SRT-2000-10 x 300mm for Purification of EDⅢ-Fc and NS1 CircRNA for Dengue LNP Vaccine Candidate

From the paper: A single-dose circular RNA vaccine prevents Zika virus infection without enhancing dengue severity in mice

Xinglong Liu 1,2,#, Zhengfeng Li 1,#, Xiaoxia Li 1,2,#, Weixuan Wu 1,2, Huadong Jiang 1,3, Yufen Zheng 1,2, Junjie Zhou 1, Xianmiao Ye 4, Junnan Lu 1, Wei Wang 5, Lei Yu 6, Yiping Li 7, Linbing Qu 1, Jianhua Wang 1, Feng Li 6, Ling Chen 1,8,?, Linping Wu 1,2,?, Liqiang Feng 1,2,?

This is a short summary on the CircRNA Purification from the above paper reference:

Introduction:

Antibodies to pre-membrane E protein generate cross-reactivity with little neutralizing capacity. This type of immune response can even enhance infection due to this non-specific response. CircRNA encoding for ED111-Fc (E pre-envelop membrane protein with epitope response) and NS1 (scaffolding/assembly protein) produced an improved response vs. trimeric and monomeric ED111. Researchers found this construct to be single dose effective in mice.

HPLC purification with SRT-2000 5um 10x300mm Column:

Sample Prep: CircRNA precipitated in LiCl; Load: ~ 20ug; HPLC Column: SRT-2000 5um 10 x 300mm– Part# 215980-10030; Elution Buffer: RNase-free TE buffer (pH 8.0); HPLC System: Agilent 1260; Flow Rate: 1.65ml/min Detection: A260nm; Concentration: Amicon Ultra-15 50Kda cut-off centrifuge filter – dissolved in RNase-free water.

Figure – 1 – Purification of NS1 Scaffold circRNA – HEK293T Expression System

Figure 1 reveals fraction 5 as the purified circRNA after 30 minutes, while figure 2 shows the circRNA infected HEK293T cell western blot with using anti ZIKV NS1 mAb 749 A4. NS1 dimer confirmed at 80Kda proving full expression

Figure 2 – Western Blot Confirmation of CircRNA Transfected HEK293 cells

Figure 3 – Optimized CircRNA Constructs using plasmid (T4 Td) – Fusion of EDⅢ-Fc and NS1 were with tPA signal

Figure 3 shows the purified fraction of CircRNA after 30 minutes. The expression of circRNA shows a much more pronounced and cleaner signal than Figure 1 using the plasmid T4Td system. Based on several publications on HPLC CircRNA purification by Alex Wesselhoeft, the impurities are most likely concatemer, nicked, pre-circular, closed circular, and free introns.

Conclusions:

20ug circRNA-LNP with EDIII-Fc and NS1 dose had exhibited maternal immunity in mice preventing the symptoms and disease manifestations. These 2 CircRNA constructs are needed to produce the most effective immunity. The purity of CircRNA obtained using SRT 2000 played an integral role (Publication by Lianghua Shen regarding circRNA encoding for PP-65 in TCR-T cells for a cytomegalovirus vaccine candidate stated that the purity of circRNA is paramount in expression and circularization efficiency) in developing such immunity.