Synthetic Biology

The use of modified RNAs as a therapeutic platform is an exciting new area of research made possible by recent advances in synthetic biology.

In the tenOever Lab, we are developing a self-amplifying RNA that is both programmable and amenable to viral packaging. We achieve this by combining synthetic biological circuits with our self-replicating RNA platform to generate biocomputers that can perform complex operations with different sets of user-defined inputs.

The core processors for these designs are novel arthropod-specific viral RNA dependent RNA polymerase (RdRp) scaffolds, which ensures both a minimalistic design and a lack of unwanted host interactions in mammalian cells. To program these constructs, we apply microRNA-enabled logic gates in conjunction with activators and repressors of replicon biology. The idea is to generate a toolbox of adaptable and tunable logic-based replicons that will serve as new biomaterial for genetic manipulation of or gene delivery into any desired cell type or eukaryotic host.

Based on current knowledge of these viruses, we synthesized the minimal components to enable their self-amplification as a starting platform. For V1, we generated a construct that can be in vitro transcribed and directly introduced into cells. For V2, we engineered bi-directional plasmids that generate both vRNA and mRNA from each individual plasmid. Once launched, each of these self-amplifying RNAs can be fitted with selection markers to enable functionality in non-insect hosts. Once a desired level of activity is achieved, logic gates can be added to generate variants to define functionality as it relates to cell lineage.

Our Synthetic Biology emphasis currently has two Aims:

Expanding on the arthropod virus-specific replicon platform
Controlling replicon biology through miRNA-based logic gates

Tool 1: Cre-Expressing Influenza Virus

We designed and synthesized influenza A virus (IAV) to encode Cre recombinase. The resulting virus retained virulence and allowed us to track infected cells through use of the TdTomato reporter mice, which indelibly labeled cells upon the introduction of Cre. These mice were infected with a near lethal dose of our Cre-containing virus, and we sorted CD45-/TdTomato+ cells at 0, 1, 2, 5, 10, and 20 days post-infection. While 95% of the cells died in the first 5 days of infection—a combination of Type I and II alveolar cells—there was a population of TdTomato+ Club cells still present on day 20. Based on these data we, and others in follow-up studies, have discovered that Club cells launch a unique antiviral response that allows for better control of virus replication.

Tool 2: Virus-encoded microRNAs

We can use the genetic real estate of RNA viruses to incorporate artificial miRNAs that we re-wire to silence host factors. These designs not only enable in vivo RNAi screening, but they have revealed a unique activity of the host microprocessor: its ability to function in the cytoplasm when a primary miRNA transcript is present. We have applied these plug-and-play moieties into both IAV and Sinbis virus, and enabled them to specifically target murine host genes. In both examples, in vivo virus passaging identified the enrichment of specific hairpins over time, indicating a selective advantage that was achieved as a result of the virus-mediated silencing of a given host factor. This experimental platform has identified numerous known components of the Type I and III interferon pathways, as well as several non-canonical targets that also limited virus replication.

Tool 3: Programmable genome editor

Our most recent viral plug-in focuses on DNA editing. The capacity to edit genomes using CRISPR-Cas systems holds immense potential for countless genetic-based diseases. However, one significant impediment preventing broad therapeutic utilization is the need for effective in vivo delivery. While genetic editing at a single cell level in vitro can be achieved with relatively high efficiency, the capacity to utilize these same biologic tools in a desired tissue in vivo remains a challenge. Non-integrating RNA virus-based vectors constitute efficient platforms for transgene expression and surpass several barriers to in vivo delivery. However, the broad tissue tropism and their propensity to induce cytotoxicity raises the concern for off-target effects and unwanted immunological responses. In an effort to circumvent this, we built a chimeric RNA virus capable of cell-specific activity and self-inactivation. We achieved this by combining host miRNA biology with the CRISPR-Cas12a RNA-guided nuclease. Exploiting the RNase activity of Cas12a, we generated a vector that self-inactivates upon delivery of Cas12a and an accompanying CRISPR RNA (crRNA). Furthermore, we engineered the virus so that maturation of the crRNA is dependent on cell-specific miRNAs, conferring further specificity. We have found that our resulting chimeric virus delivers sufficient levels of Cas12a to achieve effective genome editing whilst inducing a minimal immunological response. It can also function in a cell-specific manner, thereby facilitating in vivo editing and mitigating the risk of unwanted, off-target effects. At present, we are designing microglia- and neuron-specific editors targeted to the stop locus of TdTomato mice.