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The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Fragile X Syndrome (FXS) is a neurological disorder caused by epigenetic silencing of the FMR1 gene. Reactivation of FMR1 is a potential therapeutic approach for FXS that would correct the root cause of the disease. Here, using a candidate-based shRNA screen, we identify nine epigenetic repressors that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors, or FMR1-SFs). Inhibition of FMR1-SFs with shRNAs or small molecules reactivates FMR1 in cultured undifferentiated induced pluripotent stem cells, neural progenitor cells (NPCs) and post-mitotic neurons derived from FXS patients. One of the FMR1-SFs is the histone methyltransferase EZH2, for which an FDA-approved small molecule inhibitor, EPZ6438 (also known as tazemetostat), is available. We show that EPZ6438 substantially corrects the characteristic molecular and electrophysiological abnormalities of cultured FXS neurons. Unfortunately, EZH2 inhibitors do not efficiently cross the blood-brain barrier, limiting their therapeutic use for FXS. Recently, antisense oligonucleotide (ASO)-based approaches have been developed as effective treatment options for certain central nervous system disorders. We therefore derived efficacious ASOs targeting EZH2 and demonstrate that they reactivate FMR1 expression and correct molecular and electrophysiological abnormalities in cultured FXS neurons, and reactivate FMR1 expression in human FXS NPCs engrafted within the brains of mice. Collectively, our results establish EZH2 inhibition in general, and EZH2 ASOs in particular, as a therapeutic approach for FXS. [ABSTRACT FROM AUTHOR]

The ability to metabolically label DNA in a way that produces a latent change from one nucleobase to another would create asignal that can beamplified by PCR--this in turn would allow studies of newly synthesized DNA using high-throughput sequencing. To function as an amplifiable metabolic label, a nucleotide analogue would need to be taken up by cells and incorporated into cellular DNA; after purification of DNA, it could be converted into a different nucleobase with a different base pairing pattern. We selected 4-thiothymidine (4sT) as a candidate metabolic label: 4sT is readily taken up by a large number of polymerases in vitro, and we present a method that allows 4sT to be converted into 5-methyl-2'-deoxycytidine (5mC) after incorporation into DNA. Encouraged by these results, we treated cells with 4sT nucleoside; however, we found that 4sT is not incorporated into DNA in bacterial, yeast, or mammalian cells to useful levels under the conditions we tested. A phosphorodiamidate prodrug of 4sTMP was successfully synthesized but did not measurably improve incorporation into cellular DNA. Key words: metabolic labelling, convertible nucleobase, nucleoside prodrug, 4-thiothymidine. La capacite de realiser le marquage metabolique de l'ADN de maniere a produire la transformation latente d'une base nucleique en une autre pourrait permettre de generer un signal amplifiable par PCR, ce qui, par consequent, permettrait d'etudier de nouveaux ADN synthetises par sequençage a haut debit. Pour fonctionner comme un marqueur metabolique amplifiable, un analogue de nucleotide doit pouvoir penetrer dans les cellules et s'incorporer dans l'ADN cellulaire, puis apres purification de l'ADN, doit pouvoir etre transforme en une autre base nucleique dont l'appariement differe. Nous avons choisi la 4-thiothymidine (4sT) comme marqueur metabolique potentiel, car elle est facilement integree in vitro par un grand nombre de polymerases. Nous presentons une methode qui permet de transformer la 4sT en 5-methyl-2'-deoxycytidine (5mC) apres incorporation dans l'ADN. Encourages par ces resultats, nous avons traite des cellules avec le nucleoside 4sT. Cependant, nous avons observe que, dans les conditions evaluees, la 4sT n'est pas incorporee dans l'ADN de cellules de bacteries, de levures et de mammiferes dans une mesure suffisante pour etre utile. Nous avons reussi a synthetiser un promedicament de la 4sTMP a base de phosphorodiamidate, mais nous ne sommes pas parvenus a ameliorer de façon mesurable l'incorporation dans l'ADN cellulaire. [Traduit par la Redaction] Mots-cles : marquage metabolique, base nucleique convertible, promedicament a base de nucleoside, 4-thiothymidine., Introduction Metabolic labeling of cellular biopolymers has opened new frontiers in fields from proteomics (1) to RNA biology (2,3) to glycobiology. (4,5) In this family of approaches, cells or organisms [...]

Prime editing (PE) has gained significant attention as a next-generation gene editing technology, owing to its unique advantages. However, realizing its potential in vivo requires effective delivery strategies. While adeno-associated virus (AAV) has been employed for in vivo delivery of prime editors in research settings, it presents inherent limitations related to vector size, ongoing expression, and inability to re-dose patients. Conversely, lipid nanoparticles (LNPs) do not face these limitations and are emerging as a leading nonviral approach for the delivery of gene editors. In this study, we demonstrate successful co-delivery of chemically modified pegRNA and prime editor mRNA using LNPs for in vivo PE. We investigate the impact of pegRNA chemical modifications on editing efficiency and explore different re-dosing regimens. In a daily-repeat dose regimen, we saw striking liver toxicity and no increase in editing; by contrast, weekly repeat dosing was well tolerated and enabled 1.8-fold increase in editing efficacy. Furthermore, in the NOD scid gamma immunodeficient mouse model, the efficacy of LNP-delivered PE was enhanced by 2.8-fold. In addition, the nature of the ionizable lipids and phospholipids strongly influenced PE efficiency in vivo. Overall, these findings will greatly contribute to the future development of LNPs as a robust platform for delivering prime editors in vivo, fostering progress in PE research and therapeutic applications. [ABSTRACT FROM AUTHOR]