A cell-type specific selection system for stem cell therapy and other purposes

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A cell-type specific selection system for stem cell therapy (and other) purposes

Publication . Pólvora-Brandão, Duarte; Gontijo, Alisson Marques de Miranda Cabral

Abstract The discovery of reprogramming methods able to generate induced pluripotent stem cells (iPSCs) from adult somatic tissues was an outstanding breakthrough in the field of personalized cell-based regenerative medicine therapies. This was due both to their self-renewal capacities and their potential to differentiate into almost any cell type. Since then, these cells have been used in numerous studies, with the major focus being on disease modeling and drug screening, which can be done using patient-specific iPSCs. However, several limitations still affect the possibility of applying iPSC based technology for therapeutic purposes, one of which is the possibility of teratoma formation upon allografting of iPSCs or differentiated cells derived from them. In an optimal scenario, the perfect stem cell-based therapy would deliver/replenish a single cell type or progenitor type, and include the possibility to eliminate all other undesired or unnecessary cell types derived from the allografted stem cell. This scenario could be achieved if we had a versatile cell-type selection system that eliminates all allografted cells except our cell of interest. In order to distinguish specific cell types genetically, we can explore the transcriptional program of the cells, which reflects, at the most basic level, a unique combination of binary ON/OFF states of the regulatory elements present in their genome. The expression of these regulatory elements can be combined using intersectional genetics approaches to specifically identify a particular cell type. In this work, we developed tools to create a cell-selection system that can be used following iPSC-based therapies to select (i.e., allow the survival of) a single desired cell type derived from these iPSCs. For this, we generated an inducible cell-ablation system based on the Tet-On technology, and an intersectional genetics-based cell-type selection cassette, which produces an “antidote” against the cell-ablation system specifically in the cell of interest. For the cell-ablation system, we tested both a CRISPR/Cas9-based approach and an iCasp9-based approach, with the latter being more successful than the former. These cell-ablation systems were placed under the control of a TRE promoter, which is activated by a synthetic rtTA transcription factor in the presence of doxycycline. The antidote consists of a custom-engineered TetR*-KRAB protein, which binds to the TRE promoter independently of Doxycycline, efficiently competing with rtTA, thus preventing cell ablation. So that the TetR*-KRAB protein antidote could be driven specifically in the desired target cell using intersectional genetics, it was split into 2, 3, or 4 non-self-complementing components. Its modularity was tested by expressing the components constitutively or via cell-type specific promoter and enhancer combinations. As a proof-of-principle, we tested each component of the cell-ablation system and the TetR*-KRAB and its split forms in human HEK293T cells using constitutive promoters. For cell type specific selection, we tested iPSC and neural stem cell (NSC)-specific enhancers to guide the expression of the TetR*-KRAB in these cells. The four NSC regulatory elements that were tested led to a downregulation of the TRE promoter, showing the potential protective function of TetR*-KRAB in preventing the cell-ablation cassette activation. This system or its future derivatives can be used for selection of desired cell types during differentiation protocols or organoid cultures and even as a security measure upon future cell therapies using iPSCs or differentiated cells derived from them.

2023-05-30Tese de doutoramento

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Programa de financiamento

OE

Número da atribuição

PD/BD/135499/2018