Re-structuring lentiviral vectors to express genomic RNA via cap-dependent translation

Abstract Lentiviral vectors based on Human Immunodeficiency Virus Type I (HIV-1) package two copies of their single-stranded RNA into vector particles. Normally, this RNA genome is reverse transcribed into a double-stranded DNA provirus that integrates into the cell genome, providing permanent gene transfer and long-term expression. Integration-deficient lentivirus vectors have been developed to reduce the frequency of genomic integration and thereby limit their persistence in dividing cells. Here, we describe optimization of a reverse-transcriptase-deficient lentivirus vector, which enables direct translation of lentiviral RNA genomes upon cell entry, for transient expression of vector payloads as mRNA without a DNA intermediate. We have engineered a novel lentiviral genome arrangement in which HIV-1 sequences are removed from the 5’ end, to enable ribosomal entry from the 5’ 7-methylguanylate cap for efficient translation of the vector payload. We have shown that this lentivirally-mediated mRNA delivery platform provides transient transgene expression in vitro and in vivo. This has potential application in gene and cell therapy scenarios requiring temporary payload expression in cells and tissues that can be targeted with pseudotyped lentiviral vectors.