Establishment And Preliminary Application Of ETAT-U1A-Based MRNA Intracellular Delivery System

Introduction of functional genes to restore defects genes to physiological levels becomes the main direction of gene therapy.Because of no transcription process and the characteritsics of cytoplasmic expression,mRNA does not exhibit the difference of transfection effectives of DNA expression in the different cell lines due to nuclear pore factor interference and CpG immunogenicity.Meanwhile,mRNA has other advantages such as higher safety and lower immunogenicity.However,because of its characteristics of large molecular weight and the negatively charged,mRNA must be deliveried by some carriers through the cell membrane.The viral vector is the most widely used by virtue of its high delivery efficiency,but it is more used for the delivery of DNA.Non-viral vectors are mainly for the delivery of pDNA but also have problems such as low efficiency and large cell/body damage.Cell-penetrating peptides rely on the advantages of biocompatibility and functional diversity,and various studies have applied them to the delivery of nucleic acids.However,the combination of CPP and therapeutic genes in the previous studies relied on electrostatic force,so this non-covalent association has problems such as random and non-specific binding.In the previous study,the eTAT(enhanced TAT)-based delivery system,having a more efficient endosomal escape,was established.Meanwhile,based on the RNA-binding protein UIA,which can recognize the characteristics of the stem-loop structure formed by single-stranded RNA of specific sequence,we constructed the expression plasmids of fusion protein eTAT-U1A.eTAT-U1A was expressed in E.coli,after the exploration of the buffer conditions,it was determined that in the buffer of 20 mM PB6.0+0.3 M NaCl+7 mM MgCl2,the higher purity of and more stable eTAT-U1A could be obtained.The 3’BS EGFP mRNA with the U1A binding site was designed and synthesized in vitro.After the extracellular verification of combination of eTAT-U1A and mRNA,it was found that eTAT-U1A could delivery 3’BS EGFP mRNA into HEK-293T by observing the expression of green fluorescence,however,the efficiency was very low.Then we optimized the transduction condition through addition of the RNase inhibitor and adjustment of the molar ratio,but it was useless.Considering the steric hindrance and U1A protein binding efficiency,we redesigned the mRNA structure,and when the mRNA with six U1A binding sites,it was observed the green fluorescence ratio could increase to about 10%.In the optimized transduction conditions,the fluorescence ratio of the hard-to-transfected cell line HaCaT was also about 10%.In summary,based on the eTAT fused RNA binding protein U1A,we established a more specific mRNA transduction method,enriched the delivery of therapeutic genes,and provided a new strategy to solved the problem of foreign genes expression in difficult-to-transfect cells.