Extensive Replication of a Retroviral Replicating Vector Can Expand the A Bulge in the Encephalomyocarditis Virus Internal Ribosome Entry Site and Change Translation Efficiency of the Downstream Transgene.

We have developed retroviral replicating vectors (RRV) derived from Moloney murine gammaretrovirus with an amphotropic envelope and an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-transgene cassette downstream of the env gene. During long-term (180 days) replication of the vector in animals, a bulge of 7 adenosine residues (A's) in the J-K bifurcation domain sometimes serially added A's. Therefore, vectors with 4-12 A's in the A bulge in the J-K bifurcation domain were generated, and the impact of the variants on transgene protein expression, vector stability, and IRES sequence upon multiple infection cycles was assessed in RRV encoding yeast-derived cytosine deaminase and green fluorescent protein in vitro. For transgene protein expression, after multiple infection cycles, RRV-IRES with 5-7 A's gave roughly comparable levels, 4 and 8 A's were within about 4-5-fold of the 6 A's, whereas 10 and 12 A's were marked lower. In terms of stability, after 10 infection cycles, expansion of A's appeared to be a more frequent event affecting transgene protein expression than viral genome deletions or rearrangement: 4 and 5 A's appeared completely stable; 6, 7, and particularly 8 A's showed some level of expansion in the A bulge; 10 and 12 A's underwent both expansion and transgene deletion. The strong relative translational activity of the 5 A's in the EMCV IRES has not been reported previously. The 5A RRV-IRES may have utility for preclinical and clinical applications where extended replication is required.

MicroRNA 142-3p attenuates spread of replicating retroviral vector in hematopoietic lineage-derived cells while maintaining an antiviral immune response.

We are developing a retroviral replicating vector (RRV) encoding cytosine deaminase as an anticancer agent for gliomas. Despite its demonstrated natural selectivity for tumors, and other safety features, such a virus could potentially cause off-target effects by productively infecting healthy tissues. Here, we investigated whether incorporation of a hematopoietic lineage-specific microRNA target sequence in RRV further restricts replication in hematopoietic lineage-derived human cells in vitro and in murine lymphoid tissues in vivo. One or four copies of a sequence perfectly complementary to the guide strand of microRNA 142-3p were inserted into the 3' untranslated region of the RRV genome expressing the transgene encoding green fluorescent protein (GFP). Viral spread and GFP expression of these vectors in hematopoietic lineage cells in vitro and in vivo were measured by qPCR, qRT-PCR, and flow cytometry. In hematopoietic lineage-derived human cell lines and primary human stimulated peripheral blood mononuclear cells, vectors carrying the 142-3pT sequence showed a remarkable decrease in GFP expression relative to the parental vector, and viral spread was not observed over time. In a syngeneic subcutaneous mouse tumor model, RRVs with and without the 142-3pT sequences spread equally well in tumor cells; were strongly repressed in blood, bone marrow, and spleen; and generated antiviral immune responses. In an immune-deficient mouse model, RRVs with 142-3pT sequences were strongly repressed in blood, bone marrow, and spleen compared with unmodified RRV. Tissue-specific microRNA-based selective attenuation of RRV replication can maintain antiviral immunity, and if needed, provide an additional safeguard to this delivery platform for gene therapy applications.

Reduction of HLA class I expression by ribonucleic acid interference mitigates allogenicity of human primary and Immortalized cells.

Unmatched human leukocyte antigens (HLA) expressed by allogeneic donor cells are the major target for immunological rejection. In order to reduce the immunogenicity of allograft cells, we have developed lentiviral vectors for delivery of short hairpin ribonucleic acid (shRNA) against Class I HLA. This approach was evaluated in both an established human embryonic kidney cell line and primary human CD34+ hematopoietic stem/progenitor cells. Target cells transduced with lentiviral vectors expressing either HLA-A*0201 allele-specific or HLA-A, -B, -C consensus sequence-specific shRNA showed effective knockdown of cell surface HLA expression. Mixed lymphocyte-target cell reactions showed significantly reduced interferon-gamma production from alloreactive cytotoxic T lymphocytes and significantly reduced levels of target cell apoptosis after shRNA-mediated knockdown of HLA expression and target cell survival correlated with vector transduction efficiency. Furthermore, increasing resistance to complement-dependent cytotoxicity mediated by anti-HLA antibodies was observed to correlate with increasing levels of shRNA vector transduction in primary human CD34+ cells. Notably, non-HLA restricted killing by lymphokine-activated killer cells was not incurred after HLA knockdown. These data demonstrate the potential for genetic engineering strategies targeting incompatible HLA alleles to reduce both cellular and humoral responses and enable graft survival after transplantation of allogeneic cells and tissues.

Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function.

A vast amount of research on the regulation of gene expression has relied on plasmid reporter assays. In this study, we show that plasmids widely used for this purpose constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication. Readthrough of these RNAs into the intended transcriptional unit potently stimulated reporter activity when the inserted test sequence contained a 3' splice site (ss). We show that two human sequences, originally reported to be internal ribosome entry sites and later to instead be promoters, mimic both types of element in dicistronic reporter assays by causing these cryptic readthrough transcripts to splice in patterns that allow efficient translation of the downstream cistron. Introduction of test sequences containing 3' ss into monocistronic luciferase reporter vectors widely used in the study of transcriptional regulation also created the false appearance of promoter function via the same mechanism. Across a large number of variants of these plasmids, we found a very highly significant correlation between reporter activity and levels of such spliced readthrough transcripts. Computational estimation of the frequency of cryptic 3' ss in genomic sequences suggests that misattribution of cis-regulatory function may be a common occurrence.

Design and selection of Toca 511 for clinical use: modified retroviral replicating vector with improved stability and gene expression.

Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials.

Retroviral replicating vectors in cancer.

The use of replication-competent viruses for the treatment of cancer is an emerging technology that shows significant promise. Among the various different types of viruses currently being developed as oncolytic agents, retroviral replicating vectors (RRVs) possess unique characteristics that allow highly efficient, non-lytic, and tumor-selective gene transfer. By retaining all of the elements necessary for viral replication, RRVs are capable of transmitting genes via exponential in situ amplification. Their replication-competence also provides a powerful means whereby novel and useful RRV variants can be generated using natural selection. Their stringent requirement for cell division in order to achieve productive infection, and their preferential replication in cells with defective innate immunity, confer a considerable degree of natural specificity for tumors. Furthermore, their ability to integrate stably into the genome of cancer cells, without immediate cytolysis, contributes to long-lasting therapeutic efficacy. Thus, RRVs show much promise as therapeutic agents for cancer and are currently being tested in the clinic. Here we describe experimental methods for their production and quantitation, for adaptive evolution and natural selection to develop novel or improved RRV, and for in vitro and in vivo assessment of the therapeutic efficacy of RRVs carrying prodrug activator genes for treatment of cancer.

Optimization of enzyme-substrate pairing for bioluminescence imaging of gene transfer using Renilla and Gaussia luciferases.

BACKGROUND: Bioluminescence imaging (BLI) permits the non-invasive quantification and localization of transduction and expression by gene transfer vectors. The tendency of tissue to attenuate light in the optical region, however, limits the sensitivity of BLI. Improvements in light output from bioluminescent reporter systems would allow the detection of lower levels of expression, smaller numbers of cells and expression from deeper and more attenuating tissues within an animal. METHODS: With the goal of identifying substrates that allow improved sensitivity with Renilla luciferase (RLuc) and Gaussia luciferase (GLuc) reporter genes, we evaluated native coelenterazine and three of its most promising derivatives in BLI of cultured cells transduced with retroviral vectors encoding these reporters. Of the eight enzyme-substrate pairs tested, the two that performed best were further evaluated in mice to compare their effectiveness for imaging vector-modified cells in live animals. RESULTS: In cell culture, we observed striking differences in luminescence levels from the various enzyme-substrate combinations and found that the two luciferases exhibited markedly distinct abilities to generate light with the substrates. The most effective pairs were RLuc with the synthetic coelenterazine derivative ViviRen, and GLuc with native coelenterazine. In animals, these two pairs allowed similar detection sensitivities, which were eight- to 15-fold higher than that of the prototypical RLuc-native coelenterazine combination. CONCLUSIONS: Substrate selection can dramatically influence the detection sensitivity of RLuc and GLuc and appropriate choice of substrate can greatly improve the performance of reporter genes encoding these enzymes for monitoring gene transfer by BLI.

PTEN knockout prostate cancer as a model for experimental immunotherapy.

PURPOSE: Testing immunotherapeutic strategies for prostate cancer has been impeded by the lack of relevant tumor models in immunocompetent animals. This opportunity is now provided by the recent development of prostate specific PTEN knockout mice, which show spontaneous development of true adenocarcinoma arising from prostate epithelium and more faithfully recapitulate the human disease than any previous model. We investigated the feasibility of using tumor cells derived from this model to test tumor vaccination and adoptive immunotherapeutic strategies for prostate cancer. MATERIALS AND METHODS: PTEN-CaP8 adenocarcinoma cells derived from the biallelic PTEN knockout prostate cancer model were used to vaccinate nontumor bearing litter mates. Tumor specific effector cells were generated from splenocytes of vaccinated mice by mixed lymphocyte-tumor reactions, and antiproliferative effects and cytokine generation were examined in vitro. The effect of vaccination or adoptive immunotherapy on luciferase marked PTEN-CaP8 subcutaneous tumors was monitored by tumor volumetric measurements and noninvasive bioluminescence imaging. RESULTS: Vaccination of litter mate mice with irradiated PTEN-CaP8 cells showed a significant prophylactic effect against the subsequent tumor challenge. Effector cells harvested from vaccinated litter mates showed significant interferon-gamma secretion upon co-incubation with PTEN-CaP8 target cells and they were capable of efficient target cell growth inhibition in vitro. Intratumor adoptive transfer of effector cells resulted in significant growth inhibition of preestablished prostate tumors in vivo. CONCLUSIONS: The PTEN knockout model serves as a highly useful model in which to investigate tumor cell vaccination and adoptive immunotherapeutic strategies in the context of true adenocarcinoma of the prostate. This model should accelerate efforts to develop effective immunotherapies for human prostate cancer.

Splicing mediates the activity of four putative cellular internal ribosome entry sites.

A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5' end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3' splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3' splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity.

Highly efficient gene delivery for bladder cancers by intravesically administered replication-competent retroviral vectors.

PURPOSE: In an attempt to improve viral delivery of potentially therapeutic genes via an intravesical route, we have recently developed murine leukemia virus-based replication-competent retrovirus (RCR) vectors. EXPERIMENTAL DESIGN: We evaluated the transduction efficiency of intravesically administered RCR vectors to bladder tumor using orthotopic animal models to determine their potential as delivery vectors for bladder cancer. RESULTS: The RCR vector containing green fluorescent protein (GFP) marker gene achieved efficient in vitro transmission of the GFP transgene. Murine bladder tumor-2 mouse bladder tumors exposed to intravesically administered RCR vectors exhibited 0%, 9.2 +/- 2.9%, and 30.0 +/- 6.2% of GFP expression at 9, 18, and 27 days after exposure in the orthotopic model, respectively. Orthotopic KU-19-19 human bladder tumors exposed to intravesically administered RCR vectors exhibited 3%, 85 +/- 1.0%, and 100% of GFP expression at 7, 21, and 35 days after exposure, respectively. GFP staining was observed only in the tumor cells in the bladder. No detectable PCR products of GFP gene could be observed in distant organs. Treatment with RCR vectors containing yeast cytosine deaminase (CD) gene plus 5-fluorocytosine (5-FC) dramatically inhibited the growth of preestablished murine bladder tumor-2 tumors. A single course of 5-FC treatment resulted in a 50% animal survival in mice exposed to RCR-CD compared with a 0% survival in all controls over a 70-day follow-up period. CONCLUSIONS: Intravesically administered RCR vectors can efficiently deliver genes to orthotopic bladder tumor without viral spread in distant organs. RCR-CD/5-FC suicide gene therapy promises to be a novel and potentially therapeutic modality for bladder cancer.

Therapeutic efficacy of replication-competent retrovirus vector-mediated suicide gene therapy in a multifocal colorectal cancer metastasis model.

Replication-competent retrovirus (RCR) vectors are intrinsically incapable of infecting quiescent cells and have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. However, i.v. delivery of RCR vectors expressing therapeutic genes has never previously been tested, particularly in an immunocompetent tumor model. Therefore, in the present study, we sought to test the therapeutic effect of an RCR vector (ACE-CD) carrying the yeast cytosine deaminase (CD) gene, which converts the nontoxic prodrug 5-fluorocytosine (5FC) into the chemotoxin 5-fluorouracil, after delivery by infusion into the locoregional circulation in a multifocal hepatic metastasis model of colon cancer. After confirmation of suicide gene cytotoxicity in vitro, multifocal hepatic tumors were established in syngeneic mice with murine CT26 colorectal cancer cells expressing firefly luciferase (CT26-Luc), and the ACE-CD vector was infused via intrasplenic injection into the portal circulation. Fourteen days after locoregional infusion, systemic administration of 5FC resulted in significant inhibition of bioluminescent signals in mice whose tumors had been infected with RCR but not in control mice. Notably, there was no detectable RCR vector spread to normal liver or bone marrow by quantitative PCR analysis. Our results thus show that locoregional delivery of a suicide gene by RCR vectors infused into the portal circulation results in progressive transduction of multiple tumor foci in the liver, without evidence of spread to adjacent normal parenchyma or extrahepatic tissues, and can achieve significant tumor growth inhibition.

Adaptive evolution of a tagged chimeric gammaretrovirus: identification of novel cis-acting elements that modulate splicing.

Retroviruses are well known for their ability to incorporate envelope (Env) proteins from other retroviral strains and genera, and even from other virus families. This characteristic has been widely exploited for the generation of replication-defective retroviral vectors, including those derived from murine leukemia virus (MLV), bearing heterologous Env proteins. We investigated the possibility of "genetically pseudotyping" replication-competent MLV by replacing the native env gene in a full-length viral genome with that of another gammaretrovirus. Earlier, we developed replication-competent versions of MLV that stably transmit and express transgenes inserted into the 3' untranslated region of the viral genome. In one such tagged MLV expressing green fluorescent protein, we replaced the native env sequence with that of gibbon ape leukemia virus (GALV). Although the GALV Env protein is commonly used to make high-titer pseudotypes of MLV vectors, we found that the env replacement greatly attenuated viral replication. However, extended cultivation of cells exposed to the chimeric virus resulted in selection of mutants exhibiting rapid replication kinetics and different variants arose in different infections. Two of these variants had acquired mutations at or adjacent to the splice acceptor site, and three others had acquired dual mutations within the long terminal repeat. Analysis of the levels of unspliced and spliced viral RNA produced by the parental and adapted viruses showed that the mutations gained by each of these variants functioned to reverse an imbalance in splicing caused by the env gene substitution. Our results reveal the presence of previously unknown cis-acting sequences in MLV that modulate splicing of the viral transcript and demonstrate that tagging of the retroviral genome with an easily assayed transgene can be combined with in vitro evolution as an approach to efficiently generating and screening for replicating mutants of replication-impaired recombinant viruses.

Tumor-selective gene expression in a hepatic metastasis model after locoregional delivery of a replication-competent retrovirus vector.

PURPOSE: Replication-competent retrovirus (RCR) vectors have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. In this setting, the intrinsic inability of retroviruses to infect postmitotic normal cells, combined with their unique ability to persist through stable integration, allow further transduction of ectopic tumor foci as the infected cancer cells migrate. However, i.v. delivery of RCR vectors has never been tested previously, particularly in an immunocompetent tumor model. EXPERIMENTAL DESIGN: We combined optical imaging, flow cytometry, and molecular analysis to monitor RCR vector spread after administration via locoregional infusion in a hepatic metastasis model of colorectal cancer. RESULTS: Robust RCR replication was first confirmed in both human WiDr and murine CT26 colorectal cancer cells in vitro, with transduction levels reaching >90% in <12 days after virus inoculation at multiplicities of infection of 0.01 to 0.1. In vivo, infusion of RCR supernatant into the portal circulation resulted in progressive and significant transduction of multifocal intrahepatic CT26 tumors in syngeneic mice, averaging about 30% but with up to 60% transduction in some tumors within 4 weeks. However, immunohistochemistry and quantitative PCR analysis showed no evidence of RCR spread to adjacent normal liver or to any other normal tissues. CONCLUSIONS: Our results thus show that locoregional infusion of RCR vectors can be used to deliver therapeutic genes selectively to tumor cells in the liver while sparing normal hepatocytes and without dissemination to extrahepatic normal tissues.

Beyond oncolytic virotherapy: replication-competent retrovirus vectors for selective and stable transduction of tumors.

As cancer gene therapy employing replication-defective vectors has met with limited clinical success, there is renewed interest in using replication-competent viruses for oncolytic virotherapy. In preclinical and clinical studies, various attenuated vaccine strains and engineered virus vectors are currently being tested for their ability to achieve tumor-selective cell killing. However, significant improvements are still required in tumor selectivity, cytolytic potency, and modulating immune responses to achieve anti-tumor effects without prematurely terminating virus spread. Recently, we have developed murine leukemia virus (MLV)-based replication-competent retrovirus (RCR) vectors for highly efficient, selective, and persistent gene transfer to cancer cells, and found that such vectors may offer significant advantages as oncolytic agents. In a variety of preclinical models, RCR vectors can achieve efficient and persistent gene delivery as the virus replicates throughout an entire tumor mass after inoculation with initial multiplicities of infection as low as 0.001. When engineered to deliver suicide genes, RCR vectors achieve highly efficient and synchronized cell killing triggered by pro-drug administration, both in culture and in tumor models in vivo. Further strategies are being explored to enhance the packaging capacity, efficiency, and specificity of this vector system through the development of semi-replicative RCR vectors, adenovirus-RCR hybrids, and incorporation of tumor targeting mechanisms via modification of binding tropism and transcriptional regulation. In addition, the ability of these vectors to achieve stable transgene expression in infected tumor cells may allow therapeutic applications that move beyond oncolysis per se.

Retrovirus-mediated gene transfer to tumors: utilizing the replicative power of viruses to achieve highly efficient tumor transduction in vivo.

Vectors derived from retroviruses have been widely studied as tools for gene transfer into mammalian tissue in vivo. One application for which retroviral vectors have received particular attention is gene transfer into tumor cells for treatment of cancer. Simple retroviruses, such as murine leukemia virus (MLV), and the vectors derived from them, require cell division for infection and thus possess a degree of inherent specificity for the rapidly dividing cells of neoplastic tissue. This unique property and the ease with which retroviral vectors are manipulated and produced have provided much of the impetus for their use in experimental and clinical cancer gene-therapy studies.

Antibody-mediated targeting of replication-competent retroviral vectors.

Replication-competent murine leukemia virus (MLV) vectors can be engineered to achieve high efficiency gene transfer to solid tumors in vivo and tumor-restricted replication, however their safety can be further enhanced by redirecting tropism of the virus envelope. We have therefore tested the targeting capability and replicative stability of ecotropic and amphotropic replication-competent retrovirus (RCR) vectors containing two tandem repeats from the immunoglobulin G-binding domain of Staphylococcal protein A inserted into the proline-rich "hinge" region of the envelope, which enables modular use of antibodies of various specificities for vector targeting. The modified envelopes were efficiently expressed and incorporated into virions, were capable of capturing monoclonal anti-HER2 antibodies, and mediated efficient binding of the virus-antibody complex to HER2-positive target cells. While infectivity was markedly reduced by pseudotyping with targeted envelopes alone, coexpression of wild-type envelope rescued efficient cellular entry. Both ecotropic and amphotropic RCR vector/anti-HER2 antibody complexes achieved significant enhancement of transduction on murine target cells overexpressing HER2, which could be competed by preincubation with excess free antibodies. Interestingly, HER2-expressing human breast cancer cells did not show enhancement of transduction despite efficient antibody-mediated cell surface binding, suggesting that target cell-specific parameters markedly affect the efficiency of post-binding entry processes. Serial replication of targeted vectors resulted in selection of Z domain deletion variants, but reduction of the overall size of the vector genome enhanced its stability. Application of antibody-mediated targeting to the initial localization of replication-competent virus vectors to tumor sites will thus require optimized target selection and vector design.

Tissue-specific transcriptional targeting of a replication-competent retroviral vector.

The inability of replication-defective viral vectors to efficiently transduce tumor cells in vivo has prevented the successful application of such vectors in gene therapy of cancer. To address the need for more efficient gene delivery systems, we have developed replication-competent retroviral (RCR) vectors based on murine leukemia virus (MLV). We have previously shown that such vectors are capable of transducing solid tumors in vivo with very high efficiency. While the natural requirement of MLV infection for cell division imparts a certain degree of specificity for tumor cells, additional means for confining RCR vector replication to tumor cells are desirable. Here, we investigated the parameters critical for successful tissue-specific transcriptional control of RCR vector replication by replacing various lengths of the MLV enhancer/promoter with sequences derived either from the highly prostate-specific probasin (PB) promoter or from a more potent synthetic variant of the PB promoter. We assessed the transcriptional specificity of the resulting hybrid long terminal repeats (LTRs) and the cell type specificity and efficiency of replication of vectors containing these LTRs. Incorporation of PB promoter sequences effectively restricted transcription from the LTR to prostate-derived cells and imparted prostate-specific RCR vector replication but required the stronger synthetic promoter and retention of native MLV sequences in the vicinity of the TATA box for optimal replicative efficiency and specificity. Our results have thus identified promoter strength and positioning within the LTR as important determinants for achieving both high transduction efficiency and strict cell type specificity in transcriptionally targeted RCR vectors.

A novel, helper-dependent, adenovirus-retrovirus hybrid vector: stable transduction by a two-stage mechanism.

We have developed a novel vector system that uses a helper-dependent adenoviral vector as a carrier to deliver a fully functional retrovirus vector. The helper-dependent adenovirus (HDAd) can accommodate large inserts, provide high titers, and infect nondividing as well as dividing cells. However, adenoviral DNA is rarely integrated into the host cell genome, and its episomal expression is transient. Therefore we inserted a replication-competent, ecotropic retrovirus vector containing the green fluorescent protein (GFP) reporter gene as a second-stage component. The well-characterized host species tropism of each vector component provided a stringent biological assay system that demonstrates the two-stage transduction mechanism of the hybrid vector, because the adenovirus stage can efficiently transduce human cells but cannot replicate in murine cells, and conversely, the ecotropic retrovirus stage cannot enter human cells but can efficiently proliferate in murine cells, resulting in permanent integration and progressive spread of reporter gene expression.