGTGAGCTAGAGCTTTATTGCGGTAGTTTATCACAGTT AAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAG AAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTA AGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTG ATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCC ACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTGAATTCATGGCTGTCTG CGGTCTGGGCAGCAGACTGGGTCTTGGGTCCCGCTTGGGACTCCGAGGCTGCTTTGGTGCAGCACGCTTGCTGTATCCTCGCTTCCAGTCTAGAGGGCCACAAGGAGTAG AGGACGGTGATCGGCCCCAGCCAAGCAGCAAAACACCGAGGATACCGAAAATTT ATACAAAGACGGGAGATAAGGGGTTTAGCAGCACCTTCACGGGCGAGCGACGGC CCAAGGACGACCAAGTATTCGAGGCAGTCGGGACGACGGACGAACTGTCAAGTG CTATTGGGTTCGCTCTGGAGCTGGTTACGGAGAAGGGACATACTTTCGCGGAGGA GCTCCAAAAAATACAATGCACCCTTCAAGACGTGGGCAGTGCCCTGGCGACACC GTGTAGCAGCGCGCGCGAGGCCCACTTGAAATATACAACTTTCAAAGCAGGACC TATATTGGAACTCGAGCAGTGGATAGACAAATACACGTCCCAACTGCCTCCCCTG ACAGCTTTTATCTTGCCATCTGGCGGTAAGATAAGTTCTGCTCTTCATTTCTGTAG GGCAGTATGTAGGCGAGCGGAAAGGCGGGTTGTACCCCTGGTGCAGATGGGTGA GACTGACGCCAATGTGGCCAAGTTCTTGAACCGCCTGTCTGATTATCTGTTTACA CTCGCTAGATACGCCGCTATGAAAGAAGGAAATCAAGAGAAGATATACATGAAGAACGATCCGTCAGCGGAATCCGAGGGGCTCTGAGGCCTCGAGATAACAGGCCTATTGATTGGAAAGTTTGTCAACGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTCAGTAAACAGTATATGACCCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACCTCATCGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTATACATCGTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAATGTCATCGATATCGTCGACTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACTAGACTAGTCCTGCAGGTACGTAAGCGGCCGCGGCCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCATATGACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATTTGCCATCCAGCTGATATCCCCTATAGTGAGTCGTATTACATGGTCATAGCTGTTTCCTGGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGCTAGAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTGAATTCATGGCGGTCTGCGGATTGGGTTCACGATTGGGCCTTGGGTCTAGGCTCGGCCTGCGAGGTTGTTTCGGAGCTGCTAGATTGTTGTACCCACGATTCCAAAGCAGGGGACCACAAGGTGTGGAAGACGGTGACAGGCCTCAGCCCTCAAGCAAAACCCCTCGGATTCCGAAAATATATACAAAGACAGGAGACAAAGGCTTTTCAAGCACGTTTACTGGCGAACGAAGGCCTAAAGACGATCAAGTTTTTGAGGCGGTAGGGACAACTGACGAACTTTCATCTGCGATAGGCTTCGCGCTCGAGTTGGTGACGGAGAAGGGTCATACATTCGCTGAAGAACTTCAGAAAATTCAGTGTACGCTCCAAGATGTTGGCAGTGCGCTTGCGACACCTTGCAGCTCAGCAAGGGAAGCGCATTTGAAGTACACCACTTTCAAAGCGGGCCCGATTCTCGAACTTGAACAATGGATTGACAAATATACAAGCCAACTGCCGCCTCTTACCGCCTTCATCCTCCCTAGTGGTGGCAAAATTTCTTCCGCGCTTCATTTTTGTAGGGCCGTGTGTCGCCGCGCCGAGAGACGGGTGGTTCCGTTGGTACAGATGGGTGAAACAGATGCAAACGTGGCAAAGTTCCTTAACCGCCTCAGCGACTACTTGTTCACTCTCGCTAGGTATGCCGCCATGAAAGAAGGGAATCAAGAGAAGATCTATATGAAGAATGACCCGAGCGCTGAAAGCGAGGGACTTTGAGGCCTCGAGATAACAGGCCTATTGATTGGAAAGTTTGTCAACGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTCAGTAAACAGTATATGACCCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACCTCATCGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTATACATCGTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAATGTCATCGATATCGTCGACTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTpsc AAV EF 1 s MMAB WT BGH 5216GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCA CTAGGGGTTCCTGGAGGGGTGGAGTCGTGACCTAGGGGCTCCGGTGCCCGTCAG TGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGC AATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTC GTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAG TAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTCAG ATCAGATCTTTGTCGATCCTACCATCCACTCGACACACCCGCCAGCGGCCGCGTT GGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGG AGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTTCCTTTGTCCTGT TCCCATTTCAGAAGCTTCCGAGCTCTCGAATTAAATAAGAGAGAAAAGAAGAGT AAGAAGAAATATAAGAGCCACCATGGCTGTGTGCGGCCTGGGGAGCCGTCTTGG CCTGGGGAGCCGTCTTGGCCTGCGCGGGTGCTTCGGCGCCGCCAGGCTCCTGTAT CCCCGTTTCCAGAGCCGCGGCCCTCAGGGCGTGGAAGACGGGGACAGGCCACAG CCTTCCTCGAAGACACCCAGGATCCCCAAGATTTACACCAAAACGGGAGACAAA GGGTTTTCTAGTACCTTCACAGGAGAAAGGAGACCCAAAGATGACCAAGTGTTT GAAGCCGTGGGAACTACAGATGAATTAAGTTCAGCTATTGGGTTTGCTCTGGAAT TAGTCACAGAAAAGGGCCATACATTTGCCGAAGAGCTTCAGAAAATCCAGTGCA CATTGCAGGACGTCGGCTCGGCCCTGGCGACACCATGCTCCTCGGCCCGGGAGG CTCACTTAAAGTATACCACGTTCAAGGCGGGGCCCATCCTGGAGCTGGAGCAGT GGATCGACAAGTACACCAGCCAGCTCCCACCACTCACGGCCTTCATCCTGCCTTC GGGAGGCAAGATCAGCTCGGCGCTGCATTTCTGCCGGGCCGTGTGCCGCCGGGC CGAGAGACGTGTGGTGCCTCTTGTCCAGATGGGAGAGACCGATGCGAACGTGGC CAAGTTCTTAAACAGACTCAGTGACTATCTCTTCACGCTAGCCAGATATGCAGCC ATGAAGGAGGGGAATCAAGAGAAAATATACATGAAAAATGACCCATCGGCCGA GTCTGAGGGACTCTGAACTCGAGATAACAGGCCTATTGATTGGAAAGTTTGTCAA CGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTGGATATCCTGC TTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACTTTCTCGCCAA CTTACAAGGCCTTTCTCAGTAAACAGTATATGACCCTTTACCCCGTTGCTCGGCA ACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTG GCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACCTCAT CGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTATACATCGTTTCCATGGCTG CTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGT CGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCC GCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGCGCACCTCTCTTTACGCGGAC TCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCA CGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCAT AAGAGGACTCTTGGACTTTCAGCAATGTCATCGATATCGTCGACTCGCTGATCAG CCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAA TTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACTAGAACTAGTCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGACAGATCCGGGCCCGCATGCGTCGACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCAGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATA CTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACCTCAT CGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTATACATCGTTTCCATGGCTG CTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGT CGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCC GCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGCGCACCTCTCTTTACGCGGAC TCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCA CGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCAT AAGAGGACTCTTGGACTTTCAGCAATGTCATCTCCGATCTTTTTCCCTCTGCCAAA AATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGA AATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACACTAGTCC ACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCC GACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG GACAGATCCGGGCCCGCATGCGTCGACAATTCACTGGCCGTCGTTTTACAACGTC GTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCC TTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACA GTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCAT CTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATG CCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGAC GGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAG CTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGG CCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGA CGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTC TAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTC AATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTAT TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGA AAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGG ATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAAT GATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGT ACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTAT GCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAAC GATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGT AACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGA GCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAAC TGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCG GATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTG CTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGG GCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGC AACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAA GCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAA CTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGAC CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGTCTGGCGGTAAGATAAGTTCTGCTCTTCATTTCTGTAGGGCAGTATGTAGGCGAG CGGAAAGGCGGGTTGTACCCCTGGTGCAGATGGGTGAGACTGACGCCAATGTGG CCAAGTTCTTGAACCGCCTGTCTGATTATCTGTTTACACTCGCTAGATACGCCGCT ATGAAAGAAGGAAATCAAGAGAAGATATACATGAAGAACGATCCGTCAGCGGA ATCCGAGGGGCTCTGAACTCGAGATAACAGGCCTATTGATTGGAAAGTTTGTCAA CGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTGGATATCCTGC TTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACTTTCTCGCCAA CTTACAAGGCCTTTCTCAGTAAACAGTATATGACCCTTTACCCCGTTGCTCGGCA ACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTG GCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATA CTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACCTCAT CGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTATACATCGTTTCCATGGCTG CTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGT CGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCC GCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGCGCACCTCTCTTTACGCGGAC TCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCA CGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCAT AAGAGGACTCTTGGACTTTCAGCAATGTCATCTCCGATCTTTTTCCCTCTGCCAAA AATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGA AATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACACTAGTCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCC GACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG GACAGATCCGGGCCCGCATGCGTCGACAATTCACTGGCCGTCGTTTTACAACGTC GTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCC TTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACA GTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCAT CTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATG CCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGAC GGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAG CTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGG CCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGA CGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTC TAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTC AATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTAT TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGA AAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGG ATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAAT GATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCC GGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGT ACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAAC GATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGT AACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGATTTCGGAGCTGCTAGATTGTTGTACCCACGATTCCAAAGCAGGGGACCACAAGGT GTGGAAGACGGTGACAGGCCTCAGCCCTCAAGCAAAACCCCTCGGATTCCGAAA ATATATACAAAGACAGGAGACAAAGGCTTTTCAAGCACGTTTACTGGCGAACGA AGGCCTAAAGACGATCAAGTTTTTGAGGCGGTAGGGACAACTGACGAACTTTCA TCTGCGATAGGCTTCGCGCTCGAGTTGGTGACGGAGAAGGGTCATACATTCGCTG AAGAACTTCAGAAAATTCAGTGTACGCTCCAAGATGTTGGCAGTGCGCTTGCGAC ACCTTGCAGCTCAGCAAGGGAAGCGCATTTGAAGTACACCACTTTCAAAGCGGG CCCGATTCTCGAACTTGAACAATGGATTGACAAATATACAAGCCAACTGCCGCCT CTTACCGCCTTCATCCTCCCTAGTGGTGGCAAAATTTCTTCCGCGCTTCATTTTTG TAGGGCCGTGTGTCGCCGCGCCGAGAGACGGGTGGTTCCGTTGGTACAGATGGG TGAAACAGATGCAAACGTGGCAAAGTTCCTTAACCGCCTCAGCGACTACTTGTTC ACTCTCGCTAGGTATGCCGCCATGAAAGAAGGGAATCAAGAGAAGATCTATATG AAGAATGACCCGAGCGCTGAAAGCGAGGGACTTTGAACTCGAGATAACAGGCCT ATTGATTGGAAAGTTTGTCAACGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTT TACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAA CAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTCAGTAAACAGTATATGAC CCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCA ACCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTG TGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGC AGGTCTGGAGCAAACCTCATCGGGACCGACAATTCTGTCGTACTCTCCCGCAAGT ATACATCGTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGAC GTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGC CGCTTGGGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGC GCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTG CACTTCGCTTCACCTCTGCACGTCGCATGGAGGCCACCGTGAACGCCCACCGGAA CCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAATGTCATCTCCG ATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCT GACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTT TGTGTCTCTCACACTAGTCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC GGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGC GAGCGAGCGCGCAGAGAGGGACAGATCCGGGCCCGCATGCGTCGACAATTCACT GGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAAT CGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCA CCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCG GTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCA GTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACC CGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCT GTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAAC GCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGAT AATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACC CCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATA ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACA TTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCAAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTCAGATCAGATCTTTGTCGATCCTACCATCCACTCGACACACCCGCCAGCGGCCGCGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTTCCTTTGTCCTGTTCCCATTTCAGAAGCTTCCGAGCTCTCGAATTAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGTGTGCGGCCTGGGCAGCAGACTGGGACTGGGTTCCCGGCTGGGACTGAGAGGCTGCTTCGGCGCCGCAAGACTGCTGTACCCCAGATTCCAGAGCAGAGGACCTCAGGGCGTGGAAGATGGCGATAGACCTCAACCTAGCTCAAAGACCCCTAGAATCCCCAAGATCTACACAAAGACAGGCGACAAGGGCTTTAGCAGCACCTTTACCGGCGAAAGACGGCCTAAGGACGACCAGGTTTTCGAGGCCGTCGGAACAACCGACGAGCTGAGCTCTGCTATCGGCTTCGCCCTCGAGCTCGTGACCGAGAAGGGCCACACATTTGCCGAGGAACTGCAAAAAATTCAGTGTACCCTGCAGGACGTGGGATCTGCTCTGGCCACACCTTGCAGCAGCGCTCGGGAAGCCCACCTGAAGTACACCACCTTCAAGGCCGGCCCAATCCTGGAACTGGAACAGTGGATCGACAAGTACACCAGCCAGCTGCCACCTCTGACCGCCTTCATCCTGCCTTCTGGCGGCAAGATCAGCAGCGCCCTGCACTTCTGCAGAGCCGTGTGTAGAAGGGCCGAGCGGCGGGTGGTGCCCCTGGTGCAGATGGGCGAGACAGATGCCAACGTGGCCAAGTTCCTGAATAGACTGTCTGATTACCTGTTCACCCTGGCTCGCTACGCCGCCATGAAGGAGGGCAACCAGGAGAAAATCTATATGAAAAACGACCCCTCCGCCGAGAGCGAGGGCCTGTGAACTCGAGATAACAGGCCTATTGATTGGAAAGTTTGTCAACGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTCAGTAAACAGTATATGACCCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACCTCATCGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTATACATCGTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAATGTCATCTCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACACTAGTCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGACAGATCCGGGCCCGCATGCGTCGACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTTAAGAGGACTCTTGGACTTTCAGCAATGTCATCTCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACACTAGTCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGACAGATCCGGGCCCGCATGCGTCGACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACCTCATCGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTATACATCGTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAATGTCATCTCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACACTAGTCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGACAGATCCGGGCCCGCATGCGTCGACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACTGGATCGACAAATACACCAGCCAGCTGCCTCCTCTGACCGCCTTCATTCTGCCTTCCGGAGGCAAGATCTCCAGCGCTCTGCACTTCTGTAGAGCCGTGTGTAGAAGAGCCGAAAGAAGAGTGGTCCCTCTGGTGCAGATGGGAGAGACCGACGCCAACGTGGCCAAGTTTCTGAATAGACTGAGCGATTATCTGTTCACACTGGCTAGATACGCCGCCATGAAGGAGGGCAACCAAGAGAAGATCTACATGAAGAACGACCCCTCCGCCGAGAGCGAAGGACTGTGAACTCGAGATAACAGGCCTATTGATTGGAAAGTTTGTCAACGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTCAGTAAACAGTATATGACCCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACCTCATCGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTATACATCGTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAATGTCATCTCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACACTAGTCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGACAGATCCGGGCCCGCATGCGTCGACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTTABLESEXAMPLE 4

[0117] This example demonstrates the efficacy of the AAV synMMAB vectors at driving expression of MMAB in HEK293 MMAB KO cells.

[0118] To test the efficacy of plasmids that contain the AAV backbone designed to express synMMAB 1 and synMMAB2 transgenes in the single-stranded (ss) configuration, they were transfected into HEK293 cells and tested for expression. As assessed by western blotting, pAAV EF1L synMMAB2 displayed superior expression compared to synMMAB 1 or the wild type A7M4 / / sequence at DNA concentrations of 0.5 pg (Figure 4C), 1 pg (Figure 4B), and 2.5 pg (Figure 4C).

[0119] Similarly, to test the efficacy of plasmids that contain the AAV backbone designed to express synMMAB 1 -synMMAB 6 AAV transgenes in the self-complementary (sc) configuration, they were transfected into HEK293 cells and tested for expression. As assessed by western blotting, these studies showed that pscAAV EFIs synMMAB5 displayed superior expression compared to other synMMAB polynucleotides or the wild type MMAB sequence across the DNA concentrations of 0.5 pg (Figure 4C), 1 pg (Figure 4B), and 2.5 pg (Figure 4A).

[0120] Then western blotting for MMAB expression was performed as previously described on HEK293 MMAB WT cells, HEK 293 MMAB KO Tl-8 cells (Tl-8-0), and HEK 293 MMAB KO Tl-8 cells that were transfected with pAAV EF1L MMAB WT plasmid (Tl- 8-1), pAAV EF1L synMMAB 1 (Tl-8-2), pAAV EF1L synMMAB2 plasmid (Tl-8-3), pscAAV EFIs MMAB WT BGH plasmid (T 1-8-4), pscAAV EFIs MMAB WT RGB plasmid(T 1-8-5), pscAAV EFIs synMMABl plasmid (T 1-8-6), pscAAV EFIs synMMAB2 plasmid (T 1-8-7), pscAAV EFIs synMMAB3 plasmid (T 1-8-8), pscAAV EFIs synMMAB4 plasmid (T 1-8-9), pscAAV EFIs synMMAB5 plasmid (T 1-8- 10), and pscAAV EFIs synMMAB6 plasmid (Tl-8-11) when using 2.5 pg of plasmid (Figure 4A), 1 pg of plasmid (Figure 4B), and 0.25 pg of plasmid (Figure 4C).

[0121] Western blotting for MMAB expression was also performed on HEK293 MMAB WT cells (Tl-5), HEK 293 MMAB KO Tl-4 cells (Tl-4-0), and HEK 293 MMAB KO Tl-4 cells that were transfected with pAAV EF1L MMAB WT plasmid (Tl-4-1), pAAV EF1L synMMABl (Tl-4-2), pAAV EF1L synMMAB2 plasmid (Tl-4-3), pscAAV EFIs MMAB WT BGH plasmid (T 1-4-4), pscAAV EFIs MMAB WT RGB plasmid (T 1-4-5), pscAAV EFIs synMMABl plasmid (T 1-4-6), pscAAV EFIs synMMAB2 plasmid (T 1-4-7), pscAAV EFIs synMMAB3 plasmid (T 1-4-8), pscAAV EFIs synMMAB4 plasmid (T 1-4-9), pscAAV EFIs synMMAB5 plasmid (Tl-4- 10), and pscAAV EFIs synMMAB6 plasmid (Tl-4-11), when using 2.5 pg of plasmid (Figure 5A), 1 pg of plasmid (Figure 5B), and 0.25 pg of plasmid (Figure 5C).

[0122] These results suggest that pAAV EF IL synMMAB2 and pAAV EF 1 s synMMAB5 are the most consistently effective drivers of MMAB expression.EXAMPLE 5

[0123] This example demonstrates the efficacy of the AAV synMMAB vectors pseudoserotyped with AAV8 or AAV9 capsids at driving expression of MMAB in HEK293 MMAB KO cells.

[0124] The lead AAV plasmids, packaged as either single-stranded (pAAV EF1L synMMAB2) or self-complementary vectors (pAAV EFIs synMMAB5\ were pseudoserotyped with an AAV9 or AAV8 capsid and tested for expression in an infection study using Tl-4 and Tl-8 HEK293 MMAB knock-out cell lines. Using the respective AAV8 or AAV9 viral vectors, the Tl-4 and Tl-8 MMAB 293 KO cells were infected over a range of doses, or moiety of infection (moi), between 5xlOA4 and lxlOA6 (pAAV EF1L synMMAB2) or between approximately 2.5xlOA4 and 5xl0A5(pAAV EFIs synMMAB5). After 48 hours, the infected cells were harvested, extracted, and subjected to western blotting to detect MMAB expression from the AAV vector. Tl-8 MMAB 293 KO cells infected with AAV8 vectors showed increasing MMAB expression as moi increased (Figures 6A-6B). Tl-8 MMAB 293 KO cells infected with AAV9 vectors showed increasing MMAB expression asmoi increased (Figures 7A-7B). Tl-4 MMAB 293 KO cells infected with AAV8 vectors showed increasing MMAB expression as moi increased (Figures 8A-8B). Tl-4 and Tl-8 MMAB 293 KO cells infected with the AAV9 EF1L synMMAB2(ss) vector showed increasing MMAB expression as moi increased, with Tl-8 MMAB 293 KO cells having higher expression than Tl-4 MMAB 293 KO cells (Figures 9A-9B).

[0125] These results suggest that these plasmids could direct the expression of functional high titer AAV8 and AAV9 viral vectors in either genomic configuration, but further, that these vectors direct expression of properly processed MMAB.EXAMPLE 6

[0126] This example demonstrates the efficacy of the AAV9 single-stranded and self- complementary synMMAB vectors at rescuing isolated methylmalonic acidemia (MMA) mouse models.

[0127] To test the efficacy of the single-strand and self-complementary AAV9 MMAB vectors, a series of in vivo gene therapy experiments using newly developed MMAB mouse models were performed. These MMAB mouse models are summarized in Table 6. Two severe mutations, the orthologue of a missense mutation recurrently noted in cblB patients who received elective liver transplantation (p.Argl86Trp in MMAB in humans, MMABR180Win mice), and another that removed exons 3-7were engineered into the mouse germline. Because hepatic metabolism has been recognized as a critical to target for gene therapy in MMA, a germline rescue transgene, designed to restore expression of MMAB in the skeletal muscle under the control of the murine muscle creatine kinase (MCK) promoter, was knocked into the ROSA26 locus (ROSA26MCK-Mmab), and subsequently crossed to the mice carrying Mmab mutations. Mmab '''3''1'''3''1and MMABR180W / R180Wmice displayed lethality and massively increased plasma MMA (pMMA) levels (1150+ / - 177 uM, 360x WT), very much like patients with severe forms of cblB MMA.

[0128] MMABR180W / R180W;TgROSA26-MCK-Mmafemice survived until weaning, but manifested pronounced MMAemia (520+ / - 79 uM, 200x WT), growth retardation, increased FGF21, and decreased13C propionate oxidation.

[0129] An additional mouse strain containing the MmabA3'7allele was generated using a different genetic background Mmab '''3''1'''3''1(C57BL / 6 x FVB / N)). As depicted in Figure 21 A, Mmab33'1133'1(C57BL / 6 x FVB / N) mice survived until weaning but then survivability declines markedly. Also, as compared to wild-type mice, Mmab33'1133'1(C57BL / 6 x FVB / N)mice display severe runting (Figure 2 IB). Also, the pMMA levels for(C57BL / 6 x FVB / N) mice significantly exceed those of either heterozygotes or wild type mice (Figure 21C). A7 / w / iA3_7 A3'7(C57BL / 6 x FVB / N) mice also manifest significantly decreased 1-13C propionate oxidation as compared to wild-type mice (see Figure 22).

[0130] An additional hypomorphic mouse model strain containing the MmabA3’7allele and a transgenic MCK-Mmab construct (TgMCK'Mmab)wascreated in a C57BL6 murine background. The TgMCK-Mmabconstruct contains, from N- to C-termini, a polyA sequence, mouse Mmab CDs, and a mouse MCK promoter. This allele was introduced into a homozygotic MmabA3-7background to create the A / maZ>A3'7 / A3'7;TgMCK'Mmab(C57BL / 6) strain. This mouse model was assessed for survivability, the results of which are presented in Figure 30. In comparison with homozygotic MmabA3’7, which as discussed above is a lethal phenotype, all ATmaZ>A3'7 / A3'7;TgMCK'Mmab(C57BL / 6) mice survived well past 200 days. This A / maZ>A3'7 / A3'7;TgMCK'Mmab(C57BL / 6) strain also was assessed for plasma MMA levels (see Figure 31). In comparison with heterozygous Mmab13"1 / '133'1mice and wild-type mice with respect to the Mmab trait (both also containing the TgMCK-Mmaballele), homozygotic Mmab ''3'1mice (i.e., A / maZ>A3'7 / A3'7;TgMCK'Mmab(C57BL / 6)) exhibited markedly high pMMA. Probing the MMAB expression in liver, kidney, heart, and muscle tissue (Figure 32) revealed no expression of MMAB in ATmaZ>A3'7 / A3'7;TgMCK'Mmab(C57BL / 6)) mice in liver and kidney, whereas MMAB expression was detected in heart and muscle (see Figure 32).

[0131] The ssAAV9 EF1L synMMAB2 or scAAV9 EFIs synMMAB5 vectors were assayed for in vivo efficacy in both severe model which display lethality around weaning (MmabA3-7 A3-7) and hypomorphic model (MMABR180W / R180W,TgROSA26'MC. Neonatal treatment (At MmabA3-7 / A3-7mice (n=6) with the scAAV9 EFIs synMMAB5, systemically delivered at a dose of approximately 5el0 GC / pup on DOL1, resulted in complete rescue from lethality, and full phenotypic correction persisting greater than 300 days (Figure 12). Also, this cohort of Mmab133'1 133~1mice displayed a significant reduction in pMMA as compared to untreated MmabA3‘7 / A3'7mice (Figure 22, left panel) and an increase in 1-13C propionate oxidation to a level not statistically significantly different than wild-type mice (Figure 22, right panel).

[0132] MmabA3-7 A3-7mice also were examined histologically to assess phenotype as compared to wild-type mice and to MmabA3-7 / A3-7mice treated neonatally with scAAV9 EFIs synMMAB5 (see Figure 23). While untreated MmabA3-7 / A3-7mice exhibited Mallory -like bodies in liver tissue (Figure 23, middle panel), that of MmabA3-7 / A3-7mice treated withscAAV9 EFIs synMMAB5 resembled liver tissue of wild-type mice (Figure 23, left and right panels). Also, robust expression of MMAB was observed in liver, heart, kidney, and muscle tissue in MmabA3'7 A3'7mice six months following scAAV9 EFIs synMMAB5 (Figure 24), including a 10-, 36-, and 8-fold increase of expression over wild-type mice for liver, heart, and kidney tissue, respectively. Additional histological data concerning expression of MMAB in tissue obtained from MmabA3'7 / A3'7(C57BL / 6 x FVB / N) mice treated with scAAV9 EFIs synMMAB5 (5E10 GC / pup) are presented in Figures 25A-25D.

[0133] MMABR180W / R180W,TgROSA26'MCK'Mmafemice treated with either ssAAV9 EF1L synMMAB2 or scAAV9 EFIs synMMAB5 vectors as young adults (n=6), given at a dose of 1 el 3 GC / kg, produced equally robust responses by 2 weeks, with a 38x reduction (12.9 + / - 3.7 uM) of the circulating plasma methylmalonic acid levels (Figure 11 and Table 7) and restoration of 1-C13propionate oxidation to wild type levels (Figure 12). The ssAAV9 EF1L synMMAB2 or scAAV9 EFIs ,sj7 / A7AT4 / G-treated MMABR180W / R180W,TgROSA26'MCK'Mmafemice also showed increased transgene expression in the liver as measured by both western blot analysis (Figures 13 A and 13B) as well as RT-qPCR (figure 13C). The ssAAV9 EF1L synMMAB2 or scAAV9 EFIs synMMAB5 treated MMABR180W / R180W,TgROSA26'MCK'Mmafemice similarly showed increased transgene expression in the kidney as measured by both western blot analysis (Figures 14A and 14B) as well as RT-qPCR (Figure 14C).TABLE 6TABLE 7EXAMPLE 7

[0134] This example demonstrates the optimization of the AAV9 single-stranded and self-complementary synMMAB vectors for human translation.

[0135] To further optimize the vectors for human translation, several design improvements were performed. For the ssAAV EF1L synMMAB2 vector, an optimized 5’ untranslated region was placed before the MMAB start codon, and a CpG repeat between the polyA signal and 3TTR was removed (Figure 15). The resulting vector, pAAV EF1L MMAB Kan (ss), was transfected into the MMAB KO cell lines and compared to the parent vector. The modified vector enabled even higher expression of MMAB in both the Tl-4 MMAB KO cell line (Figure 16A) and the Tl-8 MMAB KO cell line (Figure 16B). The sequence of this vector plasmid is presented in Table 8.

[0136] A different modification of the pscAAV EFIs synMMAB5 vector was pursued. The 121 bp 5’ITR was swapped with a 130 bp version of a WT AAV2 ITR, and the antibiotic backbone was changed from ampicillin to kanamycin (Figure 17). The sequence of this vector, pscAAV EFIs MMAB Kan (sc), is presented in Table 9. The modified vector provided even higher expression of MMAB in the Tl-4 MMAB KO cell line (Figure 19).

[0137] The optimized AAV9 EF IL MMAB Kan (ss) and AAV9 EF 1 s MMAB Kan (sc)) vectors were tested for their ability to correct the expression of MMAB in an infection study using Tl-4 and Tl-8 HEK293 MMAB knock-out cell lines, similarly, as described in Example 5 above. The cells were harvested after 48 hours, and for the Western blotting, total protein was loaded at 12.5 pg / lane (Figures 26A and 26B) or 25 pg / lane (Figures 26C. 26D, 26E or 26F). The results are presented in Figures 26A-26F. The data underlying Figures 26A and 26B were generated using a vector constructed by a different manufacturing laboratory than those underlying Figures 26C-26F, and the consistency of results further reveals the activity of these optimized vectors.

[0138] The optimized AAV9 EF IL MMAB Kan (ss) and AAV9 EF 1 s MMAB Kan (sc) vectors also were tested for their impact on the survival of MmabA3'7 / A3'7(C57BL / 6 xFVB / N) mice. Neonatal treatment of MmabA3'7 / A3'7mice treated with AAV9 EF1L MMAB Kan (ss) and AAV9 EFIs MMAB Kan (sc), systemically delivered at a dose of lelO, 5el0, lei 1 on D0L1, resulted in rescue from lethality, and phenotypic correction persisting greater than 100 days (Figure 27 A ). Treatment of MmabA3'7 / A3'7mice with AAV9 EF1L MMAB Kan (ss), systemically delivered at a dose of 1E13 on DOL21(@wean), resulted in rescue from lethality, and phenotypic correction (Figure 27B ). The data in Figure 27A reveal that the sc version of this vector permitted 100% survival rate during the assay period at all doses tested, as do the data in Figure 27B for the ss version. The vectors employed in the experiment leading to Figures 27A was constructed independently of that employed for Figures 27B, and the consistency of results further reveals the activity of these optimized vectors.

[0139] The optimized AAV9 EF IL MMAB Kan (ss) and AAV9 EF 1 s MMAB Kan (sc) vectors also were tested for their ability to rescue pMMA and for the levels of FGF produced in mice. Between 28 and 36 days following the neonatal DOL1 injection of either vector (dosed at lelO, 5el0, or lei 1 GC / pup), a drastic reduction of pMMA was observed compared to untreated MmabA3'7 / A3'7(C57BL / 6 x FVB / N) mice (Figure 28A). Also, at DOL120, the FGF levels for the mice treated with the vectors (dosed at lelO GC / pup) was drastically reduced in comparison with untreated MmabA3'7 / A3'7(C57BL / 6 x FVB / N) mice (Figure 28B).

[0140] The MMAB transgene expression in liver and kidney following treatment with the AAV9 EF1L MMAB Kan (ss) and AAV9 EFIs MMAB Kan (sc) vectors also was assayed. Treated or untreated wild-type, heterozygotic, or homozygotic MmabA3'7 / A3'7(C57BL / 6 x FVB / N) mice were sacrificed at DOL120 and tissues processed and probed via Western blot analysis. The results (Figures 29A and 29B, right panels) reveal that treatment with the optimized V9 EF1L MMAB Kan (ss) and AAV9 EF Is MMAB Kan (sc)) vectors resulted in robust expression of MMAB in both tissues, dramatically exceeding the expression levels observed even for wild-type mice.

[0141] The AAV9 EF1L MMAB Kan (ss) and AAV9 EFIs MMAB Kan (sc)) vectors also were tested in the ATmaZ>A3'7 / A3'7;TgMCK'Mmab(C57BL / 6) mouse model. As revealed by the data presented in Figure 33, treatment with either of the two vectors markedly decreased plasma MMA levels when measured at 30 and 60 days from treatment, as compared with the neonatal cohort (pretreated) control.

[0142] In summary, the experiments described in these Examples have shown that a series of synthetic MMAB genes have been developed. It has been demonstrated that they have superior expression in a human MMAB knock out cell line. The new polynucleotides have been used to develop single-strand and self-complementary AAV8 and AAV9 vectors that can be used for neonatal and juvenile gene therapy for MMAB deficiency. The AAV9 vectors are potent in vivo, can produce virus well, and after systemic delivery at doses that approximate those given to humans in other AAV9 indications, mediate robust hepatorenal transgene expression in vivo. These studies provide a new class of vectors and AAV9 gene therapeutics to treat patients with MMAB deficiency, a severe form of MMA, and are suitable for immediate human translation.TABLE 8GCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGC TCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACA AAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTAC CGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTT TAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGG AGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCT TTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTT TTCTTCCATTTCAGGTGTCGTGAGCTAGAGCTTTATTGCGGTAGTTTATCACAGTT AAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAG AAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTA AGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTG ATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCC ACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTGAATTCAAATAAGAGAG AAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCGGTCTGCGGATTGGGT TCACGATTGGGCCTTGGGTCTAGGCTCGGCCTGCGAGGTTGTTTCGGAGCTGCTA GATTGTTGTACCCACGATTCCAAAGCAGGGGACCACAAGGTGTGGAAGACGGTG ACAGGCCTCAGCCCTCAAGCAAAACCCCTCGGATTCCGAAAATATATACAAAGA CAGGAGACAAAGGCTTTTCAAGCACGTTTACTGGCGAACGAAGGCCTAAAGACG ATCAAGTTTTTGAGGCGGTAGGGACAACTGACGAACTTTCATCTGCGATAGGCTT CGCGCTCGAGTTGGTGACGGAGAAGGGTCATACATTCGCTGAAGAACTTCAGAA AATTCAGTGTACGCTCCAAGATGTTGGCAGTGCGCTTGCGACACCTTGCAGCTCA GCAAGGGAAGCGCATTTGAAGTACACCACTTTCAAAGCGGGCCCGATTCTCGAA CTTGAACAATGGATTGACAAATATACAAGCCAACTGCCGCCTCTTACCGCCTTCA TCCTCCCTAGTGGTGGCAAAATTTCTTCCGCGCTTCATTTTTGTAGGGCCGTGTGT CGCCGCGCCGAGAGACGGGTGGTTCCGTTGGTACAGATGGGTGAAACAGATGCA AACGTGGCAAAGTTCCTTAACCGCCTCAGCGACTACTTGTTCACTCTCGCTAGGT ATGCCGCCATGAAAGAAGGGAATCAAGAGAAGATCTATATGAAGAATGACCCGA GCGCTGAAAGCGAGGGACTTTGAGGCCTCGAGATAACAGGCCTATTGATTGGAA AGTTTGTCAACGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTG GATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACT TTCTCGCCAACTTACAAGGCCTTTCTCAGTAAACAGTATATGACCCTTTACCCCGT TGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGT TGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGC CGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGC AAACCTCATCGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTATACATCGTTT CCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTT ACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCT CTACCGCCCGCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGCGCACCTCTCTTT ACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCA CCTCTGCACGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGCCCAAGG TCTTGCATAAGAGGACTCTTGGACTTTCAGCAATGTCATCGATATCGTCGACTCG CTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCC CCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAAT GAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGG TGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGG GATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACT AGACTAGTCCTGCAGGTACCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCATATGACCCAG CTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCAATTTGTTGCAACTABLE 9GCCAGTCTTAAGCTCGGGCCCCAAATAATGATTTTATTTTGACTGATAGTGACCT GTTCGTTGCAACAAATTGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAA AAAAGCAGGCTTCTAGACTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAA AGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGC GCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAGGCTCCG GTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGG GAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGG AAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTA TATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGA ACACAGGTCAGATCAGATCTTTGTCGATCCTACCATCCACTCGACACACCCGCCAGCGGCCGCGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAAC TGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTT CCTTTGTCCTGTTCCCATTTCAGAAGCTTCCGAGCTCTCGAATTAAATAAGAGAG AAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGTGTGTGGACTGGG CAGCAGACTGGGACTGGGATCTAGACTGGGACTGAGAGGCTGCTTCGGCGCTGC TAGACTGCTGTACCCTAGATTCCAGTCCAGAGGACCCCAAGGCGTCGAGGATGG AGATAGACCCCAGCCCTCCTCCAAGACCCCTAGAATCCCCAAGATCTACACCAA GACCGGCGACAAGGGCTTCAGCTCCACCTTTACCGGCGAGAGGAGACCCAAGGA CGATCAAGTGTTCGAGGCCGTGGGAACCACCGACGAGCTGAGCTCCGCTATCGG CTTCGCTCTGGAGCTGGTGACAGAGAAGGGCCACACCTTTGCCGAGGAGCTGCAGAAGATCCAGTGCACACTGCAAGACGTGGGCAGCGCTCTGGCTACACCTTGCAG CAGCGCTAGAGAGGCCCATCTGAAATACACCACCTTCAAAGCCGGCCCTATTCTG GAGCTGGAACAGTGGATCGACAAGTACACCAGCCAGCTGCCTCCTCTGACAGCC TTCATTCTGCCCAGCGGCGGAAAGATCTCCAGCGCTCTGCACTTCTGCAGAGCCG TCTGCAGAAGGGCTGAGAGAAGGGTGGTGCCTCTGGTGCAGATGGGCGAGACCG ATGCCAACGTGGCCAAGTTCCTCAATAGACTGTCCGATTATCTGTTCACACTGGC TAGATATGCCGCCATGAAGGAGGGCAACCAAGAGAAGATCTACATGAAGAACG ATCCCAGCGCCGAGAGCGAGGGCCTCTGAACTCGAGATAACAGGCCTATTGATT GGAAAGTTTGTCAACGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCA ATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTCAGTAAACAGTATATGACCCTTTA CCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCC ACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTC CTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCT GGAGCAAACCTCATCGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTATACAT CGTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTT TGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTG GGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGTCCGACCACGGGGCGCACCT CTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTC GCTTCACCTCTGCACGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAATGTCATCTCCGATCTTTT TCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCT GGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCT CTCACACTAGTCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGA CCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGA GCGCGCAGAGAGGGACAGATCCGGGCCCGCATGCGTCGACAATTCACTGGCCGT CGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTT GCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGAT CGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATT TTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGACCCAGCTTTCTTGTAAmino acid sequence of wild type MMAB polypeptide (NCBI Reference Sequence:NP 443077.1) SEQ ID NO: 21MAVCGLGSRLGLGSRLGLRGCFGAARLLYPRFQSRGPQGVEDGDRPQPSSKTPRIPKIYTKTGDKGFSSTFTGERRPKDDQVFEAVGTTDELSSAIGFALELVTEKGHTFAEELQKIQCTLQDVGSALATPCSSAREAHLKYTTFKAGPILELEQWIDKYTSQLPPLTAFILPSGGKISSALHFCRAVCRRAERRWPLVQMGETDANVAKFLNRLSDYLFTLARYAAMKEGNQEKIYMKNDPSAESEGLAmino acid sequence of wild type MMAB mitochondrial transit peptide SEQ ID NO: 22MAVCGLGSRLGLGSRLGLRGCFGAARLLYPRFAmino acid sequence of wild type MMAB mature enzyme SEQ ID NO: 23QSRGPQGVEDGDRPQPSSKTPRIPKIYTKTGDKGFSSTFTGERRPKDDQVFEAVGTTD ELS S AIGFALELVTEKGHTF AEELQKIQCTLQD VGS AL ATPC S SAREAHLKYTTFKAG PILELEQWIDKYTSQLPPLTAFILPSGGKISSALHFCRAVCRRAERRVVPLVQMGETD ANVAKFLNRLSDYLFTLARYAAMKEGNQEKIYMKNDPSAESEGLEXAMPLE 8

[0143] This example demonstrates resistance to lethality from dietary challenge conferred by systemic delivery of the inventive vector.

[0144] Patient mutations were identified in a large cohort of subjects with MMAB deficiency evaluated enrolled in a natural history study (NCT00078078). One mutation was selected for further modeling in mice: the orthologue of a nonsense mutation frequently found in patients (Q234X, Q228X in mouse) was bred to homozygosity (MmabQ228X / Q228X) and with mice carrying a null allele (MmabA3'7 / Q228X). Various knock-in and compound mutants were characterized and challenged with a diet consisting of a 10-fold increase in Isoleucine and Valine while maintaining total caloric intake. MMAB mice also were treated with a systemic retro-orbital injection of le+13 genome copies per kilogram of ssAAV9 EF1L MMAB (SEQ ID NO: 19) and examined for response to challenge.

[0145] The hypomorphic model, MmabA3'7 / Q228X, displayed similar survival and weight gain as compared to wild type controls, while homozygote mutant, MmabA3'7 / A3'7, showed neonatal lethality. MmabA3'7 / Q228Xmice displayed mild methylmalonic acidemia [MMA](80 + / - 77 pM, ~100x WT), very much like patients with attenuated forms of cblB MMA. When treated with the isoleucine- and valine-enriched diet, MmabA3'7 / Q228Xdemonstrated significant weight loss (-6.2 + / - ,8g n=6 over 4 days) and 100% lethality within 5 days, while controls fed the same diet thrived and gained weight. pMMA levels were drastically increased with the Isoleucine / Valine diet (1223 + / - 738 pM, ~1500x WT), reflective of severe clinical disease (Figure 20B). MmabQ228X / Q228Xmice, in contrast, survived the Isoleucine / Valine diet challenge and manifested mild MMAemia (142 + / - 74 pM) when challenged on the Isoleucine / Valine-enriched diet compared to compound MmabA3'7 / Q228Xmice.

[0146] Treatment of the MmabA3'7 / Q228Xmice with the SSAAV9.EF1LMMAB (SEQ ID NO: 19) , systemically delivered at a dose of lel3 GC / kg on DOL26, approximately two weeks prior to the high Isoleucine / Valine diet, resulted in complete rescue from lethality (p=.0081) and reduced weight loss. Additionally, treatment of the Mmcth '3'1 / Q228Xmice and MmaZ>A3-7 / A3-7;TgMCK-Mmab(C57BL / 6) mice with the ssAAV9 MMAB: AAV9.EF1LMMAB Kan vector led to a dramatic reduction in pMMA levels to near those observed for animals not challenged with the high Isoleucine / Valine diet. Data generated by the study reported in this Example are presented in Figures 20A and 20B.

[0147] These results exemplify the development of a new clinically-relevant murine MMAB mutation and a suite of new murine models to enable preclinical gene therapy studies in adult mice. The MmabA3'7 / Q228Xmice models can be used in conjunction with an Isoleucine / Valine challenge diet to induce a lethal metabolic crisis, which can be fully rescued from lethality by systemic AAV9 gene therapy using the inventive vectors. This model demonstrates that AAV9 gene therapy can be used to protect against acute lethal metabolic instability, allowing the facile assessment of vector efficacy and dose finding in the development of new gene therapeutics for patients with MMAB deficiency.

[0148] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0149] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into thespecification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0150] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS:

1. A synthetic MMAB (Metabolism Of Cobalamin Associated B) polynucleotide (synMMAB) selected from the group consisting of:(a) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-6;(b) a polynucleotide having a nucleic acid sequence with at least about 80% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-6 and encoding a polypeptide having an amino acid sequence at least about 90% identical to SEQ ID NO: 21, and having equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 7 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 7; and(c) a polynucleotide having a nucleic acid sequence with at least about 80% identity to the nucleic acid sequence of nucleotides 97-753 of any one of SEQ ID NOs: 1-6 and encoding a polypeptide having an amino acid sequence at least about 90% identical to SEQ ID NO: 23, and having equivalent or increased expression in a mammalian host cell relative to expression of nucleotides 97-753 of SEQ ID NO: 7 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 97-753 of SEQ ID NO: 7.

2. The synthetic polynucleotide of claim 1, wherein the polynucleotide has (a) at least about 90% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-6 or (b) at least about 90% identity to the nucleic acid sequence of nucleotides 97-753 of any one of SEQ ID NOs: 1-6.

3. The synthetic polynucleotide of claim 1, wherein the polynucleotide has (a) at least about 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-6 or (b) at least about 95% identity to the nucleic acid sequence of nucleotides 97-753 of any one of SEQ ID NOs: 1-6.

4. The synthetic polynucleotide of any one of claims 1-3, wherein the polynucleotide exhibits increased expression in a mammalian host cell relative to the expression SEQ ID NO: 7 or nucleotides 97-753 of SEQ ID NO: 7 in the mammalian host cell.

5. The synthetic polynucleotide of any one of claims 1-4, wherein the synthetic polynucleotide comprises codons that have been optimized relative to the naturally occurring human MMAB polynucleotide sequence of SEQ ID NO: 7 or nucleotides 97-753 of SEQ ID NO: 7.

6. The synthetic polynucleotide of claim 5, wherein the nucleic acid sequence has at least about 70% of less commonly used codons replaced with more commonly used codons.

7. A recombinant expression vector comprising the synthetic polynucleotide of any one of claims 1-6.

8. The recombinant expression vector of claim 7, wherein the recombinant expression vector is single stranded.

9. The recombinant expression vector of claim 7, wherein the recombinant expression vector is self-complementary.

10. The recombinant expression vector of any one of claims 7-9, wherein the recombinant expression vector is a viral vector.

11. The recombinant expression vector of any one of claims 7-9, wherein the recombinant expression vector is an adeno-associated viral (AAV) vector.

12. The recombinant expression vector of any one of claims 7-9, wherein the recombinant expression vector is an AAV8 or AAV9 vector.

13. The recombinant expression vector of any one of claims 7-9, wherein the recombinant expression vector is a non-viral vector.

14. The recombinant expression vector of any one of claims 7-13, wherein the recombinant expression vector is configured for expression of the synMMAB polynucleotide in one or both of hepatic tissue and renal tissue.

15. The recombinant expression vector of any one of claims 7-14, wherein the recombinant expression vector comprises the nucleic acid sequence of any one of SEQ ID NOs: 9-10 and 13-20.

16. An isolated or purified host cell comprising the recombinant expression vector of any one of claims 7-15.

17. An isolated or purified population of cells comprising the host cell of claim 16.

18. A pharmaceutical composition comprising (i) the synthetic polynucleotide of any one of claims 1-6, the recombinant expression vector of any one of claims 7-15, the host cell of claim 16, or the population of cells of claim 17 and (ii) a pharmaceutically acceptable carrier.

19. A method of treating a disease or condition mediated by MMAB (Metabolism Of Cobalamin Associated B) enzyme, comprising administering to a mammal in need thereof a therapeutic amount of the synthetic polynucleotide of any one of claims 1-6, the recombinant expression vector of any one of claims 7-15, the host cell of claim 16, the population of cells of claim 17, or the pharmaceutical composition of claim 18.

20. A method of treating a disease or condition mediated by MMAB (Metabolism Of Cobalamin Associated B) enzyme, comprising: producing the MMAB (Metabolism Of Cobalamin Associated B) enzyme by expressing the synthetic polynucleotide of any one of claims 1-6 or the recombinant expression vector of any one of claims 7-15 by a host cell, and purifying the enzyme from the host cell; and administering to a mammal in need thereof the purified MMAB (Metabolism Of Cobalamin Associated B) enzyme.

21. A method of treating a disease or condition mediated by MMAB (Metabolism Of Cobalamin Associated B) enzyme, comprising administering to a cell of a mammal in need thereof the polynucleotide of any one of claims 1-6, wherein the polynucleotide is inserted into the cell of the mammal via genome editing on the cell of the mammal using a nuclease selected from the group of zinc finger nucleases (ZFNs), transcription activator-like effectornucleases (TALENs), the clustered regularly interspaced short palindromic repeats (CRISPR / cas system) and meganuclease re-engineered homing endonucleases on a cell from the mammal.

22. The method of claim 21, wherein the method comprises administering the polynucleotide to an isolated cell of the mammal, and the method further comprises administering the cell to the mammal.

23. The method of claim 21, wherein the method comprises administering the polynucleotide to the cell of the mammal in vivo.

24. The method of any one of claims 19-23, wherein the disease or condition is methylmalonic acidemia (MMA).

25. The method of any one of claims 19-23, wherein the disease or condition is MMAB deficiency.

26. A method of detecting the presence of a synthetic MMAB (Metabolism Of Cobalamin Associated B) polynucleotide (synMMAB) in a biological sample from a mammal, the method comprising:(a) obtaining at least one test sample comprising nucleic acid from a biological sample from a mammal;(b) contacting any of the synthetic polynucleotide of any one of claims 1-6 with the at least one test sample under conditions allowing for a complex to form between the synthetic polynucleotide and the isolated nucleic acid of the test sample;(c) detecting the complex; and(d) comparing a presence of the complex in the at least one test sample with an absence of complex from a negative sample that lacks the synthetic polynucleotide, wherein detection of the complex is indicative of the presence of the synthetic polynucleotide in the biological sample from the mammal.

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