Improving Lentiviral Transduction of Human Adipose-Derived Mesenchymal Stem Cells.

Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications. Polybrene is the most commonly used reagent to improve viral gene transfer efficiency in laboratory research; however, it is not approved for human use and has also been shown to impair MSC proliferation and differentiation. Therefore, there is a need for optimized transduction protocols that can also be adapted to clinical settings. LentiBOOST (LB) and protamine sulfate are alternative transduction enhancers (TEs) that can be manufactured to current Good Manufacturing Practice standards, are easily applied to existing protocols, and have been previously studied for the transduction of human CD34+ hematopoietic stem cells. In this study, we investigated these reagents for the enhancement of lentiviral transduction of adipose-derived MSCs. We found that the combination of LB and protamine sulfate could yield comparable or even superior transduction efficiency to polybrene, with no dose-dependent adverse effects on cell viability or stem cell characteristics. This combination of TEs represents a valuable clinically compatible alternative to polybrene with the potential to significantly improve the efficiency of lentiviral transduction of MSCs for gene therapy applications.

P38 MAPK inhibition prevents polybrene-induced senescence of human mesenchymal stem cells during viral transduction.

The unique capacity of mesenchymal stem cells (MSCs) to migrate to the sites of damage, following intravenous transplantation, along with their proliferation and differentiation abilities make them promising candidates for MSC-based gene therapy. This therapeutic approach requires high efficacy delivery of stable transgenes to ensure their adequate expression in MSCs. One of the methods to deliver transgenes is via the viral transduction of MSCs. However, due to low transduction efficiency of MSCs, various polications are used to promote the association of viral particles with membranes of target cells. Among these polications polybrene is the most widely used one. Unfortunately, viral infection in presence of polybrene was shown to negatively affect proliferation rate of stem cells. The molecular mechanism underlying this effect is not yet uncovered. Therefore, the present study aimed to elucidate the mechanism of this phenomenon as well as to develop an effective approach to overcome the negative impact of polybrene on the properties of human endometrium-derived mesenchymal stem cells (hMESCs) during lentiviral infection. We found that the negative effect on proliferation observed during the viral infection in presence of polybrene is mediated by the polycation itself. Furthermore, we revealed that the treatment with polybrene alone led to the p38 MAPK-dependent premature senescence of hMESCs. These findings allowed us to develop an effective strategy to attenuate the negative polybrene impact on the hMESCs properties during lentiviral infection by inhibiting the activity of p38 MAPK. Importantly, the proposed approach did not attenuate the transduction efficiency of hMESCs, yet prevented polybrene-induced senescence and thereby restored the proliferation of the infected cells. These results provide the plausible means to reduce side effects of polybrene during the viral infection of primary cells, particularly MSCs.

Acoustic waves improves retroviral transduction in human retinal stem cells.

BACKGROUNDS: The plasticity of retinal stem cells (RSCs), a type of cells that can differentiate into neuron cells and photoreceptor cells, endows them with potential therapeutic properties that can be applied to regenerative medicine. Gene modification of these stem cells before trans-differentiation and transplantation enhances their survival and increases their therapeutic function. The different ways to effectively deliver gene into RSCs are still discussed. This study aimed to use the acoustic waves to improve the efficacy of gene delivery for RSCs. METHODS: RSCs were obtained from non-fetal human ocular pigmented ciliary margin tissues. The enhanced green fluorescent protein-encoded murine stem cell retroviruses (MSCV) were prepared and used to infect RSCs. Glass chambers containing RSCs, retroviruses, and various concentrations of polybrene (0, 0.8, 2, 4 and 8 µg/mL) were exposed under 20 or 25 Vp-p ultrasonic standing wave fields (USWF) for 5 min. The percentage of green fluorescent protein positive cells in each sample was calculated and compared to test the efficacy of gene transduction. RESULTS: Our results showed that the efficiency of gene transduction by MSCV infection was enhanced following the concentration of polybrene and the energy of USWF. The percentage of green fluorescent protein positive cells was significantly higher in chambers that contained 8 µg/mL of polybrene and was exposed to 20Vp-p of USWF for 5 min. In addition, the percentage increased in chambers contained 2, 4 and 8 µg/mL of polybrene when they were exposed to 25Vp-p of USWF. Comparing to those did not treated with ultrasound, the efficiency of retroviral transduction to RSCs increased 4-fold after exposed to USWF for 5 min. CONCLUSION: We demonstrated the ability of ultrasound standing waves to improve retroviral transduction into RSCs. We believe that this may be applied to the experimental designs of future studies and may have possible therapeutic uses.

A Systematic Evaluation of Factors Affecting Extracellular Vesicle Uptake by Breast Cancer Cells.

Extracellular vesicles (EVs) are nanometer-scale particles that are secreted by cells and mediate intercellular communication by transferring biomolecules between cells. Harnessing this mechanism for therapeutic biomolecule delivery represents a promising frontier for regenerative medicine and other clinical applications. One challenge to realizing this goal is that to date, our understanding of which factors affect EV uptake by recipient cells remains incomplete. In this study, we systematically investigated such delivery questions in the context of breast cancer cells, which are one of the most well-studied cell types with respect to EV delivery and therefore comprise a facile model system for this investigation. By displaying various targeting peptides on the EV surface, we observed that although displaying GE11 on EVs modestly increased uptake by MCF-7 cells, neuropeptide Y (NPY) display had no effect on uptake by the same cells. In contrast, neurotensin (NTS) and urokinase plasminogen activator (uPA) display reduced EV uptake by MDA-MB-231 cells. Interestingly, EV uptake rate did not depend on the source of the EVs; breast cancer cells demonstrated no increase in uptake on administration of breast cancer-derived EVs in comparison to HEK293FT-derived EVs. Moreover, EV uptake was greatly enhanced by delivery in the presence of polybrene and spinoculation, suggesting that maximal EV uptake rates are much greater than those observed under basal conditions in cell culture. By investigating how the cell's environment might provide cues that impact EV uptake, we also observed that culturing cells on soft matrices significantly enhanced EV uptake, compared to culturing on stiff tissue culture polystyrene. Each of these observations provides insights into the factors impacting EV uptake by breast cancer cells, while also providing a basis of comparison for systematically evaluating and perhaps enhancing EV uptake by various cell types.

Establishment of Green Fluorescent Protein and Firefly Luciferase Expressing Mouse Primary Macrophages for In Vivo Bioluminescence Imaging.

Macrophages play a key role in tissue homeostasis as well as in a range of pathological conditions including atherosclerosis, cancer, and autoimmunity. Many aspects of their in vivo behavior are, however, poorly understood. Bioluminescence imaging (BLI) with green fluorescent protein (GFP) and firefly luciferase (FLUC) labelled autologous reporter macrophages could potentially offer a powerful tool to study macrophage biology, but this approach has been hindered by the relative difficulty of efficient gene transfer into primary macrophages. Here we describe a straightforward method for producing large numbers of GFP/FLUC expressing mouse primary macrophages utilizing lentivirus vector, cyclosporine, and a double infection strategy. Using this method we achieved up to 60% of macrophages to express GFP with correspondingly high FLUC signal. When injected into the circulation using a mouse model of local biomaterial induced inflammation and osteolysis, macrophages were initially detectable within the lungs, followed by systemic homing to the local area of chronic inflammation in the distal femur. In addition, transduced macrophages maintained their ability to assume M1 and M2 phenotypes although the GFP/FLUC expression was altered by the polarizing signals. These reporter macrophages could prove to be valuable tools to study the role of macrophages in health and disease.

In-depth characterisation of Retinal Pigment Epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSC).

Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here, we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4, SOX2, KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology, chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65, RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane, and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together, our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases.

Adenovirus-mediated gene transfer in mesenchymal stem cells can be significantly enhanced by the cationic polymer polybrene.

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.

Polybrene inhibits human mesenchymal stem cell proliferation during lentiviral transduction.

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.

Intracellular delivery of serum-derived hepatitis C virus.

A robust and reliable cell culture system for serum-derived HCV (HCVser) has not been established yet because of the presence of neutralizing antibody and tropism for infection. To overcome this obstacle, we employed a lipid-mediated protein intracellular delivery reagent (PIDR) that permits internalization of proteins into cells. Although entry of HCVcc was not enhanced by the treatment with PIDR, entry of HCVser into hepatoma cell lines (Huh7 and HepG2) and immortalized primary hepatocytes (Hc and HuS/E2) was significantly enhanced by the PIDR treatment. The entry of HCVser into Huh7 cells in the presence of PIDR was resistant to the neutralization by an anti-hCD81 antibody, suggesting that PIDR is capable of internalizing HCVser in a receptor-independent manner. Interestingly, the PIDR-mediated entry of HCVser and HCVcc was enhanced by the addition of sera from chronic hepatitis C patients but not from healthy donors. In addition, neutralization of HCVcc infection by anti-E2 antibody was canceled by the treatment with PIDR. In conclusion, the PIDR is a valuable tool to get over the obstacle of neutralizing antibodies to internalize HCV into cells and might be useful for the establishment of in vitro propagation HCVser.

Directional spread of surface-associated retroviruses regulated by differential virus-cell interactions.

The spread of viral infections involves the directional progression of virus particles from infected cells to uninfected target cells. Prior to entry, the binding of virus particles to specific cell surface receptors can trigger virus surfing, an actin-dependent lateral transport of viruses toward the cell body (M. J. Lehmann et al., J. Cell Biol. 170:317-325, 2005; M. Schelhaas, et al., PLoS Pathog. 4:e1000148, 2008; J. L. Smith, D. S. Lidke, and M. A. Ozbun, Virology 381:16-21, 2008). Here, we have used live-cell imaging to demonstrate that for cells chronically infected with the gammaretrovirus murine leukemia virus in which receptor has been downregulated, a significant portion of completely assembled virus particles are not immediately released into the supernatant but retain long-term association with the cell surface. Retention can be attributed, at least in part, to nonspecific particle attachment to cell surface glycosylaminoglycans. In contrast to virus surfing, viruses retained at the surface of infected cells undergo a lateral motility that is random and actin independent. This diffusive motility can be abruptly halted and converted into inward surfing after treatment with Polybrene, a soluble cation that increases virus-cell adsorption. In the absence of Polybrene, particle diffusion allows for an outward flow of viruses to the infected cell periphery. Peripheral particles are readily captured by and transmitted to neighboring uninfected target cells in a directional fashion. These data demonstrate a surface-based mechanism for the directional spread of viruses regulated by differential virus-cell interactions.

Radiation increases invasion of gene-modified mesenchymal stem cells into tumors.

PURPOSE: Mesenchymal stem cells (MSCs) are multipotent cells in the bone marrow that have been found to migrate to tumors, suggesting a potential use for cancer gene therapy. MSCs migrate to sites of tissue damage, including normal tissues damaged by radiation. In this study, we investigated the effect of tumor radiotherapy on the localization of lentivirus-transduced MSCs to tumors. METHODS AND MATERIALS: MSCs were labeled with a lipophilic dye to investigate their migration to colon cancer xenografts. Subsequently, the MSCs were transduced with a lentiviral vector to model gene therapy and mark the infused MSCs. LoVo tumor xenografts were treated with increasing radiation doses to assess the effect on MSC localization, which was measured by quantitative polymerase chain reaction. MSC invasion efficiency was determined in an invasion assay. RESULTS: MSCs migrated to tumor xenografts of various origins, with few cells found in normal tissues. A lentiviral vector efficiently transduced MSCs in the presence, but not the absence, of hexadimethrine bromide (Polybrene). When LoVo tumors were treated with increasing radiation doses, more MSCs were found to migrate to them than to untreated tumors. Irradiation increased MSC localization in HT-29 and MDA-MB-231, but not UMSCC1, xenografts. Monocyte chemotactic protein-1 expression in tumors did not correlate with the basal levels of MSC infiltration; however, monocyte chemotactic protein-1 was modestly elevated in irradiated tumors. Media from irradiated LoVo cells stimulated MSC invasion into basement membranes. CONCLUSION: These findings suggest that radiation-induced injury can be used to target MSCs to tumors, which might increase the effectiveness of MSC cancer gene therapy. The production of tumor-derived factors in response to radiation stimulates MSC invasion.

Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells.

BACKGROUND: Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs. METHODS: Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes. RESULTS: Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages. CONCLUSIONS: Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs.

Detection of bovine viral diarrhea virus by amplification on polycation-treated cells followed by enzyme immunoassay / Detección del virus de la diarrea viral bovina por amplificación sobre células tratadas con policationes seguida de enzimoinmunoensayo

A bovine viral diarrhea virus (BVDV) amplification method combined with an enzyme immunoassay was developed to detect BVDV antigens in seropositive cattle. Reconstitution assays conducted by adding decreasing amounts of BVDV (Singer strain) to Madin-Darby bovine kidney (MDBK) cells showed that the sensitivity threshold of the combined assay was 10-7 TCID50. BVDV amplification was carried out in polycation (DEAE-Dextran and polybrene)- treated MDBK cells. Treated cells were able to replicate both ether-treated virus and neutralizing antibody-coated virus. Ammonium chloride decreased virus replication in polycation-treated cells, suggesting viral penetration by endocytosis. BVDV detection was tested in leukocytes from 104 seropositive cattle from 2 unvaccinated commercial closed dairy herds with high seroprevalence. Lysates and co-cultures of peripheral blood leukocytes (PBL) were tested, directly or after up to 6 blind passages in normal or polycation-treated cells. BVDV was detected in 10/104 cattle after only one co-culture of PBL in treated cells. No virus was detected in whole blood or plasma samples. BVDV positive and negative cattle were retested three times, achieving consistent results. The finding of immune carriers supports the possibility that these animals may constitute an epidemiological risk.

Se desarrolló un método de detección de antígenos del virus de la diarrea viral bovina (BVDV) combinando amplificación viral con enzimoinmunoensayo. El método combinado presentó una sensibilidad de 10-7 TCID50 en ensayos con diluciones decrecientes de BVDV cepa Singer sobre la línea celular MDBK. La amplificación del título viral se efectuó sobre células MDBK tratadas con policationes Estas células replicaron tanto el BVDV tratado con éter como el unido a anticuerpos. La replicación viral en las células tratadas disminuyó ante la presencia de cloruro de amonio, lo que sugiere la penetración viral por endocitosis. El BVDV se determinó en leucocitos de 104 bovinos seropositivos de dos rodeos en producción, cerrados y con alta seroprevalencia. Los leucocitos de sangre periférica (LSP) fueron lisados y analizados directamente o luego de hasta 6 pasajes ciegos sobre células normales o tratadas con policationes. El BVDV se detectó en 10 de los 104 animales después de solamente un cultivo de LSP en células tratadas. No se pudo detectar presencia viral en las muestras de sangre o plasma. Los estudios se repitieron tres veces en animales BVDV positivos y negativos, con resultados consistentes. El hallazgo de bovinos seropositivos portadores del virus indica la posibilidad de que estos animales puedan significar un riesgo epidemiológico.

Rapid concentration and purification of retrovirus by flocculation with Polybrene.

We have previously shown that the combined addition of Polybrene (PB) and chondroitin sulfate C (CSC) to retrovirus stocks leads to the formation of retrovirus-polymer complexes (i.e., flocs) that rapidly sediment onto cells, increases the efficiency of gene transfer, and can be used to rapidly concentrate and purify retrovirus stocks. The viruses remain associated with the polyelectrolyte complexes, however, which may complicate their use in downstream applications. In this study we determined if retrovirus could be flocculated using only one polymer (PB). We found that when retrovirus stocks were incubated with 320 microg/ml of PB, more than 70% of the viruses, and fewer than 0.3% of all other proteins, were pelleted by low-speed centrifugation. In contrast to retrovirus complexes formed with two polymers, retrovirus flocculated with PB disaggregated when they were resuspended in fresh medium. We conclude that flocculation of retroviruses with a single cationic polymer (PB) is a useful method for rapidly concentrating and purifying retroviruses, and may prove particularly useful when it is desirable to generate purified virus that is not part of a polymer complex.

Interaction between desialylated hepatitis B virus and asialoglycoprotein receptor on hepatocytes may be indispensable for viral binding and entry.

The cellular receptor for hepatitis B virus (HBV) infection has not yet been identified. The purpose of this study was to address the possibility of participation by desialylated HBV and the asialoglycoprotein receptor (ASGP-R) exclusively expressed on liver parenchymal cells, in infection. Assays for viral binding and entry were performed by culturing a hepatoblastoma cell line, HepG2, and HBV particles derived from the HBV carrier in the presence or absence of neuraminidase (NA). Viral binding and entry were clearly enhanced in the presence of NA, and the enhancement of the binding could be blocked by asialo-fetuin and ethylenediamine-tetraacetic acid (EDTA). In addition, covalently closed circular (CCC)-DNA, as a marker of infectivity, was detected in the presence of NA, but not in its absence. The optimal concentration of NA raised infectivity more than 1000 times. We concluded that this method makes it feasible to evaluate the infectivity of HBV in vitro and that ASGP-R may be a specific HBV receptor once viral particles are desialylated.

Charged polymers modulate retrovirus transduction via membrane charge neutralization and virus aggregation.

The specific mechanisms of charged polymer modulation of retrovirus transduction were analyzed by characterizing their effects on virus transport and adsorption. From a standard colloidal perspective two mechanisms, charge shielding and virus aggregation, can potentially account for the experimentally observed changes in adsorption behavior and biophysical parameters due to charged polymers. Experimental testing revealed that both mechanisms could be at work depending on the characteristics of the cationic polymer. All cationic polymers enhanced adsorption and transduction via charge shielding; however, only polymers greater than 15 kDa in size were capable of enhancing these processes via the virus aggregation mechanism, explaining the higher efficiency enhancement of the high molecular weight molecules. The role of anionic polymers was also characterized and they were found to inhibit transduction via sequestration of cationic polymers, thereby preventing charge shielding and virus aggregation. Taken together, these findings suggest the basis for a revised physical model of virus transport that incorporates electrostatic interactions through both virus-cell repulsive and attractive interactions, as well as the aggregation state of the virus.

Small increases in pH enhance retroviral vector transduction efficiency of NIH-3T3 cells.

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.

Polybrene and interleukin-4: two opposing factors for retroviral transduction of bone-marrow-derived dendritic cells.

BACKGROUND: Gene transfer using retroviral transduction offers the advantage of long-term transgene expression in developing strategies that use dendritic cells (DCs) for immunotherapy. The goal of this study was to infect DCs in an immature state in order to take advantage of their proliferating and tolerogenic potential. METHODS: Immature DCs were generated from murine bone marrow (BM) using either GM-CSF alone or GM-CSF plus IL-4. The cells were transduced directly with retroviral supernatants or by co-culture with the GP + E-86 retroviral packaging cell line in the presence of two different cationic polymers: polybrene and protamine sulfate. Phenotypic and functional characterization of the transduced cells were then performed. RESULTS: Our results show a low efficiency of retroviral infection of DCs in the presence of polybrene. This cationic polymer was found to be directly cytotoxic to murine DCs and thus favored the growth of contaminating macrophages. This effect was not observed using protamine sulfate. Furthermore, stimulation by IL-4 early in the culture increased DC differentiation, proliferation and transduction. However, we found that DCs generated in GM-CSF plus IL-4 presented a more mature phenotype with an enhanced allogeneic stimulating activity. Finally, we showed that DCs themselves down-regulated transgene expression in the co-cultured packaging cell line in a promoter-dependent manner. CONCLUSIONS: We have defined optimal conditions to generate and transduce murine BM-derived DCs. This included: the use of protamine sulfate during exposure to retroviral infectious supernatant and the addition of IL-4 at an early stage of the culture. Nevertheless, this cytokine also induced DC maturation. These findings have potential implications in experimental gene therapy.

Retroviral gene transfer to human epidermal keratinocytes correlates with integrin expression and is significantly enhanced on fibronectin.

Human epidermal keratinocytes are an important target for gene therapy because they can be easily expanded in culture and used to generate skin substitutes for the treatment of wounds, genetic diseases of the skin, and for delivery of proteins to the systemic circulation. Although retroviral transduction results in permanent genetic modification, differentiation and loss of transduced cells from the epidermis results in temporary transgene expression. To ensure permanent genetic modification, epidermal stem cells must be transduced with high efficiency. We evaluated gene transfer on two different substrates and found that the efficiency of gene transfer is substantially higher on a substrate of recombinant fibronectin (FN), when compared to tissue culture plastic (TCP). The rate of retroviral transduction on FN is four times faster than transduction on tissue culture plates and is independent of polybrene (PB). The transduction efficiency correlates with the levels of expression of integrin subunits alpha5, alpha2, and beta1, which have been shown to correlate with stem cell phenotype. Notably, cells that adhere rapidly to FN are transduced more efficiently than slowly adherent cells. In addition, integrin-blocking antibodies decrease the efficiency of gene transfer in a dose-dependent manner. Our results suggest that FN may enhance retroviral gene transfer to the least differentiated cells, thereby increasing the potential of genetically modified keratinocytes to treat short- and long-term disease states.

Efficient in vitro transduction of epithelial cells and keratinocytes with improved adenoviral gene transfer for the application in skin tissue engineering.

The adenovirus-mediated transfer of therapeutic genes into keratinocytes may be a useful approach to treat several skin diseases or to improve the graft take of in vitro generated skin equivalents used for wound coverage. However, in contrast to many other tissues, keratinocytes are relatively difficult to transduce by adenoviral vectors. To achieve high efficiency of adenoviral transduction into epithelial cells we investigated the effects of the polycation polybrene on the infection process. The human (HaCaT, A549) and rat (NBT II, MHICI) epithelial cell lines, as well as human and rat primary keratinocytes, were transduced with recombinant Ad(beta)-gal adenovirus, encoding for the reporter gene E. coli beta-galactosidase, in the presence of various polybrene concentrations. We determined the amount of beta-gal positive cells by X-gal staining and the beta-gal expression by ONPG-assay after 24 h. In all tested human and rat epithelial cell lines, as well as in human and rat primary keratinocytes, the addition of polybrene during adenoviral transduction of Ad(beta)-gal resulted in a marked increase of beta-gal positive cells and beta-gal protein expression. The efficacy of polybrene showed a clear dose dependency. The improvement of adenoviral gene transfer into various types of human and rat epithelial cells by polybrene allows us to reduce the amount of recombinant virus particles resulting in a decreased inflammation induced by this therapeutic agent. In addition, the efficient transduction and expression with enhanced adenoviral transfer of therapeutic genes into primary keratinocytes provides a powerful tool for analysing the functions and the regulation of a gene of interest in vitro.