Sehar Salman

Introduction: X Linked Severe Combined Immunodeficiency (XSCID) presents as a severe, and sometimes complete, lack of T cells, consequently resulting in B cell defects, and leading to a combined immune deficiency. It is often caused by a single genetic mutation and affects about 1 in 50,000 newborns⁶ who face high likelihood of death within the first year of life. The only potentially curative treatment is an allogeneic hematopoietic stem cell transplant (HSCT) from an HLA matched donor^1,4 – an obstacle many patients may face. The expansion in gene editing technologies however, prompts an investigation into gene correction and the potential for subsequent autologous HSCT.¹ The CRISPR/Cas9 system allows for a faster, more accurate method of gene editing that can be used in primary immune disorders like SCID. Methods: Patient derived HSCs and iPSCs were used. Site-specific double stranded breaks (DSBs) are created by Cas9, and a correction template is used to drive homology directed repair. A green fluorescent protein (GFP) marker was used to evaluate the efficacy of transduction³ and differentiation protocols were used to ensure multipotency. Flow cytometric analysis was used repeatedly to identify, sort, and count differentiated cell types. To confirm expression of the correct gene, studies used qualitative reverse transcriptase PCR and Western Blot. And lastly, to confirm that the proper T cell function, corrected cells were stimulated by anti-CD3/CD28 beads.² Results: The CRISPR/Cas9 model allowed for a site specific change of a single nucleotide progenitor (HSCs and iPSCs) cells.^2,3,5 This gene editing method was more effective than unassisted viral vector transduction.³ The corrected progenitor cells differentiated into mature erythrocytes^3,5 and functional mature T cells² that produced appropriate proteins and receptors^2,3,5. And after engraftment in a mouse host, the corrected stem cells were able to reconstitute both myeloid and lymphoid lineages, confirming the multipotency of the corrected cells.^3,5 The SCID phenotype was modeled in human iPSCs and a specific gene mutation was corrected through CRISPR/Cas9 gene editing.² This corrected stem cells showed normal T cell development and functionality.² Conclusions:  Studies have demonstrated that CRISPR/Cas 9 is a useful technique for gene correction and in the case of SCID can ultimately restore normal T cell development and maturation. Corrected HSCs can be safely engrafted in mouse models and may eventually be used in autologous transplants, which eliminate the need for a donor and decrease complications associated with allogeneic graft rejection.

1. Booth, C., Gaspar, H. B., & Thrasher, A. J. (2016). Treating immunodeficiency through HSC gene therapy. Trends in molecular medicine, 22(4), 317-327.
2. Chang, C. W., Lai, Y. S., Westin, E., Khodadadi-Jamayran, A., Pawlik, K. M., Lamb, L. S., … & Townes, T. M. (2015). Modeling human severe combined immunodeficiency and correction by CRISPR/Cas9-enhanced gene targeting. Cell reports, 12(10), 1668-1677.
3. Dever, D. P., Bak, R. O., Reinisch, A., Camarena, J., Washington, G., Nicolas, C. E., … & Uchida, N. (2016). CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature, 539(7629), 384-389.
4. Learning About Severe Combined Immunodeficiency (SCID). National Human Genome Research Institute, NIH website. https://www.genome.gov/13014325/learning-about-severe-combined-immunodeficiency-scid/. Last updated June 2, 2014. Accessed Mar 23, 2017.
5. Ou, Z., Niu, X., He, W., Chen, Y., Song, B., Xian, Y., … & Sun, X. (2016). The Combination of CRISPR/Cas9 and iPSC Technologies in the Gene Therapy of Human β-thalassemia in Mice. Scientific Reports, 6.
6. Ou, Z., Niu, X., He, W., Chen, Y., Song, B., Xian, Y., … & Sun, X. (2016). The Combination of CRISPR/Cas9 and iPSC Technologies in the Gene Therapy of Human β-thalassemia in Mice. Scientific Reports, 6.