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Incapsulated secreting transgenic cells

#1903


Preparation and Study of Encapsulated Transgenic Cells Producing Polypeptide Immunomodulators

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • BIO-CHM/Biochemistry/Biotechnology
  • MED-OTH/Other/Medicine

Status
3 Approved without Funding

Registration date
08.06.2000

Leading Institute
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany

Supporting institutes

  • Institute of Bioorganic Chemistry, Russia, Moscow\nEngelhardt Institute of Molecular Biology, Russia, Moscow

Collaborators

  • Fox Chase Cancer Center / Institute of Cancer research, USA, PA, Philadelphia\nUniversity of Turku / Finnish-Russian Joint Biotechnology Laboratory, Finland, Turku\nEcole Nationale d'Ingenieurs des Techniques des Industries Agricoles et Alimentaires, France, Nantes\nUniversität Hamburg / University Hospital Hamburg-Eppendorf, Germany, Hamburg\nQueen's University / Department of Chemical Engineering, Canada, ON, Kingston\nAbo Akademi University, Department of Biochemistry and Pharmacy, Finland, Turku

Project summary

The objectives of the project:

· To create an artificial "gland" secreting Transforming Growth Factor 1 (TGF-1) and to study the possibility of its application in vivo for gene therapy of the autoimmune disorder - multiple sclerosis - using an animal model.

· To create an artificial "gland" producing the IgG1 fragment that encompasses the amino acid sequence of the immunostimulatory peptide immunorphin and to test it in vivo using animal models for transplantable tumors.

The proposed research project is directed towards the solution of one of the most important problems of biology and medicine – the design of novel effective approaches to gene therapy for autoimmune disorders and tumors.

During the last several years, a specific line of investigation involving somatic gene therapy has been gathering force. It is aimed at the creation of an universal cell line engineered to secrete the desired recombinant gene product and not to cause immunorejection in different patients. The solution of this problem would allow to standardize gene therapy approach (therefore to lower its cost) and to make it more effective. At present, the efforts of many investigators all over the world are directed to the preparation of stable immunoprotective microcapsules (beads) prepared of inert natural or synthetic polymers. Cells entrapped in such capsules are isolated from external environmental effects, but at the same time they are viable and capable of producing recombinant proteins. The scientists from the Institute of Bioorganic Chemistry]("Polymers for biology" lab) have developed hydrogel beads capsules based on new synthetic polymers, in particular poly-N-vinyl caprolactam (PVCL) (Khim-Pharm. Journal (Russian), 1996, 1, 39-41) and composite hydrogels prepared of synthetic and natural polymers. These hydrogel beads have been successfully utilized to entrap various enzymes (Appl. Biochem. Biotechnol, 1996, 61, 75-84; Bioorgan. Khim. (Russian), 1998, 24, 4, 288-292) and several hybridomas producing monoclonal antibodies (Cell Biol., 1995, 9, 127-134; Clin. Cancer Research., 1995, 14, No. 1 (Suppl.), 212-213; Biotechnol. Techn., 1998, 12, No. 1, 11-14). It has also been shown that the structure, operational stability and membrane permeability of the beads/capsules can be varied depending on the preparation method. Within the frame of the proposed project, we plan to entrap transgenic cells (fibroblasts) into beads/microcapsules based on natural polymers (alginate, chitosan and its derivatives), and to study immunomodulator production by encapsulated cells, which are hereafter referred to as an artificial "gland".

In the Laboratory of Cell Engineering at the Institute of Molecular Biology, the original system for gene transfer and expression in mammalian and human cells using retroviral vectors was elaborated. This system was used to create transplantable transgenic cell lines secreting biologically active proteins (cytokines, hormones, oncoproteins, receptors, enzymes, structural virus-specific proteins) (Mol.Biol. (Russian), 1995, № 29, 61-70; Proc. Natl. Acad. Sci. USA, 1997, 94, 5837-5842; Bioorg. Khim. (Russian), 1998, 24, № 1, 21-24). Recently the transgenic cell line producing human interleukin-2 was developed. Implantation of these cells into experimental malignant tumors of nude mice resulted in tumor growth suppression. It was shown that the effect is from interleukin-2 activation of natural killers. In the early 1980s the scientists from the Salk Institute for Biological Studies Julliard et al. (Science, 1980, 208, 183-185), used immobilized antibodies to -endorphin as affinity absorbents in attempt to isolate this hormone from human placenta extract. A 50 кDа polypeptide was isolated. It was found to be a heavy (H) chain of immunoglobulin G (IgG). Elucidation of the causes of such an effect led to the discovery of a -endorphin-like sequence in the H-chain. It was found that the 364-377 fragment (SLTCLVKGFYPSDI) was 40% homologous to the -endorphin antigenic determinant (KSQTPLVTLFKNALKN). An artificial peptide (14 amino acid residues) corresponding to the -endorphin-like sequence in the IgG heavy chain was synthesized by Houck et al. (Science, 1980, 207, 78-79) and found to interact with opioid receptors on rat brain cells. A research group from the Institute of Immunological Engineering has synthesized and studied theNormal;Iau?iue;Iniiaiie oaeno;-endorphin-like decapeptide (H-SLTCLVKGFY-OH) corresponding to amino acid residues 364-373 of IgG H-chain and referred to as immunorphin (Immunol. Lett., 1996, 49, 21-26). The results of the study indicate that immunorphin has a potent (>100 times as potent as tuftsin) immunostimulatory activity in vitro: the peptide activates natural killers, spleniс T- and B- lymphocytes as well as lung and peritoneal macrophages of mice. In collaboration with the scientists from the Branch of Institute of Bioorganic Chemistry a receptor binding study of immunorphin has been carried out. It has been found that the peptide inhibits in a competitive manner, the binding of 125I-labeled -endorphin to naloxone-insensitive -endorphin receptors on human and mouse immunocompetent cells.

Since nonspecific immune stimulation appears to perform well for immunotherapy of oncologic disorders, we intend to use a molecular-genetic approach to increase host immunoreactivity against tumors. Retroviral vectors will be used to create transgenic fibroblasts producing the 335-373 fragment of IgG heavy chain that incorporates the amino acid sequence of immunorphin. The efficacy of gene transfer and expression will be assessed by the methods of polymerase chain reaction and radioimmunoassay. The immunorphin-producing cells will be encapsulated with a PVCL membrane and this "gland" will be implanted into animals with experimentally induced tumors. Such an approach will exclude uncontrollable distribution of non-encapsulated implanted transgenic cells and allow recovery of the “gland” from a recipient after tumor regression. Antitumor activity of the peptide produced by the "gland" will be assessed using the C57Bl/6 strain mouse models for transplantable tumors - Lewis lung carcinoma and B-16 melanoma.

Another line of investigation within the framework of the proposed project is based on the use of the rat (DA strain) model for multiple sclerosis (experimental autoimmune encephalomyelitis) already established in the Branch of the Institute of Bioorganic Chemistry (Biokhim. (Russian), 1998, 63, 1459-1469; Immunologiya (Russian), 1999, № 2, 5-8; J. Neuroimmunol., 1998, 90, 72; Multiple Sclerosis, 1998, 4, 342). We plan to use this animal model for the evaluation of this gene therapy approach to multiple sclerosis treatment based on the introduction of Transforming Growth Factor 1 (TGF-1, anti-inflammatory agent) gene into diseased animals. The gene will be transferred to primary fibroblasts, then the cells will be encapsulated and implanted into the animals. The efficacy of ТGF-1 gene transfer and expression will be confirmed by ELISA and bioassay for ТGF-1 activity using Mv1Lu cell line.

Thus, the proposed research will allow the evaluation of new original gene therapy approach to treatment disorders and tumors. It is based on the creation of artificial "glands" from PVCL-encapsulated transgenic cells producing protein bioregulators. The use of vectors with regulated promoters will allow control of the bioregulators synthesis and secretion.


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