Hyperargininemia

蛋白质替代疗法是一种医疗手段，用于治疗因基因突变导致特定蛋白质缺失或功能失常的疾病。传统的治疗方法通常涉及复杂的体外蛋白质生产和纯化过程，成本高昂。然而，随着mRNA药物技术的发展，尤其是在SARS-CoV-2疫情期间，mRNA药物的快速体内抗原生产能力得到了验证，为蛋白质替代疗法提供了新的治疗途径。这项技术的发展，包括体外转录mRNA和纳米脂质载体的应用，为临床前和临床应用中的mRNA作为治疗工具提供了新的可能性。 在这篇综述中，我们将探讨mRNA在蛋白质替代疗法中的应用，包括其在临床前和临床研究中的使用，以及生产修饰mRNA、细胞内递送系统和成品相关的关键质量属性。我们将详细讨论体外转录mRNA的优化、纳米脂质载体的设计，以及如何提高mRNA的稳定性和翻译效率，以实现更有效的蛋白质替代。此外，我们还将探讨如何通过改进mRNA的化学修饰和递送策略来最小化免疫反应，从而提高治疗效果。 在面对遗传缺陷和先天性代谢疾病时，传统的治疗方法往往集中于缓解症状而非根治病因。因此，能够针对疾病根本原因的治疗策略，如DNA疗法和新兴的RNA疗法，成为了科学界研究的热点。尽管基因编辑技术在基因治疗领域备受关注，但其他具有潜力的治疗手段，例如RNA疗法，也已经开始展现其独特的优势，相比侵入性或永久性手术，它们可能提供更为安全和可逆的治疗方案。 RNA疗法主要基于RNA分子的两大核心功能：调控和编码。在调控方面，反义寡核苷酸（ASO）技术通过与目标RNA转录物的互补序列结合，实现对其表达的精细调控。目前，已有多种ASO药物获得FDA批准，用于治疗包括杜氏肌营养不良症、家族性淀粉样多发性神经病、脊髓性肌萎缩症和家族性乳糜微粒血症综合征在内的多种疾病。 除了ASO，小干扰RNA（siRNA）也是通过RNA干扰（RNAi）机制发挥作用的分子，它通过与RNA诱导沉默复合物（RISC）结合，特异性地降解目标mRNA，从而阻断特定基因的表达。基于siRNA的药物已经成功上市，用于治疗TTR淀粉样变性和肝卟啉症等疾病。 microRNA是另一类调控RNA分子，它们通过与RISC结合，抑制目标mRNA的翻译或促进其降解，从而调控基因表达。RNA适体则代表了一种不同的调控机制，它们通过特定的三维结构与异源靶标（如蛋白质和碳水化合物）相互作用，这种结构上的多样性赋予了RNA适体广泛的应用潜力。 RNA疗法的这些调控功能为治疗遗传性疾病提供了新的策略，它们通过精确调控基因表达，有望为患者带来更为精准和有效的治疗方案。随着RNA技术的进步，未来可能会有更多的RNA疗法被开发出来，以应对各种遗传性和代谢性疾病。 信使RNA（mRNA）的编码潜能，即其指导肽或蛋白质合成的能力，已经开辟了一种全新的治疗途径，即mRNA蛋白质替代疗法。这种疗法的核心在于利用mRNA分子瞬时表达特定的蛋白质，以补充或替代体内因基因缺陷而缺乏的功能蛋白。mRNA的这一特性源于其在细胞内的基本生物学功能，即作为基因表达的中间产物，将遗传信息从DNA转录并传递给细胞的翻译机制，以合成蛋白质。 在实验室中，科学家们通过将外源mRNA直接注射到生物体内，并观察其能否诱导目标蛋白质的表达，从而验证了mRNA作为治疗手段的潜力。这种技术最初被用于研究和治疗罕见的单基因遗传疾病，这些疾病通常由单个基因的突变引起，导致相应的蛋白质功能失常或完全缺失。 尽管这些所谓的“孤儿病”在人群中的发病率较低，但它们的种类繁多，累加起来影响着全球相当数量的人群。为了鼓励制药公司开发治疗这些疾病的药物，许多国家出台了特别的立法措施，提供市场独占权和其他激励措施，以促进孤儿药的研发。这些药物虽然可能价格昂贵，但对于患者来说，它们提供了治疗罕见疾病的唯一希望。 mRNA蛋白质替代疗法的另一个重要应用是mRNA疫苗的开发。这种疫苗通过提供编码病毒抗原的mRNA，使得接种者的细胞能够产生病毒蛋白，从而激发免疫系统产生针对该病毒的免疫反应。这种方法在COVID-19疫苗的开发中取得了巨大成功，展示了mRNA技术在现代疫苗学中的潜力。 随着mRNA技术的不断进步，未来可能会有更多的mRNA蛋白质替代疗法和疫苗被开发出来，以治疗各种遗传性疾病和预防传染病。 蛋白质替代方案——为什么选择 mRNA？ 除了利用mRNA技术，还有其他几种策略可以用于蛋白质替代治疗。基因治疗是一种方法，它通过将健康的基因拷贝传递到患者体内，以补偿或纠正缺陷基因，从而恢复蛋白质的正常功能。这种方法的目的是直接在细胞内修复遗传错误，以期达到治疗遗传性疾病的效果。 另一种直接的方法是将蛋白质本身直接输送到患者体内，这种方式可以避免细胞内翻译过程的复杂性，允许更精确的剂量控制，并能够在目标组织中实现较高的蛋白浓度。这种方法已经被用于多种疾病的治疗，包括激素、酶、凝血因子和干扰素的替代疗法。然而，这种方法也有其局限性，包括蛋白质的半衰期可能较短，稳定性可能不足，以及可能引发免疫反应，这些因素都限制了其广泛应用。 此外，直接蛋白质替代疗法并不适用于所有类型的蛋白质替代，特别是那些位于细胞内的蛋白质，如转录因子和其他调节分子，因为这些蛋白质无法通过简单的递送方式进入细胞。 即使存在这些挑战，蛋白质替代治疗仍然是治疗某些遗传性疾病的有效手段，并且随着生物技术的进步，未来可能会有更多创新的方法被开发出来，以克服当前的局限性并扩大治疗范围。 利用核酸作为蛋白质替代治疗的手段，确实规避了直接蛋白质递送所面临的一些挑战，提供了治疗上的灵活性。然而，核酸治疗策略同样不是没有缺点。基因治疗的插入机制既赋予了其独特的优势，也带来了一定的局限性。 病毒载体在基因治疗中的应用能够确保目标基因被有效地整合进宿主细胞的基因组中，从而实现持久的基因表达。但这种整合可能会引起基因组的不稳定，存在潜在的致癌风险，这是一个不容忽视的问题。尽管并非所有病毒载体都会整合进基因组，它们对特定组织的选择性使得它们成为有吸引力的基因传递工具。但这种方法也可能引发其他问题，比如可能产生针对病毒载体的中和抗体，药代动力学特性可能不理想，以及在病毒载体上包装所需基因的难度。 与病毒载体相对的是非病毒递送方法，如脂质体、树状聚合物、固体脂质纳米颗粒和聚合物胶束等，它们被用来递送质粒DNA。这些非病毒方法具有较低的免疫原性和致癌风险，以及更高的组织特异性，但在转染效率上通常不如病毒载体，这是它们的主要缺点。 因此，在选择基因治疗的递送策略时，需要权衡各种方法的利弊，考虑到治疗效果、安全性和可行性等多方面因素。随着基因编辑技术的进步，未来可能会开发出更安全、更有效的基因治疗策略，以解决当前面临的挑战。 在对蛋白质替代治疗的不同方法进行比较评估后，我们可以得出结论：基于mRNA的治疗方法相较于基于蛋白质或DNA的方法，确实拥有一些显著的优势。这些优势包括： 避免细胞核插入和转录过程：mRNA治疗不需要将遗传物质插入细胞核或进行转录，这简化了治疗机制，并可能减少潜在的副作用。 增强的安全性：由于不与宿主基因组整合，mRNA治疗降低了长期基因突变的风险，提供了一种相对安全的替代方案。 作用的短暂性：mRNA在体内的半衰期有限，其表达的蛋白质水平随时间自然下降，这种短暂性有助于减少长期治疗可能带来的风险。 从监管角度来看，mRNA蛋白质替代疗法不会引起基因组的改变，因此可能比DNA疗法更容易获得立法支持和监管批准。 然而，mRNA疗法也可能引起一些副作用。例如，mRNA分子在细胞内可能会被RNase酶切割，或者通过激活内体膜上的Toll样受体（TLR）3、7和8，引发先天免疫反应，这可能导致细胞因子诱导的毒性。为了解决这一问题，研究人员开发了修饰mRNA（modRNA）技术，通过使用假尿苷替代尿苷，改变了mRNA的二级结构，从而抑制了TLR和核酸酶对mRNA的识别。这种改良的mRNA在提高蛋白质表达水平的同时，显示出了更低的免疫原性，减少了干扰素编码基因和视黄酸诱导基因的激活。 随着mRNA技术的发展，从其最初的发现到如今以COVID-19疫苗形式在全球范围内的推广，这一领域已经取得了巨大的进步。如图1所示的时间线，mRNA技术的发展经历了多个阶段，最终在疫苗开发中发挥了关键作用，为未来治疗多种疾病提供了新的可能性。 图 1. mRNA 历史上主要事件的时间表。Clin：临床，Preclin：临床前，iPSC：诱导多能干细胞，Inj：注射，CAR：嵌合抗原受体，DC：树突状细胞。 蛋白质替代疗法中 mRNA 生产的考虑因素 在现代生物技术中，用于蛋白质替代疗法的mRNA可以通过体外转录反应（IVT）在无细胞系统中高效生产。这一过程通常在严格控制的生物反应器中进行，以确保反应条件如pH值和温度的稳定性和一致性。 IVT的主要组成部分包括： 线性化DNA质粒：作为转录mRNA的模板，提供了转录所需的遗传信息。 DNA依赖性RNA聚合酶：负责从DNA模板转录出mRNA。 dNTP（脱氧核苷酸三磷酸）：作为mRNA构建的基本单元，是转录过程中不可或缺的原料。 5'帽类似物：在某些情况下，为了提高mRNA的稳定性和翻译效率，会在转录过程中添加5'帽结构。 缓冲液：如HEPES/Tris，用于维持反应环境的pH值和离子强度，为酶活性提供适宜的条件。 镁离子和氯化钠：作为辅助因子，对RNA聚合酶的活性至关重要。 RNAse抑制剂：防止RNA分子在生产过程中被降解，确保产物的完整性。 焦磷酸酶：去除反应中产生的焦磷酸盐，避免其抑制RNA聚合酶的活性。 二硫苏糖醇（DTT）：作为一种还原剂，有助于维持酶和其他蛋白质的还原状态，防止氧化。 多胺：如精胺和亚精胺，对mRNA的稳定性和细胞内处理有积极作用。 通过这些精确控制的IVT条件和辅助试剂的使用，可以显著提高mRNA的产量和质量，从而为蛋白质替代疗法提供高效、稳定的mRNA产品。这些mRNA产品在经过进一步的纯化和质量控制步骤后，可以用于开发治疗各种疾病的疗法，包括罕见遗传疾病和某些癌症。随着mRNA生产技术的不断进步，未来可能会有更多创新的治疗方案被开发出来，以满足临床需求。 成功蛋白质生产的 mRNA 架构 所得 mRNA 的翻译效率取决于其结构和化学成分，这取决于接受 IVT 的质粒的设计和反应中使用的 IVT 试剂。适合蛋白质替代疗法的 mRNA 必须具有适当的 5' 帽以及 5'–3' 非翻译区 (UTR) 和一个开放阅读框，旨在最大限度地提高蛋白质产量。用于优化 mRNA (modRNA) 的修饰如图2所示。 图 2. modRNA 的架构。对序列（质粒水平）和化学（IVT 水平）的修饰可以提高 mRNA 的翻译效率并将细胞先天免疫机制的激活降至最低。 在生产用于蛋白质替代疗法的mRNA时，5'端加帽是一个关键步骤，它对mRNA的稳定性和翻译效率至关重要。5'帽不仅保护mRNA免受细胞内核酸酶的降解，还参与mRNA的核糖体识别和翻译过程。目前，有两种主要的加帽方法用于IVT反应：共转录加帽和转录后加帽。 共转录加帽：这种方法是在mRNA的转录过程中同时进行加帽的。在IVT反应中，RNA聚合酶（如T7 RNA聚合酶）在转录mRNA的同时，将帽类似物（如“Cap 1”类似物）直接添加到新合成的mRNA分子的5'端。市面上有现成的产品，如Trilink的“Cleancap”，可以用于共转录加帽。这种方法的优点是操作简单，加帽效率高，通常可以达到90%以上。 转录后加帽：这种方法涉及两个独立的步骤。首先进行IVT反应生成mRNA，然后在后续的加帽反应中使用牛痘病毒加帽酶（vaccinia virus capping enzyme）将帽结构添加到mRNA的5'端。转录后加帽的优点是能够实现100%的加帽率，确保每个mRNA分子都具有5'帽。然而，这种方法需要额外的步骤和纯化过程，可能会增加生产成本和复杂性。 选择哪种加帽方法取决于多个因素，包括所需的mRNA质量、生产规模、成本效益以及最终应用的具体要求。共转录加帽因其操作简便和高效率而被广泛采用，而转录后加帽则适用于对mRNA质量要求极高的应用场景。在实际生产过程中，研究人员会根据实验目的和资源情况来选择最合适的加帽策略。 5'和3'非翻译区（UTR）是mRNA上位于编码区两侧的核苷酸序列，尽管它们不直接编码蛋白质，但在基因表达调控中扮演着重要角色。这些区域通过多种机制影响mRNA的稳定性、定位、翻译效率以及降解过程。 5' UTR的设计： 5' UTR区域应该避免含有起始密码子（AUG）和终止密码子，以防止过早的翻译起始或终止。 应尽量减少能够形成二级结构的序列，这些结构可能会阻碍核糖体的扫描和启动翻译过程，从而降低蛋白质的产量。 优化5' UTR的序列可以提高mRNA的翻译效率，从而增加蛋白质的产量。 3' UTR的设计： 与5' UTR不同，3' UTR区域可以包含能够形成二级结构的序列，这些结构可能有助于提高mRNA的稳定性和翻译效率。 利用已知高表达基因的3' UTR序列，如α-和β-珠蛋白的3' UTR，可以提高mRNA的稳定性和蛋白质的产量。 使用多个3' UTR拷贝或设计融合UTR，如将氨基末端增强子（AES）3' UTR与线粒体编码的12S rRNA（mtRNR1）3' UTR相结合，可以进一步提高mRNA的稳定性和翻译效率。 在设计mRNA用于蛋白质替代疗法时，对5'和3' UTR的精心设计至关重要，因为它们直接影响mRNA的生物学功能和治疗效果。通过优化这些区域，可以提高mRNA的稳定性，增强其翻译效率，从而在临床上实现更好的治疗效果。此外，随着对mRNA生物学的深入了解，未来可能会发现更多可以优化的序列特征，以进一步提高mRNA治疗策略的效率和安全性。 开放阅读框（ORF）是mRNA上的一部分，它包含了编码蛋白质的遗传信息。在质粒中，ORF通常被设计为不含内含子的连续序列，以确保mRNA能够高效地被翻译成蛋白质。以下是一些用于提高mRNA稳定性和翻译效率的策略： 密码子优化：通过选择与人类细胞中tRNA更为丰富的同义密码子，可以提高mRNA的翻译效率。这种优化有助于减少翻译过程中的停顿，从而增加蛋白质的产量。 GC/AU比率调整：通过增加GC含量，可以减少mRNA的转录后调控，使得mRNA更加稳定，不易被细胞内的降解机制识别和降解。 修饰核苷酸的使用：使用假尿苷（Ψ）和N1-甲基假尿苷（m1Ψ）等修饰核苷酸替代尿苷，可以提高mRNA的稳定性，并减少其被细胞内TLR识别的可能性。这种修饰有助于mRNA逃避细胞的先天免疫反应，从而减少mRNA的降解，增加可用于翻译的mRNA数量。 IVT反应中的修饰：在体外转录（IVT）过程中，可以通过添加含有修饰核苷酸的dNTP混合物来实现mRNA的修饰。这种方法可以在mRNA合成的同时引入所需的修饰，从而提高mRNA的质量和功能性。 通过这些策略，可以设计出更加稳定和高效的mRNA分子，用于蛋白质替代疗法。这些优化措施有助于提高mRNA的翻译效率，减少免疫反应，从而在临床应用中实现更好的治疗效果。随着mRNA技术和合成生物学的不断发展，未来可能会有更多创新的方法被开发出来，以进一步提高mRNA治疗的安全性和有效性。 Poly(A)尾巴是mRNA分子的一个重要特征，它对mRNA的稳定性和翻译效率起着至关重要的作用。以下是关于Poly(A)尾巴的一些关键点： 稳定性：Poly(A)尾巴有助于保护mRNA分子免受细胞内核酸酶的降解，从而增加其在细胞内的稳定性。 翻译效率：Poly(A)尾巴与翻译启动因子的结合有助于促进核糖体对mRNA的识别和绑定，从而提高翻译效率。 长度优化：通常认为，100-120个腺嘌呤的Poly(A)尾巴长度对于蛋白质替代疗法中的mRNA分子是优化的，可以提高翻译效率。 聚腺苷酸化过程：在细胞内，聚腺苷酸化是由聚腺苷酸聚合酶（poly(A) polymerase）催化的，而在体外转录（IVT）过程中，可以通过不同的策略来添加Poly(A)尾巴。 IVT中的聚腺苷酸化：使用T3、T7或SP6等RNA聚合酶进行IVT时，这些酶可以在mRNA的3'端添加Poly(A)尾巴。然而，这种方法可能导致Poly(A)尾巴长度不一，影响mRNA的一致性和功能。 质粒编码Poly(A)尾巴：为了避免IVT过程中的复杂性和成本增加，可以通过在质粒中编码Poly(A)尾巴序列来实现一致的Poly(A)尾巴长度。这种方法可以确保所有转录的mRNA分子都具有相同长度的Poly(A)尾巴，从而提高产品的质量和一致性。 设计策略：在质粒设计时，可以在ORF的3'端添加一个Poly(A)信号序列，如AAUAAA，这是真核生物mRNA聚腺苷酸化的信号。随后，可以添加一个或多个连续的腺苷酸序列，以形成Poly(A)尾巴。 通过这些策略，可以有效地控制mRNA分子的Poly(A)尾巴长度，从而优化其在蛋白质替代疗法中的应用。随着对mRNA生物学的进一步研究，可能会发现更多关于Poly(A)尾巴长度和功能之间关系的新见解，从而为mRNA治疗提供更多的优化机会。 IVT 反应后，必须纯化所得 mRNA，以去除污染物并获得药物级 mRNA。广泛采用的纯化程序是层析法，例如离子交换、亲和聚（dT）和离子对反相层析法。这些层析技术通常与切向流系统步骤配合。图 3总结了直到纯 mRNA 产生为止的过程。然后，产品就准备好进入下一步，即掺入构成细胞递送载体的所选递送载体中。 图 3. 基于质粒体外生产 mRNA。编码内含子缺失优化mRNA序列的质粒首先被线性化，然后与RNA聚合酶、dNTPs、缓冲系统和补充试剂一起添加到反应器中。然后通过层析和过滤技术纯化所得产物，并准备封装到递送载体中。 将 mRNA 递送至细胞的载体 为了确保mRNA能够安全有效地到达目标细胞并发挥其治疗潜力，研究人员已经开发了多种递送策略。这些方法的核心目标是保护mRNA不被降解，并确保其能够快速且精确地进入目标细胞。主要的递送方式可以分为病毒载体和非病毒载体两大类，后者进一步细分为聚合物载体、脂质载体以及聚合物-脂质混合载体。 病毒载体：利用病毒的自然能力将遗传物质递送到细胞内，但这种方法可能引起免疫反应，并存在潜在的安全性问题。 非病毒载体： 聚合物载体：使用合成或天然的高分子材料，如聚乙烯亚胺（PEI），来包裹和保护mRNA。 脂质载体：通常指脂质纳米颗粒（LNPs），它们通过形成脂质双层包裹mRNA，提高其稳定性并促进细胞摄取。 聚合物-脂质混合载体：结合了聚合物和脂质的优点，以提高mRNA的递送效率和减少副作用。 每种递送方法都有其独特的优势和局限性，选择合适的递送系统取决于治疗目标、所需mRNA的稳定性和所需的细胞类型。随着递送技术的进步，未来的mRNA疗法有望实现更精准、更高效的药物递送。 病毒疗法，特别是利用基因改造病毒的策略，已经成为研究和临床实践中的一个重要领域。这些病毒载体通常由蛋白质外壳、关键基因和控制基因整合及表达的必需元件构成，可以是逆转录病毒、腺病毒、腺相关病毒，以及其他种类。尽管病毒载体在转导效率和靶向性方面表现出色，且生产规模可以扩大，但它们在临床应用中的成功仍受到多种因素的限制，包括目标群体中可能存在的对病毒载体的预存免疫力。尽管如此，病毒载体的研究和开发仍在持续进行，以克服现有挑战并提高治疗效果。 非病毒载体技术在纳米科技领域开辟了新的可能性，特别是在 COVID-19 疫情期间，它们在两款商业化的 mRNA 疫苗中的应用尤为突出。这些载体利用阳离子聚合物纳米颗粒的静电作用，与 mRNA 分子的负电荷相结合。天然存在的聚合物如壳聚糖，以及合成聚合物如 PLL、PAMAM、PEI 和 PAA，都是构建这类载体的候选材料。在挑选合适的纳米颗粒时，需要综合考虑其电荷密度、分子量、潜在毒性以及药代动力学特性，如在体内的分布、生物降解性和清除速率。为了优化性能，这些纳米颗粒可能需要经过特定的修饰和调整。 脂质纳米颗粒（LNP）或脂质双层壳是实现mRNA有效封装和递送的另一种技术。这些LNP通常由具有水性中心的阳离子脂质构成，辅以胆固醇或聚乙二醇（PEG）等其他分子，形成最终的递送系统。阳离子脂质如1,2-二油酰基-N,N-二甲基-3-氨基丙烷（DODMA）或1,2-二油酰基-3-二甲基丙烷铵（DODAP）的使用，有助于在血液循环中减少电荷相互作用，从而延长LNP的半衰期。 LNP的关键特性，如粒径、ζ电位（聚集性）、内体释放倾向和体内分布，对其行为和稳定性至关重要，并且可以通过精心选择LNP成分来调控。例如，PEG脂质的加入可以影响颗粒的ζ电位和尺寸，提高结构稳定性，而PEG的比例和结合方式也会影响LNP的特性。高PEG比例可能导致较小LNP的形成。 此外，LNP中使用的脂质链长度也会影响其行为。例如，携带C14烷基链的1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基偶联物相比于C18链的类似物，可能在血液中循环时间更短，但细胞递送率更高。胆固醇及其衍生物的加入可以改善LNP的形态和性质，通过尾部修饰的胆固醇类似物可以改变LNP的形状，并可能赋予其特定的细胞趋向性。 最后，DODMA和DODAP这类脂质由于其低ζ电位，形成的LNP尺寸较小，且倾向于在脾脏中积聚，这使得它们在靶向该器官的疫苗或治疗剂递送中可能特别有用。通过这些策略，LNP技术在mRNA递送领域展现出巨大的潜力和灵活性。 鉴于 SARS-CoV-2 大流行，LNP 的生产已得到简化，以满足全球对 mRNA-LNP 疫苗前所未有的需求。因此，微流体等技术已被引入“战场”，以可重复地提供具有统一质量的 mRNA-LNP 物质。图 4展示了最广泛使用的 mRNA-LNP 生产技术的示意图。 图 4. 脂质纳米颗粒的形成。如今，LNP 是利用微流体技术生产的，可产生均匀且表征良好的最终产品。封装材料（各种天然或改性脂质）与 mRNA 一起被送入设备中。由此产生的 mRNA-LNP 被进一步纯化，以确保适合人类使用。 结合聚合物和脂质纳米颗粒（LNP）的方法为mRNA的封装和递送提供了一种新策略，这种方法既保持了稳定性，又具备了显著的热力学特性，能够实现在目标细胞中高效地递送mRNA。这种混合载体的设计考虑了细胞膜主要由脂质构成的特性，使其能够与细胞膜融合，并将核酸直接释放到细胞质中。通过精心选择和调整聚合物和脂质的比例，可以优化这种混合载体的性能，以提高其在体内的递送效率和生物相容性。 基于 mRNA 的蛋白质替代疗法的关键质量属性 质量分析在药品生产过程中起着至关重要的作用，它确保了不同生产批次的药品在质量上的一致性、安全性和有效性。随着全球对封装mRNA产品需求的增加，例如mRNA-LNP COVID-19疫苗，建立这些产品的关键质量属性（CQA）变得尤为重要。这些CQA同样适用于用于蛋白质替代疗法的封装mRNA产品。 封装mRNA产品，无论是作为治疗性药物还是从药学特性的角度来看，都是一种创新而独特的医疗产品。其关键质量属性的开发和标准化是一个持续进行的过程，涉及到多个方面的考量，包括但不限于： 纯度和杂质水平：确保产品中活性成分的纯度，并控制污染物和杂质的水平。 生物活性：确认产品在生物学上的有效性，包括其诱导免疫反应的能力。 物理和化学稳定性：评估产品在不同条件下的稳定性，以保证其在储存和使用过程中保持有效。 封装效率和均匀性：对于封装mRNA产品，封装材料和封装过程的效率及均匀性是关键。 递送系统的特性：评估递送系统的有效性，包括其保护mRNA不被降解和促进目标细胞吸收的能力。 关键质量属性（CQA）是确保药品质量的关键因素，对于mRNA-LNP产品，如COVID-19疫苗，CQA可以分为以下三个主要领域： 与mRNA分子相关的CQA： 序列准确性：确保mRNA分子的序列与目标治疗或疫苗的序列完全一致。 纯度：mRNA分子应该足够纯净，不含有任何非目标序列或其他杂质。 完整性：mRNA分子应无断裂或降解，保持其完整的结构以确保功能。 加帽效率：mRNA分子的5'加帽对于其稳定性和翻译效率至关重要。 磷酸化程度：mRNA分子的3'端磷酸化程度影响其在细胞内的稳定性和翻译效率。 与递送系统相关的CQA： 粒径分布：mRNA-LNP的粒径大小和分布对其在体内的分布和细胞摄取有重要影响。 封装效率：确保mRNA分子被有效封装在LNP中，以保护其不被降解并确保递送至目标细胞。 物理稳定性：LNP应保持物理稳定性，避免聚集或沉淀，以确保其在储存和使用过程中的有效性。 化学稳定性：LNP中的化学成分应保持稳定，避免氧化或其他化学反应。 表征mRNA-LNP整体特征的CQA： 包封率：mRNA分子被LNP封装的效率，这直接影响产品的疗效。 批间一致性：不同生产批次之间的mRNA-LNP产品应具有高度的一致性。 免疫原性：评估mRNA-LNP可能引起的免疫反应，确保其安全性。 热稳定性：产品在不同温度条件下的稳定性，这对于疫苗的运输和储存尤为重要。 长期稳定性：产品在长期储存后仍能保持其疗效和安全性。 这些CQA的确立和监控对于确保mRNA-LNP产品的质量和疗效至关重要，它们是药品开发和生产过程中不可或缺的部分。随着mRNA技术的发展，这些CQA可能会随着新的科学发现和生产经验而不断更新和优化。 在mRNA疫苗或治疗产品制备过程中，确保体外转录（IVT）产物的纯度至关重要。在进行封装之前，必须确保mRNA产品中不含有双链RNA（dsRNA）、截短的mRNA分子以及由DNA残留物与RNA形成的DNA-RNA杂合体。这些污染物可能会触发细胞内的外源RNA感应机制，影响产品的疗效，甚至可能激活细胞凋亡途径，增加受试者体内发生细胞因子风暴的风险。 除了确保mRNA的5'端加帽效率和Poly-A尾长度符合标准外，还应通过适当的分析方法验证mRNA的序列准确性和数量。这些质量控制措施对于确保mRNA产品的安全性和有效性至关重要。 递送载体的关键质量属性（CQA）涵盖了对其制备过程中所使用的脂质的全面分析，包括以下几个方面： 脂质的鉴定和比例：确保所使用的脂质种类正确，并验证它们在递送载体中的比例是否符合预定配方。 纯度检查：检测脂质中是否存在杂质，这些杂质可能会影响递送载体的性能和安全性。 粒径分布：递送载体的粒径大小及其分布对药物的递送效率和体内分布有重要影响。 电荷特性：脂质纳米颗粒（LNP）的表面电荷（zeta电位）可以影响其与细胞的相互作用和稳定性。 等电点：了解LNP的等电点有助于预测其在不同pH值环境下的行为。 形态学：通过电子显微镜等技术检查LNP的形态，确保其结构完整且一致。 转染效率：评估LNP将mRNA递送到目标细胞并实现基因表达的能力，这是衡量其功能性的重要指标。 在mRNA-LNP产品的开发和生产过程中，对其物理特性进行全面的表征是至关重要的。以下是一些关键的质量属性（CQA）： 粒径：LNP的粒径大小对于其在体内的分布、细胞摄取以及最终的疗效都有显著影响。 zeta电位：这是一个衡量LNP表面电荷的参数，它影响LNP在体内的稳定性和与细胞膜的相互作用。 释放属性：这涉及到mRNA从LNP中释放出来的效率，这对于确保mRNA能够在细胞内正确表达目标蛋白至关重要。 封装效率：这是指在LNP制备过程中成功封装进LNP的mRNA比例，它直接关系到产品的疗效和剂量。 储存条件：由于mRNA-LNP COVID-19疫苗对温度和其他储存条件非常敏感，因此合理的CQA设计必须考虑产品的稳定性和在不同储存条件下的稳定性。 物理和化学稳定性：LNP在储存和使用过程中的稳定性，包括防止聚集和降解，是保证产品质量和疗效的关键。 免疫原性：评估LNP可能引起的免疫反应，以确保其安全性。 图 5（此处假设存在一个图表）可能会展示上述CQA的总结，包括它们的定义、接受标准以及如何通过分析方法进行测量。这些CQA的确立和监控对于确保mRNA-LNP产品的质量和一致性至关重要，它们是药品开发和生产过程中不可或缺的部分。随着mRNA技术的发展，这些CQA可能会随着新的科学发现和生产经验而不断更新和优化。 图 5. 用于人类蛋白质替代疗法的封装 mRNA 产品生产的关键质量属性。所检查的质量与编码目标蛋白的 mRNA 分子、递送系统以及成品（在本例中为 mRNA-LNP 制剂）有关。 临床前和临床阶段基于 mRNA 的蛋白质替代疗法 mRNA基的蛋白质替代疗法为多种疾病提供了治疗潜力，包括一些至今未有有效治疗方法的疾病。这种治疗策略已在临床前和临床研究中得到验证，并已产生一些积极的研究结果。接下来，我们将探讨一些mRNA替代治疗的例子，这些例子主要关注单基因疾病在小鼠模型中的应用。 苯丙酮尿症（PKU）是一种由苯丙氨酸羟化酶（PAH）基因突变引起的常染色体隐性遗传性疾病，这种突变导致苯丙氨酸代谢受阻。PAH基因编码的酶在肝脏中负责将苯丙氨酸转化为酪氨酸。若该酶功能缺失，将导致体内苯丙氨酸水平升高，可能在血液中和/或大脑中积累，进而引发不可逆的智力损害。PKU患者需要遵循严格的低苯丙氨酸饮食，并可能需要药物治疗以控制病情。 然而，严格的饮食控制对于患者来说是一个挑战，尤其是对于需要高蛋白饮食的个体。此外，现有药物可能存在给药困难和副作用的问题。因此，研究者正在探索替代疗法，如基于mRNA的蛋白质替代疗法，以期为PKU患者提供更有效、更安全的治疗选择，从而改善他们的生活质量。这种新型治疗方法通过提供功能性PAH酶的mRNA，有可能恢复酶活性，减少血液中苯丙氨酸的积累，为PKU治疗提供新的希望。 在针对苯丙酮尿症（PKU）的小鼠模型的研究中，我们探索了mRNA替代疗法的潜力。具体来说，我们制备了编码人类苯丙氨酸羟化酶（PAH）的全长mRNA，并将其封装在脂质纳米颗粒（LNP）中，以实现向小鼠模型的肝脏细胞递送。这些小鼠模型携带有PAH基因的错义突变，模拟了人类PKU患者的遗传缺陷。 通过这种治疗，我们成功地在小鼠的肝脏细胞中表达了高水平的人PAH蛋白，有效恢复了苯丙氨酸的正常代谢过程。令人鼓舞的是，无论是在单剂量还是多次剂量的治疗后，观察到血浆中苯丙氨酸水平显著降低，且未伴随任何不良的临床症状，这表明了治疗的安全性。 这些研究成果不仅证实了mRNA替代疗法在治疗PKU方面的有效性，也为未来开发针对PKU以及其他单基因遗传疾病的新型治疗方法提供了有力的证据。随着mRNA技术和递送系统的不断进步，基于mRNA的蛋白质替代疗法有望成为治疗这类疾病的有力工具。 在另一项研究中，科学家们探索了使用脂质纳米颗粒（LNP）封装的ARG1 mRNA治疗ARG1缺陷小鼠模型的潜力。ARG1 mRNA编码的精氨酸酶是尿素循环中的关键酶，负责将氨转化为尿素，从而帮助排泄。精氨酸酶缺乏症（AD）是一种由ARG1基因突变引起的常染色体隐性遗传病，患者会出现精神运动功能障碍、痉挛性四肢瘫痪、腱反射亢进、癫痫发作和生长迟缓等症状，严重时甚至可能导致肝脏疾病。 目前，AD患者的治疗手段包括限制蛋白质摄入、使用药物降低血液中精氨酸水平，以及在某些情况下进行肝移植。Truong等人的研究显示，通过重复给予AD小鼠LNP-ARG1 mRNA治疗，可以防止体重下降，改善疾病相关的生物标志物，且未观察到肝毒性。最重要的是，这种治疗策略能够显著延长小鼠的存活时间，其中每3天接受一次治疗的小鼠在研究期间存活了77天，而每周接受一次治疗的小鼠存活了62天。 这些研究成果为遗传性代谢疾病患者及其医疗团队带来了新的希望，预示着未来可能开发出更多分子层面的治疗方法。 法布里病是一种由于α-半乳糖苷酶A（α-Gal A）缺乏引起的溶酶体贮积症，这种酶的缺失会导致脂质分子在细胞内积累，进而引发心肌病变和肾功能障碍。最近的研究表明，通过使用封装在脂质纳米颗粒（LNP）中的α-半乳糖苷酶A mRNA，可以有效改善小鼠模型和非人灵长类动物的病理状况。 此外，临床前研究也在探索mRNA蛋白替代疗法在其他代谢性疾病治疗中的应用，这些疾病目前尚缺乏有效的治疗方法。例如，Crigler-Najjar综合征、肝肾酪氨酸血症和急性间歇性卟啉症等疾病，都可能通过mRNA治疗得到改善。这些研究通常涉及将编码关键酶或蛋白的mRNA递送到患者体内，以补偿缺陷基因的功能，从而缓解或治疗疾病。 这些进展表明，mRNA治疗技术在代谢性疾病的治疗中具有广泛的应用潜力，为患者提供了新的治疗选择和希望。随着研究的深入和技术的成熟，未来可能会有更多的mRNA治疗产品被开发出来，用于治疗各种遗传性和获得性疾病。 在一篇综述文章中，作者探讨了基于mRNA的转录疗法在治疗某些肝脏疾病方面的潜力。这些临床前研究集中在使用脂质纳米颗粒（LNP）封装的mRNA分子，这些分子能够在体内翻译成治疗所需的特定蛋白质。通过这种方式，研究人员观察到，接受mRNA治疗的小鼠模型的健康状况得到了显著改善。 尽管文章中提到了mRNA替代疗法在技术实现上存在的一些挑战，比如如何确保mRNA分子有效递送到靶组织而不引起非靶组织的非特异性表达，作者仍然对这种疗法的未来持乐观态度。他们认为，随着递送技术的进步和治疗方法的优化，基于mRNA的肝脏疾病治疗策略有望在未来进入临床试验阶段，并为患者带来新的治疗选择。 这种乐观的前景基于以下几个因素： 递送技术的进步：随着LNP和其他递送系统的发展，mRNA分子的靶向性和递送效率正在不断提高。 治疗设计的创新：研究人员正在设计更精确的mRNA分子，以确保它们能够特异性地在肝脏中表达，减少潜在的副作用。 临床前研究的积极结果：在小鼠模型中观察到的治疗效果为进一步的临床研究提供了科学依据。 治疗范围的扩大：mRNA疗法的潜力不仅限于肝脏疾病，它也为其他遗传性和获得性疾病的治疗提供了新的可能性。 尽管存在挑战，但mRNA替代疗法在治疗肝脏疾病及其他领域的潜力是巨大的，值得我们持续关注和期待。 在另一篇综述文章中，探讨了修饰RNA在心脏疾病治疗中的应用。研究者们在心脏疾病动物模型中，例如缺血性心力衰竭的小鼠、猪和猴子，通过RNA蛋白替代疗法的方式，评估了mRNA作为促进心脏血管化和再生的潜在治疗工具。研究表明，这种技术在提高治疗的安全性和可扩展性、开发经济高效的临床级材料、建立稳健的递送方法和降低成本等方面具有潜力，有望成为促进心脏再生和改善心脏功能的有效治疗手段。 此外，血液疾病也成为mRNA蛋白替代治疗的另一个目标领域。例如，血友病A和B，这两种疾病分别是由于凝血因子VIII和IX缺乏引起的出血性疾病。在小鼠模型中，通过体内测试编码这些因子变体的mRNA分子，已经显示出在稳定和持久表达相应缺失因子方面的高效能。 这些研究进展表明，mRNA治疗技术在心脏疾病和血液疾病的治疗中具有广泛的应用前景，为患者提供了新的治疗选择和希望。随着mRNA治疗技术的不断发展和优化，未来可能会有更多的疾病可以通过这种创新疗法得到有效治疗。 在囊性纤维化的治疗研究中，mRNA替代疗法也展现出了积极的临床前研究成果。囊性纤维化通常被视为一种主要影响肺部的疾病，它是由囊性纤维化跨膜电导调节器（CFTR）基因的常染色体隐性突变引起的。CFTR是一种蛋白质通道，负责调控水（H2O）和氯离子（CI-）进出肺部细胞，维持肺部粘液的适宜湿度。当CFTR因突变而功能失常时，这些离子无法正常流出细胞，导致肺部粘液积聚，进而引起严重的肺部功能障碍。 研究表明，通过将CFTR mRNA递送到CFTR功能缺失的小鼠模型中，可以改善肺功能指标，效果与美国食品药品监督管理局（FDA）批准的囊性纤维化治疗药物相似。然而，为了使这种疗法在临床上具有实际应用价值，需要确保mRNA递送后CFTR蛋白的长期表达。这是因为频繁地重新给药可能会增加高剂量递送载体的毒性风险。 因此，研究人员正在探索更有效的mRNA递送系统和策略，以实现CFTR蛋白的持续表达，并减少给药频率，从而提高患者的治疗依从性和生活质量。随着mRNA技术和递送系统的进步，未来可能会开发出更安全、更有效的囊性纤维化治疗方法。 就临床试验而言，为了证明 mRNA 蛋白替代疗法的潜力，已经启动了几项临床研究。值得注意的是，大多数临床试验候选 mRNA 在其制剂中使用脂质纳米颗粒，这表明合适的载体的重要性。 Translate Bio公司正在进行一项针对囊性纤维化的治疗临床试验。该公司开发了一种新型产品，该产品包含封装在脂质纳米颗粒（LNP）中的mRNA，用于编码CFTR蛋白。在这项研究中，12名囊性纤维化患者通过雾化吸入的方式接受了不同剂量的mRNA治疗或安慰剂。初步结果显示，治疗在低剂量和中等剂量下具有良好的耐受性，并且患者的肺功能指标有所改善。 据研究估计，恢复细胞溶胶中5%的野生型CFTR mRNA水平可能足以缓解囊性纤维化的症状，尽管可能需要更高的水平来预防后期的并发症。这项研究的结果为囊性纤维化患者提供了新的治疗希望，并展示了mRNA治疗技术在遗传性疾病治疗中的潜力。 Translate Bio公司也曾开展一项针对鸟氨酸转氨甲酰酶缺乏症（OTC）的临床试验，该试验采用脂质纳米颗粒（LNP）封装的OTC编码mRNA。然而，由于药物的安全性和药代动力学特性未达到预期，该试验被迫中止。投资者和研究人员将这一结果与所使用的LNP的性能不足联系起来，这进一步凸显了开发更高效、更安全的新一代LNP载体的必要性。 这一经历强调了在mRNA治疗领域，递送系统的优化是实现治疗效果的关键因素之一。随着新型LNP技术的发展，未来可能会有更安全、更有效的mRNA药物载体问世，以满足临床治疗的需求。 Moderna公司与阿斯利康（AstraZeneca）合作，针对II型糖尿病患者和心力衰竭患者的溃疡治疗，开展了两项使用裸露mRNA的临床试验。此外，Moderna还启动了两项临床试验，评估mRNA药物的安全性和药代动力学/药效学特征，这些药物旨在治疗两种常染色体隐性遗传疾病：丙酸血症和分离性甲基丙二酸血症。这些研究中使用的是封装在LNP中的mRNA，编码治疗性酶。目前，这些临床试验正在进行中，结果尚未公布。这些研究的进展展示了mRNA治疗技术在治疗多种疾病方面的潜力和应用前景。 在mRNA治疗领域，除了针对遗传性疾病的蛋白质替代疗法外，还有针对心血管疾病的mRNA疗法也显示出了潜力。其中一种是基于编码血管内皮生长因子A（VEGF-A）的mRNA药物，VEGF-A是一种能够促进血管生成的因子，有助于刺激心肌再生和血管新生。 在阿斯利康支持的EPICCURE 2a期临床试验中，这种mRNA疗法（AZD8601）正在接受血运重建手术的心脏病患者中进行测试，这些患者左心室功能有中度损伤。AZD8601在临床前的体内试验中表现出了积极的结果，它不仅能够增加动物模型的皮肤和心脏的血流，提高血管密度，还能改善实验性心肌梗死的猪的心脏功能。 这些研究结果为mRNA在心血管疾病治疗中的应用提供了初步证据，表明这种疗法可能有助于改善患者的心脏功能和血管健康。随着临床试验的进一步开展，mRNA疗法在心血管疾病治疗中的潜力将得到更全面的评估。 结论和未来应用 总体而言，最新的临床前研究和新兴的临床试验数据持续显示，mRNA治疗是一种充满潜力的方法，它有潜力补充甚至在某些情况下取代传统的治疗方法。mRNA治疗的优势在于其能够绕过与DNA或重组蛋白治疗相关的限制，提供瞬时性蛋白表达的好处，同时避免了基因组整合的风险。 尽管mRNA治疗技术具有这些优势，但在其快速发展的初期，也展现出了一些局限性，这些局限性在一定程度上限制了其在慢性疾病治疗中的应用。例如： 瞬时蛋白表达：mRNA提供的蛋白表达是暂时性的，可能需要频繁给药以维持治疗效果，这可能对慢性疾病管理构成挑战。 免疫原性：mRNA分子可能会引发免疫反应，这可能影响治疗效果和安全性。 靶向递送：尽管mRNA治疗提供了精准医疗的潜力，但目前仍缺乏专门针对特定组织的有效递送系统。 稳定性和制造：mRNA分子的稳定性和大规模生产也是实现其临床应用需要克服的挑战。 为了克服这些挑战，研究人员正在开发新的mRNA设计、递送系统和制造技术。随着这些技术的进步，mRNA治疗在治疗慢性疾病方面的可能性将进一步增加，为患者提供更多的治疗选择。 mRNA治疗技术在心血管疾病治疗中的应用已经显示出积极的迹象。例如，Moderna与阿斯利康合作开发的mRNA药物AZD8601，它编码血管内皮生长因子A（VEGF-A），用于治疗心力衰竭。在临床试验中，这种mRNA药物通过直接注射到心脏，显示出了改善心脏功能的潜力。 尽管mRNA治疗在蛋白质替代疗法中的应用可能面临一些挑战，如需要较高剂量和频繁给药，但这些挑战正在通过技术进步得到解决。例如，冻干技术的应用可以提高mRNA药物的稳定性，使其在室温下也能保持较长时间。 此外，为了提高mRNA药物的疗效和减少剂量需求，研究人员正在探索自扩增mRNA、环状RNA等新型mRNA形式。这些新型mRNA可以在细胞内自我复制或稳定存在，从而减少给药频率和剂量。 在蛋白质替代疗法中，mRNA治疗的另一个考虑因素是编码蛋白质的大小。例如，mRNA疫苗编码的全长刺突蛋白约为180 kDa，而治疗应用中使用的mRNA编码的蛋白质大小通常较小。这可能影响递送载体的设计和药物的递送效率。 mRNA治疗的长期应用还需要解决免疫原性和递送效率的问题。随着新型递送系统和mRNA修饰技术的发展，mRNA治疗有望在未来成为治疗多种疾病的有力工具。 在将这种生物分子应用于临床实践中仍存在一些挑战，但生物分子工程的最新进展已经克服了一些重要的障碍，为这项技术的更广泛应用奠定了基础。这些进步得益于我们对这些生物分子的更深入理解、通过结构改良增强其稳定性，以及开发了更安全有效的递送系统。 mRNA技术展现出巨大的潜力，它不仅有可能彻底改变现有的治疗和诊断方法，还可能通过其成本效益和广泛应用范围，重塑药品市场的格局。尽管如此，mRNA疗法的临床应用大多还在评估阶段，其在实际应用和大规模人群中的应用效果仍需进一步观察。不过，目前得到的初步结果是非常有希望的，预示着mRNA疗法在未来可能成为医疗领域的重要工具。 原文：T.Vavilis, E.Stamoula, A.Ainatzoglou, et al., mRNA in the Context of Protein Replacement Therapy. Pharmaceutics, 2023. 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。

-Recently Held Positive Pre-BLA Meeting with FDA with Aim to Accelerate Approval Pathway of Deramiocel for the Treatment of Duchenne Muscular Dystrophy- -Phase 3, HOPE-3 Trial of Deramiocel in DMD Fully Enrolled; On Track to Report Top-Line Data from Cohort A in Q4 2024- -Reported Positive 3-Year Skeletal and Cardiac Data from HOPE-2 Open-Label Extension Trial- -Conference Call and Webcast Today at 4:30 p.m. ET- SAN DIEGO, Aug. 07, 2024 (GLOBE NEWSWIRE) -- Capricor Therapeutics (NASDAQ: CAPR), a biotechnology company developing transformative cell and exosome-based therapeutics for the treatment of rare diseases, today announced its financial results for the second quarter ended June 30, 2024 and provided a corporate update. “We continue to make significant progress across our pipeline as we move closer to the filing of our Biologics License Application (BLA) of our lead asset, deramiocel (also referred to as CAP-1002), for the treatment of Duchenne muscular dystrophy (DMD),” said Linda Marbán, Ph.D., Capricor’s chief executive officer. “We recently conducted a positive pre-BLA meeting with the U.S. Food and Drug Administration (FDA) and we will provide further updates on our plans as they become available. Furthermore, our recently announced 3-year HOPE-2 open label extension (OLE) data gives us confidence in the totality of safety and efficacy data we have seen year over year and the potential benefit that deramiocel can provide to patients with DMD. Lastly, on the corporate front, we remain focused on securing a partnership in Europe in order to support our balance sheet as we move towards major inflection points over the next several quarters.” Dr. Marbán continued, “In addition, we continue to progress our proprietary StealthX™ platform technology as part of our long-term strategy to leverage exosomes for therapeutic development. We continue to explore partnership opportunities and other non-dilutive sources of funding to advance this program.” Second Quarter 2024 and Recent Operational Highlights Deramiocel DMD Program: Deramiocel is an investigational cell therapy in Phase 3 development for the treatment of DMD. Deramiocel aims to slow disease progression through immunomodulatory, anti-inflammatory, and anti-fibrotic actions, with the goal of potentially improving skeletal and cardiac muscle function in patients with DMD. Deramiocel for the treatment of DMD has received Orphan Drug Designation and Regenerative Medicine Advanced Therapy Designation (RMAT). In addition, if Capricor receives FDA marketing approval for deramiocel for the treatment of DMD, Capricor would be eligible to receive a Priority Review Voucher (PRV) based on our previous receipt of a rare pediatric disease designation. Announced the completion of a positive pre-BLA meeting with FDA that occurred in Q3 2024, where we discussed our rolling BLA submission schedule, potential label expansion, plans for commercial manufacturing as well as other topics. We plan to announce further details from this meeting once the final meeting minutes are made available. Announced positive results from our 36-month HOPE-2 OLE study at the Parent Project Muscular Dystrophy (PPMD) Annual Conference. The 3-year results showed a statistically significant benefit (+3.7 points, p<0.001) in the PUL v2.0 total score when compared to an external comparator dataset of similar DMD patients. Data also showed improvements in multiple cardiac measures, including left ventricular ejection fraction, as well as indexed volumes (left ventricular end systolic volume and left ventricular end diastolic volume). Announced enrollment has been completed for our HOPE-3 Phase 3 (Cohorts A and B) clinical trial which enrolled 105 subjects randomized to either deramiocel or placebo in a 1:1 ratio. Next steps for Cohort A: plan to readout top-line data in the fourth quarter of 2024.Next steps for Cohort B are under consideration. Announced positive results from a Type-B clinical FDA meeting held in Q2 2024 where FDA granted our request for a pre-BLA meeting.Announced positive results from a Type-B CMC FDA meeting held in Q1 2024. In the meeting, the FDA affirmed alignment on the following topics: Comparability between drug product manufactured at our two different facilities (Los Angeles and San Diego) has been demonstrated using the provided analytical comparability data.This allowed for the use of deramiocel drug product manufactured at our San Diego manufacturing facility upon potential product approval (subject to approval of the facility). Presented at the PPMD Cardiac Workshop III. Capricor was featured in a panel on industry perspectives on cardiac monitoring in DMD clinical trials.Presented at the Cure Duchenne 2024 Futures National Conference. Capricor presented the 24-month safety and efficacy results from the HOPE-2 OLE study. StealthX™Exosome Platform: Exosomes are membrane-bound extracellular vesicles which are secreted by most cells and contain characteristic lipids, proteins and nucleic acids. They act as messengers to regulate the functions of neighboring or distant cells and have been shown to regulate functions such as cell survival, proliferation, inflammation and tissue regeneration. We are developing our exosome technology using our proprietary StealthX™ platform focused on the areas of vaccinology, targeted delivery of oligonucleotides, proteins and small molecule therapeutics to potentially treat and prevent a diverse array of diseases. Our proprietary StealthX™ exosome-based multivalent vaccine (StealthX™ vaccine) for the prevention of SARS-CoV-2 was selected to be part of Project NextGen, an initiative by the U.S. Department of Health and Human Services to advance a pipeline of new, innovative vaccines. Under the terms of the collaboration, Capricor will supply the investigational product and NIAID's Division of Microbiology and Infectious Diseases will conduct the trial.Currently, our vaccine candidate is in the manufacturing phase with plans to deliver it to NIAID by the end of 2024. Presented data at the American Society of Gene and Cell Therapy 27th Annual Meeting. Highlights from the presentation included preclinical data showing a potential exosome-based approach for the treatment of arginase-1 deficiency (ARG1-D), a rare genetic metabolic disease characterized by complete or partial lack of the enzyme arginase in the liver and red blood cells.Presented data at the International Society of Extracellular Vesicles Annual Meeting 2024. Highlights from the abstract included preclinical data showing targeted cargo delivery to the lower limbs of mice by exosomes carrying a muscle targeting moiety by intravenous injection.Presented data at the International Society for Cell & Gene Therapy 2024 Meeting. Highlights from the abstract included preclinical data showing targeted delivery of ASOs using exosomes leads to exon skipping. Corporate Updates Announced that the Company has been added to the Russell 2000 and 3000 Indexes.Presented at the Cantor Fitzgerald’s Muscular Dystrophy Symposium. Anticipated Upcoming Milestones Plan to announce the outcome from our pre-BLA meeting in the third quarter of 2024.Plan to present additional 3-year data from our HOPE-2 OLE study at a medical meeting in the fourth quarter of 2024.Plan to report topline data from HOPE-3 (Cohort A) in the fourth quarter of 2024.Continue to explore opportunities for additional partnerships outside of the U.S. and Japan to support the potential approval and commercialization of deramiocel in DMD.Plan to provide updates on our NIAID collaboration for our StealthX™ vaccine as they become available.Continue to explore opportunities for partnerships and non-dilutive sources of funding to support advancement of our StealthX™ exosome platform technology. Second Quarter 2024 Financial Results Cash position: Cash, cash equivalents and marketable securities totaled approximately $29.5 million as of June 30, 2024 compared to approximately $39.5 million as of December 31, 2023. Additionally, in the second quarter of 2024, Capricor raised approximately $2.1 million in net proceeds through issuances of common stock at an average price of approximately $6.46 per share under its at-the-market offering program. Revenues: Revenues for the second quarter of 2024 were approximately $4.0 million compared to approximately $3.9 million for the second quarter of 2023. Additionally, revenues for the first half of 2024 were approximately $8.9 million compared to approximately $6.9 million for the first half of 2023. Capricor’s primary source of revenue was from the ratable recognition of the $40.0 million (upfront and milestone payments) in accordance with its U.S. Commercialization and Distribution Agreement with Nippon Shinyaku. Expenses: Total operating expenses for the second quarter of 2024 were approximately $15.6 million compared to approximately $11.7 million for the second quarter of 2023. Total operating expenses for the first half of 2024 were approximately $30.7 million compared to approximately $22.8 million for the first half of 2023. Net loss: The Company reported a net loss of approximately $11.0 million, or $0.35 per share, for the second quarter of 2024, compared to a net loss of approximately $7.4 million, or $0.29 per share, for the second quarter of 2023. The Company reported a net loss of approximately $20.8 million, or $0.66 per share, for the first half of 2024, compared to a net loss of approximately $15.1 million, or $0.60 per share, for the first half of 2023. Financial Outlook: We believe that based on the current operating plan and financial resources, Capricor’s available cash, cash equivalents and marketable securities will be sufficient to cover anticipated expenses and capital requirements into the first quarter of 2025. This expectation excludes any additional potential milestone payments under our Commercialization and Distribution Agreements with Nippon Shinyaku, as well as any strategic use of capital not currently in the Company’s base-case planning assumptions. Upcoming Events The Company plans to participate in the following upcoming events: H.C. Wainwright 26th Annual Global Investment Conference, September 9-11, 2024, New York, NY6th Annual Exosome-Based Therapeutic Development Summit, September 17-19, 2024, Boston, MA2024 Cantor Fitzgerald Global Healthcare Conference, September 17-19, 2024, New York, NY2024 Cell & Gene Meeting on the Mesa, October 7-9, 2024, Phoenix, AZ29th Annual Congress of the World Muscle Society, October 8-12, 2024, Prague, Czech Republic2024 Maxim Healthcare Summit, October 15-17, 2024, Virtual Conference Call and Webcast To participate in the conference call, please dial 1-800-717-1738 (Domestic/Toll-Free) or 1-646-307-1865 (International) and reference the conference ID: 30827. Participants can use guest dial-in numbers above and be answered by an operator or click the Call me™ link for instant telephone access. To participate via a webcast, please click here. A replay of the webcast will be available following the conclusion of the live broadcast and will be accessible on the Company’s website. About Capricor Therapeutics Capricor Therapeutics, Inc. (NASDAQ: CAPR) is a biotechnology company dedicated to advancing transformative cell and exosome-based therapeutics to redefine the treatment landscape for rare diseases. At the forefront of our innovation is our lead product candidate, deramiocel (CAP-1002), an allogeneic cardiac-derived cell therapy. Extensive preclinical and clinical studies have shown deramiocel to demonstrate immunomodulatory, antifibrotic, and regenerative actions specifically tailored for dystrophinopathies and heart disease. Deramiocel is currently advancing through Phase 3 clinical development for the treatment of Duchenne muscular dystrophy (DMD). Capricor is also harnessing the power of our exosome technology, using our proprietary StealthX™ platform in preclinical development focused on the areas of vaccinology, targeted delivery of oligonucleotides, proteins and small molecule therapeutics to potentially treat and prevent a diverse array of diseases. At Capricor, we stand committed to pushing the boundaries of possibility and forging a path toward transformative treatments for those in need. For more information, visit capricor.com, and follow Capricor on Facebook, Instagram and Twitter. Cautionary Note Regarding Forward-Looking Statements Statements in this press release regarding the efficacy, safety, and intended utilization of Capricor’s product candidates; the initiation, conduct, size, timing and results of discovery efforts and clinical trials; the pace of enrollment of clinical trials; plans regarding regulatory filings, future research and clinical trials; regulatory developments involving products, including the ability to obtain regulatory approvals or otherwise bring products to market; manufacturing capabilities; dates for regulatory meetings; statements about our financial outlook; the ability to achieve product milestones and to receive milestone payments from commercial partners; plans regarding current and future collaborative activities and the ownership of commercial rights; scope, duration, validity and enforceability of intellectual property rights; future revenue streams and projections; expectations with respect to the expected use of proceeds from the recently completed offerings and the anticipated effects of the offerings; and any other statements about Capricor’s management team’s future expectations, beliefs, goals, plans or prospects constitute forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Any statements that are not statements of historical fact (including statements containing the words “believes,” “plans,” “could,” “anticipates,” “expects,” “estimates,” “should,” “target,” “will,” “would” and similar expressions) should also be considered to be forward-looking statements. There are a number of important factors that could cause actual results or events to differ materially from those indicated by such forward-looking statements. More information about these and other risks that may impact Capricor’s business is set forth in Capricor’s Annual Report on Form 10-K for the year ended December 31, 2023, as filed with the Securities and Exchange Commission on March 11, 2024, and in our Quarterly Report on Form 10-Q for the quarter ended March 31, 2024, as filed with the Securities and Exchange Commission on May 14, 2024. All forward-looking statements in this press release are based on information available to Capricor as of the date hereof, and Capricor assumes no obligation to update these forward-looking statements. Capricor has entered into an agreement for the exclusive commercialization and distribution of deramiocel (CAP-1002) for DMD in the United States and Japan with Nippon Shinyaku Co., Ltd. (U.S. subsidiary: NS Pharma, Inc.), subject to regulatory approval. Deramiocel is an Investigational New Drug and is not approved for any indications. None of Capricor’s exosome-based candidates have been approved for clinical investigation. For more information, please contact: Capricor Company Contact:AJ Bergmann, Chief Financial Officerabergmann@capricor.com 858.727.1755 CAPRICOR THEAPEUTICS, INC. CONSOLIDATED STATEMENTS OF OPERATIONS AND COMPREHENSIVE LOSS (UNAUDITED) Three months ended June 30, Six months ended June 30, 2024 2023 2024 2023 REVENUE Revenue$3,971,438 $3,917,467 $8,878,315 $6,904,163 TOTAL REVENUE 3,971,438 3,917,467 8,878,315 6,904,163 OPERATING EXPENSES Research and development 12,504,769 8,817,389 23,605,782 16,478,908 General and administrative 3,057,888 2,847,337 7,129,654 6,357,222 TOTAL OPERATING EXPENSES 15,562,657 11,664,726 30,735,436 22,836,130 LOSS FROM OPERATIONS (11,591,219) (7,747,259) (21,857,121) (15,931,967) OTHER INCOME (EXPENSE) Investment income 591,437 380,680 1,063,266 797,122 TOTAL OTHER INCOME (EXPENSE) 591,437 380,680 1,063,266 797,122 NET LOSS (10,999,782) (7,366,579) (20,793,855) (15,134,845) OTHER COMPREHENSIVE INCOME (LOSS) Net unrealized gain (loss) on marketable securities (152,714) 84,707 (80,826) 74,449 COMPREHENSIVE LOSS$(11,152,496) $(7,281,872) $(20,874,681) $(15,060,396) Net loss per share, basic and diluted$(0.35) $(0.29) $(0.66) $(0.60)Weighted average number of shares, basic and diluted 31,841,964 25,335,342 31,598,296 25,291,591 CAPRICOR THEAPEUTICS, INC. SUMMARY BALANCE SHEETS June 30, 2024(unaudited) December 31, 2023Cash, cash equivalents and marketable securities $29,462,030 $39,487,703 Total assets $38,277,453 $58,734,327 Total liabilities $26,775,147 $36,132,860 Total stockholders' equity - 31,983,927 and 31,148,320 common shares issued and outstanding at June 30, 2024 and December 31, 2023, respectively 11,502,306 22,601,467 Total liabilities and stockholders' equity $38,277,453 $58,734,327

–Phase 3, HOPE-3 Trial (Cohort A) of CAP-1002 in Duchenne Muscular Dystrophy Fully Enrolled; On Track to Report Top-Line Data in Q4 2024– –Positive Type-B CMC FDA Meeting held in Q1; Company Aligned with FDA on Demonstration of Non-Clinical Comparability, A Major Milestone on Path to CMC Clearance for BLA– –Received First Milestone Payment of $10 Million under U.S. Distribution and Commercialization Agreement with Nippon Shinyaku in Q1– –Recent FDA Feedback Supports Requests for a Pre-BLA Meeting and Subsequent Rolling BLA Submission at Upcoming Type-B Clinical FDA Meeting in May 2024– –Plan to Report 3-Year Data from HOPE-2 Open-Label Extension (OLE) Trial in Q2 2024– –Conference Call and Webcast Today at 4:30 p.m. ET– SAN DIEGO, May 13, 2024 (GLOBE NEWSWIRE) -- Capricor Therapeutics (NASDAQ: CAPR), a biotechnology company developing transformative cell and exosome-based therapeutics for the treatment of rare diseases, today announced its financial results for the first quarter ended March 31, 2024 and provided a corporate update. “Capricor continues to make tremendous progress across our pipeline marked by significant advancements in our CAP-1002 cell therapy program for the treatment of Duchenne muscular dystrophy (DMD),” said Linda Marbán, Ph.D., Capricor’s chief executive officer. “With enrollment completed in Cohort A of our Phase 3 pivotal trial, we further aligned with the U.S. Food and Drug Administration (FDA) on key CMC (chemistry manufacturing and controls) deliverables necessary for the filing of a Biologics License Application (BLA) including the establishment of non-clinical comparability, a major milestone on the path to clearance for a BLA submission. We are appreciative of the FDA’s continued guidance under our RMAT designation and we will provide further updates on our plans as they become available. Furthermore, we continue to work diligently with our partner, Nippon Shinyaku (U.S. subsidiary: NS Pharma, Inc.) as we prepare for the potential launch of CAP-1002. Looking ahead, later this quarter, we plan to meet with FDA to continue to discuss options for potential expedited approval pathways as well as announce the 3-year HOPE-2 OLE results.” Dr. Marbán, continued, “We continue our efforts to advance our proprietary StealthX™ exosome platform to leverage exosomes for our vaccine program as well as for therapeutic development. This quarter, we announced a collaboration for our exosome-based multivalent vaccine for the prevention of SARS-CoV-2 with the National Institute of Allergy and Infectious Diseases (NIAID) where they will conduct and fully fund a Phase 1 clinical trial, subject to regulatory approval. We continue to remain focused on advancing this program through partnerships and other non-dilutive sources of funding.” First Quarter 2024 and Recent Operational Highlights CAP-1002 Duchenne Muscular Dystrophy Program: CAP-1002 is an investigational cell therapy in Phase 3 development for the treatment of DMD. CAP-1002 aims to slow disease progression through immunomodulatory, anti-inflammatory, and anti-fibrotic actions, with the goal of potentially improving skeletal and cardiac muscle function in patients with DMD. HOPE-3, our Phase 3 study, is a multi-center, randomized, double-blind, placebo-controlled clinical trial comprised of two cohorts evaluating the safety and efficacy of CAP-1002 in participants with DMD and impaired skeletal muscle function. The trial is being conducted in the United States. Approximately 102 eligible study subjects will participate in this dual-cohort study (Cohort A and B). CAP-1002 for the treatment of DMD has received Orphan Drug Designation and Regenerative Medicine Advanced Therapy Designation (RMAT). In addition, if Capricor receives FDA marketing approval for CAP-1002 for the treatment of DMD, Capricor would be eligible to receive a Priority Review Voucher (PRV) based on our previous receipt of a rare pediatric disease designation. Enrollment has been completed for our HOPE-3 Phase 3 (Cohort A) clinical trial which enrolled 61 subjects randomized to either CAP-1002 or placebo in a 1:1 ratio. Reported a positive outcome from the interim futility analysis for Cohort A which triggered the first milestone payment of $10.0 million received in the first quarter of 2024 under our U.S. Distribution and Commercialization Agreement with Nippon Shinyaku. Announced a positive outcome from a Type-B CMC FDA meeting held in the first quarter of 2024. In the meeting, the FDA affirmed alignment on the following topics: Comparability between drug product manufactured at our two different facilities (Los Angeles and San Diego) has been demonstrated using the provided analytical comparability data. This will now allow for the use of CAP-1002 drug product manufactured at our San Diego manufacturing facility upon potential product approval (subject to approval of the facility). Granted a Type-B clinical FDA meeting scheduled to be held in May 2024 to continue discussing our pathway to BLA. The FDA advised us to include discussion and a request for a pre-BLA meeting and rolling BLA schedule at this meeting. We also plan to share with FDA our HOPE-2 OLE 3-year safety and efficacy data. Announced further specifics under our U.S. Distribution and Commercialization Agreement with Nippon Shinyaku. Capricor will receive a meaningful mid-range double-digit share of product revenue. For clarity, mid-range falls in between 30-50%, which sum will be offset by the amounts paid to us as the transfer price for the purchase of the product. As previously disclosed, there is an additional $90.0 million in potential milestone payments up to the time of approval which are triggered upon certain regulatory-based achievements. Following potential approval, there is an additional $605.0 million in potential milestones payments which may be payable to Capricor based on various sales-based targets being met. Announced we are on track to enroll 44 U.S. based patients by the end of second quarter in HOPE-3 (Cohort B) clinical trial, however at this time, we are evaluating various options, with one of such options being the opportunity for an expansion of Cohort B to include European patients. Announced the scale-up of manufacturing capacity of CAP-1002 in our new San Diego facility, intended for commercial use, subject to regulatory approval. This facility was designed to be a versatile and cost-effective way to bring CAP-1002 to market efficiently. Currently, our San Diego facility is fully operational, staffed and producing doses for clinical use. Along with BLA readiness activities, we are also actively preparing for commercial runs. Presented at H.C. Wainwright 2nd Annual Cell Therapy Conference. Presented at the Cantor Fitzgerald’s Muscular Dystrophy Symposium. Presented at the 2024 Muscular Dystrophy Association (MDA) Clinical & Scientific Conference sharing the previously presented positive 24-month results from our HOPE-2 OLE study. Key results included skeletal muscle function as measured by the Performance of the Upper Limb (PUL v2.0) showed a mean PUL v2.0 decline after 24-months of treatment with CAP-1002 was 2.8 points versus a 7.7 point decline on average observed over 24-months in the placebo patient group. Additionally, CAP-1002 revealed clinically meaningful improvements in ameliorating cardiac function Presented at the Parent Project Muscular Dystrophy (PPMD) Cardiac Workshop III. Capricor was featured in a panel on industry perspectives on cardiac monitoring in DMD clinical trials. StealthX™ Exosome Platform: Exosomes are membrane-bound extracellular vesicles which are secreted by most cells and contain characteristic lipids, proteins and nucleic acids such as mRNA and microRNAs. They act as messengers to regulate the functions of neighboring or distant cells and have been shown to regulate functions such as cell survival, proliferation, inflammation and tissue regeneration. We are developing our exosome technology, using our proprietary StealthX™ platform focused on the areas of vaccinology, targeted delivery of oligonucleotides, proteins and small molecule therapeutics to potentially treat and prevent a diverse array of diseases. Announced that our proprietary StealthX™ exosome-based multivalent vaccine (StealthX™ vaccine) for the prevention of SARS-CoV-2 was selected to be part of Project NextGen, an initiative by the U.S. Department of Health and Human Services to advance a pipeline of new, innovative vaccines. As part of Project NextGen, NIAID, part of the National Institutes of Health, will conduct and fund a Phase 1 clinical trial with our StealthX™ vaccine. Under the terms of the collaboration, Capricor will supply the investigational product and NIAID's Division of Microbiology and Infectious Diseases (DMID) will conduct the trial. If NIAID finds that our StealthX™ vaccine meets its criteria for safety and efficacy, they may consider our program for a funded Phase 2. Currently, in collaboration with an undisclosed pharmaceutical company, we are also investigating the therapeutic application of our StealthX™ exosome platform. Presented data in an oral presentation at the American Society of Gene and Cell Therapy (ASGCT) 27th Annual Meeting. The findings highlight a potential exosome-based approach for the treatment of arginase-1 deficiency (ARG1-D), a rare genetic metabolic disease characterized by complete or partial lack of the enzyme arginase in the liver and red blood cells. The data from this preclinical study further characterize our StealthX™ exosome platform. Presented data at the International Society of Extracellular Vesicles (ISEV) Annual Meeting 2024. Highlights from the abstract included data showing targeted cargo delivery to mouse lower limbs by exosomes carrying a muscle targeting moiety by intravenous injection. Anticipated Upcoming Milestones CAP-1002 DMD Program Plan to have a Type-B clinical FDA meeting in May 2024 to discuss requests for a pre-BLA meeting and rolling BLA schedule. Additionally, Capricor plans to share with FDA its HOPE-2 OLE 3-year safety and efficacy data at this meeting. Plan to report 3-year HOPE-2 OLE data in the second quarter of 2024. Plan to announce further updates with respect to next steps for HOPE-3 (Cohort B), when available. Plan to report topline data from HOPE-3 (Cohort A) in the fourth quarter of 2024. Continue to explore opportunities for additional partnerships outside of the U.S. and Japan to support the potential commercialization of CAP-1002 in DMD. StealthX™Exosome Platform Plan to provide updates on our NIAID collaboration for our StealthX™ vaccine as they become available. NIAID plans to initiate the Phase 1 clinical trial in late 2024, subject to regulatory approval. Continue to explore opportunities for partnerships and non-dilutive sources of funding to support advancement of our StealthX™ exosome platform technology. First Quarter 2024 Financial Results Cash position: Cash, cash equivalents and marketable securities totaled approximately $39.9 million as of March 31, 2024 compared to approximately $39.5 million as of December 31, 2023. In the first quarter of 2024, the Company received $10.0 million from the first milestone payment under our U.S. Distribution and Commercialization Agreement with Nippon Shinyaku and raised approximately $2.3 million in net proceeds through issuances of common stock at an average price of approximately $5.33 per share under its at-the-market offering program. Revenues: Revenues for the first quarter of 2024 were approximately $4.9 million compared with approximately $3.0 million for the first quarter of 2023. Capricor’s primary source of revenue was from the ratable recognition of the $40.0 million (upfront and milestone payments) in accordance with its U.S. Commercialization and Distribution Agreement with Nippon Shinyaku Expenses: Operating expenses for the first quarter of 2024 were approximately $15.2 million compared with approximately $11.2 million for the first quarter of 2023. Net loss: The Company reported a net loss of approximately $9.8 million, or $0.31 per share, for the first quarter of 2024, compared to a net loss of approximately $7.8 million, or $0.31 per share, for the first quarter of 2023. Financial Outlook: We believe that based on the current operating plan and financial resources, Capricor’s available cash, cash equivalents and marketable securities will be sufficient to cover anticipated expenses and capital requirements into the first quarter of 2025. This expectation excludes any additional potential milestone payments under its Commercialization and Distribution Agreements with Nippon Shinyaku, as well as any strategic use of capital not currently in the Company’s base-case planning assumptions. Upcoming Events The Company plans to participate in the following upcoming events: CureDuchenne 2024 FUTURES National Conference, May 23-26, 2024, Orlando International Society for Cell & Gene Therapy (ISCT) 2024 Meeting, May 29-June 1, 2024, Vancouver BIO International Convention 2024, June 3-6, 2024, San Diego Parent Project Muscular Dystrophy (PPMD) 30th Annual Conference, June 27-29, 2024, Orlando Conference Call and Webcast To participate in the conference call, please dial 1-888-886-7786 (Domestic/Toll-Free) or 1-416-764-8658 (International) and reference the conference ID: 01529679. Participants can use guest dial-in numbers above and be answered by an operator or click the Call me™ link for instant telephone access. To participate via a webcast, please click here. A replay of the webcast will be available following the conclusion of the live broadcast and will be accessible on the Company’s website. About Capricor Therapeutics Capricor Therapeutics, Inc. (NASDAQ: CAPR) is a biotechnology company dedicated to advancing transformative cell and exosome-based therapeutics to redefine the treatment landscape for rare diseases. At the forefront of our innovation is our lead product candidate, CAP-1002 — an allogeneic cardiac-derived cell therapy. Extensive preclinical and clinical studies have shown CAP-1002 to demonstrate immunomodulatory, antifibrotic, and regenerative actions specifically tailored for dystrophinopathies and heart disease. CAP-1002 is currently advancing through Phase 3 clinical development for the treatment of Duchenne muscular dystrophy (DMD). Capricor is also harnessing the power of our exosome technology, using our proprietary StealthX™ platform in preclinical development focused on the areas of vaccinology, targeted delivery of oligonucleotides, proteins and small molecule therapeutics to potentially treat and prevent a diverse array of diseases. At Capricor, we stand committed to pushing the boundaries of possibility and forging a path toward transformative treatments for those in need. For more information, visit capricor.com, and follow Capricor on Facebook, Instagram and Twitter. Cautionary Note Regarding Forward-Looking Statements Statements in this press release regarding the efficacy, safety, and intended utilization of Capricor’s product candidates; the initiation, conduct, size, timing and results of discovery efforts and clinical trials; the pace of enrollment of clinical trials; plans regarding regulatory filings, future research and clinical trials; regulatory developments involving products, including the ability to obtain regulatory approvals or otherwise bring products to market; manufacturing capabilities; dates for regulatory meetings; statements about our financial outlook; the ability to achieve product milestones and to receive milestone payments from commercial partners; plans regarding current and future collaborative activities and the ownership of commercial rights; scope, duration, validity and enforceability of intellectual property rights; future revenue streams and projections; expectations with respect to the expected use of proceeds from the recently completed offerings and the anticipated effects of the offerings; and any other statements about Capricor’s management team’s future expectations, beliefs, goals, plans or prospects constitute forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Any statements that are not statements of historical fact (including statements containing the words “believes,” “plans,” “could,” “anticipates,” “expects,” “estimates,” “should,” “target,” “will,” “would” and similar expressions) should also be considered to be forward-looking statements. There are a number of important factors that could cause actual results or events to differ materially from those indicated by such forward-looking statements. More information about these and other risks that may impact Capricor’s business is set forth in Capricor’s Annual Report on Form 10-K for the year ended December 31, 2023, as filed with the Securities and Exchange Commission on March 11, 2024. All forward-looking statements in this press release are based on information available to Capricor as of the date hereof, and Capricor assumes no obligation to update these forward-looking statements. Capricor has entered into a partnership for the exclusive commercialization and distribution of CAP-1002 for DMD in the United States and Japan with Nippon Shinyaku Co., Ltd. (U.S. subsidiary: NS Pharma, Inc.), subject to regulatory approval. CAP-1002 is an Investigational New Drug and is not approved for any indications. None of Capricor’s exosome-based candidates have been approved for clinical investigation. For more information, please contact: Capricor Company Contact: AJ Bergmann, Chief Financial Officer abergmann@capricor.com 858.727.1755 CAPRICOR THERAPEUTICS, INC. CONSOLIDATED STATEMENTS OF OPERATIONS AND COMPREHENSIVE LOSS (UNAUDITED) Three months ended March 31, 2024 2023 REVENUE Revenue $ 4,906,877 $ 2,986,696 TOTAL REVENUE 4,906,877 2,986,696 OPERATING EXPENSES Research and development 11,101,013 7,661,519 General and administrative 4,071,766 3,509,885 TOTAL OPERATING EXPENSES 15,172,779 11,171,404 LOSS FROM OPERATIONS (10,265,902 ) (8,184,708 ) OTHER INCOME (EXPENSE) Investment income 471,829 416,442 TOTAL OTHER INCOME (EXPENSE) 471,829 416,442 NET LOSS (9,794,073 ) (7,768,266 ) OTHER COMPREHENSIVE INCOME (LOSS) Net unrealized gain (loss) on marketable securities 71,888 (10,258 ) COMPREHENSIVE LOSS $ (9,722,185 ) $ (7,778,524 ) Net loss per share, basic and diluted $ (0.31 ) $ (0.31 ) Weighted average number of shares, basic and diluted 31,354,629 25,247,354 CAPRICOR THERAPEUTICS, INC. SUMMARY BALANCE SHEETS March 31, 2024 (unaudited) December 31, 2023 Cash, cash equivalents and marketable securities $ 39,917,078 $ 39,487,703 Total assets $ 49,444,442 $ 58,734,327 Total liabilities $ 31,009,504 $ 36,132,860 Total stockholders' equity – 31,600,183 and 31,148,320 common shares issued and outstanding at March 31, 2024 and December 31, 2023, respectively 18,434,938 22,601,467 Total liabilities and stockholders' equity $ 49,444,442 $ 58,734,327