Complement factor h gene knockout rat as a model of c3 glomerulopathy

A glomerular basement membrane and glomerular technology, applied in genetic engineering, biochemical equipment and methods, animal husbandry, etc.

Active Publication Date: 2021-06-15
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ESRD in Cfh - / - May take several months to manifest in mice, requiring long-term preclinical studies to evaluate new treatments

Method used

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  • Complement factor h gene knockout rat as a model of c3 glomerulopathy
  • Complement factor h gene knockout rat as a model of c3 glomerulopathy
  • Complement factor h gene knockout rat as a model of c3 glomerulopathy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0180] Example 1: Generation of Mice Containing an Inactivated Cfh Locus

[0181] To generate a knockout rat Cfh allele, the guide RNA was designed to cut at the end of the Cfh gene to fold (delete) the coding sequence, or to cut at a site flanking the start codon to delete the start codon . No targeting vector was used. The endogenous rat Cfh locus (exon 1 to exon 22) is shown in SEQ ID NO:1. The rat Cfh coding sequence is shown in SEQ ID NO:2. The rat CFH protein sequence is shown in SEQ ID NO: .

[0182] A CRISPR / Cas9 component comprising four guide RNAs (guide RNA target sequences listed in SEQ ID NO: 12-15 corresponding to rGU1, rGU2, rGD2 and rGD3, respectively) was introduced into Dark Agouti rat embryonic stem cells. Specifically, the 2×10 6 Rat ES cells (Dark Agouti strain DA2B) were electroporated with: 5 μg of Cas9 expression plasmid (Cas9 coding sequence shown in SEQ ID NO: 6); and 5 μg each of DNA encoding the following gRNAs: rGU1, rGU2, rGD2 and rGD3 (the ...

Embodiment 2

[0190] Example 2. Characterization of Rats Containing an Inactivated Cfh Locus

[0191] To assess whether Cfh knockout rats recapitulate the C3 glomerulopathy (C3G) phenotype, various readouts related to alternative complement pathway activation, renal pathology, and mortality were assessed. C3 glomerulopathy (C3G) is characterized by hyperactivation of alternative pathway (AP) complement activation as a result of mutations in C3 nephropathic factors and / or complement genes. Since approximately 50% of patients develop end-stage renal disease (ESRD) within 10 years of diagnosis, the survival of female and female Cfh knockout rats (20 male rats, 18 female rats) was evaluated. Such as figure 2 As shown, the median age of death of male Cfh knockout rats was 52 days The median age at death of female Cfh knockout rats was 103 days The last male Cfh knockout rat was at 122 days died, while the last female Cfh knockout rat was at 247 days die. Differences were observed be...

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Abstract

Rat cells and rats comprising an inactivated Cfh locus and methods of making and using such rat cells and rats are provided. The rats comprising an inactivated Cfh locus model C3 glomerulopathy (C3G). Methods are provided for using such rats comprising an inactivated Cfh locus to assess in vivo efficacy of putative C3G therapeutic agents.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Application No. 62 / 730,690, filed September 13, 2018, which is hereby incorporated by reference in its entirety for all purposes. [0003] References to Sequence Listings submitted as text files via EFS Web [0004] The sequence listing written in file 536658SEQLIST.txt is 294 KB, created on September 6, 2019, and is incorporated by reference. Background technique [0005] C3 glomerulopathy (C3G) is characterized by hyperactivation of alternative pathway (AP) complement activation as a result of mutations in C3 nephropathic factors and / or complement genes. Approximately 50% of patients develop end-stage renal disease (ESRD) within 10 years of diagnosis. The recurrence rate after kidney transplantation is about 80-90%. There are currently no FDA-approved treatments for C3G, and no therapies that attack the cause of C3G. [0006] Complement factor H deficient (Cfh - / - ) m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/105A01K2267/035A01K2267/0393C12N2015/8527
Inventor 基肖尔·德瓦拉拉贾-纳拉什姆哈杰弗里·D·李洛里·莫顿
Owner REGENERON PHARM INC
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