22q11 Deletion Syndrome

Tolerance Bio has launched with an oversubscribed $17.2 million seed round and a goal to develop therapies for immune-mediated diseases, from cancer to transplant rejection, using the thymus. The Philadelphia-based company plans to develop an allogeneic thymus-induced pluripotent stem cell (iPSC)-based cell therapy platform as well as thymus therapies in an effort to target immune diseases at their core. Columbus Venture Partners led the funding round. Other investors include Criteria Bio Ventures, Sessa Capital, BioAdvance and Ben Franklin Technology Partners. Tolerance is focusing on the thymus, an organ that plays an important role in training and maturing the body’s T cells to defend against immune issues like infections and cancers while also preventing autoimmunity. The T cells in the human body are developed in the first two years of life, but the thymus declines with age, leading to a higher risk for immune diseases. Tolerance hopes to delay and prevent the decline of the thymus, as well as restore the organ if it stops functioning. Tolerance CEO Francisco Leon, who co-founded Provention Bio, which was acquired by Sanofi for $2.9 billion in 2023, said that the startup plans to focus on a rare disease before turning to other indications. Tolerance’s first approach will be focused on restoring thymic function with an adult stem cell-derived thymic epithelial cell implant. “We can generate thymic cells in the lab from stem cells. And we can implant them into an animal, and they develop into a mini-thymus in the body of the animal. Then they train new T cells, and this is already done and published,” Leon told Endpoints News . As a preclinical proof-of-principle of the technology’s potential, Tolerance implanted these stem cells into mice that have no thymus, a well-known animal model of immune deficiency that has the same mutation as babies born without the thymus, a condition called DiGeorge syndrome. “By transplanting our thymic epithelial progenitors, we can generate a lot of T cells simply by implanting the thymic epithelial progenitors,” Leon added. “This is our first preclinical proof of concept.” At the same time, Tolerance is going to use these thymic cells combined with antigens for autoimmune diseases and cancer in order to explore whether they can induce tolerance in autoimmune diseases as well as tumor immunity in cancer. “This is not published yet, but we have a paper under review that shows we can educate the T cells to attack melanoma,” Leon said. “This is going to come out in a few months, probably after the review process.” A third aspect of a six-year plan is drug repositioning, where Tolerance will give people a drug to delay or prevent the involution of the thymus for those who still have a thymus. The target is still undisclosed, but it’s something the company will do in the coming months.

摘要：杆状病毒是感染鳞翅目、双翅目和膜翅目不同物种的病毒病原体，具有全球分布。由于它们的生物学特性以及由此产生的生物技术应用，杆状病毒科是自然科学中一个重要的研究和操作对象。随着RNA干扰机制的出现，已经描述了那些不编码蛋白质而是产生类似于微RNA（miRNA）的转录本的杆状病毒基因的存在。这些miRNA与病毒和宿主序列中基因表达的调控功能相关。本文全面回顾了miRNA的生物生成、功能和特性，特别关注那些在杆状病毒中鉴定出的miRNA。此外，它深入探讨了杆状病毒miRNA在调控病毒和宿主基因方面的特定作用，并提出了结构和热力学稳定性研究，这些研究有助于检测具有预测效用的共享特征。本综述旨在扩展我们对杆状病毒miRNA组的理解，有助于改进基于杆状病毒的生物杀虫剂的生产、管理害虫中的抗性现象、增强重组蛋白生产系统，并开发多样化和改进的BacMam载体以满足生物医学需求。 1.杆状病毒生物学 杆状病毒科是研究最广泛的病毒家族之一，尽管它不感染人类或对具有重要社会经济利益的动植物造成感染。相反，它主要感染各种节肢动物幼虫，包括鳞翅目、膜翅目和双翅目，其中许多被认为是农业害虫。这解释了对杆状病毒的相当大的兴趣，因为它们已经被成功地用作生物杀虫剂配方中的主要活性成分，用于控制多种农作物中的昆虫害虫。随着时间的推移，随着我们对杆状病毒的理解的增长，它们的主要生物技术应用已经多样化到其他行业，导致了作为杆状病毒重组蛋白表达载体（Baculovirus Expression Vector Systems，或BEVS）的遗传改良变种的发展。随后，出现了一种新的应用类型，涉及在哺乳动物中有用的工程病毒。尽管这些病毒在脊椎动物中不具有传染性，但它们可以转导哺乳动物细胞并携带感兴趣的重组DNA，促进疫苗和疗法的概念发展（称为BacMam应用，用于哺乳动物中的杆状病毒）。这些属性使其成为腺病毒载体的技术竞争对手，提供类似的好处，但具有更优越的免疫学特性。杆状病毒的核酸是一个大的圆形双链DNA分子，位于一个蛋白质极性核衣壳内。这个家族的名字来源于这种核衣壳的特征形态，它被脂质双层包围。这些结构被称为包涵体衍生病毒（ODVs），嵌入在称为包涵体（OB）的蛋白质副晶态基质中，后者在感染昆虫细胞的细胞核中形成。在已描述的杆状病毒中，已报告了两种主要类型的OBs：具有颗粒形态的颗粒病毒（GVs）和具有多面体形态的核多角体病毒（NPVs）。GVs通常包含一个ODV，而NPVs可以包含多个ODVs。一些NPV分离物甚至有一个脂质包膜中的ODVs含有多个核衣壳。这种形态多样性，加上宿主特异性和其他属性，导致它们被分类为四个属：Alphabaculovirus（感染鳞翅目的NPVs）；Betabaculovirus（感染鳞翅目的GVs）；Deltabaculovirus（感染双翅目的NPVs）；和Gammabaculovirus（感染膜翅目的NPVs）。目前，Baculoviridae是Lefaviridae目中的一个家族，该目还包括Nudiviridae和Hytrosaviridae，在Naldaviricetes类中（包括Nimaviridae）。值得注意的是，还有一种称为芽生病毒（BV）的病毒形态，它在感染过程中产生，至少对于alphabaculoviruses和betabaculoviruses是这样。BVs有助于将感染传播到受影响幼虫的不同组织。BVs是被脂质膜包裹的核衣壳，含有病毒蛋白，与包围ODVs的膜不同。有了这两种类型的病毒，杆状病毒的感染周期通常被分为一个由OBs和ODVs在易感幼虫的中肠细胞中主导的初级过程，以及一个由BVs（如果存在）主导的次级过程，它可以将感染传播到受影响个体的大多数组织。由杆状病毒引起的病理通常是致命的，可能涉及幼虫液化作为一种可能的结果。杆状病毒复杂的感染周期是通过其遗传内容的复杂调控实现的。国际病毒分类委员会（ICTV; https://ictv.global/taxonomy, 最后访问日期为2023年9月20日）提出了基于核心基因之间的遗传距离和其他生物学属性（如病毒形态和宿主影响）的103种杆状病毒。最近的一项研究，考虑了所有可用的杆状病毒基因组数据，仅基于遗传距离标准提出了类似的结果。这项研究探索了序列范围在81,755到178,733碱基对之间，包含89到183个编码蛋白质的基因。杆状病毒拥有自己的转录和复制机制，所有报告的成员至少共享39个同源蛋白基因。有趣的是，尽管杆状病毒基因组中存在大量的蛋白基因内容，但也观察到了非编码RNA基因的存在，包括那些编码微RNA（miRNAs）的基因，甚至转移RNA（tRNAs）。根据报告的研究，有证据表明杆状病毒基因组内存在信息重叠，其中编码蛋白质和非编码RNA分子可能在同一基因座上编码。此外，也可能存在非基因功能，如复制起点。对每个蛋白基因的功能，以及RNA基因的语法和角色的更全面理解，不仅有助于理解宿主与其寄生虫之间的自然相互作用，而且可能对人类应用产生潜在影响，包括使用这些病毒及其自然和工程变体。 2.miRNA生物生成和功能 许多原核生物和真核生物的细胞过程都受到由RNA和蛋白质分子组成的酶复合体的调控。这些复合体，称为核糖核蛋白（RNPs），通常作用于核酸底物。它们发挥各种关键作用，如通过选择性剪接促进转录本成熟，通过选择性降解或抑制转录本的翻译来调节基因表达，以及作为特定核酸酶来消除外来核酸。此外，还有参与核染色质重塑的RNPs，具有重要的表观遗传后果。这些RNPs中的RNA分子具有关键作用，因为它们通常作为指南，将酶机制导向它们的目标核酸。这些RNA分子的生物生成是通过转录通常缺乏蛋白质编码信息的基因发生的。这些RNA基因可以产生，例如，小核RNA（snRNA）、微RNA（miRNAs）或CRISPR（簇状规则间隔短回文重复序列）RNAs（crRNAs），它们参与称为剪接体、RNA诱导的沉默复合体（RISCs）或CRISPR/Cas系统的RNP复合体。长非编码RNA（lncRNAs），在真核生物的调控动态中非常丰富，也形成在基因组的特定位点特异性结合酶的复合体。对于这些例子中的任何一个，与蛋白质基因相比，RNA基因在基因组中的注释通常更具挑战性，因为我们对构成它们的句法元素的知识是有限的。开放阅读框（ORF）的缺失，尽管可能被内含子打断，不再是其完全识别的障碍。因此，细胞中发生的一些过程可能被忽视或没有完全解释，这些RNA分子作为一种“暗物质”，可能有助于更好地理解塑造和支持生物体的许多机制。 RNA干扰（RNAi）机制是大约25年前被认识到的细胞过程，并且在生化上已被成功描述，使得农业和医学的生物技术的设计和发展成为可能。这一在真核生物中高度保守的过程依赖于针对RNA分子的RNPs。该机制是由双链RNA（dsRNA，内源性产生或来自入侵核酸）的存在引发的，这触发了具有核酸酶活性或翻译抑制的RNPs的形成，使细胞能够防御病毒攻击并丰富其基因表达的调控机制。考虑到这一最后的活动，RNAi机制可能作为转录后基因沉默（PTGS）进行干预，为细胞提供了根据它们接收到的动态信号调整其功能的选项。这一保守功能是一个由参与dsRNA处理和RNPs形成的蛋白质编码基因编码的复杂系统，将充当效应核酸酶或翻译阻断剂。此外，它还包括RNA基因，如产生miRNA的miR基因，产生引导RNPs到目标分子的指南。在蛋白质编码基因中，值得一提的是那些编码III型RNases Drosha和Dicer、DGCR8（人类DiGeorge综合征关键区域8）或Pasha（非人类中的Drosha伴侣）以及称为Argonauts（AGO）的H型RNases。自本世纪初以来，已经描述并存储了数千种来自不同生物体的miRNAs，并继续对这些生物分子在生物体生理学和病理学中的作用感兴趣（图1）。 图1. 与miRNA相关的科学出版物和miRBase数据库内容。面板(A)描绘了每年关于miRNA的出版物（原创文章）数量，这些出版物来自不同的实体（除了2023年，那里的结果只是部分的）。数据是通过使用PubMed搜索（https://pubmed.ncbi.nlm.nih.gov/；最后访问日期为2023年4月3日）以及考虑的实体的对应组合关键词“microRNA”获得的。面板(B)展示了一个条形图，说明了在miRBase数据库（https://www.mirbase.org/；最后访问日期为2023年4月3日）的不同更新中报告的miRNA数量，这些miRNA来自不同的实体（当这些信息可用时）。这个图是原创的，之前没有发表过。 虽然大多数科学工作集中在理解哺乳动物miRNAs上，特别是那些涉及人类过程的miRNAs，但也有关于昆虫和病毒的报告。这些贡献丰富了我们对RNA干扰过程的全球理解，并可能为推进各种生物技术创造新的机会。通常，参与这种沉默功能的蛋白质是组成性表达的，没有显著的调节，而调节分子（如miRNA）提供可诱导的作用。因此，表征miR基因并理解它们的表达动态对于理解沉默过程的发生至关重要。MiR基因通常由RNA聚合酶II启动子和终止信号（尽管可能有其他来源，如其在内含子中的出现）界定，产生大约70-100个核苷酸的初级前体（pri-miRNA）。这种分子通常采用类似发夹的结构，在核中被Drosha/Pasha（微处理器复合体）识别，这是一种将其处理成前体miRNA的蛋白质。随后，处理过的转录本通过Exportin 5/RanGTP导出到细胞质中，在那里它被Dicer在其他RNA结合蛋白（如TRBP，人类的转录激活反应RNA结合蛋白）的支持下切割，导致形成包含成熟miRNA和AGO蛋白的RISC。这种成熟的miRNA，通常长21-25个核苷酸，源自pri-miRNA的双链区域（图2）。 当RISC复合体通过特定相互作用遇到目标转录本（通常是mRNA）时，它开始“沉默”目标。这可能涉及水解或翻译抑制，导致目标基因产物处于“关闭”状态。为了执行这一行动，需要发生RNA-RNA相互作用（miRNA-mRNA），通常涉及位于目标分子30非翻译区（UTR）的特定序列。这个区域，称为miRNA反应元件（MRE），作为基因表达的调控信号。已经观察到，RISC复合体的构象由RNP和目标RNA之间的特定结合决定，涉及A:U和G:C碱基对相互作用，以及G:U对。具体来说，这种相互作用是由位于miRNA 50端大约6到8个核苷酸的序列促进的，称为核心或种子序列。这允许预测基因组中可能被特定miRNA调节的潜在靶基因。 图2. miRNA介导的沉默机制。该图示描绘了内源性miRNA介导的沉默的经典途径。 1.从细胞基因组(gDNA)或病毒核酸(vDNA)转录生成pri-miRNA（通常由RNA聚合酶II执行）。 2,3. 与Drosha和Pasha蛋白（人类中的DGCR8）结合生成前体miRNA。 4.由Exportin5和RanGTP协助转运到细胞质。 5.与DICER和TRBP结合，连同Argonaut 2(AGO2)的存在，完成miRNA成熟并形成RNA诱导的沉默复合体(RISC)。 6.miRISC与目标转录本结合（通常位于mRNA的3' UTR）。 7.目标转录本降解或其翻译抑制；目标RNA分子数量减少和/或编码蛋白产量下降（在mRNA的情况下），导致随后对生物功能的效应。由于“杆状病毒/昆虫”系统中涉及RNA干扰的蛋白质机制来源于宿主基因，因此一些报告了bac-miRs的杆状病毒宿主的鳞翅目昆虫的主要蛋白的GenBank登录号如下：家蚕的NP_001095931.1(Ago1), NP_001036995.2(Ago2), NP_001180543.1(DICER-2), XP_012547603.2(Drosha), XP_037872173.1(Pasha)；斜纹夜蛾的XM_022976077.1(AGO), AHC98010.1(AGO2), XM_022972435.1(DICER), XP_022826597.1(Drosha), XP_022831748.1(Pasha)；草地贪夜蛾的AVK59452.1(AGO1), XP_035453543.1(AGO2), XP_035450082.2(DICER), XP_050556783.1(Drosha), XP_050553060.1(Pasha)。这个图是原创的，之前没有发表过。 3.杆状病毒编码的miRNAs 病毒是寄生实体，利用它们入侵的宿主细胞的细胞资源进行复制并在自然界中维持自己。在宿主和寄生虫之间的这种复杂关系中，发生了旨在终止感染过程或削弱宿主防御机制以促进成功入侵的分子对话。RNAi机制在这些真核生物内的相互作用中发挥着重要作用。因此，能够影响病毒过程的细胞miRNA基因的存在，以及调节自身表达级联以对抗宿主的病毒miRNA基因的出现，成为病毒感染中的显著特征。在这种情况下，杆状病毒也不例外。如前所述，它们的基因组包含miR（bac-miR）基因。全面的文献综述确定了13个有实验证据的报告bac-miRs（表1），从现在开始关注它们。其中，家蚕核多角体病毒（BmNPV；Aplphabaculovirus bomori）中有5个，加州多角体病毒（AcMNPV；Alphabaculovirus aucalifornicae）中有5个，粉纹夜蛾多角体病毒（AgMNPV；Alphabaculovirus angemmatalis）中有1个，斜纹夜蛾核多角体病毒（SpltNPV；Alphabaculovirus spliturae）中有2个。值得一提的是，所有这些病毒都属于Alphabaculovirus属。这并不一定意味着bac-miR基因的发生是这个类群所独有的，而是由于工具的可用性更大等因素，如现有的易感于这些病毒的昆虫细胞系，因此更多的研究工作已经指向这里。报告的bac-miRs与蛋白质编码区域重叠，其中一些在所有已知的杆状病毒基因组中有同源物（核心基因），包括bmnpv-miR-3、AcMNPV-miR-1、AcMNPV-miR-3、AcMNPV-miR-5、agmnpv-miR-4和Splt-NPV-miR-11698-3p。其余的bac-miRs（除了bmnpv-miR 4）不仅位于alphabaculovirus编码基因内，还位于其他属的编码基因内。它们位于Baculoviridae进化过程中保守的序列内，这一事实可能表明bac-miRs作为杆状病毒基因被低估了。鉴于此，通过进行生物信息学和实验调查来验证这一假设将是有趣的，以确定在其他杆状病毒物种的同源蛋白基因中报告的bac-miRs同源物的存在。 此外，它们的目标RNA分子不仅包括病毒mRNA，还包括宿主的mRNA，从而有助于控制它们自己的基因表达和寄生生物的基因表达。在某些情况下，它们的靶标的实验证据尚未得到证明。与鉴定bac-miRs的位置类似，报告的目标RNA由在Baculoviridae中高度保守的蛋白质编码基因的转录本组成，包括许多核心基因，如DNA聚合酶、p6.9、p40、p95、vlf-1、lef-8、DNA解旋酶、odv-e25、ac95、ac101、ac66和ac98。这进一步表明这些RNA基因可能在整个家族中的重要性，而不仅仅是在它们被描述的物种中。 此外，负责它们产生的转录机制尚未完全阐明，这阻碍了对已鉴定的前体miRNAs的典型启动子基序和下游聚腺苷酸信号的预测（表2）。对于某些bac-miRs，并假设这项工作中假设的基序所构成的限制，pri-miRNA序列可能相对较长，这与其他生物系统中的观察结果一致。 根据我们的文献搜索，这13个bac-miRs共同针对总共18个宿主mRNA分子（尽管只有三个有实验验证）。为了确定这些基因是否与特定的潜在分子途径和功能类别相关，如基因本体（GO），本研究使用ShinyGO服务器（http://bioinformatics.sdstate.edu/go/; 最后访问日期为2023年10月27日；）进行了基因列表富集分析。这使我们能够识别出31个功能丰富的类别，显著性阈值为FDR（假发现率）<0.05（详细描述见补充文件S1）。其中八个类别涉及生物过程，其余的与分子功能相关。这些类别主要涉及代谢和细胞过程，其中三个也参与生物调节（正向调节基因翻译和表达，以及细胞酰胺代谢过程）。具体来说，这些过程包括脂质和有机酸代谢、氮化合物代谢、细胞呼吸、电子传递链代谢过程和有机物质生物合成过程。值得注意的是，所有显著富集的分子功能都与催化活动或与上述生物过程不可或缺的化合物的结合有关。4.描述的细菌miRNAs的共同特征 与蛋白质编码基因中的开放阅读框类似，它们可以揭示与密码子分布相关的组成偏差，有助于预测它们的存在，miR基因也可以表现出有助于预测的组成偏差。因此，鉴定出的细菌miRNAs可以作为训练数据集，以发现循环发生、茎长和稳定性等共同特征。这些参数可以为改进预测算法提供有价值的见解。关于这一点，本研究首次对所描述的细菌miRNA在长度和GC丰富度方面进行了表征（图3）。 图3. Bac-miR常见序列特征。箱形图显示了使用Biopython 实现的专用脚本生成的杆状病毒前体miRNAs和成熟miRNAs的GC百分比(A)和长度(B)。对于这些分析，考虑了描述13个bac-miRs的出版物中报告的序列。这个图是原创的，之前没有发表过。 这份分析揭示了GC含量在前体miRNA及其成熟形式中存在显著的分散性，尽管接近甚至高于50%的值占主导地位，特别是在成熟变体中。值得注意的是，当计算成熟miRNAs中GC含量百分比与包含它们的基因组中GC含量百分比之间的比率（R=% GC miRNA/% GC genome）时，观察到的值为1.51±0.33，表明这一属性具有独特的偏差。另一方面，长度被证明是更加保守的，前体miRNAs的长度在55到138之间（90.5±16.05），miRNAs的长度在20到23之间（21.6±1.03）。这些值与其他生物系统中报告的值一致，它们可能对定义bac-miRs的预测算法有用。 另一个需要解决的问题是，是否在pri-miRNAs的二级结构中存在共同的模式，它们作为Drosha/Pasha以及DICER的底物分子（图4）。对这些特性的分析可以揭示双链区域的长度是否保守以及它们的稳定性。 图4. 杆状病毒前体miRNAs的二级结构。展示了来自AcMNPV、BmNPV、AgMNPV和SpltNPV的实验性前体miRNAs的二级结构及其相应的最小自由能（MFE）值。成熟miRNA序列以橙色突出显示。RNAStructure服务器（https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html；最后访问日期为2023年9月20日），使用299.15开尔文（26°C，可能是杆状病毒感染幼虫的温度）的温度和默认参数，用于生成结构。碱基对概率由碱基的颜色表示。这个图是原创的，之前没有发表过。 这项工作所获得的结果（与之前发表的结果非常相似）显示出潜在的共享结构偏差。为了验证这一点，我们通过评估杆状病毒前体miRNAs的结构保守程度（图5）进一步增强了我们的调查。这使我们能够识别出，在13个杆状病毒前体miRNAs中，有11个可以根据它们共享的结构被归类为两个组，尽管它们来自不同的病毒种类。值得注意的是，第一组中的前体miRNAs具有一个末端环和一个被大内环打断的长茎，而第二组中的那些则显示一个末端环和一个带有四个小内环的茎。 图5. 杆状病毒前体miRNAs二级结构的保守性。在这项工作中，使用了LocARNA-P服务器（http://rna.informatik.uni-freiburg.de/LocARNA/Inputjsp；最后访问日期为2023年9月20日），在26°C的温度设置下，对miRNAs的二级结构进行了比较分析，与实验确定的结构进行对比。生成的指导树包括所有序列(A)，并展示层次聚类以描绘相似的关系，指导结构多重比对。这棵树揭示了两个结构群体的出现，它们表现出不同程度的保守性（第一组和第二组）。在识别出这两个群体之后，分析被重复，仅使用每个群体所属的序列(B,C)，导致识别出符合典型模式的结构保守性。兼容的碱基对通过颜色编码来指示序列保守性（C-G, G-C, A-U, U-A, G-U, 或 U-G）和结构保守性（饱和度）。这个图是原创的，之前没有发表过。 另一个有趣的分析涉及预测来自pri-miRNA（前体miRNA分子）的双链RNA的末端，该末端在5'端具有较低的稳定性。这一特性被认为是决定哪条链将作为乘客链，哪条作为导向链（成熟miRNA）在RISC构象中的关键因素。因此，这些端点的理论热力学稳定性值被计算出来（表3）。 获得的结果揭示了六个bac-miRNAs（bmnpv-miR-1、bmnpv-miR-2、AcMNPV-miR-1、AcMNPV-miR-2、AcMNPV-miR-4和SpltNPV-miR-11684-3p）在成熟miRNA对应的5'端表现出不太稳定的ΔG值，无论是在外环还是在初始堆叠中。与此同时，三个miRNAs仅在第一堆叠中显示出不太稳定或同样稳定的ΔG值（BmNPV-miR-415、AcMNPV-miR-3和SpltNPV-miR-11698-3p）。只有四个分子显示出与预期趋势相矛盾的5'端稳定性特征。尽管在进行这些端点热力学稳定性预测时存在挑战（由于对由导向链和乘客链组成的小双链RNA的身份的不确定性），并且尽管并非所有情况都观察到不太稳定的5'端产生了成熟的miRNA，但检测到了这一属性保持的正向趋势。 5.用于miRNA研究的生物信息学工具 在本研究中对alphabaculoviruses中报告的bac-miRs以及其他先前发表的综合性研究的分析提供了有用的见解，以指导未来在杆状病毒基因组中表达miRNA分子的新型非编码基因的预测。为了进一步增强这一领域的研究，值得注意的是，存在许多数据库和软件工具，它们有助于miRNA的表征和目标搜索。因此，其中一些被呈现（表4）。 将先前建立的属性与报告的bac-miRs有效整合，并审慎使用已展示的工具以及定制设计的计算程序以将它们连接成一个强大的流程，有潜力预测先前未记录的bac-miRs。这代表了对这些假定的非编码基因进行后续实验验证的关键初步步骤。它极大地促进了我们对杆状病毒基因表达调控动态的理解，并进一步丰富了我们对寄生生物与其宿主在感染过程中发生的分子相互作用的理解。 6.结论 考虑到汇编的文献和进行的分析，可以得出结论，13个bac-miR基因位于Baculoviridae中进化保守序列的编码蛋白区域内，其中许多在多个属中具有同源物。它们产生初级转录本，生成大约90个核苷酸的前体miRNA（在大约50到150个核苷酸的范围内），表现出至少两种不同的构象模式（在本研究中称为第一组和第二组），导致产生大约20-23个核苷酸长度的miRNA分子。这些miRNA分子的GC含量高于它们的未处理初级转录本和它们所在基因组的GC含量。目标mRNA包括病毒基因，其中大部分在相同属的所有物种中都有同源物，以及参与细胞和代谢过程的细胞基因。将对报告的bac-miRs进行的这些分析与实验证明整合起来，可以作为预测未检测到的bac-miRs的基础。这一步骤可以作为通过转录组和功能证据确认它们的先导。 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。

WAKIX® (pitolisant) Net Revenue of $172.8 Million for Second Quarter 2024; ~29% Growth Year-over-Year Next Generation Pitolisant High-Dose (HD) Program Advances Based on Pilot Pharmacokinetic (PK) Data with PDUFA Date Expected in 2028 to Extend Pitolisant Franchise Beyond 2040 WAKIX Patent Upheld Again - U.S. Patent and Trademark Office (USPTO) Issues Final Denial After Two Attempts to Challenge the WAKIX Patent Received U.S. Food and Drug Administration (FDA) Approval and Launched WAKIX for the Treatment of EDS in Pediatric Patients with Narcolepsy Providing the First and Only Non-Scheduled Treatment Option On Track to Submit Supplemental New Drug Application for Pitolisant in Idiopathic Hypersomnia (IH) in Fourth Quarter 2024 Reiterates 2024 Net Product Revenue Guidance of $700 - $720 Million Company to Host Investor Day in New York City on October 1st Conference Call and Webcast to be Held Today at 8:30 a.m. ET PLYMOUTH MEETING, Pa., Aug. 06, 2024 (GLOBE NEWSWIRE) -- Harmony Biosciences Holdings, Inc. (Nasdaq: HRMY), today reported year-over-year net revenue growth of 29 percent for the quarter ended June 30, 2024. In addition, with the goal of addressing ongoing unmet medical needs in the narcolepsy market, the next generation pitolisant-HD development program is advancing and on track towards an expected PDUFA date in 2028. “We made substantial progress and continue to advance our pitolisant high-dose development program, targeting a PDUFA date in 2028, giving us the opportunity to extend the pitolisant franchise beyond 2040,” said Jeffrey M. Dayno, M.D., President and Chief Executive Officer of Harmony. “In addition, we are executing on our late-stage pipeline across three orphan/rare CNS franchises, which we expect to deliver at least one new product or indication launch every year over the next five years, with multi-billion-dollar revenue potential extending beyond 2040. We also delivered another strong quarter of revenue growth for WAKIX, confirming our confidence in WAKIX being a billion dollar plus market opportunity in narcolepsy alone, while gaining the approval and launching WAKIX in pediatric narcolepsy.” Key Franchise Highlights: Sleep/Wake WAKIX Net Revenue of $172.8 million in the second quarter of 2024, representing 29% growth over the same period in 2023.The average number of patients on WAKIX increased by approximately 250 patients sequentially to approximately 6,550 for the quarter ended June 30, 2024.WAKIX patent upheld again. We announced that the USPTO has issued a final denial of the petition for reexamination, which was filed by a short seller. This denial is non-appealable and reinforces our confidence in the strength of our patents, the validity of the patent portfolio, and our ability to rigorously enforce the intellectual property rights protecting WAKIX.Received FDA approval for WAKIX for the treatment of EDS in pediatric patients 6 years and older with narcolepsy on June 21, 2024, and executed commercial launch July 1, 2024.On track to submit supplemental new drug application (sNDA) for pitolisant in IH in the fourth quarter of 2024.Pitolisant Gastro-Resistant (GR): On track to initiate Dosing Optimization study in the fourth quarter of 2024 and Pivotal Bioequivalence study in the first quarter of 2025. PDUFA date expected in 2026. Provisional patent filed with the potential for patent protection out to 2044.Pitolisant HD: Pilot PK data showed meaningful differentiation with at least a ~ 20% increase in relative bioavailability and a decrease in the variability of the PK profile compared to an equivalent WAKIX dose of 35.6 mg, the highest labeled dose. Formulation optimization work continues, and we plan to study up to two times the current highest labeled dose of WAKIX, where we expect to demonstrate a further increase in relative bioavailability and decrease in variability in the PK profile. An optimized PK profile, along with a higher dose, GR coating and targeting unique symptoms (e.g., fatigue in narcolepsy, in addition to EDS and cataplexy) is expected to provide a differentiated product profile and label compared to WAKIX. PDUFA date expected in 2028. Provisional patent filed with the potential for patent protection out to 2044.Patient enrollment is ongoing in Phase 3 TEMPO study in patients with Prader-Willi syndrome (PWS).TPM-1116, a highly potent and selective oral orexin-2 receptor agonist that will be evaluated for the treatment of narcolepsy and other sleep-wake disorders. IND enabling studies are ongoing and we expect to file an IND in mid-2025 and initiate first-in-human studies in the second half of 2025.Completed pre-clinical POC study for HBS-102 in PWS with encouraging initial results; final study report and results to be shared later this year. Neurobehavioral On track for topline data from the Phase 3 RECONNECT registrational trial of ZYN002 in Fragile X syndrome (FXS) in mid-2025.Phase 3 preparation ongoing for ZYN002 in 22q11.2 deletion syndrome (22q). Rare Epilepsy EPX-100 is a potent, oral, centrally acting serotonin (5HT2) agonist, currently in a pivotal registrational trial (ARGUS) for Dravet syndrome (DS) with topline data expected in 2026.Phase 3 trial for Lennox-Gastaut syndrome (LGS), expected to initiate later this year.EPX-200 is a potent, oral, centrally acting and selective 5HT2C agonist, and is currently in IND-enabling stage. Second Quarter 2024 Financial Results Net product revenues for the quarter ended June 30, 2024, were $172.8 million, compared to $134.2 million for the same period in 2023. The 29% growth versus the same period in 2023 is primarily attributed to strong commercial sales of WAKIX driven by continued organic demand tapping into a large market opportunity (approximately 80,000 patients diagnosed with narcolepsy in the US) and the broad clinical utility of WAKIX across the approximately 9,000 HCPs that we call on (about 5,000 of whom do not participate in an oxybate REMS program). The average number of patients on WAKIX increased by approximately 250 sequentially to approximately 6,550 for the quarter ended June 30, 2024. GAAP net income for the quarter ended June 30, 2024, was $11.6 million, or $0.20 earnings per diluted share, compared to GAAP net income of $34.3 million, or $0.56 earnings per diluted share, for the same period in 2023. The decrease in GAAP net income was primarily driven by a $25.5 million upfront licensing fee paid as part of the 2024 Bioprojet Sublicensing Agreement for TPM-1116 and a $17.1 million IPR&D charge related to the acquisition of Epygenix. Non-GAAP adjusted net income was $60.6 million, or $1.05 earnings per diluted share, for the quarter ended June 30, 2024, compared to Non-GAAP adjusted net income of $45.9 million, or $0.76 per diluted share, for the same period in 2023. Reconciliations of applicable GAAP financial measures to Non-GAAP financial measures are included at the end of this press release. Harmony’s operating expenses include the following: Research and Development expenses were $63.6 million in the second quarter of 2024, as compared to $15.0 million for the same quarter in 2023, representing a 325% increase, primarily driven by a $25.5 million upfront licensing fee as part of the 2024 Bioprojet Sublicense Agreement and a $17.1 million IPR&D charge related to the acquisition of Epygenix;Sales and Marketing expenses were $28.5 million in the second quarter of 2024, as compared to $24.5 million for the same quarter in 2023, representing a 16% increase;General and Administrative expenses were $27.2 million in the second quarter of 2024, as compared to $22.8 million for the same quarter in 2023, representing a 19% increase; andTotal Operating Expenses were $119.3 million in the second quarter of 2024, as compared to $62.3 million for the same quarter in 2023, representing a 92% increase. As of June 30, 2024, Harmony had cash, cash equivalents and investments of $434.1 million, compared to $425.6 million as of December 31, 2023. Reiterates 2024 Net Product Revenue Guidance Expect full year 2024 net product revenue of $700 million to $720 million. Share Repurchase Program The remaining amount of common stock authorized for repurchases as of June 30, 2024, was $150 million. Investor Day on October 1st The company will host an investor day in New York City on October 1st. Additional event details to follow. Conference Call Today at 8:30 a.m. ET We are hosting our second quarter 2024 financial results conference call and webcast today, beginning at 8:30 a.m. Eastern Time. The live and replay webcast of the call will be available on the investor relations page of our website at https://ir.harmonybiosciences.com/. To participate in the live call by phone, dial (800) 225-9448 (domestic) or +1 (203) 518-9708 (international), and reference passcode HRMYQ224. Non-GAAP Financial Measures In addition to our GAAP results, we present certain Non-GAAP metrics including Non-GAAP adjusted net income and Non-GAAP adjusted net income per share, which we believe provides important supplemental information to management and investors regarding our performance. These measurements are not a substitute for GAAP measurements, and the manner in which we calculate Non-GAAP adjusted net income and Non-GAAP adjusted net income per share may not be identical to the manner in which other companies calculate adjusted net income and adjusted net income per share. We use these Non-GAAP measurements as an aid in monitoring our financial performance from quarter-to-quarter and year-to-year and for benchmarking against comparable companies. Non-GAAP financial measures should not be considered in isolation or as a substitute for comparable GAAP measures; should be read in conjunction with our consolidated financial statements prepared in accordance with GAAP; have no standardized meaning prescribed by GAAP; and are not prepared under any comprehensive set of accounting rules or principles. In addition, from time to time in the future there may be other items that we may exclude for purposes of our Non-GAAP financial measures; and we may in the future cease to exclude items that we have historically excluded for purposes of our Non-GAAP financial measures. About WAKIX® (pitolisant) TabletsWAKIX, a first-in-class medication, is approved by the U.S. Food and Drug Administration for the treatment of excessive daytime sleepiness or cataplexy in adult patients with narcolepsy and has been commercially available in the U.S. since Q4 2019. It was granted orphan drug designation for the treatment of narcolepsy in 2010, and breakthrough therapy designation for the treatment of cataplexy in 2018. WAKIX is a selective histamine 3 (H₃) receptor antagonist/inverse agonist. The mechanism of action of WAKIX is unclear; however, its efficacy could be mediated through its activity at H₃ receptors, thereby increasing the synthesis and release of histamine, a wake promoting neurotransmitter. WAKIX was designed and developed by Bioprojet (France). Harmony has an exclusive license from Bioprojet to develop, manufacture and commercialize pitolisant in the United States. Indications and UsageWAKIX is indicated for the treatment of excessive daytime sleepiness (EDS) or cataplexy in adult patients with narcolepsy and for the treatment of excessive daytime sleepiness (EDS) in pediatric patients 6 years of age and older with narcolepsy. Important Safety Information ContraindicationsWAKIX is contraindicated in patients with known hypersensitivity to pitolisant or any component of the formulation. Anaphylaxis has been reported. WAKIX is also contraindicated in patients with severe hepatic impairment. Warnings and PrecautionsWAKIX prolongs the QT interval; avoid use of WAKIX in patients with known QT prolongation or in combination with other drugs known to prolong the QT interval. Avoid use in patients with a history of cardiac arrhythmias, as well as other circumstances that may increase the risk of the occurrence of torsade de pointes or sudden death, including symptomatic bradycardia, hypokalemia or hypomagnesemia, and the presence of congenital prolongation of the QT interval. The risk of QT prolongation may be greater in patients with hepatic or renal impairment due to higher concentrations of pitolisant; monitor these patients for increased QTc. Dosage modification is recommended in patients with moderate hepatic impairment and moderate or severe renal impairment (see full prescribing information). WAKIX is not recommended in patients with end-stage renal disease (ESRD). Adverse ReactionsIn the placebo-controlled clinical trials conducted in patients with narcolepsy with or without cataplexy, the most common adverse reactions (≥5% and at least twice placebo) for WAKIX were insomnia (6%), nausea (6%), and anxiety (5%). Other adverse reactions that occurred at ≥2% and more frequently than in patients treated with placebo included headache, upper respiratory tract infection, musculoskeletal pain, heart rate increased, hallucinations, irritability, abdominal pain, sleep disturbance, decreased appetite, cataplexy, dry mouth, and rash. Drug InteractionsConcomitant administration of WAKIX with strong CYP2D6 inhibitors increases pitolisant exposure by 2.2-fold. Reduce the dose of WAKIX by half. Concomitant use of WAKIX with strong CYP3A4 inducers decreases exposure of pitolisant by 50%. Dosage adjustments may be required (see full prescribing information). H1 receptor antagonists that cross the blood-brain barrier may reduce the effectiveness of WAKIX. Patients should avoid centrally acting H1 receptor antagonists. WAKIX is a borderline/weak inducer of CYP3A4. Therefore, reduced effectiveness of sensitive CYP3A4 substrates may occur when used concomitantly with WAKIX. The effectiveness of hormonal contraceptives may be reduced when used with WAKIX and effectiveness may be reduced for 21 days after discontinuation of therapy. Use in Specific PopulationsWAKIX may reduce the effectiveness of hormonal contraceptives. Patients using hormonal contraception should be advised to use an alternative non-hormonal contraceptive method during treatment with WAKIX and for at least 21 days after discontinuing treatment. There is a pregnancy exposure registry that monitors pregnancy outcomes in women who are exposed to WAKIX during pregnancy. Patients should be encouraged to enroll in the WAKIX pregnancy registry if they become pregnant. To enroll or obtain information from the registry, patients can call 1-800-833-7460. The safety and effectiveness of WAKIX have not been established in patients less than 18 years of age. WAKIX is extensively metabolized by the liver. WAKIX is contraindicated in patients with severe hepatic impairment. Dosage adjustment is required in patients with moderate hepatic impairment. WAKIX is not recommended in patients with end-stage renal disease. Dosage adjustment of WAKIX is recommended in patients with moderate or severe renal impairment. Dosage reduction is recommended in patients known to be poor CYP2D6 metabolizers; these patients have higher concentrations of WAKIX than normal CYP2D6 metabolizers. Please see the Full Prescribing Information for WAKIX for more information. To report suspected adverse reactions, contact Harmony Biosciences at 1-800-833-7460 or the FDA at 1-800-FDA-1088 or www.fda.gov/medwatch. About NarcolepsyNarcolepsy is a rare, chronic, debilitating neurological disease of sleep-wake state instability that impacts approximately 170,000 Americans and is primarily characterized by excessive daytime sleepiness (EDS) and cataplexy – its two cardinal symptoms – along with other manifestations of REM sleep dysregulation (hallucinations and sleep paralysis), which intrude into wakefulness. EDS is the inability to stay awake and alert during the day and is the symptom that is present in all people living with narcolepsy. In most patients, narcolepsy is caused by the loss of hypocretin/orexin, a neuropeptide in the brain that supports sleep-wake state stability. This disease affects men and women equally, with typical symptom onset in adolescence or young adulthood; however, it can take up to a decade to be properly diagnosed. About Idiopathic HypersomniaIdiopathic Hypersomnia (IH) is a rare and chronic neurological disease that is characterized by excessive daytime sleepiness (EDS) despite sufficient or even long sleep time. EDS in IH cannot be alleviated by naps, longer sleep or more efficient sleep. People living with IH experience significant EDS along with the symptoms of sleep inertia (prolonged difficulty waking up from sleep) and 'brain fog' (impaired cognition, attention, and alertness). The cause of IH is unknown, but it is likely due to alterations in areas of the brain that stabilize states of sleep and wakefulness. IH is one of the central disorders of hypersomnolence and, like narcolepsy, is a debilitating sleep disorder that can result in significant disruption in daily functioning. About Prader-Willi syndromePWS is an orphan/rare, genetic neurological disorder with many of the symptoms resulting from hypothalamic dysfunction. The hypothalamus is the part of the brain that controls both sleep-wake state stability and signals that mediate the balance between hunger and satiety, resulting in two of the main symptoms in patients with PWS; EDS and hyperphagia (an intense persistent sensation of hunger accompanied by food preoccupations, an extreme drive to consume food, food-related behavior problems, and a lack of normal satiety). Other features include low muscle tone, short stature, behavioral problems, and cognitive impairment. Approximately 15,000 to 20,000 people in the U.S. live with PWS, and over half of them experience EDS and the majority of them have behavioral disturbances. About ZYN002ZYN002 is the first-and-only pharmaceutically manufactured synthetic cannabidiol devoid of THC and formulated as a patent-protected permeation-enhanced gel for transdermal delivery through the skin and into the circulatory system. The product is manufactured through a synthetic process in a cGMP facility and is not extracted from the cannabis plant. ZYN002 does not contain THC, the compound that causes the euphoric effect of cannabis, and has the potential to be a nonscheduled product if approved. Cannabidiol, the active ingredient in ZYN002, has been granted orphan drug designation by the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of FXS and for the treatment of 22q. Additionally, ZYN002 has received FDA Fast Track designation for the treatment of behavioral symptoms in patients with FXS. About Fragile X SyndromeFragile X syndrome (FXS) is a rare genetic disorder that is the leading known cause of both inherited intellectual disability and autism spectrum disorder. The disorder negatively affects synaptic function, plasticity and neuronal connections, and results in a spectrum of intellectual disabilities and behavioral symptoms, such as social avoidance and irritability. While the exact prevalence is unknown, upwards of 80,000 patients in the U.S. and 121,000 patients in the European Union and the UK are believed to have FXS, based on FXS prevalence estimates of approximately 1 in 4,000 to 7,000 in males and approximately 1 in 8,000 to 11,000 in females. There is a significant unmet medical need in patients living with FXS as there are currently no FDA approved treatments for this disorder. FXS is caused by a mutation in FMR1, a gene which modulates a number of systems, including the endocannabinoid system, and most critically, codes for a protein called FMRP. The FMR1 mutation manifests as multiple repeats of a DNA segment, known as the CGG triplet repeat, resulting in deficiency or lack of FMRP. FMRP helps regulate the production of other proteins and plays a role in the development of synapses, which are critical for relaying nerve impulses, and in regulating synaptic plasticity. In people with full mutation of the FMR1 gene, the CGG segment is repeated more than 200 times, and in most cases causes the gene to not function. Methylation of the FMR1 gene also plays a role in determining functionality of the gene. In approximately 60% of patients with FXS, who have complete methylation of the FMR1 gene, no FMRP is produced, resulting in dysregulation of the systems modulated by FMRP. About 22q11.2 Deletion Syndrome22q11.2 deletion syndrome (22q) is a disorder caused by a small missing piece of the 22nd chromosome. The deletion occurs near the middle of the chromosome at a location designated q11.2. It is considered a mid-line condition, with physical symptoms including characteristic palate abnormalities, heart defects, immune dysfunction, and esophageal/ GI issues, as well as debilitating neuropsychiatric and behavioral symptoms, including anxiety, social withdrawal, ADHD, cognitive impairment and autism spectrum disorder. It is estimated that 22q occurs in one in 4,000 live births, suggesting that there are approximately 80,000 people living with 22q in the U.S. and 129,000 in the European Union and the UK. Patients with 22q deletion syndrome are managed by multidisciplinary care providers, and there are currently no FDA approved treatments for this disorder. About Clemizole hydrochloride (EPX-100)EPX-100, clemizole hydrochloride, is under development for the treatment of Dravet syndrome (DS) and Lennox-Gastaut syndrome (LGS). EPX-100 acts by targeting central 5-hydroxytryptamine receptors to modulate serotonin signaling. The drug candidate is administered orally twice a day in a liquid formulation and has been developed based on a proprietary phenotype-based zebrafish drug screening platform.1 DS is caused by a loss of function mutation in the SCN1A gene, and scn1 mutant zebrafish replicate the genetic etiology and phenotype observed in the majority of DS patients. The scn1Lab mutant zebrafish model that expresses voltage gated sodium channels has been used for high-throughput screening of compounds that modulate Nav1.1 in the central nervous system. About Dravet SyndromeDravet syndrome (DS) is a severe and progressive epileptic encephalopathy that begins in infancy and causes significant impact on patient functioning. DS begins in the first year of life and is characterized by high seizure frequency and severity, intellectual disability, and a risk of sudden unexpected death in epilepsy. Approximately 85% of Dravet Syndrome cases are caused by de novo loss-of-function (LOF) mutations in a voltage-gated sodium channel gene, SCN1A1. DS has an estimated incidence rate of 1:15,700. About Lennox-Gastaut SyndromeLennox-Gastaut Syndrome (LGS) is a rare and drug-resistant epileptic encephalopathy characterized by onset in children between 3-5 years of age. The underlying cause of LGS is unknown and can be related to a wide range of factors including genetic differences and structural differences in the brain. As a result, patients experience multiple seizure types, including atonic seizures, and developmental, cognitive, and behavioral issues. LGS affects approximately 48,000 patients in the U.S. About Harmony BiosciencesAt Harmony Biosciences, we specialize in developing and delivering treatments for rare neurological diseases that others often overlook. We believe that where empathy and innovation meet, a better life can begin for people living with neurological diseases. Established by Paragon Biosciences, LLC, in 2017 and headquartered in Plymouth Meeting, PA, our team of experts from a wide variety of disciplines and experiences is driven by our shared conviction that innovative science translates into therapeutic possibilities for our patients, who are at the heart of everything we do. For more information, please visit www.harmonybiosciences.com. Forward-Looking StatementsThis press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. All statements contained in this press release that do not relate to matters of historical fact should be considered forward-looking statements, including statements regarding our full year 2024 net product revenue, expectations for the growth and value of WAKIX, plans to submit an sNDA for pitolisant in idiopathic hypersomnia; our future results of operations and financial position, business strategy, products, prospective products, product approvals, the plans and objectives of management for future operations and future results of anticipated products. These statements are neither promises nor guarantees, but involve known and unknown risks, uncertainties and other important factors that may cause our actual results, performance or achievements to be materially different from any future results, performance or achievements expressed or implied by the forward-looking statements, including, but not limited to, the following: our commercialization efforts and strategy for WAKIX; the rate and degree of market acceptance and clinical utility of pitolisant in additional indications, if approved, and any other product candidates we may develop or acquire, if approved; our research and development plans, including our plans to explore the therapeutic potential of pitolisant in additional indications; our ongoing and planned clinical trials; our ability to expand the scope of our license agreements with Bioprojet Société Civile de Recherche (“Bioprojet”); the availability of favorable insurance coverage and reimbursement for WAKIX; the timing of, and our ability to obtain, regulatory approvals for pitolisant for other indications as well as any other product candidates; our estimates regarding expenses, future revenue, capital requirements and additional financing needs; our ability to identify, acquire and integrate additional products or product candidates with significant commercial potential that are consistent with our commercial objectives; our commercialization, marketing and manufacturing capabilities and strategy; significant competition in our industry; our intellectual property position; loss or retirement of key members of management; failure to successfully execute our growth strategy, including any delays in our planned future growth; our failure to maintain effective internal controls; the impact of government laws and regulations; volatility and fluctuations in the price of our common stock; the significant costs and required management time as a result of operating as a public company; the fact that the price of Harmony's common stock may be volatile and fluctuate substantially; statements related to our intended share repurchases and repurchase timeframe and the significant costs and required management time as a result of operating as a public company. These and other important factors discussed under the caption "Risk Factors" in our Annual Report on Form 10-K filed with the Securities and Exchange Commission (the "SEC") on February 22, 2024, and our other filings with the SEC could cause actual results to differ materially from those indicated by the forward-looking statements made in this press release. Any such forward-looking statements represent management's estimates as of the date of this press release. While we may elect to update such forward-looking statements at some point in the future, we disclaim any obligation to do so, even if subsequent events cause our views to change. HARMONY BIOSCIENCES HOLDINGS, INC. AND SUBSIDIARIESCONSOLIDATED STATEMENTS OF OPERATIONS AND COMPREHENSIVE INCOME(In thousands, except share and per share data) Three Months Ended June 30, Six Months Ended June 30, 2024 2023 2024 2023Net product revenue $172,814 $134,216 $327,429 $253,342 Cost of product sold 32,144 25,008 59,628 45,788 Gross profit 140,670 109,208 267,801 207,554 Operating expenses: Research and development 63,583 14,969 85,772 28,258 Sales and marketing 28,507 24,528 55,740 47,100 General and administrative 27,224 22,809 52,900 44,871 Total operating expenses 119,314 62,306 194,412 120,229 Operating income 21,356 46,902 73,389 87,325 Other (expense) income, net 37 (31) (104) (29)Interest expense (4,404) (6,218) (8,939) (11,949)Interest income 4,705 3,442 9,133 6,528 Income before income taxes 21,694 44,095 73,479 81,875 Income tax expense (10,103) (9,795) (23,554) (18,090)Net income $11,591 $34,300 $49,925 $63,785 Unrealized (loss) income on investments (63) (491) (236) (371)Comprehensive income $11,528 $33,809 $49,689 $63,414 EARNINGS PER SHARE: Basic $0.20 $0.57 $0.88 $1.07 Diluted $0.20 $0.56 $0.87 $1.05 Weighted average number of shares of common stock - basic 56,802,357 59,974,123 56,786,873 59,853,808 Weighted average number of shares of common stock - diluted 57,541,696 60,743,953 57,571,570 60,997,410 HARMONY BIOSCIENCES HOLDINGS, INC. AND SUBSIDIARIESCONSOLIDATED BALANCE SHEETS (In thousands except share and per share data) June 30, December 31, 2024 2023ASSETS CURRENT ASSETS: Cash and cash equivalents $317,296 $311,660 Investments, short-term 29,614 41,800 Trade receivables, net 83,157 74,140 Inventory, net 5,643 5,363 Prepaid expenses 16,127 12,570 Other current assets 6,507 5,537 Total current assets 458,344 451,070 NONCURRENT ASSETS: Property and equipment, net 754 371 Restricted cash 270 270 Investments, long-term 87,178 72,169 Intangible assets, net 125,186 137,108 Deferred tax asset 180,186 144,162 Other noncurrent assets 6,465 6,298 Total noncurrent assets 400,039 360,378 TOTAL ASSETS $858,383 $811,448 LIABILITIES AND STOCKHOLDERS’ EQUITY CURRENT LIABILITIES: Trade payables $22,683 $17,730 Accrued compensation 9,641 23,747 Accrued expenses 91,644 99,494 Current portion of long-term debt 15,000 15,000 Other current liabilities 7,614 7,810 Total current liabilities 146,582 163,781 NONCURRENT LIABILITIES: Long-term debt, net 171,422 178,566 Other noncurrent liabilities 1,796 2,109 Total noncurrent liabilities 173,218 180,675 TOTAL LIABILITIES 319,800 344,456 COMMITMENTS AND CONTINGENCIES (Note 13) STOCKHOLDERS’ EQUITY: Common stock—$0.00001 par value; 500,000,000 shares authorized at June 30, 2024 and December 31, 2023, respectively; 56,833,771 and 56,769,081 shares issued and outstanding at June 30, 2024 and December 31, 2023, respectively 1 1 Additional paid in capital 632,168 610,266 Accumulated other comprehensive (loss) income (234) 2 Accumulated deficit (93,352) (143,277)TOTAL STOCKHOLDERS’ EQUITY 538,583 466,992 TOTAL LIABILITIES AND STOCKHOLDERS’ EQUITY $858,383 $811,448 HARMONY BIOSCIENCES HOLDINGS, INC. AND SUBSIDIARIESRECONCILIATION OF GAAP TO NON-GAAP FINANCIAL RESULTS(In thousands except share and per share data) Three Months Ended Six Months Ended June 30, June 30, June 30, June 30, 2024 2023 2024 2023GAAP net income $11,591 $34,300 $49,925 $63,785 Non-GAAP Adjustments: Non-cash interest expense (1) 176 424 356 840 Depreciation 91 103 254 206 Amortization (2) 5,961 5,961 11,922 11,922 Stock-based compensation expense 10,963 7,793 21,397 14,354 Licensing fee and milestone payments (3) 25,500 - 25,500 750 Transaction related costs (4) 17,095 - 17,095 - Income tax effect related to non-GAAP adjustments (5) (10,769) (2,712) (15,119) (5,252)Non-GAAP adjusted net income $60,608 $45,869 $111,330 $86,605 GAAP reported net income per diluted share $ 0.20 $ 0.56 $0.87 $1.05 Non-GAAP adjusted net income per diluted share $1.05 $0.76 $1.93 $1.42 Weighted average number of shares of common stock used in non-GAAP diluted per share 57,541,696 60,743,953 57,571,570 60,997,410 (1) Includes amortization of deferred finance charges.(2) Includes amortization of intangible asset related to WAKIX.(3) Amount represents upfront licensing fee incurred upon closing the 2024 Bioprojet Sublicense Agreement and milestone payment related to HBS102 in March 2023.(4) Includes IPR&D charge related to the acquisition of Epygenix. (5) Calculated using the reported effective tax rate for the periods presented less impact of discrete items. Harmony Biosciences Investor Contact:Brennan Doyle484-539-9700bdoyle@harmonybiosciences.com Harmony Biosciences Media Contact:Cate McCanless202-641-6086cmccanless@harmonybiosciences.com