Porphyria, Acute Intermittent

mRNA技术及其递送系统的进步，使得基于mRNA的治疗手段进入了医学的新时代。mRNA编码的蛋白具有快速、有效和短暂的特性，无需进入细胞核，不存在基因组整合的风险，使其成为治疗一系列疾病的理想工具。mRNA易于快速生产和大规模制造，成功地支撑了全球对COVID-19大流行的快速响应。然而，mRNA的稳定性、表达持续时间、递送效率和靶向性仍存在诸多挑战。近日，发表在著名医学期刊《柳叶刀》上的一篇综述总结了mRNA技术在疫苗、免疫疗法、蛋白质替代疗法和基因组编辑中的应用进展，以及mRNA递送系统的研发和应用进展。 基于mRNA的疫苗和治疗手段是一项快速发展的技术。自1990年首次在体内验证mRNA以来，mRNA平台技术已经取得了实质性进步。COVID-19大流行期间，mRNA平台展示了其在解决新兴危机方面的成本效益和速度。当前mRNA平台的关键优势包括：有效、快速和短暂的表达，无需进入细胞核，没有基因组整合的风险，设计简单，制备迅速。使得mRNA治疗成为各大药企和生物技术公司争相进入前沿领域。 mRNA药理学基础和mRNA技术的最新进展 合成的mRNA具有非常高的表达量，不需要核内递送，并且可以补充各种类型的蛋白。目前已经开发了多种修饰方法来减少mRNA的免疫原性，增强其稳定性和表达水平。合成的mRNA由五个关键元素组成：5'端帽、5'非翻译区(UTR)、编码序列区域、3'非翻译区(UTR)以及聚腺苷酸（poly[A]）尾。对这些基本区域进行优化可以增强mRNA的稳定性、翻译和免疫逃逸能力。 图1 合成的mRNA 5'端帽是mRNA的关键组成部分，对翻译启动和防止核酸酶降解至关重要。使用合成核苷酸共转录加帽是一种常见的技术，用于结合5'端帽并启动翻译。5'UTR和3'UTR的筛选和优化通常是为了提高翻译效率，减少mRNA衰减。BioNTech进行了深入的细胞库筛选，以鉴定稳定而高效的3'UTR候选者，这些候选者被用于BNT162b2 COVID-19疫苗（辉瑞-BioNTech）。除了选择最优的UTR外，还使用了各种密码子优化方法，如去除发夹结构、减少二级结构和改变鸟嘌呤-胞嘧啶含量。为了减少免疫原性和mRNA衰减，已经使用了包括假尿嘧啶（ψ）、N1-甲基假尿嘧啶（m1ψ）、5-甲基胞嘧啶（m5C）、2-硫尿嘧啶（S2U）和5-甲基尿嘧啶（m5U）在内的核苷酸修饰。m1ψ是最常用的修饰核苷酸，用于减少免疫原性和毒性。在BNT162b2和mRNA-1273（Moderna）COVID-19疫苗中使用了将尿嘧啶完全替换为m1ψ的方法。S2U和m5C的组合在蛋白表达方面显示出了有潜力的结果。为了避免mRNA直接激活免疫翻译，CureVac等公司选择使用序列工程而不是使用修饰的核苷酸。带修饰核苷酸的mRNA与序列工程化mRNA的耐受性仍在讨论中。 合成mRNA中的最后一个基本元件是聚腺苷酸（poly[A]）尾，这对调节mRNA的稳定性和翻译效率至关重要。聚腺苷酸尾可以通过酶促添加或从DNA模板转录。直接转录poly(A)操作简单，而且可以对poly(A)的长度进行精确控制，因此更为常见，而酶促技术的优势是可提供尾部修饰，从而减少对核酸外切酶的敏感性，增加mRNA半衰期的优势。 在mRNA的纯化方面，高效液相色谱法是一种常用但昂贵且耗时的方法，用于去除RNA污染物，包括可能激活免疫反应的双链（dsRNA）杂质。更简单的基于纤维素的色谱法是HPLC的替代方法，另外也有人使用其它各种基于柱的方法对mRNA进行纯化。 为传染病大流行而储备的mRNA技术 在COVID-19大流行期间，改良的mRNA-LNP疫苗平台得到了成功应用。美国国防高级研究计划局（大流行预防平台）和其他机构正在开发应对新流行病的系统。通过测序从存活患者那里获得的血液样本来识别病原体，克隆B细胞以获得中和抗体，并将最有效的中和单克隆抗体用于mRNA的递送。该方法目前已进入临床试验，且可能成为未来全球用于新流行病或疫情的疗法。 mRNA疫苗 mRNA疫苗的作用原理是通过将编码病原体抗原的合成mRNA转染到抗原呈递细胞中来触发免疫反应。这些特定的抗原被处理、提呈至细胞表面或分泌并被免疫细胞识别，从而引发保护性的体液和细胞毒性T细胞反应。 图2 mRNA疫苗诱导的免疫反应 1.针对传染病的疫苗 ①.HIV HIV-1疫苗开发的重点是诱导广泛的2层病毒株中和抗体。HIV-1 mRNA-LNP疫苗在非人灵长类动物中诱导了至少41周的持久中和抗体，目前有多个HIV-1 mRNA-LNP疫苗1期临床试验正在进行中。 值得注意的是，一项临床试验(NCT03547245)研究了一种针对生殖细胞系的靶向免疫原(eOD-GT8 60mer)，以刺激针对特定HIV-1病毒包膜蛋白区域的罕见bnAb前体B细胞。该研究使用佐剂AS01B(单磷酸脂A和皂角素)，在健康成人中诱导了VRC01类反应，记忆B细胞的中位频率为0.1%。使用修饰的mRNA-LNP编码抗原(NCT05001373)也观察到了类似的结果。这些发现支持开发增强方案以诱导VRC01类bnAbs，并鼓励将这一策略扩展到HIV和其他病原体的其他靶标。 ②.结核病 结核病是由结核分枝杆菌引起的传染性细菌性疾病。BioNTech已经启动了两项mRNA结核病候选疫苗的1期临床试验（NCT05547464）。 ③.疟疾 疟疾是由寄生虫引起的传染病，疟疾mRNA-LNP候选疫苗在小鼠中显示出强大和持久的免疫原性。BioNTech最近启动了一项PfCSP mRNA-LNP疫苗的临床试验（NCT05581641）。 ④.寨卡病毒 寨卡病毒是一种由埃及伊蚊（Stegomyia）传播的黄病毒。针对寨卡病毒的mRNA-LNP疫苗已经在临床前研究中显示了保护效果。Moderna公司的寨卡mRNA-1893疫苗正在进行2期临床试验（NCT04917861）。 ⑤.狂犬病病毒 狂犬病由狂犬病病毒引起，是一种几乎100%致死的人畜共患疾病。CV7201（NCT02241135）和CV7202（NCT03713086） mRNA-LNP疫苗在临床试验中显示出良好的安全性和耐受性。 ⑥.流感病毒 两项1期临床试验（NCT03076385和NCT03345043）评估了针对禽流感H10N8和H7N9病毒的mRNA疫苗，均在健康成人中诱导产生了强烈的体液免疫反应，并且对于特定疫苗剂量具有良好的耐受性。在2期临床试验（NCT04969276）中，对65岁或以上成年人联合接种高剂量的四价流感疫苗和mRNA-1273疫苗（COVID 19疫苗）耐受性良好 2.癌症疫苗 作为癌症治疗的新干预措施，mRNA疫苗编码肿瘤抗原，包括肿瘤相关抗原（TAAs）和肿瘤特异性抗原（TSAs），或免疫调节分子，或这些的组合，用于诱导和微调抗肿瘤免疫反应。BioNTech和Moderna等公司已经投入资源开发个性化癌症疫苗。BNT111是一种mRNA-脂质复合物候选疫苗，在不可切除的黑色素瘤患者中显示出强烈的抗原特异性免疫反应。BNT122是一种个性化的多新表位mRNA疫苗，包含多达20个患者特异性新表位，目前正在研究用于治疗黑色素瘤（NCT03815058）和结直肠癌（NCT04486378）。此外，前列腺癌（NCT01197625）、急性髓系白血病（NCT00514189）和个性化TAA 组合（NCT01334047、NCT02709616、NCT02808364和NCT02808416）的mRNA-DC疫苗也在进行临床试验。 BNT151（一种核苷修饰的IL-12 mRNA）正在1/2期临床试验（NCT04455620）中，探索作为单药治疗或与其他抗癌药物联合治疗晚期实体瘤患者的临床安全性和有效性。 另一类正在研究的mRNA癌症疫苗编码免疫调节分子，这些分子一旦注射到肿瘤部位，就会创造一个促进T细胞扩增和记忆反应的促炎性微环境。 治疗性mRNA的应用 1.单基因疾病和蛋白质替代 目前有包括Moderna在内的多家公司和研究机构正在开发mRNA疗法，用于治疗多种单基因疾病，如甲基丙二酸血症、急性间歇性卟啉病、法布里病和囊性纤维化等。 2.单克隆抗体和免疫疗法 mRNA编码的单克隆抗体用于预防和治疗病毒感染，临床试验显示了其高效表达和保护能力。mRNA技术也正在被开发用于癌症免疫治疗，形式包括mRNA编码的抗体、抗体类似物、双特异性抗体或嵌合抗原受体（CAR）T细胞疗法等。 3.免疫抑制剂 mRNA治疗手段在抑制性免疫疗法、免疫耐受和过敏等领域可能很有用。已有研究报告了一种非炎症性mRNA疫苗用于治疗实验性自身免疫性脑脊髓炎。Moderna已经开始在1期临床试验（NCT04916431）中评估mRNA-6231（一种编码人血清白蛋白-IL-2融合蛋白的mRNA-LNP）的安全性、耐受性和药代动力学。mRNA-6981是一种编码PD-L1的mRNA-LNP，在髓系细胞上增强PD-L1表面表达可用作对自身反应性淋巴细胞的共抑制信号。 4.基因组编辑器 编码基因组编辑器的mRNA具有较高的编辑效率和较低的脱靶效应，这使得更安全的体内基因组修饰成为可能。一项临床试验对TTR淀粉样变性进行了体内CRISPR-Cas9基因组编辑。用LNP递送Cas9 mRNA和sgRNA，单次给药后，TTR蛋白减少约52%（剂量为0.1 mg/kg），且在治疗后1年内TTR持续低水平，未观察到其它副反应。 用LNP向小鼠和猕猴体内递送编码腺嘌呤碱基编辑器的mRNA和靶向PCSK9的sgRNA，在小鼠中观察到高达67%的碱基编辑，在猕猴中高达34%，这导致小鼠LDL浓度稳定降低了约58%，猕猴LDL浓度降低了约14%。 图片3 基于mRNA的治疗方法示意图 肝外mRNA治疗  当通过静脉给药时，LNPs会被动靶向肝细胞和树突细胞，因此，目前静脉注射的LNPs主要用于肝脏治疗应用。但靶向其他组织和器官的mRNA治疗药物具有巨大潜力。如果需要靶向肝外其它组织，可以通过局部给药实现肝外递送。此外，还有一些研究正在尝试开发修饰的LNPs以实现选择性器官靶向。 安全性考虑和潜在挑战 mRNA疫苗的成功展示了其安全性和有效性。然而，治疗性mRNA技术的应用面临更高的安全性要求，还需要克服免疫原性和毒性问题，同时还需要优化表达和递送系统。 结论 mRNA技术已经取得了实质性的进步，尽管面临挑战，但应用前景广阔。相信mRNA技术的进步将继续推动新一代高效、靶向性更好的mRNA治疗平台的发展，为更多疾病带来革命性的干预措施。 主要参考资料： https://doi.org/10.1016/ S0140-6736(23)02444-3 关于派真生物

United Porphyrias Aims to Raise Awareness, Research and Support for Rare Disease BETHESDA, Md., May 17, 2024 /PRNewswire/ -- United Porphyrias Association (UPA) is proud to announce May 18 as Global Porphyria Day. Global Porphyria Day is intended to create awareness that prompts action for improved healthcare, increased research, and enhanced support for individuals with porphyria. The campaign encourages wearing and promoting the color purple, the color of porphyria, stemming from the Greek word "porphyra", which means purple. Continue Reading GLOBAL PORPHYRIA DAY IS MAY 18 - United Porphyrias Aims to Raise Awareness, Research and Support for Rare Disease Post this Global Porphyria Day is May 18, 2024 Porphyria is a group of disease of eight rare, genetic disorders. People living with a porphyria have changes to certain genes involved in development of heme, an essential element bringing oxygen into the bloodstream. Each type of porphyria is caused by a defect in a specific enzyme responsible for heme production, causing a buildup of other compounds, called porphyrins. The buildup of porphyrins causes different types of porphyria and associated symptoms, including significant pain. These symptoms can be broadly divided into two categories: acute (sudden and severe) and cutaneous (skin related.) Symptoms range from severe abdominal pain, nausea, vomiting, and effects on the nervous system in acute porphyria to photosensitivity which can cause skin pain, burning, tingling, itching, swelling, blistering, infections, scarring in cutaneous porphyria. UPA supports global efforts to raise awareness of porphyria improving diagnosis and treatment of these rare diseases and providing critical support to the porphyria community. UPA's internationally renowned scientific advisory board of porphyria experts works closely with the patient community to make advances. "The porphyrias are often misdiagnosed and misunderstood by the medical community," said UPA President and porphyria caregiver, Kristen Wheeden, "Our work leads directly to earlier diagnosis, better patient care, increased research, and access to therapeutics. Everyone has the right to live without pain." The United Porphyrias Association is committed to improving the quality of life of the porphyria patient community and is relentlessly focused on advancing disease awareness, research, and therapies in all the porphyrias. For more information on Porphyria, Global Porphyria Day, and the United Porphyrias Association, visit . Contact: Kristen Wheeden, President, United Porphyria Association Phone: 1-800-868-1292 Email: [email protected] SOURCE United Porphyrias Association

- ASGCT presentation demonstrates lipid nanoparticle (LNP) mediated delivery to the eye in the context of editing of the myocilin (MYOC) gene as a potential treatment for glaucoma - - Expands pipeline with new pre-clinical programs utilizing LNP mediated delivery to the liver for refractory hypertension targeting angiotensinogen (AGT) and acute hepatic porphyria (AHP) targeting 5’-aminolevulinate synthase 1 (ALAS1) - - CTX340™, targeting AGT in the liver, achieved durable editing of AGT and sustained ~30 mmHg blood pressure (BP) reduction out to 3 months in the spontaneously hypertensive rat (SHR) model, demonstrating its potential as a one-time therapeutic intervention - - CTX450™, targeting ALAS1 in the liver, achieved ~70% editing of ALAS1 and normalizes disease-associated biomarkers in acute porphyria mouse model, demonstrating its potential as a one-time therapeutic intervention - - Plans to initiate clinical trials for CTX340 and CTX450 in the second half of 2025 - ZUG, Switzerland and BOSTON, May 08, 2024 (GLOBE NEWSWIRE) -- CRISPR Therapeutics (Nasdaq: CRSP), a biopharmaceutical company focused on creating transformative gene-based medicines for serious diseases, today announced new preclinical data presented at the 27th Annual Meeting of the American Society of Cell and Gene Therapy (ASGCT) highlighting the Company’s approach to developing lipid nanoparticle (LNP) based delivery for in vivo ocular gene editing. In addition, CRISPR Therapeutics announced the expansion of its in vivo pipeline with two new programs. CTX340™ and CTX450™ utilize LNP-based delivery of CRISPR/Cas9 gene editing cargo to the liver, targeting angiotensinogen (AGT) for refractory hypertension and 5’-aminolevulinate synthase 1 (ALAS1) for acute hepatic porphyria (AHP), respectively. “Over the past two years, we have made significant progress on the development of our lipid nanoparticle platform for the delivery of CRISPR/Cas9 to the liver and are now in clinical trials with CTX310 and CTX320,” said Samarth Kulkarni, Chief Executive and Chairman of the Board of CRISPR Therapeutics. “The expansion of our in vivo pipeline speaks to the scalability of the platform and the exceptional translation capabilities of our team. We continue to add programs to treat both common and rare diseases, as we look to broaden the number of areas where CRISPR could have transformational impact.” In Vivo Pipeline Expansion CRISPR Therapeutics has established a proprietary LNP platform for the delivery of CRISPR/Cas9 to the liver. The first two in vivo programs utilizing this proprietary platform, CTX310™ and CTX320™, are directed towards validated therapeutic targets associated with cardiovascular disease, and are in on-going clinical trials. The addition of two more programs, CTX340 and CTX450, utilizing this LNP delivery technology demonstrates the modularity and scalability of the platform.Refractory hypertension is a serious unmet medical need affecting approximately 1.5 million patients in the U.S. alone. CTX340 is designed to inhibit production of hepatic angiotensinogen (AGT), a validated target to modulate the renin-angiotensin-aldosterone system (RAAS) and normalize blood pressure durably with a one-time treatment. In preclinical studies, CTX340 showed ~60% liver editing and ~90% AGT protein reduction, resulting in sustained ~30 mmHg blood pressure (BP) reduction out to 3 months in the spontaneously hypertensive rat (SHR) model.Acute hepatic porphyria (AHP) is a group of rare genetic diseases of heme biosynthesis. Symptomatic patients have acute attacks, characterized by debilitating neurovascular symptoms, as well as multiple chronic symptoms, such as pain. There are approximately 5,000 patients diagnosed with AHP in the U.S., although the disease remains underdiagnosed. CTX450 is specifically designed to inhibit production of ALAS1 in the liver, preventing accumulation of neurotoxic aminolevulinic acid (ALA) and porphobilinogen (PBG). In preclinical studies, CTX450 showed ~70% liver editing and ~97% ALAS1 protein reduction, resulting in reduction of ALA and PBG disease biomarkers to normal levels in an AHP mouse model.CRISPR Therapeutics has initiated IND/CTA-enabling studies for CTX340 and CTX450 and expects to initiate both clinical trials in the second half of 2025. ASGCT Presentation In addition to expanding the liver-targeted in vivo pipeline, CRISPR Therapeutics reported initial data demonstrating its proprietary capabilities to deliver to and edit genes in the eye, opening a potential new focus area. The data will be presented today, May 8, 2024, from 3:00 p.m. – 3:15 p.m. ET at ASGCT in an oral presentation entitled “Development of an In Vivo Non-Viral Ocular Editing Platform and Application to Potential Treatments for Glaucoma.”Glaucoma is the second leading cause of blindness worldwide. Mutations in the myocilin (MYOC) gene represent the most common genetic cause of glaucoma that affects approximately 150,000 people in the U.S. alone. In these patients, defective myocilin protein aggregates in the trabecular meshwork (TM) cells, leading to impaired outflow of aqueous humor from the anterior segment of the eye, resulting in elevated intraocular pressure. Patients with MYOC-associated glaucoma typically have an earlier onset and more rapidly progressive disease course than is seen with other causes of glaucoma. Pharmaceutical interventions carry a significant treatment burden resulting in reduced adherence to therapy and surgical interventions frequently do not lead to a durable resolution of elevated intraocular pressure (IOP). The Company has developed an LNP platform capable of delivering gene editing cargo to the TM cells in the eye. In today’s presentation, the Company presented data demonstrating efficient and specific delivery to TM cells in mouse, non-human primate, and ex vivo human eyes. The Company showed >90% editing of the MYOC gene in vitro with prioritized guide RNA, and ~90% reduction of surrogate protein expression in a mouse in vivo model after a single injection. About In Vivo ProgramsCRISPR Therapeutics has established a proprietary LNP platform for the delivery of CRISPR/Cas9 to the liver. The Company’s in vivo portfolio includes its lead investigational in vivo programs, CTX310 (directed towards angiopoietin-related protein 3 (ANGPTL3)) and CTX320 (directed towards lipoprotein(a) (Lp(a)), two validated therapeutic targets for cardiovascular disease, are in on-going clinical trials. In addition, the Company’s research and pre-clinical development candidates include CTX340 and CTX450, targeting angiotensinogen (AGT) for refractory hypertension and 5’-aminolevulinate synthase 1 (ALAS1) for acute hepatic porphyria (AHP), respectively. About CRISPR Therapeutics Since its inception over a decade ago, CRISPR Therapeutics has transformed from a research-stage company advancing programs in the field of gene editing, to a company that recently celebrated the historic approval of the first-ever CRISPR-based therapy and has a diverse portfolio of product candidates across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine, cardiovascular, autoimmune, and rare diseases. CRISPR Therapeutics advanced the first-ever CRISPR/Cas9 gene-edited therapy into the clinic in 2018 to investigate the treatment of sickle cell disease or transfusion-dependent beta thalassemia, and beginning in late 2023, CASGEVY™ (exagamglogene autotemcel) was approved in some countries to treat eligible patients with either of those conditions. The Nobel Prize-winning CRISPR science has revolutionized biomedical research and represents a powerful, clinically validated approach with the potential to create a new class of potentially transformative medicines. To accelerate and expand its efforts, CRISPR Therapeutics has established strategic partnerships with leading companies including Bayer and Vertex Pharmaceuticals. CRISPR Therapeutics AG is headquartered in Zug, Switzerland, with its wholly-owned U.S. subsidiary, CRISPR Therapeutics, Inc., and R&D operations based in Boston, Massachusetts and San Francisco, California, and business offices in London, United Kingdom. To learn more, visit www.crisprtx.com. CRISPR THERAPEUTICS® standard character mark and design logo, CTX310™, CTX320™, CTX340™ and CTX450™ are trademarks and registered trademarks of CRISPR Therapeutics AG. The CASGEVY™ word mark and design are trademarks of Vertex Pharmaceuticals Incorporated. All other trademarks and registered trademarks are the property of their respective owners. CRISPR Therapeutics Forward-Looking StatementThis press release may contain a number of “forward-looking statements” within the meaning of the Private Securities Litigation Reform Act of 1995, as amended, including statements regarding CRISPR Therapeutics’ expectations about any or all of the following: (i) its ongoing and/or planned preclinical studies, clinical trials and pipeline products and programs, including, without limitation, the status of such studies and trials, potential expansion into new indications and expectations regarding data generally (including expected timing of data releases) as well as the data that is being presented as described above; (ii) the safety, efficacy and clinical progress of its various clinical and preclinical programs including the program described in the oral presentation and poster; (iii) the data that will be generated by ongoing and planned preclinical studies and/or clinical trials, and the ability to use that data for the design and initiation of further preclinical studies and/or clinical trials; and (iv) the therapeutic value, development, and commercial potential of CRISPR/Cas9 gene editing technologies and therapies. Without limiting the foregoing, the words “believes,” “anticipates,” “plans,” “expects” and similar expressions are intended to identify forward-looking statements. You are cautioned that forward-looking statements are inherently uncertain. Although CRISPR Therapeutics believes that such statements are based on reasonable assumptions within the bounds of its knowledge of its business and operations, forward-looking statements are neither promises nor guarantees and they are necessarily subject to a high degree of uncertainty and risk. Actual performance and results may differ materially from those projected or suggested in the forward-looking statements due to various risks and uncertainties. These risks and uncertainties include, among others: the efficacy and safety results from ongoing pre-clinical studies and/or clinical trials will not continue or be repeated in ongoing or planned pre-clinical studies and/or clinical trials or may not support regulatory submissions; pre-clinical study and/or clinical trial results may not be favorable or support further development; one or more of its product candidate programs will not proceed as planned for technical, scientific or commercial reasons; future competitive or other market factors may adversely affect the commercial potential for its product candidates; uncertainties inherent in the initiation and completion of preclinical studies for its product candidates and whether results from such studies will be predictive of future results of future studies or clinical trials; uncertainties about regulatory approvals to conduct trials or to market products; uncertainties regarding the intellectual property protection for its technology and intellectual property belonging to third parties, and the outcome of proceedings (such as an interference, an opposition or a similar proceeding) involving all or any portion of such intellectual property; and those risks and uncertainties described under the heading "Risk Factors" in CRISPR Therapeutics’ most recent annual report on Form 10-K and in any other subsequent filings made by CRISPR Therapeutics with the U.S. Securities and Exchange Commission, which are available on the SEC's website at www.sec.gov. CRISPR Therapeutics disclaims any obligation or undertaking to update or revise any forward-looking statements contained in this press release, other than to the extent required by law. Investor Contact: Susan Kim +1-617-307-7503 susan.kim@crisprtx.com Media Contact: Rachel Eides +1-617-315-4493 rachel.eides@crisprtx.com