Browsing by Subject "gene therapy"

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    Molecular Therapy and Gene Therapy for Hurler Syndrome
    (2015-06) Ou, Li
    Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease which leads to systemic disease, including progressive neurodegeneration, mental retardation and death before the age of 10 years. MPS I results from deficiency of α-L-iduronidase (IDUA) and subsequent accumulation of glycosaminoglycans (GAG). IDUA enzyme acitivity is an essential assessment for research and diagnostic testing of MPS I disease. Due to different parameters (reaction time, temperature and substrate concentration) used by different labs, the enzyme levels of a certain sample varied. To solve the inconsistency of IDUA enzyme assays in this field, a standardized protocol of IDUA enzyme assay was established through adjustment by Michaelis-Menten equation (Chaper 1). In clinical practice, MPS I disease is treated by enzyme replacement therapy (ERT) and bone marrow transplantation (BMT). Clinical ERT with intravenous IDUA reverses some aspects of MPS I disease and ameliorates others. However, neurologic benefits are thought to be negligible because the blood-brain barrier (BBB) blocks enzyme from reaching the central nervous system (CNS). To address this question, high-dose IDUA (11.6 mg/kg, once per week, 4 weeks) was administered to adult MPS I mice. IDUA enzyme activity in cortex of injected mice increased to 97% of that in wild type mice (p<0.01). GAG levels in cortex were reduced by 63% of that from untreated MPS I mice (p<0.05). Water T-maze tests showed that the learning abnormality in MPS I mice was surprisingly reduced (p<0.0001). These results demonstrated the efficacy of high dose ERT in treating neurological diseases in MPS I mice (Chapter 2). Previous study in our lab showed that a single administration of lentiviral vector in neonatal MPS I mice can achieve significant metabolic correction and neurological improvements. To further improve the efficacy of lentiviral gene therapy, a total of 9 constructs were designed by codon optimization, and different combination of promoters and enhancers. The transgene expression of these 10 constructs was compared after transfection into HEK 293FT cells, and 5 constructs with the highest IDUA expression were identified (Chapter 4). These results pave the way for developing a directly applicable clinical trial of human lentiviral gene therapy for MPS I disease, and also provide evidence for vector design for treating other lysosomal diseases.
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    TARGETING THE AGMATINERGIC SYSTEM USING AN AAV-BASED GENE THERAPY FOR THE TREATMENT OF CHRONIC PAIN
    (2019-12) Pflepsen, Kelsey
    The complex mechanisms underlying chronic pain and the challenges of current pharmacotherapy leave many chronic pain patients without adequate treatment due to negative physical and social side effects. Thus, there is a critical need to develop new pain management therapies with long-term effectiveness and minimal adverse effects. Using an adeno-associated virus (AAV) gene therapy to treat chronic pain has gained interest in the last twenty years because of several therapeutic advantages. AAV gene therapy allows for selective gene expression directly to sites of interest for chronic pain treatment with the potential for sustained expression following a single injection. In an effort to treat chronic pain using an AAV gene therapy, we have created a viral vector using recombinant adeno-associated virus which encodes the biosynthetic enzyme, human arginine decarboxylase (hADC). Arginine decarboxylase (ADC) is an endogenous enzyme that catalyzes the metabolism of L-arginine into agmatine. Agmatine is an endogenous small molecule that acts as a neurotransmitter and has been previously shown to modulate neuroplastic events by antagonizing the GluN2B subunit of the N-methyl-D-aspartate (NMDA) receptor. When agmatine is delivered centrally, a reduction in pain behavior following nerve injury is observed. The central hypothesis of this body of work is that overexpression of arginine decarboxylase using an adeno-associated viral vector construct results in long-term reduction of neuropathic pain due to the production of agmatine and subsequent antagonism of the GluN2B NMDA receptor by agmatine. The experiments presented in this dissertation build upon our agmatine-based pharmacotherapy for the treatment of chronic neuropathic pain. Specifically, this work furthers our AAV-based gene therapy by understanding intrathecal AAV kinetics, evaluating how therapeutic efficacy changes with age and with the use of cell-specific promoters, and interpreting associated behavioral implications.