{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T19:39:52Z","timestamp":1787254792117,"version":"build-2736575974"},"reference-count":35,"publisher":"SAGE Publications","issue":"2","license":[{"start":{"date-parts":[[2021,4,1]],"date-time":"2021-04-01T00:00:00Z","timestamp":1617235200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.sagepub.com\/page\/policies\/text-and-data-mining-license"}],"content-domain":{"domain":["journals.sagepub.com"],"crossmark-restriction":true},"short-container-title":["The CRISPR Journal"],"published-print":{"date-parts":[[2021,4,1]]},"abstract":"<jats:p>\n                    X-linked agammaglobulinemia (XLA) is a monogenic primary immune deficiency characterized by very low levels of immunoglobulins and greatly increased risks for recurrent and severe infections. Patients with XLA have a loss-of-function mutation in the Bruton's tyrosine kinase (\n                    <jats:italic toggle=\"yes\">BTK<\/jats:italic>\n                    ) gene and fail to produce mature B lymphocytes. Gene editing in the hematopoietic stem cells of XLA patients to correct or replace the defective gene should restore B cell development and the humoral immune response. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform to precisely target integration of a corrective, codon-optimized\n                    <jats:italic toggle=\"yes\">BTK<\/jats:italic>\n                    complementary DNA (cDNA) cassette into its endogenous locus. This process is driven by homologous recombination and should place the transgenic\n                    <jats:italic toggle=\"yes\">BTK<\/jats:italic>\n                    under transcriptional control of its endogenous regulatory elements. Each integrated copy of this cDNA in BTK-deficient K562 cells produced only 11% as much BTK protein as the wild-type gene. The donor cDNA was modified to include the terminal intron of the\n                    <jats:italic toggle=\"yes\">BTK<\/jats:italic>\n                    gene. Successful integration of the intron-containing\n                    <jats:italic toggle=\"yes\">BTK<\/jats:italic>\n                    donor led to a nearly twofold increase in BTK expression per cell over the base donor. However, this donor variant was too large to package into an adeno-associated viral vector for delivery into primary cells. Donors containing truncated variants of the terminal intron also produced elevated expression, although to a lesser degree than the full intron. Addition of the Woodchuck hepatitis virus posttranscriptional regulatory element led to a large boost in\n                    <jats:italic toggle=\"yes\">BTK<\/jats:italic>\n                    transgene expression. Combining these modifications led to a\n                    <jats:italic toggle=\"yes\">BTK<\/jats:italic>\n                    donor template that generated nearly physiological levels of BTK expression in cell lines. These reagents were then optimized to maximize integration rates into human hematopoietic stem and progenitor cells, which have reached potentially therapeutic levels\n                    <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                    . The novel donor modifications support effective gene therapy for XLA and will likely assist in the development of other gene editing-based therapies for genetic disorders.\n                  <\/jats:p>","DOI":"10.1089\/crispr.2020.0080","type":"journal-article","created":{"date-parts":[[2021,4,20]],"date-time":"2021-04-20T10:05:07Z","timestamp":1618913107000},"page":"191-206","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":27,"title":["Optimizing Integration and Expression of Transgenic Bruton's Tyrosine Kinase for CRISPR-Cas9-Mediated Gene Editing of X-Linked Agammaglobulinemia"],"prefix":"10.1177","volume":"4","author":[{"given":"David H.","family":"Gray","sequence":"first","affiliation":[{"name":"Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, USA; University of California, Los Angeles, Los Angeles, California, USA."},{"name":"Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA; University of California, Los Angeles, Los Angeles, California, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isaac","family":"Villegas","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California, USA; Departments of University of California, Los Angeles, Los Angeles, California, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph","family":"Long","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California, USA; Departments of University of California, Los Angeles, Los Angeles, California, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jasmine","family":"Santos","sequence":"additional","affiliation":[{"name":"Bioengineering, University of California, Los Angeles, Los Angeles, California, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandra","family":"Keir","sequence":"additional","affiliation":[{"name":"Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, California, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alison","family":"Abele","sequence":"additional","affiliation":[{"name":"Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caroline Y.","family":"Kuo","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California, USA; Departments of University of California, Los Angeles, Los Angeles, California, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1840-6087","authenticated-orcid":false,"given":"Donald B.","family":"Kohn","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California, USA; Departments of University of California, Los Angeles, Los Angeles, California, USA."},{"name":"Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA."},{"name":"Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, California, USA; University of California, Los Angeles, Los Angeles, California, USA."},{"name":"The Eli &amp; Edith Broad Center of Regenerative Medicine &amp; Stem Cell Research, University of California, Los Angeles, Los Angeles, California, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"179","published-online":{"date-parts":[[2021,4,20]]},"reference":[{"key":"e_1_3_6_2_2","first-page":"595","article-title":"Deficient expression of Bruton's tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection","volume":"91","author":"Futatani T","year":"1998","unstructured":"Futatani T, , Miyawaki T, , Tsukada S, et al. Deficient expression of Bruton's tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection. Blood. 1998; 91:595\u2013602. DOI: 10.1182\/blood.V91.2.595.","journal-title":"Blood"},{"key":"e_1_3_6_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/361226a0"},{"key":"e_1_3_6_4_2","doi-asserted-by":"publisher","DOI":"10.1002\/eji.1830010615"},{"key":"e_1_3_6_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(19)51014-6"},{"key":"e_1_3_6_6_2","doi-asserted-by":"publisher","DOI":"10.1542\/peds.9.6.722"},{"key":"e_1_3_6_7_2","first-page":"31","article-title":"Primary hypogammaglobulinaemia: A survey of clinical manifestations and complications","volume":"86","author":"Hermaszewski RA","year":"1993","unstructured":"Hermaszewski RA, , Webster AD. Primary hypogammaglobulinaemia: A survey of clinical manifestations and complications. QJM. 1993; 86:31\u201342. 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