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.