Self-regulated apoptosis of inflammatory cells by gene therapy

A technology of apoptosis and alleles, applied in the fields of molecular biology and immunology, can solve the problems of high cost, inconvenience, danger and side effects

Inactive Publication Date: 2001-04-18
BOEHRINGER INGELHEIM PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Finally, it should be noted that these routes carry the risks and side effects often associated with conventional drug therapy and invasive surgical procedures, including high cost and inconvenience of hospitalization and recovery

Method used

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  • Self-regulated apoptosis of inflammatory cells by gene therapy
  • Self-regulated apoptosis of inflammatory cells by gene therapy
  • Self-regulated apoptosis of inflammatory cells by gene therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Generation of TNFp-granzyme B constructs

[0111] For the construction of chimeric granzyme B driven by the enhancer cis-element of the TNF promoter (single or multiple copies of the same region or different regions), identification of the region of interest that determines the optimal induction of expression of the reporter gene . Screening for TNF[alpha] promoter elements for construction of chimeric nucleic acids.

[0112] The TNFα promoter region ( image 3). Regions identified by other investigators in various other cell systems were used as references (Rhoades, et al., J. Biol. Chem., 1992, 267, 22102-22107; Leitman, et al., Mol. Cell Biol. , 1992, 12, 1352-1356; Pauli U., An important review of gene expression in eukaryotic cells, 1994, 4, 323-344). The PCR-amplified gene is then cloned upstream of a reporter gene (eg luciferase) in a commercially available promoterless vector. These constructs were tested in various cell lines such as Jurkat (T lymphoblasto...

Embodiment 2

[0128] Assay Protocol In vitro method: Luciferase assay: Luciferase activity was determined using commercially available reagents (Promega). Granzyme B gene expression:

[0129] a) Western blots of transfected cell lysates were developed using anti-granzyme B antibody.

[0130] b) Apoptosis of transfected cells: Apoptosis of transfected cells caused by granzyme B was detected by staining nuclei with propidium iodide (Krishan, A., J. Cell Biol., 66, 1994, 188-193), and determined by a commercially available cell death ELISA kit (Boehringer Mannheim). animal model

[0131] Rabbit model of IL-1β-induced arthritis (Pettipher E.R. et al, Proc. Natl. Acad. Sci., 1986, 83, 8749-8753): IL-1β was injected into knee joints of New Zealand white rabbits. Intra-articular infusion of IL-1β caused a dose-dependent infiltration of leukocytes into the joint cavity and caused loss of proteoglycans from articular cartilage.

[0132] Antigen-Induced Arthritis: Intra-articular injection of ant...

Embodiment 3

[0137] Screening for somatic variants that do not produce TNFα

[0138] Cells (THP-1, Jurkat cells) were stably transfected in vitro with TNFp-AIG chimeric nucleic acid. After several rounds of stimulation to induce apoptosis of cells expressing the TNFp-AIG gene, surviving cells were harvested. From these cells cDNA libraries were constructed for functional cloning (Legerski R and Peterson C., Nature, 1992, 359, 70-73; Jaattela M., et al., Oncogene, 1995, 10, 2297-2305).

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Abstract

The present invention relates to chimeric nucleic acids and to the therapeutic induction of apoptosis in activated inflammatory cells, or cells at a site of inflammation, by introducing into those cells the chimeric nucleic acid. The chimeric nucleic acid having at least one TNF alpha promoter enhancer attached to a functional copy of a TNF alpha promoter and further attached to at least one copy of an apoptosis-inducing gene, which is further attached to a 3'UTR. The apoptosis-inducing gene is Granzyme B. The invention also relates to methods of making and using self-regulated apoptosis chimeric nucleic acids and pharmaceutical compositions containing them for treating inflammatory diseases.

Description

technical field [0001] The invention relates to the fields of molecular biology and immunology. More specifically, the present invention relates to inducing apoptosis of inflammatory cells by introducing into said cells a gene that induces apoptosis (programmed cell death or non-necrotizing cell death). Background of the invention [0002] In many inflammatory conditions, cytokines such as IL-1β, IL-10, GM-CSF, and TNFα are overproduced due to massive aggregation and accumulation of inflammatory cells (Brennan F.M. et al., British Medical Bulletin, 1995 , 51 / 2, 368-384). The upregulation and / or dysregulation of cytokines in inflamed tissues may directly or indirectly contribute to the exacerbation of chronic inflammatory diseases. For example, the most characteristic pathology of rheumatoid arthritis (RA) occurs at sites of local inflammation (ie, synovial joints). Therefore, it appears that cytokines produced in the synovial joints of patients with RA play an important r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7088A61K38/00A61K48/00A61P1/04A61P3/10C12N15/09A61P17/06A61P25/00A61P29/00A61P31/00A61P43/00C07K14/525C12N5/10C12N9/64C12N15/85C12Q1/68
CPCC12N2830/85A61K38/00C07K14/525C12N2830/15C12N2830/00C12N2830/002C12N9/6467C12N15/85A61K48/00A61P1/04A61P17/06A61P25/00A61P29/00A61P31/00A61P43/00A61P3/10
Inventor 雷瓦蒂·J·塔泰克史蒂文·D·马林兰德尔·W·巴顿
Owner BOEHRINGER INGELHEIM PHARMA INC
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