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87. Rationally Designed Inverted Terminal Repeats Enhance Transduction of Embryonic Stem Cells
AAV Vectors I 86. AAV9 Mediated Correction of Iduronate2-Sulfatase Deficiency in the Central Nervous System to Prevent the Onset of Neurologic Deficits in a Murine Model of Mucopolysaccharidosis Type II. Kanut Laoharawee , Kelly M. Podetz-Petersen , Kelley Kitto , Lucy Vulchanova2, Carolyn Fairbanks3, Karen Kozarsky4, Walter C. Low5, R. Scott McIvor1 1 Genetics Cell Biology and Development, University of Minnesota, Minneapolis, MN, 2Neuroscience, University of Minnesota, Minneapolis, MN, 3Pharmaceutics, University of Minnesota, Minneapolis, MN, 4REGENXBIO Inc., Washington D.C., DC, 5 Neurosurgery, University of Minnesota, Minneapolis, MN 1
1
2
Mucopolysaccharidosis type II (MPS II; Hunter Syndrome) is an X-linked recessive inherited lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS) and subsequent accumulation of glycosaminoglycans (GAGs) dermatan and heparan sulphate. Affected individuals exhibit a range in severity of manifestations such as organomegaly, skeletal dysplasias, cardiopulmonary obstruction, neurocognitive deficit, and shortened life expectancy. There is no cure for MPS II at the moment. Current standard of care is enzyme replacement therapy (ELAPSRASE; idursulfase), which is used to manage disease progression. However, enzyme replacement therapy (ERT) does not show neurologic improvement. As hematopoetic stem cell transplantation (HSCT) has not shown neurologic benefit for MPS II, there is currently no clinical recourse for patients exhibiting neurologic manifestations of this disease, and new therapies are desperately needed. We have been developing the use of AAV9 vectors for delivering the human IDS coding sequence (AAV9.hIDS) into the central nervous system of MPS II mice to restore IDS levels in the brain and prevent the emergence of neurocognitive deficits in the treated animals. A series of CMV-enhancer, beta actin-regulated vectors were generated that encode human IDS with or without the human sulfatase modifying factor-1 (SUMF-1), required for activation of the sulfatase active site. Three routes of administration; Intrathecal (IT), Intracerebroventricular (ICV) and Intravenous (IV) were used in these experiments. We found no significant difference in the enzyme level between mice that were treated with AAV9 vector transducing hIDS alone and mice that were treated with AAV9 vector encoding human IDS and SUMF-1, regardless of the route of administration. IT-administrated NOD.SCID (IDS Y+) and C57BL/6 (IDS Y+) did not show elevated IDS activity in the brain and spinal cord when compared to untreated animals, while plasma showed ten-fold higher (NOD.SCID) and 150-fold higher (C57BL/6) levels than untreated animals. IDS-deficient mice intravenously administered AAV9-hIDS exhibited IDS activities in all organs that were comparable to wild type. Moreover, the plasma of IV injected animals showed enzyme activity that was 100-fold higher than wild type. IDS-deficient mice administered AAV9-hIDUA intracerebroventricularly showed IDS activities comparable to wild type in most areas of the brain and peripheral tissues, while some portions of the brain showed two- to four-fold higher activity than wild type. Furthermore, IDS activity in plasma was 200-fold higher than wild type. Surprisingly, IDS enzyme activity in the plasma of all administrated animals showed persistence for at least 12 weeks post injection; therefore, IDS enzyme was not immunogenic at least on the C57BL/6 murine background. We also conducted additional neurobehavioral testing using the Barnes maze to differentiate neurocognitive deficits of untreated MPS II animals from that of wild type littermates. We found that the learning capability of affected animals is distinctively slower than that observed in littermates. Thus, Barnes maze will be used to address the benefit of these therapies in the MPS II murine model in future
S38
experiments. These results indicate potential of therapeutic benefit of AAV9 mediated human IDS gene transfer to the CNS to prevent neurologic deficiency in the MPS II.
87. Rationally Designed Inverted Terminal Repeats Enhance Transduction of Embryonic Stem Cells Nolan Brown, Matthew Hirsch University of North Carolina, Chapel Hill, NC
Despite the prevalence of AAV vectors in gene therapy, treatments combining AAV vectors and cell therapy have not progressed into the clinic despite previous reports of successful transduction of stem cells by AAV vectors. One potential explanation is AAV vector toxicity in stem and stem-like cells, as demonstrated in previous studies. In particular, the AAV inverted terminal repeat (ITR) sequence was shown to induce apoptosis in human embryonic stem cells (hESCs) in a p53 dependent manner. A bioinformatic analysis of putative transcription factor binding sites in the AAV2 ITR sequence revealed six putative p53 binding sites. A panel of rationally designed AAV2 ITRs was generated which abrogated putative p53 binding sites and demonstrated reduced p53 binding in an in vitro binding assay. The synthetic ITRs were capable of all of the vector production requirements and were verified by sequencing directly from vector preparations. AAV vector transduction and the hESC DNA damage response was altered by the synthetic ITRs. Importantly, our ITRs demonstrated reduced toxicity in hESCs compared to an AAV vector having the AAV2 ITR sequence. The collective results allude to modulation of the host’s DNA damage response using rationally designed ITRs, which may enhance the relevancy of AAV vectors in stem cell therapies.
88. Safety and Biodistribution Study of rAAV2tYF-PR1.7-hCNGB3 in Nonhuman Primates
Guo-jie Ye1, Ewa Budzynski2, Peter Sonnentag2, T. Michael Nork3, Paul E. Miller3, Alok K. Sharma2, James N. Ver Hoeve3, Leia M. Smith4, Tara Arndt2, Roberto Calcedo5, Chantelle Gaskin1, Paulette M. Robinson1, David R. Knop1, William W. Hauswirth6, Jeffrey D. Chulay1 1 Applied Genetic Technologies Corporation, Alachua, FL, 2 Covance Laboratories Inc, Madison, WI, 3OSOD, Madison, WI, 4 LabCorp, Seattle, WA, 5University of Pennsylvania, Philadelphia, PA, 6University of Florida, Gainesville, FL Background: AGTC is developing a recombinant AAV vector expressing the human CNGB3 gene for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity and absence of color discrimination. Here we report results of a toxicology and biodistribution study of this vector administered by subretinal injection in cynomolgus macaques. Methods: Three groups of animals (n=2 males and 2 females per group) received a subretinal injection in one eye of 300 µL containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 1011 or 4 × 1012 vg/mL) and were evaluated for safety and biodistribution over a 3-month period prior to euthanasia. Toxicity assessment was based on mortality, clinical observations, body weights, ophthalmic examinations, intraocular pressure (IOP) measurements, electroretinography (ERG), visual evoked potentials (VEP), and clinical and anatomic pathology. Vector shedding and biodistribution was assessed by qPCR analyses. Immune responses to AAV and hCNGB3 were measured by ELISA, Elispot, or neutralization antibody assay for AAV2tYF. Results: There was no evidence of local or systemic toxicity and no changes in IOP, VEP responses, or hematology, coagulation or clinical chemistry parameters and no clinically important changes in ERG responses. Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
1
2
Mucopolysaccharidosis type II (MPS II; Hunter Syndrome) is an X-linked recessive inherited lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS) and subsequent accumulation of glycosaminoglycans (GAGs) dermatan and heparan sulphate. Affected individuals exhibit a range in severity of manifestations such as organomegaly, skeletal dysplasias, cardiopulmonary obstruction, neurocognitive deficit, and shortened life expectancy. There is no cure for MPS II at the moment. Current standard of care is enzyme replacement therapy (ELAPSRASE; idursulfase), which is used to manage disease progression. However, enzyme replacement therapy (ERT) does not show neurologic improvement. As hematopoetic stem cell transplantation (HSCT) has not shown neurologic benefit for MPS II, there is currently no clinical recourse for patients exhibiting neurologic manifestations of this disease, and new therapies are desperately needed. We have been developing the use of AAV9 vectors for delivering the human IDS coding sequence (AAV9.hIDS) into the central nervous system of MPS II mice to restore IDS levels in the brain and prevent the emergence of neurocognitive deficits in the treated animals. A series of CMV-enhancer, beta actin-regulated vectors were generated that encode human IDS with or without the human sulfatase modifying factor-1 (SUMF-1), required for activation of the sulfatase active site. Three routes of administration; Intrathecal (IT), Intracerebroventricular (ICV) and Intravenous (IV) were used in these experiments. We found no significant difference in the enzyme level between mice that were treated with AAV9 vector transducing hIDS alone and mice that were treated with AAV9 vector encoding human IDS and SUMF-1, regardless of the route of administration. IT-administrated NOD.SCID (IDS Y+) and C57BL/6 (IDS Y+) did not show elevated IDS activity in the brain and spinal cord when compared to untreated animals, while plasma showed ten-fold higher (NOD.SCID) and 150-fold higher (C57BL/6) levels than untreated animals. IDS-deficient mice intravenously administered AAV9-hIDS exhibited IDS activities in all organs that were comparable to wild type. Moreover, the plasma of IV injected animals showed enzyme activity that was 100-fold higher than wild type. IDS-deficient mice administered AAV9-hIDUA intracerebroventricularly showed IDS activities comparable to wild type in most areas of the brain and peripheral tissues, while some portions of the brain showed two- to four-fold higher activity than wild type. Furthermore, IDS activity in plasma was 200-fold higher than wild type. Surprisingly, IDS enzyme activity in the plasma of all administrated animals showed persistence for at least 12 weeks post injection; therefore, IDS enzyme was not immunogenic at least on the C57BL/6 murine background. We also conducted additional neurobehavioral testing using the Barnes maze to differentiate neurocognitive deficits of untreated MPS II animals from that of wild type littermates. We found that the learning capability of affected animals is distinctively slower than that observed in littermates. Thus, Barnes maze will be used to address the benefit of these therapies in the MPS II murine model in future
S38
experiments. These results indicate potential of therapeutic benefit of AAV9 mediated human IDS gene transfer to the CNS to prevent neurologic deficiency in the MPS II.
87. Rationally Designed Inverted Terminal Repeats Enhance Transduction of Embryonic Stem Cells Nolan Brown, Matthew Hirsch University of North Carolina, Chapel Hill, NC
Despite the prevalence of AAV vectors in gene therapy, treatments combining AAV vectors and cell therapy have not progressed into the clinic despite previous reports of successful transduction of stem cells by AAV vectors. One potential explanation is AAV vector toxicity in stem and stem-like cells, as demonstrated in previous studies. In particular, the AAV inverted terminal repeat (ITR) sequence was shown to induce apoptosis in human embryonic stem cells (hESCs) in a p53 dependent manner. A bioinformatic analysis of putative transcription factor binding sites in the AAV2 ITR sequence revealed six putative p53 binding sites. A panel of rationally designed AAV2 ITRs was generated which abrogated putative p53 binding sites and demonstrated reduced p53 binding in an in vitro binding assay. The synthetic ITRs were capable of all of the vector production requirements and were verified by sequencing directly from vector preparations. AAV vector transduction and the hESC DNA damage response was altered by the synthetic ITRs. Importantly, our ITRs demonstrated reduced toxicity in hESCs compared to an AAV vector having the AAV2 ITR sequence. The collective results allude to modulation of the host’s DNA damage response using rationally designed ITRs, which may enhance the relevancy of AAV vectors in stem cell therapies.
88. Safety and Biodistribution Study of rAAV2tYF-PR1.7-hCNGB3 in Nonhuman Primates
Guo-jie Ye1, Ewa Budzynski2, Peter Sonnentag2, T. Michael Nork3, Paul E. Miller3, Alok K. Sharma2, James N. Ver Hoeve3, Leia M. Smith4, Tara Arndt2, Roberto Calcedo5, Chantelle Gaskin1, Paulette M. Robinson1, David R. Knop1, William W. Hauswirth6, Jeffrey D. Chulay1 1 Applied Genetic Technologies Corporation, Alachua, FL, 2 Covance Laboratories Inc, Madison, WI, 3OSOD, Madison, WI, 4 LabCorp, Seattle, WA, 5University of Pennsylvania, Philadelphia, PA, 6University of Florida, Gainesville, FL Background: AGTC is developing a recombinant AAV vector expressing the human CNGB3 gene for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity and absence of color discrimination. Here we report results of a toxicology and biodistribution study of this vector administered by subretinal injection in cynomolgus macaques. Methods: Three groups of animals (n=2 males and 2 females per group) received a subretinal injection in one eye of 300 µL containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 1011 or 4 × 1012 vg/mL) and were evaluated for safety and biodistribution over a 3-month period prior to euthanasia. Toxicity assessment was based on mortality, clinical observations, body weights, ophthalmic examinations, intraocular pressure (IOP) measurements, electroretinography (ERG), visual evoked potentials (VEP), and clinical and anatomic pathology. Vector shedding and biodistribution was assessed by qPCR analyses. Immune responses to AAV and hCNGB3 were measured by ELISA, Elispot, or neutralization antibody assay for AAV2tYF. Results: There was no evidence of local or systemic toxicity and no changes in IOP, VEP responses, or hematology, coagulation or clinical chemistry parameters and no clinically important changes in ERG responses. Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy