The faculty of the Northwest Genome Engineering Consortium (NGEC) are working to develop and apply gene repair to the treatment of single-gene disorders of the immune system and blood cells.
This goal will be accomplished through pursuing two major scientific aims:
Aim #1: Development of Methods for LAGLIDADG Homing Endonuclease Engineering
This aim involves combining computational protein design, directed protein evolution, structural analysis and in vitro biochemical and molecular analyses. Its goal is to pioneer methods for producing artificial LAGLIDADG homing endonucleases (LHEs) for use in a variety of types of genome engineering applications. NGEC research labs focusing on this area are:
Dr. Andy Scharenberg, NGEC Co-director and Principal Investigator, seeks to take computationally designed homing endonucleases, randomly introduce mutations into their coding sequences to generate large libraries of new homing endonucleases, and then screen the libraries of mutated enzymes for those with improvement of one or more desired properties through the process of “directed evolution.”
Dr. Ray Monnat, Principal Investigator, works on identifying and characterizing the in vivo target sites of native and engineered homing endonuclease proteins, identifying new homing endonucleases from genomic sequence databases, generating new heterodimeric homing endonucleases and developing novel screens for targeted cleavage activity.
Dr. Barry Stoddard, Principal Investigator, strives to identify the structural basis for the DNA-binding specificity and catalytic activity of homing endonucleases generated by NGEC laboratories, and to compare the properties of designed enzymes to those of naturally occurring (i.e., wild-type) homing endonucleases.
Dr. David Baker, Principal Investigator, seeks to apply computational design to generate new LHEs able to recognize and cleave at high quality match sites; to refine Rosetta Design algorithms for prediction of DNA/protein interfaces by explicit incorporation of waters and rigid body framework movements; and to refine Rosetta Design algorithms for prediction of DNA/protein interfaces by explicit incorporation of structural information of novel LHE variants.
Aim #2: Application of LAGLIDADG Homing Endonucleases to Gene Repair of Hematopoietic Stem Cells
This aim will combine novel LHEs created in Aim #1 with the use of non-integrating lentiviral vectors (NIL) for the introduction of an LHE and a repair/modification template to hematopoietic stem cells for the purpose of gene repair. It will also explore new methods for manipulating endogenous DNA repair mechanisms to enhance the in situ efficacy of LHE-induced gene repair in hematopoietic stem cells, and develop methods for autologous hematopoietic stem cells engraftment optimized for gene repair applications. NGEC research labs focusing on this area are:
Dr. David Rawlings, NGEC Co-director and Principal Investigator, seeks to utilize two murine immunodeficiency disorders as model systems to evaluate methods for delivery of an LHE and donor template to induce gene repair in hematopoietic stem cells.
Dr. Nancy Maizels, Principal Investigator, strives to expand upon the current understanding of the mechanism of homology-directed repair to promote correction of genetic defects in situ by increasing the efficiency of homology-directed repair and minimizing the deleterious consequences of nuclease-targeted therapies.
Dr. Hans-Peter Kiem, Principal Investigator, seeks to initiate studies of LHE-based gene modification in canine hematopoietic stem cells as an initial step towards translation of gene repair of hematopoietic stem cells to humans.