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

Abstract

Background 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. Methods 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. Results 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. Conclusions 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.

Optimisation of a TALE nuclease targeting the HIV co-receptor CCR5 for clinical application

“…CCR5-specific nucleases have been used in CD4 + T cells and CD34 + HSCs [ 65 – 69 ]. Modification of both cell types is associated with specific advantages and shortcomings.…”

Strategies for HIV-1 suppression through key genes and cell therapy

“…Genome engineering supports the advancement of cell therapy, including a universal approach to introduce both CAR transgenes and CRISPR-Cas9 ribonucleoproteins (RNPs) into primary human T cells using engineered lentiviral particles ( 84 ). Additionally, a dual gene therapy strategy has been developed, involving a conditional suicide gene and CCR5 knockout, to overcome limitations associated with CCR5 knockout alone and receptor switching ( 85 ).…”

Targeting CCR5 as a Component of an HIV-1 Therapeutic Strategy

“…Alternatively, emergence of CXCR4-utilizing viruses can be avoided using a combinatorial approach that utilizes an HIV-1 protein-dependent suicide gene. This was accomplished by introducing a CCR5 gRNA-CRISPR/Cas9 system into TZM-bl cells to knockout CCR5 along with an HIV-1 Tat-dependent suicide gene TK-S39 ( 165 ). This novel approach allows expression of HIV-1 Tat, which can occur when a CXCR4-utilizng or CCR5 antagonist-resistant virus enters and replicates in a cell, to induce cell death and prevent cell-to-cell spread of HIV-1 in the occurrence of HIV-1 replication despite CCR5 knockdown.…”