Exploring Template-free CRISPR/Cas9 Genome Editing as a Novel Strategy for Personalized Gene Therapy of Monogenic Blood Disorders

点击翻译按钮获取中文摘要

A significant fraction of inherited monogenic disorders are caused by patient-specific mutations dispersed over the entire locus of the affected gene. Correcting these mutations by introducing healthy gene copies into the genome of the diseased cells proved successful in several clinical trials. In most of these trials the healthy genes were inserted randomly throughout the genome by retroviral vectors and were thus deprived of their endogenous control elements. As a result complications arose including gene silencing and activation of cancer causing genes. To circumvent these problems, we propose to develop a template-free CRISPR/Cas9 based genome editing approach for the in situ correction of point mutations causing chronic granulomatous (X-CGD) - or severe immunodeficiency disease (X-SCID), which are both inherited monogenic blood disorders. For this, we will generate human hematopoietic cell lines expressing single copy CYBB or IL2R-gamma genes harboring patient specific mutations and use these cell lines in a primary screen for gene correcting CRISPR/Cas9 based, integrase deficient lentiviral vectors (IDLVs). Highly correctable mutations identified in this screen will be introduced into the mouse germline by single embryo injections to obtain mouse models of X-CGD and X-SCID. These models will then be used in autologous bone marrow transplantation experiments aimed at curing the disease by ex vivo CRISPR/Cas9 IDLV delivery into hematopoietic stem cells. Overall, this project seeks to provide proof of concept for personalized gene therapy.