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Towards Optimising Lentiviral Vectors Through Structure Informed Genome Modification



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Vamva, Eirini 


Lentiviral vectors are being successfully used as therapeutic agents in a series of clinical applications of gene therapy, genome editing and cancer immunotherapy. 3rd generation HIV-1 derived lentiviral vectors are produced from 4 independent plasmids. Here, I focus on the transfer vector that contains the therapeutic gene and the cis-acting elements that drive its expression including the packaging signal (psi). Lentiviral vector particles carry two copies of transfer vector RNA that become linked via a process known as dimerisation. I focused on improving the infectivity of vectors by targeting their dimerisation and packaging properties based on the hypothesis that WT HIV-1 regulates genome encapsidation tightly by recognising dimeric RNA. The genomic RNA (gRNA) leader region is thought to act as a switch between the monomeric conformation that is associated with translation and the dimeric conformation linked with packaging. I therefore attempted to identify the structures that are important for packaging and optimise those at the expense of structures that are used by the virus for translational control. To do so, I created mutants in the 5’UTR that target regions that play important roles in the process of dimerisation including the Dimerisation Stem Loop (DSL), the U5-AUG duplex formed by sequences located at the beginning of the U5 region and nucleotides surrounding the start of the gag gene, and the polyA stem loop, a region suppressed in the 5’LTR, but known to regulate polyadenylation in the 3’LTR. The introduction of these mutations aimed to create vectors whose RNA is more likely to adopt the dimeric conformation and therefore be packaged. To evaluate this, I developed a novel competitive RT-qPCR assay to measure the relative packaging efficiencies (RPE) of transfer vectors in a competitive co-transfection environment. Biochemical characterisation showed an overall negative effect of the introduced mutations on viral infectivity. Northern Blots confirmed that the propensity of mutated vector RNA to dimerise has increased in the mutants as hypothesised. Here, I report the effect of the dimerisation-stabilising mutations on infectious and physical titres of lentiviral vectors, as well as on their packaging efficiency measured with our novel competitive qPCR assay. Our data suggest that enhancing dimerisation does not automatically lead to better packaging of vector RNA. Despite the improvement of vector RNA dimerisation efficiency and in some cases RPE, the effect of introduced mutations on the ability of the designed transfer vectors to successfully transduce 293T cells was negative, reflecting the multifunctionality of the HIV-1 leader 4 regions and the significance of RNA flexibility. Finally, I explored by Selective 2′-Hydroxyl Acylation Analysed by Primer Extension (SHAPE) the structure of psi in our vector RNAs, in particular studying the influence of regions adjacent to psi on dimerisation and packaging. SHAPE identifies the RNA backbone flexibility, which is an indication of whether nucleotides are base-paired or not. Our single nucleotide level structural analysis revealed that the presence of gag sequences stabilise the psi element of the dimeric RNA, suggesting their role in supporting a stable RNA conformation that can be packaged and offering a potential explanation for their requirement in the transfer vector plasmid for maintenance of infectious titres. These findings will give us better insights into the biology of lentiviral vectors and enable us to design more efficient vectors for a variety of clinical applications.





Lever, Andrew


lentiviral vectors, HIV-1, packaging efficiency, dimerisation, RNA structure, vector RNA


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Biotechnology and Biological Sciences Research Council (BB/N503708/1)