Molecular Therapy and Gene Therapy for Hurler Syndrome A THESIS SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Li Ou IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

Mucopolysaccharidosis type I (MPS I) is a lysosomal disease caused by α-Liduronidase (IDUA) deficiency and subsequent accumulation of glycosaminoglycan (GAG) heparan sulfate and dermatan sulfate. Lentiviral vector encoding correct IDUA sequence could be used for gene therapy treating MPS I disease. Previous study in our lab showed that intravenous injection of a lentiviral vector (CSP1) into neonatal MPS I mice achieved metabolic correction and neurological improvements. To improve the efficacy of this lentiviral gene therapy, 9 more lentiviral constructs were designed by codon optimization, different combinations of promoters and enhancers. These ten plasmids were transfected into HEK 293FT cells, and IDUA enzyme activity in cell lysates and supernatants were compared. The transfection efficiencies of each plasmid were comparable (~60%), shown by co-transfection of a plasmid encoding GFP. A total of 5 plasmids showed higher enzyme activity in cells (from 6,482 ± 1,258 to 7,358 ± 956 nmol/h/mg) than CSP1 (5,613 ± 1,047 nmol/h/mg). These 5 candidates also had the highest enzyme activity in supernatants (514 ± 213 nmol/h/mL to 861 ± 151 nmol/h/mL). Therefore, these 5 plasmids and CSP1 were selected for lentiviral vector production and further in vivo comparison. A total of 6 lentiviral vectors will be injected into temporary facial vein in neonatal MPS I mice, and tissue IDUA levels on day 30 will be compared to select the optimal vector, which has the highest IDUA levels. These results will pave a way for developing a directly applicable lentiviral gene therapy clinical trial for MPS I patients, and provide proof-of-concept evidence for treating other lysosomal diseases.