David R. Liu
Department of Chemistry and Chemical Biology, Harvard University
Howard Hughes Medical Institute
Programming Human Biology Using Next-Generation Macromolecular and Small-Molecule Tools for the Understanding and Treatment of Disease
The vast majority of current therapeutic agents function by binding to disease-associated macromolecules and modulating their activity. Recent developments, however, have made increasingly realistic the possibility of developing next-generation therapeutics that do not simply bind targets implicated in disease, but instead alter the covalent structure of genes and gene products in ways that can more effectively treat—or even cure—many diseases. While the possibility of precisely manipulating genes and proteins in mammalian cells and, eventually, in humans, has enormous potential, several major challenges must be overcome to fully realize this vision. Perhaps the most significant of these challenges are the efficient creation of the macromolecules that are needed to alter genomes or proteomes with a high degree of selectivity and potency, and the efficient delivery of macromolecules into target cells at therapeutically relevant doses. To realize a vision in which arbitrary genes or proteins can be manipulated in mammalian cells to treat disease thus requires new approaches to rapidly generating macromolecules with precise, tailor-made properties, and new approaches to delivering therapeutic macromolecules into cells.
These approaches will likely exploit recently discovered natural proteins, coupled with new technologies such as phage-assisted continuous evolution (PACE) for rapidly evolving and engineering these proteins towards uses that advance the science of therapeutics. For example, the creation of robust platforms of orthogonal and programmable CRISPR (Cas9)-based or TALE-based genome editing and transcriptional regulation tools that are capable of turning "on", turning "off", or altering the nucleotide sequence of any combination of genes or regulatory sequences in the human genome represents an ambitious but well-defined goal that would have an major impact on illuminating disease biology and potentially treating genetic diseases.
In addition to evolving and engineering macromolecules, discovering and developing small molecules that can modulate the biological activities of targets validated using genome engineering proteins is an essential activity to connect new biological insights to leads for therapeutic development. Some targets may only be addressable using macromolecular therapeutics by virtue of their binding energies and ability to catalyze transformations such as manipulating the covalent structure of genes and proteins. For other targets, however, small molecules will likely remain the most promising class of agents to modulate activities in therapeutically relevant contexts. Therefore, the development and application of new, highly efficient small-molecule discovery technologies such as the selection of DNA-encoded small-molecule libraries against many biological targets of interest in a single experiment will play crucial roles.
The activities needed to realize this vision can be classified into three stages:
Phase 1: Develop the tools. New methodologies and technologies to characterize, engineer, and evolve genome-editing proteins will be developed and applied to transform natural components such as Cas9 or TALE domains into variants with the specificity, context independence, activity level, stability, cellular compatibility, and effector functions necessary to illuminate or address human disease. These effector functions will likely include DNA cleavage, transcriptional activation, transcription repression, epigenetic modification, and recombination to insert, delete, or replace alleles. While these activities have become a focus of several laboratories, many of the key developments and insights have either not yet been reported, or have only very recently been described. Importantly, TALE- and CRISPR-based systems are programmable using a simple code that relates target DNA sequences with TALE or CRISPR protein or RNA sequences. Because this programmability alone, while crucial, is insufficient to ensure that these tools can support Phase 2 and Phase 3 activities and realize their therapeutic potential, methods to rapidly characterize, improve the specificity, and enable the regulation of these tools must also be developed.
In addition to programmable DNA-binding proteins and protein-RNA complexes, other macromolecules capable of manipulating biological information flow in human cells including antibodies, proteases, sortases, recombinases, polymerases, and nucleases are also poised to play key roles in the understanding and next-generation treatment of disease. As is the case with TALE and CRISPR systems, a primary determinant of the likely impact of these proteins is our ability to evolve or engineer therapeutically relevant levels of activity, specificity, stability, and/or cell-state dependence. Therefore, general methods that can efficiently characterize and improve diverse classes of proteins may prove especially valuable to Phase 1 efforts.
Finally, the development of rich collections of small molecules together with methods for their rapid screening or selection (in the case of DNA-encoded libraries) will power small-molecule discovery efforts that will yield new tools to validate the rapidly growing set biological targets known to play potential roles in human disease.
Phase 2: Discover the programs. Sets of evolved or engineered macromolecules or small molecules generated in Phase 1 will be used to discover and test causal relationships between genes, gene products, and disease-associated pathways in mammalian cells. As Phase 1 methods become increasingly effective, and larger and larger sets of these tools become accessible, Phase 2 activities will transition from a hypothesis-testing mode (does gene A when upregulated and protein B when inhibited induce disease if gene C is mutated?) into a hypothesis-generating mode with the goal of discovering sets of genes or proteins that when activated, repressed, or modified by macromolecules or small molecules alter the propensity of human cells to enter a diseased state.
Phase 3: Enable therapeutics. The knowledge from Phase 1 and Phase 2 will trigger new drug discovery efforts through the identification of new targets for small-molecule screening and development. In addition, the gene- and gene product-modifying tools themselves, if sufficiently specific and active, may have potential as future macromolecular therapeutics. Phase 3 efforts therefore will aim to develop both small-molecule and macromolecular therapeutics that program human cells in the ways discovered in Phase 2. The macromolecular side of this phase will require characterizing and improving macromolecular delivery (using novel technologies such as supercharged proteins), biodistribution, immunogenicity, and efficacy studies in cell culture and animal models of human disease.
Implementing this ambitious vision in a way that impacts society outside of the laboratory will require a multidisciplinary, highly collaborative culture that seamlessly integrates chemists, molecular and cell biologists, macromolecule engineering and evolution experts, clinicians, and bioinformaticists. In addition, industry experts and entrepreneurs may also play key roles in fully realizing the therapeutic potential of the resulting discoveries.
Specific examples of transformative applications include:
Š Revealing the genetic dependencies of oncogenesis, infectious disease progression, and metabolic disorders in therapeutically relevant settings
Š Programming the expression of sets of transcription factors that induce the differentiation, dedifferentiation or transdifferentiation of therapeutic cells (for example, pancreatic exocrine cells into beta cells in diabetics, white adipose tissue into brown fat in patients with metabolic disorders, or serotonergic neurons into dopaminergic neurons in Parkinson’s patients)
Š Altering the structure of the genes in infected individuals to disrupt the life cycle of infectious disease agents (as a validated example, editing CCR5 in HIV patients)
Š Developing therapeutic proteases or sortases with tailor-made specificities that can cleave or modify disease-associated proteins with high specificity and activity
Š Programming cells containing disease-associated genetic changes to undergo apoptosis
Š Implicating genes and gene combinations that grant resistance or sensitivity to known bioactive molecules for which there is no target known
Relevant references from our group
“Cationic Lipid-Mediated Delivery of Proteins Enables Efficient Protein-Based Genome Editing In Vitro and In Vivo” Zuris, J. A.; Thompson, D. B.; Shu, Y.; Guilinger, J. P.; Bessen, J. L.; Hu, J. H.; Maeder, M.; Joung, J. K.; Chen, Z.-Y.; Liu, D. R. Nature Biotechnology in press (2014).
“A System For the Continuous Directed Evolution of Proteases Rapidly Reveals Drug-Resistance Mutations” Dickinson, B. C.; Packer, M. S.; Badran, A. H.; Liu, D. R. Nature Comm. in press (2014).
“Reprogramming the Specificity of Sortase Enzymes” Dorr, B. M.; Ham, H. O.; An, C.; Chaikof, E. L.; Liu, D. R. Proc. Natl. Acad. Sci. USA in press; available online (2014).
“Anti-Diabetic Activity of Insulin-Degrading Enzyme Inhibitors Mediated by Multiple Hormones” Maianti, J. P.; McFedries, A.; Foda, Z. H.; Kleiner, R. E.; Du, X.-Q.; Leissring, M. A.; Tang, W.-J.; Charron, M. J.; Seeliger, M. A.; Saghatelian, A.; Liu, D. R. Nature 511, 94 (2014).
“Fusion of Catalytically Inactive Cas9 to FokI Nuclease Improves the Specificity of Genome Modification” Guilinger, J. P.; Thompson, D. B.; Liu, D. R. Nature Biotechnology 32, 577 (2014).
“Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity” Guilinger, J. P.; Pattanayak, V.; Reyon, D.; Tsai, S. Q.; Sander, J. D.; Joung, J. K.; Liu, D. R. Nature Methods 11, 429 (2014) (2014)
“Comprehensive Off-Target DNA Cleavage Profiling Reveals RNA-Programmed Cas9 Nuclease Specificity” Pattanayak, V.; Lin, S.; Guilinger, J.P.; Ma, E.; Doudna, J. A.; Liu, D. R. Nature Biotechnology 31, 839 (2013).
“Highly Specific, Bisubstrate-Competitive Src Inhibitors From DNA-Templated Macrocycles” Georghiou, G.; Kleiner, R. E.; Pulkoski-Gross, M.; Liu, D. R.; Seeliger, M. A. Nature Chemical Biology 8, 366 (2012).
“A System for the Continuous Directed Evolution of Biomolecules” Esvelt, K. M.; Carlson, J. C.; Liu, D. R. Nature 472, 499 (2011).
“DNA-Templated Organic Synthesis and Selection of a Library of Macrocycles” Gartner, Z. J.; Tse, B. N.; Grubina, R.; Doyon, J. B.; Snyder, T. M.; Liu, D. R. Science 305, 1601 (2004).