Rational design and construction of multi-copy biomanufacturing islands in mammalian cells.

Cell line development is a critical step in the establishment of a biopharmaceutical manufacturing process. Current protocols rely on random transgene integration and amplification. Due to considerable variability in transgene integration profiles, this workflow results in laborious screening campaigns before stable producers can be identified. Alternative approaches for transgene dosage increase and integration are therefore highly desirable. In this study, we present a novel strategy for the rapid design, construction, and genomic integration of engineered multiple-copy gene constructs consisting of up to 10 gene expression cassettes. Key to this strategy is the diversification, at the sequence level, of the individual gene cassettes without altering their protein products. We show a computational workflow for coding and regulatory sequence diversification and optimization followed by experimental assembly of up to nine gene copies and a sentinel reporter on a contiguous scaffold. Transient transfections in CHO cells indicates that protein expression increases with the gene copy number on the scaffold. Further, we stably integrate these cassettes into a pre-validated genomic locus. Altogether, our findings point to the feasibility of engineering a fully mapped multi-copy recombinant protein 'production island' in a mammalian cell line with greatly reduced screening effort, improved stability, and predictable product titers.

Multiple Alternative Promoters and Alternative Splicing Enable Universal Transcription-Based Logic Computation in Mammalian Cells.

Multi-input logic gene circuits can enable sophisticated control of cell function, yet large-scale synthetic circuitry in mammalian cells has relied on post-transcriptional regulation or recombinase-triggered state transitions. Large-scale transcriptional logic, on the other hand, has been challenging to implement. Inspired by a naturally found regulatory strategy of using multiple alternative promoters, followed by alternative splicing, we developed a scalable and compact platform for transcriptional OR logic using inputs to those promoters. The platform is extended to implement disjunctive normal form (DNF) computations capable of implementing arbitrary logic rules. Specifically, AND logic is implemented at individual promoters using synergistic transcriptional inputs, and NOT logic via microRNA inputs targeting unique exon sequences driven by those promoters. Together, these regulatory programs result in DNF-like logic control of output gene expression. The approach offers flexibility for building complex logic programs in mammalian cells.

A Workflow for In Vivo Evaluation of Candidate Inputs and Outputs for Cell Classifier Gene Circuits.

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.