234. Efficient Generation of Stable Genetically Modified Human Ipsc-Derived Macrophages for Innovative Gene and Cell Therapeutic Strategies

Hematopoietic differentiation of human induced pluripotent stem cells (iPSC) holds great promise for disease modeling, drug testing, and in particular for the development of novel gene and cell therapeutic strategies. In the past, interest has been directed primarily at the generation of reconstituting hematopoietic stem cells, a cell type as of yet problematic to obtain from hPSCs. Recently however, also long-lived mature myeloid cells such as tissue resident macrophages have been described and further characterized. Therefore, transplantation of macrophages may serve as an innovative treatment approach in several diseases such as pulmonary alveolar proteinosis (PAP) and adenosine deaminase deficiency (ADA). In this line, we have established an efficient, embryoid body-based human iPSC-differentiation protocol employing IL-3 in combination with M-SCF to continuously (up to 6 months) generate large numbers (5-10×106/week/6 well plate) of >95% pure monocyte/macrophages (iPSC-MΦ) via an intermediate “myeloid cell forming complex”. The iPSC-MΦ revealed typical morphology, surface phenotype and functionality comparable to their in vivo derived counterparts. In order to evaluate the suitability of this protocol also for gene therapy, we transduced human iPSC with 3rd generation SIN lentiviral vectors employing a minimal and safety optimized ubiquitous chromatin opening element (CBX3-UCOE) to prevent differentiation-associated epigenetic silencing. Here, GFP reporter gene expression driven by the elongation factor 1α short (EFS) promoter resulted in >90% GFP+ iPSC-MΦ after myeloid differentiation. In contrast, the EFS promoter without the CBX3-UCOE was subject to massive epigenetic silencing. Moreover, also a myeloid-biased myeloid-related protein 8 (Mrp8) promoter was efficiently protected from silencing, whilst tissue specificity was not affected by the CBX3-UCOE. Of note, the protective activity of the CBX3-UCOE was associated with decreased levels of repressive histone marks (H3K27me3, H3K9me3) and increased levels of active marks (phosphorylated polymerase). Alternatively, we also targeted an expression cassette to the AAVS1 locus employing zinc finger nuclease mediated homologous recombination. Selected clones showed targeted and robust CMV early enhancer/chicken β actin (CAG) promoter-driven fluorochrome expression in the pluripotent status as well as in terminally differentiated iPSC-MΦ. In summary, we here describe silencing resistance lentiviral vectors and genome editing strategies as well as a differentiation protocol allowing for the efficient and long-term generation of stable genetically modified iPSC-MΦ, thus paving the way for novel gene and cell based therapies.