Approximately 6 million people worldwide suffer from primary inherited immunodeficiency diseases which pose a life-long challenge for those affected by them (Bousfiha et al., 2013). Chronic Granulomatous Disease (CGD) is one such illness that impairs the ability of the innate immune system to respond to bacterial and fungal pathogens. Specifically, phagocytes are unable to produce sufficient amounts of microbicidal reactive oxygen species (ROS) to be effective (Ben-Ari et al., 2012). In addition to life-threatening infections, patients living with CGD also experience unchecked inflammation resulting in painful and clinically complicated granulomas (Rosenzweig, 2008).
In phagocytes from CGD patients, ROS production is altered by mutations in various upstream genes including CYBB (Heyworth et al., 2003). Previously, Cybb function was disrupted in C57BL/6 (B6.129S-Cybbtm1Din/J, stock# 002365) mice resulting in a mouse model of CGD (Pollock et al., 1995). However, the immunocompetent C57BL/6 genetic background prevents the study of engrafted human tissue.
A promising therapeutic approach to treat CGD involves gene therapy on hematopoietic stem cells isolated from patients, followed by autologous cell transplantation. As a key complement to this therapeutic paradigm, an appropriate pre-clinical animal model for CGD that is capable of hosting xenografted tissue is crucial for testing new therapies.
To this end, highly immunodeficient mouse lines like the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG™, Stock# 005557) are an exciting and powerful development, particularly in understanding human immunology. That these mice are capable of immune cell humanization enhances the translational relevance of the model. While NSG mice are highly deficient in lymphocytes, a limitation to using this strain is that NSG mice still maintain some phagocyte functionality, potentially confounding evaluation of therapies aimed at correcting defective phagocytes in engrafted human tissue.
In a recent article in Human Gene Therapy, Dr. Colin Sweeney and colleagues at the National Institute of Allergy and Infectious Disease created a knockout of Cybb in NSG mice; this resulted in phagocytes that are incapable of ROS production in a mouse model that is capable of engrafting human hematopoietic cells. This model is an exciting tool for CGD researchers, as an in vivo model devoid of ROS production by phagocytes is a potentially powerful platform to test novel GCD therapies.
To generate a novel CGD mouse model, Dr. Sweeney and colleagues used CRISPR/Cas9 methodology to create a 235 bp deletion in exon 1 of Cybb in the highly immunodeficient NSG mouse strain. Analysis of peripheral blood phagocytes derived from Cybb knockout mice revealed that ROS production was abrogated. Cybb knockouts also displayed increased susceptibility to pathogens and a corresponding decrease in survival rate.
As a proof of concept, CD34+ human hematopoietic stem cells from donors with or without CGD were engrafted in Cybb knockout mice. As expected, engrafted hematopoietic stem cells successfully differentiated into phagocytes such as monocytes and macrophages, as well as B cells, NK cells, and T cells. Moreover, phagocytes that arose from human CGD-affected donors failed to produce ROS, while phagocytes from healthy donors exhibited functional ROS activity. When hematopoietic stem cells from CGD patients transduced with a lentiviral vector expressing functional human CYBB were engrafted into Cybb knockout mice, ROS activity was subsequently detected in phagocytes.
This study represents an exciting step forward in the development of an in vivo model for CGD capable of engrafting healthy or diseased human hematopoietic stem cells. The authors of this study demonstrate that fusing CRISPR/Cas9 technology with the highly immunodeficient NSG mouse is an exciting paradigm for the development of preclinical testing platforms specifically designed for greater translational relevance.
Sweeney, C.L., Choi, U., Liu, C., Koontz, S., Ha, S.-K., Malech, H.L., 2017. CRISPR-Mediated Knockout of Cybb in NSG Mice Establishes a Model of Chronic Granulomatous Disease for Human Stem-Cell Gene Therapy Transplants. Hum. Gene Ther. PMID: 28264583
Ben-Ari, J., Wolach, O., Gavrieli, R., Wolach, B., 2012. Infections associated with chronic granulomatous disease: linking genetics to phenotypic expression. Expert Rev. Anti Infect. Ther. 10, 881–894. PMID: 23030328
Bousfiha, A.A., Jeddane, L., Ailal, F., Benhsaien, I., Mahlaoui, N., Casanova, J.-L., Abel, L., 2013. Primary immunodeficiency diseases worldwide: more common than generally thought. J. Clin. Immunol. 33, 1–7. PMID: 22847546
Heyworth, P.G., Cross, A.R., Curnutte, J.T., 2003. Chronic granulomatous disease. Curr. Opin. Immunol. 15, 578–584. PMID: 14499268
Pollock, J.D., Williams, D.A., Gifford, M.A., Li, L.L., Du, X., Fisherman, J., Orkin, S.H., Doerschuk, C.M., Dinauer, M.C., 1995. Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat. Genet. 9, 202–209. PMID: 7719350
Rosenzweig, S.D., 2008. Inflammatory manifestations in chronic granulomatous disease (CGD). J. Clin. Immunol. 28 Suppl 1, S67-72. PMID: 18193341