CN118389605A - Compounds and methods for enhancing transfection of lipid nanoparticle cells with polypeptides - Google Patents
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Abstract
本文公开了一种增强LNPs细胞转染的方法以及这种LNP的成分和构成，包括：提供治疗剂；提供递送载体，该递送载体为LNPs，包括源自Cofilin的肽，并将治疗剂运送到目标细胞；以及在目标细胞中释放治疗剂。还公开了一种多肽，其序列与SEQ ID NO:1至少有81.25％的同一性。且相对于SEQ ID NO:1至少包含一个替代氨基酸。
The present invention discloses a method for enhancing LNPs cell transfection and the composition and structure of such LNPs, including: providing a therapeutic agent; providing a delivery vector, which is LNPs, including a peptide derived from Cofilin, and transporting the therapeutic agent to a target cell; and releasing the therapeutic agent in the target cell. Also disclosed is a polypeptide, whose sequence is at least 81.25% identical to SEQ ID NO:1. And it contains at least one substituted amino acid relative to SEQ ID NO:1.
Description
技术领域Technical Field
本发明涉及利用多肽增强脂质纳米粒子细胞转染的组合物和方法。The present invention relates to compositions and methods for enhancing lipid nanoparticle cell transfection using polypeptides.
背景技术Background technique
RNA治疗药物，包括信使RNA(mRNA)、小干扰RNA(siRNA)和反义寡核苷酸(ASO)，在治疗各种人类疾病方面表现出巨大的潜力。随着15种获得FDA批准的RNA药物上市和众多正在进行的临床试验，RNA药物已经成为一种极具价值的治疗方法(Hammond et al.,2021；Corey et al.,2022)。值得注意的是，在抗击SARS-CoV-2大流行的斗争中，由辉瑞-拜恩泰克和莫德纳开发的mRNA疫苗取得了巨大的成功(Baden et al.,2021)。在RNA药物中，mRNA因其在药物开发中的卓越特性和相对其他技术路径较短暂的研发产业化周期而脱颖而出。首先，mRNA是一种可以根据基因组序列设计并利用体外转录技术迅速合成的线性RNA。其次，mRNA使治疗蛋白质的短暂表达成为可能，而无需担心基因组整合的问题。此外，与需要转运到细胞核的DNA载体不同，mRNA可以在转染到细胞质后立即启动蛋白质表达。此外，一旦建立了成熟的mRNA药物生产线，通过简单修改序列，后续的mRNA药物可以通过一个明确定义的工作流程迅速开发。RNA therapeutics, including messenger RNA (mRNA), small interfering RNA (siRNA), and antisense oligonucleotides (ASOs), have shown great potential in treating various human diseases. With 15 FDA-approved RNA drugs on the market and numerous ongoing clinical trials, RNA drugs have become a valuable therapeutic approach (Hammond et al., 2021; Corey et al., 2022). Notably, in the fight against the SARS-CoV-2 pandemic, the mRNA vaccines developed by Pfizer-BioNTech and Moderna have achieved great success (Baden et al., 2021). Among RNA drugs, mRNA stands out for its superior properties in drug development and its relatively short R&D and industrialization cycle compared to other technical paths. First, mRNA is a linear RNA that can be designed based on genomic sequences and rapidly synthesized using in vitro transcription technology. Second, mRNA enables transient expression of therapeutic proteins without worrying about genomic integration. In addition, unlike DNA vectors that need to be transported to the nucleus, mRNA can initiate protein expression immediately after transfection into the cytoplasm. Furthermore, once a mature mRNA drug production line is established, subsequent mRNA drugs can be rapidly developed through a well-defined workflow by simply modifying the sequence.
与小分子治疗药物能够穿越细胞膜的脂质双层不同,RNA药物，尤其是mRNA，由于其分子量较大,带电性和亲水性而面临挑战。因此，它们不能通过被动扩散穿越过细胞膜，而是依赖内吞作为细胞摄取的手段。纯mRNA很少以其“裸体”形式使用；相反，其采用传递系统来保护并转运到目标细胞。目前广泛采用的传递系统，包括选择用于目前流行的SARS-CoV-2疫苗的那些递送系统，是mRNA-脂质纳米颗粒(mRNA-LNPs)。这些LNPs包括四种脂质1)可电离的阳离子脂质(Ionizable lipid)，(2)1，2-二硬脂酰-sn-甘油基-3-磷酸胆碱(DSPC)，(3)胆固醇(Cholesterol)和(4)聚乙二醇-脂质缀合物(PEG-DMG)(Kulkarnietal.,2019a)。这些组分有助于形成均匀的纳米颗粒，增强纳米颗粒的稳定性，促进核酸的高效包裹，协助细胞摄取，并促进核酸从内体逃逸(endosomal escape)(Kulkarni et al.,2018,Kulkarni et al.,2019,Schoenmaker et al.,2021)。然而，这一过程导致约99％的RNA治疗药物被困在具有脂质双分子层屏障的内体中。只有极小部分(1％或更少)的RNA能够进入细胞质(He et al.,2021,Brown et al.,2020)。低效的内体逃逸导致需要更高的剂量，其中包括但不限于“过量”mRNA、阳离子脂质(其与mRNA需要15～18：1的w/w比)、以及mRNA相关的杂质，如dsRNAs。毒性可能来自细胞自主因素，如细胞毒性，以及非细胞自主因素，包括炎症。细胞毒性可能由各种因素引起，包括氧化应激和凋亡。因此，在RNA疗法可以有效用于治疗广泛的人类疾病之前，以无毒的方式解决内体逃逸的限速递送的挑战,通过降低剂量来减少副作用是至关重要的(Dowdy et al.,2022,Dowdy 2017)。要成功将大分子传递到细胞质中，必须解决三个关键步骤：1)细胞结合，2)诱导内吞，3)促进内体逃逸。在这些步骤中，以非细胞毒性方式从内体逃逸到细胞质被广泛认为是主要瓶颈。先前的研究表明，脂质纳米颗粒(LNPs)利用构成性和诱导性的途径，如Clathrin介导的内吞，以细胞类型特异性的方式进入细胞(Gilleron et al.,2013,Zurenko et al.,2013)。表明通过操纵和调控内吞与Actin相关的蛋白的功能，可以显著改善mRNA通过LNPs的传递效率(Gilleronet al.，2013，Zurenko et al.，2013)。Unlike small molecule therapeutics that are able to cross the lipid bilayer of the cell membrane, RNA therapeutics, especially mRNA, face challenges due to their large molecular weight, charge, and hydrophilicity. Therefore, they cannot cross the cell membrane by passive diffusion, but rely on endocytosis as a means of cellular uptake. Pure mRNA is rarely used in its "naked" form; instead, it is protected and transported to the target cell using a delivery system. The most widely used delivery system, including those selected for the currently popular SARS-CoV-2 vaccine, is mRNA-lipid nanoparticles (mRNA-LNPs). These LNPs include four lipids: (1) ionizable cationic lipids, (2) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), (3) cholesterol, and (4) polyethylene glycol-lipid conjugates (PEG-DMG) (Kulkarni et al., 2019a). These components help form uniform nanoparticles, enhance nanoparticle stability, promote efficient nucleic acid encapsulation, assist cellular uptake, and promote nucleic acid escape from endosomal (Kulkarni et al., 2018, Kulkarni et al., 2019, Schoenmaker et al., 2021). However, this process results in about 99% of RNA therapeutics being trapped in endosomes with lipid bilayer barriers. Only a very small fraction (1% or less) of RNA is able to enter the cytoplasm (He et al., 2021, Brown et al., 2020). Inefficient endosomal escape leads to the need for higher doses, including but not limited to "excess" mRNA, cationic lipids (which require a w/w ratio of 15 to 18:1 to mRNA), and mRNA-related impurities such as dsRNAs. Toxicity may come from cell-autonomous factors, such as cytotoxicity, as well as non-cell-autonomous factors, including inflammation. Cytotoxicity may be caused by various factors, including oxidative stress and apoptosis. Therefore, before RNA therapeutics can be effectively used to treat a wide range of human diseases, it is crucial to address the rate-limiting delivery challenge of endosomal escape in a non-toxic manner and reduce side effects by lowering the dose (Dowdy et al., 2022, Dowdy 2017). To successfully deliver macromolecules to the cytoplasm, three key steps must be addressed: 1) cell binding, 2) inducing endocytosis, and 3) promoting endosomal escape. Among these steps, escape from endosomes to the cytoplasm in a non-cytotoxic manner is widely considered to be the main bottleneck. Previous studies have shown that lipid nanoparticles (LNPs) use constitutive and inducible pathways, such as Clathrin-mediated endocytosis, to enter cells in a cell type-specific manner (Gilleron et al., 2013, Zurenko et al., 2013). It shows that the efficiency of mRNA delivery through LNPs can be significantly improved by manipulating and regulating the function of proteins associated with endocytosis and Actin (Gillerone et al., 2013, Zurenko et al., 2013).
细胞内吞作用是指真核细胞吞噬多种物质的过程，包括质膜蛋白和脂质、胞外分子、液体、颗粒、外泌体、病毒和细菌。这是一种在细胞内发挥多种功能的基本而保守的机制。根据所携带的物质的性质和涉及的分子机制的不同，已经确定了不同类型的内吞途径。其中包括Clathrin介导的内吞、穴突/脂质筏介导的内吞、Clathrin和穴突独立的内吞、液相内吞和吞噬作用。在这些途径中，Clathrin介导的内吞(CME)被广泛认为是哺乳动物细胞内吞的主要途径(Abouelezz et al.,2022)。内吞过程可分为几个明确定义的步骤。它始于膜内囊泡化，接着是有被覆的凹陷的形成。这些凹陷随后被分离，新形成的囊泡从膜上脱离。最后，新形成的内吞区域离开质膜进入细胞质。这些步骤由一个复杂的机制执行，包括60多种蛋白质，每个都执行特定的任务。这些任务包括膜弯曲、所携带物质的招募、支架、脂质修饰和对细胞骨架的调节。最近的研究公开了丰富的证据，证明了内吞和Actin细胞骨架之间存在着密切的联系。因此，已经确定并分类了在这种相互作用中涉及的一组特定的蛋白质，被称为内吞-Actin相关蛋白质。这些蛋白质的显著例子包括Cofilin(Lappalainenet al.,1997)，ARP2/3复合物(Sun et al.,2006)，N-WASP(Benesch et al.,2005；Innocenti et al.,2005)，cortactin(Merrifield et al.,2005；Sauvonnet et al.,2005；Zhu et al.,2005)，ABP1(Fazi et al.,2002,Polack et al.,2020)，Dynamin(Gilleron et al.,2003)和Myo1E/Myo6(Kovacs et al.,2006；Salazar et al.,2003)。这些蛋白质在促进内吞和Actin细胞骨架之间的动态相互作用方面起着至关重要的作用。Endocytosis refers to the process by which eukaryotic cells engulf a variety of substances, including plasma membrane proteins and lipids, extracellular molecules, fluids, particles, exosomes, viruses, and bacteria. It is a fundamental and conserved mechanism that performs a variety of functions within cells. Different types of endocytic pathways have been identified, depending on the nature of the substances carried and the molecular mechanisms involved. These include Clathrin-mediated endocytosis, caveolae/lipid raft-mediated endocytosis, Clathrin- and caveolae-independent endocytosis, fluid-phase endocytosis, and phagocytosis. Among these pathways, Clathrin-mediated endocytosis (CME) is widely considered to be the major pathway of endocytosis in mammalian cells (Abouelezz et al., 2022). The endocytic process can be divided into several well-defined steps. It begins with intramembrane vesicle formation, followed by the formation of coated pits. These pits are subsequently detached and the newly formed vesicles detach from the membrane. Finally, the newly formed endocytic area leaves the plasma membrane and enters the cytoplasm. These steps are executed by a complex mechanism involving more than 60 proteins, each of which performs a specific task. These tasks include membrane bending, recruitment of carried cargo, scaffolding, lipid modification and regulation of the cytoskeleton. Recent studies have disclosed abundant evidence for a close connection between endocytosis and the actin cytoskeleton. As a result, a specific group of proteins involved in this interaction have been identified and classified, termed endocytosis-actin-associated proteins. Notable examples of these proteins include cofilin (Lappalain et al., 1997), ARP2/3 complex (Sun et al., 2006), N-WASP (Benesch et al., 2005; Innocenti et al., 2005), cortactin (Merrifield et al., 2005; Sauvonnet et al., 2005; Zhu et al., 2005), ABP1 (Fazi et al., 2002, Polack et al., 2020), Dynamin (Gilleron et al., 2003), and Myo1E/Myo6 (Kovacs et al., 2006; Salazar et al., 2003). These proteins play crucial roles in promoting the dynamic interaction between endocytosis and the actin cytoskeleton.
以前的研究已经证明，致病微生物已经进化出分子机制来操纵内吞Actin相关蛋白质的活性，利用宿主细胞中的Actin细胞骨架系统，促进它们内化到靶细胞中，修饰复制小生境并促进它们的细胞内和细胞间传播。各种病原体进化出不同的机制来操纵Actin的动态重组，以增强其内体逃逸效率，促进进入、移动到宿主细胞内或从宿主细胞离开。当然，自然界进化内体逃逸的最令人印象深刻,并且可能是最有效的机制是包膜病毒(Hernandezet al.,1996)。包膜病毒在大小上与LNP相似，通过内吞作用被吸收到细胞中，并且需要内体逃逸机制。与RNA药物不到1％的内体逃逸相比，包膜病毒具有惊人的30％–70％的逃逸效率(Lagache et al.,2012,Staring J,et.al.,2018)。皮质Actin是病原体在感染时遇到的第一个障碍，并且病原体已经擅长寻找克服这种屏障的方法。包膜病毒已经熟练地找到了克服这种障碍的方法,利用诱导受体介导的信号传递，导致局部Actin扰动，以穿越皮质，随后篡夺受体介导的内吞体，通过内吞体腔的pH依赖/独立融合,利用内吞小泡的固有特性来迁移，穿越皮质。这些导致cofilin活性的调节和Actin皮层的协同装配和解装配，从而促进微生物途径的穿透和迁移。病毒已经发展了各种策略来克服皮质Actin网络代表的屏障，例如质膜处的非pH依赖性融合和受体介导的内吞作用。一旦病毒与它们的细胞受体相互作用，就触发细胞内Actin调节信号级联(Zheng et al.,2016)。HIV-1病毒是一个被广泛研究的例子(Balabanian et al.,2004；Cameron et al.,2010；Guo et al.,2011；Yoder etal.,2009)。在非循环、休眠T细胞中，HIV-1包膜蛋白gp120与病毒受体CD4和CXCR4之间的相互作用,引发了与病毒剂量相关的Actin细胞骨架重组和Cofilin活性调节。HIV-1的进入引发Cofilin的迅速磷酸化和Actin的聚合，随后是Cofilin的脱磷酸化，导致Actin解聚。Gp120与CXCR4结合会触发融合和信号转导，从而导致Rac-PAK-LIMK或Rho-ROCK-LIMK介导的cofilin失活以及F-actin稳定和聚合。这种聚合促进了细胞表面CD4/CXCR4的共吸附和簇集，为病毒进入细胞提供了便利。通过CXCR4信号传导介导的Cofilin激活引发的随后Actin活性增加了皮质Actin的动力学和Actin踏车效应，从而促进病毒前整合复合物朝核区的移动。因此，HIV包膜介导的Cofilin激活和Actin动力学改变对于病毒核定位至关重要。在受感染患者的静息CD4 T细胞中，HIV-1会诱导cofilin逐渐去磷酸化和Actin解聚，这与在迁移性T淋巴瘤细胞中检测到的cofilin激活极为相似。由于T细胞处于不同的活化状态，进一步的研究应侧重于将T细胞的活化状态与cofilin的活化状态联系起来，最好能确定一种能指示cofilin活化的细胞表面标志物。Previous studies have demonstrated that pathogenic microorganisms have evolved molecular mechanisms to manipulate the activity of endocytic actin-related proteins, exploit the actin cytoskeleton system in host cells, promote their internalization into target cells, modify replication niches and promote their intracellular and intercellular spread. Various pathogens have evolved different mechanisms to manipulate the dynamic reorganization of actin to enhance its endosomal escape efficiency and promote entry, movement into or exit from host cells. Of course, the most impressive and perhaps the most efficient mechanism for endosomal escape evolved in nature is enveloped viruses (Hernandez et al., 1996). Enveloped viruses are similar in size to LNPs, are taken up into cells by endocytosis, and require endosomal escape mechanisms. Compared with less than 1% endosomal escape of RNA drugs, enveloped viruses have an astonishing 30%–70% escape efficiency (Lagache et al., 2012, Staring J, et.al., 2018). Cortical actin is the first obstacle encountered by pathogens during infection, and pathogens have become adept at finding ways to overcome this barrier. Enveloped viruses have expertly found ways to overcome this barrier by inducing receptor-mediated signaling that results in localized actin perturbations to traverse the cortex, subsequently usurping receptor-mediated endosomes to migrate across the cortex via pH-dependent/independent fusion of the endosome lumen, and exploiting the intrinsic properties of endocytic vesicles to migrate across the cortex. These lead to modulation of cofilin activity and coordinated assembly and disassembly of the actin cortex, thereby facilitating penetration and migration of microbial pathways. Viruses have developed various strategies to overcome the barrier represented by the cortical actin network, such as pH-independent fusion at the plasma membrane and receptor-mediated endocytosis. Once viruses interact with their cellular receptors, intracellular actin-regulated signaling cascades are triggered (Zheng et al., 2016). HIV-1 virus is an extensively studied example (Balabanian et al., 2004; Cameron et al., 2010; Guo et al., 2011; Yoder et al., 2009). In non-cycling, resting T cells, the interaction between HIV-1 envelope protein gp120 and viral receptors CD4 and CXCR4 triggers viral dose-dependent reorganization of the actin cytoskeleton and regulation of cofilin activity. HIV-1 entry triggers rapid phosphorylation of cofilin and polymerization of actin, followed by cofilin dephosphorylation, leading to actin depolymerization. Gp120 binding to CXCR4 triggers fusion and signal transduction, leading to Rac-PAK-LIMK or Rho-ROCK-LIMK-mediated cofilin inactivation and F-actin stabilization and polymerization. This aggregation promotes coadsorption and clustering of CD4/CXCR4 on the cell surface, facilitating viral entry into cells. Subsequent actin activity triggered by cofilin activation mediated by CXCR4 signaling increases cortical actin dynamics and actin treadmilling, thereby promoting the movement of the viral pre-integration complex toward the nuclear region. Therefore, HIV envelope-mediated cofilin activation and changes in actin dynamics are critical for viral nuclear localization. In resting CD4 T cells from infected patients, HIV-1 induces progressive dephosphorylation of cofilin and dissociation of actin, which closely resembles the cofilin activation detected in migratory T lymphoma cells. As T cells exist in different activation states, further studies should focus on linking the activation state of T cells with that of cofilin, preferably by identifying a cell surface marker that indicates cofilin activation.
Cofilin是一种约21kDA的蛋白，在所有脊椎动物中普遍表达，并在真核细胞中自由扩散(Shishkin et al.,2016)。其功能是与Actin结合，切割Actin，导致Actin丝解聚(Wang et al.,2007；Huang et al.,2014；Chang et al.,2015)。Cofilin通过增强F-Actin的解聚和抑制G-Actin的聚合促进了Actin丝的转化，这对真核生物的Actin丝动力学至关重要。在Ser-3位点的磷酸化和脱磷酸化是Actin解聚和组装的关键机制。Rho GTPase介导的LIMK磷酸化使cofilin失活，而磷酸酯酶SSH和CIN则发挥相反的作用(Huang et al.,2008；Soosairajah et al.,2005)。一旦Cofilin通过脱磷酸化被激活，它通过与Actin结合转位进入细胞核来切割Actin。由于Cofilin是Actin组装和解组装的关键调节蛋白，因此毫不奇怪，病原体通过与Cofilin和/或Cofilin调控的信号通路的相互作以突破Actin网络屏障，从而进入宿主细胞。在先前的研究中(Yoder et al.,2009)，曾经证明包含人类Cofilin的前16个氨基酸的合成肽(S3)，包括Ser-3，与LIMK1有效竞争，因而能够激活Cofilin。这种竞争导致了Cofilin磷酸化的抑制，随后激活了Cofilin。值得注意的是，这种激活对病毒复制产生了显著影响。许多其他研究也强调了Cofilin在调节Actin动力学的时机和位置方面的关键作用。这些动力学在促进宿主与病原体之间的相互作用方面发挥着至关重要的作用，比如促进进入目标细胞、防止内体和溶酶体之间的融合，以及促进病原体的细胞内移动。Cofilin is a protein of approximately 21 kDA that is ubiquitously expressed in all vertebrates and diffuses freely in eukaryotic cells (Shishkin et al., 2016). Its function is to bind to actin, cleave actin, and lead to the disassembly of actin filaments (Wang et al., 2007; Huang et al., 2014; Chang et al., 2015). Cofilin promotes the turnover of actin filaments by enhancing the disassembly of F-actin and inhibiting the polymerization of G-actin, which is essential for the dynamics of actin filaments in eukaryotes. Phosphorylation and dephosphorylation at the Ser-3 site are key mechanisms for the disassembly and assembly of actin. Rho GTPase-mediated LIMK phosphorylation inactivates cofilin, while phosphatases SSH and CIN play the opposite role (Huang et al., 2008; Soosairajah et al., 2005). Once cofilin is activated by dephosphorylation, it translocates into the nucleus by binding to actin to cleave actin. Since Cofilin is a key regulator of Actin assembly and disassembly, it is not surprising that pathogens gain entry into host cells by breaching the Actin network barrier through interactions with Cofilin and/or Cofilin-regulated signaling pathways. In a previous study (Yoder et al., 2009), it was demonstrated that a synthetic peptide (S3) containing the first 16 amino acids of human Cofilin, including Ser-3, effectively competed with LIMK1 and was thus able to activate Cofilin. This competition resulted in the inhibition of Cofilin phosphorylation and subsequent activation of Cofilin. Notably, this activation had a significant effect on viral replication. Many other studies have also highlighted the critical role of Cofilin in regulating the timing and location of Actin dynamics. These dynamics play a crucial role in facilitating host-pathogen interactions, such as facilitating entry into target cells, preventing fusion between endosomes and lysosomes, and promoting intracellular movement of pathogens.
肽/蛋白质转导结构域(PTD)，又称为细胞穿透肽(CPPs，Pooga,M et al.,1998)，能够促进大分子通过内吞作用内化。氨基酸的疏水性R基团被认识为在病毒内体逃逸过程中发挥作用的组成部分(CPPs，Pooga，et al.，1998,Ezhevsky，et al.，1997)。源于HIV TAT蛋白并称为肽/蛋白转导结构域(PTD，Ezhevsky，et al.，1997)或细胞穿透肽(CPP)的阳离子递送肽的偶然发现，使大分子治疗剂递送领域发生了革命性变化。TAT PTD已广泛应用于将各种大分子转运到不同类型的细胞、临床前疾病模型和临床试验中(P et al.,2015,Glogau,R.et al.,2012)，包括一项重要的II期研究和正在进行的III期试验。TATPTD已广泛用于各种大分子药物的细胞内递送(P et al.,2015,Dupont,E.et al.,2015.Nakase,I.et al.,2013,Koren,E.et al.,2012)。TAT PTD和相关的PTDs/CPPs通过内吞作用将大分子输送到细胞内。TAT PTD通过两种基本机制实现细胞摄取：1)诱导巨吞作用，即一种特殊形式的内吞作用；2)促进内体逃逸。等人采用实时、定量的活细胞分裂-GFP荧光互补表型分析方法，系统地分析和优化了一系列合成的内体逃逸结构域(EED)。绿色荧光蛋白(GFP)是一种在蓝到紫外光范围内暴露时呈现出明亮绿色荧光的蛋白质。他们的研究发现，包含芳香苯基(苯丙氨酸(Phe))和吲哚环(色氨酸(Trp))的氨基酸的EED，在距离TAT-PTD-载体的六个聚乙二醇(PEG)单位的固定距离下，显著增强了胞质递送，而不引起细胞毒性。这些EED解决了在将大分子生物学肽、蛋白质和RNA治疗物递送到细胞内时内体逃逸的关键限速步骤( et al.,2016)。Peptide/protein transduction domain (PTD), also known as cell penetrating peptide (CPPs, Pooga, M et al., 1998), can promote the internalization of macromolecules by endocytosis. The hydrophobic R group of amino acids is recognized as a component that plays a role in the process of viral endosomal escape (CPPs, Pooga, et al., 1998, Ezhevsky, et al., 1997). The accidental discovery of cationic delivery peptides derived from HIV TAT protein and called peptide/protein transduction domain (PTD, Ezhevsky, et al., 1997) or cell penetrating peptide (CPP) has revolutionized the delivery of macromolecular therapeutic agents. TAT PTD has been widely used in transporting various macromolecules to different types of cells, preclinical disease models and clinical trials ( P et al., 2015, Glogau, R. et al., 2012), including an important Phase II study and an ongoing Phase III trial. TATPTD has been widely used for intracellular delivery of various macromolecular drugs ( P et al., 2015, Dupont, E. et al., 2015. Nakase, I. et al., 2013, Koren, E. et al., 2012). TAT PTD and related PTDs/CPPs deliver macromolecules into cells via endocytosis. TAT PTD achieves cellular uptake via two basic mechanisms: 1) inducing macrophagy, a specialized form of endocytosis; and 2) promoting endosomal escape. et al. used a real-time, quantitative, live-cell split-GFP fluorescence complementation phenotyping assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). Green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. Their study found that EEDs containing amino acids with aromatic phenyl groups (phenylalanine (Phe)) and indole rings (tryptophan (Trp)) significantly enhanced cytoplasmic delivery at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-vector without causing cytotoxicity. These EEDs address the key rate-limiting step of endosomal escape when delivering macromolecular biological peptides, proteins, and RNA therapeutics into cells ( et al., 2016).
需要一种能够增强LNPs细胞转染的组合物和方法。What is needed is a composition and method that can enhance cellular transfection with LNPs.
发明内容Summary of the invention
在一个实施方式中，本申请公开了一种提高液态LNPs细胞转染的方法。所述方法包括：提供治疗剂；提供递送载体，所述递送载体为液态LNPs，包括源自人类Cofilin的肽，将治疗剂输送到目标细胞；并在目标细胞中释放治疗剂。In one embodiment, the present application discloses a method for improving liquid LNPs cell transfection. The method comprises: providing a therapeutic agent; providing a delivery vehicle, wherein the delivery vehicle is liquid LNPs, comprising a peptide derived from human cofilin, delivering the therapeutic agent to a target cell; and releasing the therapeutic agent in the target cell.
在另一实施方式中，治疗剂为信使RNA、小干扰RNA或反义寡核苷酸。In another embodiment, the therapeutic agent is a messenger RNA, a small interfering RNA, or an antisense oligonucleotide.
在另一个实施方式中，所述肽包括SEQ ID NO：1；或与SEQ ID NO：1具有至少81.25％同一性并且相对于SEQ ID NO：1至少含有一个替代氨基酸。In another embodiment, the peptide comprises SEQ ID NO:1; or has at least 81.25% identity to SEQ ID NO:1 and contains at least one substituted amino acid relative to SEQ ID NO:1.
在另一实施方式中，所述肽包括N-末端部分和C-末端部分；所述N-末端部分包含SEQ ID NO：1；或与SEQ ID NO:1具有至少81.25％同一性的序列且相对于SEQ ID NO:1至少包含一个替代修饰；且所述C-末端部分包括至少一种选自由苯丙氨酸(Phe)和色氨酸(Trp)组成的组的氨基酸，且至少一种选自由甘氨酸(Gly)、亮氨酸(Leu)、异亮氨酸(Ile)和缬氨酸(Val)组成的多肽。In another embodiment, the peptide comprises an N-terminal portion and a C-terminal portion; the N-terminal portion comprises SEQ ID NO: 1; or a sequence having at least 81.25% identity with SEQ ID NO: 1 and comprising at least one substitution modification relative to SEQ ID NO: 1; and the C-terminal portion comprises at least one amino acid selected from the group consisting of phenylalanine (Phe) and tryptophan (Trp), and at least one polypeptide selected from the group consisting of glycine (Gly), leucine (Leu), isoleucine (Ile) and valine (Val).
在另一实施方式中，所述肽包括N-末端部分和C-末端部分；所述N-末端部分包含SEQ ID NO：1；或与SEQ ID NO:1具有至少81.25％同一性的序列且相对于SEQ ID NO:1至少包含一个氨基酸替代修饰；且所述C-末端部分包括至少一个选自赖氨酸(Lys)、精氨酸(Arg)和组氨酸(His)组成的组的多肽。In another embodiment, the peptide includes an N-terminal portion and a C-terminal portion; the N-terminal portion comprises SEQ ID NO: 1; or a sequence having at least 81.25% identity with SEQ ID NO: 1 and comprising at least one amino acid substitution modification relative to SEQ ID NO: 1; and the C-terminal portion comprises at least one polypeptide selected from the group consisting of lysine (Lys), arginine (Arg) and histidine (His).
在另一实施方式中，所述多肽还包括一个中间部分；中间部分位于N-末端部分和C-末端部分之间；并且中间部分包括至少一个选自由苯丙氨酸(Phe)和色氨酸(Trp)组成的多肽。In another embodiment, the polypeptide further comprises a middle portion; the middle portion is located between the N-terminal portion and the C-terminal portion; and the middle portion comprises at least one polypeptide selected from the group consisting of phenylalanine (Phe) and tryptophan (Trp).
在另一个实施方式中，递送载体还包括可电离的阳离子脂质、1，2-二硬脂酰-sn-甘油基-3-磷酸胆碱(DSPC)、胆固醇和聚乙二醇(PEG)-脂质缀合物。In another embodiment, the delivery vehicle further comprises an ionizable cationic lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and a polyethylene glycol (PEG)-lipid conjugate.
在一实施方式中，本发明公开了一种肽，其包括与SEQ ID NO：1具有至少81.25％同一性的序列且相对于SEQ ID NO:1至少包含一个氨基酸替代修饰.In one embodiment, the present invention discloses a peptide comprising a sequence having at least 81.25% identity to SEQ ID NO: 1 and comprising at least one amino acid substitution modification relative to SEQ ID NO: 1.
在另一实施方式中，所述多肽与SEQ ID NO:1相比，包含三个替代修饰。In another embodiment, the polypeptide comprises three substitution modifications compared to SEQ ID NO:1.
在另一个实施方式中，所述多肽包括与SEQ ID NO：1具有至少87.5％同一性的序列，并且包含相对于SEQ ID NO：1的两个置换修饰。In another embodiment, the polypeptide comprises a sequence that is at least 87.5% identical to SEQ ID NO:1 and comprises two substitution modifications relative to SEQ ID NO:1.
在另一个实施方式中，所述多肽包括与SEQ ID NO：1具有至少93.75％同一性的序列，并且包含相对于SEQ ID NO：1的一个氨基酸置换修饰。In another embodiment, the polypeptide comprises a sequence that is at least 93.75% identical to SEQ ID NO:1 and comprises one amino acid substitution modification relative to SEQ ID NO:1.
在另一实施方式中，所述多肽包括选自以下序列组成的组中的一个序列：SEQ IDNO:2，SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:6，SEQ ID NO:7，SEQ ID NO:8，SEQ ID NO:9，SEQ ID NO:10，SEQ ID NO:11和SEQ ID NO:12。In another embodiment, the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.
在另一实施方式中，本申请公开了一种多肽，包括一个N-末端部分和一个C-末端部分。N-末端部分包括SEQ ID NO：1；或与SEQ ID NO:1具有至少81.25％同一性的序列且相对于SEQ ID NO:1至少包含一个氨基酸替代修饰；且所述C-末端部分包括至少一种选自由苯丙氨酸(Phe)和色氨酸(Trp)组成的组的氨基酸，且至少一种选自由甘氨酸(Gly)、亮氨酸(Leu)、异亮氨酸(Ile)和缬氨酸(Val)组成的多肽。In another embodiment, the present application discloses a polypeptide comprising an N-terminal portion and a C-terminal portion. The N-terminal portion comprises SEQ ID NO: 1; or a sequence having at least 81.25% identity to SEQ ID NO: 1 and comprising at least one amino acid substitution modification relative to SEQ ID NO: 1; and the C-terminal portion comprises at least one amino acid selected from the group consisting of phenylalanine (Phe) and tryptophan (Trp), and at least one polypeptide selected from the group consisting of glycine (Gly), leucine (Leu), isoleucine (Ile) and valine (Val).
在另一实施方式中，所述多肽包括选自以下序列组成的组中的一个序列：SEQ IDNO:16、SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19。In another embodiment, the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.
在另一实施方式中，本申请公开了一种多肽，包括一个N-末端部分和一个C-末端部分。N-末端部分包括SEQ ID NO：1；或与SEQ ID NO:1具有至少81.25％同一性的序列且相对于SEQ ID NO:1至少包含一个替代修饰；且所述C-末端部分包括至少一个选自赖氨酸(Lys)、精氨酸(Arg)和组氨酸(His)组成的多肽。In another embodiment, the present application discloses a polypeptide comprising an N-terminal portion and a C-terminal portion. The N-terminal portion comprises SEQ ID NO: 1; or a sequence having at least 81.25% identity to SEQ ID NO: 1 and comprising at least one substitution modification relative to SEQ ID NO: 1; and the C-terminal portion comprises at least one polypeptide selected from lysine (Lys), arginine (Arg) and histidine (His).
在另一实施方式中，所述肽还包括一个中间部分。中间部分位于N-末端部分和C-末端部分之间；并且中间部分包括至少一个选自由苯丙氨酸(Phe)和色氨酸(Trp)组成的多肽。In another embodiment, the peptide further comprises a middle portion, the middle portion is located between the N-terminal portion and the C-terminal portion, and the middle portion comprises at least one polypeptide selected from the group consisting of phenylalanine (Phe) and tryptophan (Trp).
在另一实施方式中，所述多肽包括选自以下序列组成的组中的一个序列：SEQ IDNO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ IDNO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:31。In another embodiment, the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31.
应当理解，上述一般描述和下面的详细描述都是示例性和解释性的，旨在对所宣称的发明提供进一步的解释。It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
附图用于进一步理解本发明，已纳入并构成本说明的一部分，展示了本发明的实施例，并与说明一同用于解释本发明的原理。The accompanying drawings are provided to provide a further understanding of the present invention and are incorporated in and constitute a part of this specification. They illustrate embodiments of the present invention and together with the description serve to explain the principles of the present invention.
在图中：In the picture:
图1是AS3-EECs信号介导Cofilin激活和mRNA内体逃逸的模型。(A)AS3-EECs LNP及其各个组分的简化图解。(B)AS3-EECs信号介导Cofilin激活和mRNA内体逃逸。AS3-EECs多肽携带人Cofilin的前16个残基，并与内体逃逸结构域结合，有效地与Cofilin竞争LIMK1。该竞争抑制Cofilin磷酸化，随后激活Cofilin。这种Cofilin激活增强了Actin的动态变化，促进了内体逃逸和LNP包裹的mRNA的移动,提高翻译效率。Figure 1 is a model of AS3-EECs signal-mediated Cofilin activation and mRNA endosomal escape. (A) Simplified diagram of AS3-EECs LNP and its individual components. (B) AS3-EECs signal-mediated Cofilin activation and mRNA endosomal escape. The AS3-EECs polypeptide carries the first 16 residues of human Cofilin and binds to the endosomal escape domain, effectively competing with Cofilin for LIMK1. This competition inhibits Cofilin phosphorylation and subsequently activates Cofilin. This Cofilin activation enhances the dynamic changes of Actin, promotes endosomal escape and the movement of LNP-encapsulated mRNA, and improves translation efficiency.
图2显示AS3-EECs增强了在转染了GFP mRNA-LNPs的293T细胞中的GFP表达。转染过程是通过添加不同浓度的mRNA包裹在LNPs中进行的：8ng/mL，24ng/mL，72ng/mL，216ng/mL，648ng/mL和1944ng/mL。转染后，在不同的时间点收集细胞进行后续分析。这些时间点分别是转染后的48小时、72小时和96小时。通过流式细胞仪评估荧光强度来测量得到的GFP表达水平。数值以中位相对荧光强度(MFI)单位呈现。FIG2 shows that AS3-EECs enhance GFP expression in 293T cells transfected with GFP mRNA-LNPs. The transfection process was performed by adding different concentrations of mRNA encapsulated in LNPs: 8 ng/mL, 24 ng/mL, 72 ng/mL, 216 ng/mL, 648 ng/mL, and 1944 ng/mL. After transfection, cells were collected at different time points for subsequent analysis. These time points were 48 hours, 72 hours, and 96 hours after transfection. The resulting GFP expression levels were measured by evaluating the fluorescence intensity by flow cytometry. Values are presented in median relative fluorescence intensity (MFI) units.
图3显示AS3-EECs增强了在转染了GFP mRNA-LNPs的A549细胞中的GFP表达。转染过程是通过添加不同浓度的mRNA包裹在LNPs中进行的：8ng/mL，24ng/mL，72ng/mL，216ng/mL，648ng/mL和1944ng/mL。转染后，在不同的时间点收集细胞进行后续分析。这些时间点分别是转染后的48小时、72小时和96小时。通过流式细胞仪评估荧光强度来测量得到的GFP表达水平。数值以中位相对荧光强度(MFI)单位呈现。FIG3 shows that AS3-EECs enhance GFP expression in A549 cells transfected with GFP mRNA-LNPs. The transfection process was performed by adding different concentrations of mRNA encapsulated in LNPs: 8 ng/mL, 24 ng/mL, 72 ng/mL, 216 ng/mL, 648 ng/mL, and 1944 ng/mL. After transfection, cells were collected at different time points for subsequent analysis. These time points were 48 hours, 72 hours, and 96 hours after transfection. The GFP expression level obtained was measured by evaluating the fluorescence intensity by flow cytometry. Values are presented in median relative fluorescence intensity (MFI) units.
图4显示AS3-EECs增强了在转染了GFP mRNA-LNPs的B16细胞中的GFP表达。转染过程是通过添加不同浓度的mRNA包裹在LNPs中进行的：8ng/mL，24ng/mL，72ng/mL，216ng/mL，648ng/mL和1944ng/mL。转染后，在不同的时间点收集细胞进行后续分析。这些时间点分别是转染后的48小时、72小时和96小时。通过流式细胞仪评估荧光强度来测量得到的GFP表达水平。数值以中位相对荧光强度(MFI)单位呈现。FIG4 shows that AS3-EECs enhance GFP expression in B16 cells transfected with GFP mRNA-LNPs. The transfection process was performed by adding different concentrations of mRNA encapsulated in LNPs: 8 ng/mL, 24 ng/mL, 72 ng/mL, 216 ng/mL, 648 ng/mL, and 1944 ng/mL. After transfection, cells were collected at different time points for subsequent analysis. These time points were 48 hours, 72 hours, and 96 hours after transfection. The GFP expression level obtained was measured by evaluating the fluorescence intensity by flow cytometry. Values are presented in median relative fluorescence intensity (MFI) units.
图文详细描述Detailed description with pictures and text
现在将详细描述本发明的实施例，其示例已在附图中说明。Reference will now be made in detail to the embodiments of the present invention, examples of which are illustrated in the accompanying drawings.
鉴于Cofilin在内吞作用和Actin骨架调控中的关键作用，以及EED在促进内体逃逸中的作用，本申请的发明人已将其作为研究的重点。为了进一步研究，发明人合成了包含人类Cofilin的前16个氨基酸的多肽，并将它们与一系列内体逃逸结构域(AS3-EECs)结合。AS3-EEC多肽被FDA批准的LNP制剂包裹，并且GFP mRNA-AS3-EEC LNPs被用于转染各种细胞类型。在含有GFP mRNA-AS3-EECs的LNPs共孵育后，我们使用流式细胞仪在不同的时间点测量转染细胞的荧光。结果明确证明，含有人类Cofilin前16个氨基酸并带有EEDs的合成多肽显著提高了在所有测试的细胞类型中，包括三种人类癌细胞系，LNP包裹的mRNA的转染效率。Given the key role of Cofilin in endocytosis and Actin skeleton regulation, as well as the role of EED in promoting endosomal escape, the inventors of this application have made it a focus of research. For further study, the inventors synthesized polypeptides containing the first 16 amino acids of human Cofilin and combined them with a series of endosomal escape domains (AS3-EECs). AS3-EEC polypeptides were encapsulated by FDA-approved LNP formulations, and GFP mRNA-AS3-EEC LNPs were used to transfect various cell types. After co-incubation of LNPs containing GFP mRNA-AS3-EECs, we used flow cytometry to measure the fluorescence of transfected cells at different time points. The results clearly demonstrated that synthetic polypeptides containing the first 16 amino acids of human Cofilin and carrying EEDs significantly improved the transfection efficiency of LNP-encapsulated mRNA in all cell types tested, including three human cancer cell lines.
总结Summarize
RNA药物，包括mRNA、siRNA和ASOs，在治疗各种人类疾病方面表现出显著潜力。目前，已有15多种RNA药物获得FDA批准，同时还有大量临床试验正在进行。值得注意的是，由辉瑞-拜恩泰克和莫德纳开发的基于mRNA的疫苗在抗击SARS-CoV-2大流行方面取得了巨大成功。在RNA治疗中，mRNA因其独特的特性而脱颖而出，包括易于设计、快速合成、瞬时蛋白表达以及在细胞质中立即启动蛋白表达。RNA drugs, including mRNA, siRNA, and ASOs, have shown significant potential in treating various human diseases. Currently, more than 15 RNA drugs have been approved by the FDA, and a large number of clinical trials are ongoing. Notably, mRNA-based vaccines developed by Pfizer-BioNTech and Moderna have achieved great success in combating the SARS-CoV-2 pandemic. Among RNA therapeutics, mRNA stands out due to its unique properties, including ease of design, rapid synthesis, transient protein expression, and immediate initiation of protein expression in the cytoplasm.
然而，由于其大小和亲水性，RNA药物在细胞摄取方面面临挑战。为了克服这一障碍，利用了传递载体，如mRNA-LNPs，尽管它们从内体逃逸仍然是一个重要的障碍。成功的传递需要解决细胞关联、促进内吞作用以及促进内体逃逸等问题。在这项研究中，我们旨在探讨源自Cofilin的多肽(参与Actin动力学的蛋白质)的潜力，以提高LNPs介导的GFP mRNA的转染效率。为此，我们设计了一系列包含人类Cofilin的前16个氨基酸与内体逃逸结构域结合的多肽，称为AS3-EECs。这些AS3-EEC多肽与GFP-mRNA一同被包裹到LNPs中。在用含有多肽的LNPs转染三种不同的人类细胞系后，我们通过使用流式细胞术测量荧光强度来评估所得到的GFP表达水平。However, RNA drugs face challenges in cellular uptake due to their size and hydrophilicity. To overcome this obstacle, delivery vehicles such as mRNA-LNPs have been utilized, although their escape from endosomes remains a significant hurdle. Successful delivery requires addressing issues such as cellular association, facilitating endocytosis, and promoting endosomal escape. In this study, we aimed to explore the potential of peptides derived from cofilin, a protein involved in actin dynamics, to improve the efficiency of LNPs-mediated transfection of GFP mRNA. To this end, we designed a series of peptides containing the first 16 amino acids of human cofilin combined with an endosomal escape domain, termed AS3-EECs. These AS3-EEC peptides were encapsulated into LNPs together with GFP-mRNA. After transfection of three different human cell lines with LNPs containing the peptides, we assessed the resulting GFP expression levels by measuring fluorescence intensity using flow cytometry.
我们的发现表明，含有AS3-EECs的LNPs的流体动力学直径在不同的AS3-EEC中范围从87.8纳米到102.1纳米，略大于没有多肽的对照LNP。所有三种含有肽的LNPs中mRNA的包裹效率接近100％。这些LNPs显示出均匀的尺寸分布，多分散指数(PDI)小于0.1，几乎中性的ζ电位。这些结果表明，将Cofilin肽与mRNA一同包裹未干扰LNPs的形成，保持了其结构完整性。Our findings showed that the hydrodynamic diameters of LNPs containing AS3-EECs ranged from 87.8 nm to 102.1 nm in different AS3-EECs, slightly larger than control LNPs without peptide. The encapsulation efficiency of mRNA in all three peptide-containing LNPs was close to 100%. These LNPs showed a uniform size distribution with a polydispersity index (PDI) of less than 0.1 and an almost neutral zeta potential. These results indicate that encapsulation of Cofilin peptides with mRNA did not interfere with the formation of LNPs, maintaining their structural integrity.
此外，随着GFP-mRNA输入量在三个测试的细胞系中的增加，我们观察到GFP表达水平呈剂量依赖性增加。在转染了0.2μg/孔GFP-mRNA的293T细胞中，与SM102(mRNA-LNPs中常用的脂质)包裹的LNPs相比，GFP-LNPs包裹的AS3-EEC1(SEQ ID NO:1)、AS3-EEC2(SEQ IDNO:2)和AS3-EEC3(SEQ ID NO:14)的表达水平分别增加了21％、54％和21％。转染后72小时和96小时也观察到类似的趋势，AS3-EEC1的表达水平略有下降，而AS3-EEC2和AS3-EEC3的表达水平则保持不变。转染了0.2μg/孔GFP-mRNA的A549细胞中，GFP的最高表达水平出现在转染了AS3-EEC2的GFP-LNP中。在48小时、72小时和96小时的转染后，其表达水平分别相比SM102增加了123％、50％和56％。转染AS3-EEC1或AS3-EEC3的细胞在48小时后表达水平初步增加，随后在后续时间点回落到与SM102相似或略低的水平。在转染了0.2μg/孔GFP-LNPs包裹的AS3-EEC2的B16细胞中，与SM102相比，在转染后的48小时、72小时和96小时，表达水平分别增加了156％、60％和216％。转染了AS3-EEC1和AS3-EEC3的细胞在48小时后显示GFP表达水平上升，但在72小时时下降。然而，在96小时时，与SM102相比，表达水平恢复或甚至进一步增加。In addition, we observed a dose-dependent increase in GFP expression levels with increasing GFP-mRNA input in the three tested cell lines. In 293T cells transfected with 0.2 μg/well GFP-mRNA, the expression levels of AS3-EEC1 (SEQ ID NO: 1), AS3-EEC2 (SEQ ID NO: 2), and AS3-EEC3 (SEQ ID NO: 14) encapsulated in GFP-LNPs increased by 21%, 54%, and 21%, respectively, compared to LNPs encapsulated with SM102 (a lipid commonly used in mRNA-LNPs). Similar trends were observed at 72 and 96 hours after transfection, with a slight decrease in the expression level of AS3-EEC1, while the expression levels of AS3-EEC2 and AS3-EEC3 remained unchanged. In A549 cells transfected with 0.2 μg/well GFP-mRNA, the highest expression level of GFP was observed in GFP-LNPs transfected with AS3-EEC2. After 48, 72, and 96 hours of transfection, its expression level increased by 123%, 50%, and 56%, respectively, compared with SM102. Cells transfected with AS3-EEC1 or AS3-EEC3 showed an initial increase in expression levels after 48 hours, followed by a drop back to levels similar to or slightly lower than SM102 at subsequent time points. In B16 cells transfected with 0.2 μg/well GFP-LNPs-encapsulated AS3-EEC2, expression levels increased by 156%, 60%, and 216%, respectively, at 48, 72, and 96 hours after transfection compared with SM102. Cells transfected with AS3-EEC1 and AS3-EEC3 showed an increase in GFP expression levels after 48 hours, but a decrease at 72 hours. However, at 96 hours, expression levels recovered or even increased further compared with SM102.
综上所述，我们的结果表明，来源于Cofilin的多肽在被包裹进LNPs时显著提高了GFP mRNA的转染效率。这些发现表明，Cofilin源多肽有望作为有价值的工具，通过促进RNA药物的传递和随后的蛋白质表达，改善RNA治疗。In summary, our results demonstrate that cofilin-derived peptides significantly enhance the transfection efficiency of GFP mRNA when encapsulated into LNPs. These findings suggest that cofilin-derived peptides may serve as valuable tools to improve RNA therapeutics by facilitating the delivery of RNA drugs and subsequent protein expression.
材料和方法Materials and methods
LNP mRNA的制备和表征。Preparation and characterization of LNP mRNA.
LNPs是使用Precision NanoSystems的NanoAssemblr微流体装置采用自下而上的方法制备的。为了确保质量控制，利用核磁共振(NMR)对脂质进行了表征。LNPs were prepared using a bottom-up approach using a NanoAssemblr microfluidic device from Precision NanoSystems. To ensure quality control, lipids were characterized using nuclear magnetic resonance (NMR).
在对照组中，采用Moderna标准配方，包括SM102、Cholesterol、DSPC和PEG-DMG，按摩尔比为50:38:10:2，与无水乙醇混合在一个管中。在测试组中，多肽AS3-EEC1、AS3-EEC2或AS3-EEC3以相对于其他组分2％的摩尔比添加：AS3-EECs：SM102：胆固醇：DSPC：PEG-DMG的比例为2：49：9.8：37.7：1.5。GFP mRNA在pH为3.0的无RNase的50mM柠檬酸盐缓冲液中稀释。水溶液和乙醇溶液以3:1的体积比混合，混合速率为12ml/min，得到mRNA:脂质重量比为10:1的LNPs。随后，使用分子量截至为10kD的Slide-A-Lyzer G2透析盒(Thermo FisherScientific)进行隔夜透析。通过Malvern Panalytical的Zetasizer Nano ZS进行动态光散射(DLS)测量以确定LNPs的大小。利用RiboGreen试剂测定了包封效率和mRNA浓度。ζ电位测量在pH为7.4±0.1的条件下进行，通过将LNPs在1mM磷酸盐缓冲液中稀释，使最终mRNA浓度达到约1-2μg/ml。在稀释后测量样品的pH，并根据需要使用0.1M NaOH或0.1M HCl进行调整。这些测量使用Zetasizer Nano ZS仪器进行。报告的值使用Smolucho方程计算，代表三次测量的平均值，误差报告为标准偏差(SD)。In the control group, the Moderna standard formulation, including SM102, Cholesterol, DSPC and PEG-DMG, was mixed with anhydrous ethanol in a molar ratio of 50:38:10:2 in one tube. In the test group, the peptides AS3-EEC1, AS3-EEC2 or AS3-EEC3 were added at a molar ratio of 2% relative to the other components: AS3-EECs: SM102: Cholesterol: DSPC: PEG-DMG in a ratio of 2:49:9.8:37.7:1.5. GFP mRNA was diluted in RNase-free 50 mM citrate buffer at pH 3.0. The aqueous and ethanolic solutions were mixed in a volume ratio of 3:1 at a mixing rate of 12 ml/min to obtain LNPs with a mRNA: lipid weight ratio of 10:1. Subsequently, overnight dialysis was performed using a Slide-A-Lyzer G2 dialysis cassette (Thermo Fisher Scientific) with a molecular weight cutoff of 10 kD. Dynamic light scattering (DLS) measurements were performed by Malvern Panalytical's Zetasizer Nano ZS to determine the size of the LNPs. Encapsulation efficiency and mRNA concentration were determined using RiboGreen reagent. Zeta potential measurements were performed at pH 7.4 ± 0.1 by diluting the LNPs in 1 mM phosphate buffer to a final mRNA concentration of approximately 1-2 μg/ml. The pH of the samples was measured after dilution and adjusted as needed using 0.1 M NaOH or 0.1 M HCl. These measurements were performed using a Zetasizer Nano ZS instrument. The reported values were calculated using the Smolucho equation and represent the average of three measurements, with errors reported as standard deviations (SD).
多肽的合成Synthesis of peptides
多肽以醋酸盐的形式由Alan Scientific Inc合成，程序如下：使用SymphonyQuartet肽合成仪(Ranin)和rinda酰胺MBHA树脂作为固体支持进行Fmoc固相肽合成。合成的多肽经过标准条件(95％ TFA与水和TIS)裂解和去保护，随后用冷二乙醚沉淀。使用Agilent Prep C18mm柱的预制规模RP-HPLC进行纯化，通过质谱使用α-CHCA基质(Voyager，Applied Biosystems DE-Pro MALDI-TOF)确认多肽的纯度和大小。然后将多肽冻干，并在10mM DMSO中重新悬浮，短期存储在-20℃，长期存储在-80℃。细胞培养和转染分析。293T、B16和A549细胞系来自ATCC。这些细胞在含有10％ FBS、100U/ml青霉素和100U/ml链霉素的DMEM培养基中培养。为了实验需要，细胞以每孔2×105个细胞的密度种植在96孔板中，每孔含有200μl的培养基。为了将GFP mRNA传递到细胞中，使用了LNPs。转染过程是通过添加不同浓度的被LNPs包埋的mRNA进行的：8ng/mL，24ng/mL，72ng/mL，216ng/mL，648ng/mL和1944ng/mL。转染后，在不同的时间点收集细胞进行后续分析。具体而言，时间点分别是转染后的48小时、72小时和96小时。在实验中，使用Lipofectamine 2000作为GFPmRNA活力的阳性对照。The peptides were synthesized in acetate form by Alan Scientific Inc. The procedure was as follows: Fmoc solid phase peptide synthesis was performed using a SymphonyQuartet peptide synthesizer (Ranin) and rinda amide MBHA resin as a solid support. The synthesized peptides were cleaved and deprotected under standard conditions (95% TFA with water and TIS) and then precipitated with cold diethyl ether. Agilent Prep C18 The peptides were purified by prefabricated RP-HPLC with a 100 mm column, and the purity and size of the peptides were confirmed by mass spectrometry using an α-CHCA matrix (Voyager, Applied Biosystems DE-Pro MALDI-TOF). The peptides were then lyophilized and resuspended in 10 mM DMSO for short-term storage at -20 °C and long-term storage at -80 °C. Cell culture and transfection analysis. 293T, B16, and A549 cell lines were obtained from ATCC. These cells were cultured in DMEM medium containing 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin. For experimental needs, cells were planted in 96-well plates at a density of 2 × 105 cells per well, each well containing 200 μl of culture medium. In order to deliver GFP mRNA to cells, LNPs were used. The transfection process was performed by adding different concentrations of mRNA embedded in LNPs: 8ng/mL, 24ng/mL, 72ng/mL, 216ng/mL, 648ng/mL and 1944ng/mL. After transfection, cells were collected at different time points for subsequent analysis. Specifically, the time points were 48 hours, 72 hours and 96 hours after transfection. In the experiment, Lipofectamine 2000 was used as a positive control for GFP mRNA activity.
流式细胞术Flow Cytometry
在Coulter Epics XL流式细胞仪上进行GFP表达分析。细胞在转染后的适当时间点被胰酶化，用磷酸盐缓冲盐水(PBS)洗涤，然后固定在2％的甲醛中。使用Argon激光器的488nm线激发GFP，采集520mm(绿色荧光)和575nm(红色荧光)的发射光，以通过对角门控进行自荧光校正。使用模拟测试的细胞确定背景荧光和自荧光。排除显示减少侧向散射和前向散射的细胞碎片，以进行分析。数值以中位相对荧光强度(MFI)单位呈现。GFP expression analysis was performed on a Coulter Epics XL flow cytometer. Cells were trypsinized at appropriate time points after transfection, washed with phosphate-buffered saline (PBS), and then fixed in 2% formaldehyde. GFP was excited using the 488 nm line of the Argon laser, and emission light at 520 nm (green fluorescence) and 575 nm (red fluorescence) was collected for autofluorescence correction by diagonal gating. Background fluorescence and autofluorescence were determined using mock-tested cells. Cell debris showing reduced side and forward scatter was excluded for analysis. Values are presented in median relative fluorescence intensity (MFI) units.
结果result
LNPs的理化和结构特征。Physicochemical and structural characteristics of LNPs.
为了探讨源自Cofilin的肽是否能够提高LNPs介导的GFP mRNA转染效率，我们采用了SM102、Cholesterol、DSPC和PEG-DMG的组合，摩尔比为2:49:9.8:37.7:1.5，将GFPmRNA和多肽一同包埋。然后，我们将这些含有肽的LNPs的理化和结构性质与采用SM102、胆固醇、DSPC和PEG-DMG的FDA批准的脂质组合的LNPs进行比较，其摩尔比为50:38:10.5:1.5，被广泛认为是RNA-LNPs治疗的黄金标准。To investigate whether cofilin-derived peptides could improve the efficiency of LNPs-mediated GFP mRNA transfection, we used a combination of SM102, Cholesterol, DSPC, and PEG-DMG at a molar ratio of 2:49:9.8:37.7:1.5 to co-entrap GFP mRNA and peptides. We then compared the physicochemical and structural properties of these peptide-containing LNPs with those of LNPs using an FDA-approved lipid combination of SM102, cholesterol, DSPC, and PEG-DMG at a molar ratio of 50:38:10.5:1.5, which is widely considered the gold standard for RNA-LNPs therapy.
LNPs是使用NanoAssemblerTM微混合器制备的，这是一种微流控混合方法，确保LNPs的高批间可重复性和批内均匀性。含有肽的LNPs的水动力直径分别为AS3-EEC1为97.2nm，AS3-EEC2为88.1nm，AS3-EEC3为102.1nm，略大于没有肽的对照组，测得为87.8nm。所有三种含有肽的LNPs中mRNA的包裹效率接近100％。这些LNPs显示出均匀的尺寸分布，多分散指数(PDI)小于0.1，几乎中性的ζ电位。这些结果表明，将Cofilin肽与mRNA一同包裹未干扰LNPs的形成，保持了其结构完整性。The LNPs were prepared using a NanoAssembler TM micromixer, a microfluidic mixing method that ensures high batch-to-batch reproducibility and intra-batch uniformity of the LNPs. The hydrodynamic diameters of the peptide-containing LNPs were 97.2 nm for AS3-EEC1, 88.1 nm for AS3-EEC2, and 102.1 nm for AS3-EEC3, which were slightly larger than the control group without peptide, which was measured to be 87.8 nm. The encapsulation efficiency of mRNA in all three peptide-containing LNPs was close to 100%. These LNPs showed a uniform size distribution with a polydispersity index (PDI) of less than 0.1 and an almost neutral zeta potential. These results indicate that encapsulation of the Cofilin peptide together with the mRNA did not interfere with the formation of the LNPs, maintaining their structural integrity.
活化Cofilin的肽通过LNPs改善GFP mRNA的转染效率。Cofilin-activating peptides improved the transfection efficiency of GFP mRNA via LNPs.
使用FDA批准的LNPs配方进行高效的转染仍然是一项具有挑战性的任务。因此，我们的目标是通过调节内吞作用的与Actin相关的蛋白质，特别是Cofilin，来探索潜在的改进。我们的目标是通过操纵上游信号来调控Cofilin的活性。在人类细胞中，Cofilin在Ser-3的磷酸化通过LIM家族蛋白激酶(LIMK)的基础活性受到抑制。为了抑制Cofilin的磷酸化，我们开发了一种名为AS3的多肽，包含人类Cofilin的N末端16个氨基酸，其中包括Ser-3。这些肽被设计成与LIMK1竞争Cofilin。为了改变其亲疏水性以提高包裹效率，我们在AS3肽的C末端引入了一系列的替代突变(SEQ ID NO:1到SEQ ID NO:12)。此外，内体逃逸结构域被连接到AS3的C末端(SEQ ID NO:12到SEQ ID NO:31)，被设计成AS3-EECs。随后，我们将AS3或AS2-EECs与GFP-mRNA一起包裹到LNP中。在使用LNPs转染三种人类细胞系之后，通过使用流式细胞仪评估荧光强度来测量得到的GFP表达水平。Efficient transfection using FDA-approved LNPs formulations remains a challenging task. Therefore, we aimed to explore potential improvements by modulating Actin-related proteins involved in endocytosis, particularly cofilin. We aimed to regulate the activity of cofilin by manipulating upstream signals. In human cells, phosphorylation of cofilin at Ser-3 is inhibited by the basal activity of LIM family protein kinases (LIMKs). To inhibit the phosphorylation of cofilin, we developed a peptide named AS3 containing the N-terminal 16 amino acids of human cofilin, including Ser-3. These peptides were designed to compete with LIMK1 for cofilin. To alter its hydrophilicity to improve encapsulation efficiency, we introduced a series of substitution mutations at the C-terminus of the AS3 peptide (SEQ ID NO:1 to SEQ ID NO:12). In addition, an endosomal escape domain was attached to the C-terminus of AS3 (SEQ ID NO:12 to SEQ ID NO:31), designed as AS3-EECs. Subsequently, we encapsulated AS3 or AS2-EECs into LNPs together with GFP-mRNA. After transfection of three human cell lines with LNPs, the resulting GFP expression levels were measured by assessing fluorescence intensity using flow cytometry.
如图2所示，随着GFP-mRNA的输入增加，GFP的表达水平呈剂量依赖性增加。在293T细胞中，使用0.2μg/孔的GFP-mRNA，与SM102相比，GFP的表达水平在转染48小时后分别增加了21％，54％和21％，分别由GFP-LNPs包裹的SM102-AS3-EEC1、SM102-AS3-EEC2和SM102-AS3-EEC3。在转染后的72和96小时，与SM102相比，SM102-AS3-EEC2和SM102-AS3-EEC3的表达水平与48小时时相似，但与SM102相比，SM102-AS3-EEC1略有下降，下降了9％。As shown in Figure 2, the expression level of GFP increased in a dose-dependent manner with increasing input of GFP-mRNA. In 293T cells, using 0.2 μg/well of GFP-mRNA, the expression levels of GFP increased by 21%, 54%, and 21% at 48 h after transfection for SM102-AS3-EEC1, SM102-AS3-EEC2, and SM102-AS3-EEC3 encapsulated by GFP-LNPs, respectively, compared with SM102. At 72 and 96 h after transfection, the expression levels of SM102-AS3-EEC2 and SM102-AS3-EEC3 were similar to those at 48 h, but SM102-AS3-EEC1 decreased slightly by 9% compared with SM102.
如图3所示，在转染0.2μg/孔的GFP-mRNA的A549细胞中，GFP的最高表达水平出现在GFP-LNP包裹的SM102-AS3-EEC2转染的细胞中。与SM102相比，分别在转染后的48小时、72小时和96小时的表达水平为123％、50％和56％。在转染SM102-AS3-EEC1或SM102-AS3-EEC3的细胞中，GFP的表达水平在48小时后增加了约30％，然后下降到与SM102相似或稍低的水平。As shown in Figure 3, in A549 cells transfected with 0.2 μg/well of GFP-mRNA, the highest expression level of GFP appeared in cells transfected with SM102-AS3-EEC2 encapsulated with GFP-LNP. Compared with SM102, the expression levels at 48 hours, 72 hours, and 96 hours after transfection were 123%, 50%, and 56%, respectively. In cells transfected with SM102-AS3-EEC1 or SM102-AS3-EEC3, the expression level of GFP increased by about 30% after 48 hours and then decreased to a level similar to or slightly lower than that of SM102.
如图4所示，与A549细胞中的观察相似，在转染0.2μg/孔的GFP-LNP包裹的SM102-AS3-EEC2的B16细胞中，GFP的最高表达水平出现。在转染后的48小时、72小时和96小时，与SM102相比，表达水平分别增加了156％、60％和216％。转染GFP-LNP包裹的SM102-AS3-EEC1和SM102-AS3-EEC3的细胞，GFP的表达水平在转染48小时后分别增加了31％和55％。然而，在72小时时，与SM102相比，表达水平分别下降至-13％和2％。在96小时时，与SM102相比，表达水平再次分别增加了35％和77％。在转染三种不同AS3的细胞中观察到了类似的时间模式的GFP表达变化。表达水平在转染48小时后增加，然后在72小时后明显下降。然而，在96小时时，与SM102相比，表达水平恢复或甚至进一步增加。As shown in Figure 4, similar to the observations in A549 cells, the highest expression level of GFP occurred in B16 cells transfected with 0.2 μg/well of GFP-LNP-encapsulated SM102-AS3-EEC2. At 48 hours, 72 hours, and 96 hours after transfection, the expression levels increased by 156%, 60%, and 216%, respectively, compared with SM102. In cells transfected with GFP-LNP-encapsulated SM102-AS3-EEC1 and SM102-AS3-EEC3, the expression levels of GFP increased by 31% and 55%, respectively, 48 hours after transfection. However, at 72 hours, the expression levels decreased to -13% and 2%, respectively, compared with SM102. At 96 hours, the expression levels increased again by 35% and 77%, respectively, compared with SM102. Similar temporal patterns of GFP expression changes were observed in cells transfected with three different AS3s. The expression level increased 48 hours after transfection and then decreased significantly after 72 hours. However, at 96 h, the expression levels were restored or even further increased compared to SM102.
肽AS3-EEC1(SEQ ID NO：1)、AS3-EEC2(SEQ ID NO：2)和AS3-EEC3(SEQ ID NO：14)的实验和结果如上文在本申请中已被详细描述。对其他肽(SEQ ID NO:3、SEQ ID NO:4、SEQID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ IDNO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ IDNO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ IDNO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ IDNO:30和SEQ ID NO:31)的实验可以采用与AS3-EEC1(SEQ ID NO:1)、AS3-EEC2(SEQ ID NO:2)和AS3-EEC3(SEQ ID NO:14)相同的方法进行，获得类似的结果。The experiments and results of peptides AS3-EEC1 (SEQ ID NO: 1), AS3-EEC2 (SEQ ID NO: 2) and AS3-EEC3 (SEQ ID NO: 14) have been described in detail above in the present application. For other peptides (SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:1 9. Experiments with SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31) can be used with AS3-EEC1 (SEQ ID NO: 1), AS3-EEC2 (SEQ ID NO:2) and AS3-EEC3 (SEQ ID NO:2 NO:14) was carried out in the same manner and similar results were obtained.
熟悉该领域的技术人员将明白，可以在不偏离本发明的精神或范围的情况下对本发明进行各种修改和变化。因此，本发明的修改和变化应涵盖在附属权利要求及其等效范围内。Those skilled in the art will appreciate that various modifications and variations of the present invention may be made without departing from the spirit or scope of the present invention. Therefore, the modifications and variations of the present invention should be covered within the scope of the appended claims and their equivalents.
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