September 18, 2014
Generation of an Optimization of a Lentiviral Vector Encoding a High Expression FVIII Transgene for the Gene Therapy of Hemophilia A.
September 20, 2012
June 22, 2011
Directed Engineering of a High-Expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A
March 3, 2009
To increase the transduction efficiency of lentiviral vectors, high-throughput screening is used to identify compounds that could strengthen the transduction of target cells. Phorbol 12-myristate 13-acetate (PMA) was identified as an enhancer of transduction. When K562 cells were transduced with a fVIII encoding lentiviral vector, the transduction was four times greater in the presence of PMA.
This study focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components with the human/porcine fVIII molecule from a previous study. After finding that the hybrid human/porcine fVIII expression was enhanced by the SIV system the construction of an enhanced vector with a CMV promoter proceeded. It was shown that an optimized vector would contain the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.
A chapter of Gene Therapy: Developments and Futute Perspectives detailing different strategies on how to integrate larger transgenes into a genome
Having previously shown that a hybrid human/porcine fVIII transgene had 100-fold greater expression than wild type human fVIII we delivered this transgene by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Up to 6-100 fold expression of fVIII in the HSCs were shown compared to wild type. These cells were transplanted into hemophilia A mice with virus based vectors and resulted in long term expression at therapeutic levels.