A Pipeline for Comparison and Selection of Clinically Applicable Gene Therapy Approaches for ELANE-Associated Severe Congenital Neutropenia

Several gene editing strategies for autosomal dominant ELANE-associated severe congenital neutropenia (CN) have been proposed. All strategies efficiently correct CN with different degrees of the investigated comprehensiveness of toxicity and off-target activity. The next step now is to select the best approach for clinical translation. The first-line awareness should consider not only the universal applicability of gene editing but, most importantly, its safety. Since CN patients have a high cumulative incidence of leukemogenic transformation and pre-leukemia cells in the patient's bone marrow are detected at an early age, safety considerations are of the utmost importance.

We compared gene-editing methods head-to-head in CD34 ⁺ cells of two patients with hot-spot ELANE mutations associated with a poor response to G-CSF and high risk for leukemia development, p.A57V and p.G214V. We edited cells with the universal ELANE knockout targeting wild-type and mutant alleles in exon 2 ( uEKO). We also repaired the mutated ELANE by inducing a double-stranded break (DSB) at the position of mutation with the delivery of the WT ELANE template for homology-driven repair (HDR) by AAV6 ( HDR-AAV). We also tested the allele-specific knockout of the mutated allele ( ASKO) and the allele-specific HDR approach that relies on ssODNs to deliver the wild-type sequence for p.G214V mutation ( AS-HDR).

We investigated the effects of gene editing on the survival and granulocytic differentiation of gene-edited cells as well as editing efficiency and patient-specific genome-wide off-target profiles. Cell survival was between 74 % and 95 % in all groups post-gene editing.

For patient 1 with p.A57V mutation, we observed gene editing of 62 % in the control-AAVS1 locus, 70 % in uEKO, 76 % in ASKO, and 10.3 % HDR with an additional 49.5 % of KO in HDR-AAV. The editing led to a significant increase in differentiated neutrophils from 14.5 % in the control group to 57.4 %, 55.2 %, and 45.3 % in uEKO, ASKO and HDR-AAV groups, respectively. Gene editing also facilitated a significant increase in CFU-G and CFU-GM colonies for all treatment conditions, as compared to the control.

For patient 2 with p.G214V mutation, we measured 86 % indels in control-AAVS1, 34.4 % in uEKO and 80.25 % in HDR-AAV. In the AS-HDR group, no HDR was observed, but 92 % of mutant alleles contained frameshift indels. The edits resulted in a significant increase in neutrophils in liquid culture differentiation from 13.6 % in the control AAVS1group to 53.7 %, 57.3 %, and 62.8 % in uEKO, HDR-AAV, and AS-HDR, respectively. CFU-G and CFU-GM colonies were also enhanced.

To confirm if allele-specific frameshifts in exon 5 restored granulopoiesis, we utilized a patient-derived iPSC cell line with the p.G214R mutation. We applied ASKO gene editing to this iPSC line and achieved 24 % ASKO. The editing improved neutrophil differentiation significantly, from 2.4 % in unedited cells to 25.55 % in edited cells. CFU assays also showed a significant increase in CFU-G and CFU-GM numbers.

To detect off-targets on the patient genotype, we established GUIDE-Seq in patient-derived iPSCs with mutations p.A57V (iCN1) and p.G214R (iCN2). In iCN1, we found 10 off-targets (OT) for the uEKO sgRNA and 3 OTs for the ASKOsgRNA. For iCN2, we found 3 OTs for the HDR-AAV and the AS-HDR guides. These findings show that allele-specific guides have better OT profiles than guides targeting both alleles.

Taken together, all tested approaches have low toxicity and comparable efficacy in restoring granulopoiesis in vitro. We found that editing above 30 % efficiently restored granulopoiesis, providing evidence for the therapeutic threshold. This highlights that safety and genome toxicity considerations should guide further clinical translation. To enable clinically relevant off-target activity detection of gene-editing in the patient's genome, GUIDE-Seq in patient-derived iPSCs should be performed. Further, our observation that the introduction of allele-specific frameshift indels in exon 5 can inactivate CN-causing mutations is unexpected and warrants further investigation. Head-to-head comparison of the in vivo engraftment of CD34+ cells gene-edited using several approaches should also be performed in the future.