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Regulated expression systems

A purpose and gene technology, applied in the field of adjustable expression system, can solve problems such as high expression level

Inactive Publication Date: 2012-10-03
PROYECTO DE BIOMEDICINA CIMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its disadvantage is that it results in high expression levels under basal conditions, which makes its desired in vivo application unacceptable

Method used

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  • Regulated expression systems

Examples

Experimental program
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Effect test

Embodiment 1

[0197] Example 1. Recombinant rAAV-pTet bidi - Construction and characterization of pCMV-luc virus.

[0198] The main purpose is to obtain an inducible vector based on AAV8, which may regulate the timely expression of transgenes by changing the dose of the induced agent administered, which can specifically target the transgene expression of hepatocytes, thereby acting on the target site of our treatment And avoid the potential toxic side effects of genetic modification. Given that AAV8 primarily transduces the liver with exceptional efficiency, we set out to identify rAAV-pTet bidi - Whether the pCMV-luc system fulfills the properties explained above when administered intravenously via the tail vein.

[0199] This system was previously characterized by Chtarto et al. (Chtarto, A., et al. Gene Ther, 2003. 10:84-94), but with intracerebral administration, for which reason the biodistribution of the system after systemic administration is unknown.

[0200] For this, the lucife...

Embodiment 2

[0237] Example 2. Recombinant rAAV-pTet bidi - Construction and characterization of pAlb-luc virus.

[0238] After confirming the presence of rAAV-pTet bidi - After the vectors of the pCMV-luc system exhibited very low inducibility and significant expression outside the liver, we attempted to construct a system that allowed, on the one hand, a maximum induction rate greater than the protocol described above, and on the other hand, the transgene in Localized expression in our target organ (liver). For this, the albumin promoter (pAlb) was used instead of the minimal CMV promoter. Studies by Zabala et al. (Zabala, M., et al., Cancer Res. 2004;64:2799-2804) have shown that pAlb has a low residual expression (even lower than the minimal pCMV when it is used in other inducible systems). lower), however when it is in close proximity to the TetO7 site, it exhibits very high inducibility. On the other hand, expression of pAlb is known to be hepatocyte-specific (Frain, M., et al. M...

Embodiment 3

[0266] Example 3. Recombinant rAAV-pTet bidi - Construction and characterization of pAlb-IL12 virus

[0267] A large number of preclinical studies have shown the antitumor efficacy of IL-12 gene transfer. These data lead to a phase 1 clinical trial for the treatment of digestive system tumors with a first generation adenovirus carrying IL-12 (Sangro, B., et al. J Clin Oncol. 2004, 22:1389-1397). Clinical trials have shown that in order to obtain a therapeutic effect, a longer period of expression of IL-12 is required. In this regard, our department is working on developing a long-term expression viral vector carrying an inducible IL-12 expression system (Wang, L., et al. Gastroenterology, 2004. 126:278-289).

[0268] Among the available viral vectors, AAV is an outstanding candidate because it is a long-term expression vector and it has been demonstrated that it can be produced clinically at high doses (Meghrous, J., et al. Biotechnol Prog. 2005, 21:154 -160).

[0269] To ...

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Abstract

The application relates to gene constructs for inducible hepato-specific expression of polynucleotides of interest in response to an inducer agent, said constructs comprising (i) an inducible bi-directional operator-promoter with at least one responsive element to said inducer agent flanked by two hepato- specific promoters acting in divergent manner, (ii) a first nucleotide sequence encoding a transactivator which may be activated by said inducer agent operatively coupled to the first hepato-specific promoter, and (iii) a second nucleotide sequence operatively coupled to the second hepato-specific promoter, wherein the promoters are induced as a consequence of the binding of the transactivator to the operator region of the operator-promoter in the presence of the inducer agent.

Description

technical field [0001] The present invention is in the field of regulatable expression systems, and more specifically in the field of regulatable expression in both spatial (in a particular tissue) and temporal (in response to the addition of an inducing agent) form. The invention also relates to gene constructs and virions allowing regulation of liver-specific expression, and their use in the treatment of liver diseases. Background technique [0002] Liver functions include, inter alia, metabolism of carbohydrates and lipids, secretion of cytokines, digestion of insulin and other hormones, production of bile, and the like. Furthermore, factors affecting many genetic diseases, cardiovascular diseases, metabolic diseases, bleeding disorders and cancer are produced in the liver. Hepatocytes have a long half-life and are directly connected to the bloodstream, which facilitates the arrival of therapeutic agents. For these reasons, the liver is considered a good candidate organ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/86
CPCC12N2799/025C12N2830/008C12N2830/003C12N15/635A61P1/16A61P35/00
Inventor 格洛丽亚·冈萨雷斯阿塞古伊诺拉萨赫苏斯玛丽亚·普列托巴尔图埃纳露西娅玛丽亚·班雷利马霍
Owner PROYECTO DE BIOMEDICINA CIMA
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