Overview

Figure 1. Schematic representation of a positive feedback loop switch.

We consulted several physicians concerning different potential therapeutic applications of thistype of delivery system. Physicians by and large preferred the ability of the device to be controlled by the therapist to the autonomous switching by sensing internal physiological signals. An additional advantage of a controlled switch is that a very similar device core could be used for many different therapeutic applications.As examples of medical problems from the expertise of consulted physicians we considered wound healing, hepatitis C infection and cardiovascular ischemia.For hepatitis C and ischaemic heart disease we constructed two sets of therapeuticeffectors and tested some of these effectors’ biologic activity.

Synthetic biology has already demonstrated the ability of a genetic device to assume different stable states. However, while prokaryotic regulators appear to be an efficient tool for device construction, their number is limited and currently does not allow the scalability required for construction of complex logical functions. On the other hand, modular DNA-binding protein domains such as zinc finger proteins or Transcription-activator like (TAL) domains can be designed to bind to almost any DNA sequence, providing high orthogonality.

Therefore the challenge was to prepare an epigenetic switch based on designable DNA binding domains, such as zinc finger proteins or TAL effectors that can be designed to recognize selected exogenous DNA sequences. This type of orthogonal switch would allow the simultaneous introduction of several bistable or multistabletoggle switches into mammalian cells for advanced level of control in different applications. To our knowledge, a designed modular DNA binding protein-based toggle switch has not been implemented experimentally so far, neither in mammalian nor in bacterial cells. This endeavor has to be supported by mathematical simulationto evaluate which type of genetic switch topology has the best performance, is more robust and which parameters are most critical for its performance.

Epigenetic toggle switches havepreviously been constructed based onnatural bacterial transcription factors (Gardner et al., 2000). An epigenetic toggle switch is a genetic network, designed to memorize its state and maintain expression of a gene of choice even after the inducer has been removed (Cherry & Adler, 2000). Only when a second signal (inducer) is provided to the cells, the system will switch to a different state, shutting off expression of the first protein and/or initiating production of a second one. This type of toggle switch allows the system to assume two discrete states. For our therapeutic purposes this could mean switching the production of a single protein on and off or selecting between the production of two different protein effectors. Therefore a combination of several switches would permit selection of advanced therapeutic regimens.

If we want the switches to function independently of each other andisolated from other cellular processes, they need to be based on the orthologous DNA-binding domains. We therefore decided to construct the genetic core of our device from TAL effectors, whose code of DNA recognition has been deciphered recently (Boch et al., 2009). TAL effectors have a modular structure, with each module recognizing a single base pair. TAL effectors, fused to theFokI nuclease, were successfullyadopted as highly specific and easily customizable genome editing tools (Christian et al, 2010; Bogdanove et al.; 2011, Li et al., 2011). For synthetic biologists, TAL effectors represent perfect candidates for engineering artificial transcription factors due to their high specificity, modularity, ability to target virtually any sequence and last, but not least, they do not exhibit toxicity when expressed in cells, except in case they target an essential endogenous gene. Indeed, in addition to endonucleases, transcription factors employing TALs as DNA binding domains have also been reported recently: transcription activators were constructed by fusing TALs to the mammalian activation domain VP16 or VP64 (Zhang et al., 2011) and, when our project already started, transcription repressors were reported, based on fusion of TALs with theKRAB heterochromatin silencing domain (Garg et al., 2012; Cong et al., 2012).

References

Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A., and Bonas, U. (2009) Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors. Science. 326, 1509-1512.

Bogdanove, A. J., and Voytas, D. F. (2011) TAL Effectors: Customizable Proteins for DNA Targeting. Science. 333, 1843-1846.

Cherry, J. L. and Adler, F. R. (2000) How to make a Biological Switch. J. Theor. Biol. 203, 117-133.

Christian, M., Cermak, T., Doyle, E. L., Schmidt, C., Zhang, F., hummel, A., Bognadove, A. J., and Voytas, D. F. (2010) Targeting DNA Double-Strand Breaks with TAL Effector Nucleases. Genetics 186, 757-761.

Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F. (2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. Nat Commun.3, 968.

Gardner, T. S., Cantor, C. R., and Collins, J. J. (2000) Construction of a genetic toggle switch in Escherichia coli. Nature. 403, 339-342.

Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. Nucleic Acids Res. 40, 7584-95.

Karlsson, M., Weber, W., and Fussenegger, M. (2012) Design and construction of synthetic gene networks in mammalian cells. Methods Mol. Biol. 813, 359-376.

Li, T., Huang, S., Jiang, W. Z., Wright, D., Spalding, M. H., Weeks, D., P., and Yang, B. (2011) TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 39, 359-372.

Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149-153.