Engineering of humanized by geneti complementation

A human, endogenous technology applied in the field of engineering humanized kidneys through genetic complementation, which can solve problems such as side effects

Inactive Publication Date: 2018-11-23
RGT UNIV OF MINNESOTA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, these are subject to host rejection, and / or may cause other side effects

Method used

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  • Engineering of humanized by geneti complementation
  • Engineering of humanized by geneti complementation
  • Engineering of humanized by geneti complementation

Examples

Experimental program
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Effect test

Embodiment 1

[0141] Example 1, see Figures 4A-4D , describe experiments that successfully attempted to knock out two genes at once using HDR editing and, further, were able to select cells that were homozygous for the double gene knockout or heterozygous for each knockout. Cells are treated to introduce first and second targeting endonucleases directed to a first gene target (recombination-activated gene 2, RAG2) and a second gene target (interleukin receptor 2, gamma, IL2Rg or ILR2 gamma), respectively Enzymes (each a TALEN pair). TALENs must be designed to target a predetermined site and obtain sufficient quantities. Cell processing time is less than 5 minutes. Electroporation is used, but many other suitable methods of protein or DNA introduction are also described herein. The cells are then cultured so that they form a single colony each originating from one treated cell. Cells from different colonies were tested after 3 or 11 days. The knockout rate of RAG2 was approximately 6-f...

Embodiment 2

[0142] Example 2, see Figures 5A-5D , describing multiplex HDR editing experiments with the same target but different genes. The first gene target is the adenomatous polyposis coli gene (APC). The second gene target is p53 (TP53 gene). Cells homozygous for the double knockout and cells heterozygous for the double knockout were detected and isolated.

Embodiment 3

[0143] Example 3, see Figures 6-9, describes multiplex HDR editing to knock out 2-5 genes. There were three experiments, and the number of colonies used to test genotypes in each experiment ranged from 72 to 192. Cells were treated for different combinations, multiplexing of genes APC, p53, RAG2, low density lipoprotein receptor (LDLR), IL2Rg, kisspeptin receptor (KISSR or GPR54) and eukaryotic translation initiation factor 4GI (EIF4GI) knockout. The gene LDLR has always been less susceptible to modification than other genes. As can be seen from the results, with TALEN-specific homology-mediated repair (HDR), multiple alleles can be destroyed simultaneously. Five TALEN pairs were co-transfected in three combinations (Table A), where each of the five TALEN pairs resulted in more than 20% HDR / site and its cognate HDR template. A fraction of colonies from each replicate were positive for HDR events in at least four genes, and two colonies from repeat-A had HDR events in five g...

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Abstract

Provided here are methods for editing of a host genome to knock out or debilitate genes responsible for the growth and / or differentiation of a target organ and injecting that animal at an embryo stagewith donor stem cells to complement the missing genetic information for the growth and development of the organ. The result is a chimeric animal in which the complemented tissue (human / humanized organ) matches the genotype and phenotype of the donor. Such organs may be made in a single generation and the stem cell may be taken or generated from the patient's own body. As disclosed herein, it is possible to do so by simultaneously editing multiple genes in a cell or embryo creating a 'niche"' for the complemented tissue. Multiple genes can be targeted for editing using targeted nucleases and homology directed repair (HDR) templates in vertebrate cells or embryos.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application Serial No. 62 / 247,100, filed October 27, 2015, which is incorporated herein by reference in its entirety. [0003] The subject matter of this application may be related to the publications in International Patent Application Nos. WO 2015 / 168125A1 (published 5 November 2015) and WO 2016 / 141234 (published 9 September 2016), and in International Application No. PCT / US2016 / 040378 (filed June 30, 2016) and PCT / US2016 / 040431 (filed June 30, 2016) are the subject matter of disclosure. The entire content of the aforementioned International Application is hereby incorporated by reference. [0004] Statement of Rights to Inventions Made with Federal Funding [0005] This invention was made with Government support under Grant No. W81XWH-15-1-0393 awarded by the Department of Defense and Grant Nos. 1R43HL124781-01A1 and 1R43GM113525-01 awarded by the National Ins...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/00C12N5/07C12N15/85A01N1/02A61D19/04
CPCA01K67/00A61D19/04C12N15/85A01K67/0271A01K67/0276A01K2207/12A01K2207/15A01K2217/054A01K2217/075A01K2217/15A01K2227/105A01K2267/025C07K14/705C07K14/7155C07K14/47C07K14/4702C07K14/4705C07K14/4746A01K2227/108
Inventor S·C·法赫伦克鲁格D·F·卡尔森P·五十岚T·卡罗尔
Owner RGT UNIV OF MINNESOTA
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