Compositions and methods for targeting cancer-specific sequence variations
The present invention relates to compositions and methods for targeting cancer-specific DNA sequences, such as copy number amplifications and other types of cancer-specific sequence variations, such as cancer-specific polymorphisms, insertions, or deletions. The present invention provides hereto sequence-specific DNA targeting agents targeting a sequence within the amplified DNA region or a sequence otherwise specific for a cancer cell compared to a non-cancer cell. The invention further relates to methods for treating cancer, comprising administering such sequence-specific DNA targeting agents. The invention further relates to methods for preparing sequence-specific DNA targeting agent, as well as screening methods using the DNA targeting agents.
1. A method for preparing a sequence-specific DNA damaging agent suitable for the treatment of an epithelial cell cancer of a human subject, said method comprising identifying a cancer cell-specific copy number amplification of said epithelial cell cancer in genomic DNA of the human subject, wherein the cancer cell-specific copy number amplification comprises a non-coding, non-gene regulatory, intergenic region, and producing a sequence-specific DNA damaging agent, wherein said sequence-specific DNA damaging agent is a CRISPR-Cas system nuclease comprising a guide nucleic acid sequence that is a perfect match with a nucleic acid sequence of the non-coding, non-gene regulatory, intergenic region across a span of at least twenty nucleotides in length within said cancer cell-specific genomic DNA copy number amplification of the human subject and thereby targets the non-coding, non-gene regulatory, intergenic region within said cancer cell-specific genomic DNA copy number amplification of the human subject for damage, wherein the guide nucleic acid sequence is not a perfect match with any gene or gene expression regulatory sequence in the genome of the human subject.
2. The method according to claim 1 , which comprises identifying said cancer cell-specific copy number amplification by sequencing a sample of said epithelial cell cancer.
3. The method according to claim 1 , wherein said sequence-specific DNA damaging agent is a patient-specific DNA damaging agent, and said cancer cell-specific copy number amplification is identified based on sequencing a sample of said patient.
4. The method according to claim 1 , wherein said cancer cell-specific copy number amplification is not present in a normal cell or a non-cancerous cell.
5. The method according to claim 1 wherein the copy number amplification comprises DNA copy number amplifications of 1p22-p31, 1p32-p36, 1q, 2p13-p16, 2p23-p25, 2q31-q33, 3q, 5p, 6p12-pter, ′7p12-p13, 7q11.2, 7q21-q22, 8p11-p12, 8q, 11q13-q14, 12p, 12q13-q21, 13q14, 13q22-qter, 14q13-q21, 15q24-qter, 17p11.2-p12, 17q12-q21, 17q22-qter, 18q, 19p13.2-pter, 19cen-q13.3, 20p11.2-p12, 20q, Xp11.2-p21, or Xp11-q13.
6. The method according to claim 1 , wherein the CRISPR-Cas system nuclease is a nickase.
7. The method according to claim 1 , wherein the CRISPR-Cas system nuclease comprises one or more mutations.
8. The method according to claim 7 , wherein the CRISPR-Cas system nuclease comprises one or more mutations selected from D10A, E762A, H840A, N854A, N863A or D986A with reference to the position numbering of a Streptococcus pyogenes Cas9 (SpCas9) nuclease.
9. The method according to claim 7 , wherein the one or more mutations is in a RuvC1 domain of the CRISPR-Cas system nuclease.
10. The method according to claim 1 , wherein the CRISPR-Cas system nuclease cleaves the target sequence.
11. The method according to claim 1 , wherein the CRISPR-Cas system nuclease is a dead Cas conjugated to a DNA damaging agent.
12. The method according to claim 1 , wherein the epithelial cell cancer is selected from the group consisting of Non-Hodgkin's Lymphoma (NHL), clear cell Renal Cell Carcinoma (ccRCC), melanoma, sarcoma, leukemia, a cancer of the bladder, colon, rectum, brain, breast, head and neck, endometrium, lung, uterus, ovary, peritoneum, fallopian tubes, pancreas, esophagus, stomach, small intestine, liver, gall bladder, bile ducts and prostate cancer.