2010 Progress Report – Executive Summary
Nucleoprotein machines manage the business of information storage and transfer for the cell, including DNA replication and repair, RNA synthesis, and protein translation. The science and engineering focus of this center is on developing tools and concepts to better understand nucleoprotein machines, using as a prototype natural machines that repair DNA double-strand breaks (DSBs). The center is developing and applying bright photostable probes, super-resolution microscopy, and other physical and computational tools to investigate assembly, disassembly, and control of DNA break-repair machines. Inspired by a natural process of V(D)J recombination, which uses the double-strand break repair machine to insert, delete, and alter DNA sequences at specific sites, the Center also hopes to create engineered nucleoprotein machines to provide genetic cures for common human diseases. Such machines would be most applicable to diseases that are reasonably common, life-threatening, caused by a single-gene defect, and where correction of the defect in even a fraction of cells would provide clinical efficacy. Sickle cell disease is the initial disease target of this Center.
Consistent with the vision of NIH Roadmap Initiative in Nanomedicine, the long-term goals of this NDC are bold and ambitious: the Center investigators hope to obtain a general understanding of engineering design principles of nucleoprotein machines that carry out templated copying of nucleic acids, mediate genetic recombination, and catalyze RNA processing; they hope to establish the ability to precisely modify the information stored in DNA and RNA, thus providing novel therapeutic strategies for a wide range of human diseases, including sickle cell disease. Achieving this ability may take decades and will require significant development and clinical resources; however, initial work in developing a Pathway to Medicine (PtM) will serve as a test bed for such a vision.
The major short-term goals of our NDC are to develop new tools and approaches to characterize the dynamics of model nanomachines, the nonhomologous end joining (NHEJ) and homologous recombination (HR) complexes that repairs DNA double strand breaks. These complexes are relatively simple – they have fewer than 10 core components. Importantly, NHEJ and HR occur within self-organizing structures, or “repair foci” that are amenable to live-cell visualization using technologies being developed in the NDC. NHEJ and HR are also relevant to the long-term goals of the Nanomedicine initiative. A better understanding of the NHEJ and HR machines, including their structure-function relations and associated signaling and control loops, will be an important step toward the long-term goal of manipulating DNA and RNA at will.
Through close collaborations, biologists, bioengineers, chemists, and computational scientists in this NDC have already pushed the limits of emerging technologies to provide important scientific advances. The five most important advances include (1) development of orthogonal strategies to tag components of NHEJ and HR complexes simultaneously in vivo with different-color fluorescent labels; (2) synthesis of biocompatible quantum dots that up to 50 times less bulky than any that are commercially available; (3) development of small orthogonal fluorogenic beacons that provide a novel means to image protein-protein interactions in vivo; (4) collection of the first-ever real-time images of core NHEJ proteins migrating to sites of enzymatically induced double-strand breaks in living cells; (5) development of the first practical method for delivery of a protein therapeutic directly to the mammalian cell nucleus.
Current and future work of the NDC has been consolidated into three thrust areas:
- Development and application of protein tagging strategies. Their goal is to design, synthesize and validate optical imaging probes that are small (4–6 nm), biocompatible, photostable and potentially activatable, and to adopt these as part of orthogonal protein targeting/tagging strategies to label 4–6 components of a nanomachine with high specificity.
- Characterization of initial steps in assembly of HR and NHEJ complexes. They will induce DSBs in vivo in a controlled manner, using several complementary technical approaches. They will combine novel probes developed with high-sensitivity, high-resolution optical imaging instrumentation to achieve the ultimate goal of observing assembly of single DNA repair complexes deep inside living cells. They will elucidate the NHEJ/HR pathway choices and develop means to shift the choice. In parallel studies, they will track repair complex assembly using an in vitro single-molecule detection system based on creation of a site-specific DSB within a topologically isolated loop.
- Design, delivery, and validation of a gene correction nanomachine (PtM). The machine is based on ZFN or RAG-mediated DNA recognition and cleavage and will be used initially to correct the sickle β-globin gene. Sickle anemia, which occurs in persons who inherit two sickle β-globin alleles, is common, painful, life threatening, and presently incurable. Importantly, it is caused by a single mutation that is the same in every patient worldwide, and 100% efficient correction of the sickle β-globin gene in hematopoietic stem cells (HSCs) is not required for clinical efficacy. The center will create and optimize synthetic, engineered enzymes that safely activate the HR pathway for correcting the mutant β-globin gene. They will develop methods to introduce these enzymes together with a gene correction template, into hematopoetic stem cells. Following gene correction, the stem cells will be re-engrafted into the patient, mitigating or curing the disease.
Modern medicine – allopathic medicine – focuses on treating symptoms, commonly through small-molecule enzyme inhibitors and receptor agonists/antagonists. It does not address the underlying genetic causes of disease. Consistent with the vision of the Nanomedicine Initiative, the Center investigators envision that the allopathic model will someday be replaced by therapies that directly modify information contained in a patient’s DNA.
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