Gamma satellite insulator sequences and their use in preventing gene silencing
Inventors
Kim, Jung-hyun • Larionov, Vladimir L. • Ebersole, Tom
Assignees
US Department of Health and Human Services
Publication Number
US-9018011-B2
Publication Date
2015-04-28
Expiration Date
2028-02-15
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Abstract
Regulatory elements, specifically insulators and transgene constructs containing insulator nucleic acid sequences, are disclosed herein. Methods of using insulators and transgene constructs including insulators to inhibit, delay, or prevent gene silencing are also disclosed herein.
Core Innovation
The invention discloses regulatory elements, specifically insulator nucleic acid sequences derived from human gamma-satellite DNA arrays located in pericentromeric regions of human chromosomes, and transgene constructs containing such insulator sequences. These insulators are capable of inhibiting, delaying, or preventing gene silencing when operably linked to a coding nucleic acid sequence and integrated into a chromosomal site.
The background describes the problem of epigenetic silencing of integrated transgenes largely caused by heterochromatin spreading and position effects of surrounding chromatin. This silencing leads to expression variegation or complete suppression of transgene expression, limiting the utility of gene therapy and stable gene expression in mammalian cells. The invention addresses this challenge by identifying gamma-satellite DNA sequences that prevent or delay such silencing.
Experimental analysis revealed that human gamma-satellite DNA arrays contain clusters of CTCF transcription factor recognition sites that are not sensitive to CpG methylation, suggesting insulator activity. Transgene constructs flanked by or containing gamma-satellite insulator sequences maintain stable and persistent expression over time, resist heterochromatin spreading, and exhibit a transcriptionally permissive chromatin structure adjacent to these sequences. Thus, the core innovation is the identification and use of gamma-satellite DNA-derived insulator sequences to prevent epigenetic transgene silencing.
Claims Coverage
The patent contains two independent method claims covering methods of inhibiting silencing of coding nucleic acid sequences via integration of transgene constructs with human gamma-satellite DNA monomer repeats at chromosomal sites.
Integration of transgene constructs flanked by human gamma-satellite DNA repeats to prevent gene silencing
A method comprising introducing into a chromosomal site of a recipient cell a transgene construct containing a coding nucleic acid sequence flanked on both 5′ and 3′ ends by at least six repeats of a human gamma-satellite DNA monomer that is heterologous to the coding sequence, wherein these repeats inhibit gene silencing of the coding sequence.
Use of gamma-satellite DNA sequences flanking a coding nucleic acid sequence to inhibit gene silencing
A method comprising introducing a transgene construct into a recipient cell chromosomal site, where the construct includes a first human gamma-satellite DNA sequence, a coding nucleic acid sequence, and a second human gamma-satellite DNA sequence, with the gamma-satellite sequences heterologous to the coding sequence and inhibiting silencing of the coding sequence.
The independent claims cover methods of inhibiting gene silencing by introducing transgene constructs into recipient cells, where the coding sequence is flanked on either or both ends by multiple repeats of human gamma-satellite DNA monomers that function as insulator sequences to prevent silencing, including sequences from various human chromosomes and comprising binding sites for proteins like CTCF and Ikaros.
Stated Advantages
Gamma-satellite DNA sequences act as strong insulators that prevent epigenetic transgene silencing, enabling persistent and stable gene expression over extended periods.
The insulator sequences form open chromatin domains adjacent to the transgene, preventing heterochromatin spreading from vector backbone sequences.
Gamma-satellite DNA insulators increase the yield of stable transfectants, enhancing transgene expression efficiency.
CTCF binding sites within the gamma-satellite DNA provide methylation-insensitive insulator activity.
Documented Applications
Use of transgene constructs containing gamma-satellite DNA insulator sequences to inhibit, delay, or prevent silencing of coding nucleic acid sequences in mammalian cells, including human cells.
Ex vivo gene therapy applications involving introduction of insulator-containing transgene constructs into human CD34+ cells to maintain stable therapeutic gene expression.
In vivo or ex vivo gene transfer using viral vectors such as adenoviral, adeno-associated virus, or lentiviral vectors incorporating gamma-satellite DNA insulators to enhance transgene expression stability.
Use in human artificial chromosomes and other vector systems to maintain open chromatin and prevent transgene silencing in therapeutic and research contexts.
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