Gene silencing on a WHIM

With the report of silencing of the gain-of-function disease allele of a murine model of autosomal-dominant warts, hypogammaglobulinemia, recurrent infections, and myelokathexis (WHIM) syndrome,¹ in this issue of Blood, Gao et al open a new chapter in the remarkable story that inborn errors of immunity (IEI) have played in advancing the science of hematopoietic stem cell transplantation and autologous gene therapy.² 

Arguably, this began with the successful therapeutic hematopoietic stem cell transplant in 1968 of an infant with Wiskott-Aldrich syndrome and 2 infants with severe combined immunodeficiency (SCID). The first successful haploidentical transplant was also in a patient with SCID in the late 1970s, and the first cord blood transplant was reported in 1989 in a child with Fanconi anemia. A child with adenosine deaminase SCID was the first to receive gene addition therapy,³ and in 2016, the first license for a gammaretrovirus vector to use in gene addition therapy was granted by the European Medicines Agency,⁴ and clinical trials are underway for other IEI using lentiviral vectors, including IL2RG-, RAG1-, and artemis-deficient SCID, among others. The next refinement of genetic manipulation was gene editing (see figure), in which CRISPR DNA (CRISPR) and CRISPR-associated protein 9 (Cas9) can be used to repair a mutation by insertion of part or all of a normal copy of a gene at the appropriate physiological site. This maintains physiological control of gene expression, and with the addition of a corrected DNA template, error-free repair of the DNA break, using the ubiquitous homology-directed repair machinery, corrects the defect. This technology is being investigated for a number of IEI, some of which are close to entering clinical trials,⁵ and has entered clinical trials for sickle cell anemia. A number of IEI are caused by dominant gain-of-function mutations. Gene silencing or inactivation, using a precisely introduced nick in the mutated allele, with CRISPR/Cas9 might lead to loss of function of the allele and restoration of normal function, on condition that haploinsufficiency of the gene is not detrimental. This method is easier to perform than homology-directed mutation repair, because the nonhomologous end join repair mechanism activated by the Cas9-induced DNA break creates a loss-of-function mutation, which in this case is the desired outcome. A major issue, however, arises in specifically targeting the mutated allele but not affecting the wild-type allele.

WHIM syndrome is a rare IEI, due to germline mutations that result in gain-of-function of the chemokine receptor CXCR4 and characterized by severe neutropenia with retention of mature and apoptotic neutrophils in the bone marrow (myelokathexis), associated with hypogammaglobulinemia and susceptibility to human papillomavirus infection, leading to warts. Patients are normally diagnosed in childhood, and the presenting features may be diverse, with some diagnoses not made until adulthood. Life expectancy may be normal but is associated with significant sequalae in some patients.⁶ Conventional treatment is based on replacement of immunoglobulin, antibiotic prophylaxis, and administration of granulocyte colony-stimulating factor, usually daily. Inhibitors of CXCR4 may have a role in reducing the frequency of infection and the number of warts⁷ but require daily administration, and long-term effects of administering these agents are unknown. Hematopoietic stem cell transplantation has been performed in a few patients,⁸ and although results overall seem similar to those of transplantation for other IEI, nevertheless there are potential issues associated with graft-versus-host disease, and the risk-benefit ratio needs to be weighed carefully given the comparatively benign clinical course of the disease. In the report by Gao et al, the potential of using gene silencing to safely treat these patients has moved one step closer. Previously, the group had described the spontaneous cure of a patient with WHIM syndrome who experienced chromothripsis, likely in a single hematopoietic stem cell, in which the WHIM allele on chromosome 2 was deleted, leaving the cell hemizygous for wild-type CXCR4. Chromothripsis conferred a selective engraftment advantage to the stem cell, which led to almost all myeloid cells expressing the CXCR4^+/o phenotype and long-lived reversal of severe neutropenia and monocytopenia.⁹ Armed with the knowledge that “silencing” of the faulty gene may lead to the emergence of normal functioning hemizygous CXCR4^+/o stem cells, they used a murine model of WHIM and developed a CRISPR/Cas9-mediated Cxcr4 inactivation protocol, with a single guide RNA that did not discriminate between WHIM and wild-type Cxcr4 alleles. Having demonstrated that inactivating indels were introduced into wild-type or WHIM alleles, the group transplanted transfected hematopoietic stem and progenitor cells into lethally irradiated WHIM mice and showed that cells transduced with the active vector had a selective advantage over mock-transduced (and hence uncorrected) cells. Furthermore, WHIM allele–inactivated cells were selectively enriched, leading to hematopoietic reconstitution with mature Cxcr4^+/o leukocytes, supporting the concept that CRISPR/Cas9-edited Cxcr4^+/0 cells have a selective advantage in vivo, and this method may thus lead to a cure of the disease. There were no apparent clinical abnormalities in the transplanted mice as a result of treatment, and the authors did not examine clinical efficacy of the treatment. However, although significant preclinical work will be required before translating these findings to the clinic, including further investigation of off-target Cas9 activity and evidence of a therapeutic effect on the disease, nevertheless, with this report, the methods with which to approach treatment of genetic disorders have expanded. Gain-of-function, autosomal dominant diseases with no unfavorable influences conferred by haploinsufficiency may be less common causes of human disease, but a genetic therapeutic approach, at least for some diseases, may be on the not-too-distant horizon.

Conflict-of-interest disclosure: The author declares no competing financial interests.