Gene Editing for Primary Immunodeficiencies

Abstract

Hematopoietic stem cell (HSC) transplant from matched donors can cure patients with primary immune deficiencies (PID). For patients without such donors, gene therapy of autologous HSCs offers growing potential for clinical benefit. Current gene therapy trials employ integrating retroviral vectors to deliver normal copy(s) of the disease-causing gene, the expression of which is driven by a synthetic promoter. This approach poses risks of oncogenic transactivation of nearby genes and constitutive expression of the cargo gene that may be problematic for some. Gene editing holds promise for directing gene correction at a specific locus to improve safety and/or to restore constitutive gene regulation, however, correction of early HSCs has proved challenging. Advances in gene editing using zinc finger nucleases (ZFN), TALENs, meganucleases, or CRISPR-Cas9 have led to precise and efficient gene correction at levels that may be clinically beneficial in PID.

We evaluated targeted gene correction of CYBB (gp91phox) mutations causing X-linked chronic granulomatous disease (XCGD) due to defective phagocyte NADPH oxidase activity to reduced production of microbicidal reactive oxidative species (ROS). XCGD carriers with less than 10-20% ROS positive cells are at increased risk of infection suggesting a therapeutic target for clinical benefit. To accomplish gene therapy in XCGD, we designed a CRISPR-cas9 approach for targeted correction of a common mutation in CYBB exon 7 using a 100bp ssDNA oligo as a donor. Gene corrected XCGD patient HSCs showed gp91phox expression and ROS production following in vitro differentiation and corrected, functional multi-lineage cells were detected up to 6 months after transplant into immunodeficient NSG mice.

In XCGD, ~20% of patients have large deletions that are not amenable to repair using an oligo-donor. We utilized adeno-associated virus as a carrier for 2104bp of CYBB with homology arms, corresponding to exons 7-13. Using the same guide targeting CYBB exon 7, ~15% targeted integration of the CYBB coding region was achieved in vitro in XCGD patient HSC, accompanied by stage-appropriate gp91phox expression and ROS-producing DHR+ granulocytes. Together, these approaches for targeted repair of small (1-2 bp) mutations or large (multi-exon) deletions may offer new treatment options for patients with PID.

 

Biosketch
Suk See De Ravin’s interest in primary immune deficiency (PID) diseases began during her pediatric specialty training with a research doctorate on X-linked severe combined immunodeficiency. She subsequently worked on vector optimization and pre-clinical models for gene therapy using hematopoietic stem cells for X-SCID and other PIDs. Translation of these approaches to treatment of X-SCID patients resulted in significant clinical benefits. However, the lack of transgene regulation and random integration fueled impetus for continued search for improved gene therapy options. Using gene editing tools with Zinc fingers and CRISPR technology, she found homology repair resulted in efficient gene insertion into the safe harbor site and specific mutation repair respectively, at rates very promising for achieving significant clinical benefit. Thus, targeted gene editing of autologous HSCs has great potential for treatment of PIDs that may provide a safer genotoxic profile.