A novel protein engineering tool for rapid manufacturing of designer nucleosomes

PROJECT SUMMARYEpiCypher is pioneering the commercial development of fully defined recombinantsemi synthetic nucleosomes carrying specific histone post translational modificationsPTMstermed designer nucleosomes or dNucsTMfor epigenetics researchWe currently use native chemical ligationNCLto generate modified histones for dNuc assemblyand have developed proprietary methods to efficiently manufacturemg histone lots carrying single or combinatorial PTMs on H BH AHor Hwe are currently applying these to dNucbased applicationsincluding several high throughput inhibitor screening platformsAlphaNucTM and EpiDyneTMand the first qualitativequantitative ChIP platformSNAP ChIPTMDespite our optimization of dNuc manufacturingthe approaches are multisteplabor intensiveand requires four weeks of dedicated effort to synthesize each modified histone in the milligram scaleplus three more weeks for octamernucleosome assembly and associated QCAs suchthis pipeline is unsuitable for the manufacturing of combinatorial diversity nucleosome panels for discovery based drug development and basic research applicationsHereEpiCypher is developing a protein engineering tool for accelerated dNuc manufacturingOur innovative approach uses the Sortase ASrtAtranspeptidase to ligate modified peptides directly onto fully assembled tailless nucleosomestNucsthus generating verSaNucsTMWhile NCL is optimized to generate large quantities of highly pure histonesdNucs for applications requiring homogeneous PTM defined substratesthe verSaNuc approach enables two newfound capabilitiesathe rapid development of modified nucleosomes in small batchg scale vsmg for NCLandbthe ability to multiplex modified nucleosome synthesesThese newfound capabilities are significant as NCL lacks the throughput to deliver nucleosome substrates covering the vast PTM diversityeither of combinatorial or lesser studied marksobserved in vivoThusverSaNuc manufacturing is directly complementary to our NCL approachIn Phase Iwe have developed a ligation strategy to rapidly generate verSaNucs with modifications on histone HWe showed that verSaNucs are generally functionally equivalent in many downstream assays when compared with dNucs made using NCLIn Phase IIwe will use our verSaNuc manufacturing strategy to rapidly generate a diverse collection of high quality singlyand combinatorially modified nucleosomesAimand functionally validate these biochemical substrates using two proprietary nucleosome based assay platformsAlphaNucTM and EpiDyneTMAimAlsowe will leverage the ability to multiplex verSaNuc manufacturing to develop verSaScreenTMa high throughput discovery platform containing verSaNucs inwell format to rapidly decipher the combinatorial histone codeAimThis technology will enable the development of next generation nucleosome based tools and the expansion of modified nucleosomes into new exploratorydiscovery markets PROJECT NARRATIVESemi synthetic nucleosomes carrying specific post translational modificationsPTMsknown as designer nucleosomesdNucsrepresent powerful substrates for novel drug discovery and developmentHowevertheir potential is limited by the large investment of time and resources currently required for individual histone synthesis at milligram scaleHereEpiCypher is commercializing development of a protein engineering tool to multiplex manufacturing of nucleosomes carrying PTM combinations at the microgram scaleOur innovative technology allows for rapid commercial manufacturing of a diverse catalog of nucleosomes carrying diseaserelevant modificationsenabling the development of next generation nucleosome based tools and the expansion of such reagents into new exploratorydiscovery markets