EP-4735610-A1 - METHOD FOR CONTROLLABLE TRANSCRIPTION IN LIVER OR LIVER CANCER

Abstract

Provided is a reliable and convenient approach to achieve precise control of gene expression in the liver in vivo. The methods generally combine a doxycycline-inducible gene expression system with a Sleeping Beauty transposon vector and hydrodynamic injection, enabling efficient integration of a controllable gene expression system into the genome of hepatocytes. The doxycycline-inducible system generally allows for precise and reversible control of gene expression. In some forms, a combination of gene alterations and overexpression of Myc leads to the development of HCC in animals. The method can also use a vector that allows for the inducible expression of a tumor antigen. The method can also induce tumor antigen-specific T-cell immune responses, providing a potent tool for the development of targeted therapies for HCC.

Inventors

• MAK, TAK WAH
• ZHENG, Chunxing
• SNOW, Bryan Edward

Assignees

• The University Of Hong Kong
• Centre for Oncology and Immunology Limited
• University Health Network

Dates

Publication Date

20260506

Application Date

20240627

Claims (20)

1. A composition comprising a nucleic acid transposon vector, wherein the vector comprises: (a) a first expression segment, wherein the first expression segment comprises: (i) a promoter; (ii) a first expression sequence encoding a reverse tetracycline-controlled transactivator (rtTA) protein; (iii) a second sequence encoding a self-cleaving peptide; (iv) a third expression sequence encoding an oncogene; and (b) a second expression segment, wherein the second expression segment comprises a fourth expression sequence encoding a test gene; wherein expression of the first expression sequence is under the control of the promoter, wherein the second sequence and the third expression sequence are co-expressed with the first expression sequence, wherein the second sequence is between the first expression sequence and the third expression sequence, wherein the first expression segment and the second expression segment are in reverse trans orientation in the vector, wherein expression of the fourth expression sequence is the under the control of a tetracycline-responsive element (TRE) , and wherein the rtTA protein induces expression of the fourth expression sequence in the presence of an rtTA inducer.
2. The composition of claim 1, wherein the nucleic acid transposon vector is a sleeping beauty transposon vector.
3. The composition of claim 1 or 2, wherein the promoter is a constitutive promoter.
4. The composition of any one of claims 1-3, wherein the promoter is an EF1αpromoter, SV40 promoter, CMV promoter, UBC promoter, PGK promoter, CAGG promoter, or Alb promoter.
5. The composition of any one of claims 1-4, wherein the rtTA protein is the rtTA-Advanced protein.
6. The composition of any one of claims 1-5, wherein the self-cleaving peptide is any one of P2A, E2A, F2A, or T2A.
7. The composition of any one of claims 1-6, wherein the oncogene is c-Myc, CCND1, c-met, Yap, activated Akt (Akt1) , mutated CTNNB1, TERT, H-Ras V12 , SV40 large T antigen, or Nras (G12V) .
8. The composition of any one of claims 1-7, wherein the test gene is the chicken ovalbumin (OVA) .
9. The composition of any one of claims 1-7, wherein the test gene is a tumor antigen.
10. The composition of any one of claims 1-9, wherein the test gene encodes one or more sgRNAs, shRNAs, or combinations thereof.
11. The composition of claim 10, wherein the expression of the one or more sgRNAs, shRNAs, or combinations thereof is under the control of an H1-202 promoter.
12. A method comprising administering the composition of any one of claims 1-11 and one or more gene deletion effectors into hepatocytes of a non-human subject via hydrodynamic injection.
13. The method of claim 12, wherein, following administration, the vector is integrated into the genome of the hepatocytes.
14. The method of claim 12 or 13, wherein one or more of the gene deletion effectors targets one or more tumor suppressor genes for deletion.
15. The method of claim 14, wherein the tumor suppressor genes comprise one or more of the Trp53 gene, the AXIN1 gene, the APC gene, the ARID1A gene, the ARID2 gene, the KEAP1 gene, and the Pten gene.
16. The method of any one of claims 12-15, wherein the gene deletion effectors comprise one or more CRISPR systems, one or more TALENs, one or more Zinc Finger Nucleases, or combinations thereof.
17. The method of claim 16, wherein the CRISPR systems comprise one or more guide RNAs targeting one or more tumor suppressor genes for deletion.
18. The method of any one of claims 12-17, wherein the non-human subject is a wild-type mouse or gene-modified mouse.
19. The method of any one of claims 12-18, wherein, following administration, liver cancer is induced in the non-human subject, thereby forming a tumor model.
20. The method of claim 19, wherein the liver cancer is hepatocellular carcinoma (HCC) .

Description

METHOD FOR CONTROLLABLE TRANSCRIPTION IN LIVER OR LIVER CANCER FIELD OF THE INVENTION The present invention generally relates to a transcription technology. More specifically, the present invention relates to a controllable transcription in liver or liver cancer. BACKGROUND OF THE INVENTION The liver is a vital organ responsible for a range of essential functions, including metabolism, detoxification, and protein synthesis. Hepatocellular carcinoma (HCC) is the most common primary liver malignancy in humans and the third most common cause of cancer-related deaths worldwide. Liver gene delivery has the potential to treat various liver diseases, including liver cancer, hepatitis, and metabolic disorders, by introducing therapeutic genes into the liver cells. There are several methods of gene delivery to the liver, including viral and non-viral vectors, hydrodynamic injection, and electroporation. A significant limitation of many gene delivery approaches is the inability to precisely control gene expression. The liver is a dynamic organ that undergoes constant changes in response to environmental and physiological cues. Gene expression in the liver is highly regulated and controlled by various factors, including hormones, nutrients, and drugs. Therefore, studying gene function in the liver requires a temporal approach to capture the dynamic changes in gene expression and function over time. Temporal analysis of gene function in the liver can provide valuable insights into the molecular mechanisms of liver diseases, such as liver cancer and hepatitis. By precisely controlling the timing and duration of gene expression, gene therapy can be made more effective and safer. The use of a tetracycline-inducible gene expression is a popular choice for this purpose. The current in vivo applications of tetracycline-inducible gene expression systems are mainly focused on generating transgenic mice, transplanting in vitro modified cells, and viral transduction. There remains a need for developing convenient, efficient, liver-specific, and non-viral approach that can be applied to both wild-type and gene-modified mice without the need to generate transgenic mice or perform complicated crossbreeding. There is also a need for reliable and convenient approach for achieving precise control of gene expression in the liver in vivo. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application. Throughout this specification the word “comprise, ” or variations such as “comprises” or “comprising, ” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. SUMMARY OF THE INVENTION Disclosed are methods for achieving precise control of gene expression in liver cancer or liver in vivo, as well as composition resulting therefrom. The methods include utilizing a combination of a transposon gene delivery vector, a gene deleting tool (such as CRISPR) , and a hydrodynamic injection technique. The methods generally involve incorporation of a doxycycline-inducible gene expression system into the transposon gene delivery vector. The methods generally can be used to induce liver cancer in vivo and to achieve controlled and precise gene expression in liver cancer. Disclosed are compositions and methods for creating and using non-human models of liver cancer. Disclosed are compositions containing a nucleic acid transposon vector. Generally, the vector includes three or four sequences disposed in one or two expression segments. In some forms, the first expression sequence encodes a reverse tetracycline-controlled transactivator (rtTA) protein. In some forms, the second expression sequence encodes a self-cleaving peptide. In some forms, the third expression sequence encodes an oncogene. In some forms, the fourth expression sequence encodes a test gene. In some forms, the first expression segment includes a promoter. In some forms, the first expression segment includes the first expression sequence. In some forms, the first expression segment includes the second expression sequence. In some forms, the first expression segment includes the third expression sequence. In some forms, the first expression segment includes a promoter, the first expression sequence, the second expression sequence, and the third expression sequence. In some forms, the second expression segment includes the fourth expression sequence. In some forms, the first expression sequence is under the control of the promoter. In some forms, the second expression sequence and the third expression sequence are