The severe combined immunodeficiencies (SCID) are a set of genetic diseases in which patients are born with mutations in single genes and are unable to develop functional immune systems. While bone marrow transplantation can be curative for these diseases there remain significant limitations to this approach. Gene therapy using retroviral vectors have been used for SCID with mixed results. Tens of patients have developed functional immune systems such that they can live without having to worry about developing life-threatening infections. A handful of patients, however, have developed iatrogenic leukemia from the insertional activation of a proto-oncogene. In the next generation of viral based gene therapy trials for these diseases, the viral vectors have been engineered to decrease the probability of activating proto- oncogenes. The results of these trials, particularly the oncogenic risk, will not be known for several years. We have been working towards a third generation gene therapy approach to SCID. In contrast to using viral vectors to deliver transgenes in an uncontrolled fashion, we are working towards using homologous recombination to precisely modify the genome. We have shown that using engineered nucleases we can stimulate gene modification by homologous recombination (gene targeting) at frequencies that should be therapeutically useful (>10%) in cell lines and primary cels. The overall focus of the proposal is to translate the findings in a methodical and careful fashion to use in patient-derived umbilical cord blood derived CD34+ cells. The three specific aims are focused on translating our basic science studies into a clinical trial.
Specific aim 1 is focused o developing nuclease mediated gene targeting for the IL2RG gene.
Specific aim 2 is focused on developing nuclease mediated gene targeting for the ADA gene.
Specific aim 3 is focused on using whole genome approaches to better understand nuclease on-target activity and off-target effects. Our goal with this proposal is to complete the IND-enabling experiments necessary to initiate a first-in-man gene therapy clinical trial using homologous recombination to cure SCID.

Public Health Relevance

The severe combined immunodeficiencies (SCID) are lethal genetic diseases for which currently existing therapies have important limitations. Gene therapy using retroviral vectors for SCID has proven problematic because of the development of secondary leukemias. This proposal is focused on the pre-clinical development of homologous recombination as a more precise and safer gene therapy strategy for the cure of patients with SCID.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI097320-05
Application #
9173450
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Voulgaropoulou, Frosso
Project Start
2012-12-01
Project End
2017-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
5
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Stanford University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
Bak, Rasmus O; Dever, Daniel P; Porteus, Matthew H (2018) CRISPR/Cas9 genome editing in human hematopoietic stem cells. Nat Protoc 13:358-376
Vakulskas, Christopher A; Dever, Daniel P; Rettig, Garrett R et al. (2018) A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med 24:1216-1224
Charlesworth, Carsten T; Camarena, Joab; Cromer, M Kyle et al. (2018) Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting. Mol Ther Nucleic Acids 12:89-104
Bak, Rasmus O; Dever, Daniel P; Reinisch, Andreas et al. (2017) Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6. Elife 6:
Dever, Daniel P; Porteus, Matthew H (2017) The changing landscape of gene editing in hematopoietic stem cells: a step towards Cas9 clinical translation. Curr Opin Hematol 24:481-488
Porteus, Matthew (2016) Genome Editing: A New Approach to Human Therapeutics. Annu Rev Pharmacol Toxicol 56:163-90
Dever, Daniel P; Bak, Rasmus O; Reinisch, Andreas et al. (2016) CRISPR/Cas9 ?-globin gene targeting in human haematopoietic stem cells. Nature 539:384-389
Porteus, Matthew H (2015) Genome Editing of the Blood: Opportunities and Challenges. Curr Stem Cell Rep 1:23-30
Hendel, Ayal; Fine, Eli J; Bao, Gang et al. (2015) Quantifying on- and off-target genome editing. Trends Biotechnol 33:132-40
Porteus, Matthew H (2015) Towards a new era in medicine: therapeutic genome editing. Genome Biol 16:286

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