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Characterisation of AAV Vectors: A Review of Analytical Techniques and Critical Quality Attributes AAV 载体的表征:分析技术和关键质量属性综述
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-30 DOI: 10.1016/j.omtm.2024.101309

Standardised evaluation of adeno-associated virus (AAV) vector products for biotherapeutic application is essential to ensure the safety and efficacy of gene therapies. This includes analysing the critical quality attributes of the product. However, many of the current analytical techniques used to assess these attributes have limitations, including low-throughput, large sample requirements, poorly understood measurement variability, and lack of comparability between methods. To address these challenges, it is essential to establish higher-order reference methods that can be used for comparability measurements, optimisation of current assays, and development of reference materials. Highly precise methods are necessary for measuring the empty/partial/full capsid ratios and the titre of AAV vectors. Additionally, it is important to develop methods for the measurement of less established critical quality attributes, including post-translational modifications, capsid stoichiometry, and methylation profiles. By doing so, we can gain a better understanding of the influence of these attributes on the quality of the product. Moreover, quantification of impurities, such as host cell proteins and DNA contaminants, is crucial for obtaining regulatory approval. The development and application of refined methodologies will be essential to thoroughly characterise AAV vectors, by informing process development and facilitating the generation of reference materials for assay validation and calibration.

对用于生物治疗的腺相关病毒(AAV)载体产品进行标准化评估,对于确保基因疗法的安全性和有效性至关重要。这包括分析产品的关键质量属性。然而,目前用于评估这些属性的许多分析技术都存在局限性,包括通量低、样品需求量大、对测量变异性了解甚少以及不同方法之间缺乏可比性。为应对这些挑战,必须建立更高阶的参考方法,用于可比性测量、优化当前检测方法和开发参考材料。测量 AAV 向量的空/部分/全囊壳比率和滴度需要高精度的方法。此外,开发测量翻译后修饰、囊壳配比和甲基化特征等不太成熟的关键质量属性的方法也很重要。这样,我们就能更好地了解这些属性对产品质量的影响。此外,杂质(如宿主细胞蛋白和 DNA 杂质)的定量对于获得监管部门的批准也至关重要。开发和应用完善的方法对彻底鉴定 AAV 向量至关重要,可为工艺开发提供信息,并促进用于检测验证和校准的参考材料的生成。
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引用次数: 0
A pseudotyped adenovirus serotype 5 vector with serotype 49 fiber knob is an effective vector for vaccine and gene therapy applications 带有血清 49 型纤维钮的伪型腺病毒血清 5 型载体是疫苗和基因治疗应用的有效载体
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-30 DOI: 10.1016/j.omtm.2024.101308

Adenoviruses (Ad) have demonstrated significant success as replication-deficient (RD) viral vectored vaccines, as well as broad potential across gene therapy and cancer therapy. Ad vectors transduce human cells via direct interactions between the viral fiber knob and cell surface receptors, with secondary cellular integrin interactions. Ad receptor usage is diverse across the extensive phylogeny. Commonly studied human Ad serotype 5 (Ad5), and chimpanzee Ad-derived vector “ChAdOx1” in licensed ChAdOx1 nCoV-19 vaccine, both form primary interactions with the Coxsackie and Adenovirus Receptor (CAR), which is expressed on human epithelial cells and erythrocytes. CAR usage is suboptimal for targeted gene delivery to cells with low/negative CAR expression, including human dendritic cells (DCs) and vascular smooth muscle cells (VSMCs). We evaluated the performance of a RD Ad5 vector pseudotyped with the fiber knob of human Ad serotype 49, termed Ad5/49K vector. Ad5/49K demonstrated superior transduction of murine and human DCs over Ad5, which translated into significantly increased T cell immunogenicity when evaluated in a mouse cancer vaccine model using 5T4 tumour-associated antigen. Additionally, Ad5/49K exhibited enhanced transduction of primary human VSMCs. These data highlight the potential of Ad5/49K vector for both vascular gene therapy applications and as a potent vaccine vector.

腺病毒(Ad)作为复制缺陷(RD)病毒载体疫苗取得了巨大成功,并在基因治疗和癌症治疗方面具有广泛的潜力。广告载体通过病毒纤维钮与细胞表面受体之间的直接相互作用以及次要的细胞整合素相互作用转导人体细胞。在广泛的系统发育过程中,广告受体的用法多种多样。常见的人类 Ad 血清型 5(Ad5)和已获许可的 ChAdOx1 nCoV-19 疫苗中的黑猩猩 Ad 衍生载体 "ChAdOx1",都与柯萨奇病毒和腺病毒受体(CAR)形成主要的相互作用,后者在人类上皮细胞和红细胞上表达。对于低/阴性 CAR 表达的细胞,包括人类树突状细胞(DC)和血管平滑肌细胞(VSMC),使用 CAR 进行基因靶向递送并不理想。我们评估了以人类 Ad 血清型 49 的纤维结为假型的 RD Ad5 向量(称为 Ad5/49K 向量)的性能。与 Ad5 相比,Ad5/49K 对小鼠和人类 DC 的转导能力更强,在使用 5T4 肿瘤相关抗原的小鼠癌症疫苗模型中进行评估时,T 细胞免疫原性显著提高。此外,Ad5/49K 对原代人类 VSMC 的转导能力也有所增强。这些数据凸显了 Ad5/49K 载体在血管基因治疗应用和作为强效疫苗载体方面的潜力。
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引用次数: 0
A randomized double-blind Phase IIb trial to evaluate the efficacy of ChAd63-KH for the treatment of post kala-azar dermal leishmaniasis. 一项随机双盲 IIb 期试验,评估 ChAd63-KH 治疗卡拉-扎尔皮肤利什曼病后的疗效。
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-30 DOI: 10.1016/j.omtm.2024.101310

In a recent Phase IIa clinical trial, the candidate leishmaniasis vaccine ChAd63-KH was shown to be safe and immunogenic in Sudanese patients with post kala- azar dermal leishmaniasis. However, its value as a stand-alone therapeutic was unknown. To assess the therapeutic efficacy of ChAd63-KH, we conducted a randomized, double-blind, placebo-controlled Phase IIb trial (Clinicaltrials.gov registration: NCT03969134). Primary outcomes were safety and efficacy (≥90% improvement in clinical disease). Secondary outcomes were change in severity grade and vaccine-induced immune response. 86 participants with uncomplicated post kala azar dermal leishmaniasis of ≥ six months duration were randomised to receive ChAd63-KH (7.5x1010 viral particles once i.m.) or placebo. 75 participants (87%) completed the trial as per protocol. No severe or serious adverse events were observed. At day 90 post vaccination, 6/40 (15%) and 4/35 (11%) participants in the vaccine and placebo groups respectively showed ≥ 90% clinical improvement (RR 1.31 [95% CI, 0.40 to 4.28], p=0.742). There were also no significant differences in PKDL severity grade between study arms. Whole blood transcriptomic analysis identified transcriptional modules associated with interferon responses and monocyte and dendritic cell activation. Thus, a single vaccination with ChAd63-KH showed no therapeutic efficacy in this subset of Sudanese PKDL patients.

在最近的一项 IIa 期临床试验中,候选利什曼病疫苗 ChAd63-KH 被证明对患有卡拉扎皮肤利什曼病后的苏丹患者安全且具有免疫原性。然而,它作为一种独立疗法的价值尚不清楚。为了评估 ChAd63-KH 的疗效,我们进行了一项随机、双盲、安慰剂对照 IIb 期试验(Clinicaltrials.gov 注册:NCT03969134)。主要结果是安全性和有效性(临床疾病改善≥90%)。次要结果是严重程度等级的变化和疫苗诱导的免疫反应。86 名病程≥ 6 个月的无并发症卡拉扎后皮肤利什曼病患者被随机分配接受 ChAd63-KH(7.5x1010 病毒颗粒,一次 i.m.)或安慰剂治疗。75名参与者(87%)按照方案完成了试验。未发现严重不良反应。接种后第90天,疫苗组和安慰剂组分别有6/40(15%)和4/35(11%)的参与者临床症状改善≥90%(RR 1.31 [95% CI, 0.40 to 4.28],P=0.742)。各研究臂之间的PKDL严重程度等级也无明显差异。全血转录组分析确定了与干扰素反应以及单核细胞和树突状细胞活化相关的转录模块。因此,用 ChAd63-KH 单次接种对这部分苏丹 PKDL 患者没有疗效。
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引用次数: 0
DNA-PK inhibition enhances gene editing efficiency in HSPCs for CRISPR-based treatment of X-linked hyper IgM syndrome 抑制DNA-PK可提高HSPC中的基因编辑效率,用于基于CRISPR技术治疗X连锁高IgM综合征
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-27 DOI: 10.1016/j.omtm.2024.101297

Targeted gene editing to restore CD40L expression via homology-directed repair (HDR) in CD34+ hematopoietic stem and progenitor cells (HSPCs) represents a potential long-term therapy for X-linked hyper IgM syndrome. However, clinical translation of HSPC editing is limited by inefficient long-term engraftment of HDR-edited HSPCs. Here, we ameliorate this issue by employing a small-molecule inhibitor of DNA-PKcs, AZD7648, to bias DNA repair mechanisms to facilitate HDR upon CRISPR SpCas9-based gene editing. Using AZD7648 treatment and a clinically relevant HSPC source, mobilized peripheral blood CD34+ cells, we achieve ∼60% HDR efficiency at the CD40LG locus and enhanced engraftment of HDR-edited HSPCs in primary and secondary xenotransplants. Specifically, we observed a 1.6-fold increase of HDR-edited long-term HSPCs in primary transplant recipients without disturbing chimerism levels or differentiation capacity. As CD40L is primarily expressed in T cells, we demonstrate T cell differentiation from HDR-edited HSPCs in vivo and in artificial thymic organoid cultures, and endogenously regulated CD40L expression following activation of in-vivo-derived CD4+ T cells. Our combined findings demonstrate HDR editing at the CD40LG locus at potentially clinically beneficial levels. More broadly, these data support using DNA-PKcs inhibition with AZD7648 as a simple and efficacious addition to HSPC editing platforms.

通过同源定向修复(HDR)对CD34+造血干细胞和祖细胞(HSPCs)进行靶向基因编辑以恢复CD40L的表达,是治疗X连锁高IgM综合征的一种潜在的长期疗法。然而,HDR编辑的HSPC长期移植效率低下,限制了HSPC编辑的临床应用。在这里,我们采用DNA-PKcs的小分子抑制剂AZD7648来改善这一问题,使DNA修复机制发生偏倚,从而促进基于CRISPR SpCas9的基因编辑后的HDR。利用AZD7648和临床相关的HSPC来源--动员的外周血CD34+细胞,我们在CD40LG基因座上实现了60%的HDR效率,并增强了HDR编辑的HSPC在原代和继代异种移植中的移植效果。具体来说,我们观察到,在不影响嵌合水平或分化能力的情况下,原代移植受者中经 HDR 编辑的长期 HSPC 增加了 1.6 倍。由于 CD40L 主要在 T 细胞中表达,我们证明了 HDR 编辑的 HSPCs 在体内和人工胸腺类器官培养物中的 T 细胞分化,以及活化体内衍生的 CD4+ T 细胞后内源性调控的 CD40L 表达。我们的综合研究结果表明,CD40LG基因座的HDR编辑可能对临床有益。更广泛地说,这些数据支持使用 AZD7648 抑制 DNA-PKcs 作为 HSPC 编辑平台简单而有效的补充。
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引用次数: 0
Bio-Layer Interferometry for Adeno-Associated Virus Capsid Titer Measurement and Applications to Upstream and Downstream Process Development 用于腺相关病毒囊盖滴度测量的生物层干涉测量法及其在上下游工艺开发中的应用
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-25 DOI: 10.1016/j.omtm.2024.101306

Faster and more accurate analytical methods are needed to support the advancement of recombinant adeno-associated virus (rAAV) production systems. Recently, bio-layer interferometry (BLI) has been developed for high-throughput AAV capsid titer measurement by functionalization of the AAVX ligand onto biosensor probes (AAVX-BLI). In this work, an AAVX-BLI method was evaluated using Octet® AAVX biosensors across four rAAV serotypes (rAAV2, 5, 8, and 9) and applied in an upstream and downstream processing context. AAVX-BLI measured capsid titer across a wide concentration range (1×1010 – 1×1012 capsids/mL) for different rAAV serotypes and sample backgrounds with reduced measurement variance and error compared to an enzyme-linked immunosorbent assay (ELISA) method. Biosensors were regenerated for repeated use, with lysate samples showing reduced regeneration capacity compared to purified and supernatant samples. The AAVX-BLI method was applied in a transfection optimization study where direct capsid titer measurement of culture supernatants generated a representative response surface for total vector genome (VG) titer. For rAAV purification, AAVX-BLI was used to measure dynamic binding capacity with POROS™ CaptureSelect™ AAVX affinity chromatography, showing resin breakthrough dependence on operating flow rate. Measurement accuracy, serotype and sample background flexibility, and high sample throughput make AAVX-BLI an attractive alternative to other capsid titer measurement techniques.

重组腺相关病毒(rAAV)生产系统的发展需要更快、更准确的分析方法。最近,通过将 AAVX 配体功能化到生物传感器探针上(AAVX-BLI),开发出了生物层干涉测量法(BLI),用于高通量 AAV 胶囊滴度测量。在这项工作中,使用 Octet® AAVX 生物传感器对四种 rAAV 血清型(rAAV2、5、8 和 9)的 AAVX-BLI 方法进行了评估,并将其应用于上游和下游处理过程中。与酶联免疫吸附分析 (ELISA) 方法相比,AAVX-BLI 可在较宽的浓度范围(1×1010 - 1×1012 粒/毫升)内测量不同 rAAV 血清型和样品背景下的囊载体滴度,并降低了测量方差和误差。生物传感器可重复再生使用,与纯化样品和上清样品相比,裂解样品的再生能力较弱。AAVX-BLI 方法被应用于一项转染优化研究中,在该研究中,培养上清液的直接囊壳滴度测量为总载体基因组(VG)滴度生成了一个有代表性的响应面。在 rAAV 纯化方面,AAVX-BLI 被用于测量与 POROS™ CaptureSelect™ AAVX 亲和层析的动态结合能力,显示出树脂突破与操作流速的关系。AAVX-BLI 测量准确、血清型和样本背景灵活、样本吞吐量高,是其他噬菌体滴度测量技术的理想替代品。
{"title":"Bio-Layer Interferometry for Adeno-Associated Virus Capsid Titer Measurement and Applications to Upstream and Downstream Process Development","authors":"","doi":"10.1016/j.omtm.2024.101306","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101306","url":null,"abstract":"<p>Faster and more accurate analytical methods are needed to support the advancement of recombinant adeno-associated virus (rAAV) production systems. Recently, bio-layer interferometry (BLI) has been developed for high-throughput AAV capsid titer measurement by functionalization of the AAVX ligand onto biosensor probes (AAVX-BLI). In this work, an AAVX-BLI method was evaluated using Octet® AAVX biosensors across four rAAV serotypes (rAAV2, 5, 8, and 9) and applied in an upstream and downstream processing context. AAVX-BLI measured capsid titer across a wide concentration range (1×10<sup>10</sup> – 1×10<sup>12</sup> capsids/mL) for different rAAV serotypes and sample backgrounds with reduced measurement variance and error compared to an enzyme-linked immunosorbent assay (ELISA) method. Biosensors were regenerated for repeated use, with lysate samples showing reduced regeneration capacity compared to purified and supernatant samples. The AAVX-BLI method was applied in a transfection optimization study where direct capsid titer measurement of culture supernatants generated a representative response surface for total vector genome (VG) titer. For rAAV purification, AAVX-BLI was used to measure dynamic binding capacity with POROS™ CaptureSelect™ AAVX affinity chromatography, showing resin breakthrough dependence on operating flow rate. Measurement accuracy, serotype and sample background flexibility, and high sample throughput make AAVX-BLI an attractive alternative to other capsid titer measurement techniques.</p>","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782919","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Characterization of Residual MicroRNAs in AAV Vector Batches Produced in HEK293 Mammalian Cells and Sf9 Insect Cells 用 HEK293 哺乳动物细胞和 Sf9 昆虫细胞生产的 AAV 向量批次中残留 MicroRNA 的特征
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-25 DOI: 10.1016/j.omtm.2024.101305

With more than 130 clinical trials and eight approved gene therapy products, AAVs stand as one of the most popular vehicles to deliver therapeutic DNA in vivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell-derived RNAs, such as micro-RNAs, has not been previously investigated. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co-purified with the rAAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non-encapsidated. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.

AAV 目前已进行了 130 多项临床试验,并有 8 种基因治疗产品获得批准,是在体内输送治疗 DNA 的最常用工具之一。对 AAV 批次进行分析的一个关键质量属性是是否存在残留 DNA,因为残留 DNA 可能会带来基因毒性风险或诱发免疫反应。令人惊讶的是,以前从未调查过细胞衍生的小 RNA(如微 RNA)的存在。在这项研究中,我们检测了在哺乳动物细胞或昆虫细胞中生产的纯化 AAV 批次中是否存在 miRNA。我们的研究结果表明,无论生产细胞系或囊壳血清型(2 和 8)如何,所有批次的 AAV 中都存在 miRNA。定量检测表明,miRNA 与 rAAV 颗粒共同纯化的比例与它们在生产细胞中的丰度相关。通过免疫亲和层析纯化过程(包括切向流过滤步骤)或 RNase 处理,残留 miRNA 的水平降低了,这表明大多数 miRNA 杂质可能是非包囊化的。总之,我们首次证明了 miRNA 可与 AAV 颗粒共同纯化。要确定这些 miRNA 是否会干扰 AAV 介导的基因治疗的安全性或疗效,还需要进一步的研究。
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引用次数: 0
An amplification-free CRISPR/Cas12a assay for titer determination and composition analysis of the rAAV genome 用于 rAAV 基因组滴度测定和成分分析的无扩增 CRISPR/Cas12a 分析法
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-22 DOI: 10.1016/j.omtm.2024.101304

The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 109 and 1011 vg/mL. No significant difference was observed between the Cas12a and qPCR assay results (p﹤0.05, t-test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.

病毒基因组滴度是重组腺相关病毒(rAAV)基因治疗产品临床用药、生产和分析测试的重要指标。虽然定量 PCR 和数字 PCR 是量化 rAAV 基因组滴度的常用方法,但它们的准确性和稳健性不足。簇状规则间距短回文重复(CRISPR)-Cas12a 生物传感器正越来越多地应用于病毒检测,但目前还没有将其应用于基因治疗产品滴度测定的报道。本研究开发、优化了一种无扩增的 CRISPR-Cas12a 检测方法,并将其应用于 rAAV 基因组滴度测定。该检测方法在 4 × 109 和 1011 vg/mL 的检测范围内表现出较高的精确度和准确性。Cas12a 和 qPCR 检测结果之间无明显差异(p﹤0.05,t 检验)。此外,Cas12a 在单链和双链 DNA 底物上表现出相似的活性。根据这一特点,我们分别测定了正义链和负义链的滴度,结果显示它们的滴度与内部参考 AAV5-IN 的滴度存在显著差异。本研究首次报道了为测定滴度和分析 rAAV 基因组组成而开发的 Cas12a 检测方法。
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引用次数: 0
Atelocollagen supports three-dimensional culture of human induced pluripotent stem cells Atelocollagen 支持人类诱导多能干细胞的三维培养
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-20 DOI: 10.1016/j.omtm.2024.101302

As autologous iPSC therapy requires a custom-made small-lot cell production line, the cell production method differs significantly from the existing processes for producing allogeneic iPSC stocks for clinical use. Specifically, mass culture to produce stock is no longer necessary; instead, a series of operations from iPSC production to induction of differentiation of therapeutic cells must be performed continuously. A 3D culture method using small, closed-cell manufacturing devices is suitable for autologous iPSC therapy. Use of such devices avoids the need to handle many patient-derived specimens in a single clean room; handling of cell cultures in an open system in a cell processing facility increases the risk of infection. In this study, atelocollagen beads were evaluated as a three-dimensional biomaterial to assist 3D culture in the establishment, expansion culture, and induced differentiation of iPSCs. It was found that iPSCs can be handled in a closed-cell device with the same ease as use of 2D culture when Laminin-511 is added to the medium. In conclusion, atelocollagen beads enable 3D culture of iPSCs, and the quality of the obtained cells is at the same level as those derived from 2D culture.

由于自体 iPSC 疗法需要定制的小批量细胞生产线,因此细胞生产方法与现有的用于临床的异体 iPSC 储存生产工艺有很大不同。具体来说,不再需要大规模培养来生产储备细胞,而是必须连续进行从 iPSC 生产到诱导治疗细胞分化的一系列操作。使用小型封闭细胞制造装置的三维培养方法适用于自体 iPSC 治疗。使用这种装置可避免在一个洁净室中处理许多患者来源的标本;在细胞处理设施的开放系统中处理细胞培养物会增加感染风险。在这项研究中,对阿特劳胶原蛋白珠作为一种三维生物材料进行了评估,以协助三维培养iPSCs的建立、扩增培养和诱导分化。研究发现,在培养基中添加 Laminin-511 后,iPSCs 可在闭孔装置中进行处理,与使用二维培养一样方便。总之,atelocollagen 珠可以实现 iPSCs 的三维培养,而且获得的细胞质量与二维培养的细胞质量相同。
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引用次数: 0
Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation 通过糖基化调节可溶性 ACE2 诱饵受体的药代动力学
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-19 DOI: 10.1016/j.omtm.2024.101301

The Spike of SARS-CoV-2 recognizes a transmembrane protease, ACE2, on host cells to initiate infection. Soluble derivatives of ACE2, in which Spike affinity is enhanced and the protein is fused to Fc of an immunoglobulin, are potent decoy receptors that reduce disease in animal models of COVID-19. Mutations were introduced into an ACE2 decoy receptor, including adding custom N-glycosylation sites and a cavity-filling substitution together with Fc modifications, which increased the decoy's catalytic activity and provided small-to-moderate enhancements of pharmacokinetics following intravenous and subcutaneous administration in humanized FcRn mice. Most prominently, sialylation of native glycans increases exposures by orders of magnitude, and the optimized decoy is therapeutically efficacious in a mouse COVID-19 model. Ultimately, an engineered and highly sialylated decoy receptor produced using methods suitable for manufacture of representative drug substance has high exposure with a 5- to 9-day half-life. Finally, peptide epitopes at mutated sites in the decoys generally have low binding to common HLA class II alleles and the predicted immunogenicity risk is low. Overall, glycosylation is a critical molecular attribute of ACE2 decoy receptors and modifications that combine tighter blocking of Spike with enhanced pharmacokinetics elevate this class of molecules as viable drug candidates.

SARS-CoV-2 的 Spike 能识别宿主细胞上的跨膜蛋白酶 ACE2,从而启动感染。ACE2 的可溶性衍生物能增强 Spike 的亲和力,并与免疫球蛋白的 Fc 融合,是一种有效的诱饵受体,能减少 COVID-19 动物模型的发病率。我们在 ACE2 诱饵受体中引入了突变,包括添加定制的 N-糖基化位点和空腔填充替代以及 Fc 修饰,这些突变提高了诱饵的催化活性,并在人源化 FcRn 小鼠静脉注射和皮下注射后小幅至中度提高了药代动力学。最突出的是,原生聚糖的硅氨酰化将暴露量提高了几个数量级,优化后的诱饵在小鼠 COVID-19 模型中具有治疗效果。最终,使用适合制造代表性药物的方法生产出的工程化高度糖基化诱饵受体具有很高的暴露量,半衰期为 5 到 9 天。最后,诱饵中突变位点上的肽表位与常见的 HLA II 类等位基因的结合率一般较低,因此预测的免疫原性风险也较低。总之,糖基化是 ACE2 诱饵受体的一个关键分子属性,将更严格地阻断 Spike 与增强药代动力学相结合的修饰使这一类分子成为可行的候选药物。
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引用次数: 0
Treating late-onset Tay Sachs disease: Brain delivery with a dual trojan horse protein 治疗晚期 Tay Sachs 病:用双特洛伊木马蛋白输送大脑
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-17 DOI: 10.1016/j.omtm.2024.101300
Esther Osher, Yossi Anis, Ruth Singer-Shapiro, Nataly Urshanski, Tamar Unger, Shira Albeck, Oren Bogin, Gary Weisinger, Fortune Kohen, Avi Valevski, Aviva Fattal-Valevski, Liora Sagi, Michal Weitman, Yulia Shenberger, Nadav Sagiv, Ruth Navon, Meir Wilchek, Naftali Stern
Tay-Sachs (TS) disease is a neurodegenerative disease resulting from mutations in the gene encoding the α-subunit (HEXA) of lysosomal β-hexosaminidase A (HexA). We report that (1) recombinant HEXA alone increased HexA activity and decreased GM2 content in human TS glial cells and peripheral mononuclear blood cells; 2) a recombinant chimeric protein composed of HEXA linked to two blood-brain barrier (BBB) entry elements, a transferrin receptor binding sequence and granulocyte-colony stimulating factor, associates with HEXB ; reaches human cultured TS cells lysosomes and mouse brain cells, especially neurons, ; lowers GM2 in cultured human TS cells; lowers whole brain GM2 concentration by approximately 40% within 6 weeks, when injected intravenously (IV) to adult TS-mutant mice mimicking the slow course of late-onset TS; and increases forelimbs grip strength. Hence, a chimeric protein equipped with dual BBB entry elements can transport a large protein such as HEXA to the brain, decrease the accumulation of GM2, and improve muscle strength, thereby providing potential treatment for late-onset TS.
泰-萨克斯(Tay-Sachs,TS)病是一种神经退行性疾病,由溶酶体 β-己糖胺酶 A(HexA)α-亚基(HEXA)编码基因突变引起。我们报告说:(1) 重组 HEXA 本身会增加人 TS 神经胶质细胞和外周单核血细胞中 HexA 的活性并降低 GM2 的含量;(2) 由 HEXA 与两个血脑屏障(BBB)进入元件(转铁蛋白受体结合序列和粒细胞集落刺激因子)连接组成的重组嵌合蛋白会与 HEXB 结合;可到达人类培养的 TS 细胞溶酶体和小鼠脑细胞,尤其是神经元;降低培养的人类 TS 细胞中的 GM2;在模仿晚发性 TS 的缓慢病程对成年 TS 突变小鼠进行静脉注射(IV)时,可在 6 周内将整个大脑的 GM2 浓度降低约 40%;并增加前肢的握力。因此,具有双 BBB 入口元件的嵌合蛋白可以将 HEXA 等大分子蛋白转运到大脑,减少 GM2 的积累,并改善肌肉力量,从而为晚发性 TS 提供潜在的治疗方法。
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引用次数: 0
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Molecular Therapy-Methods & Clinical Development
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