Gene therapy has enormous potential but genotoxicity is a major concern for use of retroviral vectors in the clinic. Here we have successfully modified the integration profile of FV, resulting in significantly fewer RISs near genes and proto-oncogenes which may prove to be safer. Retargeted FVs can be produced by simply using the modified Gag and Pol helper plasmids we describe here during vector production. Retargeted FVs can be produced at high titer and efficiently transduce human CD34+ cells suggesting they will be useful for gene therapy of SCID-X1, chronic granulomatous disease, thalassemias, and potentially metabolic diseases that can be treated by hematopoietic stem cell (HSC) transplantation such as metachromatic leukodystrophy.

Of critical importance for gene therapy is the effect of modifications, such as protein fusions and amino acid substitutions, on vector titer, transduction efficiency, and transgene expression. Although the mutations needed to retarget FV modestly reduced titers, we were able to obtain clinically relevant titers (>107 TU/ml) after concentration. We also observed high levels of transgene expression relative to the control that were achieved using a low MOI in both normal human fibroblasts and cord blood CD34+ cells. There was also no evidence of vector silencing in CD34+ cells using the strong spleen focus forming virus (SFFV) promoter. The Gag-RTY modification reduced vector titer which is consistent with a study by Tobaly-Tapiero et al. in which they found fewer integrated viral copies in genomic DNA when the vector expressed Gag-RTY when compared to a control FV23. It was found Gag-RTY abolishes the affinity of FV PIC for host H2A and H2B histones, which reduces the efficiency of integration.

A surprising observation was the stronger influence of the Gag-RTY modification on RIS retargeting than the IN-CBX1 modification. Although it is known that the foamy retroviral Gag CBS interacts with H2A and H2B histones and is partially responsible for tethering the PIC to the host chromatin23, this is the first study that demonstrates mutations in the Gag CBS substantially retargets FV integration. FV Gag-RTY integration frequently occurred in H3K9me3 regions (Fig. 3), which are known to be a preferred target for CBX1. Upon further analysis, it was determined that many of the retargeted RISs were also in satellite repeat elements and near centromeres. Satellite DNA is the main component of centromeres and heterochromatin. Maskell et al. demonstrated that the ability of the FV intasome to form a stable complex with host chromatin, a prerequisite for integration, is dependent on the ability to engage nucleosomes26. It was further shown that specific amino acid substitutions in IN impede the ability of the FV intasome to interact with nucleosomes. Interestingly, these IN mutant FVs integrated more frequently within genes, which is a stark contrast to the reduction of integration within genes observed in our study with FVs harboring Gag-RTY (Table 1). The loss of the CBS functionality may alter the genomic locations accessible to the FV PIC and this may be dependent on the local chromatin structure immediately preceding integration. There may also be viral or host proteins that interact with foamy viral Gag CBS that influence integration site preferences which have yet to be determined. Our findings suggest the Gag CBS has a major role in integration site selection and Gag-RTY retargets FV integration into satellite repeats which are predominately gene sparse and frequently bear the H3K9me3 mark.

The addition of IN-CBX1 significantly increased the number of RISs within H3K9me3 regions. We also observed that FV IN-CBX1 & Gag-RTY had a greater preference for DNase I hypersensitivity, H3K9ac, and H3K4me3 sites than FV Control (Supplemental Figure S2). A possible explanation is that vectors retargeted to H3K9me3 sites or satellite elements have a preference for chromatin that is more accessible. However, it is important to consider that the state of histone methylation and acetylation during integration is not known. It has been shown that certain genes transiently bear the H3K9me3 mark during transcription activation27. IMR90 cells transduced with FV IN-CBX1 & Gag-RTY had a much greater number of RISs in satellites (Table 2) and did not have dramatically more RISs in H3K9ac regions (Supplemental Figure S2) when compared to FV Control , which suggests the retargeted FV is not being directed to H3K9me3 regions overlapping highly expressed genes. It is also important to determine if a retroviral vector has any strong integration site preferences and thus has a tendency to form integration hotspots. Hotspots were observed at much higher frequency with FV IN-CBX1 & Gag-RTY than FV Control (Fig. 4). However, FV IN-CBX1 & Gag-RTY RISs in hotspots had a strong tendency to also be near H3K9me3 regions. FV IN-CBX1 & Gag-RTY formed hotspots in putatively safe genomic regions, as H3K9me3 sites are associated with gene sparse regions. Also, we observed that RISs near H3K9me3 sites were farther away from proto-oncogene TSSs on average in CD34+ cells (Supplemental Table S1). Thus retargeted FVs have increased hotspots, but these are in gene sparse areas. This observation also provides more evidence that utilizing both the IN-CBX1 fusion protein and the Gag-RTY mutant protein results in the most efficient retargeting into gene sparse regions. The formation of integration hotspots thus appears to be a result of retargeted integration site selection rather than an indication of genotoxicity. Clonality analyses showed that all IMR90 and CD34+ cell populations were polyclonal at 23 days post vector exposure (DPVE) and 10 DPVE respectively. FV IN-CBX1 & Gag-RTY had a slight decrease in the number of clones contributing to the total cell population, with 8 clones contributing 1–2% in CD34+ cells at 10 DPVE. However, it is important to note that all of the RISs were greater than 100 kb from the nearest proto-oncogene TSS in these clones. Also, the total number of clones contributing to the retargeted FV cell populations may be slightly underrepresented due to the increased number of RISs in repetitive regions which are not included in clonality analyses. Future in vivo studies are needed to determine if FV IN-CBX1 & Gag-RTY has a dramatic effect on the resulting hematopoietic repopulating cell populations.

It has been shown that modifying the host cells to express a lentiviral IN-LEDGF fusion protein can alter the integration profile of LVs20. However, methods that require modifying the host cells prior to transduction are not a clinically relevant approach. As an alternative to manipulating tethering factors, proteins such as zinc finger and endonucleases fused directly to IN have shown weak retargeting in LVs29,30,31. Although there were only slight effects on retargeting integration, these experiments demonstrated the feasibility of modifying vectors directly. Recently, Ashkar et al. demonstrated GV integration can be targeted away from active transcription areas in the genome by introducing mutations in the IN regions that interact with bromodomain and extra-terminal (BET) proteins. GV IN interacts with host BET proteins and directs the GV PIC into or near TSSs32,33,34. Specifically in this study, single point mutations or complete truncation of the C-terminal region of GV IN resulted in an integration profile distinct from normal GV, with fewer RISs being near proto-oncogene TSSs35. However, vector titers were not expressed in TU/ml and the distributions of RISs were not analyzed in human cord blood CD34+ cells in their study. Also, while it was shown that the modified GVs had a similar percentage of RISs near proto-oncogene TSSs when compared to published normal FV data, we have demonstrated that the retargeted FVs have even less of a preference for proto-oncogene TSSs than normal FVs (Table 1). Thus, retargeted FV have a potentially safer integration profile than BET retargeted GV. Other inherent advantages of using FVs over other retroviral vectors include a lower potential to activate nearby genes and they have not been shown to exhibit read-through transcription12,22.

An important advantage of our approach is the ability to transduce target cells by simply using different helper plasmids during vector production. To obtain optimal retargeting of FV into H3K9me3 regions, both the Gag mutant and the IN-CBX1 fusion protein were necessary. This suggests that reducing the affinity of the foamy retroviral Gag CBS for H2A and H2B histones may be necessary to facilitate retargeting FVs with a modified IN. Importantly, we also show that the foamy IN can tolerate relatively large protein fusions. The CBX1 protein fused to the foamy IN is 21 kDa. Using Gag-RTY in conjunction with other FV IN fusion proteins, such as an IN-zinc finger or an IN-Cas9 fusion protein, should be considered in future work when developing retargeted FVs.

In summary the retargeted FVs we have described here integrate less frequently near proto-oncogenes than control FV. Since FV proviruses are also known to dysregulate nearby genes less than LVs or GVs36, retargeted FVs may be a safer option for HSC gene therapy. Thus, the safety of retargeted FVs should be further explored for clinical use.