Human ES Cells: A Model to test Gene Therapy Vectors

Yeshiva University, New York NY

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Linked publications & trials

Abstract

[unreadable] DESCRIPTION (provided by applicant): The overall aims of Dr. Bouhassira's project 2, one of the four components of the entire program project, are to create better experimental models to test gene therapy cassettes and vectors, and to devise anti-silencing strategies that will work at most sites of integration. Because of the considerable success that the program project as a group has had demonstrating the feasibility of gene therapy for sickle cell disease in a mouse model, we are proposing in this application for supplemental funding to develop model in which we will be able to optimize gene therapy cassettes directly in human primary cells. We propose the development of a system to produce human adult red blood cells in vitro through the directed differentiation of human embryonic stem cells. Producing human red blood cells from human ES cells rather than from circulating hematopoietic progenitors will have multiple advantages: 1) The cells will be genetically modifiable by homologous recombination and by Recombinase Mediate(Cassette Exchange. 2) The cells will be genetically homogenous. 3) We will be able to produce large quantities of cells. In order to achieve this aim, we will use a three-steps approach. First, we will find conditions in which hES cell, differentiate preferentially into adult hematopoietic cells by systematically testing co-culture with various stromal cell lines, serum from various species at different stage of development, and cocktails of recombinant cytokines known to be important for hematopoietic and erythroid differentiation. Once optimal conditions for the production of adult hematopoietic cells will have been found, we will obtain virtually pure populations of adult erythroid cells by lineage selection, using a selectable marker that is specific for expression in adult erythroid cells (Neomycin resistance gene driven by the B-globin promoter and mini LCR for instance). The marker will be stably transfected into the undifferentiated hES cells. Last, we will grow the purified adult erythroid cells into mature enucleated red blood cells, or at least orthochromatic normoblasts, using published technologies. Establishment of this novel experimental system will lay the foundations for subsequent studies that will aim at developing gene therapy cassettes designed to minimize insertional mutagenesis. [unreadable] [unreadable]