NINTH WORKSHOP ON NOVEL TECHNOLOGIES AND GENE TRANSFER FOR HEMOPHILIA THE CHILDREN’S HOSPITAL OF PHILADELPHIA FEBRUARY 22-23, 2008 Monitoring protein and transgene activities, use of Thromboelastograms and thrombin generation assays David Lillicrap, MD Monitoring the Success of Hemophilia Gene Therapy

Efficient delivery of Von Willebrand Factor and ADAMTS13 via the SV40 in vitro packaging system SV40-based in vitro packaging vectors show great promise for use in gene therapy applications: they are capable of infecting a wide variety of cells, including both dividing and quiescent cells, they do not contain any wild-type viral SV40 or packaging cell line, and can deliver both RNAi and DNA molecules up to ~18 kb in size. When prepared in vitro, the delivery system is composed of only the SV40 envelope major protein (VP1), which is produced in Sf9 insect cells. These particles do not harbor any of the wild-type SV40 genes, and no encapsidation sequences are required. The in vitro packaging vectors were shown to be successful in delivering and expressing reporter genes, genes for chemoprotection and RNAi to silence gene expression. SV40 pseudovirion delivery of Pseudomonas exotoxin A (PE38) was found to be effective either by direct injection or systemically in the treatment of human adenocarcinomas growing in mice. Expression of two genes that code for coagulation proteins, von Willebrand Factor (vWF) and ADAMTS13 (the vWF cleavage protease) was monitored for 2-6 days in transduced K562 cells. ADAMTS13 polymorphic forms were also studied in HEK293 cells. These results suggest that SV40 in vitro-packaged vectors are effective vehicles for use in gene therapy. NINTH WORKSHOP ON NOVEL TECHNOLOGIES AND GENE TRANSFER FOR HEMOPHILIA THE CHILDREN’S HOSPITAL OF PHILADELPHIA FEBRUARY 22-23, 2008 AAV serotype tropism Luk Vandenberghe, PhD NINTH WORKSHOP ON NOVEL TECHNOLOGIES AND GENE TRANSFER FOR HEMOPHILIA THE CHILDREN’S HOSPITAL OF PHILADELPHIA FEBRUARY 22-23, 2008