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

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.

Pulmonary Transplantation of Human Induced Pluripotent Stem Cell–derived Macrophages Ameliorates Pulmonary Alveolar Proteinosis

“…We consider our work as a first proof-of-concept for the feasibility and therapeutic efficacy of the intrapulmonary transplantation of iPSC-derived macrophages in the context of herPAP. However, translation of this concept into a clinical setting, will require the genetic correction and functional repair of autologous iPSC-derived macrophages from patients with herPAP, as we already have demonstrated in previous work employing lentiviral as well as nuclease technology (19,49). Moreover, clinical translation will require additional stringent and long-term safety testing in relevant animal models.…”

Integration of nano‐ and biotechnology for beta‐cell and islet transplantation in type‐1 diabetes treatment

“…Furthermore, the presence of the pCAG‐driven transgene did not alter the successful differentiation of the hiPSCs into other cell types. The same conclusions have been drawn both in case of transfection and single‐copy insertion into the AAVS1 safe harbour locus of hiPSCs . Luo et al compared the expression of eGFP under different regulation of promoters (CMW, EF1α, CAG), integrated into the AAVS1 locus of hiPS cell lines.…”

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

“…The protocol for the generation of iPSC-DMs described here is robust and allows for the production of a large number of homogeneous cells from a relatively small number of iPSCs. Following the initial differentiation of approximately 1 x 10 6 iPSCs, the subsequent cultures can be harvested every 4 days for up to 2-3 months, resulting in the production of at least 6.5 x 10 7 macrophages over that time. These in vitrogenerated human macrophages are morphologically similar to primary human macrophages, express the key macrophage cell surface markers, and exhibit phagocytic activity.…”

“…Technologies that allow for the genetic manipulation of human pluripotent stem cells (PSCs), such as Zinc Finger Nuclease, TALENS, and CRISPR-Cas9, have revolutionised medical research 1,2,3,4 . Genetic manipulation of human PSCs is a particularly attractive strategy when the primary cell of interest is difficult to expand and/or to maintain in vitro, or is difficult to genetically manipulate, such as is the case for macrophages 5,6,7,8,9 . As human iPSCs can be derived from any somatic cell, they circumvent the ethical limitations associated with ESCs, and provide a strategy for delivering personalized medicine.…”

“…As human iPSCs can be derived from any somatic cell, they circumvent the ethical limitations associated with ESCs, and provide a strategy for delivering personalized medicine. This includes patient-specific disease modelling, drug testing, and autologous cell therapy with a reduced risk of immune rejection and infection 6,8,10,11 .…”