PTSelect™-siRNA design strategies
PTSelect™ technology has two vital components; an siRNA cassette designed to contain the PTSelect™-siRNA cloned upstream of the GOI (Figure 1a) and CD4/siRNA mRNA containing an engineered RNA sequence that is complementary to the PTSelect™-siRNA, and thus targeted by the PTSelect™-siRNA. The siRNA cassette comprises three components: (I) 486 bases from intron-2 of HBG gene, which has been shown to improve transgene expression in CHO cells (Haddad-Mashadrizeh et al., 2009),(S.-Y. Kim et al., n.d.). (II) The PTSelect™-siRNA sequence that is co-transcribed with the GOI is a BART transcript of Epstein-Barr virus (EBV) (Kang et al., 2015). We used the highly expressed miR-BART10-3p (BART10), previously employed in the development of a related technology(Doyle et al., 2017). A single copy of the siRNA was used in the cassette, with an option to increase copies to reduce false positives. (III) For precursor sequences that enable siRNA processing and maturation, we tested 100-bp sequences (5’ flank and 3’ flank, Figure 1a) flanking both sides of the BART10 sequence in the EBV viral genome and two synthetic precursor sequences. Since the wild type sequences exhibited the most suppression of a reporter mRNA with a corresponding siRNA target site (data not shown), they were selected.