Compositions and methods for chimeric embryo-assisted organ production

A technology of organs and embryos, applied in the field of chimeric embryo-assisted organ preparation compositions and methods, capable of solving problems such as side effects

Inactive Publication Date: 2018-10-23
RGT UNIV OF MINNESOTA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Unfortunately, these are subject to host...

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  • Compositions and methods for chimeric embryo-assisted organ production
  • Compositions and methods for chimeric embryo-assisted organ production
  • Compositions and methods for chimeric embryo-assisted organ production

Examples

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Embodiment 1

[0350] 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

[0351] 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

[0352] 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

Human or humanized tissues and organs suitable for transplant are disclosed herein. Gene editing of a host animal provides a niche for complementation of the missing genetic information by donor stemcells. Editing of a host genome to knock out or disrupt genes responsible for the growth and/or differentiation of a target organ and injecting that animal at an embryo stage with donor stem cells tocomplement 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 U.S. Provisional Patent Applications Serial Nos. 62 / 246,926, 62 / 246,927, 62 / 246,929, 62 / 246,947, 62 / 247,092, 62 / 947,096, 62 / 247,115, 62 / 247,117, 62 / 247,118, and 62 / 247,122 Priority rights, these patents were submitted on October 27, 2015. The entire contents of the aforementioned provisional applications are incorporated herein by reference. [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...

Claims

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Application Information

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IPC IPC(8): A01K67/00C12N5/07C12N15/85A01N1/02A61D19/04
CPCA01K67/0276A01K2217/075A01K2227/108A01K2267/025C12N15/907A01K67/027
Inventor A·朝仓R·阿拉瓦利M·切尔兰J·达顿D·J·加里M·G·加里M·格雷厄姆L·李W·洛A·潘诺斯卡尔瑟斯-莫尔塔里T·奥布赖恩C·斯蒂尔J·托拉尔D·F·卡尔森S·C·法赫伦克鲁格N·古矢野-中川A·帕尔
Owner RGT UNIV OF MINNESOTA
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