455 Neonatal Delivery of scAAV9 SMN Impact of Disease Development in Two Models of Spinal Muscular Atrophy Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene[.]
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY II potentially lethal metabolic decompensations, hyperammonemia, neurological complications, and a potentially lethal cardiomyopathy A murine model of PA, created by knocking out a portion of the Pcca gene, exhibits elevated levels of metabolites and neonatal lethality within the first 48 hours We have previously demonstrated the ability of rAAV8 gene delivery to rescue the murine model of PA from lethality and decrease the levels of disease related metabolites In light of the cardiomyopathy observed in patients with PA, the cardiac histology of the Pcca-/- mice was examined to determine whether cardiac abnormalities are present Histology revealed gross abnormalities of the cardiac tissue However, Pcca-/- mice treated by rAAV8 gene delivery of the PCCA gene did not exhibit histopathological changes of the cardiac tissue compared to wildtype mice These results demonstrate the potential efficacy of rAAV8 gene delivery as an alternative treatment to transplantation for the potentially lethal cardiomyopathy observed in PA 454 AAV Mediated Gene Transfer to Skeletal Muscle Results in Sustained Reduction of Hyperbilirubinemia in an Animal Model of CriglerNajjar Syndrome Type Nunzia Pastore,1 Rosa Maria Sepe,1 Francesco Vetrini,1 Edoardo Nusco,1 Alberto Auricchio,1,2 Nicola Brunetti-Pierri.1,2 Telethon Institute of Genetics and Medicine, Naples, Italy; Pediatrics, Federico II University, Naples, Italy Crigler-Najjar syndrome is an autosomal recessive disorder characterized by severe unconjugated hyperbilirubinemia due to deficiency of the liver-specific uridine diphospho-glucuronosyl transferase A1 (UGT1A1) Current therapy relies on phototherapy to prevent life-threatening elevations of serum bilirubin levels but liver transplantation is the only permanent treatment Given the mortality and morbidity related to the transplant procedures, there is high motivation at developing gene therapy for this disorder Although correction of the deficient enzymatic activity in the affected organ, i.e the liver, would be most straightforward, expression within an ectopic tissue to clear toxic metabolites from the circulation is very attractive The muscle is the preferred tissue for this goal because of its simple and safe access through intramuscular (IM) injections Moreover, the IM route has been investigated extensively for gene therapy of various diseases and in human clinical trials as well In this study, we have investigated the efficacy of muscle-directed gene therapy for Crigler-Najjar syndrome type using Adeno Associated Viral (AAV) vectors A serotype AAV vector expressing the UGT1A1 under the control of the muscle-specific creatine kinase (MCK) promoter was injected at the dose of 3x10e12 genome copies/kg into the muscles of one month-old Gunn rats, the animal model of Crigler-Najjar syndrome type IM injections of AAV vectors resulted in the expression of functionally active UGT1A1 enzyme in the muscle as demonstrated by Western blot analysis and enzyme assay on muscle tissues AAV-injected Gunn rats showed an approximately 50% reduction of baseline serum bilirubin levels by weeks post-injection which were sustained for at least year post-injection Taken together, these data show that clinically relevant and sustained reduction of serum bilirubin levels can be achieved by simple and safe IM injections in the Gunn rats AAV-mediated muscle directed gene therapy has potential for the treatment of patients with Crigler-Najjar syndrome type Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy 455 Neonatal Delivery of scAAV9-SMN: Impact of Disease Development in Two Models of Spinal Muscular Atrophy Jacqueline Glascock,1 Monir Shababi,1 Christian Lorson.1 Molecular Microbiology and Immunology and Veterinary Pathobiology, University of Missouri, Columbia, MO Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is the leading genetic cause of infant mortality SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1) Despite its ubiquitous expression, low levels of full length SMN protein result in the preferential loss of lower motor neurons It has been shown that replacement of SMN1 using viral vectors is a viable option for the rescue of motor neurons and the treatment of SMA in the delta mouse model of SMA In this study, we used self-complementary adeno-associated virus (scAAV) carrying SMN1 to compare two different routes of viral delivery in the delta model scAAV9-SMN1 was delivered to postnatal day mice via intravenous (IV) or intracerebroventricular (ICV) injection Both routes of scAAV9-SMN1 delivery resulted in a significant increase in lifespan and weight compared to untreated mice with a subpopulation of mice surviving more than 200 days However, mice given ICV injections gained significantly more weight than their IV treated counterparts Survival analysis shows that ICV treated mice displayed fewer early deaths than IV treated animals Preliminary results have shown that SMN protein levels in the brain and spinal cord of ICV treated animals are higher than SMN protein levels in IV treated animals This study demonstrates that route of delivery is a crucial component of gene therapy treatment for SMA These results prompted a study of this vector’s (scAAV9-SMN1) ability to rescue a more severe model of disease We now show that while lifespan is significantly improved in this more severe model, the inability to fully rescue suggests that either prenatal delivery is required or that the model is too severe to be fully rescued S175 GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY II 457 Successful Use of Lipoplex for Delivery of the Small Molecules ManNAc and Sialic Acid To Rescue Hyposialylation in a Mouse Model of Hereditary Inclusion Body Myopathy (HIBM) Tal Yardeni,1,4 Carla Ciccone,1 Shelley Hoogstraten-Miller,2 Daniel Darvish,3 Yair Anikster,4 John Nemunaitis,5,6 Phil Maples,5 Chris M Jay,5 William A Gahl,1 Marjan Huizing.1 MGB, NHGRI/NIH, Bethesda, MD; 2OLAM, NHGRI/NIH, Bethesda, MD; 3HIBM Research Group, Encino, CA; 4Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel; 5Gradalis Inc., Dallas, TX; 6Mary Crowley Cancer Research Centers, Dallas, TX 456 Successful Treatment of Hypophosphatasia Model Mice by a Single Intramuscular Injection of AAV Type Vector Expressing Tissue-Nonspecific Alkaline Phosphatase Tae Matsumoto,1,2 Koichi Miyake,1 Noriko Miyake,1 Hideo Orimo,3 Sonoko Narisawa,4 José Millán,4 Yoshitaka Fukunaga,2 Takashi Shimada.1 Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan; 2Department of Pediatrics, Nippon Medical School, Tokyo, Japan; 3Division of Structural Biology and Metabolism, Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan; Sanford Children’s Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA Hypophosphatasia (HPP) is an inherited systemic skeletal disease characterized by a deficiency in tissue-nonspecific alkaline phosphatase (TNALP), which leads to abnormal mineralization of skeletal and dental tissues Patients with the infantile form may appear normal at birth, however, the prognosis of this form is poor Although enzyme replacement therapy or bone marrow transplantation was tried in these 30 years, there is no curative treatment for hypophosphatasia Recently, it was demonstrated that TNALP knockout (AKP2-/-) mice, which are good models for infantile form hypophosphatasia, could be treated by injection of a bone-targeted form of human TNALP in which a deca-aspartate sequence is linked to the C terminal end of soluble TNALP (TNALP-D10) We also reported that a single intravenous systemic injection of lentiviral or adeno-associated virus type (AAV8) vectors expressing TNALP-D10 rescued neonatal knockout mice However, the safety of a systemic injection of either lentiviral or AAV into neonates has not be established In this study, to develop a more practical gene therapy for HPP, we examined the feasibility of AAV8 vector-mediated muscle directed gene therapy for neonatal HPP Neonatal AKP2-/- mice (n=6) were treated with a single intramuscular injection of AAV8-TNALP-D10 (1x1012 vg/ body) High level of plasma ALP activity (14.6 ± 4.5 U/ml) was then detected for more than one year, thereafter X-ray analysis showed mature bone mineralization was detected in treated AKP2-/- mice but not control AAV8 vector expressing GFP injected mice Treated knockout mice lived for more than year with normal physical activity and healthy appearance, while control mice died within weeks Ectopic calcification and abnormal calcium metabolism were not detected in treated mice These findings suggest that AAV mediated intramuscular neonatal gene therapy is a highly practical option to cure the severe infantile form of HPP S176 HIBM is an adult-onset, progressive neuromuscular disorder, caused by mutations in the ubiquitously expressed, key enzyme of sialic acid (SA) synthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase, encoded by the GNE gene We created an HIBM mouse model, mimicking the Persian-Jewish founder mutation Mutant mice (-/-) unexpectedly died before day of life (P3) from severe glomerulopathy due to hyposialylation Renal findings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin Oral administration of the SA precursor, N-Acetyl-D-mannosamine (ManNAc), yielded survival beyond P3 in 43% of the mutant pups Then we assessed the efficiency and efficacy of ManNAc/SA delivery in liposomes (ManNAc-Lipoplex,SA-Lipoplex), and human GNE gene delivery in liposomes (hGNE-Lipoplex) in our HIBM mouse model Newborn pups (P1) were retro-orbitally injected with ManNAc-Lipoplex, SA-Lipoplex or hGNE-Lipoplex Mice were watched for clinical signs and survival beyond P3, and tissues were tested at P5 for sialylation, histology, glomerular disease and hGNE expression hGNE-Lipoplex injections yielded no surviving -/- pups beyond P3, however, WT survivors showed hGNE expression in tested tissues at P5, indicating no toxicity of hGNE-Lipoplex and efficient gene delivery to tissues Interestingly, -/- pups that died before P3 also showed hGNE expression in their tissues (as early as P2); treatment at P1 may not allow enough time for sufficient protein translation and SA production In contrast, ManNAc-Lipoplex injections at P1 yielded survival beyond P3 in >90% of -/- pups SA-Lipoplex yielded survival beyond P3 in >80% of -/- pups in contrast to SA in drinking water that didn’t yielded any -/- pups surviving beyond P3.The surviving mutant pups showed improved glomerular histology and improved sialylation of glomerular sialoproteins at P5 ManNAc-Lipoplex treated -/- mice continued to live beyond weaning; the oldest mice are now months The development of a muscular phenotype in these mice, similar to the symptoms of HIBM, and the effects of ManNAc treatment on these symptoms can now be assessed Our studies demonstrate: 1)retro-orbital injection in newborn mice is an efficient method for systemic delivery of compounds; 2)small molecules can be efficiently delivered in Lipoplex; 3)systemic delivery of a gene in Lipoplex yields gene expression in tissues after one day; 4)ManNAcLipoplex/SA-Lipoplex can be applied to increase sialic acid levels and systemic or intramuscular ManNAc-Lipoplex therapy should be considered for the treatment of patients with HIBM, and may also be considered for other disorders of hyposialylation (i.e.,certain cancers, certain renal disorders) Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... production In contrast, ManNAc-Lipoplex injections at P1 yielded survival beyond P3 in >90% of -/- pups SA-Lipoplex yielded survival beyond P3 in >80% of -/- pups in contrast to SA in drinking water... Renal findings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin Oral... treated by injection of a bone-targeted form of human TNALP in which a deca-aspartate sequence is linked to the C terminal end of soluble TNALP (TNALP-D10) We also reported that a single intravenous