Granted Patent
B2
US 12,234,461 · App. 18/463,589 · Granted Feb 25, 2025
Targeted non-viral DNA insertions
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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 (24)
1. An isolated viable, primary human immune cell comprising a complex comprising a ribonucleoprotein complex (RNP) and a DNA template, wherein the RNP comprises a nuclease domain and a guide RNA, wherein the size of the DNA template is greater than or equal to 300 base pairs (bp) 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, and wherein the primary cell does not comprise a viral vector for delivery of the DNA template.
2. The cell of claim 1 , wherein the size of the DNA template is greater than or equal to 1 kilobase (kb).
3. The cell of claim 1 , wherein the size of the DNA template is greater than or equal to 1.5 kb.
4. The cell of claim 1 , wherein the size of the DNA template is 300 bp to 5 kb.
5. The cell of claim 1 , wherein the DNA template is a double-stranded DNA template.
6. The cell of claim 1 , wherein the DNA template is a linear DNA template.
7. The cell of claim 1 , wherein the primary human immune cell is a primary human T cell.
8. The cell of claim 1 , wherein the DNA template comprises a gene.
9. The cell of claim 1 , wherein the DNA template encodes a chimeric antigen receptor (CAR).
10. An isolated population of cells, wherein at least 10% of the cells in the population are a cell of claim 1 .
11. A method of editing a primary human immune cell, comprising:
a. providing a complex comprising a ribonucleoprotein (RNP) and a DNA template, wherein the RNP comprises a nuclease domain and a guide RNA, wherein the size of the DNA template is greater than or equal to 300 bp 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 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.
12. The method of claim 11 , wherein non-virally introducing comprises electroporation.
13. The method of claim 11 , wherein the size of the DNA template is greater than or equal to 1 kb.
14. The method of claim 11 , wherein the size of the DNA template is greater than or equal to 1.5 kb.
15. The method of claim 11 , wherein the size of the DNA template is 300 bp to 5 kb.
16. The method of claim 11 , wherein the DNA template is a double-stranded DNA template.
17. The method of claim 11 , wherein the DNA template is a linear DNA template.
18. The method of claim 11 , wherein the primary human immune cell is a primary human T cell.
19. The method of claim 11 , wherein the DNA template comprises a gene.
20. The method of claim 11 , wherein the DNA template encodes a chimeric antigen receptor (CAR).
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Apr 2, 2024
From: ROTH, THEODORE LEE; MARSON, ALEXANDER
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, A CALIFORNIA CORPORATION
Reel/Frame 066984/0691 →
Continuity (5)
Continuation
16622843
Provisional Application
62552180
· Aug 30, 2017
Provisional Application
62520117
· Jun 15, 2017
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