A New Chapter on Targeted Gene Insertion for X-CGD: Do Not Skip the Intro(n)
Authors: Giorgia Santilli, Adrian J. Thrasher
Journal: Molecular Therapy (2017)

X-linked chronic granulomatous disease (X-CGD) is a rare, devastating primary immunodeficiency disorder (PID) characterized by a defective host defense against certain bacteria and fungi.1Roos D. de Boer M. Molecular diagnosis of chronic granulomatous disease.Clin. Exp. Immunol. 2014; 175: 139-149Crossref PubMed Scopus (108) Google Scholar It is caused by mutations in the CYBB gene, encoding the gp91phox subunit of NADPH oxidase, an enzyme complex responsible for pathogen clearance in professional phagocytic cells. Like many monogenic disorders of the immune system, X-CGD is amenable to gene therapy through gene addition. More recently, scientists have also started to explore nuclease-mediated targeted gene insertion as a strategy to restore gp91phox expression in reprogrammed induced pluripotent stem cells (iPSCs)2Zou J. Sweeney C.L. Chou B.K. Choi U. Pan J. Wang H. Dowey S.N. Cheng L. Malech H.L. Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting.Blood. 2011; 117: 5561-5572Crossref PubMed Scopus (209) Google Scholar, 3Merling R.K. Sweeney C.L. Chu J. Bodansky A. Choi U. Priel D.L. Kuhns D.B. Wang H. Vasilevsky S. De Ravin S.S. et al.An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease.Mol. Ther. 2015; 23: 147-157Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 4Flynn R. Grundmann A. Renz P. Hänseler W. James W.S. Cowley S.A. Moore M.D. CRISPR-mediated genotypic and phenotypic correction of a chronic granulomatous disease mutation in human iPS cells.Exp Hematol. 2015; 43: 838-848.e3Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar and hematopoietic stem and progenitor cells (HSPCs)5De Ravin S.S. Reik A. Liu P.-Q. Li L. Wu X. Su L. Raley C. Theobald N. Choi U. Song A.H. et al.Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease.Nat. Biotechnol. 2016; 34: 424-429Crossref PubMed Scopus (142) Google Scholar from CGD patients. In this issue of Molecular Therapy, Sweeny et al.,6Sweeney C.L. Zou J. Choi U. Merling R.K. Liu A. Bodansky A. Burkett S. Kim J.-W. De Ravin S.S. Malech H.L. Targeted repair of CYBB in X-CGD iPSCs requires retention of intronic sequences for expression and functional correction.Mol Ther. 2017; 25 (this issue): 321-330Abstract Full Text Full Text PDF Scopus (33) Google Scholar show that nuclease-mediated insertion of a CYBB minigene into exon 2, but surprisingly not exon 1, can restore gp91phox protein expression in iPSC-derived granulocytes from X-CGD patients (Figure 1). This study highlights the importance of intronic elements for the expression of the CYBB gene by the endogenous regulatory elements and cautions those developing strategies for genetic correction by targeted insertion of minigenes. HSPC gene therapy by the use of viral vectors has proven to be an effective therapeutic option for a series of PIDs. However, previous clinical trials for CGD, based on the use of gamma retroviral vectors, have, in general, failed to demonstrate significant long-term engraftment of gene-corrected cells in the absence of mutagenic proliferation of myeloid precursors.7Grez M. Reichenbach J. Schwäble J. Seger R. Dinauer M.C. Thrasher A.J. Gene therapy of chronic granulomatous disease: the engraftment dilemma.Mol. Ther. 2011; 19: 28-35Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar The recent introduction of lentiviral vectors with myeloid regulatory elements may not only improve safety by obviating enhancer-mediated mutagenesis in HSPCs, but may also drive gene expression closer to more physiological levels. Clinical trials in the US and Europe are currently underway. Alternatively, transposon-mediated integration of a bacterial artificial chromosome (BAC) vector carrying the whole genomic region of the CYBB gene has been shown to rescue the CGD phenotype in granulocytes derived from iPSCs.8Laugsch M. Rostovskaya M. Velychko S. Richter C. Zimmer A. Klink B. Schröck E. Haase M. Neumann K. Thieme S. et al.Functional restoration of gp91phox-oxidase activity by BAC transgenesis and gene targeting in X-linked chronic granulomatous disease iPSCs.Mol. Ther. 2016; 24: 812-822Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar However, clinical application of BAC transgenesis for therapy of CGD or other disorders is challenging because of the size of the payload required to deliver the necessary regulatory elements as well as the coding sequence. DNA editing techniques have opened up new exciting avenues for gene therapy of genetic disorders.9Maeder M.L. Gersbach C.A. Genome-editing Technologies for Gene and Cell Therapy.Mol. Ther. 2016; 24: 430-446Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar Scientists can now use target-directed nucleases to create a double-strand break anywhere in the genome and insert a new DNA sequence into the break by homology-directed repair (HDR). This approach has been used, for example, to insert minigenes and, among those, the CYBB minigene into the AAVS1 locus, a putative genomic safe harbor that is also resistant to silencing.2Zou J. Sweeney C.L. Chou B.K. Choi U. Pan J. Wang H. Dowey S.N. Cheng L. Malech H.L. Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting.Blood. 2011; 117: 5561-5572Crossref PubMed Scopus (209) Google Scholar, 3Merling R.K. Sweeney C.L. Chu J. Bodansky A. Choi U. Priel D.L. Kuhns D.B. Wang H. Vasilevsky S. De Ravin S.S. et al.An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease.Mol. Ther. 2015; 23: 147-157Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 5De Ravin S.S. Reik A. Liu P.-Q. Li L. Wu X. Su L. Raley C. Theobald N. Choi U. Song A.H. et al.Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease.Nat. Biotechnol. 2016; 34: 424-429Crossref PubMed Scopus (142) Google Scholar Although, in principle, safe, this strategy is still not perfect, for there is only a partial rescue of NAPDH-oxidase function in fully differentiated neutrophils. Alternatively, to capture the advantages of natural gene regulatory mechanisms, scientists can use engineered nucleases to insert a functional minigene near the transcription start site of the disease-causing gene, also taking advantage of the increased cutting efficiency of the nuclease in this part of the gene structure.10Horlbeck M.A. Witkowsky L.B. Guglielmi B. Replogle J.M. Gilbert L.A. Villalta J.E. Torigoe S.E. Tjian R. Weissman J.S. Nucleosomes impede Cas9 access to DNA in vivo and in vitro.eLife. 2016; 5: 5Crossref Scopus (229) Google Scholar The assumption here is that it will enable physiological expression of the protein of interest because transcription is driven by the gene’s own regulatory sequences. Another even more precise approach would be to repair the pathogenic mutations in situ. For example, efficient targeted repair of the beta globin gene has been achieved in HSPCs by combining the CRISPR/Cas9 system with AAV6-mediated delivery of a homologous donor template.11Dever D.P. Bak R.O. Reinisch A. Camarena J. Washington G. Nicolas C.E. Pavel-Dinu M. Saxena N. Wilkens A.B. Mantri S. et al.CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells.Nature. 2016; 539: 384-389Crossref PubMed Scopus (511) Google Scholar However, for the majority of genetic disorders where mutations can be spread across the whole gene, the wider application of this approach seems, at present, unlikely, particularly for future commercial development. The optimization of protocols to differentiate iPSCs into neutrophils3Merling R.K. Sweeney C.L. Chu J. Bodansky A. Choi U. Priel D.L. Kuhns D.B. Wang H. Vasilevsky S. De Ravin S.S. et al.An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease.Mol. Ther. 2015; 23: 147-157Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar has given impetus to study new gene therapy approaches for CGD as well as to find ways to use those cells as a potential source for therapeutic granulocyte infusion. In their paper, Sweeny et al.6Sweeney C.L. Zou J. Choi U. Merling R.K. Liu A. Bodansky A. Burkett S. Kim J.-W. De Ravin S.S. Malech H.L. Targeted repair of CYBB in X-CGD iPSCs requires retention of intronic sequences for expression and functional correction.Mol Ther. 2017; 25 (this issue): 321-330Abstract Full Text Full Text PDF Scopus (33) Google Scholar have compared different gene-editing strategies for the correction of the X-CGD phenotype using iPSC-derived granulocytes from CGD patients. First, they accurately corrected the CYBB gene in iPSCs from patients bearing a 458T > G mutation or a 461(A) deletion in exon 5. For this purpose, they nucleofected TALEN or CRISPR/Cas9 expression plasmids, targeting exon 5 together with a donor plasmid bearing the correct homologous sequence and excisable selection markers. Clones that had undergone granulocytic differentiation exhibited almost normal levels of gp91phox expression and NADPH oxidase function. Remarkably, the team also detected normal expression levels of the CD13 marker, suggesting that gene repair had contributed to normal granulocytic differentiation in CGD cells. They next devised a universal strategy to complement almost all gene mutations. For this purpose, they nucleofected TALENs or CRISPR/Cas9 plasmids targeting exon 1 or exon 2 of the CYBB locus together with a plasmid containing two versions of the CYBB mini gene (exon 1–13 or exon 2−13). They showed that TALEN-mediated insertion of a complete CYBB minigene exon 1–13 at the transcription start site of the gene (Figure 1A) did not lead to gp91phox expression or NADPH oxidase activity. In contrast, CRISPR/Cas9-mediated insertion of the CYBB exon 2–13 mini gene at the exon 2 site (Figure 1B) restored levels of gp91phox and NADPH oxidase activity that were 64%–100% and 68%–100% of those obtained in control blood, respectively. Notably, expression of gp91phox was strictly myeloid-specific, because the protein was only found in the CD13+ population. The different outcomes of the two strategies suggest that an intact exon/intron 1 is key for normal endogenous expression of gp91phox. This should not come as too much of a surprise, because it is widely recognized that intronic sequences can play a crucial role in normal gene expression. Intronic regions have also been used to boost gene expression or guide transcription in a tissue- and stage-specific manner.12Chorev M. Carmel L. The function of introns.Front. Genet. 2012; 3: 55Crossref PubMed Scopus (227) Google Scholar Although the authors did not investigate the mechanisms by which intron 1 controls expression of the CYBB gene, it is possible that the faulty correction arises from a lack of exon/intron splicing, the absence of an exon/junction complex signature, or the loss of enhancer elements that impair expression of the CYBB minigene exon 1–13 from its own promoter. Promoter-proximal introns often contain recruitment sites for enhancers or silencers. Maintaining early introns, as recently done for targeted editing of the Wiskott Aldrich Syndrome gene,13Laskowski T.J. Van Caeneghem Y. Pourebrahim R. Ma C. Ni Z. Garate Z. Crane A.M. Li X.S. Liao W. Gonzalez-Garay M. et al.Gene correction of iPSCs from a Wiskott-Aldrich syndrome patient normalizes the lymphoid developmental and functional defects.Stem Cell Reports. 2016; 7: 139-148Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar may therefore be a valid strategy for gene correction. As the authors rightly point out in the discussion, a distant upstream NF-κB binding site strongly interacts with regions in the first introns of CYBB, suggesting that these could behave as enhancers. This is in line with the observation made by others that a minimal CYBB gene promoter is not sufficient to drive normal levels of expression of the protein in the context of lentiviral vectors.14Barde I. Laurenti E. Verp S. Wiznerowicz M. Offner S. Viornery A. Galy A. Trumpp A. Trono D. Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease.Gene Ther. 2011; 18: 1087-1097Crossref PubMed Scopus (39) Google Scholar The use of selectable markers in the targeting constructs precludes evaluation of the efficiency of repair. Nevertheless, the message of this study remains strong and clear. A careful analysis of the different elements required for physiological levels of gene expression is an essential step for emerging gene-editing technologies because it will ultimately dictate the optimal strategy to achieve therapeutic levels of correction. Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional CorrectionSweeney et al.Molecular TherapyDecember 19, 2016In BriefSweeney et al. demonstrate TALEN- and CRISPR/Cas9-mediated targeted correction of CYBB mutations in iPSCs from X-CGD patients, and identify a key issue for the design of effective gene targeting and knockin strategies: that retention or inclusion of intronic elements may be necessary for expression from an endogenous promoter. Full-Text PDF Open Archive