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

Turkki V, Schenkwein D, Timonen O, Husso T, Lesch HP, Ylä-Herttuala S - Biomed Res Int (2014)

Bottom Line: For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques.Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA.In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland ; FinVector Vision Therapies Oy, 70210 Kuopio, Finland.

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.

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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 INwt, n = 17; LVV IN-I-PpoI, n = 18; and LVV INwt 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.
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fig5: 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 INwt, n = 17; LVV IN-I-PpoI, n = 18; and LVV INwt 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.

Mentions: Throughout the experiment, tumour sizes were found to remain smaller in the LVV IN-I-PpoI-injected mice than in control groups. The efficiency in tumour size reduction was similar to that obtained with LVV INwt TK (Figure 5). For the first 13 days, tumour sizes in the IN-I-PpoI group remained stable without any significant changes in tumour volumes. All tested tumours were aggressively proliferating with malignant irregularly shaped cells (Figures 6(a) and 6(d)). Flow cytometry analyses of randomly selected dissociated tumours were performed for the GFP transgene-containing LVV INwt and LVV IN-I-PpoI treated mice at 3 and 22 days after transduction (Figure 6(b)). Successful transduction and cellular entry of LVVs after the injection procedure were verified with LVVs containing INwt. Probably due to the IN-I-PpoI-carrying vector's low integration activity and its apparent cytotoxicity, at day 22, LVV-IN-I-PpoI treated tumours showed only minimal green fluorescent protein (GFP) expression (0.7–0.9%), being only slightly higher than the baseline value (0.5%) set for nontransduced tumour control. No differences in body weight loss, animal behavior, or signs of inflammation and liver or kidney failure were detected in any of the groups (Figure 6(c)). These results suggest that LVV protein transduction can be successfully used also in more difficult-to-transfect cellular environments.


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

Turkki V, Schenkwein D, Timonen O, Husso T, Lesch HP, Ylä-Herttuala S - Biomed Res Int (2014)

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 INwt, n = 17; LVV IN-I-PpoI, n = 18; and LVV INwt 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4016911&req=5

fig5: 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 INwt, n = 17; LVV IN-I-PpoI, n = 18; and LVV INwt 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.
Mentions: Throughout the experiment, tumour sizes were found to remain smaller in the LVV IN-I-PpoI-injected mice than in control groups. The efficiency in tumour size reduction was similar to that obtained with LVV INwt TK (Figure 5). For the first 13 days, tumour sizes in the IN-I-PpoI group remained stable without any significant changes in tumour volumes. All tested tumours were aggressively proliferating with malignant irregularly shaped cells (Figures 6(a) and 6(d)). Flow cytometry analyses of randomly selected dissociated tumours were performed for the GFP transgene-containing LVV INwt and LVV IN-I-PpoI treated mice at 3 and 22 days after transduction (Figure 6(b)). Successful transduction and cellular entry of LVVs after the injection procedure were verified with LVVs containing INwt. Probably due to the IN-I-PpoI-carrying vector's low integration activity and its apparent cytotoxicity, at day 22, LVV-IN-I-PpoI treated tumours showed only minimal green fluorescent protein (GFP) expression (0.7–0.9%), being only slightly higher than the baseline value (0.5%) set for nontransduced tumour control. No differences in body weight loss, animal behavior, or signs of inflammation and liver or kidney failure were detected in any of the groups (Figure 6(c)). These results suggest that LVV protein transduction can be successfully used also in more difficult-to-transfect cellular environments.

Bottom Line: For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques.Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA.In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland ; FinVector Vision Therapies Oy, 70210 Kuopio, Finland.

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.

Show MeSH
Related in: MedlinePlus