About RNA Interference and siRNAs

RNA interference (RNAi) is a sequence specific method to suppress gene expression using short double stranded RNA molecules (short interfering RNAs, siRNAs). SiRNAs first bind to and become part of a ribo-protein complex called RISC (RNA induced silencing complex) where a helicase activity dissociates one strand of the siRNA (passenger strand). The remaining strand guides RISC to complementary target RNA sequences, hybridizes and triggers degradation of the bound mRNA thereby silencing gene expression.
RNAi employs a conserved cellular mechanism used to regulate gene expression on a transscriptional level. Here, double stranded RNA precursor sequences are processed by a cellular ribonuclease, Dicer, to form natural short double stranded RNAs (microRNAs, miRNAs) which then bind and mediate degradation of targeted RNAs. Alternatively, RISC can silence gene expression by miRNA mediated translational repression at the ribosome. RNAi utilizes this process and provides the machinery with siRNA for selective silencing of target genes
As such siRNAs are artificial miRNAs designed to selectively and efficiently silence the expression of a specific gene.
About siPOOL Technology
Soon after its discovery it became evident that siRNAs are not as specific as the proposed mode of action would indicate: A thoughtfully selected sequence stretch of 20 nucleotides should statistically appear only once per genome. However, transfecting cells with different siRNAs against the same genes frequently resulted in varying phenotypes. Several independent experiments identified a stretch of 7 nuceic acids at position 2-8 in micro- and siRNAs that could mediate silencing of gene expression in a RISC dependent manner. The sequence segment is called „Seed-Sequence“ and binds as a consequence of its shortness with much higher frequence to target RNAs.
Among many efforts to overcome seed mediated off target effects the pooling strategy turned out to be very efficient. Instead of a single siRNA a complex mixture of siRNAs, all targeting the same gene was used to transfect cells. By this each siRNA and its individual set of off-targets is diluted, while all siRNAs within the pool jointly target the designated gene, resulting in a robust, selective and specific knock down of the targeted transcript.
The technology has also been shown to specifically knock down several genes in parallel without increasing off target deregulation of gene expression.

The siPOOL principle: Single siRNAs (left) hit multiple Off-targets in addition to the target transcript via their seed sequence. SiPOOLs are complex mixtures of selected siRNAs directed against the same RNA target, thereby diluting off-targets below detection limit and stabilizing the knock down of the on-target. The principle has successfully been applied to single (center) and multiple transcripts (right)
Basic Properties of the SARS-CoV-2 Virus
The Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) is believed to be of zoonotic origin and shown to be the causative agent of the current Covid19 pandemic. It is a positive-sense single-stranded RNA (+ssRNA) virus, with a single linear RNA segment. Coronaviruses infect humans, mammals and birds. They can cause illnesses ranging from the common cold to severe diseases such as Middle East respiratory syndrome (MERS, fatality rate ~34%). SARS-CoV-2 is the seventh known coronavirus to infect people, after 229E, NL63, OC43, HKU1, MERS-CoV, and the original SARS-CoV.
SARS-CoV-2 has a linear, positive-sense, single-stranded RNA genome about 30,000 bases long. With this it is the largest genome of any known RNA virus. Coronaviruses have a proof-reading exonuclease, ExoN, which explains the low mutation rate compared to influenza. This is of relevance for estimating the speed by which SARS-CoV-2 will evade the current immunization efforts.
The genome is organized as 1 single strandes positive-sense RNA that codes for 10 genes resulting in 26 proteins by cleavage of a polyprotein using proteases encoded on ORF1ab. In addition the the polyprotein encodes an RNA polymerase, the proof-reading exonuclease and several non structural proteins. The remaining genes code for structural proteins S, M, N and E. The trimeric S-protein (spike) binds with nanomolar affinity to the angiotension converting enzyme and mediates viral uptake into host cells as Type 1 & 2 pneumocytes, Alveolar Macrophages, Mucous Cells and others.

SARS-CoV-2 particles have a diameter of 100nm, contain a single stranded RNA genome of 30kb. The capsid is formed by the M (membrane) protein, the N (nucleocapsid) protein, the E (envelope) protein and the S (spike) protein. The remaining genes are generated by proteolytic cleavage from a polyprotein translated from ORF1ab. Entry into cells is estimated to take 10 minutes, first virions appear 10 hours post infection and cells burst after 24h releasing 1000 new viral particles.
Complex Pools of siRNAs (siPOOL™) Against Multiple Targets Increase Drug Potency and Convey Robustness Against Mutations
In addition to improving specificity and reducing unwanted side effects, soPOOls directed against multiple targets reduce the chances of viral resistance by mutations.
To entirely overun the RNAi mediated knock down of the RNA genome and transcripts a multitude of mutations at the sites of siRNA hybridisation are neccessary.
The multi-targeted attack on the viral genome conveys resilience against escape mutations.
The cartoon illustrates the principle with 3 siPOOLs (yellow, red, green) targeting 3 different sectors of the genome (blue) with a set of individual siRNAs.

Own Research

Which segment of the viral genome is the most promising point of attack? Do viral genomes have an achilles heel? Is the gene encoding the spike protein the best target to go after? To answer these questions we have designed more than a hundred siPOOLs covering the entire 30kb of SARS-CoV-2 genomic RNA. After transfecting SARS-CoV-2 infected Vero-cells with our siPOOLs we used a high content screening assay to monitor viral decline in cell culture. Assays were carried out at the Friedrich-Loeffler-Institute under supervision of our project partner Prof. Dr. S. Finke, an international renown virologist and cell biologist.
Our screening data clearly showed substantial differences in activity of our pools on viral viability. While some pools had little impact on viral propagation other siPOOLs entirely eradicated viruses from cells as shown by immuno fluorescence staining of S- and N-protein. In addition we monitored viability of Vero-cells to assess tolerability in host tissue. Again, some siPOOLs exhibited strong effects on cell viability whereas others did not impact growth of Vero cells.
About Delivery
Trageted delivery of siRNAs is obviously the holy grail of RNAi based therapy and in great parts responsible for the early days frustration associated with siRNA drugs. However, in the past decades great advances have been achieved using various (bio-) chemical tricks and vehicles.
Meanwhile a plethora of divergent Lipid Nano Particles are available and shown to deliver their load effectively to the designated organs and tissues.
While initially the only successfully targeted organs were the liver and to a lesser extend the kidney the number of accessible destinations has increased over the recent years. SARS-CoV-2 infection initiates in the upper respiratory tract where it invades among others type I & II pneumocytes and alveolar macrophages. Quite obviously nasopharynx, throat and lungs are easily accessible by inhalation of formulated siRNAs. At the same time the mucus and ciliated epithelia obstruct direct access to the target cells.
Currently various nanoparticles are tested for lung delivery and alpine antiviral will test successful candidate formulation for targeting siPOOLs to the designated tissues and cells. The successof these approaches will be tested on air-liquid interface cell culture, precison cut lung slices and appropriate animal models

Research Plan

By screening the SARS-CoV-2 genome with arrays of siPOOLs, we can select amongst the most potent siRNA sequences to compose an ideal antiviral siRNA pool.
siRNA delivery to several organs including the lung saw a wave of innovations in the recent years. We currently validate and compare delivery solutions of commercial and academic providers. Here we are fortunate to collaborate with Prof. Dr. O. Merkel and benefit from her expertise, experience and experimental options. We will select up to three different formulation strategies showing efficient delivery of control siRNAs in relevant cell culture and animal models for our proof of concept study. Here therapeutic siRNA mixtures will be tested in hamster being the most relevant SARS-CoV-2 infection model to date. These experiments will be carried out at the Friedrich-Loeffler-Institute on the Island Riems, which provides expertise and safety measures to work with SARS-CoV-2 viruses and infected animals. We expect to provide evidence for a general feasibility of our therapeutic strategy. The PoC is our first milestone on our way to RNAi based anti-viral remedies.