{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/353727"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/353727","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Development of a Lentivirus-mediated Gene Therapy Targeting HIV-1 RNA to Eliminate Infected Cells","abstract":"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*<sub>ΔAUG1</sub>) 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*<sub>ΔAUG1</sub> RNA in Jurkat T cells co-transduced with HIV-1<sub>NL4-3ΔE</sub> and therapeutic vectors. However, I found that translation of catalytically active polypeptides from internal AUGs in *HSVtk*<sub>ΔAUG1</sub> 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<sub>NL4-3ΔE</sub>-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<sub>NL4-3</sub> 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.","abstract_html":"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*&lt;sub&gt;ΔAUG1&lt;/sub&gt;) 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*&lt;sub&gt;ΔAUG1&lt;/sub&gt; RNA in Jurkat T cells co-transduced with HIV-1&lt;sub&gt;NL4-3ΔE&lt;/sub&gt; and therapeutic vectors. However, I found that translation of catalytically active polypeptides from internal AUGs in *HSVtk*&lt;sub&gt;ΔAUG1&lt;/sub&gt; 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&lt;sub&gt;NL4-3ΔE&lt;/sub&gt;-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&lt;sub&gt;NL4-3&lt;/sub&gt; 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.","abstract_has_math":false,"creators":["Buckingham, Amanda"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Wills, Mark","Lever, Andrew"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-03-01","date_published":"2023-03-01","updated_at":"2026-07-22T22:24:03Z","subjects":["gene therapy","gene-directed enzyme prodrug therapy","HIV-1","latency","lentiviral vector","RNA splicing","shock and kill"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ba4aa899-fd37-4d4b-b60d-e5ce92944fea/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.99785","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Wills, Mark","Lever, Andrew"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Peterhouse - Graduate Research Studentship"]},{"key":"dc:creator","label":"Author","values":["Buckingham, Amanda"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-03-01"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/353727"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["gene therapy","gene-directed enzyme prodrug therapy","HIV-1","latency","lentiviral vector","RNA splicing","shock and kill"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ba4aa899-fd37-4d4b-b60d-e5ce92944fea/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.99785"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3e0bcec5-6432-420a-bda1-8f77fc29f8d6/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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*<sub>ΔAUG1</sub>) 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*<sub>ΔAUG1</sub> RNA in Jurkat T cells co-transduced with HIV-1<sub>NL4-3ΔE</sub> and therapeutic vectors. However, I found that translation of catalytically active polypeptides from internal AUGs in *HSVtk*<sub>ΔAUG1</sub> 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<sub>NL4-3ΔE</sub>-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<sub>NL4-3</sub> 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."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["004215ab4934158441c233cb88d9df77","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["Development of a Lentivirus-mediated Gene Therapy Targeting HIV-1 RNA to Eliminate Infected Cells"]}]}],"canonical_facts":{"dc:contributor.advisor":["Wills, Mark","Lever, Andrew"],"dc:contributor.sponsor":["Peterhouse - Graduate Research Studentship"],"dc:creator":["Buckingham, Amanda"],"dc:date.issued":["2023-03-01"],"dc:description.abstract":["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*<sub>ΔAUG1</sub>) 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*<sub>ΔAUG1</sub> RNA in Jurkat T cells co-transduced with HIV-1<sub>NL4-3ΔE</sub> and therapeutic vectors. However, I found that translation of catalytically active polypeptides from internal AUGs in *HSVtk*<sub>ΔAUG1</sub> 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<sub>NL4-3ΔE</sub>-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<sub>NL4-3</sub> 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."],"dc:format.checksum.md5":["004215ab4934158441c233cb88d9df77","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.99785"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3e0bcec5-6432-420a-bda1-8f77fc29f8d6/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/353727"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ba4aa899-fd37-4d4b-b60d-e5ce92944fea/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["gene therapy","gene-directed enzyme prodrug therapy","HIV-1","latency","lentiviral vector","RNA splicing","shock and kill"],"dc:title":["Development of a Lentivirus-mediated Gene Therapy Targeting HIV-1 RNA to Eliminate Infected Cells"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:03Z"}