- Email: [email protected]
364. Neurological Correction of Mucopolysaccharidosis IIIB Mice by Haematopoietic Stem Cell Gene Therapy
Gene Therapy for CNS Diseases molecular parameters were performed. While untreated SD mice had a 16 week humane endpoint, 4 of 7 mice in higher dose group are now surviving past 56 weeks, 1 of 12 mice in the low dose cohort, and 4 of 9 mice in the mannitol cohort are surviving past 52 weeks. These increases in survival are all highly significant compared to the ~16 week humane endpoint of untreated SD mice. Behaviourally, there are no major significant differences in locomotion between the groups until after 15 weeks, when the adjunct mannitol group significantly outperforms the PBS group. Survival and behaviour monitoring, and the biochemical analyses for this study are ongoing. The preliminary results from this study show delayed onset of the SD phenotype with a single AAV9/HEXM injection and a significant benefit of a pre-injection of IV mannitol. This study is the first to show that an IV gene transfer using a scAAV/HEXM vector can provide survival and behavioural benefit in adult SD mice especially with adjunct use of mannitol. We propose that these results can advise the design of a human gene therapy trial for SD and the related Tay-Sachs disease.
364. Neurological Correction of Mucopolysaccharidosis IIIB Mice by Haematopoietic Stem Cell Gene Therapy
Stuart M. Ellison1, Rebecca Holley1, Daniel Fil1, John Mc Dermott1, Nisha Senthivel1, Alex Langford-Smith1, Fiona Wilkinson1, Simon Jones2, Rob Wynn3, Shih-hsin Kan4, Patti Dickson4, Brian Bigger1 1 Stem Cell and Neurotherapies, University of Manchester, Manchester, United Kingdom, 2Centre for Genomic Medicine, Royal Manchester Children’s Hospital, Manchester, United Kingdom, 3Paediatric Haematology, Royal Manchester Children’s Hospital, Manchester, United Kingdom, 4Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, CA Mucopolysaccharidosis Type IIIB (MPSIIIB) is a paediatric, autosomal recessive Lysosomal Storage Disease (LSD) caused by deficiency of α-N-acetylglucosaminidase (NAGLU), an enzyme in the heparan sulfate (HS) degradation pathway. Absence of NAGLU leads to the accumulation of partially degraded HS glycosaminoglycan in cell lysosomes, giving rise to cellular dysfunction with devastating clinical consequences. Individuals affected by this fatal disease exhibit severe central nervous system degeneration with progressive cognitive impairment and behavioural problems, alongside more attenuated somatic symptoms. There are currently no effective treatments available. Enzyme replacement therapy with recombinant NAGLU enzyme is ineffective for MPSIIIB since enzyme cannot cross the blood brain barrier (BBB) to where it is needed. Modified recombinant NAGLU enzymes that utilise the insulin growth factor II (IGFII) peptide to facilitate improved uptake across the BBB are currently in development. Haematopoietic stem cell gene therapy (HSCGT) is a promising therapeutic strategy that can circumvent the BBB via monocyte trafficking and engraftment in the brain, allowing delivery of enzyme by cross correction. We have developed a novel stem cell gene therapy approach to investigate the therapeutic potential of HSCGT for MPSIIIB. We designed two lentiviral vectors expressing therapeutic enzyme; the first vector expressing codon optimised NAGLU, and the second expressing a NAGLU.IGFII fusion to aid cellular uptake, both driven by the myeloid specific promoter CD11b and compared these in autologous MPSIIIB transplants against a normal WT bone marrow transplant. Here we present for the first time neurological correction of MPSIIIB mice by HSCGT. We observed correction of the MPSIIIB behavioural phenotype in treated mice to wild-type levels with normalisation of path length, average speed, frequency entering the centre and duration of speed >100mm/s in open field tests. In addition, we observed a significant correction of astrogliosis and lysosomal compartment size in the brains of CD11b. NAGLU LV treated mice with an accompanied normalisation of S146
inflammatory cytokines TNFα, IL1B and IL1RN. Furthermore, NAGLU enzyme activity was substantially increased in the brain. Interestingly, WT transplant alone was able to mediate a partial brain correction, although levels of inflammation and lysosomal storage remain high.
365. Long-Term Toxicology Evaluation of AAVrh.10hARSA Administration to the CNS of Nonhuman Primates to Treat Metachromatic Leukodystrophy
Dolan Sondhi1, Jonathan B. Rosenberg1, Bishnu P. De1, Sebastien Monette2, Alvin Chen1, Thomas Flagiello1, Elisa Caffrey1, Stephen M. Kaminsky1, Patrick Aubourg3, Ronald G. Crystal1 1 Weill Cornell Medical College, New York, NY, 2Memorial Sloan Kettering Cancer Center, New York, NY, 3INSERM U1169 and University Paris-Sud, Le Kremlin-Bicêtre, France Metachromatic leukodystrophy (MLD), a lysosomal storage disorder caused by the deficiency of the enzyme arylsulfatase A (ARSA), is a recessive, fatal neurodegenerative lysosomal storage disease associated with intracellular accumulation of sulfatides in the CNS. The disease is fatal, with no approved therapy. The focus of this study is to test the hypothesis that direct CNS administration of AAVrh.10hARSA (AAVrh.10 serotype vector coding for the human ARSA cDNA) to the CNS of nonhuman primates at doses scalable to humans has an acceptable long term safety profile. Safety of intraparenchymal administration of AAVrh.10hARSA was evaluated following its administration to the CNS of non-human primates (NHPs, African Green monkeys, n=24) at 12 locations in the white matter centrum ovale at two different doses (total dose 2.85x1010 genome copies (gc), equivalent to human clinical dose of 2.85x1011 gc previously used via a similar route of administration in humans, and 1.5x1012 gc, a 1.7-log higher dose, equivalent to a human dose of 1.5x1013 gc). NHPs administered in a similar fashion with AAVNull vector (a vector with an expression cassette without a translatable sequence) and sham (PBS) were used as controls. Target locations were determined using CAT and MRI imaging. The groups (n=6/group) were sacrificed at 1, 13, 26, and 52 wk following vector administration to determine short and long-term effects of treatment. General safety, hematologic, serum chemistry and CNS histopathology parameters were assessed at several time points up to 1 yr after vector administration. Additional in-life safety assessments included behavioral videotaping and CNS monitoring by magnetic resonance imaging (MRI) at 13, 26, and 52 wk post-administration. The vector-administered groups did not differ from the controls in any parameter of general assessment or comprehensive blood profile (complete blood count, chemistry panel). Blinded videotape analysis of NHP behavior pre-surgery and post-administration showed no discernible neurological differences. No significant adverse effects were observed in animals treated with low dose AAVrh.10hARSA; the only abnormal observation was presence of limited and reversible, minimal to mild T and B cell focal infiltrates at the CNS administration sites, findings that were corroborated by MRI. Animals treated with the higher dose of both the ARSA and Null vectors (1.5x1012 gc) demonstrated significant infiltrates of T cells, B cells and activated microglial cells and/or macrophages in the brain at the sites of administration. Similar observations were made from MRIs images, i.e., the higher dose may be associated with some local adverse effects, although these findings did not have clinical consequences on in-life data, complete blood count, serum chemistry or behavior in these NHPs. Together these findings demonstrate the safety of AAVrh.10hARSA administration at 2.85x1010 gc, equivalent to a human dose of 2.85x1011 gc and support the use of this vector as a vehicle for therapy of MLD.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
364. Neurological Correction of Mucopolysaccharidosis IIIB Mice by Haematopoietic Stem Cell Gene Therapy
Stuart M. Ellison1, Rebecca Holley1, Daniel Fil1, John Mc Dermott1, Nisha Senthivel1, Alex Langford-Smith1, Fiona Wilkinson1, Simon Jones2, Rob Wynn3, Shih-hsin Kan4, Patti Dickson4, Brian Bigger1 1 Stem Cell and Neurotherapies, University of Manchester, Manchester, United Kingdom, 2Centre for Genomic Medicine, Royal Manchester Children’s Hospital, Manchester, United Kingdom, 3Paediatric Haematology, Royal Manchester Children’s Hospital, Manchester, United Kingdom, 4Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, CA Mucopolysaccharidosis Type IIIB (MPSIIIB) is a paediatric, autosomal recessive Lysosomal Storage Disease (LSD) caused by deficiency of α-N-acetylglucosaminidase (NAGLU), an enzyme in the heparan sulfate (HS) degradation pathway. Absence of NAGLU leads to the accumulation of partially degraded HS glycosaminoglycan in cell lysosomes, giving rise to cellular dysfunction with devastating clinical consequences. Individuals affected by this fatal disease exhibit severe central nervous system degeneration with progressive cognitive impairment and behavioural problems, alongside more attenuated somatic symptoms. There are currently no effective treatments available. Enzyme replacement therapy with recombinant NAGLU enzyme is ineffective for MPSIIIB since enzyme cannot cross the blood brain barrier (BBB) to where it is needed. Modified recombinant NAGLU enzymes that utilise the insulin growth factor II (IGFII) peptide to facilitate improved uptake across the BBB are currently in development. Haematopoietic stem cell gene therapy (HSCGT) is a promising therapeutic strategy that can circumvent the BBB via monocyte trafficking and engraftment in the brain, allowing delivery of enzyme by cross correction. We have developed a novel stem cell gene therapy approach to investigate the therapeutic potential of HSCGT for MPSIIIB. We designed two lentiviral vectors expressing therapeutic enzyme; the first vector expressing codon optimised NAGLU, and the second expressing a NAGLU.IGFII fusion to aid cellular uptake, both driven by the myeloid specific promoter CD11b and compared these in autologous MPSIIIB transplants against a normal WT bone marrow transplant. Here we present for the first time neurological correction of MPSIIIB mice by HSCGT. We observed correction of the MPSIIIB behavioural phenotype in treated mice to wild-type levels with normalisation of path length, average speed, frequency entering the centre and duration of speed >100mm/s in open field tests. In addition, we observed a significant correction of astrogliosis and lysosomal compartment size in the brains of CD11b. NAGLU LV treated mice with an accompanied normalisation of S146
inflammatory cytokines TNFα, IL1B and IL1RN. Furthermore, NAGLU enzyme activity was substantially increased in the brain. Interestingly, WT transplant alone was able to mediate a partial brain correction, although levels of inflammation and lysosomal storage remain high.
365. Long-Term Toxicology Evaluation of AAVrh.10hARSA Administration to the CNS of Nonhuman Primates to Treat Metachromatic Leukodystrophy
Dolan Sondhi1, Jonathan B. Rosenberg1, Bishnu P. De1, Sebastien Monette2, Alvin Chen1, Thomas Flagiello1, Elisa Caffrey1, Stephen M. Kaminsky1, Patrick Aubourg3, Ronald G. Crystal1 1 Weill Cornell Medical College, New York, NY, 2Memorial Sloan Kettering Cancer Center, New York, NY, 3INSERM U1169 and University Paris-Sud, Le Kremlin-Bicêtre, France Metachromatic leukodystrophy (MLD), a lysosomal storage disorder caused by the deficiency of the enzyme arylsulfatase A (ARSA), is a recessive, fatal neurodegenerative lysosomal storage disease associated with intracellular accumulation of sulfatides in the CNS. The disease is fatal, with no approved therapy. The focus of this study is to test the hypothesis that direct CNS administration of AAVrh.10hARSA (AAVrh.10 serotype vector coding for the human ARSA cDNA) to the CNS of nonhuman primates at doses scalable to humans has an acceptable long term safety profile. Safety of intraparenchymal administration of AAVrh.10hARSA was evaluated following its administration to the CNS of non-human primates (NHPs, African Green monkeys, n=24) at 12 locations in the white matter centrum ovale at two different doses (total dose 2.85x1010 genome copies (gc), equivalent to human clinical dose of 2.85x1011 gc previously used via a similar route of administration in humans, and 1.5x1012 gc, a 1.7-log higher dose, equivalent to a human dose of 1.5x1013 gc). NHPs administered in a similar fashion with AAVNull vector (a vector with an expression cassette without a translatable sequence) and sham (PBS) were used as controls. Target locations were determined using CAT and MRI imaging. The groups (n=6/group) were sacrificed at 1, 13, 26, and 52 wk following vector administration to determine short and long-term effects of treatment. General safety, hematologic, serum chemistry and CNS histopathology parameters were assessed at several time points up to 1 yr after vector administration. Additional in-life safety assessments included behavioral videotaping and CNS monitoring by magnetic resonance imaging (MRI) at 13, 26, and 52 wk post-administration. The vector-administered groups did not differ from the controls in any parameter of general assessment or comprehensive blood profile (complete blood count, chemistry panel). Blinded videotape analysis of NHP behavior pre-surgery and post-administration showed no discernible neurological differences. No significant adverse effects were observed in animals treated with low dose AAVrh.10hARSA; the only abnormal observation was presence of limited and reversible, minimal to mild T and B cell focal infiltrates at the CNS administration sites, findings that were corroborated by MRI. Animals treated with the higher dose of both the ARSA and Null vectors (1.5x1012 gc) demonstrated significant infiltrates of T cells, B cells and activated microglial cells and/or macrophages in the brain at the sites of administration. Similar observations were made from MRIs images, i.e., the higher dose may be associated with some local adverse effects, although these findings did not have clinical consequences on in-life data, complete blood count, serum chemistry or behavior in these NHPs. Together these findings demonstrate the safety of AAVrh.10hARSA administration at 2.85x1010 gc, equivalent to a human dose of 2.85x1011 gc and support the use of this vector as a vehicle for therapy of MLD.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy