The overall objective of the proposed research is to use two feline models of the human mucopolysaccharidoses, feline MPS I and VI (FMPS I and FMPS VI, respectively), to develop and evaluate novel therapeutic strategies for the treatment of their respective neuronopathic and non-neuronopathic human counterparts. This proposal represents a continuation of a long standing collaboration between investigators at the University of Pennsylvania School of Veterinary Medicine (UPVM) and the Mount Sinai School of Medicine (MSSM). Previously, our laboratories have: 1) identified and established breeding colonies for the two animal models, 2) characterized the clinical course and pathophysiology of each disease, 3) developed biochemical detection assays to select appropriate matings, 4) developed purification methods for the two lysosomal enzymes which are deficient in these diseases, alpha-L-iduronidase (alphaID) and arylsulfatase B (ASB), respectively, 5) characterized the biochemical defects underlying each disease, 6) comparatively mapped the human and feline alphaID and ASB genes, 7) isolated and expressed the full-length human and feline ASB cDNAs, 8) isolated cDNAs encoding feline alphaID, and 9) constructed retroviral vectors encoding human and feline ASB.
The specific aims of the current proposal are to: 1) continue characterizing the pathophysiology of fMPS I and VI, including the ocular abnormalities using retinal pigment epithelial (RPE) cells, the skeletal pathology using chondrocyte cultures, and the central nervous system (CNS) involvement in fmPS I by magnetic resonance imaging, 2) identify the molecular lesions underlying each disease and establish molecular diagnostic methods for more efficient breeding, 3) employ mammalian overexpression systems to achieve high-level production of alphaID and ASB and evaluate enzyme replacement in the feline models, 4) construct a series of retroviral vectors containing the alphaID and ASB sequences to evaluate the metabolic correction of fMPS I and VI in vitro by retroviral-mediated gene transfer, and 5) develop somatic cell gene therapy for fMPS I and VI by: a) infecting bone marrow stem cells from affected cats with the alphalD and ASB retroviral vectors, followed by autologous bone marrow transplantation (BMT) and assessment of long term expression and clinical efficacy, b) overexpressing each enzyme in a cell line (i.e., an """"""""enzyme pump"""""""") which can be implanted into animals and used for somatic gene therapy in vivo, and c) evaluating liposome-mediated gene transfer strategies designed to deliver the alphaID or ASB gene constructs directly to sites of pathology which are not amenable to retroviral-mediated gene transfer [e.g., brain (FMPS 1) , cornea, and articular cartilage]. It is anticipated that these animal studies will facilitate the development and evaluation of enzyme replacement and somatic gene therapy for the neuronopathic and non-neuronopathic human MPS disorders.
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