US2021388362A1PendingUtilityA1

Targeted non-viral dna insertions

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Assignee: UNIV CALIFORNIAPriority: Jun 15, 2017Filed: May 20, 2021Published: Dec 16, 2021
Est. expiryJun 15, 2037(~10.9 yrs left)· nominal 20-yr term from priority
Inventors:Theodore Lee RothAlexander Marson
C12N 15/1138A61K 48/0016C12N 15/102C12N 2320/53C12N 15/90C12N 2310/20C12N 9/22C07K 14/7051C12N 15/907C12N 15/113C12N 9/226C12N 5/0636
74
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Claims

Abstract

Provided herein are methods and compositions for editing the genome of a cell. In some embodiments, a nucleotide sequence of at least 200 nucleotides in length is inserted into a target region in the genome of a cell.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A primary immune cell comprising at least one DNA template non-virally inserted into a target region of the genome of the cell, wherein the size of the DNA template is greater than or equal to about 200 base pairs (bp). 
     
     
         2 . The cell of  claim 1 , wherein the primary immune cell does not comprise a viral vector for introducing the DNA template into the primary immune cell. 
     
     
         3 . The cell of  claim 1 , wherein the size of the DNA template is greater than or equal to 1 kilobase (kb). 
     
     
         4 . The cell of  claim 1 , wherein the size of the DNA template is greater than or equal to about 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850 bp, 900 bp, 1 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9 kb, 2.0 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, 3.8 kb, 3.9 kb, 4.0 kb, 4.1 kb, 4.2 kb, 4.3 kb, 4.4 kb, 4.5 kb, 4.6 kb, 4.7 kb, 4.8 kb, 4.9 kb, 5.0 kb or any size of DNA template in between these sizes. 
     
     
         5 . The cell of  claim 1 , wherein the size of the DNA template is about 200 bp to about 500 bp, about 200 bp to about 750 bp, about 200 bp to about 1 kb, about 200 bp to about 1.5 kb, about 200 bp to about 2.0 kb, about 200 bp to about 2.5 kb, about 200 bp to about 3.0 kb, about 200 bp to about 3.5 kb, about 200 bp to about 4.0 kb, about 200 bp to about 4.5 kb, or about 200 bp to about 5.0 kb. 
     
     
         6 . The cell of  claim 1 , wherein the DNA template is a double-stranded DNA template or a single-stranded DNA template. 
     
     
         7 . The cell of  claim 1 , wherein the DNA template is a linear DNA template or a circular DNA template, optionally wherein the circular DNA template is a plasmid. 
     
     
         8 . The cell of  claim 1 , wherein the primary immune cell is a primary human immune cell. 
     
     
         9 . The cell of  claim 1 , wherein the primary immune cell is a primary T cell. 
     
     
         10 . The cell of  claim 1 , wherein the primary immune cell is a primary human T cell. 
     
     
         11 . The cell of  claim 1 , wherein the DNA template comprises a heterologous sequence. 
     
     
         12 . The cell of  claim 1 , wherein the DNA template comprises a gene. 
     
     
         13 . The cell of  claim 1 , wherein the DNA template comprises a chimeric antigen receptor (CAR). 
     
     
         14 . The cell of  claim 1 , wherein the primary immune cell is virus-free. 
     
     
         15 . A population of cells comprising a plurality of the primary immune cell of  claim 1 . 
     
     
         16 . A primary immune cell comprising at least one DNA template inserted into a target region of the genome of the primary immune cell, wherein the size of the DNA template is greater than or equal to 200 base pairs, and wherein the primary immune cell does not comprise a viral vector for introducing the DNA template into the primary immune cell. 
     
     
         17 . A viable, virus-free, primary cell comprising a ribonucleoprotein complex (RNP)-DNA template complex, wherein the RNP comprises a nuclease domain and a guide RNA, wherein the size of the DNA template is greater than or equal to 200 nucleotides in size, and wherein the 5′ and 3′ ends of the DNA template comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the primary cell. 
     
     
         18 . A method of treating a disease in a subject comprising administering the primary immune cell of  claim 1  to the subject. 
     
     
         19 . A method of editing a primary immune cell, comprising:
 a. providing a ribonucleoprotein complex (RNP)-DNA template complex, wherein the RNP comprises a nuclease domain and a guide RNA, wherein the size of the DNA template is greater than or equal to 200 nucleotides in size, and wherein the 5′ and 3′ ends of the DNA template comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the primary immune cell;   b. non-virally introducing the RNP-DNA template complex into the primary immune cell, wherein the guide RNA specifically hybridizes to a target region of the genome of the primary immune cell, and wherein the nuclease domain cleaves the target region to create the insertion site in the genome of the primary immune cell; and   c. editing the primary immune cell via insertion of the DNA template into the insertion site in the genome of the primary immune cell.   
     
     
         20 . The method of  claim 19 , wherein non-virally introducing comprises electroporation.

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