Lentiviral protein transduction with genome-modifying HIV-1 integrase-I-PpoI fusion proteins: studies on specificity and cytotoxicity

Abstract

Rare-cutting endonucleases, such as the I-PpoI, can be used for the induction of double strand breaks (DSBs) in genome editing and targeted integration based on homologous recombination. For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques. For its applications, the endonuclease needs to be transported into the target cell nucleus, where the mechanism of transport may affect its function. Here, we have studied the lentiviral protein transduction of the integrase (IN)-PpoI fusion protein using the cis-packaging method. In genome-wide interaction studies, IN-fusion proteins were verified to bind their target sequence containing 28S ribosomal RNA (rRNA) genes with a 100-fold enrichment, despite the well-documented behavior of IN to be tethered into various genomic areas by host-cell factors. In addition, to estimate the applicability of the method, DSB-induced cytotoxic effects with different vector endonuclease configurations were studied in a panel of cells. Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA. In cell studies, certain cancerous cell lines were especially prone to DSBs in rRNA genes, which led us to test the protein transduction in a tumour environment in an in vivo study. In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases.

Figure 1

Interaction of different IN proteins with the I-PpoI site on chromosome 1. The binding of IN-I-PpoI_H78A proteins (H78A in short) with the I-PpoI target site in 1p32.2 was studied using ChIP. The results (mean ± SD) represent measurements from two independent ChIP experiments. Samples were derived from three independent and nonsimultaneous transductions. No interaction with target sequence was observed in the wt IN samples studied. Results were analyzed with one-way ANOVA and Bonferroni's multiple comparison test. ***P < 0.001; **P = 0.001 to P < 0.01; *P = 0.01 to P < 0.05.

Figure 2

ChIP-sequencing-mapped IN-chromatin interactions at (a) the rDNA repeat area, (b) within the 28S rRNA gene, and (c) in the non-rDNA-localized genomic I-PpoI sites (±different window sizes around the I-PpoI site). In (a) and (c), the results are shown as a percentage of n, where n indicates the numbers of unique interactions aligning to the genome version GRCh37/Hg19. In (b), the results are shown as a percentage of all interactions aligning to the ChrUn_gl000220. “Random” represents the theoretical probability of an interaction occurring in one of the ~600 copies 43 kb rDNA repeats, in 28S RNA inside ChrUn_gl000220, or into any of the 15 bp I-PpoI recognition sites with window sizes of ±250 bp, 2.5 kb, or 25 kb surrounding the I-PpoI site. Statistical significances are calculated using the chi-square ((a) and (c)) and Fisher's exact test ((b)). Statistical analyses are conducted against wt IN (random value is not included in statistical analyses; shown for illustrative purposes only). In (a), the differences between wt IN and all the IN-I-PpoI-containing groups are significant (P < 0.001). The situation is the same in (b) and (c) (P < 0.0001), except for the IN-I-PpoI_H78A, which has a P value of 0.0174 in (b) and zero interactions localizing within the non-rDNA I-PpoI site (±0 bp) in (c). ***P < 0.001; **P = 0.001 to P < 0.01; *P = 0.01 to P < 0.05.

Figure 3

The effects of IN-I-PpoI and IN-I-PpoI_H78A protein transduction on the viability of HeLa and MRC-5 cells. Cells were transduced with 2 or 10 ng of p24 per well, and their viability was followed for three days. Significant negative deviations from untreated cells (drop in viability) are shown with black asterisks and significant differences between the cell lines at day three with gray asterisks (one-way ANOVA followed by Dunnett's multiple comparison test). ***P < 0.001; **P = 0.001 to P < 0.01; *P = 0.01 to P < 0.05.

Figure 4

A cytotoxicity study in different cell lines using two concentrations of IN-I-PpoI. Cell viabilities measured by a luminescent cell viability assay are shown as percentages of the wt IN control vector-transduced cells (mean ± SD) after a vector dose of 2 ng (a) and a vector dose of 10 ng (b) of p24. The bars represent viability after LVV mediated I-PpoI protein transduction at different time points post transduction (1, 2, 3 and 6 days, from left to right). Results were analyzed with two-way ANOVA and the Tukey's multiple comparisons test. ***P < 0.001; **P = 0.001 to P < 0.01; *P = 0.01 to P < 0.05.

Figure 5

Development of tumour volumes following LVV transduction. Volume changes (mean ± SD) of subcutaneous A549 tumours treated with wild-type control vector (wt IN), active endonuclease containing vector (IN-I-PpoI, I-PpoI in short), or thymidine kinase transgene-containing vector (wt IN+TK) are shown. Group sizes were LVV IN_wt, n = 17; LVV IN-I-PpoI, n = 18; and LVV IN_wt TK, n = 6. Differences between groups were analyzed by two-way ANOVA and Tukey's multiple comparisons test. ***P < 0.001; **P = 0.001 to P < 0.01; *P = 0.01 to P < 0.05.

Figure 6

Tumour and blood sample analysis. The amount of proliferating cells per area is shown in (a) (mean ± SD) and examples of stained tumour histological samples in (d). GFP expression (mean ± SD) in dissociated tumour cells is presented in (b) as measured by flow cytometry analysis of dissociated tumour cells or the cultured cell control. The results (mean ± SD) of blood sample analysis from the samples drawn at the end of the in vivo experiment are shown in (c) LVV IN_wt, n = 4; LVV IN-I-PpoI, n = 5; and LVV IN_wt TK, n = 3). No statistical differences were found between groups in (b) or (c). CRP: C-reactive protein, CREA: creatinine, ASAT: aspartate aminotransferase, and AFOS: alkaline phosphatase. Statistical analysis: one-way ANOVA in (a) and (b) and two-way ANOVA in (c) combined with Bonferroni's ((a) and (c)) and Tukey's (b) multiple comparisons tests. ***P < 0.001; **P = 0.001 to P < 0.01; *P = 0.01 to P < 0.05.