Designing shRNA molecules targeting DNA polymerase as a gene therapy tool against Orf virus

Contagious ecthyma (also known as orf) is an acute skin zoonosis caused by orf virus (ORFV), which affects sheep, goats and humans. As one of the typical species of the Parapoxvirus genus of the Poxviridae family, orf virus has distinctive and unique characteristics of these species. A range of immunomodulatory/ pathogenesis related genes acquired by virus that function is to limit (at least transiently) the effectiveness of host immunity during its evolution.[1] The Orf virus genome is an approximately 140-kbp double- stranded DNA containing at least 132 putative genes [2]. It shares many essential genes with vaccinia virus that are required for survival but encodes a number of unique factors that allow it to replicate in the highly specific immune environment of skin [3].Orfvirus encodes the DNA polymerase (DNA Pol), which is the core enzyme in the process of Orfvirus replication and catalyzes the replication of the viral genome [2].RNA interference (RNAi) acts to inhibit protein synthesis by targeting specific mRNAs for degradation and this process has been developed to target RNA viruses, exhibiting their potential as a therapeutic against infections[4].This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery ofsiRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA[5]. We designed two specific shRNAs to target the DNA polymerase genes of Orfvirus in order to test whether RNAi could selectively target Orf viral mRNAs or not.Materials Methods Orf virus is one of dsDNA viruses, To address the question of how shRNA sequence correlates with knockdown efficacy, 2 shRNA vectors from DNA pol gene were designed and constructed .First, DNA pol gene sequence of OV, SA00 strain (NC_005336.1)was selected from NCBI website. Coding regions of the target sequences were selected and sequences of shRNA molecules had a G/C ratio of about 50%.The shRNAs were designed to target sites that are devoid of single nucleotide polymorphisms. In order to identify conserved regions of the gene, DNA pol gene sequence was aligned with the near strains on clustal omega website. Then, to design siRNA, the target sequence was entered into three online websites, included: BLOCK-iTRNAi Designer, WI siRNA Selection Program and www.invivogen.com/sirna-wizard. The best siRNA sequences were chosen based on the folding and conserving condition. The secondary structure of mRNA was checked with CLC software. To convert siRNA to shRNA, Loop, Antisense and sense sequence were added.Results DiscussionTwo potentially effective shRNA molecules were suggested.Their sequences and target start positions included OrfshRNA-1against position 2706 of Orf virus DNA polymerase and OrfshRNA-2 against position 2913 of Orf virus DNA polymerase.that the two Sequences used in this study were specific for ORF025 and have been shown in Table 1.The locations of the shRNA molecules on the secondary structure of target RNA were shown Specific gene expression silencing by RNAi is a mechanism of transcriptional regulation in the eukaryotic cell. This is mediated by small RNA with 21-23 nucleotides length called siRNA and is conserved in terms of evolution among eukaryotes. RNAi seems to protect against not only exogenous genes such as microbial organisms including viral and bacterial genes, but also endogenous genes such as transposons. The other roles of these molecules in cells involving gene expression regulation and cell growth control have been demonstrated. Since siRNAs can have off-target effects, it is important for functional assays to make a specific mutant with one or more base mismatch within the target recognition site as a control. Verifying the sequence of an shRNA hairpin is essential since mismatch of even one nucleotide within the target sequence can ablate knockdown. One strategy recently proposed to overcome this issue involves engineering a restriction site within the loop/stem region of the hairpin to physically separate the inverted repeats by digestion, and then piecing together sequence using sense and antisense primers[6].In recent years, the use of siRNA, shRNA and miRNA has increased to inhibit the genes responsible for cancer, viral infections and autoimmune disorders. Many studies on the use of this therapeutic tool to suppress viral infections in animals and humans have been performed.For example some viruses such as BVD, FMD, BLV, EFV, HIV, HTLV1, influenza A, hepatitis C and B, dengue virus, Sindbisvirus, Ebola, Semliki Forest virus suppressed with RNAi strategies[9]. In 2003 A.Sen et al. demonstrated the effectiveness of siRNA mediated inhibition of NS5A expression in both transient as well as stable transfectants and their results demonstrated that siRNA was effective in inhibiting