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Multi-transgenic pig for xenotransplantation

A transgenic pig and transgenic technology, applied in the field of preparation of the animal, therapeutic use, and transgenic pig, can solve the problems that multiple transgenic pigs have not yet been produced

Pending Publication Date: 2018-08-31
REVIVICOR INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] Multiple transgenic pigs using polycistronic expression systems have not yet been generated that result in stable, adequate integration and expression of the transgene

Method used

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  • Multi-transgenic pig for xenotransplantation
  • Multi-transgenic pig for xenotransplantation
  • Multi-transgenic pig for xenotransplantation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0534] Example 1: Vector construction and pig production using bicistronic vectors

[0535] vector construction

[0536] Multiplex bicistronic units consisting of two (2) transgenes sharing a single promoter linked by 2A peptide sequences were synthesized. Two forms of the 2A sequence, P2A (66bp) and T2A (55bp) were utilized and a large number of both transgene units were linked to allow co-expression of both genes from one promoter. Promoters are constitutive CAG promoter (CMV enhancer, chicken actin promoter, rabbit β-globin intron 1), endothelial-specific porcine ICAM-2 promoter or Tie2 endothelial-specific enhancer with CAG promoter combination of sub. Construction of human transgene pairs (linked by 2A sequences) including thrombomodulin (TBM), CD39, EPCR, DAF, A20, CD47, CIITA, HO1, TFPI and, in some bicistronic vectors, porcine CTLA4-Ig .

[0537] Polycistronic vectors were engineered using cloning sites behind a) the porcine ICAM-2 enhancer / promoter and b) the co...

Embodiment 2

[0546] Example 2: Construction of polycistronic vectors for generation of genetically modified pigs

[0547] Multicistronic "2A" vectors (MCVs) were used to generate 6-GE pigs using a four-gene vector (two genes under the control of two promoters in each MCV) transfected into well-characterized GTKO.hCD46 cells. bicistronic), the cells are then used for somatic cell nuclear transfer. Genotypes were determined by Southern blot analysis. Gene expression was monitored by flow cytometry in PBMCs and endothelial cells, in cells and organs by immunohistochemistry, Q-PCR (quantitative polymerase chain reaction), and Western blot analysis. Transgene-specific bioactivity assays were performed to quantify and characterize complement inhibition, platelet aggregation, activated protein C formation, ATPase activity, factor Xa cleavage, mixed lymphocyte reaction (MLR), and apoptosis. Pigs with expected genotypes and robust expression of all transgenes were identified in these assays and u...

Embodiment 3

[0551] Example 3: Generation of porcine animals with six genetic modifications (6GE)

[0552] The 4-gene fragment of linear MCV (see e.g. Figure 4) was transfected into cells with GTKO (α1,3-galactosyltransferase knockout) or GTKO.CD46 (α1,3-galactosyltransferase knockout and CD46 ubiquitous expression) platform genetics in porcine fetal fibroblasts. Transfected cells were selected for expression of the two genes behind the CAG promoter by fluorescence-activated cell sorting (FAC), and these sorted cells were used as nuclear donors for somatic cell nuclear transfer (SCNT or clones). Fused embryos were transferred to multiple recipient gilts (8-10 gilts / MCV) and pregnancy was monitored until farrowing.

[0553] Pigs expressing these MCV elements were generated from several gene combinations. Four 4-gene MCV combinations that provided robust expression in live pigs included:

[0554] pREV941: EPCR-CD55-TBM-CD39

[0555] pREV971: EPCR-HO-1-TBM-CD47

[0556] pREV967: EPCR-HO-...

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Abstract

The present invention is directed to transgenic animals (e.g., transgenic porcine animals) comprising multiple genetic modifications that advantageously render these animals suitable donors for xenotransplanation. The present invention extends to organs, organ fragments, tissues and cells derived from these animals and their therapeutic use. The present invention further extends to methods of making such animals. In certain embodiments, the transgenic animals (e.g., transgenic porcine animals) lack expression of alpha gal and incorporate and express at least four transgenes under the control of at least two promoters.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application 62 / 216,225, filed September 9, 2015, and U.S. Provisional Patent Application 62 / 256,068, filed November 16, 2015, the contents of which are hereby incorporated by reference in their entirety . Background technique [0002] Pigs have been the focus of most research in xenotransplantation because pigs share many anatomical and physiological characteristics with humans. Pigs also have a relatively short gestation period, can be bred in a pathogen-free environment, and may not present the same ethical concerns associated with animals not typically used as a food source, such as primates. Scientific knowledge and expertise in the field of porcine-to-primate xenotransplantation has grown rapidly over the past decade, resulting in significantly prolonged survival of primate recipients of life-saving porcine xenografts (Cozzi et al., Xenotransplantation, 16 :203-214. 2009). Recently, significant achiev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/16C12N15/85C12N15/90C12N15/867C12N15/877A01K67/027A01N1/02
CPCA01K67/0278C12N15/8778C12N15/8509A61K35/42A61K35/34A61K35/22A61K35/407A61K35/39A61K45/06A61P11/00A61P9/12A61P9/00A61P13/12A61P1/16A61P1/18A01K2227/108A01K2267/025A61K2300/00A01N1/0226A01K67/0275C12N15/907A01K2217/052A01K2217/072A01K2217/15A01K2217/206C07K2319/50C12N2800/30C12N2840/20A61P43/00
Inventor D.L.阿亚雷斯C.菲尔普斯
Owner REVIVICOR INC
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