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1.  Retrovirus-mediated gene transfer to cystic fibrosis airway epithelial cells: effect of selectable marker sequences on long-term expression. 
Nucleic Acids Research  1993;21(3):663-669.
Retrovirus-mediated gene transfer offers the potential for stable long-term expression of transduced genes in host cells subsequent to integration of vector DNA into the host genome. Using a murine amphotropic retrovirus vector containing an interleukin-2 receptor (IL-2R) gene as a reporter and a neomycin phosphotransferase (neor) gene as a dominant selectable marker, we measured the efficiency of retrovirus-mediated gene transfer and the stability of transduced gene expression in a cystic fibrosis tracheal epithelial cell line (CFT1). The use of the IL-2R cell surface marker as a reporter of infection permitted both quantitation of vector gene expression and flow cytometric sorting of cells transduced with the vector. In initial studies, the optimal conditions for retrovirus-mediated gene transfer were determined. The presence of a polycation was required for optimal transduction efficiency. The efficiency of infection of CFT1 cells was increased by repetitive exposure to virus such that it was possible to transduce approximately 80% of the cells following three successive daily exposures. The long-term stability of expression of the non-selected IL-2R gene was also evaluated. A slow decline in the percentage of cells expressing IL-2R was seen with cells that were maintained under constant selection pressure for expression of the neor gene, which was expressed from an internal promoter. Similar results were obtained when cultures were selected initially for neor gene expression and maintained without selection thereafter. In contrast, stable expression was observed in CFT1 cells for at least one year following multiple infections in the absence of G418 selection. In conclusion, (i) transduction of foreign genes into human airway epithelial cells using an amphotropic retrovirus vector can be highly efficient in the presence of appropriate polycations and multiple exposures; and (ii) stable expression of a non-selected gene in these epithelial cells is better maintained without selection.
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PMCID: PMC309167  PMID: 7680124
2.  Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety 
Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well.
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach.
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 μg/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.
doi:10.3791/2822
PMCID: PMC3196183  PMID: 21808229
3.  Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro 
Background
Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro.
Methods
To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin® and the polycation transduction enhancer Transfectam®. The EGFP-positive transduced cells were then enriched by flow cytometry.
Results
More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes.
Conclusion
The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts.
doi:10.1186/1471-2261-6-25
PMCID: PMC1513252  PMID: 16756651
4.  Single-Step Conversion of Cells to Retrovirus Vector Producers with Herpes Simplex Virus–Epstein-Barr Virus Hybrid Amplicons 
Journal of Virology  1999;73(12):10426-10439.
We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV–Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 106 and 107 transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.
PMCID: PMC113098  PMID: 10559361
5.  Polybrene Inhibits Human Mesenchymal Stem Cell Proliferation during Lentiviral Transduction 
PLoS ONE  2011;6(8):e23891.
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.
doi:10.1371/journal.pone.0023891
PMCID: PMC3162604  PMID: 21887340
6.  Determination of Infectious Retrovirus Concentration from Colony-Forming Assay with Quantitative Analysis 
Journal of Virology  2003;77(10):5712-5720.
The colony formation assay is the most commonly used titration method for defining the concentration of replication-incompetent murine leukemia virus-derived retroviral vectors. However, titer varies with target cell type and number, transduction time, and concentration of polycation (e.g., Polybrene). Moreover, because most of the viruses cannot encounter target cells due to Brownian motion, their short half-lives, and the requirement for target cell division for activity, the actual infectious retrovirus concentration in the collected supernatant is higher than the viral titer. Here we correlate the physical viral particle concentration with the infectious virus concentration and colony formation titer with the help of a mathematical model. Ecotropic murine leukemia retrovirus supernatant, collected from the GP+E86/LNCX retroviral vector producer cell line, was concentrated by centrifugation and further purified by a sucrose density gradient. The physical concentration of purified viral vectors was determined by direct particle counting with an electron microscope. The concentrations of fresh and concentrated supernatant were determined by a quantitative reverse transcriptase activity assay. Titration of all supernatants by neomycin-resistant colony formation assay was also performed. There were 767 ± 517 physical viral particles per infectious CFU in the crude viral supernatant. However, the infectious viral concentration determined by mathematical simulation was 143 viral particles per infectious unit, which is more consistent with the concentration determined by particle counting in purified viral solution. Our results suggest that the mathematical model can be used to extract a more accurate and reliable concentration of infectious retrovirus.
doi:10.1128/JVI.77.10.5712-5720.2003
PMCID: PMC154030  PMID: 12719564
7.  Defective herpes simplex virus type 1 vectors harboring gag, pol, and env genes can be used to rescue defective retrovirus vectors. 
Journal of Virology  1997;71(5):4111-4117.
A retroviral packaging transcription unit was constructed in which the Moloney murine leukemia virus (MoMLV) gag-pol and env genes are expressed under the control of herpesvirus regulatory sequences. This transcription unit, lacking long terminal repeats, primer binding sites, and most of the retrovirus packaging signal but retaining both retroviral donor and acceptor splice sites, was cloned into a herpes simplex virus type 1 (HSV-1) amplicon plasmid, and amplicon vectors (the gag-pol-env [GPE] vectors) were generated by using a defective HSV-1 vector as helper virus. The GPE vector population was used to infect human TE671 cells (ATCC CRL 8805), harboring a lacZ provirus (TE-lac2 cells), and supernatants of infected cells were collected and filtered at different times after infection. These supernatants were found to contain infectious ecotropic lacZ retroviral particles, as shown both by reverse transcription-PCR and by their ability to transduce a beta-galactosidase activity to murine NIH 3T3 cells but not to human TE671 cells. The titer of retroviral vectors released by GPE vector-infected TE-lac2 cells increased with the dose of infectious amplicon particles. Retrovirus vector production was inhibited by superinfection with helper virus, indicating that helper virus coinfection negatively interfered with retrovirus production. Induction of retrovirus vectors by GPE vectors was neutralized by anti-HSV-1 but not by anti-MoMLV antiserum, while transduction of beta-galactosidase activity to NIH 3T3 cells by supernatants of GPE vector-infected TE-lac2 cells was neutralized by anti-MoMLV antiserum. These results demonstrate that HSV-1 GPE amplicon vectors can rescue defective lacZ retrovirus vectors and suggest that they could be used as a sort of launching ramp to fire defective retrovirus vectors from within virtually any in vitro or in vivo cell type containing defective retroviral vectors.
PMCID: PMC191567  PMID: 9094692
8.  Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral supernatant 
Arthritis Research  2002;4(3):215-219.
We are developing retroviral-mediated gene transfer to human fibroblast-like synovial cells (FLS) as one approach to characterizing genetic pathways involved in synoviocyte pathophysiology. Prior work has suggested that FLS are relatively refractory to infection by Moloney murine leukemia virus based vectors. To determine if viral titer influenced the transduction efficiency of FLS, we optimized a rapid, efficient, and inexpensive centrifugation method to concentrate recombinant retroviral supernatant. The technique was evaluated by measurement of the expression of a viral enhanced green fluorescent protein transgene in transduced cells, and by analysis of viral RNA in retroviral supernatant. Concentration (100-fold) was achieved by centrifugation of viral supernatant for four hours, with 100% recovery of viral particles. The transduction of FLS increased from approximately 15% with unconcentrated supernatant, to nearly 50% using concentrated supernatant. This protocol will be useful for investigators with applications that require efficient, stable, high level transgene expression in primary FLS.
PMCID: PMC111025  PMID: 12010573
enhanced green fluorescent protein; fibroblast-like synovial cell; gene therapy; retrovirus; titer
9.  Adenovirus-Mediated Gene Transfer in Mesenchymal Stem Cells Can Be Significantly Enhanced by the Cationic Polymer Polybrene 
PLoS ONE  2014;9(3):e92908.
Mesenchymal stem cells (MSCs) are multipotent progenitors, which can undergo self-renewal and give rise to multi-lineages. A great deal of attentions have been paid to their potential use in regenerative medicine as potential therapeutic genes can be introduced into MSCs. Genetic manipulations in MSCs requires effective gene deliveries. Recombinant adenoviruses are widely used gene transfer vectors. We have found that although MSCs can be infected in vitro by adenoviruses, high virus titers are needed to achieve high efficiency. Here, we investigate if the commonly-used cationic polymer Polybrene can potentiate adenovirus-mediated transgene delivery into MSCs, such as C2C12 cells and iMEFs. Using the AdRFP adenovirus, we find that AdRFP transduction efficiency is significantly increased by Polybrene in a dose-dependent fashion peaking at 8 μg/ml in C2C12 and iMEFs cells. Quantitative luciferase assay reveals that Polybrene significantly enhances AdFLuc-mediated luciferase activity in C2C12 and iMEFs at as low as 4 μg/ml and 2 μg/ml, respectively. FACS analysis indicates that Polybrene (at 4 μg/ml) increases the percentage of RFP-positive cells by approximately 430 folds in AdRFP-transduced iMEFs, suggesting Polybrene may increase adenovirus infection efficiency. Furthermore, Polybrene can enhance AdBMP9-induced osteogenic differentiation of MSCs as early osteogenic marker alkaline phosphatase activity can be increased more than 73 folds by Polybrene (4 μg/ml) in AdBMP9-transduced iMEFs. No cytotoxicity was observed in C2C12 and iMEFs at Polybrene up to 40 μg/ml, which is about 10-fold higher than the effective concentration required to enhance adenovirus transduction in MSCs. Taken together, our results demonstrate that Polybrene should be routinely used as a safe, effective and inexpensive augmenting agent for adenovirus-mediated gene transfer in MSCs, as well as other types of mammalian cells.
doi:10.1371/journal.pone.0092908
PMCID: PMC3962475  PMID: 24658746
10.  Retrovirus-Associated Heparan Sulfate Mediates Immobilization and Gene Transfer on Recombinant Fibronectin 
Journal of Virology  2002;76(17):8722-8728.
Recombinant retroviruses have been shown to bind to fibronectin (FN) and increase the efficiency of gene transfer to a variety of cell types. Despite recent work to optimize gene transfer on recombinant FN, the mechanism of retrovirus binding to FN and the interactions of target cells with the bound virus remain elusive. We investigated the roles of virus surface glycoprotein (gp70), cell-conditioned medium, and proteoglycans in mediating retrovirus binding to FN. We also examined the role of Polybrene (PB) in these interactions. We found that gp70 is not involved in retrovirus binding to FN. Immobilization of the virus, however, does not overcome its receptor requirement, and gp70 is still needed for successful gene transfer. Our results clearly show that retrovirus binds FN through virus-associated heparan sulfate (HS) and that binding is necessary for transduction without PB. Two distinct modes of gene transfer occur depending on PB: (i) in the presence of PB, retrovirus interacts directly with the target cells; and (ii) in the absence of PB, retrovirus binds to FN and target cells interact with the immobilized virus. PB may promote the former mode by interacting with the virus HS and reducing the negative charge of the viral particles. Interestingly, the latter mode is more efficient, leading to significantly enhanced gene transfer. A better understanding of these interactions may provide insight into virus-cell interactions and lead to a more rational design of transduction protocols.
doi:10.1128/JVI.76.17.8722-8728.2002
PMCID: PMC136998  PMID: 12163592
11.  Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells. 
Journal of Virology  1997;71(7):5663-5667.
The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.
PMCID: PMC191813  PMID: 9188645
12.  DNA-Dependent Protein Kinase Is Not Required for Efficient Lentivirus Integration 
Journal of Virology  2000;74(23):11278-11285.
How DNA is repaired after retrovirus integration is not well understood. DNA-dependent protein kinase (DNA-PK) is known to play a central role in the repair of double-stranded DNA breaks. Recently, a role for DNA-PK in retroviral DNA integration has been proposed (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644–647, 1999). Reduced transduction efficiency and increased cell death by apoptosis were observed upon retrovirus infection of cultured scid cells. We have used a human immunodeficiency virus (HIV) type 1 (HIV-1)-derived lentivirus vector system to further investigate the role of DNA-PK during integration. We measured lentivirus transduction of scid mouse embryonic fibroblasts (MEF) and xrs-5 or xrs-6 cells. These cells are deficient in the catalytic subunit of DNA-PK and in Ku, the DNA-binding subunit of DNA-PK, respectively. At low vector titers, efficient and stable lentivirus transduction was obtained, excluding an essential role for DNA-PK in lentivirus integration. Likewise, the efficiency of transduction of HIV-derived vectors in scid mouse brain was as efficient as that in control mice, without evidence of apoptosis. We observed increased cell death in scid MEF and xrs-5 or xrs-6 cells, but only after transduction with high vector titers (multiplicity of infection [MOI], >1 transducing unit [TU]/cell) and subsequent passage of the transduced cells. At an MOI of <1 TU/cell, however, transduction efficiency was even higher in DNA-PK-deficient cells than in control cells. Taken together, the data suggest a protective role of DNA-PK against cellular toxicity induced by high levels of retrovirus integrase or integration. Another candidate cellular enzyme that has been claimed to play an important role during retrovirus integration is poly(ADP-ribose) polymerase (PARP). However, no inhibition of lentivirus vector-mediated transduction or HIV-1 replication by 3-methoxybenzamide, a known PARP inhibitor, was observed. In conclusion, DNA-PK and PARP are not essential for lentivirus integration.
PMCID: PMC113232  PMID: 11070027
13.  Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation 
Background
Viral vectors provide a method of stably introducing exogenous DNA into cells that are not easily transfectable allowing for the ectopic expression or silencing of genes for therapeutic or experimental purposes. However, some cell types, in particular bone marrow cells, dendritic cells and neurons are difficult to transduce with viral vectors. Successful transduction of such cells requires preparation of highly concentrated viral stocks, which permit a high virus concentration and multiplicity of infection (MOI) during transduction. Pseudotyping with the vesicular stomatitis virus G (VSV-G) envelope protein is common practice for both lentiviral and retroviral vectors. The VSV-G glycoprotein adds physical stability to retroviral particles, allowing concentration of virus by high-speed ultracentrifugation. Here we describe a method report for concentration of virus from large volumes of culture supernatant by means of successive rounds of ultracentrifugation into the same ultracentrifuge tube.
Method
Stable retrovirus producer cell lines were generated and large volumes of virus-containing supernatant were produced. We then tested the transduction ability of virus following varying rounds of concentration by ultra-centrifugation. In a second series of experiments lentivirus-containing supernatant was produced by transient transfection of 297T/17 cells and again we tested the transduction ability of virus following multiple rounds of ultra-centrifugation.
Results
We report being able to centrifuge VSV-G coated retrovirus for as many as four rounds of ultracentrifugation while observing an additive increase in viral titer. Even after four rounds of ultracentrifugation we did not reach a plateau in viral titer relative to viral supernatant concentrated to indicate that we had reached the maximum tolerated centrifugation time, implying that it may be possible to centrifuge VSV-G coated retrovirus even further should it be necessary to achieve yet higher titers for specific applications. We further report that VSV-G coated lentiviral particles may also be concentrated by successive rounds of ultracentrifugation (in this case four rounds) with minimal loss of transduction efficiency.
Conclusion
This method of concentrating virus has allowed us to generate virus of sufficient titers to transduce bone marrow cells with both retrovirus and lentivirus, including virus carrying shRNA constructs.
doi:10.1186/1479-5876-9-137
PMCID: PMC3175463  PMID: 21849073
14.  Cationic Liposomes Enhance the Rate of Transduction by a Recombinant Retroviral Vector In Vitro and In Vivo 
Journal of Virology  1998;72(6):4832-4840.
Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application.
PMCID: PMC110029  PMID: 9573249
15.  High-Efficiency Gene Transfer into Normal and Adenosine Deaminase-Deficient T Lymphocytes Is Mediated by Transduction on Recombinant Fibronectin Fragments 
Journal of Virology  1998;72(6):4882-4892.
Primary human T lymphocytes are powerful targets for genetic modification, although the use of these targets in human gene therapy protocols has been hampered by low levels of transduction. We have shown previously that significant increases in the transduction of hematopoietic stem and progenitor cells with retroviral vectors can be obtained by the colocalization of the retrovirus and target cells on specific fibronectin (FN) adhesion domains (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). We studied the transfer of genes into primary T lymphocytes by using FN-assisted retroviral gene transfer. Activated T lymphocytes were infected for three consecutive days on the recombinant FN fragment CH-296 with a retroviral vector encoding the murine B7-1 protein. Transduced lymphocytes were analyzed for murine B7-1 expression, and it was found that under optimal conditions, 80 to 89% of the CD3+ lymphocytes were transduced. Gene transfer was predominantly augmented by the interaction between VLA-4 on the T lymphocytes and the FN adhesion site CS-1. Adenosine deaminase (ADA)-deficient primary T lymphocytes transduced on CH-296 with a retrovirus encoding murine ADA (mADA) exhibited levels of mADA activity severalfold higher than the levels of the endogenous human ADA protein observed in normal human T lymphocytes. Strikingly, the long-term expression of the transgene was dependent on the activation status of the lymphocytes. This approach will have important applications in human gene therapy protocols targeting primary T lymphocytes.
PMCID: PMC110042  PMID: 9573255
16.  Simultaneous Infection with Retroviruses Pseudotyped with Different Envelope Proteins Bypasses Viral Receptor Interference Associated with Colocalization of gp70 and Target Cells on Fibronectin CH-296 
Journal of Virology  1999;73(5):3960-3967.
Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34+ hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34+ hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols.
PMCID: PMC104174  PMID: 10196291
17.  Engraftment of NOD/SCID Mice with Human CD34+ Cells Transduced by Concentrated Oncoretroviral Vector Particles Pseudotyped with the Feline Endogenous Retrovirus (RD114) Envelope Protein 
Journal of Virology  2001;75(20):9995-9999.
Oncoretrovirus vectors pseudotyped with the feline endogenous retrovirus (RD114) envelope protein produced by the FLYRD18 packaging cell line have previously been shown to transduce human hematopoietic progenitor cells with a greater efficiency than similar amphotropic envelope-pseudotyped vectors. In this report, we describe the production and efficient concentration of RD114-pseudotyped murine leukemia virus (MLV)-based vectors. Following a single round of centrifugation, vector supernatants were concentrated approximately 200-fold with a 50 to 70% yield. Concentrated vector stocks transduced prestimulated human CD34+ (hCD34+) cells with approximately 69% efficiency (n = 7, standard deviation = 4.4%) using a single addition of vector at a low multiplicity of infection (MOI = 5). Introduction of transduced hCD34+ cells into irradiated NOD/SCID recipients resulted in multilineage engraftment with long-term transgene expression. These data demonstrate that RD114-pseudotyped MLV-based vectors can be efficiently concentrated to high titers and that hCD34+ cells transduced with concentrated vector stocks retain in vivo repopulating potential. These results highlight the potential of RD114-pseudotyped oncoretrovirus vectors for future clinical implementation in hematopoietic stem cell gene transfer.
doi:10.1128/JVI.75.20.9995-9999.2001
PMCID: PMC114573  PMID: 11559834
18.  Cationic liposomes (Lipofectin) mediate retroviral infection in the absence of specific receptors. 
Journal of Virology  1990;64(2):957-961.
We have used cationic liposomes (Lipofectin) to facilitate retrovirus infection of cells lacking the homologous viral receptor. Ecotropic murine leukemia virus and packaged retroviral vectors were shown to infect mink cells, and amphotropic packaged retroviral vectors were shown to infect hamster cells in the presence of Lipofectin but not in the presence of Polybrene. Lipofectin-mediated infection of cells lacking the homologous receptor results in a titer approximately 0.1% of the titer in cells with the homologous receptor, using the standard Polybrene protocol. The use of Lipofectin may provide a simple means to experimentally infect a wide variety of cells with viruses not normally infectious for the species, tissue, or cell type of interest.
PMCID: PMC249198  PMID: 2153257
19.  Efficient Transduction by an Amphotropic Retrovirus Vector Is Dependent on High-Level Expression of the Cell Surface Virus Receptor 
Journal of Virology  1999;73(1):495-500.
Transduction by murine leukemia virus-based retrovirus vectors is limited in certain cell types, particularly in nondividing cells. But transduction can be inefficient even in cells that divide rapidly. For example, exposure of 208F rat embryo fibroblasts to an excess of an amphotropic retrovirus vector encoding alkaline phosphatase results in a transduction efficiency of only about 10%, even though these cells divide rapidly. Here we show that transduction of 208F cells is limited by cell surface retrovirus receptor levels; overexpression of the amphotropic retrovirus receptor Pit2 markedly improved the transduction efficiency to 50%. To characterize receptor levels and binding affinity, we synthesized a fusion protein that joins the amino terminus of the amphotropic envelope protein to the Fc region of a human immunoglobulin G1 molecule for use in binding assays. In comparison to the parental cell line, the modified cell line showed an order of magnitude increase in binding sites of from 18,000 to 150,000 per cell. Thus, efficient transduction by an amphotropic retrovirus vector requires high-level expression of the retrovirus receptor Pit2. These results provide the rationale for further examination of the role of receptor levels in inefficient transduction, especially with regard to target cells for gene therapy, where a high transduction rate is often crucial.
PMCID: PMC103856  PMID: 9847355
20.  Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors 
BMC Biotechnology  2011;11:13.
Background
Monocyte-derived macrophages contribute to atherosclerotic plaque formation. Therefore, manipulating macrophage function could have significant therapeutic value. The objective of this study was to determine transduction efficiency of two HIV-based lentiviral vector configurations as delivery systems for the transduction of primary human blood monocyte-derived macrophages.
Results
Human blood monocytes were transduced using two VSV-G pseudotyped HIV-1 based lentiviral vectors containing EGFP expression driven by either native HIV-LTR (VRX494) or EF1α promoters (VRX1090). Lentiviral vectors were added to cultured macrophages at different times and multiplicities of infection (MOI). Transduction efficiency was assessed using fluorescence microscopy and flow cytometry. Macrophages transduced between 2 and 120 hours after culturing showed the highest transduction efficiency at 2-hours transduction time. Subsequently, cells were transduced 2 hours after culturing at various vector concentrations (MOIs of 5, 10, 25 and 50) to determine the amount of lentiviral vector particles required to maximally transduce human monocyte-derived macrophages. On day 7, all transduced cultures showed EGFP-positive cells by microscopy. Flow cytometric analysis showed with all MOIs a peak shift corresponding to the presence of EGFP-positive cells. For VRX494, transduction efficiency was maximal at an MOI of 25 to 50 and ranged between 58 and 67%. For VRX1090, transduction efficiency was maximal at an MOI of 10 and ranged between 80 and 90%. Thus, transductions performed with VRX1090 showed a higher number of EGFP-positive cells than VRX494.
Conclusions
This report shows that VSV-G pseudotyped HIV-based lentiviral vectors can efficiently transduce human blood monocyte-derived macrophages early during differentiation using low particle numbers that do not interfere with differentiation of monocytes into macrophages.
doi:10.1186/1472-6750-11-13
PMCID: PMC3045310  PMID: 21281514
21.  Pseudotyped Adeno-Associated Viral Vectors for Gene Transfer in Dermal Fibroblasts: Implications for Wound Healing Applications 
The Journal of surgical research  2013;184(1):691-698.
Background
Cell specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy. Pseudotyping strategy with AAV vectors has the potential to confer unique cellular tropism and transduction efficiency. We hypothesize that pseudotyped AAV vectors have differential tropism and transduction efficiency under normal and wound conditions in dermal fibroblasts.
Materials and Methods
AAV2 genome with GFP reporter was packaged in capsids of other serotypes AAV5, AAV7, and AAV8, producing pseudotyped vectors AAV2/5, AAV2/7, and AAV2/8 respectively. Murine and human dermal fibroblasts were transduced by the different pseudotypes for 24 hours at multiplicities of infection (MOI)102, 103, 104, and 105. Transduction efficiency was assessed at days 3 and 7. Experiments were repeated in a simulated wound environment by adding 10ng/ml PDGF-B to culture media.
Results
Transduction efficiency of the pseudotyped AAV vectors is dose dependent. MOI 105 results in significantly higher gene transfer. Under normal culture conditions, the pseudotyping strategy confers differential transduction of dermal fibroblasts, with significantly enhanced transduction of murine cells by AAV2/5 and AAV2/8 compared to AAV2/2. AAV2/8 is more efficacious in transducing human cells. Under wound conditions, transduction efficiency of AAV2/2, 2/5 and 2/8 is significantly lower in murine fibroblasts. At day 3 under wound conditions, all vectors demonstrate similar transduction efficiency, but by day 7 the three pseudotyped vectors transduce significantly more murine cells compared to AAV2/2. However, in human cells, there is no significant difference in transduction efficiency of each pseudotype between normal and wound conditions at both 3 and 7 days.
Conclusions
The AAV pseudotyping strategy represents a gene transfer technology that can result in differential transduction of dermal fibroblasts. The differences in transduction efficiency in murine and human dermal fibroblasts both in the normal as well as wound environment highlight issues with translatability of gene transfer techniques. These data provide a template for using pseudotyped AAV vectors in cutaneous applications.
doi:10.1016/j.jss.2013.03.051
PMCID: PMC3759652  PMID: 23590866
wound healing; gene therapy; adeno associated virus; pseudotyping; regenerative medicine
22.  Identification of parameters required for efficient lentiviral vector transduction and engraftment of human cord blood CD34+ NOD/SCID Repopulating cells 
Experimental hematology  2008;36(8):947-956.
Objectives
Human cord blood (CB) is a potential source of hematopoietic stem cells (HSC) for gene therapy to treat patients with hematopoietic disorders. However, limited numbers of CB CD34+ cells, low transduction efficiency with lentiviral vectors (LVs), and low engraftment efficiency of NOD/SCID repopulating cells (SRC), a measure of HSC, are blocks to this procedure. To optimize culture and transduction conditions, we compared various lengths of time for pre-stimulation before transduction, transduction duration, and post-transduction cell culture.
Materials and methods
We used a lentiviral vector to transduce human cord blood CD34+ cells followed by engraftment into NOD/SCID mice. We evaluated the effects of pre-stimulation and transduction time, and optimized the ex vivo cell culture duration before transplantation.
Results
We were able to achieve up to 40% transduction efficiency and up to 50% engraftment efficiency of SRC in CB CD34+ cells when CD34+ CB cells were either not pre-stimulated or pre-stimulated in 1% FBS medium for 1 hr, followed by 5 hr transduction and 3 days culture in a cocktail of growth factors after transduction. No apparent functional changes of CB CD34+ cells were noted under these conditions.
Conclusion
This gene transduction/cell expansion protocol is the first systematic study to optimize pre-stimulation time, transduction time and very importantly, ex vivo culture time after transduction, and may be of use for LV gene transduction in a gene therapy setting.
doi:10.1016/j.exphem.2008.06.005
PMCID: PMC2586287  PMID: 18640494
23.  Efficient Lentiviral Transduction of Human Mesenchymal Stem Cells That Preserves Proliferation and Differentiation Capabilities 
Stem Cells Translational Medicine  2012;1(12):886-897.
This study indicates that human mesenchymal stem cells (hMSCs) can be efficiently transduced using a protamine sulfate concentration of 100 μg/ml at low volumes over 3 days. With the capacity to easily, reliably, and inexpensively transduce hMSCs, it will be possible to efficiently use genetic engineering to add or delete a function, improve or replace an existing one, and gain insight into the biology of these cells through the use of reporter genes.
Long-term lentiviral transduction of human mesenchymal stem cells (hMSCs) greatly enhances the usefulness of these cells. However, such transduction currently requires the use of polybrene, which severely inhibits hMSC proliferation. In contrast, protamine sulfate at 100 μg/ml doubled transduction efficiencies without affecting proliferation or differentiation potential. Expression levels improved 2.2-fold with the addition of a woodchuck hepatitis post-transcriptional regulatory element. Further improvements in transduction efficiencies could be obtained by a modest increase in viral concentrations through increased viral titers or decreased transduction volumes without changing multiplicity of infection, by transducing over multiple days, or by culturing the cells in fibroblast growth factor-2. Centrifugation improved expression but had no effect on efficiency. Transgene expression was stable over 6 weeks in vitro and in vivo. Donor-to-donor and intradonor variability were observed in primary passage through passage 2 cultures, but not at passage 3. These results provide a better optimized approach for expanded use of hMSCs through genetic manipulation.
doi:10.5966/sctm.2012-0086
PMCID: PMC3659678  PMID: 23283550
Mesenchymal stem cells; Lentiviral vector; Gene therapy; Transduction; Adult human bone marrow; Adult stem cells; Cellular therapy; Osteoblast
24.  Clinical Responses to Gene Therapy in Joints of Two Subjects with Rheumatoid Arthritis 
Human Gene Therapy  2009;20(2):97-101.
Abstract
This paper provides the first evidence of a clinical response to gene therapy in human arthritis. Two subjects with rheumatoid arthritis received ex vivo, intraarticular delivery of human interleukin-1 receptor antagonist (IL-1Ra) cDNA. To achieve this, autologous synovial fibroblasts were transduced with a retrovirus, MFG-IRAP, carrying IL-1Ra as the transgene, or remained as untransduced controls. Symptomatic metacarpophalangeal (MCP) joints were injected with control or transduced cells. Joints were clinically evaluated on the basis of pain; the circumference of MCP joint 1 was also measured. After 4 weeks, joints underwent surgical synovectomy. There were no adverse events in either subject. The first subject responded dramatically to gene transfer, with a marked and rapid reduction in pain and swelling that lasted for the entire 4 weeks of the study. Remarkably, joints receiving IL-1Ra cDNA were protected from flares that occurred during the study period. Analysis of RNA recovered after synovectomy revealed enhanced expression of IL-1Ra and reduced expression of matrix metalloproteinase-3 and IL-1β. The second subject also responded with reduced pain and swelling. Thus, gene transfer to human, rheumatoid joints can be accomplished safely to produce clinical benefit, at least in the short term. Using this ex vivo procedure, the transgene persisted within the joint for at least 1 month. Further clinical studies are warranted.
doi:10.1089/hum.2008.075
PMCID: PMC2855248  PMID: 18986219
25.  Efficient and Stable Gene Expression into Human Osteoclasts Using an HIV-1–Based Lentiviral Vector 
DNA and Cell Biology  2008;27(6):315-320.
Since osteoclasts are terminally differentiated cells without proliferating activity, efficient and stable gene expression into these cells remains a difficulty. In the current study, we investigate gene transduction into human preosteoclasts by a replication defective lentivirus-based vector containing a modified HIV-1 genome. Human preosteoclasts (differentiating osteoclasts) were transduced with lentiviruses bearing an enhanced green fluorescent protein (EFGP) reporter gene. Transduction efficiencies were measured by flow cytometry for EGFP protein expression. Sorted human transduced preosteoclasts were replated and differentiated under human macrophage colony-stimulating factor and human receptor activator of NF-κB ligand. Mature osteoclasts were then analyzed by the cell viability assay, TRACP assay, and pit formation assay. Efficient gene transduction was obtained at multiplicity of infection of 10, and gene expression lasted for over 4 weeks using our protocol. Lentiviral transduction did not affect osteoclast survival, formation, or function. These results establish an efficient method for gene transduction into human preosteoclasts using a lentiviral vector. Importantly, these transduced preosteoclasts could differentiate into mature osteoclasts without a negative impact from the lentiviruses. This protocol provides a new tool for studies of osteoclast biology. Further work in this area may open new avenues for the study of osteoclast gene signaling and gene therapy of disorders of osteoclast function.
doi:10.1089/dna.2007.0656
PMCID: PMC2980781  PMID: 18363548

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