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).

Figure 1

Evaluation of the effects of electroporation of HSPC with rSB-728mR. (a) Extent of CCR5 disruption as estimated by (open box) MiSeq and (closed box) Surveyor nuclease assay (Cel-1) after electroporation with varying concentrations of research grade CCR5-specific ZFN mRNA (rSB-728mR). Regarding to MiSeq data, ~ 3,000–30,000 total sequence reads per sample were obtained and used for calculation of % CCR5 disruption (% indels). (b) Extent of modification of CCR5 and next four top off-target sequences after electroporation with varying concentrations of rSB-728mR. (c) Viability of HSPC on day 1 (D1) and day 2 (D2) after electroporation (EP). (d) The effects of electroporation on hematopoietic potential measured as colony forming units (CFU) of HSPC plated 2 days after electroporation with 50 or 150 μg/ml rSB-728mR or150 μg/ml GMP Grade SB-728 mRNA. Controls were treated with or without 150 μg/ml rSB-728mR but without electroporation (No EP). A total of 500 HSPC were plated from each sample, and CFUs were read at 14 days. *P < 0.05, **P < 0.01. CCR5, chemokine receptor 5; HSPCs, hematopoietic stem/progenitor cells; ZFNs, zinc finger nucleases.

Figure 2

Engraftment studies. CD34+ cells from two healthy donors were stimulated overnight and subjected to electroporation using 150 µg/ml of the GMP grade mRNA (SB-728mR). (a) The %CD45+ cells in bone marrow and spleen, analyzed at 20 weeks, are shown with error bars. The frequency (%) of the engrafted human CD45+ cells that were CD14+ monocytes, CD19+ B cells, CD4+ T cells and CD8+ T cells in bone marrow (b) and in spleen (c) are shown with error bars. *P < 0.05.

Figure 3

On-target and off-target sequence disruption. CD34+ cells were electroporated with 150 µg/ml SB-728mR and placed in bulk culture or methyl cellulose for CFU assay, as described in “Materials and Methods”. (a) Bulk cultures and single colonies were analyzed by miSeq and the percent of bulk or total CFU modified, percent of CFU with biallelic modifications among all CFU and percent of CFU with biallelic among all modified CFU is shown (b) The frequency of disruption among on/off target sequences are shown. CFU, colony forming units.

Figure 4

SB-728mR has no impact on HSPC engraftment. Cohorts of NSG mice were transplanted with 10⁶ HSPC per mouse from three independent donors (GLP#1, GLP#3, and GLP#4) following electroporation in the presence of 150 µg/ml SB-728mR or no electroporation and followed for percent CD45+ cells in peripheral blood at (a) 4-, 12, and 22-weeks and (b) in bone marrow (BM) at necropsy. *P < 0.05; **P < 0.01. Dotted line shows minimal threshold for defining engraftment with human cells. Note: BM samples were lost from one control and one SB-728mR-HSPC animal due to operator error. All week-22 GLP#4 control blood samples and 8 of 25 SB-728mR-HSPC blood samples were lost due to technical issues. HSPCs, hematopoietic stem/progenitor cells.

Figure 5

Percent CCR5 gene modification in mice transplanted with SB-728mR HSPC. NSG mice were transplanted with 10⁶ HSPC from three independent donors (GLP#1, GLP#3, and GLP#4) following electroporation (EP) in the presence of 150 µg/ml SB-728mR or no electroporation (No EP) and followed for percent CD45+ cells in peripheral blood mononuclear cells at 4-, 12-, and 22-weeks. CCR5, chemokine receptor 5; HSPCs, hematopoietic stem/progenitor cells.