Transcriptional Sychronisation
Transcriptional synchronisation is a principle designed to precisely and naturally regulate the transcription of virtually any possible output - be it mRNAs for proteins (e.g. transcription factors, enzymes, structural proteins) or non-coding RNAs such as tRNAs, trans-acting ribozymes, siRNAs or miRNAs.
Thus, it can be used for a multitude of genetic applications:
- An endogenous pathogen resistance gene can be synchronised with a new synthetic output that combats a targeted disease.
- Endogenous genes can be synchronised with a signal molecule for further processing e.g. by molecular logic gates. (see “cancer” below)
- The output might be used for a direct positive/negative feedback loop of the synchronised gene.
- Multiple synchronized outputs for just one endogenous gene are possible - allowing simple amplification of transcription or operon-like expression even in eukaryotes
- For analysing the function of genes - by synchronising an RNAi e.g. with a meiotic gene, this RNAi is only expressed during meiosis and could target genes, which function during meiosis is unknown.
While the use of our invention is focused on crop resistances against fungi, the same mechanism could also synchronise other genes to produce resistances against bacteria, viruses or abiotic stress factors.
Resistance against plant pathogens
Transcriptional synchronisation can also be used to create synthetic pathogen resistance in crop plants. For this, any gene, whose transcription rate is influenced by the pathogen, can be transcriptionally synchronised with a resistance output against the mentioned pathogen. For example, the FAD7 gene could be transcriptionally synchronised with a RNAi against fungal infections: If a plant cell is wounded, FAD7 expression is upregulated (Nishiuchi et al., 1999). Fungi need to penetrate the plant cell for infection, thus upregulating the expression of FAD7 (Kirsch et al., 1997). If FAD7 is now transcriptionally synchronised with e.g. an RNAi against the respective fungi, the expression of the RNAi is triggered upon infection.
Using transcriptional synchronisation against plant pathogens has at least two distinctive advantages compared to using either constitutive or additional endogenous promoters coupled with a resistance mechanism: First, a constitutive promoter for a resistance is always active – even if the pathogen is not present. This means that the plant cell is wasting its resources to produce a resistance that is not needed most of the time. This again will influence the growth and yield of crops in a negative way, depending on how many resistances are used in the plant. Also, the resistance could have unwanted off-target effects against the plant itself or other organisms [3]. Second, if the resistance is controlled by using an endogenous promoter of the plant (e.g. the FAD7 promoter), the aforementioned problem is not reliably solved: The promoter is not solely controlling the transcription rate of a gene, but the genetic context is also important, including enhancer and silencer sequences, intronic effects, etc. Thus, it would be necessary to test every resistance, which is only regulated by an endogenous promoter, if its expression is regulated as intended. Depending on the number of needed resistances, this can become time- and cost-intensive. Transcriptional synchronisation solves both problems, by using the existing transcriptional regulation pathways of the cell to express the synthetic resistance only if needed.
These advantages enable another possibility: Instead of directly synchronising only one resistance output with an endogenous gene, the endogenous gene can be synchronised with a transcription activating output, which in turn activates the transcription of multiple resistance genes. Thus, the synchronization would hardly influence the transcription time for the synchronised endogenous gene, while still being able to activate a plethora of resistance genes. Further, multiple endogenous genes can be synchronised with one (or more) synthetic resistance genes so that one pathogen triggers one (or more) resistance gene(s) by upregulating the expression of all the synchronised endogenous genes. Using multiple inputs (synchronised endogenous genes) and multiple outputs (synthetic resistances) would make it near impossible for any pathogen to adapt to the synthetic resistances by evolutionary means. All this, while having a relatively low impact on plant metabolism. Also, the number of genetic modifications can be reduced significantly compared to introducing the same number of resistances by using other methods, making transcriptional synchronisation very cost-efficient.
These advantages compared to recent approaches regarding resistance regulation in plants, could make transcriptional synchronisation a valid alternative. It could be implemented for practically every crop plant that is susceptible to diseases that are influencing the transcription rate of the plant's genes (i.e. FAD7).
A glimpse into the future: Resistance against human cancer
Disclaimer: The approach for cancer is not thought to be used as a germline therapy due to ethical reasons. However, other gene therapy methods could be used, such as viral vectors for delivery.
Moving away from our plants, a fascinating future application against human cancer could be as following: In cancer cells, the transcription of tumour suppressor genes (TSGs) is downregulated by genetic/epigenetic means [4;5] or is even suspended through gene deletions [6; 7; 8]. All this, while the expression of proto-oncogenes is increased, thus, creating oncogenes [8].
The mechanism relies on sensing these genetic alterations by comparing the transcriptional activity of (proto-)oncogenes and TSGs. This is possible by adding a DNA sequence for STAR (Short Transcription Activating RNA) after a (proto-)oncogene DNA sequence. STAR is able to activate the transcription of a target gene by binding to an upstream transcription terminator (t500), which is located right after the genes’ promoter [9]. By default, the terminator t500 prevents the transcription of the gene behind it via a stem-loop [9]. Yet, if STAR binds to the t500 terminator, transcription is enabled [9]. For maximum efficiency of STAR, a 5’ hammerhead ribozyme [10] is added to the 5’ end of the STAR DNA sequence. The 3’ end of STAR has a 3’ self-cleaving ribozyme added, e.g. the hepatitis delta virus ribozyme (HDVRz) [10]. Flanking the STAR sequence with self-cleaving ribozymes results in a STAR transcript that is independently mobile from the oncogene. Furthermore, AntiSTAR DNA sequences are added in a similar way to (multiple) TSGs. The endogenous ORFs of the (proto-)oncogenes and TSGs have poly-T sequences added directly downstream to enable nuclear export and cytoplasmic translation.
STAR and AntiSTAR inhibit each other via antisense base pairing. The mechanism is further depending on an overall equal or higher transcriptional activity of TSGs as long as the cell is healthy. Thus, for every (proto-)oncogene that has the mechanism, there is one or more TSG chosen, which (always) have a (combined) much higher transcription activity than the (proto-)oncogene. This way, the default state is that the STAR-activity is quenched by the much more abundant Anti-STAR.
This sensing mechanism is a first control and safety mechanism. By choosing TSGs that are often deleted or silenced in cancer cells, e.g. the gene for MHC 1 [11], transcription of a therapeutic output is still possible. As soon as the transcription of these genes is significantly downregulated and AntiSTAR is produced in a lower quantity than STAR, the transcription of the therapeutic output can be induced by STAR.
A second safety mechanism could be constituted by the therapeutic output itself: A proton channel could be produced and located to the cell membrane, enabling passive proton transport across the membrane. Thus, in an acidic tumour microenvironment, the channel will import protons into the cell. This will acidify cancer cells, while also reducing the extracellular proton concentration. This should result in cancer cell termination due to the now acidic cellular milieu, futile ATP consumption for proton export [12], glucose deprivation [13] and a reduced capability for tumour invasion [14]. Also, it should improve an immune answer due to the less acidic tumour microenvironment [15]. The proton channel could be the otopetrin Otop1 or Otop3 [16] or a modified viral M2 proton channel [17].
Also, a proton unloading enzyme inhibitor instead of the proton channel could be chosen: The cancerous cell couldn’t unload excess protons after the pathological gene alterations. V-ATPase unloads excess protons and prevents intracellular acidification [12]. Thus, inhibiting V-ATPase function should acidify and kill cancer cells, while other proton unloading enzymes (e.g. monocarboxylate transporters, Na+/H+ exchanger [18] and carbonic anhydrases [19] might have to be targeted too.
Also, AntiSTAR genes should be as close as possible to STAR (for direct quenching of STAR) and to the t500 terminator (close-target quenching). Thus, quenching STAR activity is less a matter of chance. So, it’s more related to differences in quantity between STAR and AntiSTAR, while stochastic problems due to diffusion area are reduced.
Additionally, the regulation via the RNA-based STAR/AntiSTAR-system should be quickly reversible due to fast RNA degradation [20]. The system described above is depicted in figure 1 below. Note, that the figure shows a simplified version of the mechanism for transcriptional synchronisation, which does not contain poly-T tails.
Figure 1: Molecular circuit against cancer development. A) Shown is the function of the mechanism in a healthy cell: Multiple TSGs (here two), which are coupled with AntiSTAR sequences, quench the transcription-activating capability of STAR. No therapeutic output (proton channel) is produced. B) In a cancerous cell, proto-oncogenes become oncogenes and experience an increased transcription. Contrary, TSG transcription is reduced. Thus, in a cancerous cell, the inhibition of STAR by AntiSTAR is suspended. C) Without AntiSTAR, STAR is able to induce the transcription of the proton channel. Note: The therapeutic output could also be a V-ATPase inhibitor or something else. Also, both STAR and AntiSTAR are flanked by a 5’ and 3’ self-cleaving ribozyme (SCRz). The 5’ SCRz here is a hammerhead ribozyme (HHRz), while the 3’ SCRz could be a hepatitis delta virus (HDV) ribozyme. The mechanism is shown simplified (e.g. without a synthetic poly-A tail).
References
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