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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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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.
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fig4: 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.

Mentions: Although excessive DNA double strand break (DSB) formation is cytotoxic, site-specific cleavage can be exploited for therapeutic purposes in the form of genome editing and gene insertion through enhanced homologous recombination (HR [23, 24]). To characterize whether cancerous cells in addition to the tested HeLa cells would be more sensitive to IN-I-PpoI-originated cytotoxicity, we conducted a viability study in a panel of cells: 293T, A549, ARPE-19, BT4C, HeLa, HepG2, HUVEC, MRC-5, U-87, and U-255 cells (see Materials and Methods for descriptions). Cells were transduced with different concentrations of the vector ranging from 2 to 10 ng of p24 per well, which had been assessed previously to be non-cytotoxic using the LVV-wt IN (Figure S2). The active endonuclease-containing LVV IN-I-PpoI caused a significant reduction in the viability of most cell lines, when compared to untreated cells (Figure 4 and supplementary Figure S3). The cell line responses to LVV IN-I-PpoI transduction varied considerably. Generally, when compared to cancer cell lines, the nontumorigenic and nontransformed MRC-5, ARPE-19, and primary HUVEC cells exhibited slightly less extensive reductions in viability after LVV-IN-I-PpoI vector treatment at the end of the study (Figures S3 and S4), although the impact on HUVEC cells was difficult to interpret due to the deviant viability at the last time point. However, overall differences between the cell lines were moderate. With a high LVV dose, especially, the A549 and BT4C cells, which likely also suffer from cancer-specific defects in their DSB repair pathways, perished or stopped dividing. Similarly, 293T cells, despite not being a cancer cell line, exhibited extensive cytotoxicity in response to LVV IN-I-PpoI transduction. HEK293-derived cell lines, such as 293T, are not suitable models for healthy cells in DSB experiments, since they are known to express the adenoviral oncoprotein E1B55K, which disrupts the functionality of the DNA damage response pathways [25].


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016911&req=5

fig4: 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.
Mentions: Although excessive DNA double strand break (DSB) formation is cytotoxic, site-specific cleavage can be exploited for therapeutic purposes in the form of genome editing and gene insertion through enhanced homologous recombination (HR [23, 24]). To characterize whether cancerous cells in addition to the tested HeLa cells would be more sensitive to IN-I-PpoI-originated cytotoxicity, we conducted a viability study in a panel of cells: 293T, A549, ARPE-19, BT4C, HeLa, HepG2, HUVEC, MRC-5, U-87, and U-255 cells (see Materials and Methods for descriptions). Cells were transduced with different concentrations of the vector ranging from 2 to 10 ng of p24 per well, which had been assessed previously to be non-cytotoxic using the LVV-wt IN (Figure S2). The active endonuclease-containing LVV IN-I-PpoI caused a significant reduction in the viability of most cell lines, when compared to untreated cells (Figure 4 and supplementary Figure S3). The cell line responses to LVV IN-I-PpoI transduction varied considerably. Generally, when compared to cancer cell lines, the nontumorigenic and nontransformed MRC-5, ARPE-19, and primary HUVEC cells exhibited slightly less extensive reductions in viability after LVV-IN-I-PpoI vector treatment at the end of the study (Figures S3 and S4), although the impact on HUVEC cells was difficult to interpret due to the deviant viability at the last time point. However, overall differences between the cell lines were moderate. With a high LVV dose, especially, the A549 and BT4C cells, which likely also suffer from cancer-specific defects in their DSB repair pathways, perished or stopped dividing. Similarly, 293T cells, despite not being a cancer cell line, exhibited extensive cytotoxicity in response to LVV IN-I-PpoI transduction. HEK293-derived cell lines, such as 293T, are not suitable models for healthy cells in DSB experiments, since they are known to express the adenoviral oncoprotein E1B55K, which disrupts the functionality of the DNA damage response pathways [25].

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