Scholarly article on topic '717. Disease Improvement with Neonatal Intracranial AAV and Systemic Lentiviral Gene Therapy in Sanfilippo Syndrome Type B Mice'

717. Disease Improvement with Neonatal Intracranial AAV and Systemic Lentiviral Gene Therapy in Sanfilippo Syndrome Type B Mice Academic research paper on "Biological sciences"

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Academic research paper on topic "717. Disease Improvement with Neonatal Intracranial AAV and Systemic Lentiviral Gene Therapy in Sanfilippo Syndrome Type B Mice"

may require only a low level of normal or gene-corrected cells for a permanent and therapeutic outcome.

715. Lentiviral Transduction of MPS1 Bone Marrow Cells Results in High Production of alpha-L-Iduronidase In Vivo

Henk Rozemuller,1 Petra Moerer,2 Niek P. van Til,3 JaapJan Boelens,1 Tom J. de Koning,4 Anton C. Martens,2 Gerard Wagemaker,3 Nico M. Wulffraat.1

'Pediatrics, University Medical Center, Utrecht, Netherlands; 2Immunology, University Medical Center, Utrecht, Netherlands; 3Hematology, Erasmus University Medical Center, Utrecht, Netherlands; Metabolic Diseases, University Medical Center, Utrecht, Netherlands.

Mucopolysaccharidosis type I (MPS1, Hurler syndrome) is caused by a deficiency of the enzyme alpha-L-iduronidase (IDUA) and is characterized by glycosaminoglycan (GAG) accumulation causing multi-organ failure. Although enzyme replacement therapy or hematopoietic stem cell (HSC) transplantation constitutes significant improvement of disease phenotype, there is still a lack of effect of those therapies on the central nervous system and the skeleton. Aim of this study is to obtain improved correction of disease in MPS1 mice, including central nervous system and skeletal defects, by overexpression of IDUA in hematopoietic cells. Lentiviral vectors with the spleen focus forming virus- (SF) and phosphoglycerate kinase- (PGK) promoters driving codon optimized human IDUA cDNA (IDUAco) expression were constructed. These vectors were used to transduce male lineage-negative (Lin-) MPS1 bone marrow cells with an average of 1 to 2 vector copies per cell, which was confirmed by quantitative PCR (Q-PCR). Transduced bone marrow cells were subsequently transplanted in 3-week-old 6 Gy irradiated MPS1 female mice. Since matched donor HSC transplantation in MPSI patients is currently the preferred treatment, control MPSI mice were transplanted with syngeneic wildtype Lin- cells. Blood and urine samples were collected monthly. On average 80% male cell chimerism was observed in peripheral blood containing 30% IDUA positive cells up to 7 months after transplantation. IDUA levels in blood were 500- and 80-fold of normal wild type levels for the SF-IDUAco and PGK-IDUAco, respectively, or normalised in wildtype bone marrow transplanted MPS1 mice. Analysis of GAG secretion in urine revealed normalization to wild type levels for all transplantation groups. At month 7, the mice were sacrificed and assessment of GAG levels in spleen, liver, lung, heart, and kidney demonstrated significant reduction of GAGs to normal levels. Most importantly, GAG accumulation was significantly decreased in brain in accordance with high levels of IDUA. CT analysis of the skeleton revealed significant improvement of the bone thickness. We conclude that the IDUAco vectors provide long-term expression of high levels of IDUA, resulting in concomitant reduction in GAG secretion in urine, reduction of GAG in other organs, including brain, and in a significant improvement of the skeletal deformations. This warrants further development towards clinical application.

716. Optimisation of Lentiviral Transduction Conditions of Haematopoietic Stem Cells for Treatment of Mucopolysaccharidosis Type IIIA (MPSIIIA)

Ana Sergijenko,1 Alex Langford-Smith,1 Kia Langford-Smith,1 Rob Wynn,2 J. Ed Wraith,3 Simon Jones,2 Fiona Wilkinson,1 Brian Bigger.1

'University of Manchester, Manchester, United Kingdom; 2Royal Manchester Children's Hospital, Manchester, United Kingdom; 3St. Mary's Hospital, Manchester, United Kingdom.

MPSIIIA is caused by a mutation in the SGSH gene, leading to disruption of the substrate degradation pathway in lysosomes and a phenotype of severe neurodegeneration. We have shown that lentiviral gene therapy using transplanted haematopoietic stem cells (HSCs) corrects the neurological phenotype in a mouse model of MPSIIIA. The aims of this project are to generate a clinically applicable vector, develop a more efficient transduction protocol for HSCs, and assess vector safety. Lentivirus production was optimised to reduce production cost by using less DNA (10.5 ug/ plate) and a PEI transfection reagent. Transduction of HSCs was also optimised. The addition of MG132 with cytokines and BSA improved transduction by 10%, and addition of valproic acid (VA) increased stem cell numbers by 35%. High titres (2x108 IU/ml) ofthree lentiviral vectors encoding GFP gene driven by a ubiquitous (PGK) or monocyte specific (CD''b and CD'8) human promoters were made and tested in vitro in a number of cell lines, and then in vivo, by transplanting transduced lineage depleted cells into wild type mice. Flow cytometry showed that over 80% of donor cells were transduced. Blood and bone marrow cells were further stained for flow cytometry to assess promoter specificity. Improved SGSH encoding cDNAs were made. It was shown that HSCs transduced with GFP encoding virus had 3 fold more copy numbers than cells transduced with SGSH-long (cDNA + 3' and 5' UTRs). The copy number was increased when using a codon-optimised SGSH (SGSH-CO) (cDNA only) and remained high when a stop mutated SGSH-CO-X was used, suggesting that vector size, not SGSH toxicity is important in HSC transduction. SGSH-CO produced more SGSH activity than the non-codon-optimised SGSH. We are currently assessing PGK-SGSH-CO, CD''b-SGSH-CO, and CD'8-SGSH-CO in a clinically relevant lentiviral backbone in MPSIIIA mice, with 6 and 12 month follow up to assess therapy and safety. We have shown that addition of MG132 and VA to HSCs increased stem cell numbers and percentage of transduced HSCs. We have also shown that vector size is important in HSC transduction, that SGSH activity is increased by codon optimisation, and therapy is more specific when using cell lineage promoters. This is a promising clinical therapy for MPSIIIA.

717. Disease Improvement with Neonatal Intracranial AAV and Systemic Lentiviral Gene Therapy in Sanfilippo Syndrome Type B Mice

Coy D. Heldermon,1 Elizabeth Qin,2 Kevin K. Ohlemiller,3 Erik D. Herzog,4 Carole Vogler,5 Mark S. Sands.2 'Medicine, University of Florida, Gainesville, FL; 2Internal Medicine, Washington University in St. Louis, St. Louis, MO; 3Otolaryngology, Washington University in St. Louis, St. Louis, MO; 4Biology, Washington University in St. Louis, St. Louis, MO; 5Pathology, St. Louis University, St. Louis, MO.

Sanfilippo Syndrome type B (MPS IIIB) is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU). We have previously shown that intracranial AAV-based gene therapy improves several aspects of the disease such as lifespan. In an attempt to correct the disease in the mouse model, MPS IIIB mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (AAV), intravenous lentiviral-NAGLU (MND)

Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy

or both (AAV/MND). Compared to untreated MPS IIIB animals, all treatments resulted in significant improvements in the clinical phenotype. AAV, MND and AAV/MND treatment results in improved motor function compared to untreated controls using a rocking rotarod paradigm. Auditory-evoked Brainstem Response measurements of hearing also demonstrate improvement in all treatment groups. Circadian rhythm analysis reveals an improvement in time to activity onset and percent of activity during daylight in the AAV group, little improvement with MND alone, and near normalization with AAV/ MND treatment. Each treatment group had a significantly increased lifespan compared to the untreated group (median survival=322 days), with the combination arm the most profound (median survival=612 days, p<0.0001), relative to AAV (463 days, p<0.001) and MND (358 days, p=0.009). In summary, intracranial AAV and systemic MND gene therapy result in disease improvement in MPS IIIB mice both alone and in combination. The combination treatment is most effective at correcting circadian activity, rotarod performance, hearing, and lifespan.

718. Aortic Dilatation in Mucopolysaccharisosis VII Mice Cannot Be Ameliorated by Deficiency of Cathepsin S or Matrix Metalloproteinase Activity, but Can Be Markedly Amelioraged by Marked Reductions in Expression of an As-yet-Unknown Gene That Prevents Upregulation of Inflamatory Pathways

Guilherme Baldo,1 Ruth Howe,1 Susan Wu,1 Russell Knuteen,2 Meera Ramamoothy,1 Robert Q. Meecham,2 Katherine Ponder.2 'Internal MEdicine, Washington Univesity School of MEdicine, St. Louis, MO; 2Cell Biology, Washington University School of Medicine, St. Loiuis, MO.

Mucopolysaccharosisis VII is due to deficient activity in P-glucuronidase (GUSB) activity, and results in accumulation of the glycosominoglycans dermatan, heparan, and chondroitin sulfate. Mice have marked dilatation (~2-fold normal diameter) and aortic regurgitation. Histologically the aortas from MPS I mice have fragmented elastin fibers, and increases in RNA and enzyme activity for cathepsin S and matrix metalloproteinase 12 (MMP12), leading us to hypothesize that one or both of these elastin-degrading enzymes led to elastin degradation and dilated aortas. To test this hypothesis, MPS VII mice were crossed with Cathepsin S and MMP-12 deficiency mice, all of which were on a 57BL/6 backgournd. Although aortas from triple-deficient mice were less dilated at 1.9+/-0.6 mm at 6 months of age than were purebred MPS VII mice (3+/-0.7 mm), they were dilated as compared with normal mice without any mutations (1.4+/-0.2 mm). We conclude that deficiency of both cathepsin S and MMP-12 are not suffieint to prevent elastin fragmentation and aortic dilatation in the MPS VII model. Cathepsin activity assay was performed under conditions where other cathepsins would also have activity. Cathepsin activity was 5-fold normal in triply-deficient aortas, which was lower than the value of 15-fold normal in mice that were just deficient in GUSB. We conclude that other cathepsins are activite in the aorta in MPS VII, and that it might be necessary to inhibit a broad spectrum of cathepsin to prevent aortic dilatation. It the process of crossing MPS VII mice with cathepsins S mice, we identified several that were expected to have dilated aortas based on the GUSB -/- status and the +/+ status other genes that were being studied, but were near-normal in diameter. These aortas had cathepsin activity was that only 3-fold normal, which was lower than the activity in the cathepsin S-deficient MPS VII mice. We conclude that these mice had another mutation that conferred protestion from aortic dilatations by reducing cathepsin activities. Microarray demonstrated that several genese were upredulated in the purebred MPS VII mice as compared with the normal mice such as CCL4 (MIP1P at 27-fold formal), PU.1 at 65-fold, and OSM1 at 104-fold, and CXCL1 at

174-fold normal. These genes are consistent with the hypothesis that GAGs bind the toll like receoptor, which activates signal transduction pathways that result in the upregulation of these genes. MPS VII mice with unexecpectedly small aortas due to this putative mutation had marked reduction in all of these genes to <10% of that in purebred MPS VII mice. We are currently trying to identify the putative gene that confers production. Our hypothesis is that it relates to signal transduction in MPS VII, and might be an excellent target to inhibit in patients with MPS VII or other types of MPS.

719. Upregulation of Inflammatory Pathways and Proteases May Lead to Aortic Valve Insufficiency in MPS VII Dogs and Can Be Prevented with Neonatal Retroviral Gene Therapy

Guilherme Baldo,1 Paul Bigg,1 Susan Wu,1 Yuli Liu,1 Mark Haskins,2 Henri Bai,1 Meg Sleeper,2 Katherine P. Ponder.1 'Internal Medicine, Washington University in Saint Louis, Saint Louis; 2Pathobiology, University of Pennsylvania, Pennsylvania, PA.

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disorder due to deficiency of P-glucuronidase (GUSB), which leads to storage of glycosaminoglycans (GAGs) and multisystemic clinical manifestations that include heart and valve disease. The pathogenesis of MPS VII valve disease is unknown, although recent studies in other organs suggest that undegraded GAGs could bind to the LPS-receptor [Toll-like receptor 4 (TLR4)], triggering inflammatory cascades and upregulating destructive proteases. In this study we aimed to address the causative mechanisms of aortic valve (AV) disease in MPS VII dogs using a microarray approach, and to determine the effects of neonatal i.v. injection with a gamma-retroviral vector (RV) expressing GUSB on correcting this aspect of the disease. Echocardiographic analyses at 6 months of age demonstrate moderate thickening (score +1.4; 0=normal and 3=severe) and regurgitation (score +2) of the AV in MPS VII dogs, which was statistically worse than in normal dogs(score +0.3 and +0.67, respectively, p< 0.05). Histological and biochemical analysis revealed a reduction in the collagen signal and a massive GAG storage (65-fold normal) in MPS VII AV. Activities of cathepsins, which are proteases that can degrade collagen and other extracellular matrix proteins, were 27-fold normal (p< 0.001). Microarray results revealed 196 genes that were >2-fold normal in MPS VII as compared with normal AV with p< 0.01, and 112 genes that were <0.5-fold normal. Pathway analysis clearly demonstrated an important inflammatory component on the disease, with the TLR4/TREM signaling pathway being one of the most upregulated (p= 2.8 x 10-7. When compared to known disease processes, MPS VII AV microarray results showed upregulation of 60 genes that are known to be increased in rheumatoid arthritis (p=1.7x10-16. Some upregulated genes included TLR4 (2.1-fold), tyrosine kinase SYK (4.3-fold), cytokines (TNFC 2.6-fold, IL8 6-fold, IL-15, 3.8-fold, IL-27 4-fold) and cathepsin S (4.8-fold), K (3.4-fold), B (3.7-fold) and MMP-12 (7.6-fold). RV-treated dogs had minimal AV thickening (score +0.5) and regurgitation (score +0.3) at 6 months, and benefits were maintained for up to 10 years. Serum GUSB activity was 3-fold normal and AV GUSB activity was 30% normal. RV treatment reduced, but did not prevent, GAG elevation in the valves (7-fold normal; 10% of MPS VII) and as well as cathepsin activities (3-fold normal; 11% of MPS VII). Our results suggest that GAG storage may activate important inflammatory pathways in the AV and increase the expression of proteases that degrade collagen and lead to valve dysfunction in MPS VII. RV gene therapy can markedly reduce AV disease in MPS VII dogs at 10 years, but the failure to completely normalize GAG levels may result in destructive changes over time. The resemblance to rheumathoid arthritis might indicate that drugs used for this disease, such as TNFa and SYK inhibitors, might also be effective in MPS VII patients to reduce valvular manifestations.

Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy