We set out to investigate the use of metformin, an FDA-approved drug, to enhance gene modification. No significant difference in growth patterns of two blood cell lines was observed in concentrations up to 10 mM. The cutting efficiency of Cas9 was significantly enhanced in a total of five guide RNAs with the use of metformin. In addition, an enhancement in targeting was observed greater than two-fold.
The modification of hematopoietic stem cells with the CRISPR/Cas9 system is inefficient. In this publication we show that histone deacetylase inhibitors, such as valproic acid (VPA) and sodium butyrate (NaB), could enhance HDR efficiency. This enhancement could be due to an increase in the accessibility of the genome-editing machinery. Overall, HDR efficiency was enhanced greater than two-fold with the use of 0.005 mM VPA. These results are promising and suggest the consideration of treatment with an HDAC inhibitor in CRISPR/Cas9 genome editing protocols.
September 18, 2014
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.
September 20, 2012
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.
June 22, 2011
A chapter of Gene Therapy: Developments and Futute Perspectives detailing different strategies on how to integrate larger transgenes into a genome
March 3, 2009
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.