- Email: [email protected]
168. Assaying Hepatic Correction Mediated by Varied AAV Vectors in a Knock-Out Transgenic Mouse Model of Methylmalonic Acidemia (MMA)
Diabetes, Metabolic and Genetic Diseases I We further show that activation of hepatic ChREBP signaling that improves glucose tolerance and insulin sensitivity is one mechanism that protects the rAAV-GPE-G6PT-treated G6pt-/- mice against agerelated obesity and insulin resistance.
166. Lentiviral Hematopoietic Stem Cell Gene Therapy for Sjögren-Larsson Syndrome
Yoon-Sang Kim1, Irudayam Maria Johnson1, Dana S’Aulis2, William B. Rizzo2, Arthur W. Nienhuis1 1 Hematology, St. Jude Children’s Research Hospital, Memphis, TN, 2 Pediatrics, University of Nebraska Medical Center, Omaha, NE Sjögren-Larsson syndrome (SLS) is a rare autosomal recessive disorder characterized by scaling skin (ichthyosis), mental retardation, and spasticity. SLS is caused by mutations in the ALDH3A2 gene, which encodes fatty aldehyde dehydrogenase (FALDH), an enzyme that is involved in the oxidation of fatty aldehyde and fatty alcohol. In SLS, FALDH deficiency and impaired fatty aldehyde oxidation results in lipid accumulation, which is responsible for the symptoms. Spurred by previous hematopoietic stem cell gene therapy trials for multi-systemic diseases, such as Adrenoleukodystrophy (ALD), Metachromatic leukodystrophy (MLD), and Cystinosis, we investigated the plausibility of hematopoietic stem cell gene transfer to correct FALDH deficiency and alleviate the phenotype in SLS. We designed the hematopoietic stem cell gene therapy using a lentiviral vector containing human ALDH3A2 cDNA based on our previous MND-WASP (Wiskott-Aldrich syndrome protein) vector design. The lentiviral vector contains the normal human ALDH3A2 coding sequence which was derived from PCR products using human mobilized peripheral blood CD34+ cDNA. The vector was tested for FALDH protein expression in transfected human HEK293T cells by western blot and the enzyme activity was confirmed using HPLC-MS/UV. A transplantation experiment was performed using the Aldh3a2-/- KO mouse model, where the lineage negative BM cells from CD45.1 Aldh3a2-/- KO mice (C57BL/6 background) were sorted and transduced with a lentiviral vector followed by transplantation into lethally irradiated CD45.2 Aldh3a2-/- KO mice (C57BL/6 background). These mice are being monitored for cell engraftment, vector copy numbers, and any phenotypic changes. An initial analysis for the grafts showed 56% transduction in the MND(a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer)-GFP group and 31% transduction in the MND-ALDH3A2 group based on the CFU-C data. Flow cytometric analysis of peripheral blood on these mice at 6 and 12 post- transplant week showed 32% transduction in MND-GFP group. Further analysis including vector copy numbers and end point analysis for various tissues including liver, spleen, and brain are underway. This work provides the foundation for moving forward with potential lentiviral hematopoietic stem cell gene therapy for non-hematological disorders.
167. Genome Editing to Generate the First Mouse Model of Alpha-One Antitrypsin Deficiency, the Leading Cause of Genetic COPD
Florie Borel1, Brynn Cardozo1, Andrew Cox1, Weiying Li1, Andrew Hoffman2, Mai ElMallah1, Christian Mueller1 1 Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, 2Tufts University, Grafton, MA Alpha-one antitrypsin (AAT) deficiency is a common autosomal codominant genetic disorder. This condition affects 1:2500 individuals of European ancestry, leading to the development of lung and liver disease. Within North American and Northern European populations, an estimated 4% of individuals are carriers of mutant alleles. AAT deficiency presents with an emphysema phenotype in the lungs of Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
older subjects. AAT deficient subjects can also suffer from liver disease of varying severity; however, lung disease is the principle cause of death. AAT is a protease inhibitor predominantly synthesized in the liver that belongs to the serine protease inhibitor (serpin) family. Upon secretion into the blood stream, AAT enters the lungs where it inactivates excess neutrophil elastase, thereby preventing damage to the alveoli. Mutations of the Serpina1 gene can lead to reduced serum levels of AAT and decreased protein functionality, allowing for unrestricted elastin breakdown, pulmonary inflammation and eventual emphysema. Currently, an animal model simulating the lung condition does not exist, which severely limits the development of therapeutics. This is due to the higher genomic complexity of mice compared to humans. Indeed due to amplification events, C57BL/6 mice have five genes that are homologous to human SERPINA1. To address this we generated a quintuple gene knockout using CRISPR/Cas9 system via zygote microinjection. We generated three founding lines in which all 5 copies of the gene were disrupted. Mice from all three lines demonstrate absence of hepatic and circulatory AAT protein as well as a reduced capability to inactivate neutrophil elastase. We also characterized the lung phenotype in response to a lipopolysaccharide challenge, where the model recapitulated many characteristics of the human lung disease including decreased elastance and increased compliance, and lung morphometry was also affected. Genomic and transcriptomic characterization will be presented. Future work will include challenges with cigarette smoke, a well-known disease accelerator in patients. Further, the ongoing generation of a new transgenic model carrying both the quintuple disruption of the murine Serpina1 genes and a single copy of the Z variant of human SERPINA1 will bring to the field the ultimate disease model that will finally allow researchers to evaluate the effects of liver-directed gene augmentation in the presence of Z-AAT polymers.
168. Assaying Hepatic Correction Mediated by Varied AAV Vectors in a Knock-Out Transgenic Mouse Model of Methylmalonic Acidemia (MMA)
Brandon T. Hubbard, Randy J. Chandler, Charles P. Venditti National Human Genome Research Institute, National Institutes of Health, Bethesda, MD Methylmalonic acidemia (MMA) is an inborn error of metabolism most commonly caused by deficient methylmalonyl-CoA mutase (MUT) activity. The disorder can have multiple clinical manifestations, including metabolic instability, stroke of the basal ganglia, pancreatitis, end-stage renal failure, growth impairment, osteoporosis and developmental delay. Unfortunately, current non-invasive therapies fail to chronically manage the disease, and patients still suffer from increased morbidity and early mortality. Solid organ transplantation, including elective liver, combined liver-kidney and isolated kidney transplantation, has been used to provide sustained benefit to patients, but the procedures come with substantial risks as well as the postoperative requirement for life-long immunosuppression. To address the large and unmet need for new therapies for patients with MMA, we have developed an effective adeno-associated viral (AAV) gene therapy that has been previously validated in a neonatal lethal mouse model of Mut deficiency (Mut-/-). The current project compares how two distinct AAV8 vectors that express the human MUT gene under the control of either the liver specific, human alpha 1-antitrypsin (hAAT) promoter, or the ubiquitous CMV-enhanced chicken β-actin (CBA) promoter differentially affect metabolite levels following systemic delivery to adult mice. The animals used in this study (Mut-/-;TgINS-MCK-Mut) express wild-type Mut in a muscle-specific fashion via a stable germline transgene and completely lack transgene expression in the liver. Mut-/-;TgINS-MCK-Mut mice accurately model the hepatorenal manifestations of MMA, but afford an opportunity to assess gene therapy vectors at or after weaning because mice are S65
Diabetes, Metabolic and Genetic Diseases I rescued from neonatal lethality, yet experience massive elevations of the characteristic metabolites (methylmalonic and 2-methylcitric acid) because of the lack of hepatic Mut activity. Disease related metabolites were measured in plasma samples derived from Mut-/-;TgINS-MCK-Mut mice (n=7) prior to AAV gene delivery. The mice were then injected via the retro-orbital route with 1.5X1011 GC of either AAV8-hAATMUT or AAV8-CBA-MUT. At 10 days and 30 days post-treatment, mice treated with either vector showed a significant reduction in methylcitrate and methymalonic acid levels, with AAV8-CBA-MUT (n=2) treated mice manifesting methylcitrate and methylmalonic acid levels that trended lower than those measured in mice injected with AAV8-hAAT-MUT (n=4) at both time points. Further studies will be needed to precisely compare the differences between the hAAT and CBA promoters in MMA mouse models, but our preliminary results demonstrate that the Mut-/-;TgINS-MCK-Mut mice can be used to easily ascertain hepatic correction of Mut deficiency, which should help inform the selection of regulatory elements that will provide maximal therapeutic efficacy to treat patients with MMA.
169. Expression Studies of Ornithine Transcarbamylase in Liver Using Minicircle Vectors for Gene Therapy: Flag-Tagging of a Mitochondrial Enzyme and Stable Expression Under Control of an Endogenous Otc PromoterEnhancer
Hiu Man Viecelli1, Sereina Deplazes1, Andrea Schlegel2, Sharon Cunningham3, Ian Alexander3, Johannes Häberle1, Beat Thöny1 1 Division of Metabolism and Children’s Research Center, University Children’s Hospital Zurich, Zurich, Switzerland, 2 Department of Sugery, Swiss HPB and Transplant Center, Zurich, Switzerland, 3Children’s Medical Research Institute, Westmead, Australia Ornithine transcarbamylase deficiency (OTCD) is the most common inherited defect of the urea cycle resulting in severe hyperammonemia and death if left untreated. We are aiming at correcting OTCD using naked-DNA minicircle (MC) vector mediated gene therapy in the spfash mouse model with low residual OTC activity. A critical parameter is delivery to periportal hepatocytes where the urea cycle is located because of metabolic zonation in the liver. To distinguish between MC-born and endogenous OTC enzyme, we generated an expression cassette with an internally Flag-tagged OTC enzyme. Internal epitope tagging is required because of the N-terminal mitochondrial import sequence. A corresponding Flag-tag sequence was introduced C-terminally of the mitochondrial import signal. The tagged protein performed similar to its non-tagged OTC enzyme upon hydrodynamic tail vein injection for liver targeting in spfash mice, indicating that the tag neither interferes with the mitochondrial import nor the formation of a functional OTC trimer. Furthermore we generated an endogenous Otc promoter-enhancer construct, termed PmO1, for potential specific or “natural” OTC transgene expression. According to others, the promoter (672 bp) sequence derived from mouse Otc was not sufficient for liver specificity (Veres et al, J Biol Chem 261: 7588-7591, 1986) and requires a corresponding enhancer sequence (Nishiyori et al, J Biol Chem 269: 1323-1331, 1994). An enhancer sequence (232 bp) originating from rat Otc was used that contains several binding sites for liver-selective transcription factors (Murakami et al, Mol Cell Biol 10: 1180-1191, 1990). The resulting MC-vector expressing OTC from this PmO1 promoter-enhancer construct was delivered to spfash mice via hydrodynamic tail vein injection, resulting in similar enzymatic activity in whole liver extracts compared to wild-type mice. Vectors designed to express Flag-tagged OTC driven from a natural Otc promoter might be useful to study biodistribution and/or long-term stable expression after MC-based gene therapy for OTCD. S66
170. PhaseRx mRNA Technology Platform Uses SMARTT Polymer Technology® to Target and Deliver mRNA to the Liver and Treat a Urea Cycle Disorder in a Mouse Disease Model Mary Prieve, Allen Li, Alex Baturevych, Eric Bell, Teri Blevins, Anna Galperin, Pierrot Harvie, Jean-Rene Ella Menye, Sean Monahan, Amber Paschal, Maher Qabar, Debashish Roy, Matt Waldheim, Mike Houston PhaseRx, Seattle, WA
Messenger RNA (mRNA) is a promising alternative in both the viral and non-viral DNA-based gene delivery fields. Current viral vectors for gene therapy are associated with serious safety concerns and nonviral vectors are limited by low gene transfer efficiency. mRNA gene expression in the liver can be used for treatment of genetic diseases involving disorders of metabolism. The majority are due to defects of single genes that code for enzymes expressed solely or predominantly in the liver. SMARTT Polymer Technology® has been developed into a robust platform for RNA therapeutics. Optimization of the mRNA technology platform has led to stepwise improvements in mRNA delivery to the liver using GalNAc targeted polymers. We demonstrated a 5,000-fold improvement in activity over our first generation delivery system. Urea cycle disorders result from single gene mutations that lead to deficiency in one of the six enzymes in the urea cycle pathway. This deficiency can trigger elevated blood ammonia levels, also known as hyperammonemia, a life-threatening illness that leads to brain damage, coma or even death in humans. The deficient protein is intracellular and IV protein therapeutics are ineffective. Liver transplantation is the only cure for urea cycle disorders but is limited by the shortage of donors and complications associated with rejection and infection of the transplant. There is a dire need for new treatment options. Using the mRNA technology platform, we have demonstrated preclinical proof of concept in a urea cycle disorder mouse disease model. Treatment with therapeutic mRNA shows normalization of blood ammonia levels in hyperammonemic mice. Therapeutic mRNA expression is detected in the liver after a single mRNA dose with good duration of expression. The treatment was well tolerated, with no toxicities associated with both single and multiple dosing regimens. This mRNA technology platform provides a significant opportunity for the treatment of urea cycle disorders and other orphan liver diseases.
171. Quantification of Human FVIII and Estimation of Its Molecular Weight: Potential Applications in Gene Therapy for Hemophilia A Rajeev Mahimkar, Barrie Carter, Gordon Vehar Biomarin Pharmaceutical Inc, Novato, CA
Current protocols for measurement of FVIII rely on chromogenic or coagulation assays, and are not designed for quantification of FVIII antigen. Development of assays for measuring FVIII antigen may be necessary and beneficial for determining the efficiency of newer therapeutic approaches for Hemophilia A such as gene therapy. Herein, we describe the development of proprietary assays for quantification of human FVIII in plasma matrices from heterologous species, and estimation of the human FVIII molecular weight. A variety of monoclonal antibodies against the FVIII heavy and light chain were screened for their ability to recognize human FVIII from normal human plasma pool, or CHO cell derived recombinant human FVIII-B domain deleted (FVIII-BDD) spiked in mouse and non-human primate (NHP) plasma. Based on the initial screen, a monoclonal each against the human FVIII heavy or the light chain was selected for enrichment, followed by detection using a sheep antiMolecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
166. Lentiviral Hematopoietic Stem Cell Gene Therapy for Sjögren-Larsson Syndrome
Yoon-Sang Kim1, Irudayam Maria Johnson1, Dana S’Aulis2, William B. Rizzo2, Arthur W. Nienhuis1 1 Hematology, St. Jude Children’s Research Hospital, Memphis, TN, 2 Pediatrics, University of Nebraska Medical Center, Omaha, NE Sjögren-Larsson syndrome (SLS) is a rare autosomal recessive disorder characterized by scaling skin (ichthyosis), mental retardation, and spasticity. SLS is caused by mutations in the ALDH3A2 gene, which encodes fatty aldehyde dehydrogenase (FALDH), an enzyme that is involved in the oxidation of fatty aldehyde and fatty alcohol. In SLS, FALDH deficiency and impaired fatty aldehyde oxidation results in lipid accumulation, which is responsible for the symptoms. Spurred by previous hematopoietic stem cell gene therapy trials for multi-systemic diseases, such as Adrenoleukodystrophy (ALD), Metachromatic leukodystrophy (MLD), and Cystinosis, we investigated the plausibility of hematopoietic stem cell gene transfer to correct FALDH deficiency and alleviate the phenotype in SLS. We designed the hematopoietic stem cell gene therapy using a lentiviral vector containing human ALDH3A2 cDNA based on our previous MND-WASP (Wiskott-Aldrich syndrome protein) vector design. The lentiviral vector contains the normal human ALDH3A2 coding sequence which was derived from PCR products using human mobilized peripheral blood CD34+ cDNA. The vector was tested for FALDH protein expression in transfected human HEK293T cells by western blot and the enzyme activity was confirmed using HPLC-MS/UV. A transplantation experiment was performed using the Aldh3a2-/- KO mouse model, where the lineage negative BM cells from CD45.1 Aldh3a2-/- KO mice (C57BL/6 background) were sorted and transduced with a lentiviral vector followed by transplantation into lethally irradiated CD45.2 Aldh3a2-/- KO mice (C57BL/6 background). These mice are being monitored for cell engraftment, vector copy numbers, and any phenotypic changes. An initial analysis for the grafts showed 56% transduction in the MND(a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer)-GFP group and 31% transduction in the MND-ALDH3A2 group based on the CFU-C data. Flow cytometric analysis of peripheral blood on these mice at 6 and 12 post- transplant week showed 32% transduction in MND-GFP group. Further analysis including vector copy numbers and end point analysis for various tissues including liver, spleen, and brain are underway. This work provides the foundation for moving forward with potential lentiviral hematopoietic stem cell gene therapy for non-hematological disorders.
167. Genome Editing to Generate the First Mouse Model of Alpha-One Antitrypsin Deficiency, the Leading Cause of Genetic COPD
Florie Borel1, Brynn Cardozo1, Andrew Cox1, Weiying Li1, Andrew Hoffman2, Mai ElMallah1, Christian Mueller1 1 Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, 2Tufts University, Grafton, MA Alpha-one antitrypsin (AAT) deficiency is a common autosomal codominant genetic disorder. This condition affects 1:2500 individuals of European ancestry, leading to the development of lung and liver disease. Within North American and Northern European populations, an estimated 4% of individuals are carriers of mutant alleles. AAT deficiency presents with an emphysema phenotype in the lungs of Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
older subjects. AAT deficient subjects can also suffer from liver disease of varying severity; however, lung disease is the principle cause of death. AAT is a protease inhibitor predominantly synthesized in the liver that belongs to the serine protease inhibitor (serpin) family. Upon secretion into the blood stream, AAT enters the lungs where it inactivates excess neutrophil elastase, thereby preventing damage to the alveoli. Mutations of the Serpina1 gene can lead to reduced serum levels of AAT and decreased protein functionality, allowing for unrestricted elastin breakdown, pulmonary inflammation and eventual emphysema. Currently, an animal model simulating the lung condition does not exist, which severely limits the development of therapeutics. This is due to the higher genomic complexity of mice compared to humans. Indeed due to amplification events, C57BL/6 mice have five genes that are homologous to human SERPINA1. To address this we generated a quintuple gene knockout using CRISPR/Cas9 system via zygote microinjection. We generated three founding lines in which all 5 copies of the gene were disrupted. Mice from all three lines demonstrate absence of hepatic and circulatory AAT protein as well as a reduced capability to inactivate neutrophil elastase. We also characterized the lung phenotype in response to a lipopolysaccharide challenge, where the model recapitulated many characteristics of the human lung disease including decreased elastance and increased compliance, and lung morphometry was also affected. Genomic and transcriptomic characterization will be presented. Future work will include challenges with cigarette smoke, a well-known disease accelerator in patients. Further, the ongoing generation of a new transgenic model carrying both the quintuple disruption of the murine Serpina1 genes and a single copy of the Z variant of human SERPINA1 will bring to the field the ultimate disease model that will finally allow researchers to evaluate the effects of liver-directed gene augmentation in the presence of Z-AAT polymers.
168. Assaying Hepatic Correction Mediated by Varied AAV Vectors in a Knock-Out Transgenic Mouse Model of Methylmalonic Acidemia (MMA)
Brandon T. Hubbard, Randy J. Chandler, Charles P. Venditti National Human Genome Research Institute, National Institutes of Health, Bethesda, MD Methylmalonic acidemia (MMA) is an inborn error of metabolism most commonly caused by deficient methylmalonyl-CoA mutase (MUT) activity. The disorder can have multiple clinical manifestations, including metabolic instability, stroke of the basal ganglia, pancreatitis, end-stage renal failure, growth impairment, osteoporosis and developmental delay. Unfortunately, current non-invasive therapies fail to chronically manage the disease, and patients still suffer from increased morbidity and early mortality. Solid organ transplantation, including elective liver, combined liver-kidney and isolated kidney transplantation, has been used to provide sustained benefit to patients, but the procedures come with substantial risks as well as the postoperative requirement for life-long immunosuppression. To address the large and unmet need for new therapies for patients with MMA, we have developed an effective adeno-associated viral (AAV) gene therapy that has been previously validated in a neonatal lethal mouse model of Mut deficiency (Mut-/-). The current project compares how two distinct AAV8 vectors that express the human MUT gene under the control of either the liver specific, human alpha 1-antitrypsin (hAAT) promoter, or the ubiquitous CMV-enhanced chicken β-actin (CBA) promoter differentially affect metabolite levels following systemic delivery to adult mice. The animals used in this study (Mut-/-;TgINS-MCK-Mut) express wild-type Mut in a muscle-specific fashion via a stable germline transgene and completely lack transgene expression in the liver. Mut-/-;TgINS-MCK-Mut mice accurately model the hepatorenal manifestations of MMA, but afford an opportunity to assess gene therapy vectors at or after weaning because mice are S65
Diabetes, Metabolic and Genetic Diseases I rescued from neonatal lethality, yet experience massive elevations of the characteristic metabolites (methylmalonic and 2-methylcitric acid) because of the lack of hepatic Mut activity. Disease related metabolites were measured in plasma samples derived from Mut-/-;TgINS-MCK-Mut mice (n=7) prior to AAV gene delivery. The mice were then injected via the retro-orbital route with 1.5X1011 GC of either AAV8-hAATMUT or AAV8-CBA-MUT. At 10 days and 30 days post-treatment, mice treated with either vector showed a significant reduction in methylcitrate and methymalonic acid levels, with AAV8-CBA-MUT (n=2) treated mice manifesting methylcitrate and methylmalonic acid levels that trended lower than those measured in mice injected with AAV8-hAAT-MUT (n=4) at both time points. Further studies will be needed to precisely compare the differences between the hAAT and CBA promoters in MMA mouse models, but our preliminary results demonstrate that the Mut-/-;TgINS-MCK-Mut mice can be used to easily ascertain hepatic correction of Mut deficiency, which should help inform the selection of regulatory elements that will provide maximal therapeutic efficacy to treat patients with MMA.
169. Expression Studies of Ornithine Transcarbamylase in Liver Using Minicircle Vectors for Gene Therapy: Flag-Tagging of a Mitochondrial Enzyme and Stable Expression Under Control of an Endogenous Otc PromoterEnhancer
Hiu Man Viecelli1, Sereina Deplazes1, Andrea Schlegel2, Sharon Cunningham3, Ian Alexander3, Johannes Häberle1, Beat Thöny1 1 Division of Metabolism and Children’s Research Center, University Children’s Hospital Zurich, Zurich, Switzerland, 2 Department of Sugery, Swiss HPB and Transplant Center, Zurich, Switzerland, 3Children’s Medical Research Institute, Westmead, Australia Ornithine transcarbamylase deficiency (OTCD) is the most common inherited defect of the urea cycle resulting in severe hyperammonemia and death if left untreated. We are aiming at correcting OTCD using naked-DNA minicircle (MC) vector mediated gene therapy in the spfash mouse model with low residual OTC activity. A critical parameter is delivery to periportal hepatocytes where the urea cycle is located because of metabolic zonation in the liver. To distinguish between MC-born and endogenous OTC enzyme, we generated an expression cassette with an internally Flag-tagged OTC enzyme. Internal epitope tagging is required because of the N-terminal mitochondrial import sequence. A corresponding Flag-tag sequence was introduced C-terminally of the mitochondrial import signal. The tagged protein performed similar to its non-tagged OTC enzyme upon hydrodynamic tail vein injection for liver targeting in spfash mice, indicating that the tag neither interferes with the mitochondrial import nor the formation of a functional OTC trimer. Furthermore we generated an endogenous Otc promoter-enhancer construct, termed PmO1, for potential specific or “natural” OTC transgene expression. According to others, the promoter (672 bp) sequence derived from mouse Otc was not sufficient for liver specificity (Veres et al, J Biol Chem 261: 7588-7591, 1986) and requires a corresponding enhancer sequence (Nishiyori et al, J Biol Chem 269: 1323-1331, 1994). An enhancer sequence (232 bp) originating from rat Otc was used that contains several binding sites for liver-selective transcription factors (Murakami et al, Mol Cell Biol 10: 1180-1191, 1990). The resulting MC-vector expressing OTC from this PmO1 promoter-enhancer construct was delivered to spfash mice via hydrodynamic tail vein injection, resulting in similar enzymatic activity in whole liver extracts compared to wild-type mice. Vectors designed to express Flag-tagged OTC driven from a natural Otc promoter might be useful to study biodistribution and/or long-term stable expression after MC-based gene therapy for OTCD. S66
170. PhaseRx mRNA Technology Platform Uses SMARTT Polymer Technology® to Target and Deliver mRNA to the Liver and Treat a Urea Cycle Disorder in a Mouse Disease Model Mary Prieve, Allen Li, Alex Baturevych, Eric Bell, Teri Blevins, Anna Galperin, Pierrot Harvie, Jean-Rene Ella Menye, Sean Monahan, Amber Paschal, Maher Qabar, Debashish Roy, Matt Waldheim, Mike Houston PhaseRx, Seattle, WA
Messenger RNA (mRNA) is a promising alternative in both the viral and non-viral DNA-based gene delivery fields. Current viral vectors for gene therapy are associated with serious safety concerns and nonviral vectors are limited by low gene transfer efficiency. mRNA gene expression in the liver can be used for treatment of genetic diseases involving disorders of metabolism. The majority are due to defects of single genes that code for enzymes expressed solely or predominantly in the liver. SMARTT Polymer Technology® has been developed into a robust platform for RNA therapeutics. Optimization of the mRNA technology platform has led to stepwise improvements in mRNA delivery to the liver using GalNAc targeted polymers. We demonstrated a 5,000-fold improvement in activity over our first generation delivery system. Urea cycle disorders result from single gene mutations that lead to deficiency in one of the six enzymes in the urea cycle pathway. This deficiency can trigger elevated blood ammonia levels, also known as hyperammonemia, a life-threatening illness that leads to brain damage, coma or even death in humans. The deficient protein is intracellular and IV protein therapeutics are ineffective. Liver transplantation is the only cure for urea cycle disorders but is limited by the shortage of donors and complications associated with rejection and infection of the transplant. There is a dire need for new treatment options. Using the mRNA technology platform, we have demonstrated preclinical proof of concept in a urea cycle disorder mouse disease model. Treatment with therapeutic mRNA shows normalization of blood ammonia levels in hyperammonemic mice. Therapeutic mRNA expression is detected in the liver after a single mRNA dose with good duration of expression. The treatment was well tolerated, with no toxicities associated with both single and multiple dosing regimens. This mRNA technology platform provides a significant opportunity for the treatment of urea cycle disorders and other orphan liver diseases.
171. Quantification of Human FVIII and Estimation of Its Molecular Weight: Potential Applications in Gene Therapy for Hemophilia A Rajeev Mahimkar, Barrie Carter, Gordon Vehar Biomarin Pharmaceutical Inc, Novato, CA
Current protocols for measurement of FVIII rely on chromogenic or coagulation assays, and are not designed for quantification of FVIII antigen. Development of assays for measuring FVIII antigen may be necessary and beneficial for determining the efficiency of newer therapeutic approaches for Hemophilia A such as gene therapy. Herein, we describe the development of proprietary assays for quantification of human FVIII in plasma matrices from heterologous species, and estimation of the human FVIII molecular weight. A variety of monoclonal antibodies against the FVIII heavy and light chain were screened for their ability to recognize human FVIII from normal human plasma pool, or CHO cell derived recombinant human FVIII-B domain deleted (FVIII-BDD) spiked in mouse and non-human primate (NHP) plasma. Based on the initial screen, a monoclonal each against the human FVIII heavy or the light chain was selected for enrichment, followed by detection using a sheep antiMolecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy