The overall objective of this project is to study LAGLIDADG homing endonuclease (LHE)-based hematopoietic stem cell (HSC) gene repair strategies in a clinically relevant large animal model, the dog. We have used the dog to develop improved HSC gene transfer strategies and to evaluate different gene transfer systems. A particular advantage of the dog model is the availability of disease models such as X-linked severe combined immunodeficiency (XSCID) and pyruvate kinase (PK) deficiency. The development of leukemia in three children with XSCID after HSC gene therapy with a gammaretroviral vector has demonstrated the risks of retrovirus-mediated gene therapy. Furthermore, our preliminary data in the dog demonstrate that all three major integrating retroviral vectors, i.e. gamma, lenti, and foamy .virus vectors carry a substantial risk of insertional mutagenesis. Thus, the development of site-specific gene replacement/gene repair strategies with reduced risks of insertional mutagenesis has become a crucially important goal for the development of safe gene therapy/repair approaches in patients with genetic disorders. To address the feasibility and safety of site-specific double strand break-induced gene targeting driven by LHEs, we will study and optimize conditions for use of self inactivating non-integrating lentiviral (NIL) vectors to drive LHE-mediated """"""""genomic marking"""""""" of dog CD34+ repopulating cells.
In Aim 1 we will target a cleavage site for an existing l-Scel variant enzyme in a non-transcribed region of the genome, while in Aim 2 we will generate dogs with an l-Scel cleavage site embedded within a fluorescent reporter. As part of Components 2-5, new LHEs capable of cleaving alternative sites will be generated;reporters with cleavage sites for these LHEs will then be developed, and the new LHEs performance benchmarked against l-Scel.
In aim 3 we will assess the feasibility of a site-specific knock-in of a selectable marker gene such as the P140K mutant of methylguanine methyltransferase (MGMT) and then use nonmyeloablative conditioning regimens in combination with in vivo selection, and in aim 4 we will evaluate the optimized conditions with LHEs created by Components 2-5 in two canine disease models, the XSCID and the PK models. We believe these studies will allow us to advance the use of LHEs in a clinically relevant large animal model.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Linked Research project Grant (RL1)
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Special Emphasis Panel (ZRR1-SRC (99))
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Thomas, John
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Fred Hutchinson Cancer Research Center
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Boissel, Sandrine; Jarjour, Jordan; Astrakhan, Alexander et al. (2014) megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Nucleic Acids Res 42:2591-601
Stone, Daniel; Kiem, Hans-Peter; Jerome, Keith R (2013) Targeted gene disruption to cure HIV. Curr Opin HIV AIDS 8:217-23
Watts, Korashon L; Nelson, Veronica; Wood, Brent L et al. (2012) Hematopoietic stem cell expansion facilitates multilineage engraftment in a nonhuman primate cord blood transplantation model. Exp Hematol 40:187-96
Certo, Michael T; Gwiazda, Kamila S; Kuhar, Ryan et al. (2012) Coupling endonucleases with DNA end-processing enzymes to drive gene disruption. Nat Methods 9:973-5
Munoz, Nina M; Beard, Brian C; Ryu, Byoung Y et al. (2012) Novel reporter systems for facile evaluation of I-SceI-mediated genome editing. Nucleic Acids Res 40:e14
Watts, Korashon Lynn; Adair, Jennifer; Kiem, Hans-Peter (2011) Hematopoietic stem cell expansion and gene therapy. Cytotherapy 13:1164-71
Beard, Brian C; Sud, Reeteka; Keyser, Kirsten A et al. (2009) Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients. Blood 113:5094-103
Trobridge, Grant D; Allen, James; Peterson, Laura et al. (2009) Foamy and lentiviral vectors transduce canine long-term repopulating cells at similar efficiency. Hum Gene Ther 20:519-23