Browsing by Author "Ou, Li"

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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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    Optimal Stoichiometry of the PS Gene-editing System
    (2023-11) Saveraid, Hanna G; Przybilla, Michael J; Ou, Li; Whitley, Chester B
    Introduction: The PS Gene-editing System has been shown to increase IDUA expression and improve therapeutic outcomes in murine models of Hurler Syndrome. However, the optimal ratio of bipartite AAV vectors in the system is unknown. Results: Mice (+/- or -/- for IDUA) treated with a 1:6 ratio of AAV1:AAV2 in the PSG System had the highest IDUA activity in plasma and liver, followed by 1:13 and then 1:27 ratio. All treated mice had higher enzyme levels than control mice. Conclusion: This work demonstrates that a 1:6 ratio is optimal; however, all ratios resulted in supraphysiological levels of IDUA activity. More research is needed to determine if other ratios (e.g., 1:1), are more effective.