Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.

Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable

Detection of recombination mediated by phiC31-int between an <i>attB</i> site contained into a NILV and a genomic <i>attP</i> site.

<p>A) Scheme of the DsRed2 PCR before and after the enzymatic restriction treatment. B) PCR DsRed2 results without restriction enzyme treatment. Lanes 1 to 3: cotransduction with CMV-Neo and CMV-PhiC31 increasing vector input of 50–150–300 ng of p24. Lanes 4 to 6: cotransduction with <i>attB</i>-CMV-Neo and CMV-PhiC31 increasing vector input of 50–150–300 ng of p24. Lane 7: <i>attB</i>-CMV-Neo. Lane 8: positive control generated by triple-transfection (CMV-phiC31-int, <i>attB</i>-CMV-Neo and CMV-<i>attP</i>-DsRed2). Lane 9: negative control without vector. Lane 10: negative control of PCR. C) PCR DsRed2 results after restriction enzyme treatment. Lanes are similar to figure B. D) Nested PCR from the product isolated from lane 6 to confirm the specificity of PCR DsRed2 amplification.</p

Analysis strategies to detect the specific integrations mediated by phiC31-int.

<p>A) Illustration of the three mechanisms of the phiC31-int mediated integration of a NILV containing an <i>attB</i> sequence. According to the type of integration, the PCR results in three different profiles: - PCRs LTR+/<i>attB</i>− : integration type (1), specific integration. - PCRs LTR−/<i>attB</i>+: integration type (2), residual integration. - PCRs LTR+/<i>attB</i>+: integration type (3), illegitimate integration. P1/P1′ are the primers used for <i>attB</i> PCR and P2/P2′ are the primers used for LTR PCR. B) Schematic representations of the inverse PCR and the adapted inverse PCR strategies used to characterize phiC31-int integration sites.</p

DNA sequence of <i>att</i> and p<i>attP</i> sites.

<p>A) Wild type <i>attP</i> and <i>attB</i> sites. After recombination two hybrids sites are formed: <i>attL</i> and <i>attR</i>. B) Recombination between <i>attB</i> site and the human locus Xq22.1 This recombination generates a p<i>attR</i> which has been isolated by inverse PCR. Xq22.1 had been described previously as a human p<i>attP</i> by MP Calos et al., who isolated the same p<i>attR</i>.</p

Repartition in 3 groups of human and murine clones according to the <i>attB</i> and LTR PCR results.

<p>Repartition in 3 groups of human and murine clones according to the <i>attB</i> and LTR PCR results.</p

Mapping and description of pa<i>ttP</i> sites isolated by iPCR on human and murine cell lines.

<p>Mapping and description of pa<i>ttP</i> sites isolated by iPCR on human and murine cell lines.</p

Hypothetical model to explain the inversion of 4.8

<p>Step 1: Integration of a NILV mediated by phiC31-int into a p<i>attP</i> site. Step2: Recombination mediated by phiC31-int between the p<i>attL</i> generated during step 1 and another p<i>att</i> site located at 4 kb.</p

Scheme of phiC31-int mediated recombination in bacterial host.

<p>PhiC31 integrase performs precise recombination between an <i>attB</i> site located in the <i>Streptomyces</i> genome and an <i>attP</i> site located on the phiC31 phage genome. The outcome is integration of the phage into the host genome.</p

Effect of NLS sequence on phiC31-int activity in NILV context.

<p>A) Cotransduction of NILVs CMV-PhiC31 and CMV-Neo or <i>attB</i>-CMV-Neo. Four p24 doses of PhiC31 vector were used (D1: 3 ng, D2: 5 ng, D3: 10 ng, D4: 33 ng). B) Cotransduction of NILVs CMV-PhiC31-NLS and CMV-Neo or <i>attB</i>-CMV-Neo. Four p24 doses of PhiC31 vector were used (D1: 3 ng, D2: 5 ng, D3: 10 ng, D4: 33 ng). No significant differences are observed between sample with or without a<i>ttB</i> sequence in the vector pTRIP-CMV-Neo. Satistics: two ways ANOVA with Bonferroni posttest (Prism 5).</p

Analysis of cell lines which constitutively expressed phiC31-int.

<p>A) PhiC31 RT-PCR on three different cell lines. HFi and Hi16 are derived from Hela cell line and TC1 from NIH-3T3 cell line. Control condition lane lacks RNA. B) PCR which detects LTR junctions or intact <i>attB</i> sites after transduction with a NILV <i>attB</i>-CMV-Neo.</p