UBE3A

环状RNA（circRNAs）是一类独特的共价闭合非编码RNA亚群，具有独特的结构特征、出色的稳定性、高保守性和丰富的表达。 近日，复旦大学研究团队在期刊《Cancer Letters》上在线发表题为“CircUBE3A(2,3,4,5) promotes adenylate-uridylate-rich binding factor 1 nuclear translocation to suppress prostate cancer metastasis”的研究论文，本研究将circUBE3A（2,3,4,5）表征为一种功能性circRNA，并将其作为预防前列腺癌转移的极具前景的靶点。 前列腺癌（PCa）是男性中主要的恶性肿瘤，其每年的发病率持续上升，2023年占据全球男性癌症新发病例的29%。手术干预和放射治疗仍然是局部PCa的有效治疗方式。然而，在转移性前列腺癌治疗中，迫切需要改进的治疗方案。因此，探索调控PCa转移的分子机制并开发有效干预措施至关重要。 环状RNA（circRNAs）是一类独特的共价闭合非编码RNA亚群，具有独特的结构特征、出色的稳定性、高保守性和丰富的表达。已经确定，侧翼内含子互补序列是导致circRNA形成的反向剪接的重要贡献者。在功能上，circRNAs可以通过作为蛋白质复合物的支架或与RNA结合蛋白（RBPs）相互作用来调节基因表达，影响基因的表达，包括它们的亲本基因。它们还可以作为microRNAs（miRNAs）的分子海绵，并展示出编码肽的能力，例如circCCDC7（15,16,17,18,19）和circ-FBXW7。越来越多证据表明，circRNAs在调控各种癌症中发挥着关键作用，使它们有望成为临床生物标志物和可行的治疗干预靶点。 这项研究利用体外筛选细胞模型和高通量circRNA测序系统，鉴定了一个与前列腺癌（PCa）相关的circRNA（circBase ID: hsa_circ_0000586）。由于它来源于泛素蛋白连接酶E3A（UBE3A）基因的四个外显子（外显子2、3、4和5），研究人员按照最新的命名指南将其命名为circUBE3A（2,3,4,5）。发现circUBE3A（2,3,4,5）在PCa组织中下调，并与肿瘤恶性相关。它通过与AUF1相互作用并促进其向细胞核的易位来抑制PCa的转移。因此，AUF1在细胞质中的缺乏导致MTHFD2 mRNA稳定性降低，导致波形蛋白表达下调。两对短倒置重复序列 （TSIR） 被鉴定为协同增强 circUBE3A（2,3,4,5） 的形成。研究发现揭示了circRNA在转移中新的调控作用，并确定了治疗PCa转移的新分子机制。 综上所述，本研究将circUBE3A（2,3,4,5）表征为一种功能性circRNA，并将其作为预防前列腺癌转移的极具前景的靶点。 参考资料：https://www.sciencedirect.com/science/article/pii/S0304383524001368 内容来源于网络，如有侵权，请联系删除。

全球新冠疫情的爆发期间，多个mRNA新冠疫苗的上市，使mRNA技术成功进入商业化应用阶段，并且随后拓展到多个其他领域被应用及研究，如肿瘤疫苗及药物、基因编辑疗法、细胞治疗等，2023年mRNA疫苗的修饰核苷技术更是荣获诺贝尔生理学或医学奖，这一系列成功且灿烂的成绩背后是长达60年的mRNA技术的积累[1]。mRNA作为一种平台化技术，有着生产工艺简单，生产周期短，灵活性高等优点，具有可编码几乎所有蛋白质的潜力，而发挥这个潜力的第一步，是如何对mRNA序列进行设计。mRNA是一种包含5’cap、5UTR、CDS、3UTR和poly(A) tail等5个元件的单链RNA结构[2]。不同的mRNA序列的免疫原性、稳定性以及表达能力存在巨大的差异，因此需要根据蛋白质序列设计出免疫原性合适、稳定性好、表达能力更强的mRNA序列，包括对帽子结构、5’UTR序列、CDS序列、3’UTR序列、Poly A、核苷酸等进行设计和优化。01帽结构及转录起始序列选择5’cap结构具有重要功能：①被翻译复合体eIF4F识别并与43S翻译起始前体结合形成48S翻译起始复合体，启动帽依赖的翻译进程。②在针对外源RNA的先天免疫系统中，可作为自身RNA的标志。③避免5’-3’外切核酸酶切割作用。④参与mRNA运输。目前常用的加帽方式有两种：一种是以Moderna的mRNA-1273疫苗为例的酶法加帽，涉及牛痘病毒加帽酶和2'-氧甲基转移酶；另一种是以Pfizer/BioNTech的BNT162b2疫苗为例的共转录加帽，涉及Cap1帽类似物[3]。两种加帽方式对翻译起始序列要求有所区别。酶法加帽序列通常采用天然T7 RNA聚合酶起始序列，以GGG作为转录起始。但共转录法加帽根据使用的帽子类型不同，会有不同的起始序列设计。例如最常使用的AG型帽类似物，通常会使用AGG型起始位点，便于起始位点与帽类似物优先结合，保证正确的转录起始。02UTR区域序列筛选及设计UTR（untranslated regions）区作为非翻译区，主要调控mRNA的翻译效率，为保证mRNA的高效表达，目前序列设计通常选用同物种来源的高表达的基因的UTR作为候选UTR，如β-globin基因[3]等。在某些情况下，过高水平的蛋白表达并不总是带来最好的效果，如在mRNA应用于蛋白补充疗法时，蛋白过度表达可能不能达到病人获益的目标，甚至可能有害：以天使综合征为例，病人因缺少UBE3A表达而出现神经系统紊乱，但过量回补该基因会导致癫痫的发生，所以UTR的选择需要结合应用场景和毒理药理实验综合评判。并且由于不同物种的对于UTR的序列偏好存在差异，其中存在的顺式调控元件可能存在很大差异，所以在不同物种的核酸药开发中，最好根据目标物种不同，选择内源UTR，避免产生预料之外的结果。从下游质控层面来看，由于加帽率检测时用到的探针结合区域位于5’UTR 20-50nt处[4]，如该区域易形成稳定二级结构可能会影响探针结合，从而影响加帽率检测。另外经常容易忽视的一个问题是5’ UTR对于体外转录（IVT）的影响，T7 RNA polymerase转录起始时为转录起始构象，与DNA模板的结合不稳定，在合成开始几十nt后转为稳定的延伸构象。如果5’转录起始存在稳定的二级结构，则可能对IVT产生抑制，影响mRNA完整性以及产量。所以应该尽量避免转录起始区域存在稳定的互补配对。03CDS区域序列优化CDS区域序列优化CDS区的设计是mRNA序列设计的重点，通常在mRNA序列中，CDS在所有功能元件中长度占比最高，不仅需要考虑序列的翻译能力，同时也要兼顾序列在体内的稳定性，而对这两个方面的评价常使用密码子适应指数CAI（codon adaptation index）和最小自由能MFE（Minimum Free Energy）作为量化指标，目前已报到的设计软件众多，但根本原则和核心参数都是CAI与MFE两个指标。将CDS序列放在选定UTR背景下，利用mRNAid、LinearDesign[5]等软件进行CDS区域的序列优化。目前这类软件通常倾向于获得高CAI低MFE的序列，但这种原则，尤其低MFE值是否与蛋白表达有相关性还存在争议。最后可利用二级结构预测软件进一步check序列中的二级结构分布情况，最终选择5‘起始端避免二级结构，CDS和3’UTR富含大片段稳定的二级结构的序列。04Poly（A）区域序列设计poly(A) tail对维持mRNA的稳定性起着重要作用，体外合成mRNA常用的加尾方式有两种：（1）在IVT（in vitro transcription）后利用poly (A) polymerase进行加尾；（2）质粒模板上设计poly (A)序列，在IVT时被转录出来从而带上poly (A)尾，由于成本低，工艺简便，这也是目前主流的方法。长片段的 poly(A)序列在质粒扩增过程中不稳定，容易发生重组导致 poly(A)序列缩短，因此需要考察质粒的poly(A)序列完整性。针对polyA尾的优化目前主要有两个方向：一是避免polyA尾缩短，研究发现把poly(A)序列间隔开来，可以显著减少质粒DNA扩增时的重组事件，维持尾巴长度，同时不影响体外转录生成的mRNA的翻译效率和半衰期[6]，如Pfizer/BioNTech的BNT162b2疫苗对poly(A)序列采用(A)30GCAUAUGACU(A)70的分子设计 [3]。另一方面，mRNA模板质粒PolyA尾稳定性不佳，对质粒PolyA尾完整性检测是非常重要的质控项目。质粒序列检测通常用sanger测序法，但过长的连续A难以稳定测通，间隔序列的引入，有助于在双端测序中找到序列比对的铆定位点，更好的进行质控阶段的测序验证。同样，在mRNA的A尾分布分析时，也有助于确立A尾的长度和分布情况。二是延长mRNA半衰期，香港科技大学旷怡团队发表了题为“Cytidine-containing tails robustly enhance and prolong protein production of synthetic mRNA in cell and in vivo”的研究，证明含C序列作为合成mRNA的尾部可提高合成mRNA的表达强度和表达时间。C取代还可以抵抗mRNA的降解，从而延长其半衰期[7]。我们内部也对C取代进行了探究，该方法是否具有普适性有待进一步探究。05总结大部分序列设计软件通过体内实验数据作为训练集获得MFE和CAI之间的相关性模型，通过模型找出高稳定高翻译（高CAI值且低MFE）的序列。但是这种量化与评分方式在实际应用中仍然存在一些问题，首先是否MFE越低，翻译水平一定越高，仍未有定论。其次低MFE的序列可能会导致上游IVT生产产量低，从而增加整体成本。此外，这种高稳定的mRNA理论上在体内翻译过程也会对核糖体的结合带来阻力，可能造成蛋白表达不理想。针对这些问题，瀚海新酶在序列设计过程引入mRNA体内降解位点预测模型，通过该模型与MFE联合评价mRNA的稳定性，优化mRNA二级结构中易降解区域的碱基，最后结合CAI值评判获得兼具强翻译能力、高稳定性和易体外合成等优势可能性大的序列。许多生物信息学工具可用于评估CAI、GC含量以及MFE。但仍未有一款软件，可以实现所优化序列的稳定性、蛋白表达和免疫原性等均达到最优。目前各种优化工具更多的价值在于给后续的湿实验验证工作提供一系列候选序列指导，序列的实际表现还当以湿实验数据为准。06瀚海新酶靶点设计与验证服务案例由于mRNA具有前文提到的生产工艺简单，生产周期短，灵活性高等特点，在疫苗或药物早研阶段，可以快速完成同一靶点或者不同靶点多个序列，包括质粒模板制备、mRNA小试制备、细胞及体内翻译水平验证、免疫原性检测等在内的前期验证工作，相较传统疫苗及药物早期靶点验证，实验周期、成本等投入大大降低。为满足定制化客户不同的应用场景，支持客户不同规模的生产需求。瀚海新酶CRO服务网站精彩上线，可全方位了解瀚海新酶mRNA相关产品、服务及最新资讯。www.hzymescro.com（点击网址/底部阅读原文 立即访问）发展至今，瀚海新酶已完成数十个概念验证项目交付。项目范围覆盖靶点氨基酸优化、mRNA分子设计优化、药物表达验证、药效药理验证。在项目A中，利用AlphaFold对融合蛋白的结构进行预测，根据多个蛋白的空间相对位置、pLDDT置信打分等，为信号肽和linker的选择提供指导。对上述氨基酸序列进行分析、序列设计和基因合成，获得高质量质粒，IVT生产的mRNA原液进行LNP包封，获得合格的LNP制剂。同时将mRNA和LNP进行细胞翻译水平检测，结果显示mRNA成功表达目的融合蛋白。在项目B中，对单条mRNA链上串联两个抗原的结构和氨基酸序列进行分析，确定mRNA分子设计如下。对上述分子进行序列设计和基因合成，获得质粒，IVT生产高纯度的mRNA原液，进行LNP包封，获得包封率＞90%，PDI＜0.2的LNP制剂。同时将mRNA进行细胞翻译水平检测，结果显示mRNA成功表达目的蛋白。随着mRNA技术应用不断的被拓展，各类管线不断增加，涉及的各项需求也越来越广阔，包括但不限于原料、设备、工艺、服务等！瀚海新酶作为拥有核心技术的原料供应商，不仅仅可以提供性价比高的mRNA原料，为了帮助客户快速地完成靶点的可行性验证，推出了概念验证服务（Proof of concept，简称POC）：瀚海新酶提供：全套mRNA疫苗&药物相关高性能酶原料、化学底物、IVT试剂盒及质控试剂盒，拥有自主质粒制备、IVT工艺开发、mRNA纯化、LNP包封及相关质检平台，可为客户提供基于原料的全套解决方案，全面的工艺开发服务，助力客户快速推进新药开发。对于早研阶段客户可提供CRO服务、概念验证服务、第三方质检服务及各类成品mRNA原液产品，编码蛋白涵盖报告基因、靶点蛋白、基因编辑蛋白和抗原蛋白等。作为深耕mRNA领域的原料与服务供应商，瀚海新酶期待与行业大众就mRNA的技术进行分享讨论，推出本篇连载专题《瀚海畅聊mRNA》第一期“一切的起始：mRNA疫苗&药物分子设计”，后续会相继就上游质粒制备、酶促体系、纯化工艺、递送系统及质量控制等方面进行分享与讨论，敬请期待！新年倍速开工  瀚海新酶元宵有礼为回馈行业新老粉丝客户的关注，助力了解RNA相关专业技术知识，瀚海新酶元宵有礼：前28位免费领取专业书籍《RNA疫苗：方法与操作》，与您共聚喜乐元宵，日日“宵”口常开！《RNA疫苗：方法与操作》包括五个部分：RNA疫苗的简介、自复制RNA载体、非复制mRNA载体、RNA疫苗的佐剂与递送、RNA疫苗临床前和临床药物开发，详细介绍了RNA疫苗研究过程中涉及的实验方法和操作技术，是一本RNA疫苗研究方面的百科《RNA疫苗：方法与操作》，能够为医学科研院所生物技术从业人员、相关专业生物检测应用人员提供较全面的参考。参与方式1. 关注瀚海新酶公众号（后续多期《瀚海畅聊mRNA》专题文章推出，请持续关注！）2. 输入框回复：RNA，登记信息活动时间2月21日—2月24日获奖说明：后台进行审核，前28名将获《RNA疫苗：方法与操作》一本，3个工作日内联系获奖，5个工作日内寄出。参与对象：行业内医学科研院所生物技术从业人员、相关专业生物检测应用人员（瀚海新酶及生物制品圈内部人员不参与）（本活动最终解释权归瀚海新酶生物科技有限公司所有）参考文献：[1] Qin S, Tang X, Chen Y, et al. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022 May 21;7(1):166.[2] Kim SC, Sekhon SS, Shin WR, et al. Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency. Mol Cell Toxicol. 2022;18(1):1-8.[3] Szabó GT, Mahiny AJ, Vlatkovic I. COVID-19 mRNA vaccines: Platforms and current developments. Mol Ther. 2022 May 4;30(5):1850-1868.[4] Beverly M, Dell A, Parmar P, et al. Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS. Anal Bioanal Chem. 2016 Jul;408(18):5021-30.[5] Zhang H, Zhang L, Lin A, et al. Algorithm for optimized mRNA design improves stability and immunogenicity. Nature. 2023 Sep;621(7978):396-403. doi: 10.1038/s41586-023-06127-z. Epub 2023 May 2. PMID: 37130545; PMCID: PMC10499610.[6] Trepotec Z, Geiger J, Plank C, et al. Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life. RNA. 2019 Apr;25(4):507-518.[7] Li CY, Liang Z, Hu Y, et al. Cytidine-containing tails robustly enhance and prolong protein production of synthetic mRNA in cell and in vivo. Mol Ther Nucleic Acids. 2022 Oct 12;30:300-310.公司介绍

GTX-102 is the first Angelman syndrome therapeutic candidate to receive PRIME designation Phase 1/2 study fully enrolled; expansion data expected in first half of 2024 NOVATO, Calif., Feb. 05, 2024 (GLOBE NEWSWIRE) -- Ultragenyx Pharmaceutical Inc. (NASDAQ: RARE) today announced that the European Medicines Agency (EMA) has granted Priority Medicine (PRIME) designation to GTX-102 for the treatment of Angelman syndrome (AS). GTX-102 is an investigational antisense oligonucleotide delivered via intrathecal administration and is designed to target and inhibit expression of UBE3A antisense transcript (UBE3A-AS). The EMA granted this designation in response to compelling early clinical data from the extension cohorts in the Phase 1/2 study of GTX-102 demonstrating clinically meaningful improvements in several neurodevelopmental domains including cognition, receptive communication and gross motor skills in individuals with Angelman syndrome. "By granting PRIME designation, the EMA is recognizing the potential for GTX-102 to address the critical need for new treatments for children and families impacted by Angelman syndrome in the EU,” said Eric Crombez, M.D., chief medical officer at Ultragenyx. “Our team is working with urgency on the development of GTX-102 for Angelman syndrome and looks forward to working closely with regulators in the U.S. and EU to bring this innovative treatment to patients.” PRIME designation is granted by the EMA to provide early and proactive support to developers of promising medicines that may offer a major therapeutic advantage over existing treatments or benefit to patients without treatment options. These medicines are considered priority medicines by the EMA, whose aim is to optimize development plans and speed up evaluations so these medicines that address significant unmet medical needs can reach patients faster. About the Phase 1/2 study The Phase 1/2, open-label, multiple-dose, dose-escalating study is evaluating the safety and tolerability of GTX-102 administered by intrathecal (IT) injection to pediatric patients with Angelman syndrome with a genetically confirmed diagnosis of full maternal UBE3A gene deletion. The study is also assessing clinical response as measured by a panel of efficacy assessments for the functional domains impacted in Angelman syndrome. Patients in the earlier extension cohorts (Cohorts 4-7) of the study have moved into long-term maintenance dosing, and the study has completed enrollment for the new expansion cohorts to verify the GTX-102 dose range and treatment regimen that will be used in the Phase 3 program. About Angelman Syndrome Angelman syndrome is a rare, neurogenetic disorder caused by loss-of-function of the maternally inherited allele of the UBE3A gene. The maternal-specific inheritance pattern of Angelman syndrome is due to genomic imprinting of UBE3A in neurons of the central nervous system (CNS), a naturally occurring phenomenon in which the maternal UBE3A allele is expressed and the paternal UBE3A is not. Silencing of the paternal UBE3A allele is regulated by the UBE3A antisense transcript (UBE3A-AS), the intended target of GTX-102. In almost all cases of Angelman syndrome, the maternal UBE3A allele is either missing or mutated, resulting in limited to no protein expression. This condition is generally not inherited but instead occurs spontaneously. It is estimated to affect one in 12,000 to one in 20,000 people globally. Individuals with Angelman syndrome have developmental delay, balance issues, motor impairment and debilitating seizures. Some individuals with Angelman syndrome are unable to walk and most do not speak. Anxiety and disturbed sleep can be serious challenges in individuals with Angelman syndrome. Although individuals with Angelman syndrome have a normal lifespan, they require continuous care and are unable to live independently. Angelman syndrome is not a degenerative disorder, but the loss of the UBE3A protein expression in neurons results in abnormal communications between neurons. Angelman syndrome is often misdiagnosed as autism or cerebral palsy. There are no currently approved therapies for Angelman syndrome; however, several symptoms of this disorder can be reversed in adult animal models of Angelman syndrome suggesting that improvement of symptoms can potentially be achieved at any age. About GTX-102 GTX-102 is an investigational antisense oligonucleotide delivered via intrathecal administration and designed to target and inhibit expression of UBE3A-AS. Nonclinical studies have shown that GTX-102 reduces levels of UBE3A-AS and reactivates expression of the paternal UBE3A allele in neurons of the CNS. Reactivation of paternal UBE3A expression in animal models of Angelman syndrome has been associated with improvements in some of the neurological symptoms associated with the condition. GTX-102 has been granted Orphan Drug Designation, Rare Pediatric Disease Designation, and Fast Track Designation from the FDA, and Orphan Designation and PRIME designation from the EMA. About Ultragenyx Pharmaceutical Inc. Ultragenyx is a biopharmaceutical company committed to bringing novel products to patients for the treatment of serious rare and ultrarare genetic diseases. The company has built a diverse portfolio of approved therapies and product candidates aimed at addressing diseases with high unmet medical need and clear biology for treatment, for which there are typically no approved therapies treating the underlying disease. The company is led by a management team experienced in the development and commercialization of rare disease therapeutics. Ultragenyx’s strategy is predicated upon time- and cost-efficient drug development, with the goal of delivering safe and effective therapies to patients with the utmost urgency. For more information on Ultragenyx, please visit the company's website at: . Ultragenyx Forward-Looking Statements and Use of Digital Media Except for the historical information contained herein, the matters set forth in this press release, including statements related to Ultragenyx's expectations and projections regarding its future operating results and financial performance, future clinical and regulatory developments for GTX-102, the clinical benefit, tolerability and safety of GTX-102, timing for enrollment, dosing and data for GTX-102 and the company’s other investigational therapies and regulatory meetings are forward-looking statements within the meaning of the "safe harbor" provisions of the Private Securities Litigation Reform Act of 1995. Such forward-looking statements involve substantial risks and uncertainties that could cause our clinical development programs, collaboration with third parties, future results, performance or achievements to differ significantly from those expressed or implied by the forward-looking statements. Such risks and uncertainties include, among others, the uncertainty of clinical drug development and unpredictability and lengthy process for obtaining regulatory approvals, the ability of the company to successfully develop GTX-102, the company’s ability to achieve its projected development goals in its expected timeframes, risk that access to PRIME for our product candidates, if granted, may not lead to faster development or regulatory review or approval process, and does not increase the likelihood that our product candidates will receive marketing approval; risks related to adverse side effects, risks related to reliance on third-party partners to conduct certain activities on the company’s behalf, the potential for any license or collaboration agreement, smaller than anticipated market opportunities for the company’s products and product candidates, manufacturing risks, competition from other therapies or products and other matters that could affect sufficiency of existing cash, cash equivalents and short-term investments to fund operations, the Company’s future operating results and financial performance, the timing of clinical trial activities and reporting results from same, and the availability or commercial potential of Ultragenyx’s products and product candidates. Ultragenyx undertakes no obligation to update or revise any forward-looking statements. For a further description of the risks and uncertainties that could cause actual results to differ from those expressed in these forward-looking statements, as well as risks relating to the business of Ultragenyx in general, see Ultragenyx's Quarterly Report on Form 10-Q filed with the Securities and Exchange Commission (SEC) on November 3, 2023, and its subsequent periodic reports filed with the SEC. In addition to its SEC filings, press releases and public conference calls, Ultragenyx uses its investor relations website and social media outlets to publish important information about the company, including information that may be deemed material to investors, and to comply with its disclosure obligations under Regulation FD. Financial and other information about Ultragenyx is routinely posted and is accessible on Ultragenyx’s Investor Relations website ( ) and LinkedIn website ( ). Contacts Ultragenyx Pharmaceutical Inc. Investors Joshua Higa +1-415-475-6370 ir@ultragenyx.com Media Carolyn Wang +1-415-225-5050 media@ultragenyx.com