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1、Messenger RNA-Based Therapeutics and Vaccines:Whats beyondCOVID-19?Dongqiao Li,Cynthia Liu,Yingzhu Li,Rumiana Tenchov,Janet M.Sasso,Di Zhang,Dan Li,Lixue Zou,Xuezhao Wang,*and Qiongqiong Zhou*Cite This:ACS Pharmacol.Transl.Sci.2023,6,943969Read OnlineACCESSMetrics&MoreArticle Recommendations*sSuppor
2、ting InformationABSTRACT:With the rapid success in the development ofmRNA vaccines against COVID-19 and with a number of mRNA-based drugs ahead in the pipelines,mRNA has catapulted to theforefront of drug research,demonstrating its substantial effective-ness against a broad range of diseases.As the
3、recent globalpandemic gradually fades,we cannot stop thinking about what theworld has gained:the realization and validation of the power ofmRNA in modern medicine.A significant amount of research hasnow been concentrated on developing mRNA drugs and vaccineplatforms against infectious and immune dis
4、eases,cancer,andother debilitating diseases and has demonstrated encouragingresults.Here,based on the CAS Content Collection,we provide alandscape view of the current state,outline trends in the researchand development of mRNA therapeutics and vaccines,and highlight some notable patents focusing on
5、mRNA therapeutics,vaccines,and delivery systems.Analysis of diseases disclosed in patents also reveals highly investigated diseases for treatments with thesemedicines.Finally,we provide information about mRNA therapeutics and vaccines in clinical trials.We hope this Review will beuseful for understa
6、nding the current knowledge in the field of mRNA medicines and will assist in efforts to solve its remainingchallenges and revolutionize the treatment of human diseases.KEYWORDS:mRNA,vaccine,therapeutic,COVID-19,infectious disease,cancerThe COVID-19 mRNA vaccines were developed andapproved at unprec
7、edented speed and have demonstratedsignificant effectiveness against infections and acute COVID intherealworld.AlthoughtheideaofusingmRNAasasimpleandpromising way to deliver vaccinesor therapeutic drugshad beenaround for decades before the onset of the recent globalpandemic,the success of mRNA vacci
8、nes against COVID hascreated huge enthusiasm around this concept and significantlyboosted development and applications of this class of medicinesin other areas.As the pandemic gradually fades,we cannot stopthinking about what the world has gained in this chaos:therealization and validation of the po
9、wer of mRNA in modernmedicine.Messenger RNA(mRNA)is the molecule that carries geneticinformation from DNA in the cell nucleus to the cytosol forsynthesizing proteins by ribosomes.While most of conventionaltherapies work by binding and inhibiting hyperactive disease-causing proteins,mRNA therapies ca
10、n restore protein activitiesfor treating diseases caused by the loss of certain proteinfunctions.Moreover,mRNAtherapyisexplicit,asdefinedbythenucleic acid sequence,and very unlikely to have an off-targeteffect.Compared to antibody or cell therapies,mRNA is alsomuch easier to synthesize and purify on
11、 large scales.Anotheradvantage is that mRNA is transient and does not enter the cellnucleus;therefore,it is very unlikely to cause any geneticmutations in cells.Many key research findings have contributed to theadvancement of mRNAs medical applications.Early researchon mRNAs stability and translatio
12、nal activity provided thefoundation for developing mRNA-based vaccines and drugs.Comprehensive exploration of nucleic acids in the 1950s and1960s brought the discovery of mRNA.13Since then,mRNAhas been the subject of systematic basic and applied researchaimed at various diseases(Figure 1).Inthefirst
13、decadesafterits discovery,theresearchwasmainlyfocused on understanding the structural and functional aspectsof mRNA and its metabolism in eukaryotic cells,in parallel withdevelopingtoolsformRNArecombinantengineering.Later,the5-cap on mRNA was discovered.4,5In the 1980s,in vitroReceived:March 8,2023P
14、ublished:July 3,2023Reviewpubs.acs.org/ptsci 2023 The Authors.Published byAmerican Chemical Society943https:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969Downloaded via 38.181.76.100 on September 20,2023 at 07:28:39(UTC).See https:/pubs.acs.org/sharingguidelines for options
15、on how to legitimately share published articles.transcription from engineered DNA templates by means of abacteriophage SP6 promoter and RNA polymerase led to theproductionofmRNAincell-freesystems.6Althoughattemptsinusing liposomes to deliver mRNA into cells to induce proteinexpression date from the
16、1970s,7,8the invention of cationiclipids9was the decisive step in enabling nucleic acid transportintocellswhichresultedinthefirstcationiclipid-assistedmRNAdelivery.9,10In the 1990s,preclinical evaluation of in vitro mRNAtranscription began for applications such as protein substitutionand cancer and
17、infectious diseases vaccinations.1119In 1992,ateam of scientists working at Scripps Research Institute usedmRNA to transiently reverse diabetes insipidus in rats.11Albeitthe concept of mRNA vaccines sounds relatively new,it wasactually first suggested in 1995,for encoding cancer antigens.13The accru
18、ed expertise was valuable in solving some of theobstacles associated with mRNA pharmaceuticals such as itsshort half-life and unfavorable immunogenicity.In 2005,a solution was found on how to prevent activation ofthe immune response against the injected mRNA per se byinserting a naturally occurring
19、modified nucleoside:pseudour-idine.20The invention of the pseudouridine modification andfurther exploration on mRNA led to the first human trial of amRNA vaccine against melanoma in 2008.21In the followingyears,numerous preclinical and clinical trials on mRNA-basedvaccines against infectious disease
20、s and cancer were com-pleted.22,23In 2009,the first trial on cancer immunotherapyusing mRNA-based vaccines in human subjects with metastaticmelanoma was conducted.21In 2010,it was shown thatpseudouridine-modified mRNA might be applied as a safeapproach for effectively reprogramming cells to pluripot
21、ency.24The first clinical trial of personalized mRNA-based cancervaccine was performed in 2017.25In 2021,a successful use oflipid nanoparticles(LNPs)comprising Streptococcus pyogenesCas9 mRNA and a CRISPR guide RNA in patients withtransthyretin amyloidosis with polyneuropathy was reported.26Two huma
22、n mRNA vaccines against COVID-19 receivedEmergencyUseAuthorizationin2020andwerefinallyapprovedin 2021.2729This was only brought about by decades ofresearch on mRNA-based therapeutics.The lessons learnedduring the COVID-19 mRNA vaccines development wererecently applied in formulating a multivalent nu
23、cleoside-modified mRNA flu vaccine.30As it became clear that mRNA vaccines provide a promisingalternative to conventional vaccine approaches due to their highefficiency,potential for rapid development,low-cost manufac-ture,and capacity for scale-up,as well as safe administration,significantefforts h
24、avebeenconcentrated ondevelopingmRNAdrug and vaccine platforms against infectious diseases,cancer,and other debilitating diseases and have demonstratedencouraging results.ThisReviewprovidesadetailedoverviewofmRNAdrugsandvaccines and considers future directions and challenges inadvancing this promisi
25、ng platform to widespread therapeuticuse.We examine data from the CAS Content Collection,46thelargest human-curated collection of published scientificinformation and analyze the publication landscape of recentresearch in order to reveal the research trends in publisheddocumentsandtoprovideinsightsin
26、tothescientificadvancesinthearea.Wealsodiscusstheevolutionofthekeyconceptsinthefield,the major technologies,and their development pipelineswithcompanyresearchfocuses,diseasecategories,developmentstages,and publication trends.We hope that this report canserve as a useful resource for understanding th
27、e current state ofknowledge in the field of mRNA medicines and the remainingchallenges to fulfill the potential of this new class of medicines.Figure1.AtimelineofthemilestonediscoveriesandkeytechnologiesleadingtothesuccessfuldevelopmentofmRNAtherapeuticsandvaccines;workfrom references 1,4,6,913,2022
28、,24,25,2845.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969944LANDSCAPE OF SCIENTIFIC PUBLICATIONSRELATED TO mRNA THERAPEUTICS ANDVACCINESTrend of mRNA Publications over Time.Based on theanalysis of CAS docum
29、ent collection,a total of 9,322 researchpapers have been published in the field of mRNA therapeuticsand vaccines.Due to the small number of published papersbefore 2000,the analysis of the development trend focused onthose papers published since 2000.As shown in the left panel ofFigure 2,the number o
30、f published papers in this area showed aslowgrowthpriorto2020followedbyasignificantincreaseeachyear afterward.From 2000 to 2019,the annual number ofpublicationsintheglobalmRNAfieldwaslessthan200,andthegrowth in publication was relatively slow.Due to the impact ofthe novel coronavirus outbreak at the
31、 end of 2019,mRNAtechnology has attracted wide attention from researchers.After2020,the number of published papers in this area has shown arapid growth trend,with the number of published papersincreasing to 3,361 in 2021 and nearly 5,000 in 2022.A total of 2,089 patents related to mRNA therapeutics
32、andvaccines have been published worldwide,according to CASdocumentcollection.Duetothesmallnumberofpatentsbefore2000,the trend analysis also focused on the analysis of patentssince 2000.As shown in the right panel of Figure 2,the numberof patents each year grew slowly from 2000 to 2010 with somefluct
33、uations,and the annual publications were all below 30.Between 2011 and 2019,the number of mRNA-related patentsworldwideincreasedfrom46to177eachyear.StimulatedbytheCOVID-19 pandemic,the annual number of patents increaseddramatically after 2020,increasing to 382 in 2021 and likely tonearly450in2022.We
34、alsoperformedtrendanalysesforpatentson mRNA therapeutic,mRNA vaccines and delivery systemsseparately.The results for those are described in the SupportingInformation.DISTRIBUTION OF RESEARCH TOPICSDistribution of Topics in Journal Publications.Thisreport then examined CAS-indexed concepts in mRNAthe
35、rapeutic and vaccine journal articles in order to revealemerging trends or the more specific focus of research anddevelopment in this field(Figure 3).From the distribution ofresearch topics based on the index concepts,it seems that theFigure 2.Annual number of published journal articles(left)and pat
36、ents(right)on mRNA therapeutics and vaccines.The data for 2022 includeextrapolated numbers for October to December 2022.Figure 3.Concepts and their co-occurrence in journal articles related to mRNA therapeutics or vaccines.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/
37、10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969945research has thus far focused on research in immunology,mechanisms of action,and disease indications.Among theimmune research(red dots),key concepts include immunoglo-bulin G,viral spike glycoproteins,neutralizing antibodies,immunogeni
38、city,etc.In terms of mechanism of action studies(green dots),key concepts include signal transduction,tran-scriptional regulation,genetic elements,RNA splicing,etc.Inthe treatment of diseases(blue dots),key concepts includediabetes,hypertension,myocarditis,and cardiovascular disease.Intermsofindicat
39、orstudies(yellowdots),keyconceptsincludec-reactive protein,leukocyte,blood platelet,etc.Conceivably,due to the outbreak of the COVID pandemic and thedevelopment of mRNA vaccines against this disease,the clustersurrounding coronavirus infection accounts for a very largeportion of journal publications
40、.Distribution of Topics in Patents.Key concepts in thefield of mRNA therapeutics and vaccines were also examinedwith a concept cluster analysis for patents within the CASContent Collection(Figure 4).Unlike journals,a significantportion of the patents focused on drug delivery such asnanoparticles(red
41、 dots)and immunotherapy in addition toSARS-CoV-2-related studies,etc.In terms of immunotherapy(green dots),key concepts include chimeric fusion proteins,chimeric antigen receptors,cancer immunotherapy,T cellreceptors,etc.In other treatment aspects(blue dots),keyconcepts include RNA vaccines,immune a
42、djuvants,etc.Thehigh occurrence of all these terms reflects the emphasis of mostR&D activities in this area.MAJOR COUNTRIES CONTRIBUTING TO THE R&DOF mRNA THERAPEUTICS AND VACCINESTop Countries/Regions.Among the top 20 countries andregions in terms of scientific research output(Figure 5),theUnited S
43、tates has the largest scientific research output in bothjournalarticlesandpatents,with29.8%and45.9%oftheworldsFigure 4.Concepts and their co-occurrence in patents related to mRNA therapeutics or vaccines.Figure 5.Major countries/regions with journal articles and patentsrelated to mRNA therapeutics o
44、r vaccines.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969946total output in these two types of publications,respectively,andsignificantly outnumbers those by other countries/regions.Germany ranks distantly t
45、he second in total scientific output,accounting for 7.4%of journal articles and 16.5%of patents.China ranks the third in scientific research output,with 6.8%ofjournalarticlesand13.4%ofpatents.Italy,Japan,andtheUnitedKingdom ranked fourth,fifth,and sixth,respectively,in totaloutput.Patent Output Tren
46、d over Time for Top Countries.Figure6showsthecontributionsbythetopfivecountriesfortheFigure 6.Journal publications(left)and patent output(right)from the top five countries during 19702019 vs 20202022.Figure7.Flowofpatentapplicationsfromthetopsixcountriestopatentofficesaroundtheworld.Note:Thecolumnon
47、theleftisforhomecountriesfrom which the patent assignees are located.The middle column represents the patent offices around the world that the priority patents in the patentfamilies were filed to.The right column represents countries/regions where the intellectual properties are protected when those
48、 patents were appliedthrough WIPO.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969947periods of 19702019 and 20202022(the period followingthe outbreak of the COVID-19 pandemic).The U.S.is in thelead position i
49、n both periods for both journal and patentpublications.The number of journal articles published by Chinafrom 1970 to 2019 was slightly lower than that of Germany,butincreased rapidly during the period of 20202022,exceedingGermany during this period.The numbers of journal articlespublished by Italy a
50、nd Japan were also small(390)during 20202022(left panel).The numbers of patent applications by organizations in theU.S.and Germanyduring 19702019werehigher thanthosein20202022,indicating the presence of sufficient R&D activitiesin these two countries before the onset of COVID-19(Figure 6,right panel
51、).While the numbers of patents from these twocountriesdecreasedduringthethree-yearperiodof20202022,the numbers were still higher than those of other countries,reflecting their leading roles.The trend of patent output fromChina appears to be different from that of other countries asindicated by the l
52、ow activity during 19702019 and 5-foldincrease during the period of 20202022,indicating that ChinaisrapidlyincreasingitstechnologicaldevelopmentinthemRNAtherapeutics and vaccines due to the impact of the COVID-19pandemic.Patent Filing Strategies Revealed by Analysis ofPatent Application Flows among
53、Major Countries/Regions.This report then examined the pattern of patentapplication flow related to technology development of mRNAtherapeutics and vaccines.Our studies show that the U.S.,Germany,the U.K.,Japan,and Italy all have applied fortechnology protection in some overseas markets(Figure 7).Inco
54、ntrast,the majority of patents initiated by organizations inChina were filed domestically to the Chinese Patent Office withsome to the World Intellectual Property Organization,indicating that those Chinese organizations have placed lessemphasis on seeking overseas protection of their intellectualTab
55、le 1.Top 15 Organizations Publishing Journal Articles on RNA Therapeutics or VaccinesaRankingOrganizationNo.of journalpublicationsCountryOrganization typeNo.of journal publications in the past 3 years(%of total)1Harvard University108U.S.University91(84.3%)2University of California84U.S.University60(
56、71.4%)2University of Pennsylvania84U.S.University52(61.9%)4Tel Aviv University74IsraelUniversity67(90.5%)5Johns Hopkins University71U.S.University58(81.7%)6Moderna62U.S.Company35(56.5%)7Washington University58U.S.University39(67.2%)8University of Oxford57U.K.University50(87.7%)9Centers for Disease C
57、ontrol andPrevention55U.S.Scientific researchinstitution52(94.5%)9National Institutes of Health55U.S.Scientific researchinstitution48(87.3%)9Yale University55U.S.University50(90.9%)12The University of Hong Kong53ChinaUniversity46(86.8%)13Cornell University51U.S.University45(88.2%)13Mount Sinai Hospi
58、tal51U.S.Scientific researchinstitution48(94.1%)13Stanford University51U.S.University35(68.6%)aData from the CAS Content Collection.Table 2.Top 15 Organizations with Patent Applications on RNA Therapeutics or VaccinesaRankingOrganizationsNo.of patentapplicationsCountryOrganization typeNo.of patent a
59、pplications in the past 3 years(%of total)1Moderna207U.S.Company61(29.5%)2CureVac150GermanyCompany15(10.0%)3BioNTech135GermanyCompany59(43.7%)4Translate Bio78U.S.Company48(61.5%)5Tron53GermanyCompany11(20.8%)6Alnylam Pharmaceuticals30U.S.Company2(6.7%)7Shire Human Genetic Therapies28U.S.Company0(0.0
60、%)8University of Pennsylvania27U.S.University13(48.1%)9Arcturus Therapeutics23U.S.Company8(34.8%)10Acuitas Therapeutics21CanadaCompany5(23.8%)11Chinese Academy of Sciences17ChinaScientific researchinstitution12(70.6%)12Massachusetts Institute ofTechnology16U.S.University4(25.0%)12University of Calif
61、ornia16U.S.University6(37.5%)14Ethris15GermanyCompany2(13.3%)15Evox Therapeutics14U.K.Company4(28.6%)aData from the CAS Content Collection.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969948properties.TheU.S.,
62、Germany,theU.K.,Japan,andItalymainlydistributed patents to other countries through the WorldIntellectualPropertyOrganization.Amongthem,thenumberofpatents distributed through the World Intellectual PropertyOrganization in the U.S.,Germany,Japan,and Italy accountedfor more than 90%,and the number of p
63、atents distributedthrough the World Intellectual Property Organization in theU.K.accounted for more than 75.0%.The U.S.ranks first in thevolume of patents distributed through the World IntellectualProperty Organization,followed by Australia,Canada,Japan,and China.Countries such as the U.S.,Australia
64、,Canada,Japan,and China are also preferred when distributing patents throughthe European Patent Office.MAJOR ORGANIZATIONS CONTRIBUTING TO THER&D OF mRNA THERAPEUTICS AND VACCINESOrganizations in Journal Publications.Among the top15 organizations in the world publishing mRNA journal articlesin this
65、area(Table 1),12 are from the U.S.,indicating that theU.S.has a predominant role in basic research.Israel,the U.K.,and China each has one organization ranking among the top 15.In terms of the nature of the top 15 organizations,11 areuniversities,3 scientific research institutions,and 1 company.The t
66、op three are Harvard University,the University ofCalifornia,and the University of Pennsylvania,with 108,84,and84journal articles,respectively.TelAviv UniversityinIsraelranks fourth globally,with 74 papers.Johns Hopkins University,Moderna,Washington University,the U.S.Centers for DiseaseControl and P
67、revention,and the U.S.National Institutes ofHealth all ranked in the top 10.The University of Oxford in theU.K.ranks eighth in the world,with 57 articles.It may be worthmentioning that the U.S.Centers for Disease Control andPrevention and Mount Sinai Hospital account for more than94%of the published
68、 journal articles in 20202022,indicatingthe high level of dedication of these two organizations to thisareainrecentyears,probablyrelatedtotheeffortonfightingtheCOVID-19.Organizations in Patent Publications.Amongthe top 15organizations with a high number of patents in this area(Table2),the dominant o
69、rganizations are mainly located in the U.S.(8outof15)followedbyGermany(4 outof15).Amongthesetoppatent applicants,companies are the main source of patents(11out of 15).Thus,as expected,the major R&D effort ofcommercialcompanieshasbeenfocusingonthedevelopmentofpatentable technologies in this area.Mode
70、rna of the U.S.hasproduced most patents,followed by CureVac and BioNTech ofGermany.The Chinese Academy of Science had the highestpercentage of patents in the past three years,accounting for70.6%,which may be indicative of an emphasis of R&D in thisarea by this organization in more recent years.PATEN
71、T DISTRIBUTION AMONG KEYTECHNOLOGIESFrom the perspective of technology classification,mRNApatents mainly include therapeutic technology,deliverytechnology,vaccine technology,and mRNA modificationtechnology(Figure 8).Out of 2,089 patents,1,129 patents arerelated to the development of mRNA therapeutic
72、s for diseasetreatment,977 related to development of delivery technology,and 659 related to vaccines.The total number is larger than2,089 because some patents covered more than one specifictechnology area.The number of patent applications in thesethree types of technology account for 93.3%of the tot
73、al patents,whereas the number of patents about mRNA modificationtechnologyisrelativelysmall,thoughthistechnologyiscrucialtothe success of mRNA vaccines and therapeutics.Analysis of Patents Related to mRNA Therapeutics.Top Countries/Regions with Published Patents Related tomRNA Therapeutics.Figure 9
74、shows the top 10 countries/regions where patent applicants in the field of mRNAtherapeutics are located.The U.S.and Germany are the toptwo countries with 550 and 214 patents,respectively.Thepatentsfromthese twocountriesaccount forabout67.7%ofthetotal global patents in this area,reflecting the predom
75、inant roleof these two countries in this area.China has filed 89 patents inthis field,ranking distantly third.Top Organizations with Published Patents Related tomRNA Therapeutics.Table 3 lists the top 11 organizations withpatents related to mRNA therapeutics.As can be seen from thetable,most of thes
76、e organizations are in the U.S.(six)andGermany(four)with one in the U.K.Most of these patentapplicants are commercial companies(nine)and only two areuniversities,indicating a heavy role of commercial companies inleading the R&D effort on mRNA therapeutics.The top fivecompanies are Moderna,BioNTech,C
77、ureVac,Translate Bio,and Tron,with Moderna holding the largest number of patents(121).Figure 8.Distribution of patents among key classes of mRNA therapeutics and vaccines.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.202
78、3,6,943969949NOTABLE PATENTS RELATED TO R&D OF mRNATHERAPEUTICSTable4highlightsseveralofthemostnotablepatentsfocusedonthe development of mRNA therapeutics.Patent application WO2020097409 by Moderna,USAfeatures methods for treating ovarian cancer,as well as othercancers such as solid tumors,lymphomas
79、,and epithelial origincancers,by administering mRNA encoding an OX40Lpolypeptide,alsoknownasCD252.Thedisclosurealsopresentspharmaceutical composition for intratumoral administrationcomprising lipid nanoparticles with a mRNA encoding a humanOX40L.The disclosure also features combination therapies,suc
80、h as the use of mRNA encoding an OX40L polypeptide incombination with a checkpoint inhibitor,such as an anti-PD-L1antibody.Patent application WO2021198157 by BioNTech,Germany,provides RNA technologies for targeting Claudin-18.2(CLDN-18.2)polypeptides.SuchRNAtechnologiescanbeusefulforthetreatment of
81、Claudin-18.2 pos.cancer,including biliary cancers,ovarian cancers,gastric cancers,gastroesophageal cancers,andpancreatic cancers.Noteworthy is a mRNA formulationencoding monoclonal IgG1,such as Zolbetuximab(Claudix-imab).Patent application WO2018160540 by Sanofi,France,andBioNTech,Germany,relates to
82、 the field of therapeutic mRNAsfor the treatment of solid tumors,including medical preparationcomprising mRNA encoding an IL-12sc protein,an IL-15 sushiprotein,anIFNprotein,andaGM-CSFprotein.Thedisclosedpharmaceutical formulations are for use in a method ofpreventing cancer metastasis.Patent applica
83、tion WO2020260685 by eTheRNA Immuno-therapies,Belgium,relatestocombinationsofmRNAsencodingCD40,caTLR4,and CD70 with mRNAs encoding tumor-associated antigens for use as therapeutic vaccine in thetreatmentofmetastaticcancerpatients,primarilywithmalignantmelanoma disease,but also to other cancer types.
84、The disclosedtherapies may further encompass the administration ofcheckpoint inhibitors.The invention provides administrationschemes focusing on administration of the therapeutic intolymph nodes,the so-called intranodal therapy.Analysis of Patents Related to mRNA Vaccines.TopCountries/Regions with P
85、ublished Patents Related to mRNAVaccines.The patent applicants in the global mRNA vaccinefield are mainly from the U.S.,China,Germany,and countriesand regions shown in Figure 10.The U.S.,China,and Germanyhave 220,144,and 134 patent applications respectively,makingthem the three countries with the st
86、rongest strength in thistechnology field.It needs to point out that China does not yethave a mRNA vaccine on market probably due to its lateinvolvement in this area and thus the patent data here does notnecessarilyreconcilewiththemarket dataand correlatewiththeimpact.Countries such as Belgium,the U.
87、K.,and France haveless than 30 patent applications in this field.Distribution of mRNA Vaccine Patents among TopOrganizations.Table 5 lists the top 12 organizations in termsof mRNA vaccine patent output.Among them,five are from theU.S.,three from Germany,two from China,and one each fromthe U.K.and Be
88、lgium.Like other technical fields,companies arethe main contributors of mRNA vaccine patents(8 out of 12).The top 3 companies are CureVac(92),Moderna(59),andBioNTech(38).Most Notable Patents Related to mRNA Vaccine R&D.Over 650 patents related to mRNA vaccine development withtheir associatedinformat
89、ionwereidentifiedin thisstudy.Tables6 and 7 list several intriguing patents focused on the mRNAvaccinesforinfectiousdiseasesandcancers,respectively,thetwomajor disease classes for which mRNA vaccines are beingdeveloped.Patent application WO2021255270(Ziphius Vaccines,Belg.;Universiteit Gent)disclose
90、s a self-amplifying COVID-19vaccine comprising a mRNA encoding SARS-CoV-2 spikeprotein,nucleocapsid protein,and alphavirus nonstructuralproteins(nsp14)in thermally stabilizing RNA vaccineformulation.Patent application WO2022129918(ImperialCollege Innovations Limited,U.K.)discloses novel uses andmeth
91、ods for thermally stabilizing RNA vaccine formulations,including self-amplifying RNA replicons derived from Ven-ezuelan Equine Encephalitis virus encoding SARS-CoV-2 spikeprotein encapsulated in lipid nanoparticles.The higher andFigure9.Distributionofthetop10countries/regionsintheglobalfieldof mRNA
92、therapeutic patents.Table 3.Top Patent Applicants for the Development ofmRNA TherapeuticsaRankingOrganizationsNo.ofpatentapplicationsCountryOrganizationtype1Moderna121U.S.Company2BioNTech92GermanyCompany3CureVac79GermanyCompany4Translate Bio36U.S.Company5Tron34GermanyCompany6Shire HumanGeneticTherap
93、ies22U.S.Company7University ofPennsylvania16U.S.University8The Ohio StateUniversity15U.S.University9ArcturusTherapeutics13U.S.Company10Ethris10GermanyCompany10Evox Therapeutics10U.K.Company10MassachusettsInstitute ofTechnology10U.S.UniversityaData from the CAS Content Collection.ACS Pharmacology&Tra
94、nslational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969950prolonged in vivo translation improves the efficacy of self-amplifying RNA vaccines even in low dose.Patent application WO2022137133(CureVac AG andGlaxoSmithKline Biologicals)dis
95、closes that the mRNA vaccineencoding variants of highly immunogenic SARS-CoV-2 spikeprotein in lipid nanoparticle formulation induces neutralizingantibodies and immune cell responses against SARS-CoV-2.Patent application WO2021155243(Moderna)discloses avaccine comprising a codon optimized human resp
96、iratorysyncytial virus(hRSV)nucleic acid encoding a stabilizedprefusion form of an hRSV F glycoprotein variant formulatedin the lipid nanoparticles.In vivo study was conducted toevaluate the immunogenicity,efficacy,and safety of the mRNAvaccine in mice and the RSV cotton rat model.Patent application
97、 WO2022116528(Suzhou CureMedBiomedical Technology)discloses a circular RNA(circRNA)vaccine comprising a specific internal ribosome entry site(IRES)element and receptor domain of SARS-CoV-2 spikeprotein without 5 or 3 ends.The covalently closed structure ofcircRNA prevents the degradation by exonucle
98、ases andimproves its biostability.They exemplified the furtherTable 4.Notable Patents on mRNA TherapeuticsPatent numberOrganizationPatent titleWO2013096709Moderna,USAIncreasing the viability or longevity of an organ or organ explant using modified mRNAs for proteins essential fororgan survivalWO2015
99、058069Moderna,USAPolynucleotides for tolerizing cellular systemsWO2016201377Moderna,USAPreparation of targeted adaptive vaccines for treatment of inflammatory disease,autoimmune disease and cancersWO2017214175Moderna,USAModified RNA encoding VEGF-A in formulations for treatment of heart failure and
100、other diseasesWO2018160540Sanofi,France;BioNTech,GermanyTherapeutic RNA and uses in treating solid tumor cancersWO2018222890Arcturus Therapeutics,USASynthesis and structure of high potency RNA therapeuticsWO2019178006SQZ Biotechnologies Co.,USAImmunogenic epitope and adjuvant-modified T cells for in
101、tracellular delivery of tumor or exogenous antigen toenhance immune response against cancer and infectionWO2020056147Moderna,USAPolynucleotides encoding glucose-6-phosphatase for the treatment of glycogen storage diseaseWO2020097409Moderna,USAUseofmRNAencodingOX40Lincombinationwithimmunecheckpointin
102、hibitortotreatcancerinhumanpatientsWO202011811Arcturus Therapeutics,USACompositions and methods for treating ornithine transcarbamylase deficiencyWO2020154189Sanofi,FranceTherapeutic RNA for treatment of advanced stage solid tumorWO2020227615Moderna,USAPolynucleotides encoding methylmalonyl-CoA muta
103、se for the treatment of methylmalonic acidemiaWO2020260685eTheRNAImmunotherapies,BelgiumAntitumor therapy comprising mRNA molecules encoding tumor-associated antigens and checkpoint inhibitorsWO2021021988Translate Bio,USATreatmentofcysticfibrosisbydeliveryofnebulizedmRNAencodingCysticFibrosisTransme
104、mbraneConductanceRegulator(CFTR)WO2021058472BioNTech and TRON,GermanyCombination treatment using therapeutic antibody and interleukin 2(IL-2)WO2021198157BioNTech,GermanymRNA compositions(RiboMab)expressing claudin-18.2-targeting antibody and anticancer uses thereofWO202120771Verve Therapeutics,USABa
105、se editing of ANGPTL3 and methods of using same for treatment of cardiovascular diseaseWO2021214204BioNTech,GermanyRNA constructs and uses thereofWO2022136266BioNTech,GermanyTherapeutic RNA for treating cancerFigure 10.Distribution of mRNA vaccine patents among countries/regions with the largest num
106、bers of patents.Table 5.Global Patent Applications by Major Institutions inthe Field of mRNA VaccinesaOrderOrganizationNo.of patentapplicationsCountryOrganizationtype1CureVac AG92GermanyCompany2Moderna59U.S.Company3BioNTech38GermanyCompany4TRON GmbH21GermanyCompany5Chinese Academyof Sciences10ChinaS
107、cientificresearchinstitution6GlaxoSmithKlineBiologicals S.A.10U.K.Company7Translate Bio7U.S.Company8CanSino Biologics6ChinaCompany8eTheRNAImmunotherapies6BelgiumCompany8University ofCalifornia6U.S.University8University ofFlorida6U.S.University8University ofPennsylvania6U.S.UniversityaData from the C
108、AS Content Collection.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969951application for making circRNAs encoding erythropoietin,anti-PD1 antibody,interleukin 15,prostate cancer specific antigenPAP,and CD16 CA
109、R receptor for protein expression in 293Tcells.Patent application US20220325255(University of Texas)disclosesantiviralcompositionsincludingmRNAencodingforaTRIM7proteinencapsulatedintoalipidnanoparticle(LNP),aswellasmethodsforimpairingenterovirusreplicationfortreatingviral infections.The disclosure p
110、rovides for the first time E3ligase targeting an enterovirus protein and the first demon-stration that a viral membrane remodeling protein is subject todegradation as a host antiviral strategy.Patent application WO2021155149 by Genentech,BioN-Tech,and Hoffmann-La Roche AG discloses mRNA vaccinescomp
111、osed of mRNAs encoding up to 20 neoepitopes(twodecatopes)deriving from cancer-specific mutations in patientsformulated in cationic liposomes(Table 7).The first-in-humanphase Ia and Ib studies of a mRNA vaccine as a monotherapyand in combination with atezolizumab were conducted inpatientswithadvanced
112、ormetastaticsolidtumors.Theyshowedinnate and neoantigen-specific immune responses induced bythe mRNA vaccine alone and combined with atezolizumab.Patent application WO2015024664 by CureVac disclosesdevelopment of a mRNA-based personalized cancer vaccineencoding prostate cancer-associated antigens,pr
113、ostate-specificantigen),PSMA(prostate-specific membrane antigen),PSCA(prostate stem cell antigen),STEAP(six transmembraneepithelial antigen of the prostate),MUC1(mucin 1)and PAP(prostatic acid phosphatase)for treating prostate cancer.Patent application WO2016180467 by BioNtech and TRONTranslationale
114、 Onkologie Mainz discloses administering to themammal T cells genetically modified to express a chimericantigen receptor(CAR)targeted to antigen.Antigen is selectedfrom claudin 18.2,claudin 6,CD19,CD20,CD22,CD33,CD123,mesothelin,CEA,c-Met,PSMA,GD-2,or NY-ESO-1.CAR-transgenic human CD8+T cells target
115、ing claudin-6proliferated in response to CLDN6-transfected autologousdendritic cells.Murine CLDN6-CAR T cells were able toproliferate strongly in response to murine BMDCs expressinghuman CLDN6 antigen after RNA transfer.Patent application WO2020097291 by Moderna disclosesmRNA cancer vaccines compose
116、d of mRNAs encoding 350neoepitopesformulatedincationiclipidnanoparticles.AphaseIstudy was undertaken to assess the safety,tolerability,andTable 6.Notable Patents Focused on the Development of mRNA Vaccines for Infectious DiseasesPatent numberOrganizationPatent titleWO2021213924BioNTech,GermanyCorona
117、virus RNA vaccine encoding SARS-CoV-2 spike protein for preventing COVID-19WO2020190750Moderna,U.S.Dept.ofHealth and Human Serv-ices,USAPreparation of HIV Env-and lentivirus Gag protein-encoding mRNA VLP vaccine to induce broad-spectrum neutralizingantibodies for treating HIV infectionWO2018151816Mo
118、derna,USAImmunogenic compositions forZikavirus including cationiclipid nanoparticles encapsulating mRNAhavingan openreadingframe encoding a viral,bacterial or parasitic antigen,a pan HLA DR-binding epitope(PADRE)and a 5 terminal capmodified to increase mRNA translation efficiencyWO2021155243Moderna,
119、USARespiratory virus vaccine compositionsWO2013055905Novartis,SwitzerlandRecombinant self-replicating polycistronic RNA molecules expressing multiple herpes virus proteins and their use in vaccinesfor inducing neutralizing antibodiesWO2021159040Moderna,USAEngineering SARS CoV-2 mRNA vaccines express
120、ing key neutralizing domains of spike protein,individually or incombination,for inducing protective immunity and immunotherapyWO2021251453Daiichi Sankyo,Universityof Tokyo,JapanNucleic acid lipid particle vaccine encapsulated with severe acute respiratory syndrome coronavirus 2 messenger ribonucleic
121、acidWO2021159130Moderna,USA;U.S.Dept.of Health and HumanServices,USAPreparation of SARS CoV-2 mRNA vaccines encoding full-length spike protein variant,stabilized into a prefusionconformation,encapsulated in a lipid nanoparticle formulationWO2021255270Ziphius Vaccines,Belgium;Universiteit Gent,Bel-gi
122、umSelf-amplifying COVID-19 RNA vaccine encoding SARS-CoV-2 Spike and Nucleocapsid protein antigen and alphavirusNonstructural proteinWO2017070613Moderna,USAHuman cytomegalovirus RNA vaccinesWO2021204179Suzhou Abogen Bioscien-ces,ChinaNucleic acid vaccines for coronavirusWO2021226436Translate Bio,USA
123、;Sanofi,FranceOptimized nucleotide sequences encoding SARS-COV-2 antigensWO2017070623Moderna,USAHerpes simplex virus RNA vaccineWO2021160346Institut Pasteur,FranceNucleic acid vaccine against severe acute respiratory syndrome coronavirus SARS-CoV-2WO2022171182Stemirna Therapeutics,ChinaVaccine reage
124、nt for treating or preventing coronavirus mutant strainCA3132188Providence TherapeuticsHoldings,CanadaCompositions and methods for the prevention and/or treatment of COVID-19WO2021183563Arcturus Therapeutics,USACoronavirus vaccine compositions and methodsWO2022150717Moderna,USASeasonal RNA influenza
125、 virus vaccinesWO2022129918Imperial College,UK Inno-vations Ltd,U.K.Engineering a thermally stabilized self-amplifying RNA vaccine based on Venezuelan Equine Encephalitis virus backboneencodingSARS-CoV-2spikeglycoproteinencapsulatedinlipidnanoparticleformulationforpreventingand/ortreatmentofCOVID-19
126、WO2022178196Sanofi Pasteur,USAMeningococcal B recombinant vaccineWO2022137133CureVac,Germany;Glax-oSmithKline BiologicalsSA,U.K.RNA vaccine against SARS-CoV-2 variantsWO2022116528Suzhou CureMed Biomed-ical Technology,ChinaCircular RNA vaccine containing circular RNA and kit for detecting novel coron
127、avirus neutralizing antibodyUS20220325255University of Texas,USACompositions and methods for treating viral infections targeting TRIM7ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969952immunogenicity of mRNA v
128、accine monotherapy in patientswith resected solid tumors and in combination withpembrolizumab in patients with unresectable solid tumors.Arandomized phase II clinical study was conducted in patientswith resected cutaneous melanoma.ANALYSIS OF PATENTS RELATED TO mRNADELIVERY SYSTEMSTop Countries/Regi
129、ons with Published Patents Re-lated to mRNA Delivery Systems.Patent applications in thefieldofmRNAdeliverysystemsaremainlyfromtheU.S.,China,Germany,and other countries,as shown in Figure 11.The U.S.has the largest number of patents(480),which equals to almostthe combined number of patents from other
130、 countries.Top Organizations with Published Patents Related tomRNADeliverySystems.AsshowninTable8,likethatinthefield of mRNA therapeutics,global patent applicants in the fieldofmRNAdeliverysystemsareprimarilylocatedintheU.S.(6oftop 10),Germany(2 out of 10),Canada(1 out of 10),and theU.K.(1 out of 10
131、.In terms of the nature of organizations,companies are the main source of these patents(9 out of 10),The top 5 patent applicants are Modena,Translate Bio,CureVac,Alnylam Pharmaceuticals,and BioNTech withModena having the largest number of patents(64).Key Technology Layout in the Field of mRNAModific
132、ation.From the annual trend,the number of globalpatentapplicationsinthefieldofmRNAmodificationfluctuates,with a significant increase and peak in 2013,followed by adecline(Supplemental Figure S3).The trend of patentapplications was relatively stable from 2015 to 2018,and aftera brief decline in 2019,
133、the number of patent applicationsshowed a slow growth trend from 2020.From the distribution of patent research topics,the globalfield of mRNA modification focuses on drug delivery types,carriermaterials,nucleicacidmodification,mechanismresearch,and other aspects(Supplemental Figure S4).Among them,in
134、Table 7.Notable Patents Focused on the Development of mRNA Vaccines for CancerPatent numberOrganizationPatent titleWO2021155149Genentech,USA;BioNTech SE,Germany;F.Hoffmann-La Roche,SwitzerlandMethodsofinducingneoepitope-specificTcellswithaPD-1axisbindingantagonistandan RNA vaccineWO2015024664CureVac
135、,GermanyComposition comprising mRNA encoding a combination of tumor antigens as vaccinefor treating prostate cancerWO2012019168ModernaTX,USAUse of modified mRNA encoding melanocyte stimulating hormone,insulin andgranulocyte colony-stimulating factor in prevention or treatment of disordersWO202009729
136、1ModernaTX,USACancer vaccines comprising mRNA(s)encoding peptide epitopes(neoepitopes)andformulated as lipid nanoparticlesWO2020141212eTheRNA Immunotherapies NV,BelgiummRNA vaccineWO2022008519BioNTech SE,Germany;TRON TranslationaleOnkologie Mainz,GermanyTherapeutic RNA for HPV-positive cancerWO20150
137、24666CureVac,GermanyRNA vaccine for treating lung cancerWO2012159643BioNTech AG,Germany;TRON TranslationaleOnkologie Mainz,GermanyIndividualized vaccines for cancerWO2015014869BioNTech AG,Germany;TRON TranslationaleOnkologie Mainz,GermanyDetermination of expression pattern of a set of tumor antigens
138、 including Cxorf61,CAGE1,PRAME and others to select cancer therapy regimenWO2022009052Janssen Biotech,USAProstate neoantigens and their usesWO2022081764RNAimmune,USAPan-ras mRNA cancer vaccinesWO2014082729BioNTech AG,Germany;Mainz GemeinnuetzigeGmbH,GermanyIndividualized vaccines for cancerWO2016180
139、467BioNtech Cell&Gene Therapies,Germany;TRON Translationale Onkologie Mainz,GermanyEnhancing the effect of car-engineered T cells by means of nucleic acid vaccinationFigure 11.Distribution of patents among the top 10 countries/regionsin the field of mRNA delivery systems.Table 8.Top Patent Applicant
140、s in the Field of mRNADelivery SystemsaNumberOrganizationsNo.ofpatentapplicationsCountryOrganizationtype1Moderna64U.S.Company2Translate Bio45U.S.Company3CureVac AG30GermanyCompany4AlnylamPharmaceuticals24U.S.Company5BioNTech22GermanyCompany6AcuitasTherapeutics19CanadaCompany7Shire HumanGeneticTherap
141、ies15U.S.Company8MassachusettsInstitute ofTechnology13U.S.University9ArcturusTherapeutics12U.S.Company9Evox TherapeuticsLtd.12U.K.CompanyaData from the CAS Content Collection.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci
142、.2023,6,943969953terms of the type of administration,key concepts includeintraperitoneal injections,pharmaceutical intravenous injec-tions,subcutaneous injections,etc.With regard to carriermaterials,nanoparticles are infused with cationic lipids,pharmaceutical nanoparticles,pharmaceutical liposomes,
143、etc.Nucleic acid modifications include oligonucleotide analogs,nucleotide analogs,peptide nucleic acids,etc.In terms ofTable 9.Notable Patents Focused on mRNA Delivery and ModificationPatent numberOrganizationPatent titleDisclosure highlightWO2013086373Alnylam Pharmaceuticals,USALipids for the deliv
144、ery of nucleic acidsLNP components for RNA deliveryWO2014093924Moderna,USAPreparation,cytotoxicity,apoptosis,andtranscriptionofmodified nucleic acid molecules and uses thereofModification of mRNAWO2016070166Arcturus Therapeutics,USATranslatable messenger RNA analogs containingunlocked nucleomonomers
145、 and with prolonged invivo half-lives for therapeutic usesmUNA or mRNA analogs with unlocked nucleomonomersUS10808242BioNTech,GermanyMethod for reducing immunogenicity of RNA byconstructing A-rich and U-poor mRNA for use intherapymRNAmodificationsfordecreasingnonspecificimmunogenicitybymRNA itselfWO
146、2017117528Acuitas Therapeutics,CanadaPreparationoflipidsandlipidnanoparticleformulationsfor delivery of nucleic acidsLNP components and formulation for nucleic acid deliveryWO2017176974The Ohio State University,USABiodegradable amino-ester nanomaterials for nucleicacid deliveryLNPs for delivery of R
147、NAs including siRNA,miRNA,and mRNAWO2017212009Curevac AG,GermanyHybrid carriers comprising cationic peptide or polymerand lipidoidA nucleic acid delivery system comprised of a cationic peptide orpolymer and a lipidoid compoundUS20180125989Translate Bio,USAImidazole cholesterol ester(ICE)-based lipid
148、 nano-particle formulation for delivery of mRNAMethods of formulating nucleic acid containing LNPWO2018009838Rubius Therapeutics,USACompositions and methods related to therapeuticerythroid cell systems expressing exogenous RNAencoding a proteinTherapeutic erythroid cell systems expressing exogenous
149、RNAencoding a proteinWO2018013525Translate Bio,USANucleic acid conjugates and uses thereofConjugates comprising sugars,folates and cell-penetrating peptidesfor delivering mRNAWO2019092145Evox Therapeutics,U.K.Exosomes comprising RNA therapeuticsMethods for using extracellular vesicles to encapsulati
150、ng nucleicacid-based therapeutics such as mRNA,circular RNA,miRNA,etc.WO2020070040Johannes Gutenberg-Uni-versity Mainz and BioN-Tech,GermanyRNA particles comprising polysarcosineLNPs for delivering mRNAsWO2020061367Moderna,USAPreparation of compounds and lipid nanoparticlecompositions for intracellu
151、lar delivery of therapeuticagentsLNPs for drug deliveryWO2020097540Arbutus Biopharma Corp.,CanadaMethods and lipid nanoparticles for delivering mRNAand siRNA in treatment of diseasesLNPs for mRNA deliveryWO2020263883Moderna,USAEndonuclease-resistant messenger RNA and usesthereofChemically modified m
152、RNA that increases mRNA stabilityCN110747214ShenzhenZhenzhiMedicalTechnology,ChinaPreparation of mRNA-antibody fusion molecule and itsuse for drug deliveryPreparation of antibodymRNA fusion/conjugate with puromycinas the linker for targeted delivery of mRNA therapeutics.WO2020160397Moderna,USAMethod
153、s of preparing lipid nanoparticlesLNP formulationWO2021001417BioNTech,GermanyRNA formulations suitable for therapySelf-amplifying RNA formulated in various polymersWO2021231854Moderna,USALipid nanoparticle compositions comprising an mRNAtherapeutic and an effector moleculeSystemthatfeaturesatethered
154、moleculetofurtherincreasetheleveland/or activity of mRNA therapeutics formulated in LNPWO2021257262Yale University,USAPoly(amine-co-ester)polymers with modified endgroups and enhanced pulmonary deliveryPEGlyated poly(amine-co-ester)polymers with modified endgroups for enhanced delivery of mRNA to th
155、e lung by inhalationWO2022032154Moderna,USACompositions for the delivery of payload molecules toairwayLNPs comprisingpayload moleculessuch as mRNA therapeutics tobe delivered to airway cellsWO2016176330University of Pennsylvania,USA;Acuitas Therapeu-tics,CanadaNucleoside-modified mRNAs encoding anti
156、gens forinducing an adaptiveModified antigen mRNA delivered in LNP induced adaptiveimmune response without inducing innate immunityWO2020191103Arcturus Therapeutics,USAMethod of making lipid-encapsulated RNA nano-particlesDetailed method for making RNA-encapsulating LNPWO2021250263eTheRNA Immunother
157、a-pie,BelgiumLipid nanoparticles comprising ionizable lipid,phos-pholipid,sterol,PEG lipid and mRNALNP components for RNA deliveryWO2022175815Pfizer,USAMethods of protecting RNAMethods of protecting RNA against degradation and componentscomprising free amino acids for this purposeWO2019246203Univers
158、ity of Texas,USALipid nanoparticle compositions for delivery of mRNAand long nucleic acidsCompositions for delivery of long nucleic acids(80 nucleotides),such as mRNAs,including cationic ionizable lipid,phospholipid,PEGylated lipid,and a steroidWO2022236093Carnegie Mellon Univer-sity,USALipid nanopa
159、rticle-mediated mRNA delivery to thepancreasLNP composition for mRNA delivery to the pancreas containing:cationic helper lipid,cholesterol analog,PEG-based compound,ionizable lipidoid,and mRNAUS20230043677Oregon State University,USAInhalable therapeuticsNanoparticles for mRNA delivery suitable for n
160、ebulization and/ordelivering mRNA by inhalationWO2022155598Tufts College and Brighamand Womens Hospital,USALipid nanoparticles for targeted delivery of mRNALNP composition for specific delivery of CRISPR-Cas9 mRNA tothe lung or liverACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps
161、:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969954mechanismresearch,keyconceptsincludetranscription,growthfactors,membrane proteins,etc.From the perspective ofpatent origin countries,the patenteesin the global field of mRNA modification are mainly from theU.S.,Germany,China,
162、Japan,and France(Supplemental FigureS5).Amongthem,theU.S.filedthelargestnumberofpatentsinthe field,Germany and China are in second and third places,with Japan and France last filing a relatively small numbers ofpatents.From the perspective of patent application institutions,globalpatent application
163、institutions in the field of mRNAmodification are mainly concentrated in the U.S.and Germany(Supplemental Table S1).Among the top 10 patent-filingorganizations,six are from the U.S.and four are from Germany.From the perspective of institutional nature,companies are stillthe main body of patent appli
164、cation,accounting for about 70%of all applications.The top five patent filers were Modena,CureVac,BioNTech,Alnylam Pharmaceuticals,and TranslateBio,which also ranked among the top 5 in the field of mRNAmodification.NOTABLE PATENTS ON mRNA DELIVERY ANDMODIFICATIONRNAs,which are hydrophilic and negati
165、vely charged,cannotdiffuseacrosscellmembranes;thus,theyrequiredeliveryvectorsand/or chemical modification to reach their targets.mRNAsmay also be quickly hydrolyzed by circulating RNases.As such,when administered systemically,RNA delivery systems need toprotect the RNA against serum nucleases,bypass
166、 theundesirable immune reaction against mRNA per se,avoidnonspecific interactions with serum proteins,and block renalclearance.47Thus,delivery vehicles and chemical modificationsare of the utmost importance for the success of the mRNAtherapeutics.Table 9 summarizes some notable patentsdisclosing ess
167、ential advances in these areas.Patent application WO2013086373 by Alnylam Pharmaceut-icals,USA,relates to novel cationic lipids that can be used incombinationwithotherlipidcomponentssuchasaneutrallipid,asterolsuchascholesterol,andaPEG-lipidconjugatecapableofreducing aggregation,to form lipid nanopar
168、ticles witholigonucleotides,to facilitate the cellular uptake and endosomalescape,and to knockdown target mRNA both in vitro and invivo.Exemplary LNP composition comprised 50%cationiclipid,10%DSPC,38.5%cholesterol,and 1.5%PEG-DMG(average PEG molecular weight of 2000).Patent application WO2020160397
169、by Moderna,USA,provides methods of producing LNP formulations and theproducedLNPformulationsthereof.Itreflectstherecenteffortstoward“bedside”and/or“point-of-care”formulations,wherebymRNAmaybeencapsulatedwithinpreformedvesiclesthatwereprepared at an earlier date.This mode of production offersadvantag
170、es in the context of clinical supply,as empty LNPvesicles may be produced and stored separately prior torecombination with mRNA in a clinical compound setting.Specifically,bedside formulations may promote increasedstability,since mRNA and empty raw materials can be storedin separately optimized cond
171、itions.Process complexity and costof goods may be reduced since the LNP preparation occursindependentofcargo,enablinga platformapproachformultiplemRNA or active agent constructs.The empty LNP plus mRNAmodality may be referred to as“post-hoc”.The concept of posthoc loading as described in the present
172、 invention may enablecontroland/oroptimizationofeachstepseparately.Further,thepost hoc loading may enable mRNA addition at timescales thatenablepoint-of-careformulation,e.g.,monthsoryearsfollowingempty LNP production.Patent application WO2017176974 by The Ohio StateUniversity,USA,relates to biodegra
173、dable amino-ester lipidnanoparticles for efficient delivery of RNAs including siRNA,miRNA,and mRNA.Provided are also compositions includingan amino-ester lipid compound of the invention,a noncationiclipid,a PEG-lipid conjugate,a sterol,and an active agent such asmRNA,which can be used to correct a m
174、utation in a genome.For example,mRNAs can be delivered to correct mutations thatcausehemophiliaduetomutationsinthegenesencodingFactorVIII(hemophilia A)or Factor IX(hemophilia B).Patent application WO2019092145 by Evox Therapeutics,UK,pertains to extracellular vesicles(Evs),specificallyexosomes,as de
175、livery vehicles for nucleic acid-based therapeu-tics.Thedistinctivepropertiesoftheextracellularvesicles(Evs),and specifically their nanosized subgroup,the exosomestheirinnate stability,low immunogenicity,biocompatibility,andgood biomembrane penetration capacityallow them tofunction as superior natur
176、al nanocarriers for efficient drugdelivery and are currently viewed as the rising star in drugdelivery.48The nucleic acid therapeutics of the presentinvention are loaded into Evs using inventive engineeringprotein and nucleic acid engineering strategies to enhanceloadingintoEvsandtofacilitaterelease
177、ofthenucleicacidcargomolecules inside target cells.Patent application US10808242 by BioNTech,Germany,isfocused on decreasing immunogenicity of RNA.The providedmethods for decreasing immunogenicity of RNA comprisemodifying the nucleotide sequence of the RNA by reducing theuridine(U)content,by elimina
178、tion of U nucleosides from thenucleotide sequence of the RNA and/or a substitution of Unucleosides by nucleosides other than U in the nucleotidesequence of the RNA.Using RNA having decreasedimmunogenicity allows administration of RNA as a drug to asubject,e.g.,in order to obtain expression of a phar
179、maceuticallyactive peptide or protein,without eliciting an immune responsewhichwouldinterferewiththerapeutic effectivenessoftheRNAor induce adverse effects in the subject.Patent application WO2020069718 by Johannes Gutenberg-University Mainz and BioNTech,Germany,relates to RNAparticles for delivery
180、of RNA to target tissues after parenteraladministration and compositions comprising such RNAparticles.Specifically,polysarcosinelipid conjugates arefeatured as suitable components for the assembly of RNAnanoparticles.By now,PEG has been the most widely used andgold standard“stealth”polymerin drugdel
181、ivery.However,PEGhas been found to exhibit some undesired effects such aslowering transfection efficiency,accelerated blood clearanceinducedbyanti-PEGantibodies,and/orcomplementactivation,as well as inducing a specific immune response.The presentinvention shows that polysarcosine-lipid conjugates av
182、oid thedisadvantages accompanied by the use of PEG.Polysarcosinelipid conjugates enable manufacturing of RNA nanoparticleswith different techniques,resulting in defined surface propertiesandcontrolledsizeranges.Manufacturingcanbedonebyrobustprocesses that are compliant with the requirements forpharm
183、aceutical manufacturing.The particles can be end-groupfunctionalized with different moieties to modulate charge or tointroduce specific molecular moieties like ligands.Patent application US20230043677 by the Oregon StateUniversity,USA,relates to nanoparticle composition forACS Pharmacology&Translati
184、onal Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969955encapsulating a therapeutic agent,such as a mRNA,suitable fornebulizationand/ordeliveryofthenebulizedformulationtothelungs by inhalation.The nanoparticles comprise an ionizablelipid,a
185、cholesterol derivative,a structural lipid,and a PEG lipid.Patent application WO2022155598 by Tufts College andBrigham and Womens Hospital,USA,discloses a highly potentnonviral LNP-mediated CRISPR-Cas9 delivery system for liverorlungdeliveryofCas9mRNA,anddemonstratesitsefficacybyFigure 12.Distributio
186、n of patents for the top 20 primary diseases.Figure 13.Analysis of proliferative diseases claimed by patents related to mRNA therapeutics and vaccines:red,more than 100 patents;orange,50100 patents;blue,less than 50 patents.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org
187、/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969956targeting the Angptl3 gene.The system is composed of a leadingtail-branched bioreducible lipidoid(306-012B)co-formulatedwith an optimized mixture of excipient lipid molecules,and itsuccessfully co-delivers SpCas9 mRNA and a single gui
188、de RNAtargeting Angptl3 via a single administration.The success of mRNA-based COVID-19 vaccines havedemonstrated the effectiveness of two key strategies fordeveloping RNA medications:the chemical modifications ofmRNA uridine to pseudouridine and 5-capping,as well as thelipid nanoparticle delivery ve
189、ctors,paving the way for furtheradvancement of mRNA therapeutics and vaccines.APPLICATION OF mRNA THERAPEUTICS ANDVACCINES IN DISEASE TREATMENT ANDPREVENTIONAnalysis of Diseases Claimed in Patents Related tomRNA Therapeutics and Vaccines among Diseases.Diseases covered in mRNA patents include 69 pri
190、mary diseaseclasses,and the top 20 disease classes are shown in Figure 12.Proliferative disorders such as neoplasm,are claimed in thelargest number of patents(600)followed by infectious diseases(358),indicating strong focus on application of mRNAmedicines in these two areas.From the anatomical persp
191、ective,digestive system diseases,immune diseases,respiratory systemFigure 14.Analysis of key concepts in patents related to neoplasm:red,more than 100 patents;orange,50100 patents;blue,less than 50 patents.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.
192、3c00047ACS Pharmacol.Transl.Sci.2023,6,943969957diseases,nervous system diseases,and glandular diseases havebeen studies with relative high frequencies in patents.Diseaseclassessuchasgeneticdisordersandothersareinvolvedinfewerthan 200 patent applications.Analysis of Proliferative Diseases Disclosed
193、in PatentsRelated to mRNA Therapeutics and Vaccines.Figure 13shows classes of diseases claimed by patents related to mRNAtherapeutics and vaccine development.The diseases arearranged hierarchically with the number range of patentsinvolved indicated by dots in different colors.As shown in thefigure,p
194、roliferative diseases such as neoplasm are the mostinvestigated diseases in the R&D of mRNA therapeutics andvaccines.Within this broader class of diseases,neoplasm is themost explored for mRNA therapeutics and vaccines andappeared in 528 patents,indicating a strong interest in applyingthis new class
195、 of medicines to cancer prevention and treatment.As shown in Figures 13 and 14,among the more specificdiseases,digestive system neoplasm,lung neoplasm,urogenitalFigure15.AnalysisofinfectiousdiseasesclaimedbypatentsrelatedtomRNAtherapeuticsandvaccines:red,morethan100patents;orange,50100patents;blue,l
196、ess than 50 patents.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969958systemneoplasm,andvariousformsofcarcinoma(i.e.,epithelialtissue-derived neoplasm)attracted the most attention,withmore than 100 patents in
197、volved in each case.PATENT DISTRIBUTION AMONG INFECTIOUSDISEASESAs shown along the hierarchical tree of infectious diseases inFigure 15,there are 16 classes and over 60 specific diseasesexplored by patents on mRNA therapeutics or vaccines.Amongthoseclassesofdiseases,viralinfectionhasbeenmostexamined
198、.Bacterial infections and parasitic infections were also of highconcern.From the anatomical system perspective,respiratorysystem infection received more attention than other systems.Mostofthesepatentsarerelatedtovaccinedevelopmentagainstinfectious diseases.Analysis of Immune Diseases Disclosed by Pa
199、tentsRelated to mRNA Therapeutics and Vaccines.Amongvarious immune diseases examined by the mRNA therapeuticand vaccine patents,autoimmune diseases,followed byhypersensitivity(exaggerated response or over reaction to anFigure 16.Analysis of key concepts in patents related to immune diseases:red,more
200、 than 100 patents;orange,50100 patents;blue,less than 50patents.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969959antigen such as in the case of allergy)and immunodeficiencydiseases,received the most attentio
201、n as shown in Figure 16.mRNA VACCINES AND THERAPEUTICS INCLINICAL TRIALSAction Mechanism of mRNA Vaccines and Therapeu-tics.When the lipid nanoparticle(LNP)encapsulating mRNAsencoding targeted protein(antigenic protein)are administeredin the body,LNP-mRNAs are engulfed by endocytosis andmRNAs are re
202、leased to cytosol through endosomal escapingmechanism in antigen-presenting cells(no shown in thefigure).49Inside the ribosomes,a cellular machinery,proteinsare translated based on the mRNAs.mRNA-encoded proteintherapeutics use synthetic mRNAs that produce the desiredproteins,such as antibodies,cyto
203、kines,and enzymes inside thehumanbody.Forvaccines,mimickingtheviralinfectionprocess,intracellular produced antigens mainly elicit cell-mediated andantibody-mediated immunities.(Figure 17)First,the protea-some degrades antigenic proteins into peptide epitopes,whicharetransportedintothe endoplasmicret
204、iculumand loadedontomajor histocompatibility complex class I molecules(MHC I).The MHC I-peptide epitope complexes are presented on thesurface of cells that bind to the T cell receptor to activate CD8+T cells and kill infected or cancer cells(cell-mediatedimmunity).The antigenic proteins are transpor
205、ted via theGolgi apparatus and released to the outside of the cells.Thesecreted proteins are endocytosed by antigen-presenting cells,degraded,and loaded onto the MHC II peptide insideendosomes.The MHC II-peptide epitope complexes arepresented on the surface of cells,which is recognized by CD4+T cell
206、s facilitating B cells to make antigen-specific antibodies(antibody-mediated immunity).50mRNA VACCINES IN CLINICAL TRIALSTop companies for mRNA vaccine research include Moderna,BioNTech,Pfizer,and CureVac.Examination of diseasesinvestigated by mRNA vaccines in clinical trials revealed thatthe vast m
207、ajority(80%)of mRNA vaccines were designed forinfectious diseases such as coronavirus infection,influenza,human immunodeficiency virus(HIV),rabies,and respiratorysyncytial virus(RSV),among others.Other mRNA vaccines inthe pipeline are being researched for various forms of cancers.Table 10 lists mRNA
208、 vaccine candidates for infectiousdiseases currently in at least phase II trials.All mRNA vaccinecandidates are developed for COVID-19 and encode the spikeproteinofSARS-CoV-2oritsreceptor-bindingdomain.mRNA-Figure 17.Mechanism of action of mRNAs vaccines and therapeutics(adapted and modified from re
209、f 50).ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969960Table 10.mRNA Vaccine Candidates for Infectious Diseases in Phase 2 or More Advanced Clinical TrialsaVaccine nameCAS RegistryNumberDisease indicationAnt
210、igenCompanyBNT-162b2(B.1.1.7+B.1.617.2)2883464-25-1COVID-19Prefusion stabilized S protein of SARS-CoV-2 B.1.1.7 andB.1.1.617.2 variantsBioNTech,PfizerBNT162b2(B.1.351)PendingCOVID-19Prefusion stabilized S protein of SARS-CoV-2 B.1.351 variantBioNTech,PfizerBNT 162b2(B.1.1.529)PendingCOVID-19Prefusio
211、n stabilized S protein of SARS-CoV-2 B.1.1.529 variantBioNTech,PfizerBNT-162b2(WT/OMIBA.1)PendingCOVID-19Prefusion stabilized S protein of SARS-CoV-2 WT and BA.1variantBioNTech,PfizerBNT-162b5(WT/OMIBA.2)PendingCOVID-19Prefusion stabilized S protein of SARS-CoV-2 WT and BA.2variantBioNTech,PfizermRN
212、A 1273PendingCOVID-19The full-length prefusion stabilized S proteinModernamRNA 1273.2112805221-47-8COVID-19Prefusion stabilized S protein of the SARS-CoV-2 and B.1.351variantModernamRNA 1273.214PendingCOVID-19bivalent of SARS-CoV-2 spike protein from Beta and DeltavariantsModernamRNA 1273.3512642373
213、-67-7COVID-19the full-length prefusion stabilized S protein of the SARS-CoV-2B.1.351 variantModernamRNA 1273.5292763208-92-8COVID-19Prefusion stabilized S protein of the SARS-CoV-2 B.1.1.529variantModernamRNA 1273.6172882950-03-8COVID-19Prefusion stabilized S protein of the SARS-CoV-2 B.1.1.617.2var
214、iantModernamRNA -77-1COVID-19SARS-CoV-2 spike protein receptor-binding domain and N-terminal fragmentModernamRNA 1283.529PendingCOVID-19Prefusion stabilized S protein of the SARS-CoV-2 B.1.1.529variantModernamRNA 1283.2112882951-80-4COVID-19PrefusionstabilizedSproteinoftheSARS-CoV-2B.1.35
215、1variantModernaLVRNA 009PendingCOVID-19SARS-CoV-2 spike proteinAIM VaccineARCT 1652714576-70-0COVID-19Self-Transcribing and Replicating mRNA encoding SARS-CoV-2 spike protein variantsArcturusTherapeuticsARCT 1542698334-90-4COVID-19Self-Transcribing and Replicating mRNA encoding SARS-CoV-2 spike prot
216、einArcturusTherapeuticsARCT 0212541451-24-3COVID-19Self-Transcribing and Replicating mRNA encoding SARS-CoV-2 spike protein variantsArcturusTherapeuticsBCD 250b2756425-11-1COVID-19The receptor-binding domain of SARS-CoV-2 spike proteinBiocadCOVID-19 mRNAvaccinePendingCOVID-19SARS-CoV-2 spike protein
217、CanSino BiologicsSYS 6006PendingCOVID-19SARS-CoV-2 spike proteinCSPCPharmaceuticalDS 56702749556-96-3COVID-19SARS-CoV-2 spike proteinDaiichi SankyoHDT 3012437182-02-8COVID-19Self-amplifying RNA encoding SARS-CoV-2 spike proteinEmcurePharmaceuticalsEG-COVIDPendingCOVID-19SARS-CoV-2 spike proteinEyeGe
218、nePTX-COVID19-B2726459-47-6COVID-19SARS-CoV-2 spike proteinProvidenceTherapeuticsSW-BIC-2132699076-70-3COVID-19The full-length SARS-CoV-2 spike proteinStemirnaTherapeuticsABO1009-DPPendingCOVID-19SARS-CoV-2 omicron variant spike proteinSuzhou AbogenBiosciencesARCoV2543878-98-2COVID-19The receptor-bi
219、nding domain of SARS-CoV-2 spike proteinSuzhou AbogenBiosciencesAwcornaPendingCOVID-19SARS-CoV-2 spike protein receptor-binding domainWalvaxBiotechnologyComirnaty2417899-77-3COVID-19The full-length prefusion stabilized S proteinBioNTechSpikevax2430046-03-8COVID-19The full-length prefusion stabilized
220、 S proteinModernamRNA -92-0COVID-19+influenzaPrefusionstabilizedSproteinofSARS-CoV-2andhemagglutininModernamRNA -31-3Respiratory syncytial virusinfectionRSV prefusion stabilized F glycoproteinModernaBNT 1612760529-48-2InfluenzaHemagglutinin from H1N1 and B/Yamagata influenza st
221、rainsBioNTechmRNA -87-3InfluenzaHemagglutinin from four seasonal influenza strainsModernamRNA -90-8InfluenzaHemagglutinin and neuraminidase antigensModernamRNA -91-9InfluenzaHemagglutinin and neuraminidase antigensModernaMRT 54072900351-99-5InfluenzaQuadrivalent infl
222、uenza vaccineSanofimRNA -97-7Zika virus infectionStructural proteins of Zika virusModernamRNA -55-4Zika virus infectionStructural proteins of Zika virusModernamRNA -59-8CMV infectionSixmRNAscodingforpentamerviralantigenandglycoproteinBof CMVModernaaData from the CAS
223、Content Collection,Clinicaltrials.gov,and Pharmaprojects.bVaccine no longer in development.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,9439699611283 is a potential refrigerator-stable COVID-19 vaccinecomprising
224、mRNA encoding a SARS-CoV-2 spike protein N-terminal fragment and receptor-binding domain formulated inlipid nanoparticles.It is considered as the“next generation”vaccine candidate aiming for a pan-human coronavirus domainvaccine.In addition to mRNA vaccines for SARS-CoV-2 infection,several mRNA vacc
225、ine candidates for infection by other virusessuch as RSV,influenza virus,Zika virus,and cytomegalovirus(CMV)have entered clinical trials.In January 2023,Modernaannounced that RSV vaccine,mRNA-1345,demonstratedvaccine efficacy of 83.7%at preventing symptoms in olderadults in randomized phase III tria
226、l.51Moderna plans to submitmRNA-1345 for regulatory approval in the first half of 2023.52Two research groups examined levels of neutralizing anti-bodies and differences in CD4+or CD8+T cell responsesinduced by monovalent and bivalent COVID-19 boostervaccines for protecting against omicron variants.N
227、eithergroup observed superior immune responses to bivalent boostervaccinescomparedtomonovalentvaccines.Mostofneutralizingantibodies elicited by vaccines targeting newer variants stillrecognize only the original virus because of“immuneimprinting”in which the body repeats its immune response tothe fir
228、st variant encountered.53,54However,fine-tuning thedosage of booster vaccines might increase their efficacy ofprotection again immune-escape COVID-19 variants.55The success of COVID-19 mRNA vaccine has revealed theapplication potential of mRNA platform not only for expansionto other infectious disea
229、ses but also for cancers(Table 11),especially as therapeutic vaccines The clinical study of mRNAvaccines has shown good efficacy in the treatment of melanoma,non-small-cell lung cancer(NSCLC),and prostate cancer.Ref.Autogene cevumeran(RO 7198457,BNT122),jointlydeveloped by BioNTech and Genentech,is
230、a mRNA-basedindividualized neoantigen specific immunotherapy(iNeST).Itencodes up to 20 neoepitopes defined by the patients tumor-specific mutations delivered in an RNA-lipoplex formulation.Acombination of intravenously administered autogene cevumer-an combined with anti-PD-L1 immune checkpoint inhib
231、itoratezolizumab is conducted in patients with locally advanced ormetastatic solid tumors has entered a first-in-human phase Istudy.It has induced strong CD4+and CD8+T cell immunityagainst neoantigens.Randomized phase II studies of autogenecevumeran for patients with melanoma in combination withpemb
232、rolizumab,for individuals with non-small-cell lung cancer(NSCLC)in combination with atezolizumab,and forindividuals with colorectal cancer(CRC)are currently ongoing.BNT111 developed with the BioNTechs FixVac platformencodes four tumor-associated antigens(TAAs),the cancer-testis antigen NYESO-1,the h
233、uman melanoma-associatedantigen A3(MAGE-A3),tyrosinase,and putative tyrosine-protein phosphatase(TPTE)and is encapsulated in an RNA-lipoplexformulation.IthasenteredphaseIIclinicaltrialstotreatadvanced melanoma and has gained FDA fast track designationin 2021.A report from a phase II trial showed tha
234、t the use ofBNT111 alone or in combination with PD-1 antibody caninduce tumor antigen-specific CD4+and CD8+T cell immuneresponses.CV9201,developedbyCureVac,isanRNA-basedtherapeuticvaccine encoding five NSCLC antigens.The first-in-human,multicenter,phase I/IIa study was conducted in 7 patients withlo
235、cally advanced NSCLC and 39 patients with metastaticNSCLC.The result demonstrated that specific immuneresponses against at least one antigen were detected in 63%ofpatients after treatment and the frequency of activated IgD+CD38hi B cells increased by more than 2-fold in 60%ofevaluated patients.56Mod
236、ernas personalized mRNA cancer vaccine,mRNA-4157,encodes 34 unique neoantigen genes that may stimulate specificT cell responses.Phase I trials showed that this vaccine is safeand tolerable in monotherapy or in combination withpembrolizumab.57In December 2022,Moderna and Merckannounced that mRNA-4157
237、 in combination with anti-PD-1antibody,pembrolizumab reduced the risk of recurrence ordeath by 44%in patients with stage III/IV melanoma comparedwith pembrolizumab monotherapy based on their randomizedphase IIb trial.58mRNA THERAPEUTICS IN CLINICAL TRIALSTop companies for mRNA therapeutic research i
238、ncludeBioNTech,Moderna,Arcturus Therapeutics,AstraZeneca,and Sanofi.mRNA therapeutics have a broad range of targeteddiseases.Consistent with the patent-disease analysis data above,mRNA therapeutics are being developed largely for cancers,followed by metabolic,cardiovascular,infectious,immunolog-ical
239、,and respiratory diseases.Table 11.mRNA Vaccines in Phase 2 or More Advanced Clinical Trials for CancersaVaccine nameCAS RegistryNumberDisease indicationAntigenCompanyAutogenecevumeran2365453-34-3Melanoma;colorectal cancerPatient-specific neoantigensBioNTechmRNA 41572741858-84-2MelanomaUp to 34 neoa
240、ntigensModernamRNA 43592900354-08-5Melanoma;non-small-cell lung carcinomaIDO and PD-L1ModernaBNT 1112755828-88-5MelanomaMix of four melanoma-associated antigensBioNTechBNT 1122900354-09-6Prostate cancerMix of five prostate cancer-specific antigensBioNTechBNT 1132882951-85-9PV16+head-and-neck squamou
241、s carcinomaHPV16-derived tumor antigens(oncoprotein E6 andE7)BioNTechCV 92021665299-76-2Non-small-cell lung cancerNY-ESO-1,MAGE C1,MAGE C2,TPBG(5T4),survivin,MUC1CureVacCV 91032882951-83-7Prostate cancerMix of four prostate cancer-associated antigensCureVacSW 1115C32882951-82-6Non-small-cell lung ca
242、ncer;esophageal cancerPatient-specific neoantigensStemirnaTherapeuticsRocapuldencel T;AGS 0032396421-01-3Non-small-cell lung cancer;lung cancer;bladder and renal cancerAutologous tumor antigen and CD40L-loadeddendritic cell immunotherapyArgosTherapeuticsaData from the CAS Content Collection,Clinical
243、trials.gov,and Pharmaprojects.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969962mRNA therapeutic products currently in clinical trials areexamined in Table 12 to reveal a landscape view of the currentprogress
244、 in mRNA therapeutics in the clinical developmentpipeline.AselectfewarealsoexaminedinfurtherdetailbelowtoshowcasethevarietyofmRNAtherapeutics,theirmechanismofactions,and their targeted disease indications.ArcturusTherapeuticsdevelopedandiscurrentlyevaluatingamRNA therapeutic for the treatment of orn
245、ithine trans-carbamylase(OTC)deficiency that currently has no FDA-approvedtreatments.TheureacycleenzymeOTChelpsremoveammonia from liver cells,and a deficiency leads to highammonia levels.Utilizing their lipid-mediated nucleic aciddelivery system(LUNAR),Arcturus is currently researchingLUNAR-OTC in a
246、 phase II clinical trial(NCT05526066)toevaluate its safety and tolerability in participants with OTCdeficiency.59AZD8601 is a mRNA therapeutic developed through acollaborationbetweenAstraZenecaandModerna.ItencodesforVEGF-A,a paracrine factor important for new blood vesselformation and progenitor cel
247、l division that contributes to repairand regeneration of the heart.A phase II clinical trial(NCT03370887)examined the safety,tolerability,and explor-Table 12.mRNA Therapeutic Products in Clinical Trials62amRNA drugnameCAS RegistryNumberDisease indicationsDescriptionCompanyA-001;Tri-Mix-MEL;ECL-006;E
248、011-MEL2877674-59-2MelanomaA mixture of three mRNAs encoding constitutively activated CLT4,CD40L,and TLR4 plusmRNAs for five melanoma-associated antigens(tyrosinase,gp100,MAGE-A3,MAGE-C2,and PRAME),which activate key immune cells against cancereTheRNA Immu-notherapiesARCT-810;LUNAR-OTC2877704-48-6Or
249、nithine trans-carba-mylase deficiencymRNA encoding ornithine transcarbamylase formulated in a lipid nanoparticle to correct theenzyme deficiencyArcturus Therapeu-ticsAZD-86012603440-18-0Heart failure and is-chemic cardiovascu-lar diseasesmRNA encoding vascular endothelial growth factor A to stimulat
250、e new vascular blood vesselformation and repair as well as regenerate heart cellsAstraZenecaBD-1112901016-63-3Herpetic viral keratitisViral-like particle drug delivery system used to transduce cas9 mRNA that directly targets andcuts the viral genome of herpes simplex virus 1 to effectively remove th
251、e virusBD GeneBNT-1412877707-22-5Solid tumorsmRNA encoding a monoclonal antibody targeting claudin 18,a protein commonly expressedin multiple cancersBioNTechBNT-1422877707-34-9Solid tumorsmRNAencodingabispecificantibodytargetingCD3,aproteininvolvedinactivationofcertaintypes of T cells,and claudin 6(
252、CLDN6),a protein highly expressed in certain cancersBioNTechBNT-1512877709-82-3Solid tumorsA nucleoside-modified,cationic lipoplexes-loaded mRNA encoding an interleukin-2(IL-2)variant to stimulate anti-cancer T cellsBioNTechBNT-1522877709-92-5Solid tumorsA nucleoside-modified mRNA encoding interleuk
253、in-7 to stimulate anti-cancer T cellsBioNTechBNT-1532877709-93-6Solid tumorsA nucleoside-modified mRNA encoding interleukin 2(IL-2)to stimulate anti-cancer T cellsBioNTechLioCynx-M004;Lion TCR2901015-92-5Hepatitis B virus-re-lated hepatocellularcarcinomaA genetically modified autologous cell therapy
254、 derived from T cells transfected with mRNAencoding to express a T-cell receptor that recognizes the hepatitis B surface antigen on thesurface of HBV-related cancer cellsLion TCRMEDI--03-1Solid tumorsLNP-encapsulated mRNA encoding IL-12 to increase intratumor production of IL-2 viaintratu
255、moral injectionModernamRNA-27522878461-50-6Solid tumorsLipid nanoparticle-encapsulated mRNAs encoding human T cell co-stimulator,OX40L,andproinflammatory cytokines,IL-23 and IL36,for intratumoral injectionModernamRNA-37052878470-78-9Methylmalonic acide-miaLipid nanoparticle-encapsulated mRNA encodin
256、g the mitochondrial enzyme methyl-CoAmutase that is deficient in methylmalonic acidemiaModernamRNA-37452878574-58-2Glycogen storage dis-ease type 1aLipid nanoparticle-encapsulated mRNA encoding glucose 6-phosphatase to restore thedeficient enzyme responsible for converting glycogen into glucose for
257、treatment of type 1aglycogen storage diseaseModernamRNA-39272878577-32-1Propionic acidemiaLipid nanoparticle-encapsulated mRNAs encoding propionyl-CoA carboxylase subunit and subunit to restore the deficient enzyme and reduce toxic buildup of some substances inpropionic acidemiaModernamRNA-6231b2878
258、577-39-8Autoimmune diseasesLipid nanoparticle-encapsulated modified mRNA encoding a mutated form of human IL-2fused to human serum albumin to increase its half-life to restore IL-2-and T-cell-mediatedimmune homeostasisModernaMRT-5005b2328142-67-0Cystic fibrosisInhalable form of engineered mRNA varia
259、nt encoding fully functional cystic fibrosistransmembrane conductance regulator protein for restoring lung function in cystic fibrosis;Phase I/II clinical trial(NCT03375047)showed no improvement in lung function but diddiscover that repeated inhaled doses of mRNA are safe63Translate Bio(ac-quired by
260、 Sanofi)SAR-44-17-2Solid tumorsMixture of four mRNAs encoding IL-2 single chain,IL-15 fused to the sushi domain of IL-15R,GM-CSF,and interferon 2b,which have been reported as mediators of tumorregressionSanofi,BioNTechSQZ-eAPC-HPV2879306-51-9HPV and solid tumorsmRNA-based cell therapeutic
261、 agent that delivers five mRNAs for HPV16 protein antigens andimmune-stimulating proteins,including CD86 and membrane bound IL-2 and IL-12,intofourdifferenttypesofengineeredimmunecells(monocytes,T-cells,B-cells,andNKcells)ofcancer patients in a single stepSQZ Biotechnolo-giesUX053;LUNAR-GSDIII290100
262、3-30-1Glycogen storage dis-ease type IIILipid nanoparticle-encapsulated mRNA therapeutic encoding the glycogen debranchingenzyme;replacingthedefectiveAGLgeneproductallowscellstobreakdownglycogenusingnormal pathwaysUltragenyx,Arctu-rus TherapeuticsVerve-1012894841-30-4Heterozygous familialhypercholes
263、terolemiamRNA-based lipid nanoparticle therapeutic that targets the liver and base-editing in thePCSK9 gene to disrupt PCSK9 protein production to lower LDL cholesterol and treatcardiovascular diseaseVerve TherapeuticsaData from the CAS Content Collection,Clinicaltrials.gov,and Pharmaprojects.bDrug
264、no longer in development.ACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969963atory efficacy of an intra-myocardium AZD8601 injection inpatients with moderately impaired systolic function undergoingcoronary arte
265、ry bypass grafting surgery.Trial results revealed noserioussideeffectsandarisingtrendinefficacyforthetreatmentgroup.60SQZ Biotechnologies,in collaboration with Roche,hasdeveloped a mRNA-based cell therapeutic called SQZ-eAPC-HPV.SQZ-eAPC-HPVdeliversfivemRNAsforHPV16proteinantigensand immune-stimulat
266、ing proteins,including CD86 andmembrane-bound IL-2 and IL-12,into four different types ofengineered immune cells(monocytes,T-cells,B-cells,and NKcells).A phase I/II clinical trial(NCT05357898)is currentlyrecruitingtoassesssafetyandtolerability,antitumoractivity,andimmunogenic and pharmacodynamic eff
267、ects of SQZ-eAPC-HPV as monotherapy and in combination with pembrolizumabin patients with recurrent,locally advanced,or metastaticHPV16+solid tumors.BioNTech developed BNT141,a mRNA that encodessecreted IgG antibodies targeting claudin 18.2,a proteincommonly expressed on certain solid tumors.It is b
268、eingresearched in phase I/II clinical trial number NCT04683939,looking at its safety and pharmacokinetics in patients withClaudin 18.2-positive solid tumors.61METHODSThisstudyusedtheCASContentCollectionastheprimarydatasource,which aggregates and connects key scientific detailsdisclosed in more than
269、50,000 journals and patents from 64issuing authorities,as well as many other sources to cover thescopeofsciencefromchemistryandlifesciencesto engineering,materials science,and agriculture.A search query that searchedfor words or phrases in titles,abstracts,keywords,CAS indexterms,and substances was
270、developed to extract all the scientificjournal articles and patents related to mRNA vaccines andtherapeutics.The resulting patents were then intellectuallyreviewedto1)removefalsedropsand2)classifyeachdocumentinto one or more of the following classes:a)vaccines,b)therapeutics,and/or c)delivery system
271、 as well as mRNAmodification methods.During this review process,the likelyintriguing/notable/highly influential patents were also taggedfor further analyses.Once such reviews were done,the answer set containing over2,000 patents and more than 9,000 journal articles was furtheranalyzed for patents an
272、d journal articles separately for thecategories of mRNA vaccines and mRNA therapeutics.Specificanalyses included the trends over time;landscape analysesincluding country distribution,patent office distribution,flow ofpatents from applicant home countries to patent offices,andmost influential organiz
273、ations;and topic cluster analyses.Mostnotable patents,diseases involved,and specific mRNAtherapeutics and vaccines in clinical trials were also examinedbased on both CAS-licensed data and publicly availableinformation.The analysis tools include Thomson Data Analyze(DDA),VOSviewer,OmniGraphSketcher,a
274、nd ECharts.At the document level,this report focused more on patentsthanjournal articles.Agroupofpatentapplicationscoveringthesame or similar technical content is called a patent family.Patents for the same technological invention may be filed inmultiple countries or regions,and the CAS database mai
275、ntainsthese related applications as one record for indexing;this recordis referred to as the basic patent for most analyses in this report.Individual patent applications from the same patent family maybe applied for in different countries and/or patent offices.Thisproperly represents the distributio
276、n of patent applications filedby applicants countries(i.e.,applicants from different countriesor organizations);individual patent applications from the samefamily are counted separately for geographic distribution andpatent flow analysis.The country of origin of a patentedtechnology is determined ba
277、sed on the country location of thepatent assignee(or applicant/inventor).The country analysisinthis report is based on the country location of the patentapplicant for determination of the country of origin of thetechnology or the actual country owning the technology.OUTLOOK AND PERSPECTIVESThe remar
278、kable success of mRNA vaccines against COVID-19has strongly motivated interest in mRNA as a way of deliveringtherapeutic proteins.However,a variety of challenges remain tobe resolved before mRNA can be verified as a commontherapeutic modality with wide-ranging relevance to both rareand common diseas
279、es.The challenges in developing bettermRNA formulations are as follows:Enhancing specific protein expression:Elaboration of themRNAcargotoaugmentthetimeandamountofproteinproductionin vivo,including advances in the design of the primary chemicalstructure of the mRNA,novel forms of circular and self-a
280、mplifyingmRNA,and improved purification strategies.The most criticaladvances in mRNA vaccines and therapeutics are due to thediscovery that the insertion of chemically modified nucleosides,specifically in uridine moieties,can significantly increase proteinexpression.Sofar,over130chemicalmodification
281、sofRNAhavebeen described,and the properties and effects of multiple otherRNA chemical modifications remain to be explored.20,64Allparts of the mRNAthe cap,5 and 3 regions,open readingframe,and polyadenylated tailcan be optimized to augmentprotein expression.65In addition to the amount of proteinexpr
282、ession,a crucial hurdle of mRNA therapeutics for chronicdiseasesistherelativelyshorttimeperiodofproteinproduction,which requires repetitive administrations.Self-amplifyingmRNAs(saRNAs)make use of the self-replication of an RNAalphavirus,which can amplify RNA transcripts in thecytoplasm.66,67Circular
283、 mRNAs(circRNAs)provide anotherapproach for extending the duration of protein production.Thecircular structure shields circRNA from exonuclease attacks,which prolongs the RNA lifespan,thus raising the total proteinyield.68 Improving mRNA packaging and delivery systems,includingionizable LNPs,peptide
284、-based nanoparticles,cells,and cell-basedextracellular vesicles.The rapid clinical implementation ofmRNA-based vaccines and therapies has become possible dueto the development of efficient delivery vehicles to protect andtransport the highly unstable mRNAs.Various smart deliverysystems have been inv
285、ented to improve key features such ascirculation time in blood stream,biodistribution,cargo loading,and release.Lipid-based nanoparticles are currently the most widelystudied and clinically advanced vehicles for mRNA deliv-ery.47,6971ThemostextensivelyusedarecationicandionizableLNPs.72Although catio
286、nic lipid-based nanoparticles haveshown promise in certain therapies,their possible cytotoxicity73and relatively short circulation time74have somehow impededtheir clinical application.To deal with these issues,a variety ofnovel lipidPEG conjugates and ionizable lipids have beensynthesized and tested
287、.Ionizable lipids remain neutral atphysiological pH,which reduces their toxicity and increases tosome degree their circulation half-life.75Furthermore,theACS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969964prot
288、onation of ionizable lipids at acidic pH not only allowssuccessful condensation and encapsulation of mRNAs,but alsoallows the escape of mRNAs from the acidic endosomes.70,76ThePEGshellsetupbyPEGylatedlipidsconsiderablyprolongsthe circulation half-life of the LNPs and reduces theiraggregation;it also
289、 cut back unfavorable interactions withserum proteins.77Another promising approach uses extracellular vesicles andespecially their smallest version,the exosomes,as a deliveryvehicle.48Exosomes are nanosized vesicles enclosed by a lipidbilayer membrane.They are produced by most eukaryotic cellsand su
290、bsequently released in the extracellular space,providingmeans of efficient intercellular communication and signaling,including transport of bioactive molecules such as proteins,nucleic acids,and metabolites between cells and acrossbiological barriers.78,79As natural delivery vehicles,exosomespossess
291、 multiple benefits,such as biocompatibility and lowimmunogenicity.It has been reported that exosomes originatedfrom various cell types do not provoke toxicity and are well-tolerated after repetitive dosing.80Thus,they can be viewed aspromising mRNA delivery systems in cases in which repeateddosing i
292、s required.Peptides represent other versatile,biocompatible,andtargetable RNA carriers.From this class,cell-penetratingpeptides(CPPs)that can penetrate the cellular membraneand transfer to the cytoplasm are used most frequently for RNAdelivery by forming a variety of nanocomplexes depending onthefor
293、mulationsettingsandthepropertiesoftheusedCPPsandRNAs.81,82A further novel biological alternative to lipid nanoparticlecarriers is the use of cell-based delivery systems,exploiting thecellular paracrine communication ability to directly deliverproteins synthesized by mRNA brought into cells ex vivo.I
294、tprovides certain advantages,such as biocompatibility,lack oftoxicity,and extended half-life in circulation.Variousmodifications are feasible by introducing mRNA into cellssuch as immune cells,blood cells,and others.8385Recently,abacteria-mediated vehicle for oral mRNA vaccine delivery hasbeen also
295、developed.This multiple-target vaccine successfullyexplored engineered Salmonella-based vector to deliver mRNAvaccine against Delta and Omicron variants of COVID-19.86,87TargetingmRNAtherapeuticstocertaintissuesandengineeringof delivery systems with tissue-specific affinity.Fulfilling thepotential o
296、f mRNA therapeutics requires proper targeting.Liveris the largest internal organ in human body,performing vitalroles in various physiological activities,thus large amount ofdisease targets potentially receptive to mRNA therapies residewithin the liver hepatocytes.A promising liver-targeted N-acetylg
297、alactosamine(GalNAc)ligand has been assessed invarioustrialsforenhancingthecellularuptakeandtissuespecificdelivery of mRNAs.88GalNAc-based delivery relies on theability of the hepatocytes to express the asialoglycoproteinreceptor,a high-capacity,rapidly internalizing receptor thatbinds glycoproteins
298、 via receptor-mediated endocytosis.Inrecent years,encouraging progress has been made in the fieldof GalNAc conjugates,underscoring the value of targetingmoieties.89,90The conjugation of GalNAc moieties tooligonucleotides represents a promising and safe approach forliver-targeted delivery of nucleic
299、acid therapeutics.Selective delivery of mRNA therapeutics to organs other thanliver requires specifically developed packaging and delivery systemswithappropriate affinity.Therecenteffortsondeveloping betterdelivery systems and/or administration routes for this purposehave produced some exciting resu
300、lts.For example,DanielSiegwarts team has recently developed a method of SelectiveORgan Targeting(SORT)that uses various engineered lipidnanoparticles to selectively deliver mRNA to extrahepaticorgans.91Qiaobing Xus team has identified an endogenouslylymph node-directing lipid nanoparticle system.92K
301、athryn A.Whiteheadsteamdevisedastrategyofdeliveringcationichelperlipid-containing lipid nanoparticles by intraperitoneal admin-istration to deliver mRNA to pancreas,especially the insulin-producing cells.93Gaurav Sahays lab has developed a LNPsystem with increased polyethylene glycol(PEG)and inclusi
302、onof a cholesterol analog,-sitolsterol to optimize the delivery ofthe nebulized,LNP-based mRNA for fibrosis transmembraneconductance regulator(CFTR)protein as potential inhalableLNP-based mRNA therapies.94 Strategies for allowing repeated dosing for the treatment ofchronic conditions.The development
303、 of mRNA therapeuticsraises further challenges as compared to those with mRNAvaccines.While immunization requires only a small amount ofprotein production,as the immune system will amplify theantigenic signal,mRNA therapeutics require much higher levelof protein to reach a therapeutic level.The tiss
304、ue bioavailability,half-life in circulation,and carrier efficiency to deliver to thetargeted tissue remain challenging.Another major difficulty isthe repeated dosing,which is typically required in the treatmentof chronic diseases and which activates innate immunity in thelong run,with a resultant re
305、duction of therapeutic proteinexpression.65Regardless of these hurdles,however,a variety ofemerging technologies are currently under development withthe effort to deal with them.26,9597Despite these challenges,impressive progress has been madein the research and development of mRNA therapeutics andv
306、accines.Most recently,Luis A.Rojas et al.have reported thepromising result of a Phase 1 clinical trial in which personalizeduridine mRNA neoantigen vaccines induced substantial T cellactivity in postsurgery patients with pancreatic ductaladenocarcinoma.98The remarkable success of the mRNAvaccines fo
307、r COVID-19,along with recent expansion of thistechnology into treatment of most lethal cancers,has validatedthe great promise of this new class of medications andsignificantly enhanced the chance to witness their extensiveapplications in the near future.ASSOCIATED CONTENT*sSupporting InformationThe
308、Supporting Information is available free of charge athttps:/pubs.acs.org/doi/10.1021/acsptsci.3c00047.SI-1,trend over time for patents related to mRNAtherapeutics;SI-2,trend over time for patents related tomRNA vaccines;SI-3,trend over time for patents onmRNA delivery systems;SI-4,trend over time fo
309、r patentsrelated to mRNA modification;SI-5,analysis of researchtopics in mRNA therapeutics patents;SI-6,analysis ofresearchtopicsinmRNAvaccinepatents;SI-7,analysisofresearch topics in patents related to mRNA modification;and SI-8,top countries and organizations in the field ofmRNA modification(PDF)A
310、CS Pharmacology&Translational Sciencepubs.acs.org/ptsciReviewhttps:/doi.org/10.1021/acsptsci.3c00047ACS Pharmacol.Transl.Sci.2023,6,943969965AUTHOR INFORMATIONCorresponding AuthorsXuezhaoWangInformationCenter,NationalScienceLibrary,Chinese Academy of Science,Beijing 100190,P.R.China;Email:Qiongqiong
311、 Zhou CAS,Columbus,Ohio 43202,UnitedStates;orcid.org/0000-0001-6711-369X;Email:qzhoucas.orgAuthorsDongqiao Li Information Center,National Science Library,Chinese Academy of Science,Beijing 100190,P.R.ChinaCynthia Liu CAS,Columbus,Ohio 43202,United States;orcid.org/0000-0003-3858-1501Yingzhu Li CAS,C
312、olumbus,Ohio 43202,United States;orcid.org/0000-0002-4946-7272Rumiana Tenchov CAS,Columbus,Ohio 43202,UnitedStates;orcid.org/0000-0003-4698-6832Janet M.Sasso CAS,Columbus,Ohio 43202,United States;orcid.org/0000-0002-1156-5184Di Zhang Information Center,National Science Library,Chinese Academy of Sci
313、ence,Beijing 100190,P.R.ChinaDanLiInformationCenter,NationalScienceLibrary,ChineseAcademy of Science,Beijing 100190,P.R.ChinaLixue Zou Information Center,National Science Library,Chinese Academy of Science,Beijing 100190,P.R.ChinaComplete contact information is available at:https:/pubs.acs.org/10.10
314、21/acsptsci.3c00047Author ContributionsD.L.and C.L.contributed equally to this paper.NotesThe authors declare no competing financial interest.ACKNOWLEDGMENTSThe authors sincerely appreciate the CAS Data,Analytics&Insights team for their assistance in data extraction.The authorswant to thank Caroline
315、 Ma and Sunny Yu for their effort infacilitating collaboration and Dharmini Patel for projectmanagement.The authors are grateful to the leadership teamfrom their two organizations:Xiwen Liu,Manuel Guzman,Gilles Georges,Michael Dennis,Craig Stephens,and DawnRiedel.We are also grateful to the consulti
316、ng experts for theirguidance during the research process.The Chinese NationalScience Library(NSL)work was funded by the YouthInnovation Promotion Association for Chinese Academy ofScience(No.2023182),NSTL“Research on Key Technologiesof mRNA Vaccine R&D Pipeline”(E11Z0416),and ChineseAcademy of Scien
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