Genetically Modified Cell Lines Including a TP53 Modification and Methods of Use

• US 20190309272A1
• Filed: 04/08/2019
• Published: 10/10/2019
• Est. Priority Date: 04/09/2018
• Status: Active Application

• Current Assignee: University of South Carolina
• Original Assignee: University of South Carolina
• Inventors: BANISTER, CAROLYN E., WARRINGTON, JOHN, LIU, CHANGLONG, BUCKHAULTS, PHILLIP
• Application Number:
  US16/377,960
• Publication Number:
  US 20190309272A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1024 In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Y 207/01037 Protein kinase (2.7.1.37) C12Y207/11001, C12Y207/11008 - C12Y207/11013, C12Y207/11021, C12Y207/11022, C12Y207/11024, C12Y207/11025, C12Y207/1103, C12Y207/12001 take precedence
• CPC Class Codes: G01N 2333/4748 p53
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity

Abstract

The present disclosure is directed to genetically engineered cell lines which include a modification to knockout a portion of the TP53 gene. Embodiments disclosed herein provide aspects of the knockout cell lines, methods for producing the knockout cell lines, in vitro assays using the knockout cell lines, and kits including the knockout cell lines. In certain implementations, the embodiments can provide doctors and patients improved tools for determining a treatment or for comparing treatments for patients having tumors that include a TP53 mutation.

Claims

1. A knockout cell line, wherein each cell of the knockout cell line comprises a MCF7 breast cancer cell having a deletion of at least one coding region in a gene, and wherein the gene includes a TP53 gene having a nucleotide sequence corresponding to Seq. ID No. 1.

2. The knockout cell line of claim 1, wherein the coding region comprises a part of an exon.

3. The knockout cell line of claim 1, wherein the coding region comprises one or more of exons 1-11.

4. The knockout cell line of claim 3, wherein each of exons on 1-11 include an nucleotide sequence from seq. ID No. 1, and wherein the nucleotide sequence corresponds to a range of base numbers for each exon selected from the group:

the nucleotide sequence for exon 1 comprises base numbers 1-162, the nucleotide sequence for exon 2 comprises base numbers 10917-11018, the nucleotide sequence for exon 3 comprises base numbers 11136-11157, the nucleotide sequence for exon 4 comprises base numbers 11267-11545, the nucleotide sequence for exon 5 comprises base numbers 12303-12486, the nucleotide sequence for exon 6 comprises base numbers 12568-12680, the nucleotide sequence for exon 7 comprises base numbers 13249-13358, the nucleotide sequence for exon 8 comprises base numbers 13702-13838. the nucleotide sequence for exon 9 comprises base numbers 13931-14004, the nucleotide sequence for exon 10 comprises base numbers 16824-16930, the nucleotide sequence for exon 11 comprises base numbers 17849-19137.

5. The knockout cell line of claim 4, wherein the MCF7 breast cancer cell includes a deletion of the nucleotide sequence for exon 4, and wherein the nucleotide sequence for exon 4 comprises:

6. The knockout cell line of claim 4, wherein the MCF7 breast cancer cell includes a deletion of the nucleotide sequence for exon 10, and wherein the nucleotide sequence for exon 10 comprises:

7. An in vitro assay for determining efficacy of a treatment in breast cancer cells that include a TP53 gene mutation, comprising:

providing the treatment to a plurality of cells having a deletion of at least one coding region in the TP53 gene, and

measuring a result.

8. The in vitro assay of claim 7, further comprising:

providing the treatment to a plurality of wild type MCF7 breast cancer cells;

measuring a wild type result; and

comparing the wild type result to the result.

9. The in vitro assay of claim 8, wherein comparing the wild type result to the result comprises:

determining a first quantitative measurement describing the number of live wild type MCF7 breast cancer cells included in the plurality of wild type MCF7 breast cancer cells to which the treatment was provided;

determining a second quantitative measurement describing the number of live cells included in the plurality of cells having a deletion of at least one coding region in the TP53 gene to which the treatment was provided, and wherein the treatment provided to the wild type MCF7 breast cancer cells and the treatment provided to the plurality of cells having the deletion of at least one coding region in the TP53 gene are the same.

10. The in vitro assay of claim 7, wherein measuring the result comprises determining a quantitative measure of cell death.

11. The in vitro assay of claim 7, wherein providing the treatment comprises administering a drug to the plurality of cells derived from the knockout cell line.

12. The in vitro assay of claim 11, wherein the drug comprises one or more of the drugs from the group consisting of:

Abiraterone, Afatanib, Alectinib, Allopurinol, Altretamine, Amifostine, Aminolevulinic Acid, Anastrozole, Arsenic Trioxide, Axitinib, Azactidine, Belinostat, Bendamustine Hydrochloride, Bleomycin Sulfate, Bortezomib, Bosutinib, Busulfan, Cabazitaxel, Cabozantinib, Capecitabine, Carboplatin, Carfilzomib, Carmustine, Celecoxib, Ceritinib, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Clyclopamine, Cobimetinib, Crizotinib, Cyclophosphamide, Cytarabine Hydrochloride, Dabrafenib Mesylate, Dacarbazine, Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Decitabine, Dexrazoxane, DMSO, Docetaxel, Doxorubicin Hydrochloride, Enzalutamide, Epirubicin Hydrochloride, Erismodgib, Erlotinib Hydrochloride, Estramustine Phosphate Sodium, Etoposide, Everolimus, Exemestane, Floxuridine, Fludarabine Phosphate, Fluorouracil, Fulvestrant, Gefintinib, Gemcitabine Hydrochloride, Hydroxyurea, Ibrutinib, Idarubicin Hydrochloride, Idelalisib, Ifosfamide, Imatinib, Imiquimod, Irinotecan Hydrochloride, Ixabepilone, Ixazomib, Laptinib, Lenalidomide, Letrozole, Lomustine, Mechlorethamine Hydrochloride, Megestrol Acetate, Melphalan Hydrochloride, Mercaptopurine, Methotrexate, Methoxsalen, Mitomycin, Mitotane, Mitoxantrone, Nelarabine, Nilotinib, Nutlin3, Olaparib, Omacetaxine Mepesuccinate, Osimertinib, Oxaliplatin, Paclitaxel, Palbociclib, Panobinostat, Pazopanib Hydrochloride, Pemetrexed Disodium Salt Heptahydrate, Pentostatin, Pipobroman, Plerixafor, Plicamycin, Pomalidomide, Ponatinib, Pralatrexate, Procarbazine Hydrochloride, Raloxifene, Regorafenib, Rom idepsin, SenexinB, Sirolimus, Sorafenib, Streptozocin, Sunitinib, Tamoxifen Citrate, Temozolomide, Temsirolimus, Teniposide, Thalidomide, Thioguanine, Thiotepa, Topotecan Hydrochloride, Trametinib, Tretinoin, Triethylenemelamine, Trifluridine, Uracil Mustard, Valrubicin, Vandetanib, Vemurafenib, Vinblastine Sulfate, Vincristine Sulfate, Vinorelbine Tartrate, Vismodegib, Vorinostat, Zoledronic Acid 2.

13. A method for producing a knockout cell line from a wild type cell line, the method comprising deleting a portion of the TP53 gene in a cell derived from the wild type cell line, wherein deleting the portion of the TP53 gene comprises delivering a guide RNA to the cell.

14. The method of claim 13, wherein the wild type cell line comprises human MCF7 breast cancer cells.

15. The method of claim 13, wherein the portion of the TP53 gene comprises at least one coding region included in exons 4-10.

16. The method of claim 13, wherein delivering the guide RNA to the cell includes delivering an expression cassette to the cell, wherein the expression cassette includes a DNA sequence for expressing the guide RNA.

17. The method of claim 16, wherein delivering the guide RNA to the cell further includes delivering a second expression cassette to the cell, wherein the second expression cassette includes a DNA sequence for expressing Cas9.

18. The method of claim 13, further comprising selecting for a genetically modified cell, wherein selecting for the genetically modified cell comprises culturing the cell in the presence of an agent.

19. The method of claim 18, wherein the agent comprises Nutlin3.

20. The method of claim 13, wherein the guide RNA comprises one or more of the sequences: