Nuclease-mediated repeat expansion

Abstract

Nuclease-mediated methods for expanding repeats already present at a genomic locus are provided. Non-human animal genomes, non-human animal cells, and non-human animals comprising a heterologous hexanucleotide repeat expansion sequence inserted at an endogenous C9orf72 locus and methods of making such non-human animal cells and non-human animals through nuclease-mediated repeat expansion are also provided. Methods of using the non-human animal cells or non-human animals to identify therapeutic candidates that may be used to prevent, delay or treat one or more neurodegenerative disorders associated with repeat expansion at the C9orf72 locus are also provided.

We claim: 
     
         1 . A non-human animal or non-human animal cell comprising in its genome a heterologous hexanucleotide repeat expansion sequence inserted at an endogenous C9orf72 locus, wherein the heterologous hexanucleotide repeat expansion sequence comprises more than about 100 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1. 
     
     
         2 . The non-human animal or non-human animal cell of  claim 1 , wherein the heterologous hexanucleotide repeat expansion sequence comprises at least about 300 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1. 
     
     
         3 . The non-human animal or non-human animal cell of  claim 2 , wherein the hexanucleotide repeat expansion comprises at least about 500 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1. 
     
     
         4 . The non-human animal or non-human animal cell of  claim 3 , wherein the hexanucleotide repeat expansion comprises at least about 600 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1. 
     
     
         5 . The non-human animal or non-human animal cell of  claim 1 , wherein the repeats are contiguous in the heterologous repeat expansion sequence. 
     
     
         6 . The non-human animal or non-human animal cell of  claim 1 , wherein the heterologous hexanucleotide repeat expansion sequence is located between the first non-coding endogenous exon and exon 2 of the endogenous C9orf72 locus. 
     
     
         7 . The non-human animal or non-human animal cell of  claim 1 , wherein the endogenous C9orf72 locus comprises a human C9ORF72 nucleotide sequence. 
     
     
         8 . The non-human animal or non-human animal cell of  claim 7 , wherein the human C9ORF72 nucleotide sequence comprises SEQ ID NO: 46 and/or SEQ ID NO: 47. 
     
     
         9 . The non-human animal or non-human animal cell of  claim 1 , wherein the non-human animal is a rodent or the non-human animal cell is a rodent cell. 
     
     
         10 . The non-human animal or non-human animal cell of  claim 9 , wherein the rodent is a rat or a mouse or wherein the non-human animal cell is a rat cell or a mouse cell. 
     
     
         11 . The non-human animal or non-human animal cell of  claim 1 , wherein the non-human animal or non-human animal cell is heterozygous for the heterologous hexanucleotide repeat expansion sequence. 
     
     
         12 . The non-human animal or non-human animal cell of  claim 1 , wherein the non-human animal or non-human animal cell exhibits:
 (a) increased expression of C9orf72 transcripts that retain intron sequence compared to a control non-human animal or control non-human animal cell comprising a wild type C9orf72 locus; and/or   (b) an increased number of RNA foci compared to a control non-human animal or control non-human animal cell comprising a wild type C9orf72 locus; and/or   (c) an increased level of dipeptide repeat proteins compared to a control non-human animal or control non-human animal cell comprising a wild type C9orf72 locus.   
     
     
         13 . The non-human animal cell of  claim 1 , wherein the non-human animal cell is an embryonic stem cell, an embryonic stem-cell-derived motor neuron, a brain cell, a cortical cell, a neuronal cell, a muscle cell, a heart cell, or a germ cell. 
     
     
         14 . The non-human animal cell of  claim 1 , wherein the non-human animal cell is a one-cell stage embryo. 
     
     
         15 . The non-human animal cell of  claim 1 , wherein the non-human animal cell is in vitro. 
     
     
         16 . The non-human animal cell of  claim 1 , wherein the non-human animal cell is in vivo. 
     
     
         17 . The non-human animal of  claim 1 , wherein the non-human animal comprises in its germline genome the heterologous hexanucleotide repeat expansion sequence inserted at the endogenous C9orf72 locus. 
     
     
         18 . A method for assessing a therapeutic candidate for the treatment of a disease or a condition associated with a hexanucleotide repeat expansion sequence at a C9orf72 locus, comprising:
 (a) administering a candidate agent to the non-human animal or non-human animal cell of  claim 1 ;   (b) performing one or more assays to determine if the candidate agent has an effect on one or more signs or symptoms associated with the disease or condition; and   (c) identifying the candidate agent that has an effect on the one or more signs or symptoms associated with the disease or condition as a therapeutic candidate.   
     
     
         19 . The method of  claim 18 , wherein the candidate agent is administered in vivo to the non-human animal, and the one or more assays are performed in vitro in cells isolated from the non-human animal after administration of the candidate agent. 
     
     
         20 . The method of  claim 18 , wherein the candidate agent is administered in vitro to a non-human embryonic-stem-cell-derived motor neuron. 
     
     
         21 . The method of  claim 18 , wherein the one or more assays comprise quantitative polymerase chain reaction (qPCR) to detect intron-containing C9orf72 RNA transcripts. 
     
     
         22 . The method of  claim 18 , wherein the one or more assays comprise measuring RNA foci comprising a C9orf72 sense or antisense RNA transcript, optionally wherein the RNA foci are measured by fluorescence in situ hybridization. 
     
     
         23 . The method of  claim 18 , wherein the one or more assays comprise measuring the accumulation of dipeptide repeat proteins, optionally wherein the dipeptide repeat proteins are polyGA dipeptide repeat proteins or polyGP dipeptide repeat proteins, and optionally wherein the accumulation of dipeptide repeat proteins is measured by immunohistochemistry.