Delivery methods and compositions for nuclease-mediated genome engineering
The present disclosure is in the field of genome engineering, particularly targeted modification of the genome of a cell.
1. A method of introducing a donor nucleic acid molecule into an isolated cell, the method comprising:
administering to the cell at least one adeno-associated virus (AAV) vector comprising a donor nucleic acid molecule in the presence of at least one inhibitor of Platelet-derived growth factor receptor under conditions sufficient to increase efficiency of viral delivery to the cell, wherein the AAV vector has an AAV6 capsid.
2. The method of claim 1 , wherein the donor molecule comprises a transgene that is expressed in the cell.
3. The method of claim 2 , wherein the transgene is integrated into the genome of the cell.
4. The method of claim 3 , wherein the transgene encodes a chimeric antigen receptor (CAR).
5. The method of claim 3 , further comprising introducing at least one nuclease into the cell, wherein the transgene is integrated into one or more genes of the cell following cleavage of the one or more genes by the nuclease.
6. The method of claim 5 , wherein the nuclease cleaves a programmed cell death 1 (PD1) gene, a Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) gene, a beta 2-microglobulin (B2M) and/or a T-cell receptor alpha (TRAC) gene.
7. The method of claim 6 , wherein the transgene encodes a CAR.
8. The method of claim 7 , wherein the CAR is integrated into a PD1, CTLA-4 or TRAC gene following cleavage of the gene by a nuclease.
9. The method of claim 1 , wherein the cell is a hematopoietic stem cell, a T-cell, a B-cell or an NK cell.
10. A method of treating a cancer in subject,
the method comprising introducing a donor nucleic acid molecule into a cell according to the method of claim 1 , wherein donor nucleic acid molecule comprises a sequence encoding a CAR such that the cell expresses the CAR; and
administering the cell to the subject.
11. The method of claim 1 , wherein the AAV vector comprises an AAV2 inverted terminal repeat (ITR).
12. A method of introducing a donor nucleic acid molecule into an isolated cell, the method comprising:
administering to the cell at least one adeno-associated virus (AAV) vector comprising a donor nucleic acid molecule in the presence of at least one inhibitor of Platelet-derived growth factor receptor under conditions sufficient to increase efficiency of viral delivery to the cell, wherein the inhibitor is selected from the group consisting of CP-673451 and Crenolanib and wherein the AAV vector has an AAV6 capsid.
13. The method of claim 12 , wherein the AAV vector comprises an AAV2 inverted terminal repeat (ITR).