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5. Columbus Children’s Research Institute - Dr. Haiyan Fu Global neurological degeneration is the primary cause of high mortality and premature death in MPS IIIB (Sanfilippo B) patients. Therefore, one of the critical issues in developing therapies for MPS IIIB is to be able to deliver therapeutic reagents (including gene delivery vectors or enzymes) to the entire central nervous system (CNS), and not just to a localized brain area. The presence of the blood-brain barrier (BBB) prevents the IV injected vectors and enzymes from entering the CNS. Our long-term goal is to develop gene therapy using adeno-associated viral (AAV) vector to treat MPS IIIB patients. To achieve this goal, our efforts have been focused on two major aspects. First, we must achieve widespread distribution of the recombinant AAV (rAAV) gene delivery vectors in the CNS. Second, we must maximize the therapeutic benefits of the recombinant gene delivered by the rAAV vector for the CNS disorders in the MPS IIIB mouse model. In our previous studies, we have made rAAV vectors (means of carrying genes into cells) containing the gene of normal human -N-acetylglucosaminidase (NaGlu), the enzyme missing in MPS IIIB patients. We have previously shown that our AAV vectors mediated the production of functional NaGlu and corrected the storage of glycosaminoglycan (GAG, previously called mucoploysaccharides) in cultured MPS IIIB cells. We also delivered the AAV vector into MPS IIIB mouse brain by direct injection, and demonstrated the long-term, but only localized, production of the missing enzyme and the localized correction of lysosomal storage in the brain area related to the injection sites. In our further efforts, we developed two non-surgical approaches to deliver rAAV vectors into the CNS of adult mice. We demonstrated a broad spread of rAAV vector in the CNS by an intracisternal (IC) injection. We also established an intravenous (IV) injection procedure following mannitol pretreatment to temporarily disrupt the BBB, and achieved a global distribution of AAV vector in mouse CNS. We believe that these vector delivery approaches will provide a solid foundation for gene therapy for MPS IIIB, as well as other types of MPS with CNS manifestations. We also anticipate that combining IV and IC injection may enhance the CNS delivery of rAAV vectors and provide therapy for other affected tissues and organs as well. We have used these new approaches in our ongoing therapeutic studies over the last few years. We have treated young adult MPS IIIB mice with rAAV2-hNaGlu vector by an IV injection, an IC injection, or a combination of IV and IC injection. We pretreated the mice with an IV infusion of mannitol. After these treatments, we observed a decrease of pathological lysosomal storage in the brains of all the treated MPS IIIB mice, with the NaGlu activity reaching 10-100% of normal levels. The IV vector injection lead to the clearance of lysosomal storage in liver, and partial correction of storage in other somatic tissues. Most importantly, these treatments significantly prolonged the lifespan of MPS IIIB mice to 11.1-19.5 months (IV+IC injected), 9.2-15.9 months (IV injected), and 10.3-17.5 months (IC injected), compared to 7.9-11.0 months in non-treated MPS IIIB mice. Furthermore, we also observed improved behavioral performance in 72% of IV+IC treated MPS IIIB mice. These behavioral improvements included increased activity, normalized swimming speed, and nearly normalized performance in a visual cue task in a water maze, which indicates improvements in learning ability. These results demonstrated a positive therapeutic impact of IV+IC delivery of rAAV vector carrying the correct NaGlu gene for the treatment of the CNS disorders in MPS IIIB mice, though it did not effect a complete cure. We also wish to emphasize that we designed our therapeutic experiments in mice to fit the requirements, in terms of feasibility and scale, for human application. Furthermore our recent experiments demonstrated that the optimal timing for IV injected rAAV vector to enter the CNS was 8 minutes after mannitol pretreatment. The number of transduced brain cells was increased approximately 10-fold when the vector was IV injected at 8 minutes after mannitol infusion, compared to that at 10 minutes after mannitol pretreatment, which was the timing we used in our previous therapeutic experiments, as described above. We anticipate that using this optimal timing will further improve the therapeutic impacts of our rAAV gene therapy procedures on MPS IIIB. Our studies showed a great potential to treat MPS IIIB with rAAV gene delivery vectors. However, more effort is needed to achieve the desired therapeutic goals. Our ongoing research will be focused on improving the efficacy of rAAV gene therapy for the CNS disorders of MPS IIIB, by further optimizing our vector delivery strategies and enhancing the enzymatic functions of rAAV-expressed NaGlu. We are also working toward the future clinical application of AAV gene therapy for treating MPS IIIB patients. |
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