Methods and compositions for treatment of a genetic condition
Methods and compositions comprising a CRISPR/Cas nuclease that cleaves a T cell receptor (TCR) gene and/or human leukocyte (HLA) gene, the nuclease comprising a cleavage domain and a single guide RNA that binds to a target site in the TCR and/or HLA gene.
1. A CRISPR/Cas nuclease comprising a cleavage domain and a single guide RNA (sgRNA), wherein the sgRNA binds to a target site as shown in any of SEQ ID NO:96-108 in a T cell receptor (TCR) gene or a target site as shown in any of SEQ ID NO:118-148 in a human leukocyte (HLA) gene or HLA accessory gene; wherein the nuclease cleaves and inactivates the TCR and/or HLA gene.
2. The nuclease of claim 1 , wherein the TCR gene is a T-cell receptor alpha (TRAC) gene or a T-cell receptor beta (TRBC) gene.
3. The nuclease of claim 1 , wherein the HLA gene is an HLA-A gene, an HLA B gene, an HLA C gene, an HLA-DPA gene, an HLA-DQ gene, an HLA-DRA gene and/or a beta-2 microglobulin gene.
4. The nuclease of claim 1 , wherein the cleavage domain comprises a Type IIS restriction enzyme nuclease domain or a Cas protein.
5. A mammalian cell comprising the nuclease of claim 1 .
6. The mammalian cell of claim 5 , wherein the mammalian cell is a stem cell.
7. The mammalian cell of claim 6 , wherein the stem cell is a hematopoietic stem cell.
8. The mammalian cell of claim 5 , further comprising a transgene encoding a chimeric antigen receptor (CAR).
9. The mammalian cell of claim 5 , further wherein one or more additional genes are inactivated in the cell.
10. The mammalian cell of claim 9 , wherein the one or more additional inactivated genes comprise a PD1 gene, a CTLA-4 gene, a Tap1 gene, a Tap2 gene, a tapasin gene, a CIITA gene and/or an RFX5 gene.
11. A method of inactivating an endogenous TCR and/or HLA gene in a cell, the method comprising the steps of:
administering to the cell one or more CRISPR/Cas nucleases according to claim 1 , wherein the nucleases cleave and inactivate the TCR and/or HLA genes.
12. The method of claim 11 , comprising a Type IIS restriction enzyme nuclease domain or a Cas protein.
13. The method of claim 11 , wherein the cell is a mammalian cell.
14. The method of claim 13 , wherein the mammalian cell is a stem cell.
15. The method of claim 14 , wherein the stem cell is a hematopoietic stem cell.
16. The method of claim 11 , further comprising integrating one or more CAR transgenes into the cell.
17. A cell made by method of claim 11 .
18. A method of treating a disorder in a subject in need thereof, the method comprising
administering to the subject one or more CRISPR/Cas nucleases according to claim 1 , wherein the nucleases cleave and inactivate the TCR and/or HLA genes in a cell of the subject, such that the disorder is treated.
19. The method of claim 18 , wherein the disorder is a cancer.
20. The method of claim 18 , wherein the cell is stem cell.
21. The method of claim 20 , wherein the stem cell is a hematopoietic stem cell.