IP Library › Granted Patent US 12,611,470
Granted Patent B2
US 12,611,470 · App. 17/606,408 · Granted Apr 28, 2026

Chromatin-opening element for stable long term gene expression

Inventors: Christina Smolke (Stanford, CA); Shireen S. Rudina (Stanford, CA)
Assignees: CZ Biohub SF, LLC; The Board of Trustees of the Leland Stanford Junior University
A61K48/0066C12N15/85C12N2830/46
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Quick Facts
Patent No.
US 12,611,470
App. No.
17/606,408
Granted
Apr 28, 2026
Kind
B2
Abstract

A novel ubiquitous chromatin opening element (UCOE) named SRF-UCOE and methods for its use are provided. Compositions including recombinant and synthetic SRF-UCOE nucleic acid molecules, DNA constructs and vectors comprising the SRF-UCOE nucleic acid molecules, host cells comprising the DNA constructs or vectors, and cell culture systems comprising such host cells. SRF-UCOE polynucleotide sequences can be used in DNA constructs or expression cassettes for transformation and expression in cells or organisms of interest. The compositions and methods provided are useful for increasing and/or maintaining expression of a gene of interest. Transgenic cells, tissues, and animals comprising a SRF-UCOE nucleotide sequence are also provided. Methods are provided for increasing and/or maintaining expression of a gene of interest and for treating a subject via gene therapy.

Claims (34)

1 . A recombinant nucleic acid molecule for controlling expression of a gene of interest (GOI) in a target cell, the nucleic acid comprising

(a) a ubiquitous chromatic opening element (UCOE) polynucleotide comprising a nucleic acid sequence having at least 90 percent sequence identity over the length of the nucleic acid sequence set forth in SEQ ID NO:5; and

(b) a heterologous promoter;

wherein the UCOE and the promoter are each arranged in the recombinant nucleic acid to be operably linked to a GOI in a target cell so as to control expression of the GOI.

2 . The recombinant nucleic acid molecule of claim 1 , comprising a nucleic acid sequence having at least 90 percent sequence identity to the nucleic acid sequence set forth in any of SEQ ID NOs: 1, 2, 3, or 4.

3 . The recombinant nucleic acid molecule of claim 1 , comprising a nucleic acid sequence having at least 95 percent sequence identity to the nucleic acid sequence set forth in any of SEQ ID NOs: 1, 2, 3, 4, or 5.

4 . The recombinant nucleic acid molecule of claim 1 , further comprising said GOI.

5 . The recombinant nucleic acid molecule of claim 1 , wherein the heterologous promoter is a eukaryotic promoter or a viral promoter.

6 . The recombinant nucleic acid molecule of claim 1 , wherein the heterologous promoter is a mammalian promoter.

7 . The recombinant nucleic acid molecule of claim 1 , wherein the heterologous promoter is a tissue-specific promoter.

8 . A vector comprising the recombinant nucleic acid molecule of claim 1 .

9 . A host cell comprising the recombinant nucleic acid molecule of claim 1 .

10 . The host cell of claim 9 , wherein the host cell is a eukaryotic cell.

11 . The host cell of claim 9 , wherein the host cell is a bacterial cell.

12 . A composition comprising the recombinant nucleic acid molecule of claim 1 formulated in a pharmaceutically acceptable carrier.

13 . A recombinant nucleic acid molecule for controlling expression of a gene of interest (GOI) in a target cell, the nucleic acid comprising:

(a) a ubiquitous chromatic opening element (UCOE) polynucleotide comprising the nucleic acid sequence of positions 479-780 of SEQ ID NO: 1 up to the full length of SEQ ID NO: 1; and

(b) a heterologous promoter;

wherein the UCOE and the promoter are each configured in the recombinant nucleic acid to be operably linked to a GOI in a target cell so as to control expression of the GOI.

14 . A recombinant nucleic acid molecule for controlling expression of a gene of interest (GOI) in a target cell, the nucleic acid comprising:

(a) a ubiquitous chromatic opening element (UCOE) polynucleotide comprising a nucleic acid sequence having at least 90 percent sequence identity over the length of positions 479-780 of SEQ ID NO: 1 up to at least 90 percent sequence identity of the full length of SEQ ID NO: 1; and

(b) a heterologous promoter,

wherein the UCOE and the promoter are each configured in the recombinant nucleic acid to be operably linked to a GOI in a target cell so as to control expression of the GOI.

15 . The recombinant nucleic acid molecule of claim 14 , wherein the UCOE polynucleotide has 90% sequence identity to SEQ ID NOs: 1, 2, 3, or 4.

16 . The recombinant nucleic acid molecule of claim 14 , wherein the UCOE polynucleotide has 95% sequence identity to SEQ ID NOs: 1, 2, 3, or 4.

17 . The recombinant nucleic acid molecule of claim 1 , wherein the UCOE resists transgene silencing, resists DNA methylation, and/or resists histone deacetylation of the GOI.

18 . A method of producing a product encoded by a gene of interest (GOI), comprising:

(a) introducing into a target cell the recombinant nucleic acid molecule of claim 4 comprising a UCOE and a heterologous promoter operably linked to said GOI; and

(b) culturing

the target cell to produce the product encoded by the GOI.

19 . A method of increasing expression of an endogenous gene in the genome of a target cell, comprising:

(a) introducing the recombinant nucleic acid molecule of claim 1 into the genome of

said cell such that the UCOE and the promoter of the recombinant nucleic acid are operably linked to said endogenous gene; and

(b) culturing said target cell to produce a product encoded by said endogenous gene.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 7, 2021
From: THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY
To: CHAN ZUCKERBERG BIOHUB, INC.; THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY
Reel/Frame 058325/0494 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 26, 2021
From: SMOLKE, CHRISTINA; RUDINA, SHIREEN S.
To: THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY
Reel/Frame 057914/0137 →
Continuity (2)
Provisional Application 62841151 · Apr 30, 2019
Related Publication 20220193266A1 · Jun 23, 2022
References Cited (80)
US 6949361B2 · Antoniou et al. · 2005 [cited by applicant]
US 7442787B2 · Antoniou et al. · 2008 [cited by applicant]
US 7632661B2 · Simpson et al. · 2009 [cited by applicant]
US 20090018031A1 · Trinklein et al. · 2009 [cited by applicant]
US 20090111144A1 · Bebbington · 2009 [cited by applicant]
US 20140186890A1 · Otte · 2014 [cited by examiner]
EP 2348118A1 · 2011 [cited by applicant]
WO 2000005393A2 · 2000 [cited by applicant]
WO 2002024930A2 · 2002 [cited by applicant]
WO 2006123097A1 · 2006 [cited by applicant]
WO 2018204764A1 · 2018 [cited by applicant]
Huxley et al. “The mouse surfeit locus contains a cluster of six genes associated with four Gp G-rich islands in 32 kilobases of genomic DNA,” Mol. Cell Biol., 1990, 10(2), 605-614 (Year: 1990). [cited by examiner]
NCBI Blastn sequence alignment, NCBI Blast: Nucleotide Sequence, accessed Apr. 12, 2025, instant SEQ ID No. 1 aligned with SEQ ID No. 12129 of Trinklein et al. (US 2009/0018031 A1) (Year: 2025). [cited by examiner]
NCBI Blastn sequence alignment, NCBI Blast: Nucleotide Sequence, accessed Apr. 12, 2025, instant SEQ ID No. 5 aligned with SEQ ID No. 12129 of Trinklein et al. (US 2009/0018031 A1) (Year: 2025). [cited by examiner]
PCT/US2020/030093 , “International Search Report and Written Opinion”, Aug. 17, 2020, 10 pages. [cited by applicant]
ENCODE Project Consortium, An Integrated Encyclopedia of DNA Elements in the Human Genome, Nature, vol. 489, No. 7414, Sep. 9, 2012. [cited by applicant]
Adamson et al., A Multiplexed Single-cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response, Cell, vol. 167, No. 7, Dec. 15, 2016, pp. 1867-1882. [cited by applicant]
Alhaji et al., Silencing of Transgene Expression in Mammalian Cells by DNA Methylation and Histone Modifications in Gene Therapy Perspective, Biotechnology and Genetic Engineering Reviews, vol. 35, No. 1, Apr. 2019, pp.… [cited by applicant]
Allen et al., Correlation of DNA Methylation with Histone Modifications Across the HNRPA2B1-CBX3 Ubiquitously-Acting Chromatin Open Element (UCOE), Epigenetics, vol. 2, No. 4, Oct.-Dec. 2007, pp. 227-236. [cited by applicant]
Altschul et al., Basic Local Alignment Search Tool, Journal of Molecular Biology, vol. 215, No. 3, Oct. 5, 1990, pp. 403-410. [cited by applicant]
Altschul et al., Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Programs, Nucleic Acids Research, vol. 25, No. 17, Sep. 1, 1997, pp. 3389-3402. [cited by applicant]
Anderson, Human Gene Therapy, Nature, vol. 392, Apr. 30, 1998, pp. 25-30. [cited by applicant]
Antoniou et al., Transgenes Encompassing Dual-Promoter CpG Islands from the Human TBP and HNRPA2B1 Loci are Resistant to Heterochromatin-Mediated Silencing, Genomics, vol. 82, No. 3, Sep. 2003, pp. 269-279. [cited by applicant]
Bannister et al., Regulation of Chromatin by Histone Modifications, Cell Research, vol. 21, No. 3, Mar. 2011, pp. 381-395. [cited by applicant]
Benton et al., The Use of UCOE Vectors in Combination with a Preadapted Serum Free, Suspension Cell Line Allows for Rapid Production of Large Quantities of Protein, Cytotechnology, vol. 38, Jan. 2002, pp. 43-46. [cited by applicant]
Brendel et al., Physiological Regulation of Transgene Expression by a Lentiviral Vector Containing the A2UCOE Linked to a Myeloid Promoter, Gene Therapy, vol. 19, No. 10, Oct. 2012, pp. 1018-1029. [cited by applicant]
Chen et al., Genome-Wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis, Cell, vol. 160, No. 6, Mar. 12, 2015, pp. 1246-1260. [cited by applicant]
Chen et al., Reactivation of Silenced, Virally Transduced Genes by Inhibitors of Histone Deacetylase, Proceedings of the National Academy of Sciences, vol. 94, No. 11, May 27, 1997, pp. 5798-5803. [cited by applicant]
Chowdhury et al., Long-term Improvement of Hypercholesterolemia After Ex Vivo Gene Therapy in LDLR-deficient Rabbits, Science, vol. 254, No. 5039, Dec. 20, 1991, pp. 1802-1805. [cited by applicant]
Crane-Robinson et al., Chromosomal Mapping of Core Histone Acetylation by Immunoselection, Methods, vol. 12, No. 1, May 1, 1997, pp. 48-56. [cited by applicant]
Dighe et al., Long-term Reproducible Expression in Human Fetal Liver Hematopoietic Stem Cells with a UCOE-based Lentiviral Vector, Public Library of Science One, vol. 9, No. 8, Aug. 12, 2014, pp. 1-8. [cited by applicant]
Duhig et al., The Human Surfeit Locus, Genomics, vol. 52, No. 1, Aug. 1998, pp. 72-78. [cited by applicant]
Ehrhardt et al., Episomal Vectors for Gene Therapy, Current Gene Therapy, vol. 8, No. 3, Jun. 2008, pp. 147-161. [cited by applicant]
Ellis et al., Silencing and Variegation of Gammaretrovirus and Lentivirus Vectors, Human Gene Therapy, vol. 16, No. 11, Nov. 1, 2005, pp. 1241-1246. [cited by applicant]
Emerson, Ex Vivo Expansion of Hematopoietic Precursors, Progenitors, and Stem Cells: The Next Generation of Cellular Therapeutics, Blood, vol. 87, No. 8, Apr. 15, 1996, pp. 3082-3088. [cited by applicant]
Ernst et al., Mapping and Analysis of Chromatin State Dynamics in Nine Human Cell Types, Nature, vol. 473, No. 7345, Mar. 2011, 7 pages. [cited by applicant]
Flotte et al., Stable in Vivo Expression of the Cystic Fibrosis Transmembrane Conductance Regulator with an Adeno-Associated Virus Vector, Proceedings of the National Academy of Sciences, vol. 90, No. 22, Nov. 15, 1993,… [cited by applicant]
Gill et al., Increased Persistence of Lung Gene Expression Using Plasmids Containing the Ubiquitin C or Elongation Factor1α Promoter, Gene Therapy, vol. 8, No. 20, Nov. 2001, pp. 1539-1546. [cited by applicant]
Guttman et al., Ab Initio Reconstruction of Cell Type-specific Transcriptomes in Mouse Reveals the Conserved Multi-Exonic Structure of lincRNAs, Nature Biotechnology, vol. 28, No. 5, May 2, 2010, pp. 503-510. [cited by applicant]
Haenseler et al., Lentiviral Gene Therapy Vector with UCOE Stably Restores Function in iPSC-Derived Neutrophils of a CDG Patient, Matters, vol. 2018, May 2018, pp. 1-11. [cited by applicant]
Hitt et al., Human Adenovirus Vectors for Gene Transfer Into Mammalian Cells, Advances in Pharmacology, vol. 40, 1997, pp. 137-206. [cited by applicant]
Hsu et al., The UCSC Known Genes, Bioinformatics, vol. 22, No. 9, May 2006, pp. 1036-1046. [cited by applicant]
Jost et al., Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent, Molecular Cell, vol. 68, No. 1, Oct. 5, 2017, pp. 210-223. [cited by applicant]
Jostock et al., Mammalian Stable Expression of Biotherapeutics, Chapter 15, Therapeutic Proteins: Methods and Protocols, vol. 899, Jan. 2012, pp. 227-238. [cited by applicant]
Karlin et al., Applications and Statistics for Multiple High-Scoring Segments in Molecular Sequences, Proceedings of the National Academy of Sciences, vol. 90, No. 12, Jun. 15, 1993, pp. 5873-5877. [cited by applicant]
Karlin et al., Methods for Assessing the Statistical Significance of Molecular Sequence Features by Using General Scoring Schemes, Proceedings of the National Academy of Sciences, vol. 87, No. 6, Mar. 1990, pp. 2264-226… [cited by applicant]
Kazuki et al., Human Artificial Chromosomes for Gene Delivery and the Development of Animal Models, Molecular Therapy, vol. 19, No. 9, Sep. 1, 2011, pp. 1591-1601. [cited by applicant]
Kent et al., The Human Genome Browser at UCSC, Genome Research, vol. 12, No. 6, Jun. 1, 2002, pp. 996-1006. [cited by applicant]
Khan et al., JASPAR 2018: Update of the Open-access Database of Transcription Factor Binding Profiles and Its Web Framework, Nucleic Acids Research, vol. 46, No. D1, Jan. 4, 2018, pp. D260-D266. [cited by applicant]
Knight et al., Safer, Silencing-resistant Lentiviral Vectors: Optimization of the Ubiquitous Chromatin-opening Element Through Elimination of Aberrant Splicing, Journal of Virology, vol. 86, No. 17, Sep. 1, 2012, pp. 90… [cited by applicant]
Kunkiel et al., The CpG-Sites of the CBX3 Ubiquitous Chromatin Opening Element Are Critical Structural Determinants for the Anti-Silencing Function, Scientific Reports, vol. 7, No. 1, Aug. 2017, pp. 1-13. [cited by applicant]
Kuriyama et al., Expression of a Retrovirally Transduced Gene Under Control of an Internal Housekeeping Gene Promoter Does Not Persist Due to Methylation and is Restored Partially by 5-Azacytidine Treatment, Gene Therap… [cited by applicant]
Liu et al., Delivery Strategies of the CRISPR-Cas9 Gene-Editing System for Therapeutic Applications, Journal of Controlled Release, vol. 266, Nov. 28, 2017, pp. 17-26. [cited by applicant]
Miller et al., Recombinant Adeno-Associated Virus (rAAV)-Mediated Expression of a Human Gamma-Globin Gene in Human Progenitor-Derived Erythroid Cells, Proceedings of the National Academy of Sciences, vol. 91, No. 21, Oc… [cited by applicant]
Milone et al., Clinical Use of Lentiviral Vectors, Leukemia, vol. 32, No. 7, Jul. 2018, pp. 1529-1541. [cited by applicant]
Muller-Kuller et al., A Minimal Ubiquitous Chromatin Opening Element (UCOE) Effectively Prevents Silencing of Juxtaposed Heterologous Promoters by Epigenetic Remodeling in Multipotent and Pluripotent Stem Cells, Nucleic… [cited by applicant]
Naso et al., Adeno-Associated Virus (AAV) as a Vector for Gene Therapy, BioDrugs, vol. 31, No. 4, Jul. 1, 2017, pp. 317-334. [cited by applicant]
Needleman et al., A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins, Journal of Molecular Biology, vol. 48, No. 3, Mar. 1970, pp. 443-453. [cited by applicant]
Pearson et al., Improved Tools for Biological Sequence Comparison, Proceedings of the National Academy of Sciences of the United States of America, vol. 85, No. 8, Apr. 1988, pp. 2444-2448. [cited by applicant]
Perez et al., CRISPR-Cas Systems: Ushering in the New Genome Editing Era, Bioengineered, vol. 9, No. 1, Jul. 2018, pp. 214-221. [cited by applicant]
Philip et al., Efficient and Sustained Gene Expression in Primary T Lymphocytes and Primary and Cultured Tumor Cells Mediated by Adeno-associated Virus Plasmid DNA Complexed to Cationic Liposomes, Molecular and Cellular… [cited by applicant]
Pikaart et al., Loss of Transcriptional Activity of a Transgene is Accompanied by DNA Methylation and Histone Deacetylation and is Prevented by Insulators, Genes & Development, vol. 12, No. 18, Aug. 5, 1998, pp. 2852-28… [cited by applicant]
Rein et al., Identifying 5-methylcytosine and Related Modifications in DNA Genomes, Nucleic Acids Research, vol. 26, No. 10, May 15, 1998, pp. 2255-2264. [cited by applicant]
Rice et al., EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics, vol. 16, No. 6, Jun. 2000, pp. 276-277. [cited by applicant]
Russell et al., Adeno-associated Virus Vectors Preferentially Transduce Cells in S Phase, Proceedings of the National Academy of Sciences, vol. 91, No. 19, Sep. 13, 1994, pp. 8915-8919. [cited by applicant]
Saunders et al., Chromatin Function Modifying Elements in an Industrial Antibody Production Platform-Comparison of UCOE, MAR, STAR and cHS4 Elements, Public Library of Science One, vol. 10, No. 4, Apr. 7, 2015, pp. 1-20. [cited by applicant]
She et al., Definition, Conservation and Epigenetics of Housekeeping and Tissue-Enriched Genes, Brihanmumbai Municipal Corporation Genomics, vol. 10, Jun. 17, 2009, pp. 1-12. [cited by applicant]
Silva et al., Meganucleases and Other Tools for Targeted Genome Engineering: Perspectives and Challenges for Gene Therapy, Current Gene Therapy, vol. 11, No. 1, Feb. 1, 2011, pp. 11-27. [cited by applicant]
Smith et al., Comparison of Biosequences, Advances in Applied Mathematics, vol. 2, No. 4, Dec. 1, 1981, pp. 482-489. [cited by applicant]
Uchiyama et al., Foamy Virus Vector-Mediated Gene Correction of a Mouse Model of Wiskott-Aldrich Syndrome, Molecular Therapy, vol. 20, No. 6, Jun. 1, 2012, pp. 1270-1279. [cited by applicant]
Walsh et al., Regulated High Level Expression of a Human Gamma-globin Gene Introduced into Erythroid Cells by an Adeno-associated Virus Vector, Proceedings of the National Academy of Sciences of the United States of Ame… [cited by applicant]
Wang et al., Factorbook.Org: A Wiki-based Database for Transcription Factor-binding Data Generated by the ENCODE Consortium, Nucleic Acids Research, vol. 41, No. D1, Jan. 2013, pp. D171-D176. [cited by applicant]
Weth et al., CTCF Induces Histone Variant Incorporation, Erases the H3K27me3 Histone Mark and Opens Chromatin, Nucleic Acids Research, vol. 42, No. 19, Oct. 2014, pp. 11941-11951. [cited by applicant]
Williams et al., CpG-Island Fragments from the HNRPA2B1/CBX3 Genomic Locus Reduce Silencing and Enhance Transgene Expression from the hCMV Promoter/Enhancer in Mammalian Cells, Brihanmumbai Municipal Corporation Biotech… [cited by applicant]
Wilson et al., Ex Vivo Gene Therapy of Familial Hypercholesterolemia, Human Gene Therapy, vol. 3, No. 2, Apr. 1, 1992, pp. 179-222. [cited by applicant]
Wu, Analysis of Hypersensitive Sites in Chromatin, Methods in Enzymology, vol. 170, Jan. 1, 1989, pp. 269-289. [cited by applicant]
Xiang et al., Massively Parallel RNA Device Engineering in Mammalian Cells With RNA-seq, Nature Communications, vol. 10, No. 1, Sep. 23, 2019, pp. 1-16. [cited by applicant]
Yang et al., DNA Methylation Contributes to Loss in Productivity of Monoclonal Antibody-producing CHO Cell Lines, Journal of Biotechnology, vol. 147, Nos. 3-4, Jun. 1, 2010, pp. 180-185. [cited by applicant]
Zhang et al., A Ubiquitous Chromatin Opening Element (UCOE) Confers Resistance to DNA Methylation-Mediated Silencing of Lentiviral Vectors, Molecular Therapy, vol. 18, No. 9, Sep. 2010, pp. 1640-1649. [cited by applicant]
Zufferey et al., Production of High-Titer Lentiviral Vectors, Current Protocols in Human Genetics, vol. 26, No. 1, Aug. 2000. [cited by applicant]