Non-Human Animals Comprising A Humanized TTR Locus And Methods Of Use

Abstract

Non-human animal genomes, non-human animal cells, and non-human animals comprising a humanized TTR locus and methods of using such non-human animal genomes, non-human animal cells, and non-human animals are provided. Non-human animal cells or non-human animals comprising a humanized TTR locus express a human transthyretin protein or a chimeric transthyretin protein, fragments of which are from human transthyretin. Methods are provided for using such non-human animals comprising a humanized TTR locus to assess in vivo efficacy of human-TTR-targeting reagents such as nuclease agents designed to target human TTR. Methods are also provided for making such non-human animals comprising a humanized TTR locus.

1 . A non-human animal comprising in its genome a genetically modified endogenous Ttr locus comprising a human TTR sequence comprising both TTR coding sequence and non-coding sequence. 
     
     
         2 . The non-human animal of  claim 1 , wherein a region of the endogenous Ttr locus comprising both Ttr coding sequence and non-coding sequence has been deleted and replaced with a corresponding human TTR sequence comprising both TTR coding sequence and non-coding sequence. 
     
     
         3 . The non-human animal of  claim 1 , wherein the genetically modified endogenous Ttr locus comprises the endogenous Ttr promoter, wherein the human TTR sequence is operably linked to the endogenous Ttr promoter. 
     
     
         4 . The non-human animal of  claim 1 , wherein at least one intron and at least one exon of the endogenous Ttr locus have been deleted and replaced with the corresponding human TTR sequence. 
     
     
         5 . The non-human animal of  claim 1 , wherein the entire Ttr coding sequence of the endogenous Ttr locus has been deleted and replaced with the corresponding human TTR sequence. 
     
     
         6 . The non-human animal of  claim 5 , wherein the region of the endogenous Ttr locus from the Ttr start codon to the Ttr stop codon has been deleted and replaced with the corresponding human TTR sequence. 
     
     
         7 . The non-human animal of  claim 1 , wherein the genetically modified endogenous Ttr locus comprises a human TTR 3′ untranslated region. 
     
     
         8 . The non-human animal of  claim 1 , wherein the endogenous Ttr 5′ untranslated region has not been deleted and replaced with the corresponding human TTR sequence. 
     
     
         9 . The non-human animal of  claim 1 , wherein the region of the endogenous Ttr locus from the Ttr start codon to the Ttr stop codon has been deleted and replaced with a human TTR sequence comprising the corresponding human TTR sequence and a human TTR 3′ untranslated region, and
 wherein the endogenous Ttr 5′ untranslated region has not been deleted and replaced with the corresponding human TTR sequence, and 
 wherein the endogenous Ttr promoter has not been deleted and replaced with the corresponding human TTR sequence. 
 
     
     
         10 . The non-human animal of  claim 9 , wherein:
 (i) the human TTR sequence at the genetically modified endogenous Ttr locus comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 18; or   (ii) the genetically modified endogenous Ttr locus encodes a protein comprising a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 1;   (iii) the genetically modified endogenous Ttr locus comprises a coding sequence comprising a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 90; or   (iv) the genetically modified endogenous Ttr locus comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 14 or 15.   
     
     
         11 . The non-human animal of  claim 1 , wherein the genetically modified endogenous Ttr locus encodes a transthyretin precursor protein comprising a signal peptide, and the region of the endogenous Ttr locus encoding the signal peptide has not been deleted and replaced with the corresponding human TTR sequence. 
     
     
         12 . The non-human animal of  claim 11 , wherein the first exon of the endogenous Ttr locus has not been deleted and replaced with the corresponding human TTR sequence. 
     
     
         13 . The non-human animal of  claim 12 , wherein the first exon and first intron of the endogenous Ttr locus have not been deleted and replaced with the corresponding human TTR sequence. 
     
     
         14 . The non-human animal of  claim 11 , wherein the region of the endogenous Ttr locus from the start of the second Ttr exon to the Ttr stop codon has been deleted and replaced with the corresponding human TTR sequence. 
     
     
         15 . The non-human animal of  claim 11 , wherein the genetically modified endogenous Ttr locus comprises a human TTR 3′ untranslated region. 
     
     
         16 . The non-human animal of  claim 11 , wherein the region of the endogenous Ttr locus from the second Ttr exon to the Ttr stop codon has been deleted and replaced with a human TTR sequence comprising the corresponding human TTR sequence and a human TTR 3′ untranslated region, and
 wherein the endogenous Ttr 5′ untranslated region has not been deleted and replaced with the corresponding human TTR sequence, and 
 wherein the endogenous Ttr promoter has not been deleted and replaced with the corresponding human TTR sequence. 
 
     
     
         17 . The non-human animal of  claim 16 , wherein:
 (i) the human TTR sequence at the genetically modified endogenous Ttr locus comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 19; or   (ii) the genetically modified endogenous Ttr locus encodes a protein comprising a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 2;   (iii) the genetically modified endogenous Ttr locus comprises a coding sequence comprising a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 91; or   (iv) the genetically modified endogenous Ttr locus comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 16 or 17.   
     
     
         18 . The non-human animal of  claim 1 , wherein the genetically modified endogenous Ttr locus does not comprise a selection cassette or a reporter gene. 
     
     
         19 . The non-human animal of  claim 1 , wherein the non-human animal is homozygous for the genetically modified endogenous Ttr locus. 
     
     
         20 . The non-human animal of  claim 1 , wherein the non-human animal is a mammal. 
     
     
         21 . The non-human animal of  claim 20 , wherein the non-human animal is a rat or mouse. 
     
     
         22 . The non-human animal of  claim 21 , wherein the non-human animal is a mouse. 
     
     
         23 . A method of assessing the activity of a human-TTR-targeting reagent in vivo, comprising:
 (a) administering the human-TTR-targeting reagent to the non-human animal of  claim 1 ; and   (b) assessing the activity of the human-TTR-targeting reagent in the non-human animal.   
     
     
         24 .- 40 . (canceled) 
     
     
         41 . A method of optimizing the activity of a human-TTR-targeting reagent in vivo, comprising:
 (I) performing the method of claim  23 a first time in a first non-human animal comprising in its genome a genetically modified endogenous Ttr locus comprising a human TTR sequence comprising both TTR coding sequence and non-coding sequence;   (II) changing a variable and performing the method of step (I) a second time with the changed variable in a second non-human animal comprising in its genome the genetically modified endogenous Ttr locus comprising the human TTR sequence comprising both TTR coding sequence and non-coding sequence; and   (III) comparing the activity of the human-TTR-targeting reagent in step (I) with the activity of the human-TTR-targeting reagent in step (II), and selecting the method resulting in the higher activity.   
     
     
         42 .- 51 . (canceled) 
     
     
         52 . A method of making the non-human animal of  claim 1 , comprising:
 (a) introducing into a non-human animal embryonic stem (ES) cell:
 (i) a nuclease agent that targets a target sequence in the endogenous Ttr locus; and 
 (ii) a targeting vector comprising a nucleic acid insert comprising the human TTR sequence flanked by a 5′ homology arm corresponding to a 5′ target sequence in the endogenous Ttr locus and a 3′ homology arm corresponding to a 3′ target sequence in the endogenous Ttr locus, 
   wherein the targeting vector recombines with the endogenous Ttr locus to produce a genetically modified non-human ES cell comprising in its genome the genetically modified endogenous Ttr locus comprising the human TTR sequence;   (b) introducing the genetically modified non-human ES cell into a non-human animal host embryo; and   (c) gestating the non-human animal host embryo in a surrogate mother, wherein the surrogate mother produces an F0 progeny genetically modified non-human animal comprising in its genome the genetically modified endogenous Ttr locus comprising the human TTR sequence.   
     
     
         53 .- 57 . (canceled)