Watson’s Nov­el CAP Attracts Glob­al Atten­tion in the mRNA Industry

Back­ground of Cap

mRNA vac­cines work by deliv­er­ing mRNA that encodes for an anti­gen into human cells, where it is trans­lat­ed to pro­duce the cor­re­spond­ing anti­gen pro­tein, there­by induc­ing an effec­tive immune response. The sta­bil­i­ty of mRNA, trans­la­tion effi­cien­cy, and inher­ent immuno­genic­i­ty are key to the suc­cess of mRNA as a ther­a­peu­tic. The 5’ cap struc­ture of mRNA pre­vents degra­da­tion by exonu­cle­as­es, recruits trans­la­tion ini­ti­a­tion fac­tors (eIF4E) to pro­mote ribo­some recog­ni­tion and ini­ti­a­tion of trans­la­tion, and pre­vents recog­ni­tion by innate immune recep­tors. There­fore, opti­miz­ing the mRNA cap struc­ture (cap) is cru­cial for enhanc­ing the bioac­tiv­i­ty of mRNA vac­cines and reduc­ing their immunogenicity.

Cur­rent­ly, co-tran­scrip­­tion­al cap­ping (“one-step” cap­ping) is the pre­ferred method for in vit­ro cap­ping of mRNA. How­ev­er, the com­plex­i­ty of the prepa­ra­tion process and the struc­tural­ly patent­ed cap ana­log mate­ri­als have become a ​“bot­tle­neck” in the mRNA sup­ply chain. New cap analogs that are free from patent bar­ri­ers, effec­tive in cap­ping, and cost-effi­­cient are the top choice for mRNA vaccine/​drug man­u­fac­tur­ing com­pa­nies, yet they also rep­re­sent a chal­leng­ing prob­lem that mRNA mate­r­i­al com­pa­nies urgent­ly need to solve !

Wat­son Bio, as an upstream mate­r­i­al sup­pli­er for mRNA vac­cines with core tech­nolo­gies, aims to achieve a top-down indus­tri­al chain sub­sti­tu­tion in the mRNA sec­tor. Its strong R&D team has been ded­i­cat­ed to devel­op­ing and opti­miz­ing new cap ana­log struc­tures and con­duct­ing R&D on cap ana­log mate­r­i­al prod­ucts with inde­pen­dent intel­lec­tu­al prop­er­ty rights, thus solv­ing prob­lems for mRNA researchers world­wide ! Pre­vi­ous­ly, Wat­son Bio had shared some data on its self-devel­oped cap ana­log prod­ucts. This post will specif­i­cal­ly show­case the research find­ings of Watson’s inde­pen­dent­ly devel­oped new cap ana­log, Cap5 (ENE), aim­ing to deliv­er a report card to the mRNA indus­try pro­fes­sion­als con­cerned with the exper­i­men­tal effects of new cap ana­log products !

Project Back­ground of Cap

Her­pes Zoster (HZ), also known as shin­gles, is caused by the reac­ti­va­tion of the latent Var­i­cel­la-Zoster Virus (VZV), which typ­i­cal­ly caus­es chick­en­pox in chil­dren. After recov­ery from chick­en­pox, the virus remains dor­mant in the body for a long time. With aging, as the immune func­tion grad­u­al­ly declines, the virus can reac­ti­vate and cause shin­gles. Unlike chick­en­pox, shin­gles does not confer ​“once infect­ed, life­time immu­ni­ty,” and over 90% of adults har­bor the latent Var­i­cel­la-Zoster Virus. To date, there are four shin­gles vac­cines approved for mar­ket­ing glob­al­ly, with the main tech­nol­o­gy plat­forms being live atten­u­at­ed vac­cines and recom­bi­nant pro­tein vac­cines. Live atten­u­at­ed vac­cines are less effec­tive in the elder­ly and can­not be used in immuno­com­pro­mised indi­vid­u­als ; while sub­unit vac­cines per­form bet­ter in old­er patients, they have more sig­nif­i­cant side effects. mRNA, as a new gen­er­a­tion vac­cine tech­nol­o­gy, with its rapid devel­op­ment advan­tages and plat­­form-based pro­duc­tion process­es, is poised to become a new option for the next gen­er­a­tion of her­pes virus vaccines.

Exper­i­men­tal Procedure

Wat­son col­lab­o­rates with part­ners on the VZV vac­cine project to design and devel­op mRNA vac­cines. Through a step-by-step exper­i­men­tal val­i­da­tion process — anti­gen selec­tion, sequence design, in vit­ro tran­scrip­tion, mRNA-LNP prepa­ra­tion, and ani­mal exper­i­ments — the designed VZV_​mRNA1 and VZV_​mRNA2 shin­gles mRNA vac­cines demon­strat­ed very strong immune effects in mice. Addi­tion­al­ly, in syn­the­siz­ing VZV_​mRNA1 and VZV_​mRNA2, Watson’s pro­pri­etary new cap ana­log Cap5(ENE) and the tra­di­tion­al m7GpppAmG were specif­i­cal­ly used to assess the exper­i­men­tal effec­tive­ness of Cap5(ENE).

Exper­i­men­tal Results of Cap

• Both mRNA and LNP met deliv­ery stan­dards.: Wat­son Bio pos­sess­es capa­bil­i­ties in the prepa­ra­tion and test­ing of mRNA/​LNP, and offers com­pre­hen­sive solu­tions in the devel­op­ment and process research of inno­v­a­tive ani­mal mRNA vac­cines in the field of vet­eri­nary bio­prod­ucts, such as RABV, FMDV, PEDV, PRV, PRRSV, ASFV, etc., with rich expe­ri­ence and mul­ti­ple suc­cess­ful cas­es. In this joint devel­op­ment of the VZV mRNA vac­cine, dif­fer­ent sequences were used for the prepa­ra­tion of mRNA/​LNP. Upon test­ing, the VZV-mRNA vac­cines all met the deliv­ery standards !

• Cell trans­fec­tion — high lev­els of tar­get pro­tein expres­sion : HEK293T cells were trans­fect­ed with 10μg of VZV_​mRNA1 and VZV_​mRNA2 LNP for­mu­la­tions, respec­tive­ly. 48 hours post-tran­s­fec­­tion, the expres­sion of the tar­get pro­tein gE was val­i­dat­ed via West­ern Blot using a spe­cif­ic anti­body (ab272686). The results indi­cat­ed that the gE pro­tein expres­sion lev­els of the Cap5(ENE) VZV mRNA vac­cines were sig­nif­i­cant­ly high­er than those of the CAP m7GpppAmG VZV mRNA vac­cines, with the over­all pro­tein expres­sion rate being 2.04 times and 1.72 times that of m7GpppAmG, respectively.

• Humoral immu­ni­ty — high titers of spe­cif­ic anti­bod­ies : Mice were inject­ed intra­mus­cu­lar­ly with 10μg of VZV_​mRNA1 and VZV_​mRNA2 vac­cines and a con­trol com­mer­cial vac­cine on D0 and D21. Blood was col­lect­ed from the orbit on D7 and D14 after each immu­niza­tion to iso­late serum for the detec­tion of serum gE-spe­­cif­ic IgG anti­body lev­els. The results showed that the anti­body titers of the Cap5(ENE) mRNA vac­cines were high­er than those of the CAP m7GpppAmG. Specif­i­cal­ly, the IgG anti­body lev­els of the VZV_​mRNA2 (Cap5(ENE)) vac­cine were 4.0 times (D7), 1.43 times (D14), 1.46 times (D28), and 2.24 times (D35) high­er than those of the VZV_​mRNA2 (CAP m7GpppAmG).

• Cel­lu­lar immu­ni­ty eval­u­a­tion — high IFNγ secre­tion : Mice were inject­ed intra­mus­cu­lar­ly with 10μg of VZV_​mRNA1 and VZV_​mRNA2 vac­cines and a con­trol com­mer­cial vac­cine on D0 and D21. The spleens were har­vest­ed on D7 and D14 after each immu­niza­tion, and splenic lym­pho­cytes were iso­lat­ed for the detec­tion of spleen gE-spe­­cif­ic lym­pho­cyte IFNγ secre­tion lev­els. The results indi­cat­ed that the secre­tion lev­els of IFNγ from the Cap5(ENE) mRNA vac­cines were high­er than those from CAP m7GpppAmG. Specif­i­cal­ly, the cytokine IFNγ secre­tion lev­els of the VZV_​mRNA2 (Cap5(ENE)) vac­cine were 1.07 times (D14), 1.06 times (D28), and 1.84 times (D35) high­er than those of the VZV_​mRNA2 (CAP m7GpppAmG).

Con­clu­sion

Cap5(ENE), prod­uct num­ber CAP5752

• The Cap5(ENE) mRNA vac­cine is con­sis­tent with the CAP m7GpppAmG mRNA vac­cine in terms of CMC prepa­ra­tion parameters.
• The tar­get pro­tein expres­sion lev­el of the Cap5(ENE) mRNA vac­cine at the cel­lu­lar lev­el is high­er than that of the CAP m7GpppAmG mRNA vaccine.
• In mice, both the humoral immu­ni­ty lev­el (IgG) and the cel­lu­lar immu­ni­ty lev­el (IFNγ) of the Cap5(ENE) mRNA vac­cine are high­er than those of the CAP m7GpppAmG mRNA vaccine.

Com­pared to the cap ana­log CAP m7GpppAmG, Wat­son Bio’s pro­pri­etary cap ana­log Cap5(ENE) employs the same method for in vit­ro tran­scrip­tion prepa­ra­tion of mRNA, requir­ing only a replace­ment of the cap ana­log dur­ing co-tran­scrip­­tion. Addi­tion­al­ly, Wat­son Bio has con­duct­ed a series of bio­log­i­cal val­i­da­tions and phar­ma­co­log­i­cal eval­u­a­tions of the cap ana­log, which show that mRNA pre­pared with Cap5(ENE) not only has high­er lev­els of tar­get pro­tein expres­sion but also exhibits high­er lev­els of both humoral and cel­lu­lar immu­ni­ty in mice than the com­mon­ly used cap ana­log CAP m7GpppAmG. Let’s look for­ward to the lat­er exper­i­men­tal results of Cap5(ENE), at which time Wat­son Bio will also share the data.