Generation of chimeric antigen receptor (car)-primary nk cells for cancer immunotherapy using a combination of cas9/rnp and aav viruses
AssigneeResearch Institute At Nationwide Children's Hospital
InventorsDean Anthony LEE, Kathrin Christine MEYER, Meisam Naeimi KARAROUDI, Shibi LIKHITE
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Abstract
Disclosed are methods for genetically engineering cells using Adeno-associated viral (AAV) delivery of a CRISPR/CAS9 system and ribonucleoproteins for integration. Said methods include a DNA-free technique for the genome editing of primary and expanded human cells (including NK cells) utilizing Cas9 ribonucleoprotein complexes (Cas9/RNPs). In some aspects, disclosed herein are AAV plasmids for performing said methods.
Claims20
A plasmid for use with clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) integration systems wherein the plasmid comprises in order a left homology arm, a splice acceptor, a transgene, and a right homology arm;
- wherein the left and right homology arms are each 800 bp in length or less.
5-7. (canceled)
A plasmid for use in CRISPR/Cas9 integration methods consisting of 1 or 2 protospacer adjacent motifs (PAMs) and CRISPR RNAs (crRNAs) and a transgene;
- wherein the order of the encoded nucleic acid comprises a PAM, a crRNA and a transgene;
- and wherein when 2 PAMs and crRNAs are used, of the encoded nucleic acid comprises a first PAM, a first crRNA, a transgene, a second PAM, and second gRNA.
20-35. (canceled)
Dependencies are inferred from the claim text.