Splicing-based synthetic gene circuits for universal logic computation in mammalian cells

Abstract

Synthetic biology and biomolecular computing research, in the past two decades, has enabled design of genetic logic circuits that reprogram cellular behavior to achieve a certain desired outcome. Multi-input gene circuits have mostly relied on post-transcriptional or recombinase-triggered state transitions. My focus has particularly relied on building multi-input logic circuitry at transcriptional level in mammalian systems. One of the particular challenges is to implement OR logic at transcriptional level in mammalian cells. There are a few shortcomings with the current approaches of OR logic implementation. Firstly, there is redundancy in genetic information which leads to increased payload size making it unsuitable for design of gene therapy vectors (Kramer et al., 2004; Leisner et al., 2010). Circuit complexity increases with increase in input number (Gaber et al., 2014). Experimentally, the redundant genetic information results in multi-valued logic with twice the output obtained when both constructs are active compared with a single active construct (Kramer et al., 2004; Rinaudo et al., 2007). In prokaryotic systems, the solution has been shown by using multiple promoters with their own transcription initiation sites (Tamsir et al., 2011). However, the same cannot be applicable in mammalian systems as there are secondary interactions involved between the promoter elements (Angelici et al., 2016; Lohmueller et al., 2012). In eukaryotic systems, nature has devised its own mechanism: alternative promoters coupled with alternative splicing to mitigate this problem. Inspired by this regulatory strategy of using multiple alternative promoters and alternative splicing, I develop synthetic gene circuit designs that implement multi-input transcriptional OR logic while mitigating genetic redundancy, circuit complexity and secondary interactions between inputs . Firstly, as a proof-of-concept, OR logic implementation was tested by building a polychromatic reporter using alternative promoters. The small library of polychromatic genetic circuits tested allowed me to figure out the design features that govern the functionality of such a circuit. The learnings from the polychromatic system-aided further in the implementation of two and three-input bona fide OR gates that are scalable and compact. Finally, universal disjunctive normal form logic was realized by the use of synergistic transcription factor inputs (AND logic) for activating alternative promoters and miRNA inputs (NOT logic) directed at the alternating exons. These DNF-like circuits, thus, permit the implementation of complex, multi-input logic for the control of gene expression. In the end, I discuss the design guidelines and design features that are particularly important for developing an application-relevant synthetic gene circuit in mammalian systems at the transcriptional level using alternative promoters. Furthermore, the thesis highlights the opportunities for additional logic integration and biological parts improvement that exist in alternative promoter-based synthetic OR logic circuits. All in all, the circuit designs presented in the thesis serve as a foundation for the design of gene therapy vectors that are relevant for targeting of heterogeneous population i.e., simultaneous expression of the same output upon presence of cell-specific molecular markers.