NGEC Co-Director and Principal Investigator
Director, Center for Immunity and Immunotherapies
Head, Section of Immunology
Seattle Children's Research Institute
Professor, Pediatrics
Adjunct Professor, Immunology
University of Washington School of Medicine
Seattle Children's Research Institute
M/S C9S-7
1900 9th Avenue
Seattle, WA 98101
Tel: (206) 987-7450 Fax: (206) 884-7310
E-mail Dr. Rawlings
Dr. Rawlings graduated magna cum laude with a BS in biological sciences from Davidson College, where he was elected a member of Phi Beta Kappa, and earned his MD from the University of North Carolina, School of Medicine. He completed residency and chief residency in pediatrics at the University of California, San Francisco, and pediatric rheumatology/immunology subspecialty training at Children's Hospital Los Angeles. Dr. Rawlings pursued postdoctoral research as an intramural fellow at the NIH and at HHMI, University of California, Los Angeles.
Formerly a member of the UCLA faculty, he joined the University of Washington as the head of the Section of Pediatric Immunology in 2001, and now directs the Center for Immunity and Immunotherapies at Seattle Children's Research Institute.
Dr. Rawlings is co-director of the NGEC and a principal investigator in the area of Gene Repair in Murine Immunodeficiency Models.
“The NGEC provides a tremendous opportunity to establish the engineering tools and delivery systems to test the feasibility of targeted gene repair in hematopoietic disorders. I am very excited to be part of these unique efforts. Given the broad local expertise and established infrastructure for treatment of such diseases, we will be well positioned to move from proof-of-principal studies into translational work.”For its role in the NGEC, the Rawlings lab will 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. In both models, a selective advantage for gene-corrected lymphoid stem cells will provide a useful initial system in which to evaluate both feasibility and efficiency of various repair strategies.
In collaboration with the Scharenberg lab, Dr. Rawlings and his team have generated a novel murine model of X-linked severe combined immunodeficiency (XSCID) that will enable the lab to evaluate gene repair under near ideal conditions using the well-characterized LHE, I-SceI. This model will allow the Rawlings lab to evaluate a range of NIL vector designs, transduction protocols, and HSC target populations in order to identify optimal methods for in vivo gene repair.
Following establishment of successful gene repair, the Rawlings lab will then utilize the identical LHE-XSCID animal model and delivery systems to evaluate the functional activity of an engineered I-AniI enzyme.
Subsequently, the lab will evaluate a second engineered LHE, designed to cut within the Btk gene at the X-linked immunodeficiency (XID) locus. NIL vectors, carrying the XID-specific LHE and a repair template with or without a cis-linked selection marker, will be used to determine if drug selection can enrich gene repaired stem cells and, thereby, enhance the rate of immune reconstitution in this animal model.
The combined studies of the Rawlings lab will provide a key benchmark against which to judge gene repair approaches using engineered LHEs and NIL delivery systems.
Key lab personnel assisting this work include:
Alexander Astrakhan, BS (pre-doctoral student): Will characterize the I-SceI-XSCID knock in mice; generate various NIL vector constructs, purify and transduce bone marrow cell populations, and transplant cells into I-SceI-XSCID animal model. He will also perform assays to characterize mouse HSC, T, B, and myeloid populations following transplantation.
Hannah Kerns, BS (research scientist): Will utilize her proficiency in lentiviral production, cell culture techniques, flow cytometry, and animal studies to assist in conducting transplantation into mice, harvesting blood and tissue samples, and examining functions in specific cell lineages after transplantation.
Socheath Khim, BS (research scientist): Will manage all of the animal colonies/experimental animals involved and will participate in experimentation, including genotyping, cellular analysis, cell culture work, and viral transduction studies.
Brigid Stirling, PhD (senior research scientist): Using her experience of carrying out lentiviral gene delivery in immune deficient animal models, Dr. Stirling will construct and evaluate NIL vectors, and characterize the gene-repaired populations by various functional assays in Btk deficient, XID models.
Yupeng Wang, PhD (postdoctoral fellow): Will utilize his expertise in cell signaling and lentiviral gene delivery in various animal models and carry out analysis of NIL vectors in ES cell lines and primary HSC cells and hematopoietic cell lines in vitro and in vivo. Dr. Wang will also characterize the gene repaired populations by various functional assays in the I-SceI-XSCID and other models and carry out the multi-parameter immuno-phenotyping and FACS sorting of cell populations.
Yupeng Wang, postdoctoral fellow in the Rawlings lab
The Rawlings lab uses expertise in basic and clinical immunology, signal transduction, and lymphocyte developmental biology to understand how altered signals can lead to immunologic diseases. This work is relevant to a range of related outcomes because dysregulation of lymphocyte growth and differentiation can lead, alternatively, to immunodeficiency states, or to autoimmunity and malignancy. Ultimately, Dr. Rawlings and his team seek to utilize this information to develop translational therapies capable of specifically modulating these disorders.
As a postdoctoral fellow in Dr. Owen Witte’s laboratory, Dr. Rawlings initially characterized the role of Bruton’s tyrosine kinase (Btk) in B-cell development and how deficient function of this enzyme led to the related immune disorders, X-linked immunodeficiency (XID; in mice) and X-linked agammaglobulinemia (XLA; in humans), respectively. Subsequently, his laboratory has worked to define the molecular events that link Btk to B-cell receptor signaling.
His group identified the downstream requirement for Btk in calcium signaling via activation of phosholipase C gamma (PLCγ), and in the NFκB cascade through protein kinase C-beta (PKCβ) and the adapter protein, CARMA1. In parallel, his laboratory has pioneered studies demonstrating rescue of B cell development in animal models of XLA using viral vectors for gene therapy and is working to move this approach into clinical trials.
Tsukada S, Saffran DC, Rawlings DJ, Parolini O, Allen RC, Klisak I, Sparkes RS, Kubagawa H, Mohandas T, Quan S, Belmont JW, Cooper MD, Conley ME, Witte ON. Deficient expression of a B-cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. (1993) Cell 72:279-290.
Rawlings DJ, Saffran DC, Tsukada S, Largaespada DA, Grimaldi JC, Cohen L, Mohr RN, Bazan JF, Howard M, Copeland NG, Jenkins NA and Witte ON. Mutation of the BPK tyrosine kinase unique region in X-linked immunodeficiency. (1993) Science 261:358-361.
Rawlings DJ, Quan SG, Kato RM, and Witte ON. Establishment of a Long-Term Culture System for Human B Cell Progenitors. (1994) Proc. Nat. Acad. Sci. 92:1570-1574.
Rawlings DJ, Scharenberg AM, Park H, Wahl MI, Lin S, Kato RM, Fluckiger A-C, Witte ON, Kinet J-P. Activation of Btk by a phosphorylation mechanism initiated by src family kinases. (1996) Science 271:822-825.
Fluckiger A-C, Sanz E, Garcia-Lloret M, Su T, Hao QL, Kato RM, Quan S, de la Hera A, Crooks G, Witte ON, Rawlings DJ. In vitro reconstitution of human B cell ontogeny: From CD34+ multipotent progenitors to Ig secreting cells. (1998) Blood 92:4509-4520.
Fluckiger A-C, Li Z, Wahl MI, Kato RM, Kinet JP, Longnecker R, Witte ON, Scharenberg, Rawlings DJ. Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B cell receptor activation. (1998) EMBO 17: 1973-85.
Guo B, Kato RM, Garcia-Lloret M, Wall M and Rawlings DJ: Engagement of the human pre-B cell receptor (pre-B) generates a lipid raft-dependent calcium signaling complex. (2000) Immunity 13:243-253.
Kang, SW, Wahl, MI, Leitges M, Tarakhovsky, A, Tabuchi, R, Kato,R, Turck, CW, Kawakami, T, Witte ON, Rawlings, DJ. PKCß modulates antigen receptor signaling via regulation of Btk membrane localization. (2001) EMBO 20:5692-5702
Su TT, Rawlings DJ. Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development. (2002) J. Immunol. 168: 2101-2110.
Su TT, Guo B, Kawakami Y, Sommer K, Chae K, Humphries LA, Kato RM, Kang S, Patrone L, Wall R, Teitell M, Leitges M, Kawakami T, Rawlings DJ. PKCβ controls IκB kinase lipid raft recruitment and activation in response to BCR signaling. (2002) Nature Immunol 3:780-786.
Saito K, Tolias KF, Saci A, Koon HB, Humphries LA, Scharenberg A, Rawlings DJ, Kinet J-P, Carpenter CL. Btk regulates PtdIns-4,5-P2 synthesis: importance for calcium signaling and PI3K activity. (2003) Immunity 19:669-678.
Yu PW, Tabuchi RS, Kato RM, Astrakhan A, Chae K, Ellmeier W, Witte ON, Rawlings DJ. Sustained correction of B cell development and function in a murine model of X-linked agammaglobulinemia (XLA) using retroviral gene transfer. (2004) Blood 104(5):1281-1290.
Humphries LA, Dangelmaier C, Kato RM, Griffith N, Irene Bakman I, Christoph W. Turk CW, Daniel JL, Rawlings DJ. Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the PLC? ?SH2-SH3 linker. (2004) J Biol Chem 279(36):37651-37661.
Karen Sommer, Beichu Guo, Joel L. Pomerantz, Ashok D. Bandaranayake, Miguel E. Moreno-García, Yulia L. Ovechkina, and David J. Rawlings. Phosphorylation of the CARMA1 Linker Controls NF-?B Activation. (2005) Immunity 23 (6): 561-574.
Humblet-Baron S, Anover S, Kipp K, Zhu Q, Ye P, Zhang W, Ovechkina Y, Khim S, Astrakhan A, Strom T, Kohn D, Candotti F, Vyas Y, Ochs H, Miao C, Rawlings D. Lentiviral Vector-Mediated Gene Therapy as Treatment for Wiskott-Aldrich Syndrome (WAS): Pre-Clinical Studies in Human Cell Lines and WASP -/- Mice. Molecular Therapy, 11(1): S133, 2005.
Miao, CH, Ye, P, Thompson, AR, Rawlings, DJ, Ochs, HD. Immunomodulation of transgene responses following naked DNA transfer of human factor VIII into hemophilia A mice. (2006) Blood 1;108(1):19-27.
Gomez TS, McCarney SD, Carrizosa E, Labno CM, Comiskey EO, Nolz JC, Zhu P, Freedman BD, Clark MR, Rawlings DJ, Billadeau DD, Burkhardt JK. HS1 Functions as an Essential Actin-Regulatory Adapter Protein at the Immune Synapse. (2006) Immunity 24:1-12.
Zhang W, Humblet-Baron S, Kipp K, Khim S, Jarjour J, Sommer K, Stirling B, Pernell L, Rawlings DJ. Rescue of B Cell Development in an Animal Model of X-Linked Agammaglobulinemia (XLA) Via B Lineage-Specific Lentiviral Gene Therapy. Molecular Therapy, 13(1): S64, 2006.
Humblet-Baron S, Sather B, Anover S, Becker-Herman S, Kasprowicz DJ, Khim S, Nguyen T, Hudkins-Loya K, Alpers CE, Ziegler SF, Ochs H, Torgerson T, Campbell DJ, Rawlings DJ. Wiskott-Aldrich syndrome protein is required for regulatory T cell homeostasis. (2007) J Clin Invest. 1;117(2):407-418.
Humblet-Baron S, Zhang W, Kipp K, Khim S, Jarjour J, Sommer K and Rawlings DJ. B lineage-Specific Lentiviral Gene Therapy Rescues B cell Development in an Murine Model of X-linked Agammaglobulinemia(XLA). XIIth Meeting of the European Society for Immunodeficiencies, Abstract book, 2006.
Astrakhan A, Omori M, Nguyen T, Becker-Herman S, Iseki M, Aye T, Hudkins-Loya K, Dooley J, Farr A, Alpers CE, Ziegler S, and Rawlings DJ. Local increase in thymic stromal lymphopoietin induces systemic alterations in B cell. (2007) Nature Immunology 8(5):522-531.
Evans JG, Chavez-Rueda KA, Eddaoudi A, Meyer-Bahlburg A, Rawlings DJ, Ehrenstein MR, Mauri C. Novel suppressive function of transitional 2 B cells in experimental arthritis. (2007) J Immunol 178(12):7868-7878.
Rawlings DJ, Sommer K, Moreno-García ME. The CARMA1 signalosome links the signalling machinery of adaptive and innate immunity in lymphocytes. (2006) Nat Rev Immunol 6(11):799-812.
Scharenberg AM, Humphries LA, and Rawlings DJ. Calcium signalling and cell-fate choice in B cells. (2007) Nat Rev Immunol. 7(10):778-789
Dorshkind K and Rawlings DJ. Immunologic Basics of Hematology”, Part 2: B-Cell Immunity. (2006) Hematology: Basic Principles and Practice. 5th Edition. (In Press)