- Email: [email protected]
46. The First Viable Mouse Model of cblC Type Combined Methylmalonic Acidemia and Homocysteinemia: AAV Gene Therapy Rescues Neonatal Lethality and Provides Insight into Disease-Associated Retinal Degeneration
Diabetes, Metabolic and Genetic Diseases protected cells by enabling efficient selection only of CCR5-modified T-cells. The mutant human dihydrofolate reductase (mDHFR) chemoselection system has been used to render cells resistant to lymphotoxic concentrations of the drug methotrexate (MTX). We tested our experimental approach by transducing cells with lentiviral vector encoding a mDHFR cassette followed by chemoselection in MTX at 0.02uM. This approach resulted in a six fold enrichment of gene modified primary CD4+ T cells ex vivo. Previously we have shown that combining megaTAL treatment with adeno-associated virus (AAV) transduction produces very high rates of homologydriven repair (HDR) in primary human T cells. Hence, we combined megaTAL/AAV treatment to integrate the mutant DHFR into the CCR5 locus, producing a population of MTX-resistant CD4+ cells that also lack CCR5. Primary human CD4+ T cells were transfected with CCR5-megaTAL mRNA and transduced with AAV6 containing a mutant DHFR donor template flanked by 0.6kb CCR5 homology arms. They demonstrated a greater than five-fold enrichment in MTX compared to untreated controls ex vivo. Next, we have transplanted NSG mice with 1x106 gene modified cells/ mouse to assess the therapeutic potential of our approach. Mice that engraft effectively will be treated with daily injections of 0, 0.5 and 2 mg/kg of MTX to monitor preferential selection and enrichment of our target cell population. Subsequent studies will assess the long term control of viremia in these mice following HIV challenge. In conclusion, the CCR5-megaTAL nuclease platform produces very high levels of genemodified CD4+ T-cells and protects these cells from subsequent HIV infection in vivo. Furthermore, combining targeted integration and chemical selection results in the specific selection of gene modified primary human T cells. To our knowledge we are the first group to report MTX-mediated chemoselection and expansion of CD4+ T cells following targeted integration at the CCR5 locus.
decreases in intrinsic excitability, dendritic arborization complexity, and synapses in motor cortex layer V neurons. These findings show that 1) the intrinsic excitability of neurons of homozygous Arg1 knockout mice is abnormal and that, unexpectedly, heterozygous neurons (single copy loss) exhibit an intermediate phenotype compared to wild type and homozygous knockouts (double copy loss) (Fig . A); corresponding loss of Arg1 decreased the frequency of miniature excitatory postsynaptic currents and the amplitude of miniature inhibitory postsynaptic currents; 2) neuronal branching and spine phenotypes differ between genotypes with, unexpectedly, an intermediate phenotype for heterozygotes (Fig. B); and 3) with electron microscopic analysis and comparison of layer V synapses from arginase 1 knockout, heterozygous, and wild type mice, there is a very low density of excitatory (i.e. asymmetrical) synapses (Fig. C) in the knockout and decreased number of inhibitory (perisomatic) synapses (i.e. symmetrical) on somata of pyramidal cells, both dramatic findings. Finally, changes in synaptic morphology and abnormal ultrastructural features were found in knockout mice, also suggesting neuronal degeneration and inflammation. Neonatal intravenous administration on the second postnatal day with AAV expressing arginase 1 by a hepatocyte-specific promoter led to a nearresolution of these abnormalities when administered to homozygous Arg1 knockout animals. Summary: Our studies suggest that arginase 1 deficiency leads to severe and specific changes to intrinsic excitability and synaptic connectivity of motor cortical circuits. Importantly, we find that neonatal AAV-based Arg1 gene expression is effective in reversing both the physiological and anatomical hallmarks of the disorder.
Diabetes, Metabolic and Genetic Diseases 45. Rescue of the Functional Alterations of Motor Cortical Circuits in Arginase 1 Deficiency with AAV-Based Gene Therapy
Gloria Cantero1, Xiao-Bo Liu2, Steven D. Cederbaum1, Peyman Golshani1, Gerald S. Lipshutz1 1 UCLA, Los Angeles, CA, 2UC Davis, Davis, CA The urea cycle is the main mechanism for terrestrial mammals to detoxify excess nitrogen. Disorders of the proximal urea cycle characteristically have periodic episodes of hyperammonemia leading to often severe and permanent neurological deterioration & disability. Ammonia has been implicated by compromising potassium buffering of astrocytic membranes and causing clinical neurological abnormalities by impairing cortical inhibition. Complete arginase 1 (Arg1) deficiency, a distal urea cycle disorder, is the least severe of these abnormalities, demonstrating neurological impairment including spasticity, loss of ambulation and seizures; while characterized by the presence of hyperargininemia, hyperammonemia is not a frequent clinical finding. While mortality is unfortunately common due to acute episodes of hyperammonemia in proximal urea cycle disorders, patients with hyperargininemia often are long-lived, however, suffering from progressive intellectual disability and spastic diplegia, and the mechanisms underlying the neurological dysfunction are not understood. To gain better insight on how the loss of arginase expression causes dysfunction in the developing brain, and if gene therapy could prevent these abnormalities, we studied how the excitability and functional and anatomical connectivity of motor cortical neurons are altered in the disorder using the murine knockout model. In addition, we examined if AAV expressing Arg1, administered IV on postnatal day 2, could rescue these findings. Results: Single- and double-copy loss of Arg1 caused dose-dependent S20
46. The First Viable Mouse Model of cblC Type Combined Methylmalonic Acidemia and Homocysteinemia: AAV Gene Therapy Rescues Neonatal Lethality and Provides Insight into Disease-Associated Retinal Degeneration
Madeline Arnold1, Jennifer L. Sloan1, Nathan P. Achilly1, Gene Elliot1, Ighovie F. Onojafe2, Brian P. Brooks1,2, Charles P. Venditti1 1 National Human Genome Research Institute, Bethesda, MD, 2 National Eye Institute, Bethesda, MD Combined methylmalonic acidemia and homocysteinemia, cblC type (cblC), is the most common inborn error of cobalamin metabolism and is caused by mutations in the MMACHC gene. MMACHC transports and processes intracellular cobalamin (vitamin B12) into its two active cofactors, 5’-adenosylcobalamin and methylcobalamin, necessary for the enzymatic reactions of methylmalonyl-CoA mutase and methionine synthase, respectively. Mutations in MMACHC result in methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia. Disease manifestations include growth failure, anemia, heart defects, developmental delay and a progressive Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Diabetes, Metabolic and Genetic Diseases maculopathy and pigmentary retinopathy that causes blindness, usually by the end of the first decade. Despite the use of conventional therapies including cobalamin injections and other cofactors, the manifestions of cblC, in particular the eye disease, remain unresolved with treatment. In order to explore disease pathophysiology and develop AAV gene therapy for cblC deficiency, we first created a viable mouse model using TALENs to edit the murine Mmachc gene, near the location of the most common mutation in humans, c.271dupA. Two alleles were investigated: c.165_166delAC p.P56CfsX4 (Δ2) and c.162_164delCAC p.S54_T55delinsR (Δ3). MmachcΔ2/Δ2 and MmachcΔ3/Δ3 homozygous mutant mice displayed reduced survival, severe growth retardation, and massive metabolic perturbations. The median survival was less than 7 days with 90% of the mutant mice perishing before 3 weeks (Δ2 n=13; Δ3 n=42). The weights of MmachcΔ3/Δ3 mice were reduced relative to heterozygote and wild type littermates (n=15, p<0.0001). MmachcΔ2/Δ2 and MmachcΔ3/Δ3 mice (n=4, n=6) recapitulated the biochemical features of cblC, with significantly elevated plasma methylmalonic acid, homocysteine, cystathionine and decreased methionine compared to littermate controls (n=7) (p<0.05 for all metabolites). To assess the potential for gene therapy as a treatment for cblC, we generated two AAV vectors: rAAVrh10CBA-mMmachc and rAAV9-CBA-hMMACHC and compared AAV with biweekly injections of OH-cobalamin, the standard therapy. MmachcΔ3/Δ3 mice were then treated with a single vector dose (1 x 1011 GC) delivered via intrahepatic injection in the neonatal period. MmachcΔ3/Δ3 mice treated with AAV vectors (AAVrh10 n=11, AAV9 n=5) displayed dramatically improved clinical appearance with improved growth (p= 0.0568), and increased survival (p<0.0001 for both vectors), with the oldest treated mutants currently living beyond 9 months. Successful gene therapy in the MmachcΔ3/Δ3 mice also enabled us to model, for the first time, cblC associated ocular pathology: surviving MmachcΔ3/Δ3 mice displayed thinning of the outer nuclear layer and shortening of photoreceptor outer segments, consistent with the pathology described in patients. Our results demonstrate that AAV gene delivery of MMACHC represents a new therapy for cblC which can treat the systemic, and possibly ocular, manifestations of this relatively common and devastating inborn error of metabolism.
47. Gene Transfer-Mediated Diversion Towards Non-Toxic Metabolites for Therapy of Primary Hyperoxaluria Type 1 Nicola Brunetti-Pierri, Raffaele Castello, Patrizia Annunziata, Roberta Borzone Telethon Institute of Genetics and Medicine, Pozzuoli, Italy
Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to mutations of the AGXT gene encoding the peroxisomal enzyme alanine:glyoxylate-aminotransferase (AGT) which catalyzes the conversion of glyoxylate to glycine. In PH1 patients, glyoxylate cannot be efficiently converted into glycine and is instead oxidized to oxalate resulting in systemic oxalosis with deposition of insoluble calcium oxalate in kidneys and in other tissues, leading to nephrolithiasis, nephrocalcinosis, kidney failure, and systemic tissue damage. Combined liver/kidney transplantation is the only therapeutic strategy available to prevent disease progression. We hypothesize that overexpression of specific genes encoding enzymes involved in glyoxalate metabolism will steer glyoxylate towards alternative pathways to diminish oxalate production. To test this hypothesis, we overexpressed murine glyoxylate reductase/ hydroxypyruvate reductase (GRHPR), that converts glyoxylate into glycolate, by a helper-dependent adenoviral vector (HDAd-GRHPR) in livers of Agxt−/− mice. The intravenous injection of HDAd-GRHPR resulted in significant reduction of hyperoxaluria and concomitant increase of serum glycolate that was not associated with signs of toxicity. Glutamate-pyruvate transaminase (GPT) in the cytosol Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
transaminate glyoxylate using glutamate and alanine as aminogroup donors. We hypothesize that GPT overexpression will steer glyoxylate towards transamination to diminish oxalate production. The intravenous injection of a helper-dependent adenoviral vector expressing murine GPT (HDAd-GPT) in Agxt−/− mice also resulted in significant and sustained reduction of hyperoxaluria. Interestingly, co-administration of both HDAd-GRHPR and HDAd-GPT resulted in further reduction and normalization of hyperoxaluria. Glycolate is one of the substrates leading to glyoxylate production, via peroxisomal glycolate oxidase (GO). We also show that an HDAd expressing a short hairpin RNA against GO resulted in reduction of hyperoxaluria in Agxt−/− mice. In summary, the results of this study show that metabolic diversion towards non-toxic metabolites has potential for treatment of PH1 and potentially other forms of hyperoxalurias, both primary and secondary. The metabolic diversion could be also obtained by RNA-based molecules expressing GRHPR and/or GPT or inhibiting GO activity. In addition, this study shows that HDAd vectors can be used to functionally validate therapeutic enzyme targets in inherited metabolic diseases.
48. Treatment of Methylmalonic Acidemia by Promoterless Gene-Targeting Using AdenoAssociated Viral (AAV) Mediated Homologous Recombination
Randy J. Chandler1, Adi Barzel2, Mark A. Kay2, Charles P. Venditti1 1 MGMMGB, National Institutes of Health, Bethesda, MD, 2 Departments of Pediatrics and Genetics, Stanford University, Stanford, CA Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of metabolism most typically caused by mutations in methylmalonyl-CoA mutase (MUT). While the hallmark of this disease is elevated levels of methylmalonic acid in the plasma, other disease related metabolites such as methylcitrate are elevated in the plasma as well. Patients with MMA suffer from frequent and potential lethal bouts of metabolic instability that can be eliminated by liver transplantation. Adeno-associated viral (AAV) gene therapy has shown great promise as the treatment for MMA in a murine model of the disease. However, a majority of the AAV-treated mice developed hepatocellular cancer, which was determined to arise from AAV-mediated insertional mutagenesis. In an attempt to create a safer gene therapy platform for the treatment of MMA, we created a novel vector for site-specific gene addition of human MUT into the mouse albumin (Alb) locus. This promoterless AAV vector contains a 2A-peptide coding sequence proximal to a codon-optimized human MUT gene. The 2A-MUT sequence is flanked by arms of homology immediately upstream of mouse Alb stop codon. Since albumin is expressed exclusively in hepatocytes, we prepared an AAV serotype 8 vector to take advantage of the murine liver tropism conferred by this capsid. This newly created vector was named AAV8-Alb-A2MUT. After site-specific integration of the vector into the Alb locus in the liver, ribosomal skipping generates both Alb and MUT as separate proteins derived from the same transcript. We delivered a dose of 2.5e12 GC of AAV8-Alb-2A-MUT to five mice with MMA by intraperitoneal injection at birth. At one month post-injection, we observed increased hepatic expression of the MUT by western blot, improved growth and a significant reduction of disease related metabolites, in the treated MMA mice (Table 1). This gene delivery approach is anticipated to provide permanent hepatic transgene expression while reducing the risk of off-target integration and vectormediated insertional mutagenesis.
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decreases in intrinsic excitability, dendritic arborization complexity, and synapses in motor cortex layer V neurons. These findings show that 1) the intrinsic excitability of neurons of homozygous Arg1 knockout mice is abnormal and that, unexpectedly, heterozygous neurons (single copy loss) exhibit an intermediate phenotype compared to wild type and homozygous knockouts (double copy loss) (Fig . A); corresponding loss of Arg1 decreased the frequency of miniature excitatory postsynaptic currents and the amplitude of miniature inhibitory postsynaptic currents; 2) neuronal branching and spine phenotypes differ between genotypes with, unexpectedly, an intermediate phenotype for heterozygotes (Fig. B); and 3) with electron microscopic analysis and comparison of layer V synapses from arginase 1 knockout, heterozygous, and wild type mice, there is a very low density of excitatory (i.e. asymmetrical) synapses (Fig. C) in the knockout and decreased number of inhibitory (perisomatic) synapses (i.e. symmetrical) on somata of pyramidal cells, both dramatic findings. Finally, changes in synaptic morphology and abnormal ultrastructural features were found in knockout mice, also suggesting neuronal degeneration and inflammation. Neonatal intravenous administration on the second postnatal day with AAV expressing arginase 1 by a hepatocyte-specific promoter led to a nearresolution of these abnormalities when administered to homozygous Arg1 knockout animals. Summary: Our studies suggest that arginase 1 deficiency leads to severe and specific changes to intrinsic excitability and synaptic connectivity of motor cortical circuits. Importantly, we find that neonatal AAV-based Arg1 gene expression is effective in reversing both the physiological and anatomical hallmarks of the disorder.
Diabetes, Metabolic and Genetic Diseases 45. Rescue of the Functional Alterations of Motor Cortical Circuits in Arginase 1 Deficiency with AAV-Based Gene Therapy
Gloria Cantero1, Xiao-Bo Liu2, Steven D. Cederbaum1, Peyman Golshani1, Gerald S. Lipshutz1 1 UCLA, Los Angeles, CA, 2UC Davis, Davis, CA The urea cycle is the main mechanism for terrestrial mammals to detoxify excess nitrogen. Disorders of the proximal urea cycle characteristically have periodic episodes of hyperammonemia leading to often severe and permanent neurological deterioration & disability. Ammonia has been implicated by compromising potassium buffering of astrocytic membranes and causing clinical neurological abnormalities by impairing cortical inhibition. Complete arginase 1 (Arg1) deficiency, a distal urea cycle disorder, is the least severe of these abnormalities, demonstrating neurological impairment including spasticity, loss of ambulation and seizures; while characterized by the presence of hyperargininemia, hyperammonemia is not a frequent clinical finding. While mortality is unfortunately common due to acute episodes of hyperammonemia in proximal urea cycle disorders, patients with hyperargininemia often are long-lived, however, suffering from progressive intellectual disability and spastic diplegia, and the mechanisms underlying the neurological dysfunction are not understood. To gain better insight on how the loss of arginase expression causes dysfunction in the developing brain, and if gene therapy could prevent these abnormalities, we studied how the excitability and functional and anatomical connectivity of motor cortical neurons are altered in the disorder using the murine knockout model. In addition, we examined if AAV expressing Arg1, administered IV on postnatal day 2, could rescue these findings. Results: Single- and double-copy loss of Arg1 caused dose-dependent S20
46. The First Viable Mouse Model of cblC Type Combined Methylmalonic Acidemia and Homocysteinemia: AAV Gene Therapy Rescues Neonatal Lethality and Provides Insight into Disease-Associated Retinal Degeneration
Madeline Arnold1, Jennifer L. Sloan1, Nathan P. Achilly1, Gene Elliot1, Ighovie F. Onojafe2, Brian P. Brooks1,2, Charles P. Venditti1 1 National Human Genome Research Institute, Bethesda, MD, 2 National Eye Institute, Bethesda, MD Combined methylmalonic acidemia and homocysteinemia, cblC type (cblC), is the most common inborn error of cobalamin metabolism and is caused by mutations in the MMACHC gene. MMACHC transports and processes intracellular cobalamin (vitamin B12) into its two active cofactors, 5’-adenosylcobalamin and methylcobalamin, necessary for the enzymatic reactions of methylmalonyl-CoA mutase and methionine synthase, respectively. Mutations in MMACHC result in methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia. Disease manifestations include growth failure, anemia, heart defects, developmental delay and a progressive Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Diabetes, Metabolic and Genetic Diseases maculopathy and pigmentary retinopathy that causes blindness, usually by the end of the first decade. Despite the use of conventional therapies including cobalamin injections and other cofactors, the manifestions of cblC, in particular the eye disease, remain unresolved with treatment. In order to explore disease pathophysiology and develop AAV gene therapy for cblC deficiency, we first created a viable mouse model using TALENs to edit the murine Mmachc gene, near the location of the most common mutation in humans, c.271dupA. Two alleles were investigated: c.165_166delAC p.P56CfsX4 (Δ2) and c.162_164delCAC p.S54_T55delinsR (Δ3). MmachcΔ2/Δ2 and MmachcΔ3/Δ3 homozygous mutant mice displayed reduced survival, severe growth retardation, and massive metabolic perturbations. The median survival was less than 7 days with 90% of the mutant mice perishing before 3 weeks (Δ2 n=13; Δ3 n=42). The weights of MmachcΔ3/Δ3 mice were reduced relative to heterozygote and wild type littermates (n=15, p<0.0001). MmachcΔ2/Δ2 and MmachcΔ3/Δ3 mice (n=4, n=6) recapitulated the biochemical features of cblC, with significantly elevated plasma methylmalonic acid, homocysteine, cystathionine and decreased methionine compared to littermate controls (n=7) (p<0.05 for all metabolites). To assess the potential for gene therapy as a treatment for cblC, we generated two AAV vectors: rAAVrh10CBA-mMmachc and rAAV9-CBA-hMMACHC and compared AAV with biweekly injections of OH-cobalamin, the standard therapy. MmachcΔ3/Δ3 mice were then treated with a single vector dose (1 x 1011 GC) delivered via intrahepatic injection in the neonatal period. MmachcΔ3/Δ3 mice treated with AAV vectors (AAVrh10 n=11, AAV9 n=5) displayed dramatically improved clinical appearance with improved growth (p= 0.0568), and increased survival (p<0.0001 for both vectors), with the oldest treated mutants currently living beyond 9 months. Successful gene therapy in the MmachcΔ3/Δ3 mice also enabled us to model, for the first time, cblC associated ocular pathology: surviving MmachcΔ3/Δ3 mice displayed thinning of the outer nuclear layer and shortening of photoreceptor outer segments, consistent with the pathology described in patients. Our results demonstrate that AAV gene delivery of MMACHC represents a new therapy for cblC which can treat the systemic, and possibly ocular, manifestations of this relatively common and devastating inborn error of metabolism.
47. Gene Transfer-Mediated Diversion Towards Non-Toxic Metabolites for Therapy of Primary Hyperoxaluria Type 1 Nicola Brunetti-Pierri, Raffaele Castello, Patrizia Annunziata, Roberta Borzone Telethon Institute of Genetics and Medicine, Pozzuoli, Italy
Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to mutations of the AGXT gene encoding the peroxisomal enzyme alanine:glyoxylate-aminotransferase (AGT) which catalyzes the conversion of glyoxylate to glycine. In PH1 patients, glyoxylate cannot be efficiently converted into glycine and is instead oxidized to oxalate resulting in systemic oxalosis with deposition of insoluble calcium oxalate in kidneys and in other tissues, leading to nephrolithiasis, nephrocalcinosis, kidney failure, and systemic tissue damage. Combined liver/kidney transplantation is the only therapeutic strategy available to prevent disease progression. We hypothesize that overexpression of specific genes encoding enzymes involved in glyoxalate metabolism will steer glyoxylate towards alternative pathways to diminish oxalate production. To test this hypothesis, we overexpressed murine glyoxylate reductase/ hydroxypyruvate reductase (GRHPR), that converts glyoxylate into glycolate, by a helper-dependent adenoviral vector (HDAd-GRHPR) in livers of Agxt−/− mice. The intravenous injection of HDAd-GRHPR resulted in significant reduction of hyperoxaluria and concomitant increase of serum glycolate that was not associated with signs of toxicity. Glutamate-pyruvate transaminase (GPT) in the cytosol Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
transaminate glyoxylate using glutamate and alanine as aminogroup donors. We hypothesize that GPT overexpression will steer glyoxylate towards transamination to diminish oxalate production. The intravenous injection of a helper-dependent adenoviral vector expressing murine GPT (HDAd-GPT) in Agxt−/− mice also resulted in significant and sustained reduction of hyperoxaluria. Interestingly, co-administration of both HDAd-GRHPR and HDAd-GPT resulted in further reduction and normalization of hyperoxaluria. Glycolate is one of the substrates leading to glyoxylate production, via peroxisomal glycolate oxidase (GO). We also show that an HDAd expressing a short hairpin RNA against GO resulted in reduction of hyperoxaluria in Agxt−/− mice. In summary, the results of this study show that metabolic diversion towards non-toxic metabolites has potential for treatment of PH1 and potentially other forms of hyperoxalurias, both primary and secondary. The metabolic diversion could be also obtained by RNA-based molecules expressing GRHPR and/or GPT or inhibiting GO activity. In addition, this study shows that HDAd vectors can be used to functionally validate therapeutic enzyme targets in inherited metabolic diseases.
48. Treatment of Methylmalonic Acidemia by Promoterless Gene-Targeting Using AdenoAssociated Viral (AAV) Mediated Homologous Recombination
Randy J. Chandler1, Adi Barzel2, Mark A. Kay2, Charles P. Venditti1 1 MGMMGB, National Institutes of Health, Bethesda, MD, 2 Departments of Pediatrics and Genetics, Stanford University, Stanford, CA Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of metabolism most typically caused by mutations in methylmalonyl-CoA mutase (MUT). While the hallmark of this disease is elevated levels of methylmalonic acid in the plasma, other disease related metabolites such as methylcitrate are elevated in the plasma as well. Patients with MMA suffer from frequent and potential lethal bouts of metabolic instability that can be eliminated by liver transplantation. Adeno-associated viral (AAV) gene therapy has shown great promise as the treatment for MMA in a murine model of the disease. However, a majority of the AAV-treated mice developed hepatocellular cancer, which was determined to arise from AAV-mediated insertional mutagenesis. In an attempt to create a safer gene therapy platform for the treatment of MMA, we created a novel vector for site-specific gene addition of human MUT into the mouse albumin (Alb) locus. This promoterless AAV vector contains a 2A-peptide coding sequence proximal to a codon-optimized human MUT gene. The 2A-MUT sequence is flanked by arms of homology immediately upstream of mouse Alb stop codon. Since albumin is expressed exclusively in hepatocytes, we prepared an AAV serotype 8 vector to take advantage of the murine liver tropism conferred by this capsid. This newly created vector was named AAV8-Alb-A2MUT. After site-specific integration of the vector into the Alb locus in the liver, ribosomal skipping generates both Alb and MUT as separate proteins derived from the same transcript. We delivered a dose of 2.5e12 GC of AAV8-Alb-2A-MUT to five mice with MMA by intraperitoneal injection at birth. At one month post-injection, we observed increased hepatic expression of the MUT by western blot, improved growth and a significant reduction of disease related metabolites, in the treated MMA mice (Table 1). This gene delivery approach is anticipated to provide permanent hepatic transgene expression while reducing the risk of off-target integration and vectormediated insertional mutagenesis.
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