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.

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.

Viral Transduction of DRG Neurons.

The manipulation of gene expression is an essential tool to study the function of genes or signaling pathways. Uniform and robust gene manipulation is crucial for successful assays. However, neuronal cells are generally difficult-to-transfect cells with conventional DNA/RNA transfection reagents. Therefore, virus-mediated gene delivery is a primary choice for the studies of gene functions in neurons. In this chapter, we will describe the methods for lentivirus-mediated gene expression or knockdown in DRG neurons.

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.

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.

Scavenging Circulating Mitochondrial DNA as a Potential Therapeutic Option for Multiple Organ Dysfunction in Trauma Hemorrhage.

Trauma is a leading cause of death worldwide with 5.8 million deaths occurring yearly. Almost 40% of trauma deaths are due to bleeding and occur in the first few hours after injury. Of the remaining severely injured patients up to 25% develop a dysregulated immune response leading to multiple organ dysfunction syndrome (MODS). Despite improvements in trauma care, the morbidity and mortality of this condition remains very high. Massive traumatic injury can overwhelm endogenous homeostatic mechanisms even with prompt treatment. The underlying mechanisms driving MODS are also not fully elucidated. As a result, successful therapies for trauma-related MODS are lacking. Trauma causes tissue damage that releases a large number of endogenous damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs released in trauma, such as mitochondrial DNA (mtDNA), could help to explain part of the immune response in trauma given the structural similarities between mitochondria and bacteria. MtDNA, like bacterial DNA, contains an abundance of highly stimulatory unmethylated CpG DNA motifs that signal through toll-like receptor-9 to produce inflammation. MtDNA has been shown to be highly damaging when injected into healthy animals causing acute organ injury to develop. Elevated circulating levels of mtDNA have been reported in trauma patients but an association with clinically meaningful outcomes has not been established in a large cohort. We aimed to determine whether mtDNA released after clinical trauma hemorrhage is sufficient for the development of MODS. Secondly, we aimed to determine the extent of mtDNA release with varying degrees of tissue injury and hemorrhagic shock in a clinically relevant rodent model. Our final aim was to determine whether neutralizing mtDNA with the nucleic acid scavenging polymer, hexadimethrine bromide (HDMBr), at a clinically relevant time point in vivo would reduce the severity of organ injury in this model. CONCLUSIONS: We have shown that the release of mtDNA is sufficient for the development of multiple organ injury. MtDNA concentrations likely peak at different points in the early postinjury phase dependent on the degree of isolated trauma vs combined trauma and hemorrhagic shock. HDMBr scavenging of circulating mtDNA (and nuclear DNA, nDNA) is associated with rescue from severe multiple organ injury in the animal model. This suggests that HDMBr could have utility in rescue from human trauma-induced MODS.

The effects of polycations on vascular permeability in the rat. A proposed role for charge sites.

This study investigated whether charge sites in the walls of the microvasculature may play a role in maintaining the impermeability of the nonrenal capillaries to albumin. All experiments were performed in nephrectomized rats, studied in the awake state. The intravenous injection of protamine sulfate (4 mg/100 g body wt dissolved in 0.9% saline) was followed by a mean increase of 29.1% in hematocrit and a decrease of 28.4% in plasma albumin concentration over a 10-min period, indicating a significant 50-60% loss of albumin from the vascular space; a finding confirmed by studies using exogenous 125I-labeled albumin. Changes persisted for the remaining 80 min of observation, and could be reproduced by the injection of two other polycations, hexadimethrine and poly-l-lysine. These effects were not prevented by the antihistamine diphenhydramine hydrochloride. In contrast to 125I-labeled albumin, 14C-labeled neutral dextran of comparable size was not confined to the vascular space; its apparent volume of distribution progressively increased during the 90 min of observation. Intravenous injection of protamine sulfate was followed by a significantly smaller loss of 14C-dextran (36.5%) than albumin (59.1%) from the vascular space (P less than 0.01). Protamine sulfate could not be demonstrated to result in any changes in the physicochemical characteristics of albumin. These observations suggest that the negative charge sites present in nonglomerular capillary walls have functions similar to equivalent sites present in the glomerular capillaries. Thus, charge sites could contribute to the low permeability of the microvasculature to negatively charged macromolecules such as albumin. This may be an important mechanism for retaining albumin in the vascular space and preventing edema formation in health.

New procedure for DNA transfection with polycation and dimethyl sulfoxide.

A new procedure for DNA transfection has been developed in a system of chicken embryo fibroblast cells and cloned Rous sarcoma virus DNA by using a polycation reagent as a mediator to adsorb DNA to the cell surface and dimethyl sulfoxide as an agent to facilitate the uptake of adsorbed DNA by the cells. In this new, simple, and convenient polycation-dimethyl sulfoxide transfection, which requires no carrier DNA even with small amounts of DNA, the number of transformed cell foci induced by Rous sarcoma virus DNA was proportional to the dose of the transfecting DNA, and chicken embryo fibroblast cells were successfully transformed by v-src-containing subgenomic DNA as well.

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.

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.

Detection of bovine viral diarrhea virus by amplification on polycation-treated cells followed by enzyme immunoassay.

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.

In Vitro-Pooled shRNA Screening to Identify Determinants of Radiosensitivity.

Short hairpin RNA (shRNA)-pooled screening is a valuable and cost-effective tool for assaying the contribution of individual genes to cell viability and proliferation on a genomic scale. Here we describe the key considerations for the design and execution of a pooled shRNA screen to identify determinants of radiosensitivity.

Neoplastic transformation of human keratinocytes by polybrene-induced DNA-mediated transfer of an activated oncogene.

Polybrene, in conjunction with dimethyl sulfoxide (DMSO) shock has been shown to increase the frequency of DNA-mediated gene transfer to mammalian cells as compared with the frequency obtained with calcium phosphate transfection. We have successfully adapted this procedure for use with human epidermal keratinocytes. Non-tumorigenic human epidermal epithelial cells immortalized by SV40 tumor antigen were neoplastically transfected, using Polybrene at a concentration of 10 micrograms ml-1, followed by a 4 min shock, with 30% DMSO, with a plasmid carrying the activated H-ras gene from the EJ bladder carcinoma cell line. The transfected cells showed morphological alterations and induced carcinomas when transplanted into nude mice. They contained integrated copies of the transfected H-ras gene and expressed high levels of the p21 protein. Polybrene-induced DNA transfection, therefore, offers the opportunity to transfer genes effectively into human epidermal keratinocytes and should accelerate the study of the interaction between oncogenes and human epithelial cells. This study appears to represent the first neoplastic conversion of nontumorigenic, immortalized human epidermal keratinocytes by an activated human oncogene.

Conditions of formation of the heparin-fibronectin-collagen complex and the effect of plasmin.

The formation and composition of the insoluble heparin-fibronectin-collagen complex and its degradation by proteolysis was investigated. At fixed concentrations of the other molecular components of the complex, the maximal rate of complex formation, measured turbidimetrically, was reached at a concentration of 4 microM heparin and 0.9 microM collagen, while the rate of complex formation was linearly related to concentrations of fibronectin as high as 3 microM. Heparin was incorporated into the complex in a saturable manner, and was released in active anticoagulant form by plasmin but not by urokinase. The complex formation was inhibited by 5 mM calcium or 250 mM NaCl as well as by polybrene or spermin. It is suggested that fibronectin binds both heparin and collagen cooperatively to form an insoluble ternary complex of the extracellular matrix.

Effects of acidic and basic macromolecules on the activity of protein phosphatase-1.

The dephosphorylation of phosphorylase a by the catalytic subunit of protein phosphatase-1 obtained from rabbit skeletal muscle is inhibited by heparin in a noncompetitive manner with respect to phosphorylase a (Ki = 8 micrograms/ml). The inhibitory effect of heparin is also observed in the presence of effectors (e.g., glucose and AMP) modifying the dephosphorylation of phosphorylase a. Heat-stable protein inhibitors of protein phosphatase-1 can develop their inhibitory effect of the activity of protein phosphatase-1 even in the presence of heparin. The inhibitory effect of heparin and the heat-stable inhibitor-2 of phosphatase is additive. Polybrene, a heparin antagonist, prevented phosphatase-1 from the inhibition caused by heparin or the inhibitors. Proteins with basic character, histone fractions (H1, H3) and protamine sulfate, can counteract with the inhibitory effect of heparin, but they cannot intercept the actions of inhibitor-1 or -2.

Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex.

The predominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of beta-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent Mr of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly(D-lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac.

Reversible inhibition by protamine of human synovial and rabbit platelet secretory phospholipase A2.

We investigated the effects of protamine on the release and the activity of 14 kDa type II phospholipase A2 (sPLA2). Protamine blocks both release and activity of sPLA2 from thrombin-stimulated platelets in a concentration-dependent manner. Heparin, an anionic sulfate polysaccharide which has a high affinity for this enzyme, has no inhibitory effect on sPLA2 by itself but it is able to reverse the inhibitory effect of protamine. The liberation by thrombin of platelet factor 4, an alpha-granule constituent, unlike to that of ATP stored in dense bodies, was suppressed by protamine. Platelet aggregation, determined in parallel, was not affected by protamine. Also, protamine did not inhibit platelet arachidonic acid liberation, which is mainly produced by cytosolic PLA2. The non-proteinaceous polycationic hexadimethrine and acidic protein casein failed to inhibit platelet sPLA2 activity. By contrast, the basic polypeptides poly(L-arginine) and poly(L-lysine) potently inhibited sPLA2 activity, indicating the important role of basic amino acids in the inhibitory effect evoked by protamine. Activities of the human recombinant sPLA2 and the unpurified synovial enzyme of patients with rheumatoid arthritis were also inhibited by the same range of protamine, poly(L-arginine) and poly(L-lysine) concentrations. Our results demonstrate that protamine, unlike heparin, blocks platelet sPLA2 release and exerts a reversible inhibitory effect on its activity, probably through the interaction of basic amino acids with the enzyme.

Serum protein media are important factors in the manual hexadimethrine bromide (polybrene) test, experience in China.

BACKGROUND AND OBJECTIVES: The use of the manual hexadimethrine bromide (polybrene) test in routine cross-matching after accurately detecting cell grouping and irregular antibodies is prevalent in China. This article reports the importance of serum protein mediums in the performance of the manual hexadimethrine bromide test. MATERIALS AND METHODS: Blood group O red blood cells and Blood group AB and Rh positive serum were collected at random from healthy blood donators, IgG anti-D serum separated from pregnant woman, then tested with each other by the manual hexadimethrine bromide methods in routine tests and some designed corresponding tests with IgG, IgM anti-D monoclonal diagnostic reagents and some serum protein components. RESULTS: Red blood cells that were adjusted to 3-5% suspension by normal saline then only added in 0.7 ml low ionic medium (LIM) and two drops of polybrene solution adhere to the surface of test tubes' bottom when centrifuged, so it was difficult to perform the next approach, but the adherence disappeared when red blood cells' concentrations exceeded 20-30%. Rh positive red blood cells coated by anti-D have the same phenomenon. This adherence can be prevented by serum medium diluted from 1:128 to 1:1024 times by normal saline and hemoglobin medium diluted from 1:32 to 1:128 times, but not by albumin or immunoglobulin medium. The denary logarithm values of the greatest inhibited dilutions of serum and hemoglobin elution between antibody sensitizing red blood cells and the same pre-sensitizing red blood cells tests were no significant difference (P value > 0.05). CONCLUSIONS: The whole serum or serum protein mediums are important factors that can influence successfully performance of the manual hexadimethrine bromide test. So appliance of the manual hexadimethrine bromide test to immunohematology laboratory, such as when performing titrations of serum or plasma, or when testing eluates for antibody activity, this adherence must be considered.

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.

Helper virus-free herpes simplex virus type 1 amplicon vectors for granulocyte-macrophage colony-stimulating factor-enhanced vaccination therapy for experimental glioma.

Subcutaneous vaccination therapy with glioma cells, which are retrovirally transduced to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF), has previously proven effective in C57BL/6 mice harboring intracerebral GL261 gliomas. However, clinical ex vivo gene therapy for human gliomas would be difficult, as transgene delivery via retroviral vectors occurs only in dividing cells and ex vivo glioma cells have a low growth fraction. To circumvent this problem, a helper virus-free herpes simplex virus type 1 (HSV-1) amplicon vector was used. When primary cultures of human glioblastoma cells were infected with HSV-1 amplicon vectors at an MOI of 1, more than 90% of both dividing and nondividing cells were transduced. When cells were infected with an amplicon vector, HSVGM, bearing the GM-CSF cDNA in the presence of Polybrene, GM-CSF secretion into the medium during the first 24 hr after infection was 1026 ng/10(6) cells, whereas mock-infected cells did not secrete detectable GM-CSF. Subcutaneous vaccination of C57BL/6 mice with 5 x 10(5) irradiated HSVGM-transduced GL261 cells 7 days prior to intracerebral implantation of 10(6) wild-type GL261 cells yielded 60% long-term survivors (>80 days), similar to the 50% long-term survivors obtained by vaccination with retrovirally GM-CSF-transduced GL261 cells. In contrast, animals vaccinated with the same number of nontranduced GL261 cells or with GL261 cells infected with helper virus-free packaged HSV-1 amplicon vectors carrying no transgene showed only 10% long-term survivors. In conclusion, helper virus-free HSV-1 amplicon vectors appear to be effective for cytokine-enhanced vaccination therapy of glioma, with the advantages that both dividing and nondividing tumor cells can be infected, no viral proteins are expressed, and these vectors are safe and compatible with clinical use.