40. Enhanced Factor IX Delivery from Bioengineered Hybrid Human Skeletal Muscle Co-Expressing VEGF

Abstract

The continuous development of efficient and safe gene delivery approaches is a prerogative to move the field of gene therapy forward. We have recently been focusing on the development of novel gene delivery approaches based on the use of genetically engineered adult human stem cells to establish a novel ex vivo gene therapy paradigm at the cross-roads of gene therapy, stem cell technology and tissue engineering. Adult human skeletal muscle stem cells are easy to obtain by needle biopsy and can be efficiently transduced ex vivo with third generation lentiviral vectors. With GFP as a marker gene, nearly 100% of cells were shown to be transduced and using the human blood clotting factor IX (FIX) as a transgene, unprecedented levels (>5 |[mu]|g /106 cells/day) of FIX were obtained in vitro, whereas only 0.4 |[mu]|g rhFIX/106 cells/day was secreted when retroviral vectors were employed. These cells can be efficiently bioengineered in vitro into postmitotic muscle fibers and implanted subcutaneously as bioartificial muscles (BAMs). The bio-engineered tissues can be removed in the event of an adverse reaction, which significantly improves the safety margin of this technology and which distinguishes this potential |[ldquo]|reversible|[rdquo]| gene therapy approach from most other gene therapy strategies. Implantation of BAMs transduced ex vivo with lentiviral-GFP vectors resulted in robust and long-term GFP expression in NOD-SCID mice. FIX secreting BAMs implanted subcutaneously into NOD-SCID mice, secreted FIX into the circulation for greater than 90 days but the plasma levels were below therapeutic levels after 20 days. When the muscle fibers were bioengineered from human GFP myoblasts and human myoblasts genetically transduced to express vascular endothelial growth factor (VEGF), significantly more fluorescence was detected after 1 month compared to BAMs without VEGF secretion. A network of new blood vessels was established around the hybrid GFP-VEGF BAMs within 1 month, concomitant with a localized increased vascular permeability around these hybrid BAMs. Using hybrid FIX-VEGF BAMs, the circulating levels of FIX increased significantly and plasma levels of FIX were maintained above therapeutic levels long term. The present study suggests that a limited angiogenic response may contribute to the inability to obtain long-term transgene expression levels following ex vivo gene therapy, as previously shown in a phase I/II clinical trial for hemophilia (Roth et al., N Engl J Med. 344:1735, 2001). This study provides novel insights into the mechanisms that improve the outcome of tissue engineering and gene therapy, particularly by modulating angiogenesis. Implantable tissues bioengineered from genetically engineered muscle cells may provide an alternative and safer approach to in vivo gene transfer for chronic protein delivery. To our knowledge, this is the first demonstration that adult human stem cells transduced with lentiviral vectors can give rise to prolonged circulating clotting factor levels in vivo.