Abstract
Retroviral gene therapy offers immense potential to treat many genetic diseases and has already shown efficacy in clinical trials. However, retroviral vector mediated genotoxicity remains a major challenge and clinically relevant approaches to reduce integration near genes and proto-oncogenes are needed. Foamy retroviral vectors have several advantages over gammaretroviral and lentiviral vectors including a potentially safer integration profile and a lower propensity to activate nearby genes. Here we successfully retargeted foamy retroviral vectors away from genes and into satellite regions enriched for trimethylated histone H3 at lysine 9 by modifying the foamy virus Gag and Pol proteins. Retargeted foamy retroviral vectors integrated near genes and proto-oncogenes less often (p < 0.001) than controls. Importantly, retargeted foamy retroviral vectors can be produced at high, clinically relevant titers (>107 transducing units/ml), and unlike other reported retargeting approaches engineered target cells are not needed to achieve retargeting. As proof of principle for use in the clinic we show efficient transduction and retargeting in human cord blood CD34+ cells. The modified Gag and Pol helper constructs we describe will allow any investigator to simply use these helper plasmids during vector production to retarget therapeutic foamy retroviral vectors.
Highlights
Retroviral vector gene therapy offers immense potential to treat many genetic diseases[1] and has shown efficacy in clinical trials[2,3,4]
The Foamy retroviral vectors (FVs) Pol helper plasmid expresses chromobox protein homolog 1 (CBX1) fused to the C-terminus of IN (IN-CBX1) with a flexible glycine-serine, (GS)[3], linker (Fig. 1a,b)
The unconcentrated titers of FVGag-RTY and FVIN-CBX1 & Gag-RTY were between 4 and 5 fold lower than control unmodified FV (Fig. 2), all modified FVs could be produced at clinically relevant titers, >107 transducing units (TU)/ml, after a 100-fold concentration
Summary
Retroviral vector gene therapy offers immense potential to treat many genetic diseases[1] and has shown efficacy in clinical trials[2,3,4]. Vector mediated genotoxicity results when the integrated vector dysregulates or disrupts host genes which can lead to oncogenic transformation[7]. Despite these risks gene therapy remains a promising alternative to allogeneic transplantation[8]. Gijsbers et al showed that lentiviral vector (LV) integration can be retargeted towards gene-poor regions in the genome by modifying LEDGF20 In their approach, host cell lines were modified to express chromobox protein homolog 1 (CBX1) fused to LEDGF. Regions in the human genome[21] This approach was successful in retargeting LV integration, it requires modifying a target cell line to express a chimeric LEDGF-CBX1 protein, which is not practical for clinical use. Mutations in the CBS negatively affect the ability of the foamy virus PIC to interact with host chromatin
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