Preclinical development and qualification of ZFN-mediated CCR5 disruption in human hematopoietic stem/progenitor cells

Abstract

Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC). The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinical in vitro and in vivo testing to demonstrate the feasibility and (preclinical) safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo. Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov).

The changing landscape of gene editing in hematopoietic stem cells: a step towards Cas9 clinical translation

“…With the first genome editing of autologous HSCs clinical trial being approved by the FDA for the treatment of HIV using ZFNs targeting CCR5 40 , the new paradigm within cell and gene therapy has begun. Now with the variety of genome editing tools in the toolbox, we can easily typeset the genetic code with base pair precision to: 1) reactivate silenced genes through enhancer disruption, 2) replace good genes with bad genes and 3) revert disease-causing nucleotide substitutions, and with that ease of modification, comes great responsibility for all the researchers and clinicians involved.…”

Hematopoietic Stem Cell Gene Therapy: Progress and Lessons Learned

“…Wang et al, 2015). Trials targeting BCL11A are approaching the clinic (Chang et al, 2017) and several early phase clinical trials have been completed using ZFNs to modify the CCR5 gene in HIV-infected patient peripheral blood T-cells (Tebas et al, 2014) or HSCs (DiGiusto et al, 2016). …”

Recent advances in the use of ZFN-mediated gene editing for human gene therapy

“…Editing HSPCs instead of CD4 + T cells has the potential to provide a long-lasting source of modified cells. Success of this strategy has been established in preclinical studies [23] and a recent clinical trial (#NCT02500849) has been initiated using this approach. Programs to disrupt CCR5 in T cells and HSPCs, using the other nuclease platforms that include TALENs, CRISPR/Cas9 and megaTALs (a meganuclease fused to TAL effector modules), are also underway; these are at the pre-clinical stage.…”

“…The method of ZFN delivery could also vary depending on the human cell types. For example, Ad5/F35-mediated delivery of ZFNs was very efficient in CD4 + T cells while it was less efficient in HSPCs [23]. The nontoxic mRNA electroporation has been efficient for the introduction of ZFNs into HSPCs.…”