Elizabeth Andrews

Introduction. Primary immunodeficiency disorders (PID) are commonly caused by point mutations and indels that cause defects in immune development and function, predisposing the body to various infections and secondary diseases^]. It is estimated that the prevalence of PID is about 1:100,000 3. Chronic granulomatous disease (CGD) and severe combined immunodeficiency (SCID) are excellent examples of two immunodeficiencies caused by point mutations or indels in a single gene. Symptoms tend to appear in the first few years of life and treatment options for these individuals are limited. Novel research into gene-editing technologies, like CRISPR-Cas9, have enabled ways in which the sequence of the human genome can be precisely manipulated to achieve a targeted therapeutic effect. CRISPR-Cas9 is an RNA-guided nuclease system that produces a targeted double-stranded break at a specific site on the DNA strand. This break is then repaired by imperfect non-homologous end joining (NHEJ) to produce a knockout genes or homology-directed repair (HDR) for precise sequence replacement. Studies are being done to enhance the HDR mechanism so that loss-of-function mutations can be replaced by a DNA-template containing the correct information10^,11. Research has shown that CRISPR-Cas9 can be effectively delivered to target cells and correct point mutations with little to no off-target cleavage in both CGD and SCID. Methods. Optimized CRISPR-Cas9 was applied to CD34+ hematopoietic stem cells (HSPCs) isolated from two X-linked CGD patients and using electroporation. Cell viability, proliferative capacity, and restoration of protein expression were determined by flow cytometry12. Whole genome sequencing was used to detect insertions or deletions outside of the targeted gene after correction12^,13^,5^,11. CRISPR-Cas9 optimized for SCID was tested for by nucleofecting induced pluripotent stem cell’s (IPSC) from skin keratinocytes of patients with this disease5. Results. CRISPR-Cas9 can be delivered efficiently into CD34⁺ HSPCs with low toxicities using a scalable electroporation system. The optimized CRISPR methodology converted a single base pair mutation to the normal sequence in CD34+ HSPCs of X-linked CGD patients, thereby restoring function of the targeted protein. No off-target sites were detected12. The SCID study demonstrated the correction of the human JAK3 mutation by CRISPR-Cas9, which restored the differentiation potential of early T cell progenitors. No off-target mutations were noted5. Conclusions: Proof of concept is shown for the CRISPR-Cas9 system’s targeted approach for gene mutation repair with little to no off-target activity. Currently, the NHEJ repair rate is greater than the HDR mechanism. However, with more research into the enhancement of the HDR mechanism, precise sequence replacement can be used to treat many different loss-of-function mutations causing primary immunodeficiencies. Applications for the CRISPR-Cas9 system in the treatment of disease are incredibly versatile, fast, and easy compared to other gene-editing technologies and has the potential to be used for a wide range of diseases caused by genetic mutations.

1. Sheikhbahaei S, Sherkat R, Roos D, Yaran M, Najafi S, Emami A. Gene mutations responsible for primary immunodeficiency disorders: A report from the first primary immunodeficiency biobank in Iran. Allergy, Asthma & Clinical Immunology. 2016; 12(1).
2. Al-Herz W, Aldhekri H, Barbouche M, Rezaei N. Consanguinity and Primary Immunodeficiencies. Human Heredity. 2014; 77(1-4), 138-143.
3. Kobrynski L, Powell RW, Bowen S. Prevalence and Morbidity of Primary Immunodeficiency Diseases, United States 2001-2007. Journal of Clinical Immunology. 2014; 34(8): 954-961.
4. Wu J, Wang WF, Zhange YD, Chen TX. Clincial features and genetic analysis of 48 patients with chronic granulomatous disease in a single center study from shanghai, china (2005-2015): new studies and a literature review. Journal of Immunology Research. 2017; 2017:8745254.
5. Chang CW, Lai YS, Westin E, Khodadadi-Jamayran A, et al. Modeling human severe combined immunodeficiency and correction by CRISPR-Cas9-enhanced gene targeting. Cell Reports. 2015; 12(10): 1668-1677.
6. Kang EM, Marciano BE, DeRavin S, Zarember KA, Holland SM, Malech HL. Chronic granulomatous disease: overview and hematopoietic stem cell transplantation. Journal of Allergy and Clinical Immunology. 2011; 127(6): 1319-1326.
7. Chiriaco M, Salfa I, Matteo GD, Rossi P, Finocchi A. Chronic granulomatous disease: clinical, molecular, and therapeutic aspects. Pediatric Allergy and Immunology. 2016; 27(3): 242-253.
8. Pellagatti A, Dolatshad H, Valletta S, Boultwood J. Application of CRISPR/Cas9 genome editing to the study and treatment of disease. Archives of Toxicology. 2015; 89(7): 1023-1034.
9. Ma Y, Zhang L, Huang X. Genome modification by CRISPR/Cas9. The FEBS Journal. 2014; 281(23): 5186-5193.
10. Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nature Biotechnology. 2015; 33: 538-542.
11. Lin S, Staahl BT, Alla RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Weigel D, ed. eLife. 2014;3:e04766.
12. Ravin, S.S., et al. (2017). CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-inked chronic granulomatous disease. Science Translational Medicine, 9. (372).
13. Lin Y, Cradick TJ, Brown MT, et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research. 2014;42(11):7473-7485
14. Maeder ML, Gersbach CA. Genome-editing technologies for gene and cell therapy. Molecular Therapy. 2016; 24(3): 430-446.
15. Sheikhbahaei S, Sherkat R, Roos D, Yaran M, Najafi S, Emami A. Gene mutations responsible for primary immunodeficiency disorders: A report from the first primary immunodeficiency biobank in Iran. Allergy, Asthma & Clinical Immunology. 2016; 12(1).
16. Al-Herz W, Aldhekri H, Barbouche M, Rezaei N. Consanguinity and Primary Immunodeficiencies. Human Heredity. 2014; 77(1-4), 138-143.
17. Kobrynski L, Powell RW, Bowen S. Prevalence and Morbidity of Primary Immunodeficiency Diseases, United States 2001-2007. Journal of Clinical Immunology. 2014; 34(8): 954-961.
18. Wu J, Wang WF, Zhange YD, Chen TX. Clincial features and genetic analysis of 48 patients with chronic granulomatous disease in a single center study from shanghai, china (2005-2015): new studies and a literature review. Journal of Immunology Research. 2017; 2017:8745254.
19. Chang CW, Lai YS, Westin E, Khodadadi-Jamayran A, et al. Modeling human severe combined immunodeficiency and correction by CRISPR-Cas9-enhanced gene targeting. Cell Reports. 2015; 12(10): 1668-1677.
20. Kang EM, Marciano BE, DeRavin S, Zarember KA, Holland SM, Malech HL. Chronic granulomatous disease: overview and hematopoietic stem cell transplantation. Journal of Allergy and Clinical Immunology. 2011; 127(6): 1319-1326.
21. Chiriaco M, Salfa I, Matteo GD, Rossi P, Finocchi A. Chronic granulomatous disease: clinical, molecular, and therapeutic aspects. Pediatric Allergy and Immunology. 2016; 27(3): 242-253.
22. Pellagatti A, Dolatshad H, Valletta S, Boultwood J. Application of CRISPR/Cas9 genome editing to the study and treatment of disease. Archives of Toxicology. 2015; 89(7): 1023-1034.
23. Ma Y, Zhang L, Huang X. Genome modification by CRISPR/Cas9. The FEBS Journal. 2014; 281(23): 5186-5193.
24. Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nature Biotechnology. 2015; 33: 538-542.
25. Lin S, Staahl BT, Alla RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Weigel D, ed. eLife. 2014;3:e04766.
26. Ravin, S.S., et al. (2017). CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-inked chronic granulomatous disease. Science Translational Medicine, 9. (372).
27. Lin Y, Cradick TJ, Brown MT, et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research. 2014;42(11):7473-7485.
28. Maeder ML, Gersbach CA. Genome-editing technologies for gene and cell therapy. Molecular Therapy. 2016; 24(3): 430-446.