Journal of Clinical PracticeJournal of Clinical PracticeКлиническая практика2220-30952618-8627Eco-Vector11506310.17816/clinpract115063ReviewsНаучные обзорыReview ArticlePractical application of massively parallel reporter assay in biotechnology and medicineПрактическое применение массового параллельного репортерного анализа в биотехнологии и медицинеhttps://orcid.org/0000-0002-5989-575657201430841N-6935-20153387-6944RomanovStanislav E.РомановСтанислав Евгеньевич
Russian Federation
romanov@mcb.nsc.ruhttps://orcid.org/0000-0003-2174-649657191597308N-7957-20157579-3460LaktionovPetr P.ЛактионовПетр Павлович
Russian Federation

PhD

к.б.н.

laktionov@mcb.nsc.ruNovosibirsk State UniversityНовосибирский национальный исследовательский государственный университетInstitute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of SciencesИнститут молекулярной и клеточной биологии Сибирского отделения Российской академии наук060120232401202313474870512202217122022Copyright ©; 2023, Eco-VectorCopyright ©; 2023, Эко-Вектор2023Eco-VectorЭко-Векторhttps://creativecommons.org/licenses/by-nc-nd/4.0https://clinpractice.ru/clinpractice/article/view/115063

The development and viability of an organism relies on tissue-specific gene programs. The genome regulatory elements play a key role in the regulation of such programs, whereas its disfunction can lead to the establishment of various pathologies, including cancer, congenital disorders, and autoimmune diseases. The development of high-throughput approaches in genomics has led to the emergence of massively parallel reporter assays (MPRA), which enable genome-wide screening and functional verification of regulatory elements. Although MPRA was originally used for investigation of fundamental aspects of epigenetics, it also has a great potential for clinical and practical biotechnology. Currently, MPRA is used for validation of clinically significant mutations, identification of tissue-specific regulatory elements, identification of the favorable loci for transgene integration, as well as represents an essential tool for creating highly efficient expression systems, with a wide range of applications from protein production and design of novel therapeutic antibody super-producers to gene therapy. In this review, the basic principles and areas of practical application of high-throughput reporter assays will be discussed.

Развитие и жизнедеятельность организма опирается на тканеспецифичные генные программы. Ключевую роль в регуляции таких программ играют регуляторные элементы генома, а нарушения в их работе могут приводить к развитию различных патологий, включая пороки развития, онкологические и аутоиммунные заболевания. Развитие высокопроизводительных геномных исследований привело к появлению методов массового параллельного репортерного анализа (МПРА), которые позволяют проводить функциональную проверку и идентификацию регуляторных элементов в масштабе генома. Изначально МПРА применялся в качестве инструмента для исследования фундаментальных аспектов эпигенетики, однако этот подход также имеет огромный потенциал для клинической и практической биотехнологии. На текущий момент МПРА используют для валидации клинически значимых мутаций, идентификации тканеспецифичных регуляторных элементов, поиска наиболее перспективных для интеграции трансгена локусов. МПРА является также незаменимым инструментом для создания высокоэффективных экспрессионных систем, спектр применения которых распространяется от подходов для наработки белков и конструирования суперпродуцентов терапевтических антител нового поколения до генной терапии. В настоящем обзоре предложены к рассмотрению основные принципы и области практического применения методов высокопроизводительного репортерного анализа.

massively parallel reporter assayMPRAexpression systemsbiotechnologyclinical bioengineeringмассовый параллельный репортерный анализМПРАэкспрессионные системыбиотехнологияклиническая биоинженерияМинистерство науки и высшего образования Российской Федерации и Экспертный институт социальных исследованийMinistry of Science and Higher Education of the Russian FederationСоглашение № 075-15-2021-1086, контракт № RF----193021X0015, 15.ИП.21.0015Oudelaar AM, Higgs DR. The relationship between genome structure and function. Nat Rev Genet. 2021;22(3):154–168. doi: 10.1038/s41576-020-00303-xPreissl S, Gaulton KJ, Ren B. Characterizing cis-regulatory elements using single-cell epigenomics. Nat Rev Genet. 2023; 24(1):21–43. doi: 10.1038/s41576-022-00509-1Bradner JE, Hnisz D, Young RA. Transcriptional addiction in cancer. Cell. 2017;168(4):629–643. doi: 10.1016/j.cell.2016.12.013Chatterjee S, Ahituv N. Gene regulatory elements, major drivers of human disease. Annu Rev Genomics Hum Genet. 2017;18: 45–63. doi: 10.1146/annurev-genom-091416-035537Corradin O, Saiakhova A, Akhtar-Zaidi B, et al. Combinatorial effects of multiple enhancer variants in linkage disequilibrium dictate levels of gene expression to confer susceptibility to common traits. Genom Res. 2014;24(1):1–13. doi: 10.1101/gr.164079.113Maurano MT, Humbert R. Rynes E, et al. Systematic localization of common disease-associated variation in regulatory DNA. Science. 2012;337(6099):1190–1195. doi: 10.1126/science.1222794Claringbould A, Zaugg JB. Enhancers in disease: molecular basis and emerging treatment strategies. Trends Mol Med. 2021; 27(11):1060–1073. doi: 10.1016/j.molmed.2021.07.012Ernst J, Kheradpour P, Mikkelsen TS, et al. Mapping and analysis of chromatin state dynamics in nine human cell types. Nature. 2011;473(7345):43–49. doi: 10.1038/nature09906ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012;489(7414): 57–74. doi: 10.1038/nature11247Lonsdale J, Thomas J, Salvatore M, et al. The genotype-tissue expression (GTEx) project. Nat Genet. 2013;45(6):580–585. doi: 10.1038/ng.2653Lee CP, Ko AM, Chiang SL, et al. Regulatory elements in vectors containing the ctEF-1α first intron and double enhancers for an efficient recombinant protein expression system. Scie Rep. 2018;8(1):15396. doi: 10.1038/s41598-018-33500-0Ingusci S, Verlengia G, Soukupova M, et al. Gene therapy tools for brain diseases. Front Pharmacol. 2019;10:724. doi: 10.3389/fphar.2019.00724Pan D, Büning H, Ling C. Rational design of gene therapy vectors. Mol Ther Method Clin Dev. 2019;12:246–247. doi: 10.1016/j.omtm.2019.01.009Xu D, Wang X, Jia Y, et al. SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells. J Cell Mol Med. 2018; 22(4):2231–2239. doi: 10.1111/jcmm.13504Romanov SE, Kalashnikova DA, Laktionov PP. Methods of massive parallel reporter assays for investigation of enhancers. Vavilovskii Zhurnal Genet Selektsii. 2021;25(3):344–355. doi: 10.18699/VJ21.038Thurman RE, Rynes E, Humbert R, et al. The accessible chromatin landscape of the human genome. Nature. 2012;489(7414):75–82. doi: 10.1038/nature11232Andersson R, Sandelin A. Determinants of enhancer and promoter activities of regulatory elements. Nat Rev Genet. 2020;21(2):71–87. doi: 10.1038/s41576-019-0173-8Furlong EE, Levine M. Developmental enhancers and chromosome topology. Science. 2018;361(6409):1341–1345. doi: 10.1126/science.aau0320Pang B, van Weerd JH, Hamoen FL, Snyder MP. Identification of non-coding silencer elements and their regulation of gene expression. Nat Rev Mol Cell Biol. 2022. doi: 10.1038/s41580-022-00549-9Chen D, Lei EP. Function and regulation of chromatin insulators in dynamic genome organization. Curr Opin Cell Biol. 2019;58: 61–68. doi: 10.1016/j.ceb.2019.02.001Haberle V, Stark A. Eukaryotic core promoters and the functional basis of transcription initiation. Nat Rev Mol Cell Biol. 2018;19(10):621–637. doi: 10.1038/s41580-018-0028-8Lettice LA, Williamson I, Devenney PS, et al. Development of five digits is controlled by a bipartite long-range cis-regulator. Development. 2014;141(8):1715–1725. doi: 10.1242/dev.095430Pennacchio LA, Bickmore W, Dean A, et al. Enhancers: five essential questions. Nat Rev Gen. 2013;14(4):288–295. doi: 10.1038/nrg3458Dickel DE, Ypsilanti AR, Pla R, et al. Ultraconserved enhancers are required for normal development. Cell. 2018;172(3):491–499.e15. doi: 10.1016/j.cell.2017.12.017Sur I, Taipale J. The role of enhancers in cancer. Nat Rev Canc. 2016;16(8):483–493. doi: 10.1038/nrc.2016.62Bell AC, West AG, Felsenfeld G. The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell. 1999;98(3):387–396. doi: 10.1016/s0092-8674(00)81967-4Kellum R, Schedl P. A position-effect assay for boundaries of higher order chromosomal domains. Cell. 1991;64(5):941–950. doi: 10.1016/0092-8674(91)90318-sDavidson IF, Peters JM. Genome folding through loop extrusion by SMC complexes. Nat Rev Mol Cell Biol. 2021;22(7):445–464. doi: 10.1038/s41580-021-00349-7Hnisz D, Day DS, Young RA. Insulated neighborhoods: structural and functional units of mammalian gene control. Cell. 2016;167(5):1188–1200. doi: 10.1016/j.cell.2016.10.024Dixon JR, Selvaraj S, Yue F, et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature. 2012;485(7398):376–380. doi: 10.1038/nature11082Flavahan WA, Drier Y, Johnstone SE, et al. Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs. Nature. 2019;575(7781):229–233. doi: 10.1038/s41586-019-1668-3Hnisz D, Weintraub AS, Day DS, et al. Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science. 2016;351(6280):1454–1458. doi: 10.1126/science.aad9024Rhie SK, Perez AA, Lay FD, et al. A high-resolution 3D epigenomic map reveals insights into the creation of the prostate cancer transcriptome. Nat Commun. 2019;10(1):4154. doi: 10.1038/s41467-019-12079-8Sun JH, Zhou L, Emerson DJ, et al. Disease-associated short tandem repeats co-localize with chromatin domain boundaries. Cell. 2018;175(1):224–238.e15. doi: 10.1016/j.cell.2018.08.005Harris MB, Mostecki J, Rothman PB. Repression of an interleukin-4-responsive promoter requires cooperative BCL-6 function. J Biol Chem. 2005;280(13):13114–13121. doi: 10.1074/jbc.M412649200Lanzuolo C, Roure V, Dekker J, et al. Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex. Nat Cell Biol. 2007;9(10): 1167–1174. doi: 10.1038/ncb1637Mori N, Stein R, Sigmund O, Anderson DJ. A cell type-preferred silencer element that controls the neural-specific expression of the SCG10 gene. Neuron. 1990;4(4):583–594. doi: 10.1016/0896-6273(90)90116-wLi L, He S, Sun JM, Davie JR. Gene regulation by Sp1 and Sp3. Biochem Cell Biol. 2004;82(4):460–471. doi: 10.1139/o04-045Srinivasan L, Atchison ML. YY1 DNA binding and PcG recruitment requires CtBP. Genes Dev. 2004;18(21):2596–2601. doi: 10.1101/gad.1228204Tiwari VK, McGarvey KM, Licchesi JD, et al. PcG Proteins, DNA methylation, and gene repression by chromatin looping. PLoS Biol. 2008;6(12):e306. doi: 10.1371/journal.pbio.0060306Segert JA, Gisselbrecht SS. Bulyk ML. Transcriptional silencers: driving gene expression with the brakes on. Trends Genet. 2021;37(6):514–527. doi: 10.1016/j.tig.2021.02.002Andersson R, Gebhard C, Miguel-Escalada I, et al. An atlas of active enhancers across human cell types and tissues. Nature. 2014;507(7493):455–461. doi: 10.1038/nature12787Bernstein BE, Stamatoyannopoulos JA, Costello JF, et al. The NIH roadmap epigenomics mapping consortium. Nat Biotechnol. 2010;28(10):1045–1048. doi: 10.1038/nbt1010-1045ENCODE Project Consortium; Birney E, Stamatoyannopoulos JA, Dutta A, et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature. 2007;447(7146):799–816. doi: 10.1038/nature05874FANTOM Consortium and the RIKEN PMI and CLST (DGT); Forrest AR, Kawaji H, Rehli M, et al. A promoter-level mammalian expression atlas. Nature. 2014;507(7493):462–470. doi: 10.1038/nature13182Kellis M, Wold B, Snyder MP, et al. Defining functional DNA elements in the human genome. Proc Natl Acad Sci USA. 2014;111(17):6131–6138. doi: 10.1073/pnas.1318948111Roadmap Epigenomics Consortium; Kundaje A, Meuleman W, Ernst J, et al. Integrative analysis of 111 reference human epigenomes. Nature. 2015;518(7539):317–330. doi: 10.1038/nature14248Bernstein BE, Kamal M, Lindblad-Toh K, et al. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell. 2005;120(2):169–181. doi: 10.1016/j.cell.2005.01.001Spicuglia S, Vanhille L. Chromatin signatures of active enhancers. Nucleus. 2012;3(2):126–131. doi: 10.4161/nucl.19232Visel A, Blow MJ, Li Z, et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature. 2009;457(7231): 854–858. doi: 10.1038/nature07730Rada-Iglesias A, Bajpai R, Swigut T, et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature. 2011;470(7333):279–283. doi: 10.1038/nature09692Whyte WA, Orlando DA, Hnisz D, et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell. 2013;153(2):307–319. doi: 10.1016/j.cell.2013.03.035Barski A, Cuddapah S, Cui K, et al. High-resolution profiling of histone methylations in the human genome. Cell. 2007; 129(4):823–837. doi: 10.1016/j.cell.2007.05.009Pang B, Snyder MP. Systematic identification of silencers in human cells. Nat Genet. 2020;52(3):254–263. doi: 10.1038/s41588-020-0578-5Dixon JR, Jung I, Selvaraj S, et al. Chromatin architecture reorganization during stem cell differentiation. Nature. 2015; 518(7539):331–336. doi: 10.1038/nature14222Nora EP, Goloborodko A, Valton AL, et al. Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell. 2017;169(5): 930–944.e22. doi: 10.1016/j.cell.2017.05.004Rao SS, Huang SC, Glenn St Hilaire B, et al. Cohesin loss eliminates all loop domains. Cell. 2017;171(2):305–320.e24. doi: 10.1016/j.cell.2017.09.026Johnson DS, Mortazavi A, Myers RM, Wold B. Genome-wide mapping of in vivo protein-DNA interactions. Science. 2007; 316(5830):1497–1502. doi: 10.1126/science.1141319Rhee HS, Pugh BF. Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell. 2011;147(6):1408–1419. doi: 10.1016/j.cell.2011.11.013John S, Sabo PJ, Thurman RE, et al. Chromatin accessibility pre-determines glucocorticoid receptor binding patterns. Nat Genet. 2011;43(3):264–268. doi: 10.1038/ng.759Doni Jayavelu N, Jajodia A, Mishra A, Hawkins RD. Candidate silencer elements for the human and mouse genomes. Nat Commun. 2020;11(1):1061. doi: 10.1038/s41467-020-14853-5Xu B, Wang H, Wright S, et al. Acute depletion of CTCF rewires genome-wide chromatin accessibility. Genome Biol. 2021;22(1):244. doi: 10.1186/s13059-021-02466-0Song L, Zhang Z, Grasfeder LL, et al. Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity. Genome Res. 2011;21(10):1757–1767. doi: 10.1101/gr.121541.111Valouev A, Johnson SM, Boyd SD, et al. Determinants of nucleosome organization in primary human cells. Nature. 2011; 474(7352):516–520. doi: 10.1038/nature10002Song L, Crawford GE. DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells. Cold Spring Harb Protoc. 2010; 2010(2):pdb.prot5384. doi: 10.1101/pdb.prot5384Pajoro A, Muiño JM, Angenent GC, Kaufmann K. Profiling nucleosome occupancy by MNase-seq: experimental protocol and computational analysis. Methods Mol Biol. 2018;1675: 167–181. doi: 10.1007/978-1-4939-7318-7_11Buenrostro JD, Giresi PG, Zaba LC, et al. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods. 2013;10(12):1213–1218. doi: 10.1038/nmeth.2688Giresi PG, Kim J, McDaniell RM, et al. FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin. Genome Res. 2007; 17(6):877–885. doi: 10.1101/gr.5533506Lu L, Liu X, Huang WK, et al. Robust Hi-C maps of enhancer-promoter interactions reveal the function of non-coding genome in neural development and diseases. Mol Cell. 2020;79(3): 521–534.e15. doi: 10.1016/j.molcel.2020.06.007Ngan CY, Wong CH, Tjong H, et al. Chromatin interaction analyses elucidate the roles of PRC2-bound silencers in mouse development. Nat Genet. 2020;52(3):264–272. doi: 10.1038/s41588-020-0581-xSun F, Chronis C, Kronenberg M, et al. Promoter-enhancer communication occurs primarily within insulated neighborhoods. Mol Cell. 2019;73(2):250–263.e5. doi: 10.1016/j.molcel.2018.10.039Li Z, McGinn O, Wu Y, et al. ESR1 mutant breast cancers show elevated basal cytokeratins and immune activation. Nat Commun. 2022;13(1):2011. doi: 10.1038/s41467-022-29498-9Lieberman-Aiden E, van Berkum NL, Williams L, et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009;326(5950): 289–293. doi: 10.1126/science.1181369Fullwood MJ, Ruan Y. ChIP-based methods for the identification of long-range chromatin interactions. J Cell Biochem. 2009; 107(1):30–39. doi: 10.1002/jcb.22116Mumbach MR, Rubin AJ, Flynn RA, et al. HiChIP: Efficient and sensitive analysis of protein-directed genome architecture. Nat Methods. 2016;13(11):919–922. doi: 10.1038/nmeth.3999Fang R, Yu M, Li G, et al. Mapping of long-range chromatin interactions by proximity ligation-assisted ChIP-seq. Cell Res. 2016;26(12):1345–1348. doi: 10.1038/cr.2016.137Banerji J, Rusconi S, Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell. 1981;27(2 Pt 1):299–308. doi: 10.1016/0092-8674(81)90413-xKvon EZ, Kazmar T, Stampfel G, et al. Genome-scale functional characterization of Drosophila developmental enhancers in vivo. Nature. 2014;512(7512):91–95. doi: 10.1038/nature13395Patwardhan RP, Lee C, Litvin O, et al. High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis. Nat Biotechnol. 2009;27(12):1173–1175. doi: 10.1038/nbt.1589Patwardhan RP, Hiatt JB, Witten DM, et al. Massively parallel functional dissection of mammalian enhancers in vivo. Nat Biotechnol. 2012;30(3):265–270. doi: 10.1038/nbt.2136Melnikov A, Murugan A, Zhang X, et al. Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nat Biotechnol. 2012;30(3): 271–277. doi: 10.1038/nbt.2137Kwasnieski JC, Mogno I, Myers CA, et al. Complex effects of nucleotide variants in a mammalian cis-regulatory element. Proc Natl Acad Sci USA. 2012;109(47):19498–19503. doi: 10.1073/pnas.1210678109McAfee JC, Bell JL, Krupa O, et al. Focus on your locus with a massively parallel reporter assay. J Neurodev Disord. 2022; 14(1):50. doi: 10.1186/s11689-022-09461-xKheradpour P, Ernst J, Melnikov A, et al. Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay. Genome Res. 2013;23(5): 800–811. doi: 10.1101/gr.144899.112Kwasnieski JC, Fiore C, Chaudhari HG, Cohen BA. High-throughput functional testing of ENCODE segmentation predictions. Genome Res. 2014;24(10):1595–1602. doi: 10.1101/gr.173518.114Arnold CD, Gerlach D, Stelzer C, et al. Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science. 2013; 339(6123):1074–1077. doi: 10.1126/science.1232542Nguyen TA, Jones RD, Snavely AR, et al. High-throughput functional comparison of promoter and enhancer activities. Genome Res. 2016;26(8):1023–1033. doi: 10.1101/gr.204834.116Hansen TJ, Hodges E. ATAC-STARR-seq reveals transcription factor-bound activators and silencers within chromatin-accessible regions of the human genome. Genome Res. 2022; 32(8):1529–1541. doi: 10.1101/gr.276766.122Pang B, Neijssen J, Qiao X, et al. Direct antigen presentation and gap junction mediated cross-presentation during apoptosis. J Immunol. 2009;183(2):1083–1090. doi: 10.4049/jimmunol.0900861Straathof KC, Pulè MA, Yotnda P, et al. An inducible caspase 9 safety switch for T-cell therapy. Blood. 2005;105(11):4247–4254. doi: 10.1182/blood-2004-11-4564Bergman DT, Jones TR, Liu V, et al. Compatibility rules of human enhancer and promoter sequences. Nature. 2022;607(7917): 176–184. doi: 10.1038/s41586-022-04877-wDavis JE, Insigne KD, Jones EM, et al. Dissection of c-AMP response element architecture by using genomic and episomal massively parallel reporter assays. Cell Syst. 2020;11(1):75–85.e7. doi: 10.1016/j.cels.2020.05.011Ribeiro-dos-Santos AM, Hogan MS, Luther RD, et al. Genomic context sensitivity of insulator function. Genome Res. 2022; 32(3):425–436. doi: 10.1101/gr.276449.121Rosenberg AB, Patwardhan RP, Shendure J, Seelig G. Learning the sequence determinants of alternative splicing from millions of random sequences. Cell. 2015;163(3):698–711. doi: 10.1016/j.cell.2015.09.054Cheung R, Insigne KD, Yao D, et al. A multiplexed assay for exon recognition reveals that an unappreciated fraction of rare genetic variants cause large-effect splicing disruptions. Mol Cell. 2019;73(1):183–194.e8. doi: 10.1016/j.molcel.2018.10.037Mikl M, Hamburg A, Pilpel Y, Segal E. Dissecting splicing decisions and cell-to-cell variability with designed sequence libraries. Nat Commun. 2019;10(1):4572. doi: 10.1038/s41467-019-12642-3Matreyek KA, Starita LM, Stephany JJ, et al. Multiplex assessment of protein variant abundance by massively parallel sequencing. Nat Genet. 2018;50(6):874–882. doi: 10.1038/s41588-018-0122-zSample PJ, Wang B, Reid DW, et al. Human 5' UTR design and variant effect prediction from a massively parallel translation assay. Nat Biotechnol. 2019;37(7):803–809. doi: 10.1038/s41587-019-0164-5Vainberg Slutskin I, Weinberger A, Segal E. Sequence determinants of polyadenylation-mediated regulation. Genome Res. 2019;29(10):1635–1647. doi: 10.1101/gr.247312.118Safra M, Nir R, Farouq D, et al. TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code. Genome Res. 2017;27(3): 393–406. doi: 10.1101/gr.207613.116Mulvey B, Lagunas T, Dougherty JD. Massively parallel reporter assays: defining functional psychiatric genetic variants across biological contexts. Biol Psych. 2021;89(1): 6–89. doi: 10.1016/j.biopsych.2020.06.011Zopf CJ, Quinn K, Zeidman J, Maheshri N. Cell-cycle dependence of transcription dominates noise in gene expression. PLoS Comput Biol. 2013;9(7):e1003161. doi: 10.1371/journal.pcbi.1003161Akita H, Ito R. Kamiya H, et al. Cell cycle dependent transcription, a determinant factor of heterogeneity in cationic lipid-mediated transgene expression. J Gene Med. 2007;9(3):197–207. doi: 10.1002/jgm.1010Cooper LJ, Topp MS, Pinzon C, et al. Enhanced transgene expression in quiescent and activated human CD8 + T cells. Human Gene Therapy. 2004;15(7):648–658. doi: 10.1089/1043034041361217Brightwell G, Poirier V, Cole E, et al. Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector. Gene. 1997;194(1): 115–123. doi: 10.1016/s0378-1119(97)00178-9Dutton RL, Scharer J, Moo-Young M. Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line. Cytotechnology. 2006;52(1):55–69. doi: 10.1007/s10616-006-9041-4Burns WR, Zheng Z, Rosenberg SA, Morgan RA. Lack of specific γ-retroviral vector long terminal repeat promoter silencing in patients receiving genetically engineered lymphocytes and activation upon lymphocyte restimulation. Blood. 2009; 114(14):2888–2899. doi: 10.1182/blood-2009-01-199216Rosenqvist N, Segerstad C, Samuelsson C, et al. Activation of silenced transgene expression in neural precursor cell lines by inhibitors of histone deacetylation. J Gene Med. 2002; 4(3):248–257. doi: 10.1002/jgm.268Kim M, O’Callaghan PM, Droms KA, James DC. A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies. Biotechnol Bioeng. 2011;108(10):2434–2446. doi: 10.1002/bit.23189Brooks AR, Harkins RN, Wang P, et al. Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle. J Gene Med. 2004;6(4):395–404. doi: 10.1002/jgm.516Laker C, Meyer J, Schopen A, et al. Host cis-mediated extinction of a retrovirus permissive for expression in embryonal stem cells during differentiation. J Virol. 1998;72(1):339–348. doi: 10.1128/JVI.72.1.339-348.1998Yao S, Sukonnik T, Kean T, et al. Retrovirus silencing, variegation, extinction, and memory are controlled by a dynamic interplay of multiple epigenetic modifications. Mol Ther. 2004;10(1):27–36. doi: 10.1016/j.ymthe.2004.04.007Cranston A, Dong C, Howcroft J, Clark AJ. Chromosomal sequences flanking an efficiently expressed transgene dramatically enhance its expression. Gene. 2001;269(1–2): 217–225. doi: 10.1016/s0378-1119(01)00459-0Tripathi NK, Shrivastava A. Recent developments in bioprocessing of recombinant proteins: expression hosts and process development. Front Bioeng Biotechnol. 2019;7:420. doi: 10.3389/fbioe.2019.00420Nair RR, Blankvoort S, Lagartos MJ, Kentros C. Enhancer-driven gene expression (edge) enables the generation of viral vectors specific to neuronal subtypes. Science. 2020;23(3): 100888. doi: 10.1016/j.isci.2020.100888Gruh I, Wunderlich S, Winkler M, et al. Human CMV immediate-early enhancer: a useful tool to enhance cell-type-specific expression from lentiviral vectors. J Gene Med. 2008;10(1): 21–32. doi: 10.1002/jgm.1122Montaño-Samaniego M, Bravo-Estupiñan DM, Méndez-Guerrero O, et al. Strategies for targeting gene therapy in cancer cells with tumor-specific promoters. Front Oncol. 2020;10:605380. doi: 10.3389/fonc.2020.605380Sano KI, Maeda K, Oki M, Maéda Y. Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence. FEBS Lett. 2002;532(1–2):143–146. doi: 10.1016/s0014-5793(02)03659-1Eisenhut P, Mebrahtu A, Moradi Barzadd M, et al. Systematic use of synthetic 5'-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories. Nucleic Acids Res. 2020;48(20): e119–e119. doi: 10.1093/nar/gkaa847Peyret H, Brown JK, Lomonossoff GP. Improving plant transient expression through the rational design of synthetic 5' and 3' untranslated regions. Plant Methods. 2019;15(1):108. doi: 10.1186/s13007-019-0494-9Moritz B, Becker PB, Göpfert U. CMV promoter mutants with a reduced propensity to productivity loss in CHO cells. Sci Rep. 2015;5(1):16952. doi: 10.1038/srep16952Ho SC, Koh EY, Soo BP, et al. Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells. BMC Biotechnol. 2016;16(1):71. doi: 10.1186/s12896-016-0300-yKim JM, Kim JS, Park DH, et al. Improved recombinant gene expression in CHO cells using matrix attachment regions. J Biotechnol. 2004;107(2):95–105. doi: 10.1016/j.jbiotec.2003.09.015Phi-Van L, von Kries JP, Ostertag W, Strätling WH. The chicken lysozyme 5’ matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. Mol Cell Biol. 1990;10(5):2302–2307. doi: 10.1128/mcb.10.5.2302-2307.1990Grosveld F, van Assendelft GB, Greaves DR, Kollias G. Position-independent, high-level expression of the human β-globin gene in transgenic mice. Cell. 1987;51(6):975–985. doi: 10.1016/0092-8674(87)90584-8Pikaart MJ, Recillas-Targa F, Felsenfeld G. Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev.1998;12(18):2852–2862. doi: 10.1101/gad.12.18.2852Neville JJ, Orlando J, Mann K, et al. Ubiquitous chromatin-opening elements (UCOEs): Applications in biomanufacturing and gene therapy. Biotechnol Adv. 2017;35(5):557–564. doi: 10.1016/j.biotechadv.2017.05.004Li CL, Xiong D, Stamatoyannopoulos G, Emery DW. Genomic and functional assays demonstrate reduced gammaretroviral vector genotoxicity associated with use of the cHS4 chromatin insulator. Mol Ther. 2009;17(4):716–724. doi: 10.1038/mt.2009.7Browning D, Trobridge G. Insulators to improve the safety of retroviral vectors for HIV gene therapy. Biomedicines. 2016;4(1):4. doi: 10.3390/biomedicines4010004Hayashi H, Kubo Y, Izumida M, Matsuyama T. Efficient viral delivery of Cas9 into human safe harbor. Sci Rep. 2020; 10(1):21474. doi: 10.1038/s41598-020-78450-8Papapetrou EP, Schambach A. Gene insertion into genomic safe harbors for human gene therapy. Mol Ther. 2016;24(4): 678–684. doi: 10.1038/mt.2016.38Sadelain M, Papapetrou EP, Bushman FD. Safe harbours for the integration of new DNA in the human genome. Nat Rev Canc. 2011;12(1):51–58. doi: 10.1038/nrc3179Wang W, Guo X, Li Y, et al. Enhanced transgene expression using cis-acting elements combined with the EF1 promoter in a mammalian expression system. Eur J Pharm Sci. 2018; 123:539–545. doi: 10.1016/j.ejps.2018.08.016Schlabach MR, Hu JK, Li M, Elledge SJ. Synthetic design of strong promoters. Proc Natl Acad Sci USA. 2010;107(6): 2538–2543. doi: 10.1073/pnas.0914803107Morgan RA, Ma F, Unti MJ, et al. Creating new β-globin-expressing lentiviral vectors by high-resolution mapping of locus control region enhancer sequences. Mol Ther Methods Clin Dev. 2020;17:999–1013. doi: 10.1016/j.omtm.2020.04.006Vaknin I, Amit R. Molecular and experimental tools to design synthetic enhancers. Curr Opin Biotechnol. 2022;76:102728. doi: 10.1016/j.copbio.2022.102728Wu MR, Nissim L, Stupp D, et al. A high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS). Nat Commun. 2019;10(1):2880. doi: 10.1038/s41467-019-10912-8Yu TC, Liu WL, Brinck MS, et al. Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems. Nat Commun. 2021;12(1):325. doi: 10.1038/s41467-020-20094-3Shlyueva D, Stelzer C, Gerlach D, et al. Hormone-responsive enhancer-activity maps reveal predictive motifs, indirect repression, and targeting of closed chromatin. Mol Cell. 2014; 54(1):180–192. doi: 10.1016/j.molcel.2014.02.026Johnson GD, Barrera A, McDowell IC, et al. Human genome-wide measurement of drug-responsive regulatory activity. Nat Commun. 2018;9(1):5317. doi: 10.1038/s41467-018-07607-xPardi N, Hogan MJ, Porter FW. Weissman D. mRNA vaccines--a new era in vaccinology. Nat Rev Drug Dis. 2018;17(4): 261–279. doi: 10.1038/nrd.2017.243Leppek K, Byeon GW, Kladwang W, et al. Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. Nat Commun. 2022;13(1):1536. doi: 10.1038/s41467-022-28776-wPeng T, Zhai Y, Atlasi Y, et al. STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells. Genome Biol. 2020;21(1):243. doi: 10.1186/s13059-020-02156-3Glaser LV, Steiger M, Fuchs A, et al. Assessing genome-wide dynamic changes in enhancer activity during early mESC differentiation by FAIRE-STARR-seq. Nucleic Acids Res. 2021; 49(21):12178–12195. doi: 10.1093/nar/gkab1100Van Arensbergen J, FitzPatrick VD, de Haas M, et al. Genome-wide mapping of autonomous promoter activity in human cells. Nat Biotechnol. 2017;35(2):145–153. doi: 10.1038/nbt.3754Maricque BB, Dougherty JD, Cohen BA. A genome-integrated massively parallel reporter assay reveals DNA sequence determinants of cis-regulatory activity in neural cells. Nucleic Acids Res. 2017;45(4):e16. doi: 10.1093/nar/gkw942Inoue F, Kreimer A, Ashuach T, et al. Identification and massively parallel characterization of regulatory elements driving neural induction. Cell Stem Cell. 2019;25(5):713–727.e10. doi: 10.1016/j.stem.2019.09.010Dekkers CM. Application of genomics tools to animal breeding. Curr Genom. 2012;13(3):207–212. doi: 10.2174/138920212800543057Xiao Y, Liu H, Wu L, et al. Genome-wide association studies in maize: praise and stargaze. Mol Plant. 2017;10(3):359–374. doi: 10.1016/j.molp.2016.12.008Mackay TF, Richards S, Stone EA, et al. The drosophila melanogaster genetic reference panel. Nature. 2012;482(7384): 173–178. doi: 10.1038/nature10811Husby A, Kawakami T, Rönnegаrd L, et al. Genome-wide association mapping in a wild avian population identifies a link between genetic and phenotypic variation in a life-history trait. Proc Biol Sci. 2015;282(1806):20150156. doi: 10.1098/rspb.2015.0156Tseng CC, Wong MC, Liao WT, et al. Genetic variants in transcription factor binding sites in humans: triggered by natural selection and triggers of diseases. Int J Mol Sci. 2021; 22(8):4187. doi: 10.3390/ijms22084187Jung S, Liu W, Baek J, et al. Expression quantitative trait loci (eQTL) mapping in korean patients with crohn’s disease and identification of potential causal genes through integration with disease associations. Front Genet. 2020;11:486. doi: 10.3389/fgene.2020.00486Alsheikh AJ, Wollenhaupt S, King EA, et al. The landscape of GWAS validation; systematic review identifying 309 validated non-coding variants across 130 human diseases. BMC Med Genomics. 2022;15(1):74. doi: 10.1186/s12920-022-01216-wKlein JC, Keith A, Rice SJ, et al. Functional testing of thousands of osteoarthritis-associated variants for regulatory activity. Nat Commun. 2019;10(1):2434. doi: 10.1038/s41467-019-10439-yMyint L, Wang R, Boukas L, et al. A screen of 1,049 schizophrenia and 30 Alzheimer’s‐associated variants for regulatory potential. Am J Med Genet B Neuropsychiatr Genet. 2020; 183(1):61–73. doi: 10.1002/ajmg.b.32761Cooper YA, Teyssier N, Dräger NM, et al. Functional regulatory variants implicate distinct transcriptional networks in dementia. Science. 2022;377(6608):eabi8654. doi: 10.1126/science.abi8654Choi J, Zhang T, Vu A, et al. Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma. Nat Commun. 2020;11(1):2718. doi: 10.1038/s41467-020-16590-1McAfee JC, Lee S, Lee J, et al. Systematic investigation of allelic regulatory activity of schizophrenia-associated common variants. medRxiv. 2022. Available from: https://www.researchgate.net/scientific-contributions/Jessica-Lee-Bell-2203854579Slatkin M. Linkage disequilibrium--understanding the evolutionary past and mapping the medical future. Nat Rev Gen. 2008;9(6):477–485. doi: 10.1038/nrg2361Ulirsch JC, Nandakumar SK, Wang L, et al. Systematic functional dissection of common genetic variation affecting red blood cell traits. Cell. 2016;165(6):1530–1545. doi: 10.1016/j.cell.2016.04.048Khetan S, Kales S, Kursawe R, et al. Functional characterization of T2D-associated SNP effects on baseline and ER stress-responsive β-cell transcriptional activation. Nat Commun. 2021;12(1):5242. doi: 10.1038/s41467-021-25514-6Dietrich P, Dragatsis I. Familial dysautonomia: mechanisms and models. Genet Mol Biol. 2016;39(4):497–514. doi: 10.1590/1678-4685-GMB-2015-0335Fujimoto A, Furuta M, Totoki Y, et al. Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer. Nat Genet. 2016;48(5):500–509. doi: 10.1038/ng.3547Rheinbay E, Nielsen MM, Abascal F, et al. Analyses of non-coding somatic drivers in 2,658 cancer whole genomes. Nature. 2020;578(7793):102–111. doi: 10.1038/s41586-020-1969-6Lim Y, Arora S, Schuster SL, et al. Multiplexed functional genomic analysis of 5’ untranslated region mutations across the spectrum of prostate cancer. Nat Commun. 2021;12(1):4217. doi: 10.1038/s41467-021-24445-6Lagunas T, Plassmyer S, Friedman R, et al. A cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of genetic variants in vivo. bioRxiv. 2021. doi: 10.1101/2021.05.17.444514Griesemer D, Xue JR, Reilly SK, et al. Genome-wide functional screen of 3'UTR variants uncovers causal variants for human disease and evolution. Cell. 2021;184(20):5247–5260.e19. doi: 10.1016/j.cell.2021.08.025