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. In some aspects, disclosed herein are AAV plasmids for performing said methods
Claims35
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.
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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.
A method of genetically modifying a cell by homologously directed repair comprising
- a) obtaining a ribonucleoprotein (RNP) complex comprising a class 2 CRISPR/Cas endonuclease (Cas9) complexed with a corresponding CRISPR/Cas guide RNA and an AAV vector comprising a plasmid comprising a transgene (such as, for example, a chimeric antigen receptor for a tumor antigen);
- wherein the transgene is flanked by homology arms;
- and wherein the homology arms are 800 bp in length or less;
- and
- b) introducing the transgene and the RNP complex into the cell;
- wherein the transgene is introduced into the cell via infection with the Adeno-associated virus (AAV) into the cell;
- wherein the RNP complex hybridizes to a target sequence within the genomic DNA of the cell and the cell's DNA repair enzymes insert the transgene into the host genome at the target sequence within the genomic DNA of the cell thereby creating a modified cell.
A method of genetically modifying at least one cell by homology directed repair (HDR) comprising:
- (a) introducing into the cell (i) a ribonucleoprotein (RNP) complex comprising a class 2 CRISPR/Cas endonuclease complexed with a CRISPR/Cas guide RNA encoding a target sequence in the cell genome, and (ii) an AAV vector comprising a plasmid comprising a transgene flanked by first and second homology arms each having a length of no more than 800 bp, wherein the transgene encodes a chimeric antigen receptor;
- and
- (b) maintaining the cell for a time and under conditions sufficient for (i) the RNP complex to hybridize to the target sequence and introduce a double-stranded break (DSB) in the target sequence;
- and (ii) the cell to insert the transgene into the cell genome at the target sequence and repair the break by HDR, thereby incorporating the transgene into the cell genome at the site of the DSB.
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Dependencies are inferred from the claim text.