Transferrin receptor transgenic models

A transferrin, transgenic animal technology, applied in the direction of transferrin, genetic engineering, animal/human protein, etc., can solve the problem of unsuitable mouse model

Pending Publication Date: 2019-10-25
DENALI THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As a result, these existing mouse models are unsuitable as tools for evaluating therapeutic agents capable of crossi

Method used

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  • Transferrin receptor transgenic models
  • Transferrin receptor transgenic models
  • Transferrin receptor transgenic models

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1: Generation and Characterization of HUTFR Mice

[0097] Methods for generating knock-in / knock-out mice have been published in the literature and are well known to those skilled in the art. Briefly, C57B16 mice were used to generate a knock-in of the human apical TfR mouse line via pronuclear microinjection into one-cell embryos followed by transfer of the embryos to pseudopregnant females. Specifically, Cas9, sgRNA of SEQ ID NO: 10-11 and donor DNA of SEQ ID NO: 4 were introduced into the embryo. The donor DNA comprised the human apical domain coding sequence SEQ ID NO: 2 that had been codon optimized for expression in mice. The apical domain coding sequence is flanked by a left homology arm (nucleotides 1-817 of SEQ ID NO:4) and a right homology arm (nucleotides 1523-2329 of SEQ ID NO:4). The donor sequence was designed in such a way that the apical domain was inserted after the fourth mouse exon and flanked just at its 3' end by the ninth mouse exon. Co...

Embodiment 2

[0098] Example 2: Generation of Tool Antibodies for Monitoring Antibody Brain Uptake

[0099] Tool antibodies targeting human TfR or human / mouse BACE1 were generated by transforming Expi293 or ExpiCHO cells with expression plasmids containing DNA encoding the heavy and light chains and using protocols familiar to those skilled in the art. Bispecific antibodies were generated using the "knobs-into-holes" technique; the knob and hole half-antibodies were expressed separately and then ligated using published methods. Antibodies were purified first with protein A and then by size exclusion chromatography. Antibodies generated for these studies are as follows:

[0100] Anti-TfR: A human IgG1 antibody that binds to the apical domain of human TfR.

[0101] Anti-BACE1: Human IgG1 antibody that binds to human BACE1 and cross-reacts with mouse BACE1. This antibody inhibits the enzymatic activity of BACE1.

[0102] Anti-TfR / BACE1: human IgG1 knob bispecific antibody that binds to t...

Embodiment 3

[0103] Example 3: HUTFR 顶端的+ / - and HUTFR 顶端的+ / + Blood analysis of mice

[0104] From wild-type C57B16, huTfR 顶端的+ / - and huTfR 顶端的+ / + Blood was collected from mice (n=3 / group) and subjected to standard complete blood count (CBC) analysis. No genotype-specific differences were observed in any red blood cell parameter, including total red blood cell, hemoglobin, and hematocrit levels (Figure 1).

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Abstract

In some aspects, the present invention provides chimeric transferrin receptor (TfR) polynucleotides and polypeptides. In other aspects, this invention provides chimeric TfR transgenic animal models and methods of using the animal models to identify therapeutics that can cross the blood-brain barrier.

Description

[0001] Background of the invention [0002] The blood-brain barrier (BBB) ​​prevents most macromolecules from entering the brain from the periphery, and thus limits the use of macromolecular therapeutics that require brain exposure. The transferrin receptor (TfR) is highly expressed at the BBB and can be used to transport such therapeutic agents across the BBB via receptor-mediated transcytosis. Mouse models have previously been developed in which the mouse TfR is replaced by full-length human TfR cDNA with the aim of assessing the ability of potential therapeutic agents to cross the BBB. However, these transgenic mice were unhealthy and exhibited abnormally high TfR expression, low red blood cell counts and high serum iron concentrations. Yu et al., Science Trans. Med., 6(261):261ra154 (2014). As a result, these existing mouse models are not suitable as tools for evaluating therapeutic agents capable of crossing the BBB to treat brain diseases; models more representative of e...

Claims

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Application Information

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IPC IPC(8): C07K14/705G01N33/68C12N5/10A01K67/027C12N15/63
CPCA01K67/0275C07K14/70582C07K14/79G01N33/68A01K2217/00G01N2500/10C12N15/8509C12N2015/8527A01K2227/105A01K2267/03A01K67/0278A01K2217/072A01K2217/075C07K14/705C07K2319/00G01N33/5088G01N33/5308
Inventor 马克·S·丹尼斯亚当·P·西尔弗曼乔伊·余·祖切罗
Owner DENALI THERAPEUTICS INC
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