- Email: [email protected]
165. Liver-Directed Gene Therapy for Murine Glycogen Storage Disease Type IB
Diabetes, Metabolic and Genetic Diseases I 162. Functional Correction of Mucopolysaccharidosis I in Adult Mice by a Systemic rAAV9-IDUA Gene Delivery
Aaron Meadows, Ricardo Pineda, Marybeth Camboni, Kathryn Waligura, Darren Murrey, Douglas M. McCarty, Haiyan Fu Center for Gene Therapy, Research Institute at Nationwide Children’s Hospital, Columbus, OH Mucopolysaccharidosis (MPS) I is a lysosomal storage disease caused by autosomal recessive defect in iduronidase (IDUA). The lack of IDUA activity results in the accumulation of GAGs in cells in virtually all organs, leading to profound somatic and neurological disorders. No treatment is currently available for the neurological disorders of MPS I. In this study, we have developed a self-complementary (sc) AAV9 vector expressing human IDUA, targeting the root cause. A single intravenous injection of scAAV9hIDUA vector at 5x1012vg/kg led to the rapid and persistent restoration of IDUA activity and the clearance of lysosomal storage pathology throughout the CNS, peripheral nervous system (PNS) and broad peripheral tissues, as well as the correction of astrocytosis in the CNS and PNS. Furthermore, we demonstrate that a single systemic scAAV9-IDUA gene delivery provides long-term neurological benefits in MPS I mice, resulting in significant improvement in cognitive and motor function, and extension of survival (ongoing). More importantly, functional benefits were also achieved in MPS I mice that were treated at advanced disease stages. These data demonstrate the promising clinical potential of systemic scAAV9hIDUA gene delivery for treating MPS I and other neurogenetic diseases.
163. Hematopoietic Stem Cells Transplantation Can Normalize Thyroid Function in a Cystinosis Mouse Model
Heloise P. Gaide Chevronnay1, Virginie Janssens1, Patrick Van Der Smissen1, Celine J. Rocca2, Xiao-Hui Liao3, Samuel Refetoff3, Christophe E. Pierreux1, Stephanie Cherqui2, Pierre J. Courtoy1 1 Cell Biology Unit, de Duve Institute & Université catholique de Louvain, Brussels, Belgium, 2Pediatrics, UCSD, La Jolla, CA, 3 Medicine, The University of Chicago, Chicago, IL Cystinosis is a multi-systemic lysosomal storage disease caused by defective transmembrane cystine transporter, cystinosin (CTNS gene). In the mouse model of cystinosis, Ctns-/- mice, it has already been shown that hematopoietic stem and progenitor cell (HSC) transplantation provides long-term protection of kidneys and eyes, which are affected early on in cystinosis. Tissue repair involves transfer of cystinosin-bearing lysosomes from HSPCs differentiated as macrophages into deficient adjacent cells, via tunnelling nanotubes (TNTs). Hypothyroidism is the most frequent and earliest endocrine complication in cystinotic patients and was recently shown as a complication in the Ctns-/- mice too. Here, we are evaluating the benefit of HSPC transplantation in the thyroid of Ctns-/- mice. Abundant engraftment of bone marrow-derived cells in Ctns-/- thyroid correlated with drastic decreased of cystine content, normalization of TSH level and correction of the structure of a large fraction of thyrocytes. In the thyroid microenvironment, HSPCs differentiated into a distinct, mixed macrophage/dendritic cell lineage expressing CD45 and MHCII, but not-detectably CD11b and F4/80. Like in cystinotic kidneys, HSPC-derived cells produced TNT-like extensions capable of crossing the follicular basement laminae. Interestingly, HSPCs themselves further squeezed into follicles, allowing extensive contact with thyrocytes, but did not transdifferentiate into Nkx2.1expressing cells, the hallmark of thyrocytes. This is the first report
S64
demonstrating the potential of HSPC transplantation to correct thyroid disease, and supports a major multisystemic benefit of stem cells therapy for cystinosis.
164. Improvement of Gene Therapy for Wilson Disease
Oihana Murillo1, Daniel Moreno1, Cristina Gazquez1, Iñigo Navarro-Blasco2, Jesus Prieto1, Ruben Hernandez-Alcoceba1, Gloria Gonzalez-Aseguinolaza1 1 Gene therapy, FIMA, Pamplona, Spain, 2Biochemestry, University of Navarra, Pamplona, Spain Wilson’s disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs, which may cause side effects and do not restore normal copper metabolism. We have recently demonstrated that the administration of an AAV vector expressing ATP7B under the control of a liver specific promoter induces full restoration of copper homeostasis in a mouse model of Wilson’s disease. However, the size of the vector genome surpasses the optimal size for AAV packaging, limiting the use of large promoters or additional regulatory sequences such as introns or bigger poly A sequences. In the present work we have designed two truncated versions of ATP7B protein (T1 and T2), and have analyzed their functionality and therapeutic efficacy. ATP7B contains 6 metal binding sites (MBS) in the N-terminal region. T1 presents a deletion of 1 MBS and a fragment of the adjacent one, whereas T2 lacks the first 4 MBS. Both proteins were active since they were able eliminate intracellular excess of copper in an in vitro system. The administration of recombinant AAV vectors carrying the truncated proteins under the control of a liver specific promoter was able to reduce liver damage and urinary copper excretion in the murine model, but T1 was less efficient than the WT ATP7B gene. In contrast, T2 showed full capacity to normalize these parameters. Furthermore, T2 was more efficient than WT in restoring ceruloplasmin oxidase activity in serum. In conclusion, we have identified a fully functional truncated version of ATP7B protein that will reduce the size of the therapeutic AAV vector genome and allow the introduction of additional regulatory elements.
165. Liver-Directed Gene Therapy for Murine Glycogen Storage Disease Type IB
Joonhyun Kwon1, Jun-Ho Cho1, Young Mok Lee2, Goo-Young Kim1, Javier Anduaga1, Janice Chou1 1 NICHD/NIH, Bethesda, MD, 2University of Florida, Gainesville, FL Glycogen storage disease type Ib (GSD-Ib) deficient in the glucose-6-phosphate transporter (G6PT or SLC37A4) is characterized impaired glucose homeostasis, myeloid dysfunction, and long-term complication of hepatocellular adenoma (HCA). We have shown that gene therapy mediated by a recombinant (r) AAV8 vector expressing G6PT directed by the chicken β-actin promoter/CMV enhancer enabled the G6pt-/- mice lived to over 51 weeks but all 5 transduced G6pt-/- mice expressed only low levels of hepatic G6PT activity and two developed multiple HCAs with one undergoing malignant transformation. We now examined the safety and efficacy of rAAV8-GPE-G6PT, a rAAV8 vector expressing G6PT directed by the gluconeogenic tissue-specific human G6PC promoter/enhancer (GPE). Of the fifteen rAAV8-GPE-G6PT-treated G6pt-/- mice that lived over age 60 weeks expressed 2-62% of wild-type hepatic G6PT activity with only one developed HCA. The treated mice, including the HCA-bearing mouse exhibit a leaner phenotype along with normal blood metabolite, display normal glucose tolerance profiles, maintain normoglycemia over a 24-hour fast, and retain insulin sensitivity. Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
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
Aaron Meadows, Ricardo Pineda, Marybeth Camboni, Kathryn Waligura, Darren Murrey, Douglas M. McCarty, Haiyan Fu Center for Gene Therapy, Research Institute at Nationwide Children’s Hospital, Columbus, OH Mucopolysaccharidosis (MPS) I is a lysosomal storage disease caused by autosomal recessive defect in iduronidase (IDUA). The lack of IDUA activity results in the accumulation of GAGs in cells in virtually all organs, leading to profound somatic and neurological disorders. No treatment is currently available for the neurological disorders of MPS I. In this study, we have developed a self-complementary (sc) AAV9 vector expressing human IDUA, targeting the root cause. A single intravenous injection of scAAV9hIDUA vector at 5x1012vg/kg led to the rapid and persistent restoration of IDUA activity and the clearance of lysosomal storage pathology throughout the CNS, peripheral nervous system (PNS) and broad peripheral tissues, as well as the correction of astrocytosis in the CNS and PNS. Furthermore, we demonstrate that a single systemic scAAV9-IDUA gene delivery provides long-term neurological benefits in MPS I mice, resulting in significant improvement in cognitive and motor function, and extension of survival (ongoing). More importantly, functional benefits were also achieved in MPS I mice that were treated at advanced disease stages. These data demonstrate the promising clinical potential of systemic scAAV9hIDUA gene delivery for treating MPS I and other neurogenetic diseases.
163. Hematopoietic Stem Cells Transplantation Can Normalize Thyroid Function in a Cystinosis Mouse Model
Heloise P. Gaide Chevronnay1, Virginie Janssens1, Patrick Van Der Smissen1, Celine J. Rocca2, Xiao-Hui Liao3, Samuel Refetoff3, Christophe E. Pierreux1, Stephanie Cherqui2, Pierre J. Courtoy1 1 Cell Biology Unit, de Duve Institute & Université catholique de Louvain, Brussels, Belgium, 2Pediatrics, UCSD, La Jolla, CA, 3 Medicine, The University of Chicago, Chicago, IL Cystinosis is a multi-systemic lysosomal storage disease caused by defective transmembrane cystine transporter, cystinosin (CTNS gene). In the mouse model of cystinosis, Ctns-/- mice, it has already been shown that hematopoietic stem and progenitor cell (HSC) transplantation provides long-term protection of kidneys and eyes, which are affected early on in cystinosis. Tissue repair involves transfer of cystinosin-bearing lysosomes from HSPCs differentiated as macrophages into deficient adjacent cells, via tunnelling nanotubes (TNTs). Hypothyroidism is the most frequent and earliest endocrine complication in cystinotic patients and was recently shown as a complication in the Ctns-/- mice too. Here, we are evaluating the benefit of HSPC transplantation in the thyroid of Ctns-/- mice. Abundant engraftment of bone marrow-derived cells in Ctns-/- thyroid correlated with drastic decreased of cystine content, normalization of TSH level and correction of the structure of a large fraction of thyrocytes. In the thyroid microenvironment, HSPCs differentiated into a distinct, mixed macrophage/dendritic cell lineage expressing CD45 and MHCII, but not-detectably CD11b and F4/80. Like in cystinotic kidneys, HSPC-derived cells produced TNT-like extensions capable of crossing the follicular basement laminae. Interestingly, HSPCs themselves further squeezed into follicles, allowing extensive contact with thyrocytes, but did not transdifferentiate into Nkx2.1expressing cells, the hallmark of thyrocytes. This is the first report
S64
demonstrating the potential of HSPC transplantation to correct thyroid disease, and supports a major multisystemic benefit of stem cells therapy for cystinosis.
164. Improvement of Gene Therapy for Wilson Disease
Oihana Murillo1, Daniel Moreno1, Cristina Gazquez1, Iñigo Navarro-Blasco2, Jesus Prieto1, Ruben Hernandez-Alcoceba1, Gloria Gonzalez-Aseguinolaza1 1 Gene therapy, FIMA, Pamplona, Spain, 2Biochemestry, University of Navarra, Pamplona, Spain Wilson’s disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs, which may cause side effects and do not restore normal copper metabolism. We have recently demonstrated that the administration of an AAV vector expressing ATP7B under the control of a liver specific promoter induces full restoration of copper homeostasis in a mouse model of Wilson’s disease. However, the size of the vector genome surpasses the optimal size for AAV packaging, limiting the use of large promoters or additional regulatory sequences such as introns or bigger poly A sequences. In the present work we have designed two truncated versions of ATP7B protein (T1 and T2), and have analyzed their functionality and therapeutic efficacy. ATP7B contains 6 metal binding sites (MBS) in the N-terminal region. T1 presents a deletion of 1 MBS and a fragment of the adjacent one, whereas T2 lacks the first 4 MBS. Both proteins were active since they were able eliminate intracellular excess of copper in an in vitro system. The administration of recombinant AAV vectors carrying the truncated proteins under the control of a liver specific promoter was able to reduce liver damage and urinary copper excretion in the murine model, but T1 was less efficient than the WT ATP7B gene. In contrast, T2 showed full capacity to normalize these parameters. Furthermore, T2 was more efficient than WT in restoring ceruloplasmin oxidase activity in serum. In conclusion, we have identified a fully functional truncated version of ATP7B protein that will reduce the size of the therapeutic AAV vector genome and allow the introduction of additional regulatory elements.
165. Liver-Directed Gene Therapy for Murine Glycogen Storage Disease Type IB
Joonhyun Kwon1, Jun-Ho Cho1, Young Mok Lee2, Goo-Young Kim1, Javier Anduaga1, Janice Chou1 1 NICHD/NIH, Bethesda, MD, 2University of Florida, Gainesville, FL Glycogen storage disease type Ib (GSD-Ib) deficient in the glucose-6-phosphate transporter (G6PT or SLC37A4) is characterized impaired glucose homeostasis, myeloid dysfunction, and long-term complication of hepatocellular adenoma (HCA). We have shown that gene therapy mediated by a recombinant (r) AAV8 vector expressing G6PT directed by the chicken β-actin promoter/CMV enhancer enabled the G6pt-/- mice lived to over 51 weeks but all 5 transduced G6pt-/- mice expressed only low levels of hepatic G6PT activity and two developed multiple HCAs with one undergoing malignant transformation. We now examined the safety and efficacy of rAAV8-GPE-G6PT, a rAAV8 vector expressing G6PT directed by the gluconeogenic tissue-specific human G6PC promoter/enhancer (GPE). Of the fifteen rAAV8-GPE-G6PT-treated G6pt-/- mice that lived over age 60 weeks expressed 2-62% of wild-type hepatic G6PT activity with only one developed HCA. The treated mice, including the HCA-bearing mouse exhibit a leaner phenotype along with normal blood metabolite, display normal glucose tolerance profiles, maintain normoglycemia over a 24-hour fast, and retain insulin sensitivity. Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
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