Study On The Design Of Antisense With Conformational Dynamic Transformation Based On Prodrug Strategy

Nucleic acid drugs are a new type of biotechnology drugs that have been paid much attention in the world at present.They are characterized by convenient design,wide application,strong specificity and low resistance to drugs.Nucleic acid drugs target nucleic acid sequences such as mRNA which are closely related to the process of disease.Due to specificity of protein sequence coding and base paring,nucleic acid drugs have the advantages of “treating the symptoms and treating the root causes” and have a great application prospect.The listed nucleic acid drugs include Si RNA,antisense nucleic acid,aptamer nucleic acid,other nucleic acid drugs also include ribozyme,sa RNA,etc.As an important class of nucleic acid drugs,antisense nucleic acid is the most mature.Antisense nucleic acids regulate mRNA and protein expression through antisense mechanisms,including hybridization with mRNA to block translation process or activation of RNase H to degrade target mRNA,or binding with splicing sites on pre-mRNA to change exon content in mature mRNA.Since 1998,the first antisense drug Vitravene TM was approved by the US FDA for marketing.Since 2013,Mipomersen,Eteplirsen,Nusinersen and Tegesedi have also been approved for marketing.Despite great advances,nucleic acid is rapidly cleared by the liver due to the electronegativity of nucleic acid drugs,enzymatic degradability,and multiple biological barriers in vivo that result in inefficient cytoplasmic delivery.Up to now,the effective delivery of nucleic acid drugs is still a major problem to be solved.The main strategies delivery of nucleic acid drugs is still a major problem to be solved.The main strategies developed in recent years are the use of various delivery carriers and chemical modifications for nucleic acid drugs.The delivery carriers mainly include cationic lipids,polyrthylenimines or liposome preparations,which interact with negatively charged oligonucleotides through electrostatic interaction and self-assemble in a hydrophilic medium.In this strategy,oligonucleotides are compressed into polymers,which are released after endocytosis into cell.Currently,available transfection reagents include Oligofectamine 2000(Invitrogen),Jetpei(Polyplus Transfection),K2(Biontex),etc.However,the potential biotoxicity of transfection reagents remains to be further clarified.Chemical modification is mainly at the molecular level,through the base,ribose,phosphate or terminal structure modification of oligonucleotide to improve its physical and chemical properties,in addition,including PEG-conjugation strategies,to achieve long-term cycling and targeting in vivo.These advances have provided a large number of unique new biomacromolecules,but chemical modifications also have potential hazards such as reduced binding affinity and adverse reactions.It should be noted that there are still important opportunities to expand the composition diversity of modifications and the structure-activity relationships of more effective and diverse products of nucleic acid drugs.The treatment of oligonucleotide is still in its infancy in clinical practice,and the combination with advanced diagnosis treatment will become the development trend of oligonucleotide therapy in the future.At the molecular level,the design and optimization of novel functionalized oligonucleotides is the key to clinical success and also the core of the oligonucleotide therapy revolution.Study found that the nucleic acid sequence from into senior structure by complementary base pairing in the human body naturally,some senior conformation of nucleic acid molecules,such as hairpin,ring,four spiral,due to its special conformational change(the arrangement of the bases and the phosphoric acid and base pile),adjust the physical and chemical properties,including improving resistance to enzymatic hydrolysis stability,etc.Based on the changes of physical and chemical properties brought by conformation,the further design of nucleic acid drugs with new structural characteristics or mechanism of action can provide new ideas and more possibilities for the research and application of related fields,which has important scientific value.In this study,from the perspective of molecular conformation,the design concept of nucleic acid of new structural molecules were designed,including: 1.Design of a self-assembled end-hairpin structure based on base recognition.2.Design of a self-assembled end-hairpin conjugated PEG structure based on base recognition.3.Design of stapled end-hairpin based on chemical bond.These three new structural molecules not only have the basic characteristics of prodrug,that is,increase the stability of drugs,promote the long-term effect of drugs,but also can be transformed into active drug structures by sensitives bond breaking in response to specific environment in vivo.1.Structural design of self-assembled end hairpin based on base recognitionUsing anti-tumor antisense nucleic acid GTI-2040 as the model molecule,we designed and synthesized a series of prodrug nucleic acid molecules with conditionally responsive and breakable disulfide bonds,which can self-assemble into a terminal hairpin structure after solution annealing.These nucleic acid molecules have the following two characteristics:(1)the end of the hairpin structure raised enzyme resistant ability of the nucleic acid drug molecules from the space conformation.(2)under the condition of high concentration of GSH reducing tumor environment,sensitive bond is broken and molecular hairpin structure at the end of the screw(stem)zone formed by folding change from a chain base pairing to two chains base pairing,spiral thermal stability(Tm)fell sharply under physiological conditions of temperature,which prompted the antisense nucleic acids dissociate out from advanced structure and further combine with target.Through a series of enzymatic hydrolysis experiments(serum,plasma and DNase I),we confirmed that the new structural molecule protected by hairpin structure showed better resistance to enzymatic hydrolysis than the natural nucleic acid.Under the reducing condition of simulated tumor microenvironment,disulfide bond fracture occurred in all prodrug molecules,and the non-denatured gel electrophoresis experiment showed that the nucleic acid active agent sequence could be further dissociated.A preferred molecule(O2)effectively improved the anti-tumor cell proliferation and anti-solid tumor activity in vivo in cell experiments and tumor models in nude mice.2.Design of a self-assembled end-hairpin conjugated PEG structure based on base recognitionOn the basis of the structural design work in the first part,we further explore the efficient synthesis method of introducing short chain PEG into the curling end of molecular hairpin structure and the stability change of resistance to enzymatic hydrolysis.By two different synthetic methods,PEG chains can be conjugated efficiently.In vitro stability experiments confirmed that PEG-conjugated hairpin molecules could further increase their resistance to enzymolysis.Under simulated tumor reducing conditions in vitro,disulfide bond breakage occurred in both of the two PEG-conjugated hairpin molecules.Non-denatured gel electrophoresis experiments showed that the active nucleic acid sequences could be further dissociated.3.Design of stapled structure based on chemical bond.In this part of our work,we designed an intramolecular ring of prodrug antisense nucleic acid with stimulus-response characteristics by a simple and practical chemical method.In other words,the bis(2-hydroxyethyl)disulfide modified by two ends of carbonyl bromide is used as the binding molecule to react with the mercaptans modified by thiophosphoric acid(PS)in the nucleic acid sequence to form the local or global ring structure within the nucleic acid molecule from the two thiophosphoric acid position.The formation of the stapled structure was confirmed by mass spectrometry and non-denaturing gel electrophoresis.The stability experiment of enzyme hydrolysis in vitro showed that the antisense nucleic acid of the stapled structure showed good serum stability.The disulfide bond in the binding molecule is broken after DTT treatment and can be converted into a linear nucleic acid molecule combining with the target to play a role.Anti-tumor cell proliferation experiments showed that the antisense nucleic acid with the booklet structure had higher antisense activity than the natural sequence.To sum up,the design of this project is carried out from the perspective of nucleic acid molecular conformation,and the classical prodrug principle is fused into the antisense nucleic acid structure.The new molecule designed has unique conditional release and other mechanisms.This study can provide a valuable reference for the study of the relationship between conformation and stability of nucleic acid drugs,and also provide a new practice for the design of more nucleic acid molecular structure.