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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 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.

Retroviral (RV) vector mediated gene therapy offers immense potential for permanently treating a broad spectrum of genetic conditions and has shown efficacy in clinical trials. However, vector proviruses can dysregulate nearby host genes, referred to as genotoxicity, which can lead to oncogenic transformation. Studies on the integration profiles and the mechanisms responsible for targeting integration have been carried out, resulting in a better understanding of the safety of RV vectors. Integration is influenced by the pre-integration complex (PIC) and its interactions with host proteins. Retargeting RV vectors to safe locations in the genome is an important step in reducing potential genotoxic effects. Although the RV PIC and its interactions are poorly defined, studies have shown that it is possible to retarget RV vector integration. It has been demonstrated that lentiviral vector integration can be retargeted using artificial tethering factors. A modified cell line was produced that expressed lens epithelium–derived growth factor (LEDGF) fused to chromobox protein homolog 1 (CBX1) in place of the wild type LEDGF. The LEDGF-CBX1 fusion protein targeted lentiviral vector integration into methylated regions in the human genome. However, this strategy requires modifying a cell to express a fusion protein and is therefore not practical for clinical use. Other studies have also shown it is possible to retarget RV vector integration, but these studies resulted in reduced transgene expression or impaired vector titers. A clinically relevant method for retargeting integration to safer genomic locations is needed.Here we demonstrate an approach to retarget foamy viral (FV) vector integration into safer heterochromatic regions that does not require a modified cell line. Foamy viruses have a broad tropism, a large packaging capacity, and efficiently transduce hematopoietic stem cells, making them well suited for gene therapy. We describe a FV vector with a modified Pol which expresses an integrase-CBX1 (IN-CBX1) fusion protein and a modified Gag which expresses a mutated chromatin binding site (CBS) domain. CBX1 binds to trimethylated histone H3K9 (H3K9me3), so we hypothesized the IN-CBX1 fusion protein would target FV vector integration into heterochromatin. The integration sites of the modified FV vector (n = 1494) and the control FV vector were compared in normal human fibroblast cells. We analyzed the retargeting ability by comparing the integration sites to normal human fibroblast H3K9me3 ChIP-Seq data. Significant retargeting to H3K9me3 regions was observed with the modified FV vector (P 107 transducing units/ml). This study demonstrates that FV vectors can be efficiently retargeted using foamy integrase fusion proteins. Since this approach does not require engineering the target cells, it is promising for use in the clinic, including hematopoietic stem cell gene therapy.

Hematopoietic stem cell gene therapy offers immense potential to treat many genetic diseases and has already shown efficacy in clinical trials. However, retroviral vector mediated gene dysregulation, known as genotoxicity, remains a major challenge and clinically relevant approaches to reduce integration near genes and proto-oncogenes are needed. The frequency at which a retroviral vector integrates near proto-oncogenes is a major factor in determining the safety of the vector. Here we describe a clinically relevant method to retarget foamy retroviral vector (FV) integration away from proto-oncogenes using modified Gag and Pol helper constructs. The chromatin binding site (CBS) of FV Gag was altered using a previously described triple alanine substitution mutation of RTY (Gag-RTY) shown to drastically reduce chromatin binding of the FV pre-integration complex. The modified FV Pol construct expresses chromobox protein homolog 1 fused to the C-terminus of integrase (CBX1-IN). CBX1 interacts with tri-methylated lysine 9 of histone H3 (H3K9me3) and is associated with gene sparse regions in the human genome. We hypothesized a modified FV expressing Gag-RTY and CBX1-IN would have an altered integration site profile and be retargeted to H3K9me3 regions and away from genes and proto-oncogenes. FV integration sites from modified and control FVs were compared in normal human fibroblasts and in CD34+ human cord blood cells. We observed retargeting of FV integration into H3K9me3 regions with the modified FV (Figure 1Figure 1). Importantly, retargeting FV integration significantly reduces the number of integration sites near proto-oncogene transcription start sites (Table 1Table 1). Retargeted FVs can be produced at clinically relevant titers (> 107 transducing units / ml), resulting in efficient transgene expression (75 % of control in CD34+ cells) with no evidence of vector silencing. Another published method to retarget lentiviral vector integration required using a modified cell line, which is not practical for clinical use. Our approach is cell line independent and the modified Gag and Pol helper constructs will allow an investigator to simply use these modified helper plasmids during vector production to retarget any therapeutic FV in any target cell. Retargeted FVs integrate less frequently near proto-oncogenes than gammaretroviral vectors and unmodified FVs, and may be the safest current option for hematopoietic stem cell gene therapy.Table 1Retargeted FV integrates further away from genes and proto-oncogenes than control FV.Percent of FV integration sites in human CD34*** cellsVectorTotal sitesIn RefSeq genes< 10 kb from TSS< 50 kb from proto-oncogene TSSControl FV421740.924.54.7Retargeted FV153430.6***12.5***2.2****statistically significant at p < 0.001 compared to control vector. FV, foamy retroviral vector; TSS, transcription start site.View Large Image | Download PowerPoint Slide