TALEN-mediated functional correction of human iPSC-derived macrophages in context of hereditary pulmonary alveolar proteinosis

Alexandra Kuhn,Mania Ackermann,Claudio Mussolino,Nico Lachmann,Toni Cathomen,Thomas Moritz

doi:10.1038/s41598-017-14566-8

Abstract

Hereditary pulmonary alveolar proteinosis (herPAP) constitutes a rare, life threatening lung disease characterized by the inability of alveolar macrophages to clear the alveolar airspaces from surfactant phospholipids. On a molecular level, the disorder is defined by a defect in the CSF2RA gene coding for the GM-CSF receptor alpha-chain (CD116). As therapeutic options are limited, we currently pursue a cell and gene therapy approach aiming for the intrapulmonary transplantation of gene-corrected macrophages derived from herPAP-specific induced pluripotent stem cells (herPAP-iPSC) employing transcriptional activator-like effector nucleases (TALENs). Targeted insertion of a codon-optimized CSF2RA-cDNA driven by the hybrid cytomegalovirus (CMV) early enhancer/chicken beta actin (CAG) promoter into the AAVS1 locus resulted in robust expression of the CSF2RA gene in gene-edited herPAP-iPSCs as well as thereof derived macrophages. These macrophages displayed typical morphology, surface phenotype, phagocytic and secretory activity, as well as functional CSF2RA expression verified by STAT5 phosphorylation and GM-CSF uptake studies. Thus, our study provides a proof-of-concept, that TALEN-mediated integration of the CSF2RA gene into the AAVS1 safe harbor locus in patient-specific iPSCs represents an efficient strategy to generate functionally corrected monocytes/macrophages, which in the future may serve as a source for an autologous cell-based gene therapy for the treatment of herPAP.

Highlights

The reprogramming of human somatic cells into induced pluripotent stem cells[1] in combination with precise genome engineering technologies employing zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or RNA-guided nucleases, has rapidly advanced our options for disease modeling, drug screening and cell replacement therapies[2,3,4,5,6,7] and has had a marked impact on personalized medicine including gene and cell therapy strategies[8]
Pulmonary macrophage transplantation has been described as a highly effective therapeutic approach for Hereditary pulmonary alveolar proteinosis (herPAP) in two murine disease models[26,27]. Given this background we aimed to develop a gene therapy approach for herPAP using induced pluripotent stem cells (iPSCs) and transcriptional activator-like effector nucleases (TALENs) technology to correct the CSF2RA-mediated form of the disease
Targeted insertion of the CSF2RAcoop gene into the AAVS1 safe harbor locus by homologous recombination using TALENs and herPAP patient-derived iPSCs. Programmable nucleases such as TALENs represent a promising technology to introduce DNA double-strand breaks and allow for a homology-directed repair process precisely targeting the gene of interest into a pre-defined genomic locus. We here adapted this technology to develop a cell and gene therapy approach for herPAP based on macrophages derived from gene corrected PAP-disease specific iPSCs

Summary

Introduction

The reprogramming of human somatic cells into induced pluripotent stem cells (iPSCs)[1] in combination with precise genome engineering technologies employing zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or RNA-guided nucleases, has rapidly advanced our options for disease modeling, drug screening and cell replacement therapies[2,3,4,5,6,7] and has had a marked impact on personalized medicine including gene and cell therapy strategies[8]. Within the lympho-hematopoietic system, designer nuclease-mediated homology-directed repair (HDR) has already been applied for genetic correction of disorders such as chronic granulomatous disease[7,9,10], ß-hemoglobinopathies[11,12,13] and severe combined immunodeficiency (SCID)[14,15] In this context, the intron 1 of the PPP1R12C gene on human chromosome 19, originally described as the favored integration site for adeno-associated viruses and referred to as AAVS1 locus, has been exploited as a predefined “safe harbor” integration site allowing for the stable expression of transgenic sequences in pluripotent stem cells (PSCs) and thereof differentiated progeny[7,10,16,17,18]. We here describe for the first time the TALEN-mediated genetic integration of a codon-optimized CSF2RA transgene (CSF2RAcoop) into the AAVS1 locus of herPAP patient-derived iPSCs restoring GM-CSF receptor functionality and correcting the in vitro disease phenotype of herPAP iPSC-derived monocytes/macrophages

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Conclusion