Chromatin-opening element for stable long term gene expression
Inventors
SMOLKE, Christina • Rudina, Shireen S.
Assignees
Leland Stanford Junior University • CZ Biohub SF LLC • Biohub
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Publication Number
US-12611470-B2
Publication Date
2026-04-28
Expiration Date
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.
Core Innovation
The invention describes a ubiquitous chromatin opening element (UCOE) referred to as SRF-UCOE from the human Surfeit locus (chr9; SURF1/SURF2 region) that modularly confers long-term anti-transgene silencing when operably linked to heterologous promoters. The SRF-UCOE candidates are screened and characterized, including Candidate 6 and truncations 6-1, 6-2, and 6-3, for resistance to silencing in stable transfections and lentiviral transductions across promoters.
The recombinant nucleic acids include an SRF-UCOE polynucleotide and a heterologous promoter, wherein the UCOE and the promoter are arranged to be operably linked to a gene of interest in a target cell to control expression of the gene of interest. Candidate 6, and especially truncation 6-3, delays or limits expression loss versus negative controls, including comparison to A2UCOE/3′UCOE, across multiple promoters including Ef1α, CMV, RSV, and PGK.
The document supports an epigenetic mechanism for the anti-silencing activity by reporting that rescue of silenced expression is achieved using a DNA methylation inhibitor (5-aza-cytidine) and an HDAC inhibitor (TSA), consistent with resistance to DNA methylation and histone deacetylation. The invention further characterizes sequence and functional attributes, including being different from A2UCOE, being small size, and lacking synthetic or standalone promoter activity, while maintaining performance across promoters and delivery contexts.
Claims Coverage
The provided claim set includes three independent claims, all directed to recombinant nucleic acids that control expression of a gene of interest in a target cell by operably linking a ubiquitous chromatin opening element polynucleotide with a heterologous promoter.
Operably linked UCOE polynucleotide with heterologous promoter to control gene of interest expression
A recombinant nucleic acid molecule for controlling expression of a gene of interest in a target cell, comprising a ubiquitous chromatin opening element polynucleotide with at least 90 percent sequence identity over the length of the nucleic acid sequence set forth in SEQ ID NO:5, and a heterologous promoter, wherein the UCOE and the promoter are arranged to be operably linked to a gene of interest in a target cell so as to control expression of the gene of interest.
UCOE defined by positions 479-780 of SEQ ID NO: 1 with heterologous promoter
A recombinant nucleic acid molecule for controlling expression of a gene of interest in a target cell, comprising a ubiquitous chromatin opening element 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 a heterologous promoter, wherein the UCOE and the promoter are each configured in the recombinant nucleic acid to be operably linked to a gene of interest in a target cell so as to control expression of the gene of interest.
UCOE with identity constraints to positions 479-780 through full length of SEQ ID NO: 1 and heterologous promoter
A recombinant nucleic acid molecule for controlling expression of a gene of interest in a target cell, comprising a ubiquitous chromatin opening element 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 a heterologous promoter, wherein the UCOE and the promoter are each configured to be operably linked to a gene of interest in a target cell so as to control expression of the gene of interest.
Across the independent claims, the core coverage is a recombinant nucleic acid that includes a UCOE polynucleotide with defined sequence identity constraints or defined SEQ ID NO: 1 positional and identity boundaries and a heterologous promoter, configured so the UCOE and promoter are operably linked to a gene of interest in a target cell to control gene of interest expression.
Stated Advantages
Delays or limits expression loss by resistance to transgene silencing.
Supports resistance to DNA methylation and histone deacetylation, consistent with long-term anti-transgene silencing.
Provides performance across multiple promoters and delivery contexts.
UCOE sequence is different from A2UCOE.
Small size.
Lack of synthetic or standalone promoter activity.
Favorable comparison to A2UCOE/3′UCOE.
Documented Applications
Controlling expression of a gene of interest in a target cell by operably linking the UCOE and heterologous promoter to the gene of interest.
Increasing expression of an endogenous gene in a target cell by introducing a recombinant nucleic acid into the cell genome with an operably linked UCOE and promoter and culturing the cells to produce the product encoded by the endogenous gene.
Gene-therapy administration.
Transduction using lentiviral transductions for evaluation of expression maintenance.
Stable transfection-based evaluation of resistance to silencing.
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