Elsevier

Biomaterials

Volume 69, November 2015, Pages 191-200
Biomaterials

Leading opinion
TALEN-mediated functional correction of X-linked chronic granulomatous disease in patient-derived induced pluripotent stem cells

https://doi.org/10.1016/j.biomaterials.2015.07.057Get rights and content

Abstract

X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes.

Introduction

X-CGD (OMIM reference number: 300481) is a rare inherited disease of the immune system, which is caused by mutations in the NOX2 gene, encoding gp91phox [1]. The glycoprotein gp91phox is part of the NADPH oxidase (NOX) complex of phagocytes that generates reactive oxygen species (ROS), leading to a microbicidal oxidative burst. This mechanism is absent in X-CGD patients, leading to severe, recurrent and life-threatening bacterial and fungal infections. Allogeneic hematopoietic stem cell transplantation is an option for treating patients with X-CGD [2] but many patients lack a human leukocyte antigen (HLA) compatible donor. Another curative approach is autologous transplantation of hematopoietic stem and precursor cells (HSPCs) after ex vivo gene therapy. Conventional gene therapy approaches for X-CGD are based on the transfer of a therapeutic transgene into autologous HSPCs via retroviral vectors that semi-randomly integrate in the genome. Although retroviral-based gene therapy trials for X-CGD initially led to clinical improvement in the patients, methylation of the viral promoter resulted in transgene silencing over time and therefore loss of therapeutic benefit. Furthermore, insertional activation of the EVI1, PRDM16 and SETBP1 genes led to clonal dominance of corrected cells, eventually leading to myelodysplasia [3], [4]. Additionally, it is speculated that ectopic gp91phox expression in HSPCs caused inadequate ROS production in the stem cell compartment, thus contributing to the low levels of engraftment of gene-corrected cells [5]. Together, these observations highlight the need for alternative strategies that combine tissue specific expression of the therapeutic transgene with targeted genome editing.

The possibility to target therapeutic transgenes into a predefined genomic safe harbor, like the adeno-associated virus integration site 1 (AAVS1), should avoid the risk of insertional mutagenesis and prevent unwanted positional effects, which lead to variegation of transgene expression levels [6], [7]. Particularly the insulator regions identified in vicinity of AAVS1 seem to protect endogenous promoters from being affected by potential enhancer effects of the transgene promoters, and vice versa, so allowing for stable and long-term expression of the transgene [8], [9]. Designer nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have been exploited to modify the genomes of complex organisms in a targeted manner, including the integration of therapeutic transgenes to desired loci [10], [11], [12], [13], [14]. The latter is achieved by introducing a sequence-specific DNA double-strand break (DSB) in the target locus to enhance the homologous recombination (HR) frequency with an exogenous DNA donor cassette [8], [15], [16]. In the context of X-CGD, targeted transgene insertion into the AAVS1 locus has the advantage that a single set of validated reagents are sufficient for clinical treatment of all X-CGD patients, regardless of their underlying mutation [17]. Current challenges to designer nuclease-mediated targeted integration of a transgene in HSPCs include the low HR frequency in these multipotent stem cells and the lack of appropriate technologies to select for a “clean” clone due to the limited self-renewal properties of HSPCs in vitro[18], [19]. Nonetheless, two recent reports demonstrated the feasibility of ZFN-mediated genome editing in a polyclonal human HSPC population by applying optimized culture conditions [19], [20]. In contrast, because of their unlimited self-renewal in vitro, designer nuclease-mediated gene targeting in induced-pluripotent stem cells (iPSCs) allows for a thorough analysis of individually selected clones [9], [21], [22]. Previously, ZFNs have been employed to integrate a therapeutic gp91phox expression cassette under transcriptional control of a constitutively active CMV early enhancer/chicken β-actin (CAG) promoter into the AAVS1 site of X-CGD patient-derived iPSCs [9]. This approach successfully corrected the ROS-deficient phenotype in iPSC-derived granulocytes, but inadequate ROS production in hematopoietic cell types, especially the stem cell compartment, may have detrimental effects for future clinical applications.

In this study, we aimed at targeting the integration of a miR223 promoter driven gp91phox transgene to the AAVS1 site of X-CGD patient-derived iPSCs to restore function of the NOX complex in derived granulocytes while avoiding inadequate ROS production in the stem cell compartment [23]. We showed in an X-CGD model cell line that AAVS1-specific TALENs and ZFNs have similar activity but TALENs have a superior cytotoxicity profile than ZFNs. In line with this observation, we found that the TALEN pair supported the generation of correctly targeted iPSC clones more efficiently. Corrected X-CGD iPSCs retained their pluripotency and no designer nuclease-associated mutations were detected in the top-ten predicted off-target sites. Myeloid differentiation of corrected X-CGD iPSCs led to a 35-fold up-regulation of gp91phox mRNA, reaching levels similar to healthy control cells. On a functional level, reconstitution of ROS production and formation of neutrophil extracellular traps (NETs) were achieved, both indicative of functional correction of the cellular disease phenotype.

Section snippets

Plasmids

The generation of AAVS1-specific ZFN [24] and TALEN [25] expression plasmids and donor DNA is described in the Supplementary Material.

Transfection of PLB-985 cells and cytotoxicity assays

Transfection of X-CGD PLB-985 cells was performed using the Neon-Transfection System (Invitrogen/Life Technologies) according to manufacturer's instructions. For gene targeting, 0.6 μg of each nuclease expression vector and 3.8 μg of donor plasmid was used. AnnexinV (BD Biosciences) staining for apoptotic cells was performed two days post-transfection of the

Assessment of designer nucleases targeting AAVS1

The overall goal of this study was to develop a safe genome editing strategy to ensure tissue specific gp91phox transgene expression in a cellular disease model of X-CGD. To this end, we first compared the cleavage activity and nuclease-associated cytotoxicity of a ZFN and a TALEN pair targeted to the AAVS1 site [24], [25] in a PLB-985 based X-CGD model cell line [30]. Increasing amounts of nuclease expression plasmids were used to transfect X-CGD PLB-985 cells. Cleavage activity of the

Discussion

Disease modeling based on patient-derived iPSCs is particularly useful when studying rare genetic disorders, like X-CGD, for which patient cells are not easily accessible. Furthermore, in combination with targeted genome engineering and efficient differentiation protocols, the iPSC technology enables researchers to correlate particular genotypes with specific cellular phenotypes in an isogenic setting. The goals of our study were to identify a well-suited nuclease platform for targeting the

Funding

This work was supported by grants from the German Research Foundation [SFB 738–C9 to A.S. and T.C.; SPP1230–Ca311/2 to T.C.; the REBIRTH Cluster of Excellence to A.S. (EXC 62/1) and A.-K.D. (fellowship)], the Federal Ministry of Education and Research [BMBF 01EO0803 to T.C.; IFB-Tx to A.S.], the German Academic Exchange Service [Modern Applications in Biotechnology to A.S.], the Gebert Rüf Stiftung [J.R.], and the “Rare Diseases – New Approaches” program [GRS-046/10 to J.R.].

Conflict of interest

T.C. is a consultant for TRACR Hematology.

Notes

T.C. and A.S. contributed equally to this work.

Acknowledgments

We would like to thank Theresa Buchegger, Silke Glage, Daniela Lenz, Heimo Riedel, Susanne Rittinghausen and Marcel Tauscher for experimental support, Christopher Baum, Claudio Mussolino, and Philipp Henneke for scientific advice, and Nasreen Hoque and Michael Morgan for proofreading the manuscript.

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