Transgenic Ungulates Expressing CTLA4-IG and Uses Thereof

a technology of ungulates and transgenic ungulates, applied in the field of transgenic ungulates, can solve the problems of antigen specific unresponsive state, difficult to overcome obstacles, and significant number of people dying, and achieve the effect of reducing or eliminating cell-mediated rejection of a xenotransplan

Inactive Publication Date: 2009-07-23
AYARES DAVID LEE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0030]In a sixth aspect, the present invention is a method of reducing or eliminating cell mediated rejection of a xenotransplant in a recipient comprising providing xenogenic cells, tissues or organs to a recipient which have been genetically modified to express ...

Problems solved by technology

A significant number of people die while awaiting an organ.
However, there are several obstacles that must be overcome before the transfer of these organs or tissues into humans can be successful.
The immunological barriers to xenografts differ from those to allografts because of the greater molecular incompatibility between host and donor tissue.
In the absence of costim...

Method used

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  • Transgenic Ungulates Expressing CTLA4-IG and Uses Thereof
  • Transgenic Ungulates Expressing CTLA4-IG and Uses Thereof
  • Transgenic Ungulates Expressing CTLA4-IG and Uses Thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Production of the pREV785 Construct

[0189]A construct was assembled containing the CTLA4-Ig transgene. The pREV785 construct contains the CTLA4-Ig transgene driven by the chicken beta actin promoter (ubiquitous expression) with additional enhancer and MAR sequences (FIG. 4). Complete sequence of the pREV785 plasmid comprising the pREV785 construct and associated vector backbone is shown in FIG. 5 (SEQ D NO. 4).

example 2

Production of the pREV792 Construct

[0190]A second construct was assembled containing the CTLA4-Ig transgene. The pREV792 construct contains the CTLA4-Ig transgene, driven by the rat insulin II promoter (tissue specific expression), with additional enhancer and insulator sequences (FIG. 6). Complete sequence of the pREV792 plasmid comprising the pREV792 construct and associated vector backbone is shown in FIG. 7 (SEQ ID NO. 5).

example 3

Transfection of Cells with the pREV785 Construct

[0191]A fetal fibroblast cell line (DPFA11) was isolated from a fetus at day 40 of gestation. Fetuses were mashed through a 60-mesh metal screen using curved surgical forceps slowly so as not to generate excessive heat. The cell suspension was then pelleted and resuspended in DMEM containing 20% fetal calf serum, 4 ng / ml basic fibroblast growth factor. Cells were cultured three days, and cryopreserved.

[0192]For transfection, three different linearized DNA vectors were simultaneously introduced into cells by electroporation. One was the pREV785 construct described in Example 1 (2 ug, linearized with PacI / Swa I), and one was a construct containing a human CD55 gene driven by the chicken beta actin promoter (pPL675) (lug). The third (pREV 784) was a construct comprising the hTFPI (human tissue factor pathway inhibitor) gene, the chicken beta actin promoter and the CMV enhancer.

[0193]Twenty-four hours following transfection, human CD55 tra...

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Abstract

The present invention provides ungulates, including pigs, expressing CTLA4-Ig, as well as tissue, organs, cells and cell lines derived from such animals. Such animals, tissues, organs and cells can be used in research and medical therapy, including xenotransplanation. In addition, methods are provided to prepare organs, tissues and cells expressing the CTLA4-Ig for use in xenotransplantation, and nucleic acid constructs and vectors useful therein.

Description

[0001]This application claims priority to U.S. Provisional Patent Application No. 60 / 706,843, filed Aug. 9, 2005.FIELD OF THE INVENTION[0002]The present invention provides transgenic ungulates, such as pigs, expressing cytoxic T-lymphocyte associated protein 4 (CTLA4) fused to an immunoglobulin (Ig) (CTLA4-Ig), as well as organs, tissues, cells and cell lines derived therefrom. The invention also provides transgenic ungulates, and organs, tissues, cells and cell lines derived therefrom, expressing CTLA4 under the control over a tissue-specific promoter. These ungulates, including organs, tissues, cells and cell lines derived therefrom, can be used in research and medical therapy, including xenotransplantation. In addition, methods are provided to prepare organs, tissues, and cells expressing CTLA4-Ig for use in xenotransplantation, as well as nucleic acid constructs and vectors useful therein.BACKGROUND[0003]The success of allogeneic (human to human) organ transplantation has been e...

Claims

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Application Information

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IPC IPC(8): A61K35/12A01K67/027C12N5/10A01N1/02C12N15/87C12N15/88C07H21/00
CPCA01K2217/05A01K2227/108A01K2267/02A01K2267/025C12N15/85C07K2319/30C12N15/8509C12N2830/008A61K38/1774C07K14/70521A61P37/06
Inventor AYARES, DAVID LEE
Owner AYARES DAVID LEE
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