Self-Amplifying mRNA Ancestral-strain Monovalent Vaccine(Arcturus Therapeutics)

导读：体外转录RNA（IVT RNA）包括mRNA、自复制RNA(saRNA)、反式扩增RNA(taRNA)和环状RNA(circRNA)，近年来在预防和治疗用生物制剂的开发中备受关注。IVT RNA是一种精心设计的序列，以模拟天然RNA转录本发挥翻译蛋白的生物学功能。IVT RNA的开发领域正在从人类拓展至动物，包括畜牧业、水产业和宠物领域。在多种类型的IVT RNA中，mRNA最为经典，且在大规模人群中验证了其疗效和安全性。mRNA序列由5个关键结构元件组成：5'帽结构（Cap）、5'非翻译区（UTR）、编码区（CDS）、3'非翻译区（UTR）和poly（A）尾巴。mRNA结构极其不稳定，容易被降解。“万变不离其宗”，合理的序列设计是提高mRNA稳定性的一种有效策略。本期文章，菌菌将以BioNTech和辉瑞联合开发的mRNA疫苗BNT162b2为实例，分享mRNA疫苗的序列设计策略。01 BNT162b2的编码序列设计mRNA的编码区（CDS），也称为开放阅读框（ORF），在体内翻译产生具有生物活性的目标蛋白，如病原体关键抗原、肿瘤抗原、抗体或其他功能性蛋白，CDS序列的设计和优化至关重要。CDS的设计策略主要为密码子优化，以提升蛋白翻译效率。密码子的优化包括两个水平，分别为复合密码子家族和家族内密码子优化。1.1 复合密码子家族优化例如，鉴于宿主锌指抗病毒蛋白（ZAP，基因名 ZC3HAV1）以病毒 RNA 中的 CpG 二核苷酸为靶标，并招募细胞RNA降解复合物来降解病毒RNA基因组，因此避免使用 CGN 密码子在病毒进化上是合理的。与之对应地，NC_045512中的 SARS-2-Spike 包含42个Arg残基，其中30个由 AGR 密码子编码，只有12个由 CGN 密码子编码。然而，人类基因使用CGN的频率高于AGR密码子。辉瑞/BioNTech采用了典型的复合密码子优化策略，在BNT-162b2的CDS区域减少了8个AGR密码子，对应增加了CGN密码子。mRNA疫苗的高CpG同时赋予其较高的稳定性和安全性。Moderna开发的mRNA-1273也使用了这一策略，但在数量上有所差异，菌菌将在下期文章中对mRNA-1273序列进行详细解析，敬请关注。类似地，高表达的人类核糖体蛋白中，81%的亮氨酸通过CUN密码子编码。因此，在mRNA疫苗BNT-162b2中，多数的UUR被替换为CUN。1.2 家族内密码子优化家族内密码子优化的原则是替换为高表达基因优选且被最丰富的tRNA解码的最佳密码子。基于上述原则，在编码Arg的密码子中，CGG被认为是CGN家族中的最优解，BNT-162b2和mRNA-1273也基于此进行了优化。而在Glu编码密码子的优化方面，辉瑞/BioNTech采用了自由优化策略。BNT-162b2保留了14/34个GAA密码子，而没有选择全部替换为tRNA丰度最高的GAG密码子。当前密码子优化主要以CAI（密码子适应指数）为基本的优化参数。BNT-162b2的CAI约为0.95，相比天然新冠病毒S蛋白编码区域（CAI＜0.7）大幅改善。02 BNT162b2的非编码区序列设计2.1 BNT162b2 5’ UTR序列设计5’UTR为非编码区域，位于mRNA编码区（CDS）上游，是核糖体起始翻译的结合位点。5’UTR主要发挥2个重要功能：提升mRNA稳定性和起始翻译。在mRNA疫苗研发过程中，有三种 5’UTR序列优化策略。第一种也是最简单的，直接选取高表达的人类基因 5’UTR，例如人alpha珠蛋白的 5’UTR。这一策略在mRNA序列设计中较为常见，BNT162b2直接选取了人alpha珠蛋白基因的5’UTR，在此基础上针对Kozak序列进行了优化，具体为使用GCCACCAUG替换了ACCAUG。第二种方法是使用病原体mRNA自身的5’UTR，这一策略目前主要用于自复制RNA(saRNA)的序列设计，如日本厚生劳动省（PMDA）获批的ARCT-154。关于ARCT-154的序列解析可戳链接查看：全球首款自复制RNA(saRNA)传染病疫苗ARCT-154的序列解析第三种是通过指数富集（SELEX）的方式来实现 5’UTR的系统演化，这种方法有一定的筛选难度较大。不同的5’UTR序列可能适用于不同的mRNA编码序列、不同的靶细胞。为此，耀海生物mRNA制备平台建立了丰富的天然UTR库、突变UTR库，为客户CDS序列定制化筛选最适合的UTR序列。如感兴趣可扫描文末二维码联系菌菌~2.2 BNT162b2 3’UTR序列设计3’UTR位于mRNA编码区（CDS）下游，与5’UTR的功能和设计原则较为相似。辉瑞/BioNTech融合了天然序列和SELEX筛选序列。BNT162b2 3’UTR的部分片段来自人类线粒体12S rRNA（mtRNR1），其余片段为人AES/TLE5基因。AES/TLE5上存在2个C→U(Ψ)突变以提高结合能量。BNT162b2的CDS与3’UTR中间存在6个碱基），随后是136 nt的AES突变序列片段，再下游是139 nt的mtRNR1序列。2.3 BNT162b2 poly(A)序列设计为维持mRNA疫苗的稳定性，BNT162b2采用了分段式poly(A)序列：长度为110 nt的poly(A)由30个腺苷、10个非纯A序列、以及下游的70个腺苷组成。03 总结随着mRNA工艺路线的不断升级，无疑推动了各类体外转录RNA（IVT RNA）疫苗/药物的开发进程，深刻地影响生物医药领域的格局。IVT RNA的开发前景广阔，当前正在从人用药领域拓展至畜牧养殖、水产养殖和宠物领域。合理的序列设计是mRNA开发的首要目的。基于多年的技术沉淀，耀海生物开发了自有的mRNA体外转录质粒载体、建立了天然UTR和改造UTR库、自主研发了mRNA纯化工艺和检测方法，全力推动mRNA制品的研发进程。参考文献[1] Xia X. Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and Moderna mRNA Vaccines. Vaccines (Basel). 2021 Jul 3;9(7):734. doi: 10.3390/vaccines9070734. [2] WHO. Messenger RNA vaccines.识别微信二维码，添加生物制品圈小编，符合条件者即可加入生物制品微信群！请注明：姓名+研究方向！版权声明本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。

Commercial manufacture of Kostaive® on track for delivery of initial 4 million doses in Q3 Kostaive® European Marketing Authorization Application approval decision expected Q3 Multiple Kostaive® Phase 3 trials further demonstrate breadth and durability of STARR® vaccine platform ARCT-2138 (LUNAR-FLU) Phase 1 topline immunogenicity and safety data, anticipated in Q3 ARCT-810 (LUNAR-OTC) Phase 2 and ARCT-032 (LUNAR-CF) Phase 1b interim data and update to be provided on July 1st JP Morgan engaged to monetize investment in ARCALIS JV in Japan Investor conference call at 4:30 p.m. ET today SAN DIEGO--(BUSINESS WIRE)-- Arcturus Therapeutics Holdings Inc. (the “Company”, “Arcturus”, Nasdaq: ARCT), a global messenger RNA medicines company focused on the development of infectious disease vaccines and opportunities within liver and respiratory rare diseases, today announced its financial results for the first quarter ended March 31, 2024, and provided corporate updates. “Arcturus continues to make encouraging progress in both our vaccine and therapeutics pipeline,” said Joseph Payne, President & CEO of Arcturus. “In collaboration with our global vaccine partner CSL and their partner Meiji Seika Pharma, we are excited to begin the commercialization of Kostaive this year.” Mr. Payne continued, “We also look forward to providing meaningful clinical study data updates July 1st, for each of our flagship mRNA therapeutic programs, ARCT-810 (OTC deficiency) and ARCT-032 (CF).” “I am pleased to announce that we have engaged JP Morgan to monetize our stake in ARCALIS, our JV manufacturing operation in Japan,” stated Andrew Sassine, Chief Financial Officer of Arcturus Therapeutics. “Additionally, I am pleased to announce we will begin to qualify for commercial milestones under our CSL collaboration upon commencement of Kostaive revenues in Japan this year. Finally, our cash runway remains strong for at least three years into the first quarter of fiscal year 2027.” Recent Corporate Highlights In March, Meiji Seika Pharma announced plans to supply Japan with 4 million doses of Kostaive for fall/winter season of 2024. To support this effort, Arcturus along with CDMO partners are on track to deliver the initial 4 million commercial doses of Kostaive in Q3. In March, the Company, along with partners CSL and Meiji, announced that the Company’s bivalent COVID-19 Vaccine candidate, ARCT-2301 (Wuhan strain and Omicron BA.4/5), met the primary endpoint (non-inferiority) in a Phase 3 clinical study in Japan. The study enrolled 930 healthy adults and individuals with comorbidities, who previously received three to five doses of mRNA COVID-19 vaccines, including the last booster at least three months prior to recruitment. Both the geometric mean titer (GMT) ratio and seroresponse rate (SRR) difference of neutralizing antibodies against SARS-CoV-2 (Omicron BA.4/5) and Wuhan strains met pre-specified non-inferiority and superiority criteria versus a licensed mRNA vaccine comparator. There were no causally-associated serious adverse events with ARCT-2301. In March, Arcturus and CSL initiated a Phase 3 pivotal study with the ARCT-2303 candidate vaccine containing the Omicron XBB.1.5 variant. The purpose of this study is to generate additional immunogenicity and safety data to support product licensure in the U.S. The study will also assess the co-administration of ARCT-2303 with the age-appropriate seasonal influenza vaccines. Approximately 1,680 young and older adults are planned to be recruited in the southern hemisphere. The Company has filed a Marketing Authorization Application (MAA) for Kostaive to the European Medicines Agency (EMA), with the European Commission (EC) expected to provide an approval decision in Q3. ARCT-2138 (LUNAR-FLU, Quadrivalent Seasonal Influenza), is progressing well through Arcturus’ partner CSL. As of May 1, 2024, 84 healthy young adults were recruited in a Phase 1 dose-finding and immunogenicity study and received one of four dose levels of the study vaccine or a licensed influenza vaccine. The recruitment of older adults is ongoing. In April, the Company presented Phase 1 single ascending dose (SAD) studies for ARCT-810, an mRNA therapeutic candidate for ornithine transcarbamylase (OTC) deficiency, at the Society for Inherited Metabolic Diseases (SIMD) annual conference. ARCT-810-01 was a Phase 1 SAD study that enrolled 30 healthy adults, randomized 2:1 to receive ARCT-810 (0.1, 0.2, 0.3 or 0.4 mg/kg) or placebo as an intravenous infusion. ARCT-810-02 is a recently completed Phase 1b SAD study that enrolled 16 adults with mild OTC deficiency, randomized 3:1 to receive single doses of ARCT-810 (0.2, 0.3, 0.4, or 0.5 mg/kg) or placebo as an intravenous infusion. The results showed that ARCT-810 was generally well tolerated with no serious or severe adverse events in both studies. The results from ARCT-810-01 and ARCT-810-02 studies facilitated the initiation of a Phase 2 multiple ascending dose study of ARCT-810 (ARCT-810-03) in OTC deficiency adolescents and adults which is ongoing in the UK and EU. Subjects are randomized to receive 6 doses of ARCT-810 or placebo (randomized 3:1) administered every 14 days. The Company will share a progress update on the Phase 2 study on July 1, 2024. Arcturus is advancing ARCT-032, an inhaled mRNA therapeutic for cystic fibrosis. The Company remains on track to share Phase 1b interim data on July 1, 2024. Each CF patient in this trial receives two inhaled administrations of ARCT-032. Financial Results for the three months ended March 31, 2024 Revenues in conjunction with strategic alliances and collaborations: Arcturus’ primary sources of revenues were from license fees, consulting and related technology transfer fees, reservation fees and collaborative payments received from research and development arrangements with pharmaceutical and biotechnology partners. For the three months ended March 31, 2024, revenues were $38.0 million compared with $80.3 million for the three months ended March 31, 2023. The decrease was primarily attributable to the CSL agreement as $78.2 million total revenue was recognized during the first quarter of 2023 upon the achievement of a conditional payment and multiple milestones, compared to $32.4 million total revenue related to CSL during the first quarter of 2024, resulting in a decrease of $45.8 million. The total decrease was primarily offset by an increase in revenue of $4.9 million related to the agreement with BARDA. Operating expenses: Total operating expenses for the three months ended March 31, 2024, were $68.4 million compared with $65.5 million for the three months ended March 31, 2023. Research and development expenses: Research and development expenses consist primarily of external manufacturing costs, in-vivo research studies and clinical trials performed by contract research organizations, clinical and regulatory consultants, personnel-related expenses, facility-related expenses and laboratory supplies related to conducting research and development activities. Research and development expenses were $53.6 million for the three months ended March 31, 2024, compared with $51.8 million in the comparable period last year. The increase in research and development expenses were primarily driven by the CSL and BARDA programs as well as Arcturus’ internal OTC and Cystic Fibrosis programs. Additionally, investments increased in early stage and discovery technologies, including the initiation of preclinical research related to its Lyme Disease and Gonorrhea vaccine discovery programs. General and Administrative Expenses: General and administrative expenses primarily consist of salaries and related benefits for executive, administrative, legal and accounting functions and professional service fees for legal and accounting services as well as other general and administrative expenses. General and administrative expenses were $14.9 million for the three months ended March 31, 2024, compared with $13.8 million in the comparable period last year. The increase in expenses resulted primarily from increased personnel expenses due to increased salaries, increased travel and consulting expenses as well as escalated rent expense associated with facilities. Net Loss: For the three months ended March 31, 2024, Arcturus reported a net loss of approximately $26.8 million, or ($1.00) per diluted share, compared with a net income of $50.8 million, or $1.87 per diluted share in the three months ended March 31, 2023. Cash Position and Balance Sheet: Cash, cash equivalents and restricted cash were $345.3 million as of March 31, 2024, and $348.9 million on December 31, 2023. Arcturus achieved a total of approximately $420.1 million in upfront payments and milestones from CSL as of March 31, 2024, and expects to continue to receive future milestone payments from CSL supporting the ongoing development of the COVID and flu programs and three additional vaccine programs by CSL. The expected cash runway extends at least three years based on the current pipeline and programs through the first quarter of fiscal year 2027. Earnings Call: Wednesday, May 8, 2024 @ 4:30 pm ET Domestic: 1-888-886-7786 International: 1-416-764-8658 Conference ID: 96934019 Webcast: Link About Arcturus Therapeutics Founded in 2013 and based in San Diego, California, Arcturus Therapeutics Holdings Inc. (Nasdaq: ARCT) is a global mRNA medicines and vaccines company with enabling technologies: (i) LUNAR® lipid-mediated delivery, (ii) STARR® mRNA Technology (sa-mRNA) and (iii) mRNA drug substance along with drug product manufacturing expertise. Arcturus developed the first self-amplifying messenger RNA (sa-mRNA) COVID vaccine (Kostaive®) in the world to be approved. Arcturus has an ongoing global collaboration for innovative mRNA vaccines with CSL Seqirus, and a joint venture in Japan, ARCALIS, focused on the manufacture of mRNA vaccines and therapeutics. Arcturus’ pipeline includes RNA therapeutic candidates to potentially treat ornithine transcarbamylase deficiency and cystic fibrosis, along with its partnered mRNA vaccine programs for SARS-CoV-2 (COVID-19) and influenza. Arcturus’ versatile RNA therapeutics platforms can be applied toward multiple types of nucleic acid medicines including messenger RNA, small interfering RNA, circular RNA, antisense RNA, self-amplifying RNA, DNA, and gene editing therapeutics. Arcturus’ technologies are covered by its extensive patent portfolio (over 400 patents and patent applications in the U.S., Europe, Japan, China, and other countries). For more information, visit . In addition, please connect with us on Twitter and LinkedIn. Forward-Looking Statements This press release contains forward-looking statements that involve substantial risks and uncertainties for purposes of the safe harbor provided by the Private Securities Litigation Reform Act of 1995. Any statements, other than statements of historical fact included in this press release, are forward-looking statements, including those regarding strategy, future operations, the likelihood of success and continued advancement of the Company’s pipeline (including ARCT-032 and ARCT-810) and partnered programs (including the COVID-19 and flu programs partnered with CSL Seqirus), the likelihood of delivery of doses of Kostaive (including timing and volume thereof), the anticipated commercialization of Kostaive and the timing thereof, the likelihood and timing of a European Marketing Authorization application approval decision for Kostaive, the monetization of Arcturus’ interests in ARCALIS JV in Japan, that preclinical or clinical data will be predictive of future clinical results, the likelihood and timing of clinical study updates (including for ARCT-2138 (LUNAR-FLU), ARCT-032 (LUNAR-CF)), the qualification for commercial milestones under the CSL collaboration, the continuation and expected recruitment in the Phase 3 pivotal study of ARCT-2303 candidate vaccine containing the Omicron XBB.1.5 variant, the ongoing recruitment in the ARCT-2138 (LUNAR-FLU) Phase 1 study, the likelihood or timing of collection of accounts receivables including expected future milestone and other payments from CSL, its current cash position and expected cash burn and runway, and the impact of general business and economic conditions. Arcturus may not actually achieve the plans, carry out the intentions or meet the expectations or projections disclosed in any forward-looking statements such as the foregoing and you should not place undue reliance on such forward-looking statements. These statements are only current predictions or expectations, and are subject to known and unknown risks, uncertainties, and other factors that may cause our or our industry’s actual results, levels of activity, performance or achievements to be materially different from those anticipated by the forward-looking statements, including those discussed under the heading "Risk Factors" in Arcturus’ most recent Annual Report on Form 10-K, and in subsequent filings with, or submissions to, the SEC, which are available on the SEC’s website at . Except as otherwise required by law, Arcturus disclaims any intention or obligation to update or revise any forward-looking statements, which speak only as of the date they were made, whether as a result of new information, future events or circumstances or otherwise. Trademark Acknowledgements The Arcturus logo and other trademarks of Arcturus appearing in this announcement, including LUNAR® and STARR®, are the property of Arcturus. All other trademarks, services marks, and trade names in this announcement are the property of their respective owners. ARCTURUS THERAPEUTICS HOLDINGS INC. AND ITS SUBSIDIARIES CONDENSED CONSOLIDATED BALANCE SHEETS March 31, 2024 December 31, 2023 (in thousands, except par value information) (unaudited) Assets Current assets: Cash and cash equivalents $ 288,396 $ 292,005 Restricted cash 55,000 55,000 Accounts receivable 27,057 32,064 Prepaid expenses and other current assets 5,335 7,521 Total current assets 375,788 386,590 Property and equipment, net 11,763 12,427 Operating lease right-of-use asset, net 29,413 28,500 Non-current restricted cash 1,885 1,885 Total assets $ 418,849 $ 429,402 Liabilities and stockholders’ equity Current liabilities: Accounts payable $ 9,144 $ 5,279 Accrued liabilities 34,770 31,881 Deferred revenue 71,516 44,829 Total current liabilities 115,430 81,989 Deferred revenue, net of current portion 11,795 42,496 Operating lease liability, net of current portion 27,652 25,907 Other non-current liabilities — 497 Total liabilities 154,877 150,889 Stockholders’ equity Common stock, $0.001 par value; 60,000 shares authorized; issued and outstanding shares were 26,917 at March 31, 2024 and 26,828 at December 31, 2023 27 27 Additional paid-in capital 658,628 646,352 Accumulated deficit (394,683 ) (367,866 ) Total stockholders’ equity 263,972 278,513 Total liabilities and stockholders’ equity $ 418,849 $ 429,402 ARCTURUS THERAPEUTICS HOLDINGS INC. AND ITS SUBSIDIARIES CONSOLIDATED STATEMENTS OF OPERATIONS AND COMPREHENSIVE (LOSS) INCOME Unaudited Three Months Ended March 31, December 31, (in thousands, except per share data) 2024 2023 2023 Revenue: Collaboration revenue $ 32,598 $ 79,729 $ 25,078 Grant revenue 5,414 556 5,777 Total revenue 38,012 80,285 30,855 Operating expenses: Research and development, net 53,573 51,768 36,620 General and administrative 14,851 13,762 12,507 Total operating expenses 68,424 65,530 49,127 (Loss) income from operations (30,412 ) 14,755 (18,272 ) Loss from foreign currency (53 ) (328 ) (54 ) Gain on debt extinguishment — 33,953 — Finance income, net 4,016 2,477 6,881 Net (loss) income before income taxes (26,449 ) 50,857 (11,445 ) Provision for income taxes 368 103 262 Net (loss) income $ (26,817 ) $ 50,754 $ (11,707 ) (Loss) earnings per share Basic $ (1.00 ) $ 1.91 $ (0.44 ) Diluted $ (1.00 ) $ 1.87 $ (0.44 ) Weighted-average shares used in calculation of (loss) earnings per share: Basic 26,879 26,555 26,628 Diluted 26,879 27,149 26,628 Comprehensive (loss) income: Net (loss) income $ (26,817 ) $ 50,754 $ (11,707 ) Comprehensive (loss) income $ (26,817 ) $ 50,754 $ (11,707 ) View source version on businesswire.com: Contacts IR and Media Contacts Arcturus Therapeutics Neda Safarzadeh VP, Head of IR/PR/Marketing (858) 900-2682 IR@ArcturusRx.com Source: Arcturus Therapeutics Holdings Inc. 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