Abstract
Lentiviral vectors based on HIV-1 were developed as a gene therapy tool due to their ability to facilitate efficient transduction and long-term stable transgene expression in both dividing and non-dividing cells. Lentiviral vectors contain large regions of HIV-1 derived sequences, including the 5’ R-U5-leader region, a 5’ portion of gag, a region of env containing the Rev-response element (RRE), the central polypurine tract (cPPT) and the 3’ LTR with a 400-nucleotide deletion that abolished its promoter activity. However, it remains unclear whether all of the HIV-1 sequence in vector genomes is essential. Furthermore, the introduction of non-HIV-1 sequences, such as the internal promoter and transgene, as well as the woodchuck hepatitis virus post-transcriptional element (WPRE) alters the nucleotide and dinucleotide bias of the vector genome, often increasing CpG dinucleotide abundance. Herein, we characterize the functional relevance of the viral RNA elements in the lentiviral vector genome, and investigate the susceptibility of lentiviral vectors to ZAP, an antiviral protein that targets CpG dinucleotides.An optimal lentiviral vector genome would include only the viral sequences required for transduction efficiency and gene expression to minimize the amount of foreign sequence inserted into a patient’s genome. To determine which viral sequences are required, we performed a systematic deletion analysis, which showed that most of the gag region and over 50% of the env region could be deleted. We demonstrate that Rev and the RRE functions at the level of vector genome packaging, whilst the WPRE is important for efficient transgene expression. Because the splicing profile for lentiviral vectors is poorly characterized, we used long read sequencing to determine how canonical and cryptic splice sites were used. Deleting a substantial amount of env sequence reduced the number of pre-mRNA splicing events per transcript and increased the proportion of unspliced genomes. Furthermore, we show that ZAP does not have a substantial effect on lentiviral vector titre, transgene expression or virion production, even for a codon-optimized Gag-Pol packaging construct or a vector genome expressing Cas9 that contain a large number of CpG dinucleotides. Overall, the findings presented in this study provides an insight into the biology of lentiviral vectors and may enable new lentiviral vector designs with improved biosafety and increased transgene capacity to accommodate large and complex transgenes for a variety of clinical applications.
| Date of Award | 1 Dec 2021 |
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| Original language | English |
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| Supervisor | Chad Swanson (Supervisor) & Conrad A. Vink (Supervisor) |