Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout

• Published in: Virology journal Vol. 18; no. 1; p. 31
• Main Authors: Mehmetoglu-Gurbuz, Tugba, Yeh, Rose, Garg, Himanshu, Joshi, Anjali
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 30-01-2021; BioMed Central; BMC
• Subjects: Acquired immune deficiency syndrome; AIDS; Antibodies; Antiviral drugs; Care and treatment; CCR5; CCR5 protein; Chemokines; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CXCR4; CXCR4 protein; Cytotoxicity; Data analysis; Deoxyribonucleic acid; Development and progression; DNA; Flow cytometry; Ganciclovir; Gene Editing - methods; Gene Knockout Techniques; Gene therapy; Genes, Transgenic, Suicide; Genetic aspects; Genetic engineering; Genetic Therapy - methods; Health aspects; HEK293 Cells; Hematopoietic stem cells; HIV; HIV cure; HIV infection; HIV Infections - therapy; Human immunodeficiency virus; Humans; Immune system; Infections; Insertion; Methods; Patient outcomes; Reagents; Receptors, CCR5 - genetics; Reporter gene; Software; Stem cells; Suicide; Suicide genes; Suicides & suicide attempts; Tat protein; Thymidine; Thymidine kinase; TK-SR39; Transduction, Genetic; Viruses; United States; CRISPR; Ganciclovir; HIV; CXCR4; Conditional; CCR5; Cytotoxic; Gene therapy; TK-SR39; HIV cure
• ISSN: 1743-422X; 1743-422X
• DOI: 10.1186/s12985-021-01501-7

Gene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone.
A two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein.
Transduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir.
Our study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.