IP Library › Granted Patent US 12,509,683
Granted Patent B2
US 12,509,683 · App. 17/698,912 · Granted Dec 30, 2025

Methods and compositions for modulating gene expression

Inventors: David Arthur Berry (Chestnut Hill, MA); Rahul Karnik (Cambridge, MA); Laura Gabriela Lande (Chestnut Hill, MA)
Assignee: FLAGSHIP PIONEERING INNOVATIONS V, INC.
C12N15/113A61K35/12C12N9/00C12N9/1007C12N9/22C12N9/80C12N15/63C12N15/85C12N15/87C12N15/90C12N15/907C12Y201/01037C12Y201/01043C12Y305/01098A01K2217/056A01K2227/706A01K2267/0318C07K2319/00C07K2319/85C12N2310/14C12N2310/20
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Quick Facts
Patent No.
US 12,509,683
App. No.
17/698,912
Granted
Dec 30, 2025
Kind
B2
Abstract

The present disclosure provides compositions with a modulating gene expression and methods for modulating transcription.

Claims (24)

1 . A method of decreasing expression of a gene within a cell, the gene being within an anchor sequence-mediated conjunction that comprises a first anchor sequence, a second anchor sequence, and an internal enhancing sequence, the method comprising a step of:

contacting the cell with a composition comprising a targeting element or a nucleic acid encoding the targeting element,

wherein the targeting element comprises an enzymatically inactive Cas nuclease and a gRNA which targets the first anchor sequence, the second anchor sequence, or both of the first anchor sequence and the second anchor sequence,

wherein the first and second anchor sequences each comprise a CTCF binding site, and wherein the CTCF binding site has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

2 . The method of claim 1 , wherein neither the first nor second anchor sequences are located within an internal enhancing sequence or a promoter.

3 . The method of claim 1 , wherein the gene is FOXJ3, MYC, SHMT2, or CDK6.

4 . The method of claim 1 , wherein the gRNA comprises a sequence of any of SEQ ID NOs: 40-47, 86, or 87.

5 . The method of claim 4 , wherein the cell is a human cell.

6 . The method of claim 1 , wherein the Cas nuclease comprises Cas9.

7 . The method of claim 1 , wherein contacting comprises transfecting the cell with the composition.

8 . The method of claim 1 , wherein the gRNA comprises a sequence at least 90% complementary to the first and/or second anchor sequence.

9 . The method of claim 1 , wherein the composition physically interferes with formation and/or maintenance of the anchor sequence-mediated conjunction.

10 . The method of claim 1 , wherein the gene is an oncogene, a tumor suppressor, or a gene associated with a nucleotide repeat.

11 . The method of claim 1 , wherein the targeting agent is linked to an effector domain with an amino acid linker.

12 . The method of claim 11 , wherein the effector domain comprises a DNA methyltransferase, histone methyltransferase, acetyltransferase, or deacetylase.

13 . The method of claim 1 , wherein the CTCF binding site has the sequence of SEQ ID NO: 1.

14 . The method of claim 1 , wherein the CTCF binding site has the sequence of SEQ ID NO: 2.

15 . The method of claim 1 , wherein the gene is separated from the first anchor sequence or the second anchor sequence by about 1 kb to about 100 Mb.

16 . The method of claim 1 , wherein the gene is separated from the internal enhancing sequence by about 100 kb to about 750 kb.

17 . The method of claim 1 , wherein the first anchor sequence is located at least 400 base pairs away from any transcription start site.

18 . The method of claim 1 , wherein the anchor sequences are between 20-80 nucleotides.

19 . The method of claim 1 , wherein the targeting element binds the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

20 . The method of claim 1 , wherein the targeting element binds a sequence proximal to SEQ ID NO: 1 or SEQ ID NO: 2.

21 . The method of claim 1 , which comprises contacting the cell with the nucleic acid encoding the targeting element.

Assignments (6)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 15, 2025
From: FLAGSHIP PIONEERING, INC.
To: FLAGSHIP PIONEERING INNOVATIONS V, INC.
Reel/Frame 073213/0846 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 26, 2025
From: KARNIK, RAHUL
To: OMEGA THERAPEUTICS, INC.
Reel/Frame 070639/0331 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 26, 2025
From: LANDE-DINER, LAURA GABRIELA; BERRY, DAVID ARTHUR
To: FLAGSHIP PIONEERING, INC.
Reel/Frame 070639/0392 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 26, 2025
From: OMEGA THERAPEUTICS, INC.
To: FLAGSHIP PIONEERING, INC.
Reel/Frame 070639/0514 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 26, 2025
From: FLAGSHIP PIONEERING, INC.
To: FLAGSHIP PIONEERING INNOVATIONS V, INC.
Reel/Frame 070639/0543 →
SECURITY INTEREST Recorded Feb 10, 2025
From: OMEGA THERAPEUTICS, INC.
To: PIONEERING MEDICINES 08- B, INC.
Reel/Frame 070167/0243 →
Continuity (7)
Continuation 15821632 · Nov 22, 2017
Continuation PCTUS2017050553 · Sep 7, 2017
Provisional Application 62542703 · Aug 8, 2017
Provisional Application 62439327 · Dec 27, 2016
Provisional Application 62416501 · Nov 2, 2016
Provisional Application 62384603 · Sep 7, 2016
Related Publication 20230174978A1 · Jun 8, 2023
References Cited (146)
US 8513207B2 · Brown · 2013 [cited by applicant]
US 9801877B2 · Yao et al. · 2017 [cited by applicant]
US 10023922B2 · Stelzer et al. · 2018 [cited by applicant]
US 11312955B2 · Berry et al. · 2022 [cited by applicant]
US 11382950B2 · Lande et al. · 2022 [cited by applicant]
US 11434476B2 · Jaenisch et al. · 2022 [cited by applicant]
US 11624065B2 · Lande et al. · 2023 [cited by applicant]
US 11951154B2 · Lande et al. · 2024 [cited by applicant]
US 20120115227A1 · Cohen-Haguenauer et al. · 2012 [cited by applicant]
US 20140273226A1 · Wu · 2014 [cited by applicant]
US 20140322707A1 · He et al. · 2014 [cited by applicant]
US 20150071906A1 · Liu et al. · 2015 [cited by applicant]
US 20150376612A1 · Lee et al. · 2015 [cited by applicant]
US 20160010076A1 · Joung et al. · 2016 [cited by applicant]
US 20160024474A1 · Conway et al. · 2016 [cited by applicant]
US 20160186208A1 · Jaenisch et al. · 2016 [cited by applicant]
US 20160215280A1 · Fanucchi et al. · 2016 [cited by applicant]
US 20160340749A1 · Stelzer et al. · 2016 [cited by applicant]
US 20170014449A1 · Bangera et al. · 2017 [cited by applicant]
US 20170130247A1 · Dowen et al. · 2017 [cited by applicant]
US 20170362649A1 · Lieberman-Aiden et al. · 2017 [cited by applicant]
US 20180245079A1 · Lieberman Aiden et al. · 2018 [cited by applicant]
US 20190005191A1 · Hnisz et al. · 2019 [cited by applicant]
US 20190024086A1 · Lande et al. · 2019 [cited by applicant]
US 20190194764A1 · Stelzer et al. · 2019 [cited by applicant]
US 20190241964A1 · Hunter et al. · 2019 [cited by applicant]
US 20190255106A1 · Lande et al. · 2019 [cited by applicant]
US 20190309291A1 · Lee et al. · 2019 [cited by applicant]
US 20190352648A1 · Young et al. · 2019 [cited by applicant]
US 20190359959A1 · Jaenisch et al. · 2019 [cited by applicant]
US 20200002558A1 · Iwasaki et al. · 2020 [cited by applicant]
US 20200149039A1 · Schuijers et al. · 2020 [cited by applicant]
US 20200224274A1 · Bernstein et al. · 2020 [cited by applicant]
US 20200255828A1 · Aiden et al. · 2020 [cited by applicant]
US 20210322577A1 · Lande et al. · 2021 [cited by applicant]
US 20220024999A1 · Berry et al. · 2022 [cited by applicant]
US 20220267756A1 · Lande et al. · 2022 [cited by applicant]
US 20220348893A1 · Lunardi et al. · 2022 [cited by applicant]
US 20220380760A1 · Tadin Strapps et al. · 2022 [cited by applicant]
US 20220403387A1 · Lande et al. · 2022 [cited by applicant]
US 20230054672A1 · Berry et al. · 2023 [cited by applicant]
US 20230096554A1 · Jaenisch et al. · 2023 [cited by applicant]
US 20230374549A1 · Beech et al. · 2023 [cited by applicant]
US 20230399640A1 · Kennedy et al. · 2023 [cited by applicant]
US 20240132559A1 · Witt et al. · 2024 [cited by applicant]
WO 2009146033A2 · 2009 [cited by applicant]
WO 2012019168A2 · 2012 [cited by applicant]
WO 2015038892A1 · 2015 [cited by applicant]
WO 2015191780A2 · 2015 [cited by applicant]
WO 2015196128A2 · 2015 [cited by applicant]
WO 2016022363A9 · 2016 [cited by applicant]
WO 2016063264A1 · 2016 [cited by applicant]
WO 2016070037A2 · 2016 [cited by applicant]
WO 2016073990A2 · 2016 [cited by applicant]
WO 2016081798A1 · 2016 [cited by applicant]
WO 2016089920A1 · 2016 [cited by applicant]
WO 2016103233A2 · 2016 [cited by applicant]
WO 2016115326A1 · 2016 [cited by applicant]
WO 2016154330A1 · 2016 [cited by applicant]
WO 2016164356A1 · 2016 [cited by applicant]
WO 2016174250A1 · 2016 [cited by applicant]
WO 2017011710A2 · 2017 [cited by applicant]
WO 2017031370A1 · 2017 [cited by applicant]
WO 2017040793A1 · 2017 [cited by applicant]
WO 2017064546A1 · 2017 [cited by applicant]
WO 2017075406A1 · 2017 [cited by applicant]
WO 2017106290A1 · 2017 [cited by applicant]
WO 2017143042A2 · 2017 [cited by applicant]
WO 2018035495A1 · 2018 [cited by applicant]
WO 2018049073A1 · 2018 [cited by applicant]
WO 2018049075A1 · 2018 [cited by applicant]
WO 2018049077A1 · 2018 [cited by applicant]
WO 2018049079A1 · 2018 [cited by applicant]
WO 2018111944A1 · 2018 [cited by applicant]
WO 2018129544A1 · 2018 [cited by applicant]
WO 2019036430A1 · 2019 [cited by applicant]
WO 2019071054A1 · 2019 [cited by applicant]
WO 2018204764A1 · 2019 [cited by applicant]
Amabile et al., “Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing,” Cell (2016) vol. 167, pp. 219-232. [cited by applicant]
Banani et al., “Biomolecular condensates: organizers of cellular biochemistry,” Nature Reviews Molecular Cell Biology (2017) vol. 18, No. 5, pp. 285-298. [cited by applicant]
Cho et al., “Antisense Transcription and Short Article Heterochromatin at the DMI CTG Repeats Are Constrained by CTCF,” Molecular Cell (2005) vol. 20, pp. 483-489. [cited by applicant]
Cong et al., “Multiplex Genome Engineering Using CRISPR/Cas Systems,” Science (2013) vol. 339, pp. 819-823. [cited by applicant]
De Groote et al., “Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes,” Nucleic Acids Res. (2012) vol. 40, No. 21, pp. 10596-10613. [cited by applicant]
De Souza et al., “DNA Methylation Profiling in Human Huntington's Disease Brain,” Human Molecular Genetics (2016) vol. 15, No. 10, pp. 2013-2030. [cited by applicant]
Deng et al., “Controlling Long-Range Genomic Interactions at a Native Locus by Targeted Tethering of a Looping Factor,” Cell (2012) vol. 149, pp. 1233-1244. [cited by applicant]
De Wit et al., “CTCF Binding Polarity Determines Chromatin Looping,” Molecular Cell (2015) vol. 60. No. 4, pp. 676-684. [cited by applicant]
Dunham et al., “An Integrated Encyclopedia of DNA Elements in the Human Genome,” Nature (2012) vol. 6; 489 (7414):57-74. [cited by applicant]
Ecker et al., “Genomics: Encode explained,” Nature Sep. 6, 2012; vol. 489 (7414) pp. 52-55. [cited by applicant]
Extended European Search Report for Application No. 17849560.2 dated Mar. 31, 2020. [cited by applicant]
Flavahan et al., “Insulator dysfunction and oncogene activation in IDH mutant gliomas,” Nature (2016) vol. 529, No. 7584, 110-4 (Epub: Dec. 23, 2015). [cited by applicant]
Guo et al., “YY1 Target DB: an intergral information resource for Yin Yang 1 target loci,” Database (2013) pp. 1-10. [cited by applicant]
Herold et al., “CTCF: insights into insulator function during development,” Development (2012) vol. 139, pp. 1045-1057. [cited by applicant]
HSU, “Completion of a Programmable DNA-Binding Small Molecule Library,” Thesis, California Institute of Technology (2008) retrieved from thesis.library.caltech.edu/4398, 153 pages. [cited by applicant]
International Search Report and Written Opinion issued in PCT/US2017/050553, mailed Jan. 30, 2018. [cited by applicant]
Jinek et al., “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science (2012) vol. 337, pp. 816-821. [cited by applicant]
Kearns et al., “Functional annotation of native enhancers with a Cas9-histone demethylase fusion,” Nature Methods (2015) vol. 12, No. 5, pp. 401-403. [cited by applicant]
Kim et al., “Genome-wide target specificities of CRISPR RNA-guided programmable deaminases,” Nat Biotechnol (2017) vol. 35, pp. 475-480. [cited by applicant]
Koferle et al., “Brave new epigenomes: the dawn of epigenetic engineering,” Genome Medicine (2015) vol. 7, No. 59, pp. 1-3. [cited by applicant]
Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature (2016) vol. 533(7603), pp. 420-424, Supplement. [cited by applicant]
Krylov et al., “A general method to design dominant negatives to B-HLHZip proteins that abolish DNA binding,” PNAS (1997) vol. 94, No. 23, pp. 12274-12279. [cited by applicant]
Lei et al., “Targeted DNA methylation in vivo using an engineered dCas 9-MQ1 fushion protein,” Nature Communications (2017) vol. 16026, pp. 1-10. [cited by applicant]
Li et al., “An alternative CTCF isoform antagonizes canonical CTCF occupancy and changes chromatin architecture to promote apoptosis,” Nature Communications (2019) vol. 10, Article 1535, 13 pages. [cited by applicant]
Lin et al., “Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins,” Molecular Cell (2015) vol. 60, pp. 208-219. [cited by applicant]
Ling et al., “Long-range DNA interactions are specifically altered by locked nucleic acid-targeting of a CTFC binding site,” Biochimica et Biophysica Acta (BBA)—Gene Regulatory Mechanisms (2011) vol. 1809, No. 1. pp. 24… [cited by applicant]
Liu et al., “Editing DNA methylation in the mammalian genome,” Cell (2016) vol. 167 (1), pp. 233-247. [cited by applicant]
Ma et al.,“Targeted Gene Suppression by Inducing De Novo DNA Methylation in the Gene Promotor,” Epigenetics & Chromatin (2014) vol. 7:20, pp. 1-11. [cited by applicant]
Mcdonald et al., “Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation,” Biology Open (2016) vol. 5, No. 6, pp. 866-874. [cited by applicant]
Morgan et al., “Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping,” Nature Communications (2017) vol. 8, Article 15993, 9 pages. [cited by applicant]
Patterson et al., “DNA Methylation: Bisulphite Modification and Analysis,” J Vis Exp. (2011) vol. 56, e3170, pp. 1-9. [cited by applicant]
Rada-Iglesias et al.,“Whole-genome maps of USFI and USF2 binding and histone H3 acetylation reveal new aspects of promoter structure and candidate genes for common human disorders,” Genome Research (2008) vol. 18 (3), p… [cited by applicant]
Ran et al., “Genome engineering using the CRISPR-Cas9 system,” Nature Protocols (2013) vol. 8, No. 11, pp. 2281-2308. [cited by applicant]
Sabari et al., “Coactivator condensation at super-enhancers links phase separation and gene control,” Science (2018) doi: 10.1126/science.aar3958, pp. 1-16. [cited by applicant]
Shin et al., “Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets,” Cell (2017) vol. 168, No. 1-2, pp. 159-171. [cited by applicant]
Thakore et al., “Highly Specific Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements,” Nature Medicine (2015) vol. 12 No. 12, pp. 1143-1149. [cited by applicant]
Torres et al., “Potent and sustained cellular inhibition of miR-122 by lysine-derivatized peptide nucleic acids (PNA) and phosphorothioate locked nucleic acid (LNA)/2'O-methyl (OMe) mixmer anti-miRs in the absence of tr… [cited by applicant]
Varun Narenda et al., “CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation,” Science (2015) vol. 347, No. 6225, pp. 1017-1021. [cited by applicant]
Viscidi et al., “Novel Chemical Method for the Preparation of Nucleic Acids for Nonisotopic Hybridization,” J Clin Microbiol (1986) vol. 23, No. 2, pp. 311-317. [cited by applicant]
Vojta et al., “Repurposing the CRISPR-Cas9 System for Targeted DNA Methylation,” Nucleic Acids Research (2016) vol. 44, No. 12, pp. 5615-5628. [cited by applicant]
Woloszynska-Read et al., “DNA Methylation-dependent Regulation of BORIS/CTCFL Expression in Ovarian Cancer,” Cancer Immunity (2007) vol. 7, No. 1, pp. 1-10. [cited by applicant]
Wu et al., “MicroRNAs direct rapid deadenylation of mRNA,” Proc Natl Acad Sci (2006) vol. 103, pp. 4034-4039. [cited by applicant]
Xu et al., “A CRISPR-based approach for targeted DNA demethylation,” Cell Discovery (2016) vol. 2, No. 16009, pp. 1-12, doi:10.1038/celldisc.2016.9. [cited by applicant]
Ziebarth et al., “CTCFBSDB 2.0: a database for CTCF-binding sites and genome organization,” Nucleic Acid Research (2013) vol. 41; D188-94. [cited by applicant]
International Search Report for PCT/US2017/50553 (Methods and Compositions for Modulating Gene Expression, filed Sep. 7, 2017), issued by ISA/US, 6 pages (dated Jan. 30, 2018). [cited by applicant]
Written Opinion for PCT/US2017/50553 (Methods and Compositions for Modulating Gene Expression, filed Sep. 7, 2017), issued by ISA/US, 12 pages (dated Jan. 30, 2018). [cited by applicant]
Hinisz, D. et al., “Super-Enhancers in the Control of Cell Identity and Disease,” Cell (2013) 155:934-947. [cited by applicant]
Ji, X. et al., “3D Chromosome Regulatory Landscape of Human Pluripotent Cells,” Cell Stem Cell (2016) 18:262-275. [cited by applicant]
Ji, X. et al., “Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions,” PNAS (2015) 112(12):3841-3846. [cited by applicant]
Mansour, M. et al., “An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element” Science, downloaded from www.sciencemag.org/content/early/recent, 9 pages (Nov. 13, 2014). [cited by applicant]
Spencer, R. et al., “A Boundary Element Between Tsix and Xist Binds the Chromatin Insulator Ctcf and Contributes to Initiation of X-Chromosome Inactivation,” Genetics (2011) 189:441-454. [cited by applicant]
Szabo, P. et al., “Role of CTCF Binding Sites in the lgf2/H19 Imprinting Control Region,” Molecular and Cellular Biology (2004) 24(11):4 791-4800. [cited by applicant]
Tang, Z. et al., “CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription,” Cell (2015) 163:1-17. [cited by applicant]
International Search Report for PCT/US2017/50556 (Methods and Compositions for Modulating Gene Expression, filed Sep. 7, 2017), issued by ISA/US, 5 pages (Dec. 26, 2017). [cited by applicant]
Written Opinion for PCT/US2017/50556 (Methods and Compositions for Modulating Gene Expression, filed Sep. 7, 2017), issued by ISA/US, 8 pages (Dec. 26, 2017). [cited by applicant]
International Search Report for PCT/US2017/50558 (Methods and Compositions for Modulating Gene Expression, filed Sep. 7, 2017), issued by ISA/US, 5 pages (Dec. 18, 2017). [cited by applicant]
Written Opinion for PCT/US2017/50558 (Methods and Compositions for Modulating Gene Expression, filed Sep. 7, 2017), issued by ISA/US, 6 pages (Dec. 18, 2017). [cited by applicant]
International Search Report for PCT/US2017/50561 (Methods and Compositions for Modulating Gene Expression, filed Sep. 7, 2017), issued by ISA/US, 5 pages (Dec. 18, 2017). [cited by applicant]
Written Opinion for PCT/US2017/50561 (Methods and Compositions for Modulating Gene Expression, filed Sep. 7, 2017), issued by ISA/US, 6 pages (Dec. 18, 2017). [cited by applicant]
Barrera, L. et al., “Survey of variation in human transcription factors reveals prevalent DNA binding changes,” Science (2016) 351(6280):1450-1454. [cited by applicant]
Dowen, J. et al., “Control of Cell Identity Genes Occurs in Insulated Neighborhoods in Mammalian Chromosomes,” Cell (2014) 159:374-387. [cited by applicant]
Hnisz, D. et al., “Activation of proto-oncogenes by disruption of chromosome neighborhoods,” Science (2016) 351 (6280):1454-1458. [cited by applicant]
Hnisz, D. et al., “Convergence of Developmental and Oncogenic Signaling Pathways at Transcriptional Super-Enhancers,” Molecular Cell (2015) 58:362-370. [cited by applicant]
Guo, C. et al., “CTCF Binding Elements Mediate Control of V(D)J Recombination,” Nature (2011) 477(7365):424-430. [cited by applicant]
Extended European Search Report for European Patent Application No. 17849560.2 dated Mar. 31, 2020. [cited by applicant]
Farumashia, 2015, vol. 51, No. 4, pp. 305-309. [cited by applicant]
Gombert, W. M., & Krumm, A. “Targeted deletion of multiple CTCF-binding elements in the human C-MYC gene reveals a requirement for CTCF in C-MYC expression.” PloS One (2009) vol. 4,7 e6109. [cited by applicant]
Witcher, M., and B. M. Emerson. “Epigenetic silencing of the p16(INK4a) tumor suppressor is associated with loss of CTCF binding and a chromatin boundary.” Molecular Cell vol. 34,3 (2009): 271-84. [cited by applicant]