Recombinant vectors for use in position-independent transgene expression within chromatin

Abstract

The embodiments of the present invention are directed to discrete, cis-acting regulatory elements that include a barrier element, an insulating element, a silencing element, and matrix attachment regions (“MARs”). Additional embodiments of the present invention are directed to nucleic acid molecules that are useful for facilitating stable transgene expression within a chromatin environment. Additional embodiments of the present invention are directed to recombinant expression vectors including nucleic acid molecules of the present invention that can be incorporated into artificial chromosomes, eukaryotic cell-lines, non-human transgenic animals, and transgenic plants, to improve recombinant protein production in a broad range of eukaryotic hosts, and pharmaceutical compositions including nucleic acid molecules of the present invention that are also useful for gene therapy in the treatment of various genetic diseases.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a continuation-in-part of application Ser. No. 10 / 853,581, filed May 24, 2004, which claims the benefit of provisional application No. 60 / 473,109, filed May 23, 2003.SEQUENCE PROGRAM LISTING APPENDIX [0002] Two identical CDs identified as “Copy 1 of 2” and “Copy 2 of 2,” containing the sequence listing for the present invention, is included as a sequence listing appendix. TECHNICAL FIELD [0003] This present invention relates to recombinant DNA technology involving compositions containing DNA elements identified within the mammalian c-myc loci that enables more efficient transgene expression. Methods for the application of the compositions to develop various expression vectors that are useful for enhancing transgene expression in eukaryotic hosts are also provided. BACKGROUND OF THE INVENTION [0004] Eukaryotic gene expression is governed at multiple levels, involving local modes of regulation with short-range effects a...

AI Technical Summary

Benefits of technology

[0007] In various embodiments of the present invention, compositions and methods for using DNA sequences identified from human and murine c-myc loci are provided. Nucleic acid molecules with these DNA sequences are useful for facilitating stable expression a transgene. Various embodiments of the present invention are directed to recombinant expression vectors that include the present-nucleic-acid-molecule compositions of the present invention, and that can be incorporated into eukaryotic cell-lines, artificial chromosomes, transgenic animals, and transgenic plants to improve the production of recombinant proteins in a broad range of eukaryotic hosts. Pharmaceutical compositions that include the nucleic-acid-molecule compositions of the present invention within expression vectors are also useful for gene therapy in the treatment of various genetic diseases.

[0008] In one embodiment, the nucleic-acid-molecule compositions of the present invention can function as barrier elements that preclude silencing of transgenes by stabilizing host heterochromatin, and that enhance transgene expression levels when incorporated into expression vectors that further include a transgene, a promoter, and / or an enhancer. Expression vectors including the nucleic-acid-molecule compositions of the present invention can be inserted into an artificial chromosome or a chromosome of a host cell to improve transcriptional efficiency.

[0009] In another embodiment, the nucleic-acid-molecule compositions of the present invention can function as insulating elements that facilitate transcriptional efficiency by blocking enhancer-promoter interactions, and that preclude the effect of neighboring regulatory sequences when incorporated into expression vectors that further include a transgene, a promoter, and / or an enhancer.

[0010] In another embodiment, the nucleic-acid-molecule compositions of the present invention can mediate the attachment of chromatin to a nuclear matrix, and can facilitate the regulation of transcription when incorporated into an expression vector that further includes a transgene, a promoter, and / or an enhancer. The compositions of the present invention are directed to several, discrete, cis-acting regulatory elements, that include a barrier element, an insulating element, a silencing element, and matrix-attachment regions (“MAR”), which have not been previously characterized. Each nucleic-acid-molecule compositions of the present invention can be used individually, can be combined together in various combinations, and can be used with heterologous insulators and MARs in various combinations within a broad vector-design scheme for which stable transgene expression in a chromatin environment is desired.

Problems solved by technology

Because a substantial portion of the eukaryotic genome exists in the transcriptionally inactive heterochromatin state, random integration of exogenous genes into a heterochromatin environment likely results in transgene silencing.

For example, gene therapy has only been partially successful, because of position-effect variegation that results from random insertion of transgenes into heterochromatin regions, leading to transgene suppression [G. H. Karpin, Curr. Opin. Genet. Dev. 4: 281-291 (1994)].

Unpredictable and low yields in recombinant protein production, which significantly reduces the rate at which therapeutic proteins are brought to the market, have also been attributed to position-effect variegation.

Production yields from cultivated mammalian cells, and from transgenic plants and animals, are still not sufficient to meet growing demands.

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