CRISPR-Cas9 and Transposon System for Recurrent HCC Models
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Solution Overview
Problem
Current research lacks effective animal models for recurrent hepatocellular carcinoma (HCC), hindering the development of therapeutic strategies and the understanding of molecular mechanisms driving HCC recurrence.
Innovation Solution
A composition including a CRISPR-Cas expression vector, a transposon expression vector, and a transposase expression vector is used to generate animal models of recurrent HCC. This system modifies liver cells to inhibit Tp53 expression and increase c-Myc oncogene expression, leading to the development of focal HCC tumors that can recur after surgical resection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If orthotopic implantation of human HCC cells in immune-deficient mice is used to generate focal HCC tumors, then the tumor formation is achieved, but the model lacks an intact immune system which limits its translational value
Solution Approach 1:
The invention segments the model generation approach by using CRISPR-Cas9 to modify specific genes (Tp53 knockout and c-Myc overexpression) in the liver of immune-competent mice, rather than implanting cells from immune-deficient mice. This allows the immune system to remain intact while still achieving reliable HCC tumor formation through genetic manipulation.
Solution Approach 2:
The invention replaces the mechanical cell implantation method with a genetic engineering approach using CRISPR-Cas9. Instead of physically implanting tumor cells, the system uses targeted gene editing to induce endogenous tumor development, thereby maintaining immune system functionality while achieving tumor formation.
2Adaptability or versatility
If syngeneic mouse HCC cell lines are used after orthotopic implantation in immune competent mice, then the immune system remains intact, but the tumors regress and fail to develop recurrent tumors
Solution Approach 1:
The invention changes critical parameters of the HCC model by implementing specific genetic modifications: Tp53 knockout and c-Myc overexpression. These parameter changes in the genetic makeup of the liver cells enable sustained tumor growth and recurrence capability in immune-competent mice, overcoming the regression issue seen with unmodified syngeneic cell lines.
Solution Approach 2:
The invention performs preliminary genetic modification of the liver cells before tumor development occurs. By pre-knocking out Tp53 and pre-overexpressing c-Myc in the liver, the system ensures that when tumors develop, they possess the necessary genetic characteristics for sustained growth and recurrence from the outset, rather than attempting to modify them later.
3Adaptability or versatility
If liver surgery is performed in small animals to create recurrent HCC models, then clinical translatability is improved, but the procedure becomes complicated and technically challenging
Solution Approach 1:
The invention enables the liver to serve itself by using CRISPR-Cas9 delivered in vivo to modify liver cells directly within the animal. This self-service approach eliminates the need for complex surgical procedures to implant cells or perform resections, as the genetic modification and subsequent tumor development occur naturally within the living animal system.
Solution Approach 2:
The invention introduces CRISPR-Cas9 as an intermediary that mediates the complex process of HCC model generation. Instead of requiring complex surgical manipulation, the CRISPR-Cas9 system delivers the genetic modification function, simplifying the overall procedure while maintaining clinical translatability through the use of immune-competent mice that undergo minimal intervention.
Data Source
AI summary
Compositions including recurrent hepatocellular carcinoma (HCC) model generating systems are disclosed. The HCC model generating system contains a CRISPR-Cas expression vector, a transposase expression vector, and a transposon expression vector. Also provided is a non-human animal model of recurrent HCC containing genetic modifications introduced by integration of the model generating system into target liver cells of target animals. Upon stable integration, the model generating system modifies expression of Tp53 and expresses the oncogene in the target liver cells of the target animal, resulting in the development of a HCC tumors in the liver. Methods of using the non-human animal model of recurrent HCC are also provided. The non-human animal model of recurrent HCC can be used for research purposes such as investigating the molecular and genetic mechanisms underlying recurrent HCC tumor development, identifying potential therapeutic targets, and evaluating/screening potential compounds for the treatment of recurrent HCC.