Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection

Abstract

Significance Patients homozygous for the C-C chemokine receptor type 5 (CCR5) gene with 32-bp deletions (Δ32) are resistant to HIV infection. Using the piggyBac technology plus transcription activator-like effector nucleases or clustered regularly interspaced short palindromic repeats-Cas9, the authors report, to our knowledge, for the first time in induced pluripotent stem cells (iPSCs) the efficient and seamless derivation of a homozygous CCR5Δ32 mutation, exactly mimicking the natural mutation. Monocytes and macrophages differentiated from these mutated iPSCs in vitro are resistant to HIV infection. This approach can be applied in the future toward the functional cure of HIV infection. The findings are also of great interest to researchers in many fields who wish to correct or introduce mutations in specific genes.

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

“…Owing to the indispensable role of CCR5 for HIV-1 entry and the natural presence of the CCR5 D32 variant, a number of gene editing studies have targeted CCR5 by various approaches (Badia et al, 2014;Holt et al, 2010;Huang et al, 1996;Mussolino et al, 2014;Perez et al, 2008;Saito et al, 2014;Ye et al, 2014). In a recently finished Phase I clinical trial, autologous CD4 + T-cells with CCR5 modified by ZFN showed promise in alleviating the disease condition of some HIV-1 patients (Tebas et al, 2014).…”

“…In eukaryotes, the DSBs are more commonly repaired by the mechanism of error-prone non-homologous end joining (NHEJ), therefore generating sequence changes, for instance insertions and deletions (indels), around the DSBs . Owing to the simplicity of manipulation and versatility, the CRISPR/Cas9 system has been utilized as an attractive tool for various applications, such as genome-wide screening (Shalem et al, 2014;Zhou et al, 2014), gene repression and activation (Cheng et al, 2013;Doench et al, 2014;Gilbert et al, 2014), targeted fluorescence imaging (Tanenbaum et al, 2014) and novel approaches against pathogens including hepatitis B virus (Lin et al, 2014a;Seeger & Sohn, 2014), human papillomavirus (Kennedy et al, 2014), Epstein-Barr virus (Wang & Quake, 2014;Yuen et al, 2015), malaria (Wagner et al, 2014) and HIV-1 (Ebina et al, 2013;Hu et al, 2014;Ye et al, 2014).…”

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…TALENs and CRISPR have not yet been trialed in humans. However, the results from in vitro studies are very promising [223] , with CRISPR editing able to excise the provirus from infected cells, and thus able to target latent proviruses [224] . ZFNs have also been used to target the provirus, using lentivirus to achieve stable expression of the nucleases [225] .…”

Engineering HIV-Resistant, Anti-HIV Chimeric Antigen Receptor T Cells

“…36,37 Individuals with an allelic variant that is not functional (CCR5D32) are protected from CCR5-tropic HIV infection. 38 Hematopoietic stem cell transplant using a CCR5D32 donor led to the only known cure of HIV-1 infection, 39,40 and T cells treated with engineered nucleases that introduce mutations at the CCR5 locus are resistant to HIV, [41][42][43][44][45] accelerating ongoing efforts to develop gene editing-and cell-based therapeutic agents for HIV. 11,46 Using new gene-editing techniques, it has recently become possible to achieve high rates of homology-directed recombination (HDR) of therapeutic cassettes into targeted loci, including CCR5 in primary T cells.…”