SYNAPTA: An artificial genetic system and its application for the generation of novel nucleic acid therapeutics

Life's diversity is largely based on the versatility of two polymers: polyeptides (i.e. proteins) and polynucleotides (nucleic acids). Nucleic acids in particular display unique properties beyond their ability to encode genetic information, which make them important tools in chemistry, biotechnology, nanotechnology and medicine. Nucleic acids also have enormous potential as therapeutics but suffer from systemic constraints inherent in DNA and RNA chemistry such as poor serum / nuclease stability. Aptamers are a promising class of biomolecular therapeutics based on structured single-stranded nucleic acids with the potential to rival antibodies in some clinical settings. A broad spectrum of both RNA- and DNA-based aptamers have been described directed against a wide-range of targets and several are currently undergoing in clinical trails underlining their potential. However, reagents based on natural nucleic acids such as RNA or DNA are not optimal with respect to a number of desirable properties for clinical reagents and therapeutics, notably in vivo stability and bioavailability. In principle, aptamers may be stabilized (post-selection) by medicinal chemistry approaches and this approach has been validated by the Macugen, the 1st aptamers based drug, which has been approved for the treatment of macular degeneration. However, post-selection modifications can alter and / or weaken aptamer structure and target interactions and may modify aptamer specificity. Therefore direct selections using modified nucleic acid chemistries would be desirable. Many novel nucleic acid structures have been built with a view towards increased orthogonality. The challenge here is to design scaffolds that lead to minimal interaction / interference with the cellular genetic machinery while simultaneously maintaining an ability to communicate with it. A different approach towards chemically orthogonal nucleic acids involves the modification of the backbone but leaves the informational nucleobases intact. Replacement of the canonical ribofuranose with other pentoses (or hexoses and tetroses) can indeed have dramatic effects on helical conformation and duplex stability and formation. We have selected two unnatural nucleic acid architectures, Hexitol nucleic acid (HNA) and Cyclohexenyl nucleic acids (CeNA) as our backbone structures. Both HNA and CeNA are completely resistant to nuclease degradation and appear not to be substrates for DNA or RNA modifying enzymes. Significantly, they are non-toxic to cells as nucleotides and therefore appear to be not recognized as substrates by the cellular replication, transcription and translation machine. This proposal aims to develop the platform technologies needed to deliver tailor-made 'designer' ligands of high serum stability, defined compact structure and expanded functionality based on these novel chemistries with obvious potential as a novel class of bio-therapeutics. These novel polymers will also provide insights into the parameters of molecular information storage and propagation through the study of artificial genetic systems entirely based on unnatural chemistry.

Our proposal targets one of the key goals of synthetic biology, the construction of an artificial genetic system. The orthogonal nucleic acid chemistry and its cognate replicases developed as part of this proposal will realize such an artificial genetic system in vitro and provide key components for it establishment in vivo. The latter will be pursued as a long-term goal in collaboration with our associate partner at Genoscope, France (Part C). The artificial genetic system, while worthwhile of study in its own right, provides the key enabling technology for our second goal, the selection of nucleic acid therapeutics based on orthogonal nucleic acid chemistry. In pursuit of these goal we are also developing a suite of platform technologies for the synthesis, replication and evolution of nucleic acid polymers with expanded chemistry with applications ranging from the economical production of nucleic acid therapeutics and diagnostics, nanotechnology, material science as well as molecular tagging and coding and computing applications. We will use a hierarchical forward engineering approach to: 1) generate a technology platform for synthetic genetics i.e. for the enzymatic synthesis, replication and evolution of unnatural nucleic acids with beneficial properties for nucleic acid therapeutic design (e.g. serum stability, nuclease resistance). This will be based on further development of a novel emulsion-based selection technology already established in the laboratory of PI1 to use substrates synthetically developed by PI2; 2) build an artificial genetic system based on orthogonal chemistry and study said system with respect to information transfer, heredity and evolution; 3) exploit said system to expand aptamer technology and to isolate aptamers (and aptazymes) based on orthogonal chemistry using methodologies developed i.a. by PI3.

Our collaboration targets an application and will develop technologies of potential strategic importance for the pharmaceutical industry that could give rise to a host of novel applications and promises to deliver novel ways for the economic production of nucleic acid based therapeutics with potentially important consequences for the competitivity of European pharmaceutical industries in the face of aggressive competition from US and Asia in this area. The ability to custom produce novel nucleic acid-like polymers of programmable sequence would also have fundamental enabling benefits for the emerging fields of nanotechnology, nucleic acid computing and material science.