Evolution of complexity in non-viral oligonucleotide delivery systems: from gymnotic delivery through bioconjugates to biomimetic nanoparticles.

From the early days of research on RNA biology and biochemistry, there was an interest to utilize this knowledge and RNA itself for therapeutic applications. Today, we have a series of oligonucleotide therapeutics on the market and many more in clinical trials. These drugs - exploit different chemistries of oligonucleotides, such as modified DNAs and RNAs, peptide nucleic acids (PNAs) or phosphorodiamidate morpholino oligomers (PMOs), and different mechanisms of action, such as RNA interference (RNAi), targeted RNA degradation, splicing modulation, gene expression and modification. Despite major successes e.g. mRNA vaccines developed against SARS-CoV-2 to control COVID-19 pandemic, development of therapies for other diseases is still limited by inefficient delivery of oligonucleotides to specific tissues and organs and often prohibitive costs for the final drug. This is even more critical when targeting multifactorial disorders and patient-specific biological variations. In this review, we will present the evolution of complexity of oligonucleotide delivery methods with focus on increasing complexity of formulations from gymnotic delivery to bioconjugates and to lipid nanoparticles in respect to developments that will enable application of therapeutic oligonucleotides as drugs in personalized therapies.

Extending the biosynthetic repertoires of cyanobacteria and chloroplasts.

Chloroplasts in plants and algae and photosynthetic microorganisms such as cyanobacteria are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals and complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power, and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the levels of production to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the available synthetic biology tools and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts.

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.