Design And Evaluation Of Antisense Oligonucleotides With New Mechanism And Structure

Objective:Nucleic acid drugs are the third pharmaceutical industry boom after traditional small molecule drugs and antibody drugs.As of December 2022,a total of 17 nucleic acid drugs have been approved by the FDA.Nucleic acid drugs can intervene at the source by inhibiting or up-regulating disease-related gene expression or by providing transcripts encoding target antigens or immunogens,achieving the effect of“curing the symptoms and the root cause”.However,due to the inherent limitations of nucleic acid molecules,such as easy degradation and difficult cellular uptake,overcoming these limitations is the main challenge for drug development in this field.Continuously innovating scientific concepts and designing new mechanism and structure nucleic acid molecules can provide novel solutions for key scientific questions such as anti-enzyme degradation,delivery,and therapeutic efficacy,which can help achieve breakthroughs.This study focused on antisense nucleic acids and aimed to improve stability against enzyme degradation and cellular uptake by applying multiple modification strategies and designing new structured and mechanism-based antisense nucleic acid drugs.Methods:Taking anti-tumor as a research model,three antisense oligonucleotide molecules with new mechanism and structure were designed,including:dumbbell-shaped antisense oligonucleotide prodrug based on dynamic conformational transformation strategy;anisamide-antisense oligonucleotide conjugate;loaded doxorubicin hairpin conformation anisamide-antisense oligonucleotide prodrug.We used ~1H-NMR and MS to confirm its structure,circular dichroism and T_m value to confirm its secondary structure,and simulated serum and DNase Ⅰenzyme to verify its anti-enzymatic stability.Fluorescence experiments and flow cytometry experiments were used to verify its cell uptake ability.In vitro anti-tumor experiments(such as tumor cell inhibition experiment,cell apoptosis experiment,and RT-qPCR experiment,etc.)were used to evaluate its tumor inhibitory activity,and then in vivo anti-tumor activity test further verify its anti-tumor activity.Results:1.In the work of dumbbell antisense oligonucleotide prodrug based on dynamic conformational transformation strategy,we propose a novel antisense oligonucleotide prodrug with dumbbell conformation and disulfide bond response switch function.The antisense oligonucleotides can be self-assembled to form a"dumbbell"conformational antisense oligonucleotide under solution annealing,and its structure was confirmed by CD.When in the"dumbbell"conformation,both ends of the antisense sequence are protected by the hairpin conformation,which can effectively improve the stability of anti-nuclease degradation.After the current drug molecules enter the tumor cells,the high concentration of glutathione environment will cause the response switch region(disulfide bond)to break,the"dumbbell"conformation transformed into a"chain"conformation,releasing the antisense functional region,namely"prodrug",and then recognize and bind to the target m RNA to play a therapeutic role.A series of degradation experiments(serum and DNase Ⅰenzyme)showed that the dumbbell antisense oligonucleotide prodrug had higher anti-enzymatic degradation stability than the natural or single-ended hairpin antisense oligonucleotide prodrug.When in the reductive microenvironment of the tumor,the disulfide bond is broken and the natural antisense oligonucleotides are dissociated.In addition,dumbbell antisense oligonucleotide prodrugs showed strong anti-tumor activity in-vitro and in-vivo(p<0.05).2.In the work of design of anisamide-antisense oligonucleotide conjugate,we proposed a conjugate of anisamide-antisense oligonucleotide,in which anisamide ligand molecules have high affinity for Sigma receptors on tumor cell surface.Modification of AA molecules on ASON is helpful to increase ASON uptake by tumor cells.In this work,the reaction of thio-modified mercaptan with anisamide ligand modified by carbonyl bromide was used to achieve efficient and flexible connection between small molecular ligands and antisense oligonucleotides in solution.The ligand was successfully linked to antisense oligonucleotides by MS.In addition,through in-vitro stability test,cell uptake test and in-vitro anti-tumor activity test,it was found that the binding site and number of anisamide ligand molecules on antisense oligonucleotides had effects on its anti-enzymolysis stability and anti-tumor activity.Among them,the antisense oligodeoxynucleotides(T6)conjugated with double-ended anisamide ligands have better anti-enzymolysis and cell uptake ability(p<0.05)and anti-tumor activity.Furthermore,the anti-tumor activity of T6 was verified by anti-tumor activity in-vivo and in-vitro(p<0.05).3.In the work of design of hairpin conformational anisamide-antisense oligonucleotide prodrug with doxorubicin,according to the previous research,antisense oligonucleotide prodrug with hairpin structure can improve its anti-enzymolysis ability and bring better anti-tumor activity.On this basis,this part of the work is further designed to introduce anisamide ligands at the end of the hairpin loop,and use the double helix structure of antisense oligonucleotide prodrug as a drug carrier to load anthracycline drugs,in order to achieve multi-mechanism synergistic treatment of tumor.In this part of the work,we chose doxorubicin(DOX)as the loading drug,and designed the functional molecules loaded with DOX hairpin conformational anisamide-antisense oligonucleotide prodrug.As in the first part of the work,with the disulfide bond breaking in the highly reducing tumor microenvironment,the"hairpin"conformation is transformed into a"chain"conformation,and the"original drug"and DOX are also released.The structure of functional molecules was confirmed by MS and fluorescence spectrophotometer,and the cellular uptake of anisamide was further verified by confocal microscopy.The better anti-tumor activity of functional molecules was also verified by anti-tumor activity test in-vivo and in-vitro.Conclusion:To sum up,in order to solve the scientific problems of cell entry ability and stability of nucleic acid drugs,the concepts of dynamic conformational transformation and conjugates are integrated into the structure of antisense oligo nucleotides,and three kinds of nucleic acid drugs with new structures and new mechanisms are designed.The new design of nucleic acid-based therapy in this study provides better stability and bioavailability(p<0.05).