The biomedical science has long sought to correct disease-causing mutations, disable the genomes of invading pathogens, arm immune cells to attack tumors, and enable countless other therapeutic opportunities through the modification of genomes. Genome editing broadly represents the diverse technologies that make different genomic alterations in various contexts.

In recent years, many user-programmable genome editing techniques and technologies have been developed. These include homologous recombination, zinc-finger nucleases (ZFNs), meganucleases, and transcription activator-like effector nucleases (TALENs). Perhaps the most well-known of these technologies are CRISPR-Cas systems, which target gene sequences with easily programmable designer RNA guides.

These platforms operate through the repair of nuclease-induced breaks in the genome that can be exploited to induce genome edits, either gene knockouts or precise correction through homology-directed repair. Some editing approaches involve the insertion of vector-derived cargo sequences into the genome. Other strategies use nuclease-inactive forms that can be fused to enzymes to alter chromatin without changing the DNA sequence (such as base editing and prime editing).

Genome editing of somatic cells is carried out either ex vivo, followed by the reintroduction of edited cells into the patient, or in vivo, by delivering the editing machinery to tissues within the body. Clinical trial data have demonstrated that ex vivo editing of allogeneic T cells can be used to fight cancer and autologous hematopoietic stem cells to eliminate the need for blood transfusions in patients with sickle cell disease. However, ex vivo editing is logistically complex, expensive, and hard to scale. Therefore, the biomedical community is developing in vivo approaches for the editing of somatic cells.

To achieve success with in vivo approaches, a number of new technologies must be developed that offer better control of precise genomic changes at targeted sites, with reduced potential for unintended modifications at both targeted and nontargeted sites, and with a better understanding of the biological consequences of unintended editing events. The consortium also aims to develop new technologies that enable sequence-specific alterations (base editing and prime editing) and methods to detect unwanted genomic events with increased predictive ability and sensitivity, as well as establish new safe and effective delivery strategies.

Despite the potential of these therapeutic genome editing tools, significant challenges still prevent potential therapies from being fully realized. The NIH formed the SCGE consortium in 2018 and allocated to the program $190 million in funds over six years. To date, the consortium includes 72 principal investigators from 38 institutions who are pursuing 45 projects.

"NIH realized it was important for all of us who are investigating gene editing to work together toward a common goal," said Danith Ly, PhD, a Carnegie Mellon University professor who joined the consortium in 2019, in a statement. "We're designing molecules that can go into the cell and we're cataloging each and every one. What we'll end up with is a very valuable, rigorously evaluated resource for those who want to bring gene editing to patients."

Innovating genome editing

With the overall value of transparency, the research conducted by the consortium will result in tools, reagents, methods, and best practices that will be available to the larger research community and the public.

The consortium seeks to discover new editors and build upon existing editors in part by tuning them for increased precision. This includes new CRISPR-Cas systems as well as gene editors that are based solely on nucleic acid analogues that do not require protein cofactors (helicases, nucleases, transposases, or recombinases).

Moreover, the consortium will continue to develop and improve engineered platforms (base editing techniques). These include base editors that can catalyze C-to-T transitions (cytosine base editors [CBEs]), A-to-G transitions (adenine base editors [ABEs]), or C-to-G transversions with broader targeting capabilities and increased specificity. As an example, prime editing, which eliminates limitations in changes to the targetable nucleotides, was partly developed through the SCGE consortium. It is also exploring epigenome editing modalities that can extend the genome engineering toolbox by altering gene expression or reprogramming cell phenotypes.

One example of this is peptide nucleic acids (PNAs), which are relatively small, synthetic molecules that recognize specific DNA sequences through triplex formation and subsequently induce editing. Additionally, the engineering of editors that target mitochondrial DNA could open up genome editing therapies for the treatment of mitochondrial diseases, which affect one in approximately 5,000 people, according to the authors.

In terms of delivery systems, the SCGE consortium is working on 20 distinct projects that will explore new methods for the delivery of genome editing machinery to specific tissue types in vivo. In these projects, existing viral vectors and nanoparticles are being improved, along with the examination of other promising strategies for better in vivo delivery.

Benchmarks

The consortium is working to generate in vivo reporter systems that are broadly applicable to many delivery systems and editing technologies and are independent of the target cell or tissue type, or the specific disease to be corrected. They are being developed in the context of small and large animal models to support preclinical research. The systems will have the ability to detect activity of multiple nucleases, as well as other types of editors (ABEs, CBEs, and PNA-based editing systems). The consortium is also developing techniques for in vivo cell tracking using advanced imaging methods.

To test products in humans, the consortium is working to develop human cell-based and organoid platforms to define the unintended biological effects of editing. Projects use human primary cells when possible. These systems can be scaled up to enable studies at higher throughput than would be feasible in animal models, and they can also facilitate deeper molecular characterization of the various outcomes after editing different human cell types within a tissue, the consortium said.

Importantly, each project is integrated and requires multiple phases, including an initial phase that establishes proof of principle; an intermediate phase that involves testing in small animals, followed by technology scale-up; and finally, testing in large animal models.

At the end of the project, the consortium will compile new genome editors, delivery technologies, and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, into an SCGE toolkit that will be shared with the biomedical research community.

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