Uncontrolled genomic insertion of transgenes, leading to insertional mutagenesis, remains a primary challenge for safe genetic therapies. Lentiviral vectors offer an effective gene delivery route, but also suffer from prevalent gene insertion into transcriptionally active regions (Schröder et al., 2002; Modlich et al., 2009). Novel strategies to attain targeted gene integration (TGI) into predetermined genomic loci are highly warranted. TGI using lentiviral vectors can be achieved by delivering donor sequences for insertion by homologous recombination (HR) into double-strand breaks (DSBs) generated by tailored endonucleases (Lombardo et al., 2011). Development of programmable nucleases, ZFNs, TALENs, and now CRISPR/Cas9, has made effective gene disruption feasible, even in stem cells, but genetic operations involving TGI in stem cells are still challenged by low membrane permeability, low HR efficiency, and high sensitivity to external manipulation (Genovese et al., 2014; Hockemeyer et al., 2011; Hendel et al., 2015; Tay et al., 2013; Tiyaboonchai et al., 2014).

We previously established genomic editing in cells treated with ‘all-in-one’ lentiviral particles loaded with locus-targeted ZFN proteins and a donor sequence flanked by homology arms (Cai et al., 2014a). Here, building on these findings, we report a simple method for establishing TGI by treating cells with integrase-defective lentiviral vectors (IDLVs) packaged with both the transgene-containing donor template and ZFN proteins fused to the N-terminus of lentiviral Gag and GagPol polypeptides. Locus-directed cleavage and TGI is evident in cell lines, CD34⁺ hematopoietic stem cells, and induced pluripotent stem cells (iPSCs) upon transduction with ZFN-loaded vectors. Notably, the prevalence of TGI in two safe harbors in the human genome, the CCR5 and AAVS1 loci, is high in iPSCs treated with ZFN-loaded lentiviral vectors, resulting in non-mosaic clones harboring a site-directed gene insertion and no additional cutting at top-ranked off-target sites.

IDLVs harboring both ZFN protein and vector RNA with the transgene flanked by homology arms are here designated ‘IDLV-ZFN(locus)/gene’, where ‘gene’ refers to the transgene cassette flanked by homology arms and transferred by the vector. Principles of production, transduction, and integration of such vectors are illustrated in Figure 1A. Together with a dimer of lentiviral vector RNAs, approximately 5000 copies of Gag and GagPol polypeptides (of which about half carry a ZFN fused to the matrix protein) form the capsid, which is wrapped in a segment of the membrane during budding from the producer cells. ZFNs are released from Gag and GagPol upon cleavage by the viral protease during maturation of the virus particle. The mature virus particles contain two types of ZFNs, left and right, which upon DNA binding in the target cell form a catalytically active dimer. After viral entry in the target cells and endosomal escape, vector RNA is reverse transcribed to DNA, which can serve as donor for homology-directed insertion at targeted breakage sites generated by the co-delivered ZFNs (Figure 1A, lower panel).

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To visualize the enrichment of ZFN-containing lentiviral polypeptides at the membrane of the producer cells, we transfected HEK293T cells with GagPol plasmid harboring either the coding sequence for a HA-tagged ZFN (HA-ZFN(gfp)) recognizing the egfp gene or the egfp gene itself fused to the 5’-end of gag gene and analyzed the virus-producing cells using confocal microscopy (Figure 1B and Figure 1—figure supplement 1A). By staining with an antibody specific for the tag, we found that the ZFNs were highly enriched at the cell membrane (Figure 1B). We then visualized the lentiviral particles (designated LPs since the vector was not included) and found for both LP-HA-ZFN(gfp) and LP-eGFP co-localization of the fusion protein with viral p24 protein (Figure 1C and Figure 1—figure supplement 1B and C), suggesting that ZFN (or eGFP) proteins were indeed packaged into the LPs. To measure the lifespan of LP-delivered ZFNs in transduced cells, HEK293 cells were synchronized at 4°C for 60 min before fixation at different time points (1, 12, 24, and 48 hr, respectively) after transduction with ZFN-loaded LPs. Already within the first hour after exposure to LP-HA-ZFN(gfp), the ZFNs were easily detectable inside the cells, whereas after 24 hr essentially all ZFNs had been degraded or diluted (Figure 1D). These findings showed that nucleases delivered by LPs were immediately available after transduction and that the time of action was restricted due to decay of the proteins.

We then performed proof-of-efficacy studies by transducing HEK293T cells with IDLVs (500 ng p24), with or without AAVS1-directed ZFNs, carrying an eGFP expression cassette (IDLV-ZFN(AAVS1)/egfp). Initially, three days post-transduction, a higher frequency of eGFP⁺ cells was observed among cells transduced with IDLV/egfp than for cells transduced with IDLV-ZFN(AAVS1)/egfp (59.1% versus 44.7%), indicating that the overall transduction potential was higher for IDLVs that did not carry ZFNs (Figure 1E and Figure 1—figure supplement 1D and 1E). Nevertheless, at later time points 2.5% of the cells were eGFP⁺ after treatment with IDLV-ZFN(AAVS1)/egfp, whereas the percentage dropped to below 1% in IDLV/egfp-treated cells (Figure 1E and Figure 1—figure supplement 1E). PCR amplification and sequencing of the 5’ and 3’ junction sites including part of the donor sequence and the regions flanking the inserted transgene cassette showed that events of correct integration into AAVS1 locus were evident after treatment with IDLV-ZFN(AAVS1)/egfp, whereas PCR products indicative of site-directed insertion did not appear for IDLV/egfp (Figure 1F). However, DSBs were not repaired by HR only, and mismatches introduced by non-homologous end joining (NHEJ) were detected in 5% of the AAVS1 alleles, as measured by Surveyor nuclease assay (Figure 1G). To evaluate the levels of TGI with another reporter gene, we treated HEK293T cells with IDLV-ZFN(AAVS1)/fluc carrying AAVS1-targeted ZFNs and a firefly luciferase (fluc) expression cassette. Indeed, fluc gene expression was maintained for up to 18 days after transduction with IDLV-ZFN(AAVS1)/fluc (Figure 1H), although the levels dropped from the initial 3-day time point, where episomal forms were still likely to support expression. For IDLV/fluc, in contrast, luminescence dropped to almost undetectable levels. TGI of the fluc gene into the AAVS1 locus was verified by PCR (Figure 1I), and the rate of NHEJ estimated to be 8% (Figure 1J).

Next, we created ZFN-loaded IDLVs carrying the puromycin resistance gene (IDLV-ZFN(AAVS1)/puro) allowing us to easily isolate single puromycin-resistant clones and further scrutinize AAVS1-targeted integration events by Southern blot analysis (Figure 1K). Among 16 clones generated with IDLV-ZFN(AAVS1)/puro, a 2.2-kb fragment, indicative of precise TGI by HR, was detected in 11 clones (Figure 1L). Other types of integrations, resulting in lower-mobility bands, were evident in the five remaining clones. Notably, two clones (clone 5 and clone 13) showed a 3.7-kb band, and these clones therefore were among clones that were analyzed by Southern blotting using another restriction enzyme and an AAVS1-specific probe (Figure 1—figure supplement 1F and 1G). A common 6.4-kb band was observed for clones 5 and 13 (Figure 1—figure supplement 1G), suggesting that the full-length lentiviral DNA in these two cases was captured and inserted by chance into the ZFN-induced DSB at the AAVS1 locus. These findings demonstrated that site-directed gene insertion into ZFN-generated DSBs also occurred, although less frequently, in an HR-independent fashion.

We analyzed 15 additional puromycin-resistant clones by PCR and found that 13 out of these clones yielded PCR products indicative of TGI (Figure 1—figure supplement 1H). In total, 84% (26/31) of the analyzed clones contained a targeted insertion of the puro gene in the AAVS1 locus. In contrast, the fragments detected by Southern blot analysis of IDLV/puro-treated clones had variable sizes (Figure 1M), indicating that the integrations were random events that occurred at different genomic locations.

To demonstrate the feasibility of this approach in stem cells, we isolated CD34⁺ hematopoietic stem cells from human cord blood and focused initially on optimizing lentiviral transduction rates for optimal ZFN delivery. We first verified that ZFN-loaded VSV-G-pseudotyped LPs loaded with AAVS1-directed ZFNs resulted in targeted cleavage upon transduction of CD34⁺ cells. Hence, indels introduced through repair by NHEJ were found in 5% of the alleles (Figure 2—figure supplement 1A). Next, we compared the capacity of recombinant human fibronectin (RetroNectin) and EF–C peptides, the latter recently discovered artificial peptide nanofibrils, to support virus entry to CD34⁺ cells (Yolamanova et al., 2013; Lump et al., 2015). In our setting, the presence of EF-C allowed transduction of 85.1% of the cells with a standard eGFP-encoding lentiviral vector, whereas between 27.5 and 30.8% of the cells were transduced in the presence of RetroNectin (Figure 2—figure supplement 1B). Therefore, we chose to include EF-C for targeted integration in CD34⁺ cells.

In the first of two protocols (Figure 2A, orange labeling), we transduced CD34⁺ cells derived from two different donors with IDLV-ZFN(AAVS1)/egfp or IDLV/egfp (MOI = 5) and analyzed for eGFP⁺ cells 9 days post-transduction (Figure 2B). On average, treatment with IDLV-ZFN(AAVS1)/egfp resulted in (0.49 ± 0.14)% eGFP⁺ cells, which was significantly higher than the percentage of eGFP⁺ cells (0.07 ± 0.03)% measured after transduction with the IDLV/egfp control without ZFNs (Figure 2B). PCR analyses confirmed events of TGI by IDLV-ZFN(AAVS1)/egfp, but not by IDLV/egfp (Figure 2C). Notably, IDLV-ZFN(AAVS1)/egfp transduction only slightly affected the cell viability (Figure 2D), and cell proliferation was not disturbed by the virus treatment (Figure 2E). Encouraged by this, we reasoned that the efficiency could be further increased (i) by using higher MOIs, (ii) by pretreating CD34⁺ cells with Vpx-loaded lentiviral particles (to degrade the reverse transcription inhibitor SAMHD1 [Laguette et al., 2011]), and (iii) by treating the cells with an extra dose of ZFNs delivered by LP-ZFNs. Indeed, using this improved protocol 2 (Figure 2A, green labeling), we were able to increase the percentage of eGFP⁺ cells to (0.98 ± 0.13)% after transduction with IDLV-ZFN(AAVS1)/egfp (Figure 2F). Also based on this protocol, TGI in the bulk was confirmed by PCR analysis (Figure 2G). Notably, IDLV-ZFN(AAVS1)/egfp treatment did not compromise the colony-forming capacity of CD34⁺ cells (Figure 2H). In accordance with the flow cytometry data, three eGFP⁺ colonies (example shown in Figure 2—figure supplement 1C) were identified out of a total of 302 analyzed colonies.

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Ways of engineering cells by TGI may be of particular interest for therapeutic modification and use of iPSCs. To establish TGI in iPSCs using lentiviral protein transduction, we first evaluated the permissiveness of iPSCs to LPs loaded with ZFNs targeting the AAVS1 and CCR5 loci, respectively. LP-ZFN(AAVS1) and LP-ZFN(CCR5) disrupted 4% and 8%, respectively, of the targeted alleles in iPSCs (Figure 3A).

We then transduced iPSCs with IDLV-ZFN(AAVS1)/puro. Of the approximately 500 puromycin-resistant clones formed from 50,000 transduced iPSCs (Figure 3B), we initially randomly picked, isolated and expanded eight clones (clones 1–8) for further analysis. From a subsequent experiment we picked 15 additional clones (clones 9–23). PCR amplification of the 5’ and 3’ junction sites showed that TGI had occurred in 19 out of the 23 clones (Figure 3C and D). Findings based on Southern blot analysis of clones 9–23 were consistent with the PCR analysis and confirmed that 11 of these 15 clones contained an AAVS1-directed insertion (Figure 3D). Eight out of the 11 clones harbored a precise, homology-directed transgene insertion, whereas only one of the two junction sites in the remaining 3 clones (clones 9, 17, and 18) was intact. Hence, the 5’ junction site was intact in clone 17, whereas clones 9 and 18 contained an intact 3’ junction site, indicating that site-directed insertion in these cases involved both HR and NHEJ. Sequence analysis of the 5’ and 3’ junction sites amplified from clones 1–8 showed junctions sequences indicative of precise, homology-driven insertion (Figure 3E and Supplementary file 1A). For one particular clone, clone 7, we were not able to amplify the 469-bp AAVS1 fragment, suggesting that both alleles in this clone contained a TGI (Figure 3C). Interestingly, sequencing of the AAVS1 locus provided evidence of biallelic cleavage in at least one of the clones (clone 5), resulting in a 1-bp insertion in the allele that did not contain the transgene (Supplementary file 1A). Stemness of TGI-modified iPSCs was verified by characterization of a single representative clone using alkaline phosphatase (AP) and the TRA-1-60 pluripotency marker (Figure 3F). Also, genomic integrity was confirmed by karyotyping (Figure 3G).

To detect potential off-target cleavage by the virus-delivered ZFNs, we first sequenced the three top-ranked off-target sites in clones 1–8, but did not identify indels in any of these loci (Supplementary file 1B). We then isolated genomic DNA from a pool of more than 100 puromycin-resistant iPSC clones and confirmed by PCR analysis the presence of TGI events in the pool (Figure 3H, insert). Then, next-generation sequencing was performed on PCR amplicons representing the on-target AAVS1 locus as well as seven top-ranked off-target sites. Among this pool of clones, most of which carry an AAVS1-directed gene insertion, we were not able to detect indels at off-target positions, whereas 3% of the AAVS1 alleles that were not already targeted by transgene insertion contained indels (Figure 3H and Figure 3—figure supplement 1A). Altogether, our findings demonstrated high levels of ZFN specificity and precise TGI in the AAVS1 locus in iPSCs treated with IDLV-ZFN(AAVS1)/puro vectors, resulting in at least one TGI event in 19 of 23 analyzed iPSC clones.

To demonstrate that such features of the ‘all-in-one’ gene delivery system was not limited to insertion into the AAVS1 locus, we produced IDLVs (designated IDLV-ZFN(CCR5)/puro) carrying ZFNs targeting the human CCR5 locus as well as vector RNA carrying the PGK-puro cassette flanked by homology arms matching the CCR5 locus. Transduction of iPSCs with IDLV-ZFN(CCR5)/puro resulted in effective colony formation (Figure 3I). Among the puromycin-resistant clones, we randomly picked, isolated and expanded 15 clones (clones 1–15) and showed by PCR analysis of each of these colonies that the transgene cassette in all 15 clones (100%) had been inserted site-specifically into the CCR5 locus (Figure 3J). For a single representative clone, stemness was verified using alkaline phosphatase (AP) and the TRA-1-60 pluripotency marker (Figure 3K). Once again, we pooled more than 100 puromycin-resistant iPSC clones and performed next-generation sequencing on PCR amplicons amplified from the on-target locus and nine high-ranked off-target sites in the pooled DNA sample (Figure 3L and Figure 3—figure supplement 1B). Notably, we identified an indel in approximately 19% of the CCR5 alleles that did not already carry a TGI, demonstrating that the second CCR5 allele was cleaved by ZFNs in approximately one of every five clones harboring a CCR5-directed insertion. Importantly, off-target cleavage and indel formation by NHEJ was not evident in any of the nine analyzed off-target candidate loci. Hence, not even the CCR2 locus (off-target 3), which is highly homologous to the CCR5 site and a known off-target cleavage site for CCR5-targeted ZFNs, harbored detectable levels of indels.

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In conclusion, lentivirus-based co-delivery of nuclease proteins and a HR donor represents a powerful and safe approach for inserting transgenes into predetermined ‘safe harbors’ in the human genome.

On average, four out of five lentiviral integrations land in genes (Mitchell et al., 2004). It is still unclear to which extent this biased integration pattern induces toxicity due to insertional mutagenesis, but new approaches to alter the integration properties of lentiviral vectors remain attractive. Different strategies have been employed to reprogram the integration machinery of lentiviral vectors including (i) addition of a DNA-binding zinc-finger protein to the integrase (Bushman and Miller, 1997), (ii) fusion of a chromosomal binding domain to LEDGF, a cellular co-factor of the integrase (Gijsbers et al., 2010), and (iii) insertion of lentiviral DNA intermediates by transposases and recombinases (Vink et al., 2009; Staunstrup et al., 2009; Moldt et al., 2011; Cai et al., 2014b). These approaches successfully alter the integration preference, but do not allow site-specific insertion into a preferred locus. Here, we present an alternative homology-directed insertion strategy facilitated by nuclease-directed DNA cleavage. By co-packaging designer nucleases and donor sequences into lentiviral vectors and utilizing cellular HR repair pathways to integrate the transgene, we can guide lentiviral cargo toward a safe harbor in up to 84% of HEK293T cells and 89% of iPSCs that were transduced with the ‘all-in-one’ vector system. The efficiency may likely be further improved by exploiting heterodimer-forming rather than homodimer-forming FokI domains (Doyon et al., 2011).

TGI into the AAVS1 and CCR5 loci of stem cells has previously been achieved using HR-based strategies exploiting two- or three-component systems based on delivery of plasmids, IDLVs, and baculovirus vectors as well as combinations of mRNA encoding ZFNs and IDLVs serving as the HR donor (Lombardo et al., 2011; Genovese et al., 2014; Tay et al., 2013; Tiyaboonchai et al., 2014; Hockemeyer et al., 2009; Lei et al., 2011). Despite the potential of genetic therapies based on TGI in stem cells, like CD34⁺ cells and iPSCs, existing technologies remain insufficient and highly toxic (Genovese et al., 2014; Hoban et al., 2015; Merling et al., 2015). Exploiting the capacity of lentiviruses to co-deliver nucleases and a transgene cassette, we showed TGI in CD34⁺ cells transduced with ZFN-loaded IDLVs without affecting the viability, proliferation, or colony-forming capacity of the cells. However, the efficiency was only modest, suggesting that a selective growth advantage of the TGI-engineered cells, as in SCID-X1, would be required for successful engraftment. Possibly, efficacy could be boosted by combining virus treatment with exposure to chemicals enhancing stem cell self-renewal, by supplying NHEJ inhibitors (Chu et al., 2015), or by manipulating cell cycle conditions in favor of HR (Marniemi et al., 1975).

Stable transduction rates with ZFN-loaded IDLVs leading to TGI are undoubtedly reduced relative to conventional lentiviral gene delivery strategies. In iPSCs, however, transduction with IDLVs carrying ZFNs targeting either the AAVS1 or the CCR5 locus generated a high number of puromycin-resistant clones. Remarkably, 34 out of a total of 38 analyzed clones contained the transgene expression cassette inserted precisely into the locus targeted by the ZFNs. Also, as typically observed in relation to the standard use of IDLVs, we found that a few clones contained an additional, randomly inserted transgene cassette, which could be of relevance for potential future therapeutic use. Importantly, however, we found that lentiviral ZFN protein delivery did not lead to detectable off-target cleavage. Hence, cleavage in a total of 16 predicted off-target loci (7 for AAVS1 and 9 for CCR5) did not appear as analyzed by deep sequencing of large pools of clones carrying the transgene cassette. If desired, this approach can be combined with removal of the selection marker by seamless excision, exploiting for example lentiviral protein transduction of piggyBac transposase (Cai et al., 2014b). Remarkably, in a large pool of iPSCs, most if not all carrying a site-targeted gene insertion in the CCR5 locus, off-target cleavage and indel formation at the neighboring CCR2 locus was not evident after deep sequencing analysis. This is in contrast to higher levels of CCR2 targeting reported in previous studies using other delivery strategies based on adenoviral vectors, plasmid transfection, or administration of recombinant proteins (Perez et al., 2008; Gaj et al., 2012), suggesting that lentiviral delivery of site-targeted endonucleases supports safer engineering of the genome. Together, our findings demonstrate that lentivirus particles can ferry programmable nucleases, facilitating homology-driven, site-directed genomic insertion of a co-delivered transgene. Such co-delivery ensures time-limited exposure of the treated cell to nucleases and, yet, results in frequent TGI with particular applicability in iPSCs.

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Author details

1. Yujia Cai

   Department of Biomedicine, Aarhus University, Aarhus, Denmark

   Contribution

   YC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Anders Laustsen

   Department of Biomedicine, Aarhus University, Aarhus, Denmark

   Contribution

   AL, Revising article, Final approval, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Yan Zhou

   Department of Biomedicine, Aarhus University, Aarhus, Denmark

   Contribution

   YZ, Revising article, Final approval, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Chenglong Sun

   Department of Biomedicine, Aarhus University, Aarhus, Denmark

   Contribution

   CS, Revising article, Final approval, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. Mads Valdemar Anderson

   1. Department of Biomedicine, Aarhus University, Aarhus, Denmark
   2. Technical University of Denmark, Lyngby, Denmark

   Contribution

   MVA, Revising article, Final approval, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

6. Shengting Li

   1. Department of Biomedicine, Aarhus University, Aarhus, Denmark
   2. Beijing Genomics Institute, Shenzhen, China

   Contribution

   SL, Revising article, Final approval, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

7. Niels Uldbjerg

   Department of Clinical Medicine, Aarhus University, Aarhus, Denmark

   Contribution

   NU, Revising article, Final approval, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

8. Yonglun Luo

   Department of Biomedicine, Aarhus University, Aarhus, Denmark

   Contribution

   YL, Revising article, Final approval, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-0007-7759

9. Martin R Jakobsen

   Department of Biomedicine, Aarhus University, Aarhus, Denmark

   Contribution

   MRJ, Revising article, Final approval, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

10. Jacob Giehm Mikkelsen

    Department of Biomedicine, Aarhus University, Aarhus, Denmark

    Contribution

    JGM, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    giehm@biomed.au.dk

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-1322-3209

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