Compositions and methods of treating head and neck cancer
The present invention provides methods related to the treatment of head and neck squamous cell carcinoma (HNSCC) and its associated premalignant lesions. In particular, the invention features methods which may specifically target HNSCC-associated genes and alter gene expression to treat or alleviate a symptom of HNSCC, or its related premalignant lesions. These methods may involve decreasing the function of an HNSCC-associated gene with aberrant gain-of-function; or increasing the function of an HNSCC-associated gene with aberrant loss-of-function.
1. A method of altering expression of a genomic locus of interest in a mammalian cell,
comprising contacting the genomic locus with a non-naturally occurring or engineered composition comprising a deoxyribonucleic acid (DNA) binding polypeptide comprising:
(a) a N-terminal capping region
(b) a DNA binding domain comprising at least five or more TALE monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest, and
(c) a C-terminal capping region
wherein (a), (b) and (c) are arranged in a predetermined N-terminus to C-terminus orientation,
wherein the polypeptide includes at least one or more effector domains, and
wherein the polypeptide is encoded by and translated from a codon optimized nucleic acid molecule so that the polypeptide preferentially binds to DNA of the genomic locus; and
wherein the genomic locus of interest is associated with TP63, CCND1, CCNE1, YAP1, HRAS, PIK3CA, PIK3CG, NOTCH1, NOTCH 2, NOTCH 3, IRF6, CDKN2A, TP53, CASP8, PTEN, FAT1, RIPK4, EZH1, EZH2, MED1, MLL2, CDH1, FBXW7, PCLO, RIMS2, RB1, NSD1 or NFE2L2.
2. The method according to claim 1 , wherein the at least one or more effector domains is selected from the group consisting of: transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, nuclear-localization signal domains, transcription-protein recruiting domain, cellular uptake activity associated domain, nucleic acid binding domain and antibody presentation domain.
3. The method according to claim 1 , wherein the at least one or more effector domains is a nuclease domain.
4. The method according to claim 3 , wherein the nuclease domain is a non-specific FokI endonuclease catalytic domain.
5. The method according to claim 1 , wherein altering expression of a genomic locus is repressing expression of the genomic locus and wherein the at least one or more effector domains included in the polypeptide is at least one or more repressor domains.
6. The method according to claim 5 , wherein the genomic locus is associated with a gene selected from the group consisting of: TP63, CCND1, CCNE1, YAP1, HRAS, PIK3CA, PIK3CG and NFE2L2.
7. The method according to claim 5 , wherein the polypeptide includes at least one mSin interaction domain (SID) repressor domain.
8. The method according to claim 7 , wherein the polypeptide includes at least four SID repressor domains.
9. The method according to claim 5 , wherein the polypeptide includes a Krüppel-associated box (KRAB) repressor domain or a fragment thereof.
10. The method according to claim 1 , wherein altering expression of a genomic locus is activating expression of the genomic locus and wherein the at least one or more effector domains included in the polypeptide is at least one or more activator domains.
11. The method according to claim 10 , wherein the genomic locus is associated with a gene selected from the group consisting of: NOTCH1, NOTCH 2, NOTCH 3, IRF6, CDKN2A, TP53, CASP8, PTEN, FAT1, RIPK4, EZH1, EZH2, MED1, CDH1, FBXW7, PCLO, RIMS2, RB1 and NSD1.
12. The method according to claim 10 , wherein the polypeptide includes a VP16 activator domain.
13. The method according to claim 10 , wherein the polypeptide includes a VP64 activator domain.
14. The method according to claim 10 , wherein the polypeptide includes a p65 activator domain.
15. The method according to claim 1 wherein the DNA binding domain comprises (X 1-11 -X 12 X 13 -X 14-33 or 34 or 35 ) z ,
wherein X 1-11 is a chain of 11 contiguous amino acids,
wherein X 12 X 13 is a repeat variable diresidue (RVD),
wherein X 14-33 or 34 or 35 is a chain of 21, 22 or 23 contiguous amino acids,
wherein z is at least 5 to 40, and
wherein at least one RVD is selected from the group consisting of NI, HD, NG, NN, KN, RN, NH, NQ, SS, SN, NK, KH, RH, HH, HI, KI, RI, SI, KG, HG, RG, SD, ND, KD, RD, YG, HN, NV, NS, HA, S*, N*, KA, H*, RA, NA, and NC, wherein (*) means that the amino acid at X 13 is absent.
16. The method according to claim 15 , wherein z is at least 10 to 26.
17. The method according to claim 15 , wherein
at least one of X 1-11 is a sequence of 11 contiguous amino acids set forth as amino acids 1-11 in a sequence (X 1-11 -X 14-34 or X 1-11 -X 14-35 ) of FIG. 15 or
at least one of X 14-34 or X 14-35 is a sequence of 21 or 22 contiguous amino acids set forth as amino acids 12-32 or 12-33 in a sequence (X 1-11 -X 14-34 or X 1-11 -X 14-35 ) of FIG. 15 .
18. The method according to claim 15 , wherein the at least one RVD is selected from the group consisting of (a) HH, KH, NH, NK, NQ, RH, RN, SS for recognition of guanine (G); (b) SI for recognition of adenine (A); (c) HG, KG, RG for recognition of thymine (T); (d) RD, SI) for recognition of cytosine (C); (e) NV for recognition of A or G; and (f) H*, HA, KA, N*, NC, NS, RA, S* for recognition of A or T or G or C, wherein (*) means that the amino acid at X 13 is absent.
19. The method according to claim 18 , wherein
the RVD for the recognition of G is RN, NH, RH or KH; or
the RVD for the recognition of A is SI; or
the RVD for the recognition of T is KG or RG; and
the RVD for the recognition of C is SD or RD.
20. The method according to claim 15 , wherein at least one of the following is present
[LTLD] (SEQ ID NO: 1) or [LTLA] (SEQ ID NO: 2) or [LTQV] (SEQ ID NO 3) at X 1-4 , or
[EQHG] (SEQ ID NO: 4) or [RDHG] (SEQ ID NO: 5) at positions X 30-33 or X 31-34 or X 32-35 .
21. The method according to claim 1 wherein
the N-terminal capping region or fragment thereof comprises 147 contiguous amino acids of a wild type N-terminal capping region, or
the C-terminal capping region or fragment thereof comprises 68 contiguous amino acids of a wild type C-terminal capping region, or
the N-terminal capping region or fragment thereof comprises 136 contiguous amino acids of a wild type N-terminal capping region and the C-terminal capping region or fragment thereof comprises 183 contiguous amino acids of a wild type C-terminal capping region.