Development of a Lentivirus-mediated Gene Therapy Targeting HIV-1 RNA to Eliminate Infected Cells

Over 38.4 million people worldwide are living with HIV-1, the etiological agent of acquired immunodeficiency syndrome and the cause of over 40.1 million deaths. The latent HIV-1 reservoir is the major roadblock to cure and necessitates lifelong antiretroviral therapy (ART), which is vulnerable to drug resistance. I contributed towards the development of a novel therapeutic strategy that aims to eliminate cells actively expressing HIV-1 and those harbouring HIV-1 reactivated from latency. This approach hijacks the currently unexploited HIV-1 alternative RNA splicing process to functionalize a defective cell suicide enzyme (*HSVtk*_ΔAUG1) through targeted RNA *trans*-splicing with the HIV-1 D4 splice site. In-frame *HSVtk* translation is initiated from the start codon of the HIV-1 *tat1* donor exon in chimeric mRNA. HSVtk activates the prodrug ganciclovir (GCV), with cytotoxic metabolite GCV-triphosphate (TP) disrupting DNA synthesis to selectively kill HIV-1-expressing cells. Following proof-of-principle transfection studies, therapeutic constructs were engineered for lentivirus-mediated delivery to HIV-1-infected cells in vitro. I first optimized production of a panel of VSV-G-pseudotyped D4-targeting lentivectors for high infectious titre and low transfer plasmid carryover. I confirmed trans-splicing between HIV-1 *tat1* and *HSVtk*_ΔAUG1 RNA in Jurkat T cells co-transduced with HIV-1_NL4-3ΔE and therapeutic vectors. However, I found that translation of catalytically active polypeptides from internal AUGs in *HSVtk*_ΔAUG1 caused dose-dependent cytotoxicity with GCV in uninfected cells. Modification of internal AUGs in D4 opt 2 effectively mitigated this major off-target effect. Based on the MTT assay, D4 opt 2 in combination with GCV reduced the viability of HIV-1_NL4-3ΔE-expressing Jurkat T cells by approximately 50% with no impact on uninfected cells. For improved therapeutic potential, I replaced the promoter in D4 opt 2 with that for the human *EF1α* gene, resulting in 4.4-times more RNA payload. EF1α-driven D4 opt 2 with GCV reduced the viability of HEK293T cells expressing full-length HIV-1_NL4-3 by up to 70%. I developed a panel of lentivectors delivering opt 2 with low (ScrambleV2), moderate (ScrambleV1), or high (D4) affinity predicted in silico for target HIV-1 pre-mRNA and found that the strength of interaction between opt 2 and HIV-1 correlated with the propensity for *trans*-splicing and elimination of HIV-1-expressing cells by the opt 2 cell suicide system *in vitro*. Having demonstrated that this HIV-1-targeted cell suicide system could eliminate cells actively expressing HIV-1, I next investigated its potential against latent HIV-1 in J- Lat 10.6 cells. I found LRAs exerted class and dose-dependent effects on viability which could affect the outcome of shock and kill. Despite their superior reactivation potential, NF-κB agonists made J-Lat 10.6 cells more difficult to eliminate. In contrast, the 26S proteasome inhibitor bortezomib (BTZ), known to sensitise HIV-1-infected cells to death, enhanced shock and kill. I discovered that the nucleoside analogue DNA methyltransferase inhibitor decitabine (DAC) is an adjuvant of the HIV-1-targeted cell suicide system, with DAC-TP and GCV-TP known to synergise. A ≥65% reduction in J-Lat 10.6 viability was achieved when cells were stimulated with BTZ, DAC, or a TNFα/DAC ‘shocktail’ prior to treatment with EF1α-driven D4 opt 2 and GCV. My work supports further development of our HIV-1-targeted cell suicide system against cells actively expressing HIV-1 and those chronically infected when combined with appropriate LRAs.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Buckingham, Amanda

Advisors dc:contributor.advisor

• Wills, Mark
• Lever, Andrew

• gene therapy
• gene-directed enzyme prodrug therapy
• HIV-1
• latency
• lentiviral vector
• RNA splicing
• shock and kill

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ba4aa899-fd37-4d4b-b60d-e5ce92944fea/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.99785

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/353727