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, but instead alter the covalent structure of genes and gene products in ways that can more effectively treat — or even cure — 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 (1) the efficient creation of the macromolecules that are needed to alter genomes or proteomes with a high degree of selectivity and potency, and (2) the efficient delivery of macromolecules into target cells at therapeutically relevant doses in vitro and, especially, in vivo. To realize a vision in which arbitrary genes or proteins can be manipulated in mammalian cells to treat disease will require novel approaches to rapidly generating macromolecules with precise, tailor-made properties and to delivering therapeutic macromolecules into cells.
These approaches will interface in powerful ways with recently discovered genome-editing proteins, such as CRISPR/Cas9 and TALE repeat arrays, that enable the targeted manipulation of disease-associated genes in live animals. New technologies, such as phage-assisted continuous evolution (PACE), can rapidly evolve these proteins toward variants with increased therapeutic relevance and enhanced abilities to illuminate disease biology. In addition, laboratory-evolved forms of other macromolecules – including proteases, recombinases, and sortases – that selectively manipulate the covalent structure of specific genes and proteins will also play key roles in enabling next-generation therapeutics.
Complementing the development of macromolecular tools and therapeutics, the development of small molecules that can modulate the biological activities of targets implicated in human diseases, especially those without an underlying genetic basis, will continue to connect new biological insights with leads for therapeutic development. Some targets may only be addressable using macromolecules 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 accessible 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 synthesis and selection of DNA-encoded small-molecule libraries against many disease-associated targets in a single experiment, will be crucial.
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, DNA modification, transcriptional activation, transcription repression, chromatin modification, and recombination to insert, delete, or replace genes or gene fragments. Because the properties of naturally occurring genome-editing proteins are insufficient to enable these tools to 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 genome-editing proteins, other macromolecules capable of manipulating the structure of genes and gene products in human cells, including proteases, recombinases, and sortases, are also poised to play key roles in the understanding and next-generation treatment of human disease. As 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.
The development of methods for the production and rapid screening or selection (in the case of DNA-encoded libraries) will power new small-molecule discovery efforts that will yield tools to validate the rapidly growing set of biological targets known to play potential roles in human disease.
Phase 2: Discover the programs. Sets of 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 upregulation of gene A and inhibition of protein B 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 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 promising new drug discovery efforts through the identification of new targets for small-molecule screening and development. In addition, the gene- and protein-modifying macromolecular tools themselves, if sufficiently specific and active, may have potential as future next-generation 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, biodistribution, immunogenicity, and efficacy studies in cell culture and animal models of human disease.
Implementing this ambitious vision in a way that has an effect on society outside of the laboratory will require a multidisciplinary, highly collaborative culture that seamlessly integrates chemists, molecular and cell biologists, disease experts, macromolecule engineering and evolution experts, and bioinformaticists. In addition, industry experts and entrepreneurs may also play key roles in realizing the full therapeutic potential of the resulting discoveries.
Specific examples of transformative applications include
- revealing the genetic dependencies of oncogenesis, infectious disease progression, and metabolic disorders in therapeutically relevant settings;
- 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 patients with Parkinson's disease);
- altering the structure of the genes in infected individuals to disrupt the life cycle of infectious disease agents (as a partially validated example, editing CCR5 in patients with HIV);
- developing therapeutic proteases or sortases with tailor-made specificities that can cleave or modify disease-associated proteins with high specificity and activity;
- programming cells containing disease-associated genetic changes to undergo apoptosis; and
- implicating genes and gene combinations that grant resistance or sensitivity to known bioactive molecules for which there is no target known.
This work is supported in part by grants from the Defense Advanced Research Projects Agency and the National Institutes of Health.
As of March 23, 2016