Characterization and utilization of simple replication-competent retroviral vectors

Retroviral vectors are commonly used for gene delivery. Replication-defective vectors, which are widely used, have been shown to exhibit poor transduction efficiency in-vivo. Replication-competent retroviral (RCR) vectors circumvent this problem, yet these vectors are not well characterized.;These studies attempted to further characterize replication-competent vectors based on the murine leukemia virus (MTV) and the gibbons ape leukemia virus (GALV). Retroviruses have been shown to naturally alter their tropism via incorporation of foreign envelope glycoproteins. Creation of a genetically pseudotyped RCR vector may reveal key regulatory elements that allow such events to occur and ultimately may result in safer vector design in the future. Construction of a genetically pseudotyped GALV enveloped MLV RCR resulted in the discovery of novel RNA splicing regulatory elements located within the viral long terminal repeat (LTR).;RCR vectors must be engineered in a manner that will allow transgene expression without having a deleterious effect on their replication. Current RCR designs rely on the usage of internal ribosome entry sites (IRES) to drive expression of the transgene. The most well studied IRES is from the encephalomyocarditis virus (EMCV) and this IRES is commonly used in gene therapy vectors. Many attempts have been made to create a genetically stable RCR that can express transgenes over multiple infection cycles. The vectors used herein have an IRES inserted immediately downstream of the env stop codon. Three RCR vectors employing this strategy were analyzed for their replication kinetics and genomic stability in multiple tumor cell lines.;For any RCR to be effective an adequate amount of therapeutic transgene must be expressed. Recently, evidence suggested that mammalian mRNAs posses sequences with IRES capabilities, many of which claimed to be extremely robust and very short in length. These putative IRES sequences may create a more stable and better expressing RCR. Insertion of six putative cellular IRESs into the RCR system revealed that the apparent activity of these alleged IRESs appears to be the result of RNA splicing.;While RCRs offer tremendous hope for gene therapy, these vectors can also be used as experimental tools to further probe molecular biology.