The Berlin patient is a unique case and dependent on

The Berlin patient is a unique case and dependent on the histocompatibility-matched CCR5Δ32/Δ32 homozygous donors. These donors occur at a little higher rate in the European Caucasian population, but they are very rare in Asians and Africans.15, 16, 17 The recently developed genome-editing technology, such as zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALENs), and CRISPR and CRISPR-Cas9, provides powerful approaches for CCR5 artificial modification.8, 18, 19 These approaches are making it possible to modify self-cells to resist HIV infection, and they represent promising approaches for autologous cell-based therapy. Positive results have been obtained in both preclinical and clinical trials in which HIV-infected patients were transplanted with autologous ZFN-disrupted CCR5 CD4+ cells.20, 21, 22 Investigators have also ablated the entire CCR5 gene, but the long-term adverse effects of CCR5-disrupted cell transplantation are unknown. In contrast, naturally occurring CCR5Δ32/Δ32 homozygotes are healthy in the population. Thus, CCR5Δ32/Δ32 induction may be a much better approach than CCR5 disruption. Ye et al. successfully created mutant CCR5Δ32/Δ32 homozygotes in induced pluripotent stem LY2584702 using the combination of piggyBac transposon technology and TALENs or CRISPR-Cas9 technology. While exciting, their technological approach was complicated. In this paper, we have developed a more simple approach to induce CCR5Δ32/Δ32 homozygotes in human cells using only the CRISPR-Cas9 technology and a pair of single-guide RNAs.
Results
Discussion In this study, we successfully converted wild-type CCR5 genes into CCR5Δ32/Δ32 homozygotes in a CD4+ Jurkat cell line and in primary human CD4+ cells by using CRISPR-Cas9 technology. This approach is simple and effective, better than the previous combination use of piggyBac transposon technology and TALENs or CRISPR-Cas9 technology. This approach should be easily transferred to hematopoietic stem and progenitor cells (HSPCs), which are longer-lasting than primary CD4+ cells. Ye et al. employed the induced pluripotent stem cells (iPSCs). However, the safety of iPSCs is still under study. CRISPR-Cas9 safety is another concern before applying the technique for clinical use. Dual safety problems are enough to prohibit the clinical use of this therapy. Thus, we did not try it in iPSCs. In other studies, adenovirus delivery system or mRNA electroporation was applied, in contrast to the lentiviral delivery system in this study. This CRISPR-Cas9 approach in this study is easily transferred to mRNA electroporation. The lentiviral delivery system can be used ex vivo, but not in vivo. Following analysis of the Berlin patient, Dr. Hutter reported another six HIV-infected patients received bone marrow transplantation from donors of CCR5Δ32/Δ32 homozygotes. But none of these transplantations succeeded. All patients died either from infections or from the relapse of lymphoma within 1 year. Kordelas and colleagues also reported a failed case, where the patient died from the CXCR4-tropic variant rapid rebound after the transplantation. Therefore, CXCR4-tropic or dual-tropic HIV viruses are enough to ruin the goal of an HIV/AIDS cure based on CCR5-targeting therapy. The Berlin patient benefited from the primary presence of the CCR5-tropic viruses. Disrupting the CXCR4 gene may not be a practical approach, because CXCR4 plays an important role in hematopoietic stem cell homing and retention. However, we do find CCR5-only-tropic virus infections. Circulating Recombinant Form 07_BC (CRF07_BC) is the most predominant epidemic HIV-1 viruses in China. Until now, all of isolated CRF07-BC viruses are CCR5 tropic, and CXCR4-tropic (X4) viruses have thus far not been found in CRF07_BC. Moreover, Zhang et al. tried to artificially mutate CRF07-BC Env V3 loop into CXCR4-like amino acids, such as two amino acid insertions between positions 13 and 14, as well as an arginine substitution at position 11 or 16 (IG insertion and P16R mutation or MG insertion and S11R mutation). These CXCR4-like mutations in CRF07-BC completely abrogated virus infectivity. Therefore, natural or artificial mutated CRF07-BC viruses are all CCR5 tropic. This is good news for this study. At least we can apply a CCR5-targeting strategy in CCR5-tropic virus, such as HIV-1 CRF07-BC.