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Published on: February 24, 2023
A Workflow for In Vivo Evaluation of Candidate Inputs and Outputs for Cell Classifier Gene Circuits
Margaux Dastor1, Joerg Schreiber1, Laura Prochazka1
1Department of Biosystems Science and Engineering, ETH Zurich , Mattenstrasse 26, 4058 Basel, Switzerland.
Abstract:
Cell classifier gene circuits that integrate multiple molecular inputs to restrict the expression of therapeutic outputs to cancer cells have the potential to result in efficacious and safe cancer therapies. Preclinical translation of the hitherto developments requires creating the conditions where the animal model, the delivery platform, in vivo expression levels of the inputs, and the efficacy of the output, all come together to enable detailed evaluation of the fully assembled circuits. Here we show an integrated workflow that addresses these issues and builds the framework for preclinical classifier studies using the design framework of microRNA (miRNA, miR)-based classifier gene circuits. Specifically, we employ HCT-116 colorectal cancer cell xenograft in an experimental mouse metastatic liver tumor model together with Adeno-associated virus (AAV) vector delivery platform. Novel engineered AAV-based constructs are used to validate in vivo the candidate inputs miR-122 and miR-7 and, separately, the cytotoxic output HSV-TK/ganciclovir. We show that while the data are largely consistent with expectations, crucial insights are gained that could not have been obtained in vitro. The results highlight the importance of detailed stepwise interrogation of the experimental parameters as a necessary step toward clinical translation of synthetic gene circuits.
Insights
This study presents a workflow for preclinical evaluation of microRNA (miRNA)-based gene circuits for cancer therapy. The integrated approach validates circuit components in vivo, crucial for advancing synthetic gene circuit clinical translation.
Area of Science:
- Biotechnology
- Molecular Biology
- Oncology
Background:
- Synthetic gene circuits offer targeted cancer therapy by integrating molecular inputs to control therapeutic outputs.
- Preclinical translation requires a comprehensive evaluation of animal models, delivery systems, and in vivo expression dynamics.
Purpose of the Study:
- To establish an integrated workflow for preclinical assessment of microRNA (miRNA)-based classifier gene circuits.
- To validate novel Adeno-associated virus (AAV)-based constructs for in vivo evaluation of miRNA inputs and cytotoxic outputs.
Main Methods:
- Utilized a HCT-116 colorectal cancer cell xenograft in a mouse metastatic liver tumor model.
- Employed Adeno-associated virus (AAV) vectors for delivery of engineered constructs.
- Validated candidate microRNA (miR)-122 and miR-7 inputs and the HSV-TK/ganciclovir cytotoxic output in vivo.
Main Results:
- Demonstrated the feasibility of an integrated workflow for evaluating synthetic gene circuits in vivo.
- Gained crucial insights into in vivo performance not obtainable through in vitro studies.
- Confirmed largely expected data while highlighting the necessity of stepwise parameter interrogation.
Conclusions:
- The developed workflow provides a framework for preclinical classifier studies of miRNA-based gene circuits.
- Stepwise interrogation of experimental parameters is essential for the clinical translation of synthetic gene circuits.
- In vivo validation is critical for assessing the efficacy and safety of complex gene circuit designs.
