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

A Uniquely Stable Replication-Competent Retrovirus Vector Achieves Efficient Gene Delivery in Vitro and in Solid Tumors

A major obstacle in cancer gene therapy is the limited efficiency of in vivo gene transfer by replication-defective retrovirus vectors in current use. One strategy for circumventing this difficulty would be to use vectors capable of replication within tumor tissues. We have developed a replication-competent retrovirus (RCR) vector derived from murine leukemia virus (MuLV). This vector utilizes a unique design strategy in which an internal ribosome entry site–transgene cassette is positioned between the env gene and the 3′ long terminal repeat (LTR). The ability of this vector to replicate and transmit a transgene was examined in culture and in a solid tumor model in vivo . The RCR vector exhibited replication kinetics similar to those of wild-type MuLV and mediated efficient delivery of the transgene throughout an entire population of cells in culture after an initial inoculation with 1 plaque-forming unit (PFU) of vector per 2000 cells. After injection of 6 × 10 3 PFU of vector into established subcutaneous tumors, highly efficient spread of the transgene was observed over a period of 7 weeks, in some cases resulting in spread of the transgene throughout the entire tumor. MuLV-based RCR vectors show significant advantages over standard replication-defective vectors in efficiency of gene delivery both in culture and in vivo . This represents the first example of the use of an RCR vector in an adult mammalian host, and their first application to transduction of solid tumors. Low efficiency of gene delivery by currently used viral vectors remains a significant barrier to the success of gene therapy of cancer. To examine the possibility of achieving superior transduction efficiency utilizing retroviral vectors that are capable of replication, we inserted an IRES–transgene cassette between the env gene and 3′ LTR of an infectious full-length murine leukemia virus (MuLV) clone. We found that the resulting vector replicated with kinetics similar to those of wild-type MuLV, and was stable through multiple serial passages in cultured cells. Injection of this vector into established subcutaneous tumors in mice resulted in highly efficient transmission of the transgene and, in some cases, transduction of entire tumor masses. These results demonstrate the potential utility of replication-competent retroviral vectors for cancer gene therapy

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

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

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. 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. 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. 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

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

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

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. 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. 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. 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

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

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

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.FLuc), 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