Slov Vet Res | Vol 60 No 3 | 123 DOI 10.26873/SVR-1879-2023 UDC 577.2:615.371:06.05NOBEL:172.4 Pages: 123–26 Editorial In the Spotlight A 2023 Nobel Prize in Physiology or Medicine: Pathway for Next Generation of Vaccines This year’s Nobel Prize in Physiology or Medicine has been awarded to Katalin Karikó and Drew Weissman for discov- eries that enabled the development of messenger RNA (mRNA) vaccines against COVID-19. mRNA is a transient molecule in the cell that conveys the instructions for syn- thesis of a protein from the nucleus, where instructions are stored as a genetic code in the DNA, to the cell’s pro- tein making machinery (ribosomes) in the cytoplasm. It took several decades of research to uncover how mRNA could be used to deliver an antigen into cells and trigger the body’s own immune response. Traditional vaccine development, which used a weakened or dead virus to stimulate an immune response against the disease, is lengthy and costly. Progress in molecular biol- ogy enabled the development of vaccines based on individ- ual viral components, where parts of the viral genetic code are used to make proteins that stimulate the formation of virus-blocking antibodies. The most recently developed mRNA vaccines contain viral mRNA that, when injected into the body, instructs the cells to produce parts of viral proteins that trigger the immune response. Since mRNA can be quickly synthesized and modified, the development of mRNA vaccines can be much faster than traditional vaccines. By discovering how to make mRNA stable and prevent immune activation by the mRNA itself, the semi- nal discoveries of his year’s Nobel Prize laureates were es- sential to the development and implementation of mRNA vaccines. Letošnja Nobelova nagrada za fiziologijo ali medicino je bila podeljena Katalin Karikó in Drewu Weissmanu za odkritja, ki so omogočila razvoj cepiv proti COVID-19 na osnovi spo- ročilne RNA (mRNA). mRNA je prehodna molekula v celi- ci, ki posreduje navodila za sintezo proteinov iz jedra, kjer so navodila shranjena kot genetski kod v DNA, celičnemu sistemu za izdelovanje proteinov (ribosomov) v citoplazmi. Potrebnih je bilo več desetletij raziskav, da bi odkrili, kako uporabiti mRNA za prenos antigenov celicam in začetek te- lesu lastnega imunskega odziva. Tradicionalni razvoj cepiv, ki je uporabljal oslabljene ali mrtve viruse za stimulacijo imunskega odziva proti bolez- nim, je drag in dolgotrajen. Napredek v molekularni biologiji je omogočil razvoj cepiv na osnovi posameznih virusnih delov. Pri tem so deli virusnega genetskega koda upora- bljeni za proizvodnjo proteinov, ki stimulirajo tvorbo protite- les proti virusom. Najsodobnejša cepiva na osnovi mRNA vsebujejo del virusne mRNA, ki ob injiciranju v telo celicam posredujejo navodila za proizvodnjo delov virusnih protei- nov, ki sprožijo imunski odziv. Ker je lahko mRNA hitro sin- tetizirana in nadgrajena, je lahko tudi proizvodnja cepiv na osnovi mRNA precej hitrejša kot proizvodnja tradicionalnih cepiv. Z odkritjem, kako molekulo mRNA napraviti stabilnej- šo in kako preprečiti imunsko aktivacijo s samo molekulo mRNA, so bila temeljna odkritja letošnjih Nobelovih nagra- jencev, ključna za razvoj in uporabo cepiv na osnovi mRNA. Nobelova nagrada 2023 za fiziologijo ali medicino: pot do naslednjega rodu cepiv Uroš Rajčević1*, Klementina Fon Tacer2** 1Novartis Pharmaceutical Manufacturing LLC, Ljubljana, Slovenia, 2Texas Tech University School of Veterinary Medicine and Texas Center for Comparative Cancer Research, Amarillo, Texas, USA *Co-Editor, uros.rajcevic@novartis.com **Editor-in-Chief, fontacer@ttu.edu Accepted: 28 September 2023 124 | Slov Vet Res | Vol 60 No 3 Gene therapy produces a therapeutic effect by genetically modifying cells through introduction of a new gene or re- construction of existing defective genetic material. In the case of mRNA vaccines, the genetic code for an antigen (i.e., a viral protein) triggers an immune response that ulti- mately leads to immune protection against the virus. Gene therapy has been gaining momentum in human medicine as a versatile therapeutic approach in the past decade, follow- ing the first gene therapy approvals in Europe in 2012 and 2016 for Glybera (to reverse lipoprotein lipase deficiency) and Strimvelis [to treat severe combined immunodeficien- cy due to adenosine deaminase deficiency (ADA-SCID)], re- spectively (1, 2), and the first approval in the USA in 2017 for Kymriah (to treat acute lymphoblastic leukemia)(3). Besides offering hope for a cure to patients with rare diseases, cer- tain types of cancer, and developmental genetic diseases, gene therapy also has enormous potential in preventing diseases via mRNA vaccines. In the post-genomic era, the development of technology and the availability of genomic data enables the rational and targeted design and clinical testing of various kinds of gene therapeutics. Genes can be delivered to the cell of interest by DNA plas- mids, viral vectors, mRNA, or other methods. Although gene transfer technology using mRNA was investigated for decades, several roadblocks, including a lack of mRNA stability and immune rejection of RNA molecules, hindered the efficiency of mRNA delivery. Through several decades of research, Katalin Karikó and Drew Weissman made vital discoveries that were instrumental in breaching these road- blocks. First, Karikó and Weissman discovered that the in- troduction of chemical modifications in mRNA nucleoside bases almost abolished inflammation when base-modified mRNA was delivered to dendritic cells (4). This discovery was a paradigm shift in our understanding of how cells recognize and respond to different forms of mRNA, as it showed that in vitro transcribed mRNA containing modi- fied bases evades innate immune recognition. Additionally, they found that base modification of mRNA increased its stability and, consequently, protein production in cells (5, 6). These two discoveries, along with the unprecedented investment by the pharmaceutical industry, enabled the rapid development of the mRNA vaccine during recent pan- demics and opened the flood doors for novel gene therapy opportunities, including vaccine development and cancer immunotherapy (7, 8). Compared to the viral vectors currently used in most ap- proved gene therapies, mRNA gene transfer is a safer al- ternative because mRNA is devoid of potentially toxic viral genes and regulatory elements present in the viral vectors. Furthermore, mRNA does not integrate into the genome, is non-replicative, and decays within a few days. This tempo- rary therapeutic expression of the encoded protein is desir- able in vaccine development and has been implemented in certain CAR-T therapy research applications (8, 9). Genska terapija povzroči terapevtski učinek z genskim spreminjanjem celic z vnašanjem novih genov ali popra- vljanjem obstoječega, okvarjenega genetskega materiala. V primeru cepiv na osnovi mRNA genetski kod za antigen (npr. virusni protein) sproži imunski odziv, kar v končni fazi privede do imunske zaščite proti virusu. Genska terapija v zadnjem desetletju pridobiva na pomembnosti kot razno- vrstni terapevtski pristop, po prvih odobritvah tovrstne te- rapije v Evropi leta 2012 in 2017 za Glybero (ki je zdravila pomanjkanje lipoproteinske lipaze) ter Strimvelis (ki zdravi hudo kombinirano imunsko pomanjkljivost zaradi pomanj- kanja adenozinske deaminaze (ADA-SCID)) (1, 2) in po prvi odobritvi v ZDA za Kymriah (ki zdravi akutno limfoblastič- no levkemijo in ne-Hodgkinove limfome) (3). Poleg tega, da lahko ponudi upanje za ozdravitev bolnikom z redkimi boleznimi, nekaterimi oblikami raka in razvojnimi bolezni- mi, kaže genska terapija velikanski potencial pri preventivi bolezni s cepivi na osnovi mRNA. V po-genomski eri razvoj tehnologije in dostopnost genomskih podatkov omogočata racionalno in ciljno načrtovanje in klinično preizkušanje raz- ličnih oblik genske terapije. Geni so lahko v ciljno celico dostavljeni s plazmidi DNA, z virusnimi vektorji, mRNA ter z drugimi metodami. Čeprav je bila tehnologija genskega prenosa z uporabo mRNA predmet raziskovanj že več desetletij, so številne ovire, vključno s pomanjkljivo stabilnostjo mRNA in imunskim zavračanjem molekul mRNA, zadrževale učinkovito dostav- ljanje mRNA v celico. Med večdesetletnimi raziskavami sta Katalin Karikó in Drew Weissman prišla do ključnih odkritij, nujnih za odpravo teh ovir. Najprej sta odkrila, da je uvaja- nje kemijskih sprememb v nukleozidne baze mRNA skoraj odpravilo vnetne procese, kadar je bila mRNA z modificira- nimi bazami uvedena v dendritične celice (4). To odkritje je pomenilo premik v načinu razmišljanja, kako celice prepoz- najo in odgovorijo na različne oblike mRNA. Ugotovila sta, da se in vitro prepisane mRNA, ki vsebujejo modificirane baze, izognejo prirojenemu imunskemu prepoznavanju. Poleg tega sta ugotovila tudi, da modifikacija baz pri mRNA poveča njeno stabilnost in posledično produkcijo proteinov v celici (5, 6). Ti dve odkritji sta, skupaj z do tedaj nepredsta- vljivimi investicijami v farmacevtski industriji, omogočili hi- ter razvoj cepiv na osnovi mRNA med nedavno pandemijo in na stežaj odprli vrata priložnostim novih genskih terapij, vključno z razvojem cepiv in imunoterapij raka (7, 8). V primerjavi z virusnimi vektorji, ki so trenutno najpogosteje uporabljeni vektorji pri večini odobrenih genskih terapij, je genski prenos z mRNA varnejši pristop zato, ker mRNA ne vsebuje potencialno nevarnih virusnih genov in regulatornih elementov, ki se nahajajo v virusnih vektorjih. Poleg tega se mRNA ne vključuje v genom, se ne replicira in razpade v ne- kaj dneh. Tako začasno terapevtsko izražanje kodiranega proteina je zaželeno pri razvoju cepiv in je bilo uporabljeno tudi pri nekaterih oblikah razvoja terapij s CAR-T (8, 9). V veterinarski medicini, tudi zaradi manj stroge zakonoda- je v primerjavi s humano medicino, genska terapija ni nič Slov Vet Res | Vol 60 No 3 | 125 Due to the less stringent regulations in veterinary medicine compared to human medicine, gene therapy is not new in veterinary medicine. Rather, comparative medicine is lead- ing the way in the development of novel approaches, such as combining gene therapy with electrochemotherapy in cancer treatment to improve the therapeutic outcome, a technique pioneered by and also mastered through the collaborative efforts of Slovenian veterinarians, medical doctors, and comparative researchers (10-13). In horses and dogs, interleukin (IL)-12 based gene therapy improved the electrochemotherapy antitumor effects on spontane- ously occurring tumors in large and companion animals (10-13) and has potential in translating to human medi- cine. Furthermore, mRNA vaccines hold great promise in veterinary medicine. In past years, several mRNA vaccines have entered clinical trials. Due to their low risk of inser- tional mutagenesis, high potency, and potential for low-cost manufacturing, mRNA vaccines promise solutions to com- bat emerging and re-emerging infectious diseases, such as rabies, Zika, and influenza (14, 15) The exciting discoveries leading to this year’s Nobel Prize in Physiology or Medicine will impact future human and vet- erinary medicine and may be critical to help combat current and future zoonotic diseases. Funding: K.F.T. is funded by the Texas Tech University start- up, Cancer Prevention and Research Institute (CPRIT) of Texas Scholar Award RR200059, the Foundation for Prader– Willi Syndrome (FPWR) Grants 22-0321 and 23-0447. Acknowledgments: We thank Dr. Rebecca Gee for the lan- guage editing. novega. Primerjalna medicina nas vodi v razvoju novih pris- topov, kot je kombinacija genske terapije z elektrokemote- rapijo raka za izboljšanje terapevtskega učinka, tehnik, ki so jih s skupnimi napori vzpostavili in vodili slovenski veteri- narji, zdravniki in primerjalni raziskovalci (10–13). Pri konjih in psih je genska terapija z interlevkinom (IL)-12 izboljšala protitumorske učinke elektrokemoterapije pri spontanih tumorjih velikih živali in hišnih ljubljenčkov (10–13) in ima tudi potencial translacije v humano medicino. Poleg tega v veterinarski medicini veliko obljubljajo tudi cepiva na osnovi mRNA. Številna cepiva se v zadnjih letih že klinično preiz- kušajo. Zaradi nizkega tveganja za insercijsko mutagenezo, visokih zmogljivosti in potencialno nizkih stroškov proizvo- dnje cepiva na osnovi mRNA obetajo rešitve za boj proti novim in obstoječim kužnim boleznim, kot so steklina, zika in gripa (14, 15). Vznemirljiva odkritja, ki so privedla do letošnje Nobelove nagrade za fiziologijo ali medicino, bodo vplivala na huma- no in veterinarsko medicino in bodo lahko tudi ključno pri- pomogla k boju z zoonozami danes in v prihodnosti. Financiranje: K.F.T. je financirana s sredstvi ‘Texas Tech University start-up’, ‘Cancer Prevention and Research Institute (CPRIT) of Texas Scholar Award RR200059’ in s sredstvi ‘Foundation for Prader–Willi Syndrome (FPWR) Grants 22-0321 and 23-0447’. References 1. Glybera. Amsterdam: European medicines agency, 2023. https://www. ema.europa.eu/en/medicines/human/EPAR/glybera (15. 11. 2023) 2. Strimvelis. Amsterdam: European medicines agency, 2023. https:// www.ema.europa.eu/en/medicines/human/EPAR/strimvelis (15. 11. 2023) 3. FDA approval brings first gene therapy to the United States. Silver Spring: Food and Drug Administration, 2023. https://www.fda.gov/ news-events/press-announcements/fda-approval-brings-first-gene- therapy-united-states (15. 11. 2023) 4. Karikó K, Buckstein M, Ni H, Weissman D. Suppression of RNA recogni- tion by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 2005; 23: 165–75. 5. Karikó K, Muramatsu H, Welsh FA, et al. Incorporation of pseudouri- dine into mRNA yields superior nonimmunogenic vector with in- creased translational capacity and biological stability. Mol Ther 2008; 16: 1833–40. 6. Anderson BR, Muramatsu H, Nallagatla SR, et al. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res 2010; 38: 5884–92. 7. 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