Design
In a 2017 Cell paper (1), L. Nissim and colleagues in the Timothy Lu team at the MIT presented a revolutionary logic AND gate device comprising a two-module RNA-based circuit. Using this circuit they reported the expression of a multi-component immunostimulatory cassette only in cancer cells in which two distinct promoters are active and demonstrated a potent in-vivo antitumor response elicited by the targeted expression of this cassette.
In ‘Nissim’s’ system basic design (Fig. 1A), the expression of the gene of interest (GOI), whether an experimental fluorescent protein such as mKate2 or a master transcription factor (TF) that controls the expression of multiple therapeutic genes, is governed by the coordinated activity of two separate modules, each driven by one of two synthetic promoters, P1 and P2. Module 1 comprises an mRNA transcript driven by promoter P1, harboring two exons which encode the intact protein product of the GOI. Upon splicing, a sequence in the intron separating these exons functions as a microRNA (miRNA, here miR1) which targets the 3’ end of module 1 transcripts in-trans for degradation, thus preventing translation. The transcript of module 2 is synthesized from promoter P2 and contains multiple miR1 reverse complementary binding sites, which function as ‘sponge’ sequences that relieve the autoinhibition of module 1.
In state [1,1] (Fig. 1B), the P2 promoter expresses multiple copies of a sponge RNA (light green), which binds miR1 and prevents its binding to its target sequence at the 3’ end of module 1 (dark green). This enables sequestration of miR1 away from inhibiting the mKate2 transcript expressed by P1, thus allowing for mKate2 expression.
Fig. 1 (copied from Fig. 2 in (1)). The Basic RNA-Only Single-Output AND Gate Design.
Indeed, as shown by Nissim and colleagues (1) in spite of laborious optimization attempts, at least 15% of the product of module 1 that is synthesized in the presence of the sponge can still be detected in the absolute absence of the sponge. The default state of module 1 is in fact not ‘0’, as desired, but rather an incompletely attenuated ‘1’. A severe safety concern therefore emerges.
Our goal was to devise a new strategy, which locks the basal output of the entire circuit at ABSOLUTE ZERO, thus paving the way for its safe implementation in the clinical setting.
Our solution
We have chosen to address the safety challenge by exploiting the phenomenon of mRNA trans-splicing (TS), namely, the bimolecular joining of exons from different mRNA species, which is well documented in numerous organisms and is currently explored for diverse gene therapy applications (2). TS can be targeted to pre-selected transcripts (referred to as ‘targeted TS’) by promoting base pairing between corresponding introns flanking the exons to be trans-spliced (see, for example, (3–5) and our own previous work (6)).
Fig. 2 Scheme of the proposed trans splicing-governed circuit, based on (1). GOI, gene of interest; Ex. Exon
Similarly to Nissim et al., we present a two-module system (Fig. 2): Module 1, driven by a synthetic promoter P1, encodes the 5’ end of the GOI (including the translation initiation codon) that is referred to as exon 1, followed by the TS ‘target’ intron. Critically, NO mature, potentially hazardous protein can be translated from this module, ensuring the desired ‘0’ state. Module 2, governed by promoter P2, encompasses the TS ‘guide’ intron, followed by exon 2, which encodes the rest of the protein. Similarly, module 2 cannot produce any functional protein and, standing alone, is permanently in state ‘0’ too. In analogy to the multiple sequence repetitions in the sponge design, module 2 can possess tandem repeats of the TS guide sequences to increase the prospects for productive TS events. Using this design, the system becomes fully dependent on the synchronized function of both promoters P1 and P2.
Genetic design
In our project we have designed and assembled two circuits, each comprising two TS modules (Fig. 3):
Circuit 1, modules TS1 and TS2
Module TS1 (BBa_k2946010): Exon 1 of mKate2 followed by the first 198 bp of the miR1 intron employed by Nissim et al. (1), including the 5’ basal stem to serve as the TS guiding sequence to module TS2.
Module TS2 (BBa_k2946011): Downstream to a 40 bp linker (positions 421-460 in the kanamycin resistance gene of thermophilic bacillus from plasmid pTB913 (GenBank accession K02551.1)) we have inserted the remaining 219 bp of the miR1 intron, including the 3’ basal stem as the TS1 TS binding domain (BD) and the full acceptor splice site, followed by mKate2 exon 2, and a 40 bp linker (positions 361-400 in the kanamycin resistance gene).
Module TS2 was designed to facilitate optimal targeted TS with the transcript of module TS1 to generate the intact mKate2 coding sequence.
Circuit 2, modules TS11 and TS12
Module TS11 (BBa_k2946013): Here we have chosen to adapt the optimized TS elements which have recently been reported in (7) in the context of the alpha-fetoprotein (AFP) gene. To avoid TS with endogenous AFP transcript in HEK293T cells we have chosen to invert the optimized 50 bp sequence derived from intron 5 of AFP (NCBI Reference Sequence: NG_023028.1). The inverted sequence also preserves an artificial 2-base mismatch (7) to prevent formation of long nuclear double-stranded RNA. This TS guiding sequence is followed by a 34 bp linker preceding the HSV1pA poly A site (1).
Module TS12 (BBa_K2946014): Downstream to a 40 bp linker from the kanamycin resistance gene (positions 421-460) we have inserted a TS binding domain (BD) which is the reverse complementary of the AFP-based TS guiding sequence of module TS11. This TS BD is followed by the 34 bp linker which precedes the HSV1pA poly A (1) and the intron splice enhancer, branch point, polypyrimidine tract and the acceptor splice site, all taken from (7), continuing with mKate2 exon 2 and a 40 bp linker (positions 361-400 in the kanamycin resistance gene) and the HSV1pA poly A site.
The main differences between these two circuits lie in the size, base composition and the actual sequence of the TS-guiding elements incorporated into their respective modules.
Fig. 3 Schematic representation of Trans-Splicing for our system.
Work plan
- Gene design- Constructing the different genes with two sets of TS modules (BBa_k2946010, BBa_k2946011, BBa_k2946013, BBa_K2946014).
- Cloning- The genes were cloned into a plasmid vector system in order to produce the necessary DNA.
Transformation of plasmids into E.coli Top10 competent cells. - Production of DNA from bacterial culture – DNA was obtained in large quantity using MaxiPrep DNA purification kit.
- DNA transfection- The purified DNA was transfected into HEK293T cells, using FuGENE HD Transfection Reagent.
- Flow Cytometry analysis (FACS) – Transfected cells were analyzed for the GOI expression by fluorescence measurement using FACS analysis. Reporter gene (mKate2) was used to evaluate the expression of the GOI.
Expectations
We expect to show:
a) Safety: Each of the four modules created in our study will exhibit zero mKate2 fluorescence in transfected cells in the absence of its respective partner.
b) Efficacy: Co-transfection of the two modules comprising each of the two circuits will result in clear mKate2 fluorescence.
Reference
1. Nissim, L., M.-R. Wu, E. Pery, A. Binder-Nissim, H. I. Suzuki, D. Stupp, C. Wehrspaun, Y. Tabach, P. A. Sharp, and T. K. Lu. 2017. Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy. Cell 171: 1138-1150.e15.
2. Yang, Y., and C. E. Walsh. 2005. Spliceosome-Mediated RNA Trans-splicing. Mol. Ther. 12: 1006–1012.
3. Puttaraju, M., S. F. Jamison, S. G. Mansfield, M. A. Garcia-Blanco, and L. G. Mitchell. 1999. Spliceosome-mediated RNA trans-splicing as a tool for gene therapy. Nat. Biotechnol. 17: 246–252.
4. Puttaraju, M., J. DiPasquale, C. C. Baker, L. G. Mitchell, and M. A. Garcia-Blanco. 2001. Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing. Mol. Ther. 4: 105–14.
5. Suñé-Pou, M., S. Prieto-Sánchez, S. Boyero-Corral, C. Moreno-Castro, Y. El Yousfi, J. Suñé-Negre, C. Hernández-Munain, and C. Suñé. 2017. Targeting Splicing in the Treatment of Human Disease. Genes (Basel). 8: 87.
6. Schlesinger, J., D. Arama, H. Noy, M. Dagash, P. Belinky, and G. Gross. 2003. In-cell generation of antibody single-chain Fv transcripts by targeted RNA trans-splicing. J Immunol Methods 282: 175–186.
7. Poddar, S., P. S. Loh, Z. H. Ooi, F. Osman, J. Eul, and V. Patzel. 2018. RNA Structure Design Improves Activity and Specificity of trans -Splicing-Triggered Cell Death in a Suicide Gene Therapy Approach. Mol. Ther. - Nucleic Acids 11: 41–56.