Retroviral vector-mediated gene transfer into keratocytes: in vitro effects of polybrene and protamine sulfate

Seitz, Berthold; Baktanian, Edwin; Gordon, Erlinda M.; Anderson, W. French; LaBree, Laurie; McDonnell, Peter J.

doi:10.1007/s004170050129

Cited by 31 publications

(20 citation statements)

References 42 publications

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“…Several investigators have directly compared the effectiveness of Pb and PS on retroviral vector transduction enhancement and have reported results ranging from similar enhancement (7) to higher efficiencies with PS (23) or higher efficiencies with Pb (6,24). In our studies Pb was more effective than PS, although the difference was less extensive with HI serum.…”

Section: Discussionmentioning

confidence: 51%

Small Increases in pH Enhance Retroviral Vector Transduction Efficiency of NIH‐3T3 Cells

Jensen

Chen

Miller

2003

Biotechnology Progress

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Increases in pH between 7.1 and 7.7 increase the efficiency of polybrene (Pb)- and protamine sulfate (PS)-aided retroviral transduction of NIH-3T3 cells in a serum-lot-dependent manner. The increase in Pb-aided transduction efficiency at pH 7.7, relative to the value at pH 7.33, ranged from 13% to 49% for three serum lots. For a constant Moloney murine leukemia virus (MMLV) vector dilution at pH 7.33, three different serum lots resulted in absolute transduction efficiencies ranging from 29% to 53% using Pb. At the same vector dilution, PS-aided transduction was less effective on an absolute basis than Pb-aided transduction, but the benefit of elevated pH was more pronounced with PS. There was a similar enhancement with PS at elevated pH for a murine stem cell virus (MSCV) vector as for the MMLV vector. The benefit at pH 7.7 for PS-aided transduction was partially due to greater PS stability at elevated pH. Heat inactivating the serum supplement or adding protease inhibitors helped to stabilize PS. This increased the absolute transduction efficiency but decreased the relative benefit of elevated pH to a level similar to that for Pb-aided transduction. Incubating Pb with the vector at pH 7.1 for 10 min, prior to readjusting to pH 7.7 and transducing the cells, was sufficient to abrogate the beneficial effects of transduction at pH 7.7. In contrast, prior exposure of PS with vector at pH 7.1 did not affect subsequent transduction at pH 7.7. These results indicate that pH is an important variable in retroviral transduction and that the relative benefits of Pb or PS on retroviral vector transduction will vary with the pH, polymer addition method, and serum lot.

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Section: Discussionmentioning

confidence: 51%

Small Increases in pH Enhance Retroviral Vector Transduction Efficiency of NIH‐3T3 Cells

Jensen

Chen

Miller

2003

Biotechnology Progress

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“…This adsorption does not depend on the type of receptor and viral envelope and is also temperature-independent. Polybrene is considered non-toxic at low concentrations, but has been found to negatively affect cell proliferation in some cell types, such as keratinocytes, at concentrations greater than 10 µg/mL [16]. In our attempts to transduce hMSCs with lentivirus, we observed that hMSC proliferation was also affected by polybrene.…”

Section: Introductionmentioning

confidence: 79%

Polybrene Inhibits Human Mesenchymal Stem Cell Proliferation during Lentiviral Transduction

et al. 2011

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Human mesenchymal stem cells (hMSCs) can be engineered to express specific genes, either for their use in cell-based therapies or to track them in vivo over long periods of time. To obtain long-term expression of these genes, a lentivirus- or retrovirus-mediated cell transduction is often used. However, given that the efficiency with these viruses is typically low in primary cells, additives such as polybrene are always used for efficient viral transduction. Unfortunately, as presented here, exposure to polybrene alone at commonly used concentratons (1–8 µg/mL) negatively impacts hMSC proliferation in a dose-dependent manner as measured by CyQUANT, EdU incorporation, and cell cycle analysis. This inhibition of proliferation was observable in culture even 3 weeks after exposure. Culturing the cells in the presence of FGF-2, a potent mitogen, did not abrogate this negative effect of polybrene. In fact, the normally sharp increase in hMSC proliferation that occurs during the first days of exposure to FGF-2 was absent at 4 µg/mL or higher concentrations of polybrene. Similarly, the effect of stimulating cell proliferation under simulated hypoxic conditions was also decreased when cells were exposed to polybrene, though overall proliferation rates were higher. The negative influence of polybrene was, however, reduced when the cells were exposed to polybrene for a shorter period of time (6 hr vs 24 hr). Thus, careful evaluation should be done when using polybrene to aid in lentiviral transduction of human MSCs or other primary cells, especially when cell number is critical.

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“…As polycations improve adenovirusmediated gene transfer, less virus would have to be used, which would improve the therapeutic index by reducing unwanted responses associated with high doses of virus. On the other hand, multiple reports indicate that polycations could exhibit nonspecific cytotoxicity in vivo as well as in vitro (73,74), with some studies demonstrating unacceptable cytotoxicity for DEAE-dextran (74,75) and Polybrene (76,77), at least under some experimental conditions. Therefore, while our study conceptually demonstrates the feasibility of the polycation-mediated improvement of VSV-based OV therapy in vitro, future studies are needed to compare Polybrene and DEAE-dextran to other polycations that could be used safely and effectively in vivo in combination with VSV and ruxolitinib.…”

Section: Figmentioning

confidence: 99%

Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

Felt

Droby

Grdzelishvili

2017

J Virol

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Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma cell (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of resistance are still unclear. JAK1/2 inhibitors (such as ruxolitinib and JAK inhibitor I) strongly stimulate VSV replication and oncolysis in all resistant cell lines but only partially improve the susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication rather than on receptor specificity, with several ubiquitously expressed cell surface molecules playing a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDAC cells. Our data show a dramatically weaker attachment of VSV to HPAF-II cells, the most resistant human PDAC cell line. Although sequence analysis of low-density lipoprotein (LDL) receptor (LDLR) mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels but did not improve VSV attachment or LDL uptake in HPAF-II cells. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with Polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and Polybrene or DEAE-dextran successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication.IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC) cells. Although VSV is effective against most PDAC cells, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDAC cells. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line, HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (which inhibits antiviral signaling) successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple-combination approach could be used in the future to treat PDAC tumors that are highly resistant to OV therapy.KEYWORDS DEAE-dextran, combination treatment, oncolytic virus, pancreatic cancer, Polybrene, polycation, resistance, vesicular stomatitis virus, virus attachment, type I interferon, ruxolitinib

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Retroviral vector-mediated gene transfer into keratocytes: in vitro effects of polybrene and protamine sulfate

Cited by 31 publications

References 42 publications

Small Increases in pH Enhance Retroviral Vector Transduction Efficiency of NIH‐3T3 Cells

Small Increases in pH Enhance Retroviral Vector Transduction Efficiency of NIH‐3T3 Cells

Polybrene Inhibits Human Mesenchymal Stem Cell Proliferation during Lentiviral Transduction

Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

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