CRISPR/Cas-mediated genome editing to treat EGFR-mutant lung cancer
View Patent ↗The invention relates to a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas guide RNA (gRNA) comprising a targeting domain that is complementary to human genomic Epidermal Growth Factor Receptor (EGFR) DNA, and a vector system including one or more packaged vector(s) including: (a) a first regulatory element operably linked to a gRNA, and (b) a second regulatory element operably linked to a nucleic acid encoding a Cas protein. Also disclosed are methods of altering a nucleic acid sequence encoding EGFR in a cell including contacting the cell with a vector system, methods of treating lung cancer, and methods of selectively inducing apoptosis in a cell including administering a gRNA to the cell.
1. A clustered regularly interspaced short palindromic repeats (CRISPR)/Cas guide RNA (gRNA) comprising a targeting domain that is complementary to human genomic Epidermal Growth Factor Receptor (EGFR) DNA, wherein the targeting domain is configured to:
(a) restore or destroy a wild type sequence at E746-A750 in exon 19,
(b) restore or destroy a threonine residue at position 790 in exon 20, or
(c) restore or destroy a leucine at position 858 in exon 21.
2. The gRNA according to claim 1 , further comprising:
a first complementarity domain;
a linking domain;
a second complementarity domain, which is complementary to the first complementarity domain;
a proximal domain; and
optionally, a tail domain.
3. The gRNA according to claim 1 comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14.
4. The gRNA according to claim 1 comprising a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25.
5. The gRNA according to claim 1 comprising a sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40.
6. A vector system comprising one or more packaged vector(s) comprising:
(a) a first regulatory element operably linked to a sequence encoding a gRNA according to claim 1 , and
(b) a second regulatory element operably linked to a nucleic acid encoding a Cas protein.
7. The vector system according to claim 6 , wherein the Cas protein is a Cas9 protein.
8. The vector system according to claim 7 , wherein the Cas9 protein is selected from the group consisting of Streptococcus pyogenes Cas9, Streptococcus thermophilus Cas9, Staphylococcus aureus Cas9 and Neisseria meningitides Cas9.
9. The vector system according to claim 6 , wherein said one or more packaged vectors is/are selected from the group consisting of retroviruses, lentiviruses, adenovirus, adeno-associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses.
10. The vector system according to claim 6 comprising only a single packaged vector.
11. A method of altering a nucleic acid sequence encoding EGFR in a cell comprising contacting said cell with a vector system according to claim 6 .
12. A method of treating lung cancer in a subject comprising administering a vector system according to claim 6 to said subject.
13. The method according to claim 12 , wherein said lung cancer is a non-small cell lung cancer (NSCLC).
14. A method of selectively inducing apoptosis in a cell comprising administering a gRNA according to claim 1 to said cell.