Novel manganese superoxide dismutase regulatory elements and uses therefore

a technology of manganese superoxide dismutase and regulatory elements, applied in the field of new transcriptional regulatory elements, can solve the problems of inability to adapt to and control the expression of foreign genes, lack of specificity, repeated administration of recombinant ad based vectors, etc., and achieve the effect of increasing the transcription/expression level of heterologous polynucleotides

Inactive Publication Date: 2005-07-28
NICK HARRY +2
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  • Abstract
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Benefits of technology

[0043] The inducible expression system can also include a heterologous polynucleotide operably linked to the MnSOD regulatory element such that, upon activation of the MnSOD regulatory element by the compound, transcription or expression of the heterologous polynucleotide is induced. The inducible expression system can further include a heterologous promoter (i.e., a promoter which is not derived from the MnSOD regulatory element itself) to further increase transcription / expression levels of the heterologous polynucleotide.

Problems solved by technology

However, vectors developed from these viruses all lack some level of specificity which presents an obstacle for appropriate and controlled expression of foreign genes (Friedmann, 1996).
On the other hand, repeated administration of recombinant Ad based vectors, is often limited by host immune responses against viral structural proteins.
One of the main issues in potential clinical application of gene therapy is the need for increased gene transfer efficiency and target specificity associated with regulated expression at therapeutically relevant levels in vivo (Chow et al., 1997).
Unfortunately, almost all of the presently available inducible promoters used in gene therapy vectors require exogenous stimulation by non-physiological or artificial substances such as tetracycline (No et al., 1996), ecdysone (Delort and Capecchi, 1996), or RU486 (Massie et al., 1998).
One of the disadvantages of these systems is that they all require expression of two gene products.
This is usually accomplished by co-transfection of separate plasmids into mammalian cells, thus potentially limiting the size of the transgene when a viral vector system is used for transgene delivery.
The disadvantages of these systems include the continuous treatment with the exogenous substance and slow clearance from the organism, which interferes with quick and precise induction.
However, available inducible promoters do not allow the cell to control the timing of expression of the transgene or its fold induction.

Method used

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  • Novel manganese superoxide dismutase regulatory elements and uses therefore
  • Novel manganese superoxide dismutase regulatory elements and uses therefore
  • Novel manganese superoxide dismutase regulatory elements and uses therefore

Examples

Experimental program
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Effect test

example 1

Characterization of the Rat MnSOD Promoter

[0224] The following studies were performed to characterize the region of the rat MnSOD gene responsible for induction of gene expression in response to inflammatory mediators.

The Rat MnSOD Promoter does not Contain all the DNA Elements Necessary for Cytokine-Inducible Expression

[0225] MnSOD gene expression is stimulated by inflammatory mediators, including LPS, TNF-α, and IL-1β, and is due at least in part to de novo gene transcription. To identify sequences in rat MnSOD which confer specificity for LPS-, TNF-α- and IL-1β-dependent gene induction, a series of MnSOD promoter deletion constructs were generated using the promoterless human growth hormone vector pØGH (FIG. 1A). Unique restriction sites within the rat MnSOD 5′ flanking sequence were used to create vectors containing the promoter deletions. Each 5′ promoter deletion construct contained sequences which incorporated the MnSOD transcriptional start site. Cells transfected with a...

example 2

Characterization of MnSOD Enhancer Elements

[0227] The studies described in Example 1 showed that the rat MnSOD gene 5′ proximal promoter was not solely responsible for inducible gene expression. Accordingly, the following studies were performed to identify and characterize further transcriptional regulatory elements within the MnSOD gene itself (e.g., within intron sequences).

A Novel Inducible Cis-Acting Enhancer Element Exists within the Rat and Human MnSOD Genes

[0228] To determine whether the remaining DNaseI hypersensitive sites described in Example 1 (within the MnSOD gene) contained regulatory function, pØGH expression vectors were created that contained both a 2.5 Kb fragment of the MnSOD promoter (Hind / E) and a 6.1 kb HindIII internal fragment of the MnSOD gene which contains all of the other Dnase I hypersensitive sites (FIG. 2A). Expression of hGH mRNA in cells transfected with this vector was compared to expression of hGH in cells transfected with a pØGH construct cont...

example 3

Construction of Recombinant Adeno-Associated Virus (AAV) Vectors Containing MnSOD Prohancer Elements

[0244] The novel MnSOD prohancer elements of the invention can also be used to generate delivery vectors suitable for inducible heterologous gene expression. A series of AAV vectors can be engineered containing the MnSOD prohancer element. To do this, the PCMV promoter is removed from pTR-UF2 using KpnI and XhaI, and the MnSOD prohancer, either alone or coupled to a minimal promoter, is inserted (FIG. 10). The recombinant AAV plasmids are packaged using the current method for isolating rAAV as developed by Hermonat and Muzyczka (1984). Human cells (e.g., 293 cells) are transfected with the plasmid which consists of a transgene flanked by the AAV terminal repeats (TR), the only AAV sequences required for viral DNA replication, packaging and integration. The cells are also transfected with a complementing plasmid that is defective for packaging, but supplies the wild type AAV rep and c...

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Abstract

A novel transcriptional regulatory element which was isolated from the MnSOD gene and which exhibits promoter-enhancer activity is disclosed. The promoter-enhancer activity of the element is further modulated by inflammatory mediators to regulate transcription. Methods of using the promoter-enhancer element to regulate gene expression, and therapeutic uses involving the promoter-enhancer element are also described.

Description

RELATED APPLICATIONS [0001] This application is a Divisional Application of application Ser. No. 09 / 856,766, filed on Aug. 28, 2001, which is a § 371 Application of PCT / US99 / 2833 1, filed on Nov. 30, 1999, which claims priority to U.S. Ser. No. 60 / 110,334, filed on Nov. 30, 1998. The contents of all of the aforementioned application(s) are hereby incorporated herein in their entirety by this reference.GOVERNMENT FUNDING [0002] This invention was made with government support under grant HL-39593 awarded by the National Institutes of Health. The United States government has certain rights in the invention.FIELD OF THE INVENTION [0003] The invention relates to novel transcriptional regulatory elements derived from manganese superoxide dismutase (MnSOD) genes, as well as methods of identifying and using the regulatory elements to control gene expression. BACKGROUND OF THE INVENTION [0004] Precise control of regulated gene expression has multiple potential applications including inducibl...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C07K14/47C12N9/02
CPCA61K48/00C12N9/0089C07K14/4705
Inventor NICK, HARRYROGERS, RICHARDVALENTINE, JOHN
Owner NICK HARRY
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