Tool Building

The building of reverse genetic systems for diverse RNA viruses has laid the framework to add biological plug-n-play moieties capable of influencing replication activity. We use these systems to build viruses that can be controlled by desired cellular or external cues as a means of answering specific biological questions.

Tool 1: L7Ae –  We generated libraries of viruses that could be tracked by individual barcodes containing a small individual genetic signature and have now applied this to Influenza A virus, coxsackie virus, and SARS-CoV-2. As insertion was fitness neutral in each example, these libraries could be used to follow individual virions through both tissues and different transmission events to ascertain the dynamics and bottlenecks of viral spread.

Tool 2: Defective interfering particles – (text to be provided) We designed and synthesized IAV to encode Cre recombinase.  The resulting virus maintained virulence but 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. These data demonstrated that while 95% of the cells die in the first 5 days of infection – a combination of Type I and II alveolar cells – there is a sustained population of TdTomato+ Club cells that are still present on day 20.  Based on these data we, and others in subsequent follow up studies, have discovered that Club cells launch a unique antiviral response that renders them better able to control virus replication.

Tool 3: We also 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 a means to perform an in vivo RNAi screen, but they have revealed a unique activity of the host microprocessor which can function in the cytoplasm in the presence of a primary miRNA transcript.   We have applied these plug-n-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 specific hairpins that enriched over time, indicating the selective advantage that was achieved as a result of the virus-mediated silencing of a given host factor.  This experimental platform has identified many known factors involved in both the Type I and III interferon pathways and revealed a number of non-canonical targets that also limited virus replication.

Tool 4: 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 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 challenging. 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 that exhibits cell-specific activity and self-inactivates.  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 can be made dependent on cell-specific miRNAs, which confers further specificity. We have found 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 microglial and neuronal specific editors targeted to the stop locus of the TdTomato mice.