Barry L. Stoddard

Appointments and Affiliations

 
 
Fred Hutchinson Cancer Research Center
Basic Sciences Division
Program in Structural Biology
Full Member
University of Washington
School of Medicine
Biochemistry
Affiliate Professor
Professional Headshot of Barry L. Stoddard

Mailing Address

Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N.
A3-025
Seattle, Washington 98109-1024
United States

Contact

Degrees

Postdoctoral Fellow, University of California, Berkeley, MCB, Divison of Biochemistry, 1992.
Ph.D., Massachusetts Institute of Technology, Biophysical Chemistry, 1990.
B.A., Whitman College, Chemistry, 1985.

Research Interests

The goal of this laboratory is to understand the structure/function relationships of several interesting biological systems at the atomic level. The tools employed are X-ray crystallography, computer modelling, and genetic manipulation of the molecules of interest. Three projects are summarized below:

PROJECT 1: Structure, function and engineering of intron-encoded protein factors.

Intron-encoded proteins ('homing endonucleases'), found in eubacteria, archea, and single cell eukaryotes, recognize DNA sequences with very high specificity and promote the gene-specific transfer of introns and inteins within host genomes. In addition, many of these proteins also act as specific cofactors for intron-splicing (displaying 'maturase' activity), by forming tightly bound ribonucleoprotein complexes to their cognate introns.

Over the past five years, we have determined the structure and mechanism of several naturally occuring homing endonucleases. More recently, we have begun to address the problem of re-engineering these proteins to recognize DNA target sequences of our own choice and design. The development of single-protein gene-specific reagents (SP-GSRs) would be a major step towards the creation of artificial biomolecules that could be used as diagnostic reagents for genetic disorders, sensors against bacterial and viral pathogens, and research tools for molecular and cellular biology. An SP-GSR is a protein molecule which can recognize and bind a single unique target site located within an entire complex genome. The protein could also be engineered to carry out a variety of chemical processes at the DNA target site. Such a molecule would have to be extraordinarily specific, because the size of the human genome is quite large--approximately four billion base pairs in length. An arsenal of such molecules, directed against DNA sequences of an investigator's own choosing, does not currently exist.

We have initiated a project to combine genetic, biochemical and computational experiments in order to design and engineer artifical SP-GSRs from homing endonucleases. It should be possible to create new DNA specificities for these proteins by combining genetic selection strategies with computational protein redesign methods. New methods of computational molecular design and of genetic selection must be invented and then used in a rational design cycle for this project to succeed.

PROJECT 2: Structure, function and engineering of nucleotide synthesis and salvage enzymes for directed anticancer gene therapy.

Nucleotide synthesis and salvage enzymes are well-documented targets for the design of inhibitors for chemotherapeutic and antibiotic drug development, and for the creation of altered enzymatic catalysts for gene-therapy applications. Our lab is working on structural and mechanistic studies of several enzymes from this broad metabolic area, including tetrahydrofolate synthetase, cyclohydrolase and dehydrogenase, cytosine deaminase (CD), deoxycytidine kinase (dCK) and human ribonucleotide reductase. In particular, the selection and engineering of CD and dCK variants that act efficiently on nucleotide analogues is an important goal, for the purpose of creating enhanced catalysts for anticancer prodrug-gene therapy.

Prodrug gene therapy is a therapeutic strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme which is capable of converting a nontoxic prodrug into a potent cytotoxin. Such a method allows selective eradication of tumor cells while sparing normal tissue from significant cell killing. The effectiveness of this strategy is dependent on a bystander effect in which untransfected tumor cells are killed through active or passive transport of the cytotoxic enzyme product. Several enzyme/prodrug combinations are under active investigation, demonstrating effectiveness in both tissue culture and animal models. However, the combination of low transfection efficiencies and poor turnover of prodrug substrates limit the efficiency of cell killing in the tumor. In order to improve such therapies, enzyme variants must be selected and engineered for enhanced turnover of the prodrug substrate. In this project, a collaboration of two laboratories are optimizing cytosine deaminase and deoxycytidine kinase for prodrug suicide gene therapy, using a combination of structural biology and directed evolution screens, and to test the efficacy of enzyme variants in cell line and animal models.

PROJECT 3: Protein Engineering (in collaboration with Drs. David Baker and Ray Monnat, Jr. at the University of Washington and Dr. Andrew Scharenberg at the Childrens Hospital Research Institute in Seattle).

As described in projects 1 and 2 above, a major focus of our research is the selection and engineering of a variety of enzyme catalysts for altered substrate specificities, and for altered biophysical properties such as enhanced stability and catalytic lifetimes. In 2002, we carried out what was (for us) a seminal experiment in which we created a fully active, chimeric homing endonuclease capable of recognizing and cleaving a complementary chimeric target site (article (3) below). As part of that project, we discovered that computational algorithms being developed by the laboratory of David Baker at the UW could be successfully applied to such a problem, by repacking a protein interface and creating a novel domain packing architecture. Subsequently, we have used similar algorithms with great success for a variety of purposes, such as the thermostabilization of an enzyme catalyst for prodrug gene therapy.

Our current and long-term aims is to continue to develop and exploit computational engineering algorithms to alter substrate binding specificities for homing endonucleases and cytosine deaminase enzymes. Both projects involve the repacking and optimization of protein-nucleic acid interfaces, which poses substantial challenges relative to protein cores and interfaces.

Additional Experience

Member, Defense/Science StudyGroup (DSSG) 2000-2001

Consultant, New England Biolabs (Ipswitch, MA) 2005 - present

Consultant and Member of Scientific Advisory Board, Targeted Growth, Inc (Seattle, WA) 2006 - present.

Intellectual Property Law Consultant and Expert Witness
(Kenyon & Kenyon, LLP; Finnegan-Henderson LLP; Kaye Scholer LLP) 2008 - Present

Industry

Co-founder, Precision Genome Engineering Inc. 2006 - present

Languages

(Reading, Writing, Speaking)

English: (Fluent, Fluent, Fluent)

Memberships

American Association for the Advancement of Science
American Crystallographic Association

Previous Positions

1990-1992, Postdoctoral Research Fellow, University of California, Berkeley, College of Arts and Sciences, Molecular and Cellular Biology, Biochemistry

Recent Publications

2017
2016
2015
Siegel JB, Smith AL, Poust S, Wargacki AJ, Bar-Even A, Louw C, Shen BW, Eiben CB, Tran HM, Noor E et al..  2015.  Computational protein design enables a novel one-carbon assimilation pathway.. Proceedings of the National Academy of Sciences of the United States of America. 112(12):3704-9. Abstract
2014
2013

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