CRISPR-Cas9 Engineering of the RAG2 Locus Via Complete Coding Sequence Replacement for Therapeutic Applications

Overview

Journal Nat Commun

Specialty Biology

Date 2023 Oct 27

PMID 37891182

Authors

Daniel Allen

Orli Knop

Bryan Itkowitz

Nechama Kalter

Michael Rosenberg

Ortal Iancu

Katia Beider

Yu Nee Lee

Arnon Nagler

Raz Somech

Ayal Hendel

Affiliations

Soon will be listed here.

Abstract

RAG2-SCID is a primary immunodeficiency caused by mutations in Recombination-activating gene 2 (RAG2), a gene intimately involved in the process of lymphocyte maturation and function. ex-vivo manipulation of a patient's own hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9/rAAV6 gene editing could provide a therapeutic alternative to the only current treatment, allogeneic hematopoietic stem cell transplantation (HSCT). Here we show an innovative RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) for the purpose of preserving the critical endogenous spatiotemporal gene regulation and locus architecture. Expression of the corrective transgene leads to successful development into CD3TCRαβ and CD3TCRγδ T cells and promotes the establishment of highly diverse TRB and TRG repertoires in an in-vitro T-cell differentiation platform. Thus, our proof-of-concept study holds promise for safer gene therapy techniques of tightly regulated genes.

Citing Articles

Identification of tacrolimus-related genes in familial combined hyperlipidemia and development of a diagnostic model using bioinformatics analysis.

Xu Y, He H, Li H Heliyon. 2025; 11(3):e41705.

PMID: 39916852 PMC: 11800081. DOI: 10.1016/j.heliyon.2025.e41705.

Large Donor CRISPR for Whole-Coding Sequence Replacement of Cell Adhesion Molecule LRRTM2.

Pollitt S, Levy A, Anderson M, Blanpied T J Neurosci. 2025; 45(7).

PMID: 39824639 PMC: 11823385. DOI: 10.1523/JNEUROSCI.1461-24.2024.

Homology-Directed-Repair-Based Genome Editing in HSPCs for the Treatment of Inborn Errors of Immunity and Blood Disorders.

Allen D, Kalter N, Rosenberg M, Hendel A Pharmaceutics. 2023; 15(5).

PMID: 37242571 PMC: 10220672. DOI: 10.3390/pharmaceutics15051329.

References

Wilson A, HELD W, MacDonald H . Two waves of recombinase gene expression in developing thymocytes. J Exp Med. 1994; 179(4):1355-60. PMC: 2191458. DOI: 10.1084/jem.179.4.1355. View

Cowan M, Gennery A . Radiation-sensitive severe combined immunodeficiency: The arguments for and against conditioning before hematopoietic cell transplantation--what to do?. J Allergy Clin Immunol. 2015; 136(5):1178-85. PMC: 4641002. DOI: 10.1016/j.jaci.2015.04.027. View

Notarangelo L, Kim M, Walter J, Lee Y . Human RAG mutations: biochemistry and clinical implications. Nat Rev Immunol. 2016; 16(4):234-46. PMC: 5757527. DOI: 10.1038/nri.2016.28. View

Barron M, Makhija M, Hagen L, Pencharz P, Grunebaum E, Roifman C . Increased resting energy expenditure is associated with failure to thrive in infants with severe combined immunodeficiency. J Pediatr. 2011; 159(4):628-32.e1. DOI: 10.1016/j.jpeds.2011.03.041. View

de Ravin S, Wu X, Moir S, Anaya-OBrien S, Kwatemaa N, Littel P . Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency. Sci Transl Med. 2016; 8(335):335ra57. PMC: 5557273. DOI: 10.1126/scitranslmed.aad8856. View

Nelson C, Wu Y, Gemberling M, Oliver M, Waller M, Bohning J . Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nat Med. 2019; 25(3):427-432. PMC: 6455975. DOI: 10.1038/s41591-019-0344-3. View

Gilpatrick T, Lee I, Graham J, Raimondeau E, Bowen R, Heron A . Targeted nanopore sequencing with Cas9-guided adapter ligation. Nat Biotechnol. 2020; 38(4):433-438. PMC: 7145730. DOI: 10.1038/s41587-020-0407-5. View

Booth C, Gaspar H, Thrasher A . Treating Immunodeficiency through HSC Gene Therapy. Trends Mol Med. 2016; 22(4):317-327. DOI: 10.1016/j.molmed.2016.02.002. View

Canny M, Moatti N, Wan L, Fradet-Turcotte A, Krasner D, Mateos-Gomez P . Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat Biotechnol. 2017; 36(1):95-102. PMC: 5762392. DOI: 10.1038/nbt.4021. View

10.

Gardner C, Pavel-Dinu M, Dobbs K, Bosticardo M, Reardon P, Lack J . Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System. J Clin Immunol. 2021; 41(5):852-862. PMC: 8254788. DOI: 10.1007/s10875-021-00989-6. View

11.

Amit I, Iancu O, Levy-Jurgenson A, Kurgan G, McNeill M, Rettig G . CRISPECTOR provides accurate estimation of genome editing translocation and off-target activity from comparative NGS data. Nat Commun. 2021; 12(1):3042. PMC: 8144550. DOI: 10.1038/s41467-021-22417-4. View

12.

Kisielow P, Miazek A, Cebrat M . NWC, a new gene within RAG locus: could it keep GOD under control?. Int J Immunogenet. 2008; 35(4-5):395-9. DOI: 10.1111/j.1744-313X.2008.00791.x. View

13.

Yu W, Misulovin Z, Suh H, Hardy R, Jankovic M, Yannoutsos N . Coordinate regulation of RAG1 and RAG2 by cell type-specific DNA elements 5' of RAG2. Science. 1999; 285(5430):1080-4. DOI: 10.1126/science.285.5430.1080. View

14.

Wang D, Tai P, Gao G . Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019; 18(5):358-378. PMC: 6927556. DOI: 10.1038/s41573-019-0012-9. View

15.

Kang H, Bartholomae C, Paruzynski A, Arens A, Kim S, Yu S . Retroviral gene therapy for X-linked chronic granulomatous disease: results from phase I/II trial. Mol Ther. 2011; 19(11):2092-101. PMC: 3222528. DOI: 10.1038/mt.2011.166. View

16.

Themeli M, Chhatta A, Boersma H, Prins H, Cordes M, de Wilt E . iPSC-Based Modeling of RAG2 Severe Combined Immunodeficiency Reveals Multiple T Cell Developmental Arrests. Stem Cell Reports. 2020; 14(2):300-311. PMC: 7013232. DOI: 10.1016/j.stemcr.2019.12.010. View

17.

Shapiro J, Tovin A, Iancu O, Allen D, Hendel A . Chemical Modification of Guide RNAs for Improved CRISPR Activity in CD34+ Human Hematopoietic Stem and Progenitor Cells. Methods Mol Biol. 2020; 2162:37-48. DOI: 10.1007/978-1-0716-0687-2_3. View

18.

Shapiro J, Iancu O, Jacobi A, McNeill M, Turk R, Rettig G . Increasing CRISPR Efficiency and Measuring Its Specificity in HSPCs Using a Clinically Relevant System. Mol Ther Methods Clin Dev. 2020; 17:1097-1107. PMC: 7251314. DOI: 10.1016/j.omtm.2020.04.027. View

19.

Gil-Farina I, Fronza R, Kaeppel C, Lopez-Franco E, Ferreira V, DAvola D . Recombinant AAV Integration Is Not Associated With Hepatic Genotoxicity in Nonhuman Primates and Patients. Mol Ther. 2016; 24(6):1100-1105. PMC: 4923321. DOI: 10.1038/mt.2016.52. View

20.

Miyazaki K, Watanabe H, Yoshikawa G, Chen K, Hidaka R, Aitani Y . The transcription factor E2A activates multiple enhancers that drive expression in developing T and B cells. Sci Immunol. 2020; 5(51). DOI: 10.1126/sciimmunol.abb1455. View