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. 2021 Jan 30;18(1):31.
doi: 10.1186/s12985-021-01501-7.

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

Tugba Mehmetoglu-Gurbuz  1 , Rose Yeh  2 , Himanshu Garg  1 , Anjali Joshi  3
Affiliations

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

Tugba Mehmetoglu-Gurbuz et al. Virol J. .

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.

Keywords: CCR5; CRISPR; CXCR4; Conditional; Cytotoxic; Ganciclovir; Gene therapy; HIV; HIV cure; TK-SR39.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Fig. 1
Fig. 1
Strategy of combination gene therapy approach for HIV and plasmid maps. a Incorporating CCR5 KO along with conditional suicide gene to achieve HIV cure. Killing of HIV infected cells is achieved only in the presence of GCV providing an additional layer of safety for in vivo use. b Vector maps for the dual transduction strategy for HIV cure. Vector 1 incorporates TK-SR39 gene along with selection marker GFP downstream of an IRES. Vector 2 expresses the tat gene along with CCR5 gRNA-CRISPR/Cas9 cassette. c Strategy for packaging the vector 1 using an integrating lentivirus system and vector 2 using a non-integrating lentivirus resulting in permanent TK-SR39 integration, CCR5 KO and transient GFP expression for cell enrichment
Fig. 2
Fig. 2
Characterization of Tat expression and CCR5 KO via vector 2. a TZM cells were transfected with CCR5gRNA-CRISPR/Cas9 Tat vector, Control-gRNA-CRISPR/Cas9 Tat vector or pCDNA3.1 empty vector. Luciferase activity was determined 48 h post transfection. b TZM-TKSR39 cells were transfected as in part A above. GFP expression was measured 48 h post transfection via flow cytometry. c TZM cells were transfected as in part A above. Six days post transfection, cells were analyzed for CCR5 expression by flow cytometry. d DNA was isolated from control TZM cells or TZM cells transfected with the CCR5gRNA-CRISPR/Cas9 Tat vector. Genetic disruption of the CCR5 locus was determined by PCR followed by T7EI digestion
Fig. 3
Fig. 3
Dual transduction of TZM cells with TK-SR39 vector followed by CCR5gRNA-CRISPR/Cas9 Tat vector results in GFP expression and CCR5 KO. a Strategy for dual transduction of TZM cells with integrating lentiviral vector 1 to deliver Tat dependent TK-SR39 gene and with non-integrating lentiviral vector 2 to deliver Tat and CCR5gRNA-CRISPR/Cas9. GFP expression and CCR5 KO was determined over a period of time. b TZM cells were transduced with vector 1 followed by vector 2 as in part A. Down regulation of surface CCR5 expression was monitored over a period of time by flow cytometry
Fig. 4
Fig. 4
Generation of a stable cell clone with TK-SR39 integration and CCR5 KO. a 293T cells were transfected to generate CCR5gRNA CRISPR/Cas9 Tat NIL particles. TZM-TK-SR39 cells were transduced with the NIL lentivirus and the cells monitored for GFP expression and CCR5 down regulation. CCR5 negative cells were sorted 3 times to generate sort 1–3 (S1, S2, S3) followed by single cell cloning. b Flow cytometry analysis of (b). The TZM-TK-SR39 cells transduced with control gRNA packaged lentivirus or CCR5 gRNA lentivirus were stained for CCR5 expression prior to cell sorting. c The sorted cells (Sort 1–3) were assayed for surface CCR5 expression by flow cytometry. d Sort-3 cells were further subjected to single cell cloning and several potential clones screened for CCR5 downregulation. The selected clone H7 was stained for CCR5 and compared to the parental Sort-3 cells
Fig. 5
Fig. 5
Characterization of H7 cell line for resistance to HIV infection by CCR5 tropic HIV a TZM-TKSR39 or H7 cells were infected with HIV reporter virus particles pseudotyped with Lai or YU-2 Envelope. Infection was determined 24 h post infection by measuring luciferase activity in the cultures. b DNA was isolated from TZM-TKSR39 cell line or H7 clone and site of CRISPR mediated CCR5 disruption amplified using specific primers. PCR product was digested using the T7EI and bands were resolved on an agarose gel. c TZM, TZM-TK-SR39 or H7 cells were stained for CD4, CXCR4 and CCR5 expression using specific antibodies and analyzed by flow cytometry
Fig. 6
Fig. 6
H7 cell line shows Tat dependent TK gene expression and cytotoxicity in the presence of GCV. a TZM, TK-SR39 or H7 cells were transduced with control or Tat expressing lentivirus particles. Cells were lysed 48 h post infection and TK gene expression determined by western blotting. b TZM, TK-SR39 or H7 cells were transduced as in part A above and treated with the indicated concentrations of GCV. Percent viability was determined 48 h post transduction using the Cell Titer Glo luciferase based assay
Fig. 7
Fig. 7
H7 cells can control replication of both R5 and X4 tropic HIV. a TZM, TK-SR39 or H7 cells were infected with replication competent Lai or YU-2 virus in the presence or absence of 5 µg/ml GCV. Culture supernatants were harvested at day 6 post infection and an equal volume of supernatant used to infect TZM cells. Percent infection in TZM cells was determined 24 h post infection. b H7 cells were infected with HIV Lai or YU-2 virus as in part A. Images of whole wells were acquired using the Cytation5 imager
Fig. 8
Fig. 8
H7 cells resist R5 HIV infection and control X4 HIV via GCV treatment. (A) TZM, TK-SR39 or H7 cells were infected with replication competent Lai or YU-2 virus in the presence or absence of 5 µg/ml GCV. Cells were fixed at day 3 post infection and images acquired using the Nikon Eclipse Ti microscope
Fig. 9
Fig. 9
Overview of combination gene therapy for HIV combining CCR5 knock-out with a suicide gene
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