A Winn-funded grant progress report
The cause of feline infectious peritonitis is a mutant form of feline coronavirus (FCoV) that replicates efficiently in monocytes and macrophages. At the same time, there is dysregulation of the host’s cell-mediated immunity. The virus replicates unchecked and this efficient method of systemic viral replication plays a critical role in FIP pathogenesis.
A previous study demonstrated that RNA interference (RNAi) which targets highly conserved viral genes could be utilized to inhibit FCoV replication in vitro. It appears RNAi when mediated by small interfering RNAs (siRNAs) or microRNAs (miRNAs) may have therapeutic potential if these small RNA molecules are delivered in sufficient quantity to the monocytes and macrophages of a cat with FIP.
siRNA is delivered to cells and has a transient effect, only lasting a few days. It has been noted that the amount of siRNA delivered to cells is dependent on the delivery vehicle used. This is the biggest obstacle in delivering these molecules to target cells in vivo. In comparison, cells can be manipulated to produce miRNA. This introduction of mi RNA which expresses DNA into the host cell genome can be accomplished with the use of lentivirus vectors. In this process, miRNA can be expressed for the lifetime of the cell, allowing this silencing effect to occur indefinitely.
The authors in this study define the feasibility of generating an RNAi response against feline coronavirus in feline hematopoietic stem cells (HSCs) by introduction of DNA that codes for anti-FCoV miRNAs using non-replicating lentivirus vectors. Along with this objective, another objective is to assess the ability of the expressed miRNA to inhibit FCoV replication in HSCs in vitro. The HSCs were obtained from feline bone marrow and replicated in a laboratory setting.
In describing their methodology, the authors constructed three lentiviruses, each expressing a different anti-FCoV miRNA. The HBCs were stably transduced with with miRNA-expressing lentivirus vectors. They then infected the HSCs with two different strains of FCoV to test the effectiveness of the transduction and the expression of the anti-FCoV miRNA. Real-time, reverse transcription PCR was used to determine the quantification of inhibition of coronavirus replication.
Considerations and observations of the authors from the study were noted. Recent advances in stem cell therapy allow for genetic modification of these cells which makes these cells a potential delivery system for small RNA molecules. HSCs can self-renew and differentiate into all hematopoietic lineages, including monocytes and macrophages. The potential result is a considerable number of genetically engineered cells. The authors were able to use lentiviruses to successfully integrate miRNA into HSC genomes. The lentivirus vectors are effective tools for gene transfer and have been used successfully in dogs and cats. An additional benefit noted was the inability of the lentivirus used in this study to replicate.
The authors also commented that both virus strains tested in the study were Serotype II but they believe the designed miRNA should be able to recognize and target various strains of FCoVs circulating in the field, including both serotypes I and/or II.
In conclusion, they state this study suggests that genetic modification of HSCs for production of anti-coronavirus miRNAs will reduce feline coronavirus replication. This work will potentially lead to the use of the genetically modified HSCs for the prevention and treatment of FIP. Reinfusion of these cells could potentially minimize viral load which could restore the cat’s immune response where virus clearance is improved. The authors also believe these modified HSCs may protect cats with a high risk of developing FIP through inhibition of FCoV replication therefore reducing the likelihood of mutation and expansion of the virus population containing this deleterious mutation. (VT)
[A Winn-funded project, W15-030 – “Immune modulation using small interfering RNA for treatment of feline infectious peritonitis” – is a continuation of project MT13-008.]
Anis EA, Wiles RP, Kania SA, Legendre AM, Kennedy MA. Effect of small interfering RNAs on in vitro replication and gene expression of feline coronavirus. Am J Vet Res. 2014 Sep;75(9):828-34.