HAP1

Findings To Be Presented During the European Society of Gene & Cell Therapy 30th Annual Meeting BRUSSELS--(BUSINESS WIRE)-- Be Biopharma Inc. (“Be Bio”), a company pioneering the discovery and development of Engineered B Cell Medicines (BCMs), will present research from preclinical studies during the European Society of Gene & Cell Therapy (ESGCT) 30th Annual Meeting. In an oral presentation on Friday, October 27, from 11:00 to 13:00 CEST, Be Bio demonstrates that BCMs are capable of producing a variety of therapeutic protein classes using precision gene editing. The data include durable in vivo production of the human clotting factor IX, prevention of tumor growth in a patient-derived xenograft model via a CD19xCD3 bispecific antibody, and engraftment without preconditioning in immunocompetent non-human primates (NHPs). Also being presented are findings from a preclinical study showing BCMs are capable of producing highly active acid sphingomyelinase, the gene defective in Niemann-Pick disease, and correct the disease phenotype in knockout cells. “The data we are presenting give further evidence that our precision B cell engineering platform enables the expression of diverse therapeutic proteins and represents a promising approach for a new class of medicines with potentially broad therapeutic utility,” said Rick Morgan, Ph.D., chief scientific officer, Be Biopharma. “These data highlight the therapeutic modularity and unique advantages of our BCM platform.” Today’s gene and cell therapies are potentially transformative medicines which can treat previously intractable diseases. However, there are barriers to the broad adoption of these therapies as current platforms struggle with drawbacks including inability to titrate dose and reliance on toxic preconditioning, among other challenges. Terminally differentiated human plasma cells (PCs) derived from genetically engineered B cells -- termed B Cell Medicines, or BCMs -- potentially offer natural longevity1, capacity for high levels of protein secretion (thousands of Ig molecules/cell/sec)2, ability to engraft without preconditioning, and the ability to redose. Study Summary: “Development of an ex vivo precision gene engineered B cell medicine platform and demonstration of engraftment without preconditioning in non-human primates” BCMs are produced via a CRISPR/Cas9 engineering platform that achieves gene knockouts with greater than 90% efficiency as well as targeted HDR-mediated gene insertions at frequencies as high as 60%. A BCM prototype engineered to express firefly luciferase with a phenotype of >90% CD27+CD38+ was injected via IV into immunodeficient mice, demonstrating rapid bone-marrow-homing and durable engraftment (>100 days). Further illustrating the modularity of the BCM platform, B cells were engineered to produce either lysosomal storage disease (LSD) enzyme acid sphingomyelinase (ASM), clotting factor IX (FIX), or an anti-CD19/CD3 bispecific T cell engager. These examples demonstrated that BCMs can produce proteins with specific activity much higher than standard recombinant proteins (ASM), are stably expressed for over 4 months in vivo (FIX) and show efficacy in tumor treatment (anti-CD19/CD3 scFv). In order to assess engraftment in an immunocompetent model, an ex vivo method was developed to engineer, expand and differentiate non-human primate (NHP) peripheral blood B lymphocytes into PCs. Using zirconium-89-oxine cell radiolabeling and high sensitivity PET/CT tracking, ex vivo expanded and differentiated autologous PCs demonstrated rapid homing and engraftment in PC niches in unconditioned NHPs. Details for the oral presentation of this study are as follows: Title: “Development of an ex vivo precision gene engineered B cell medicine platform and demonstration of engraftment without preconditioning in non-human primates” Lead Author: Hanlan Liu, Ph.D., MBA, Senior VP, Pipeline and Non-clinical Development, Be Biopharma Presenter: Rick Morgan, Ph.D., Chief Scientific Officer, Be Biopharma Oral Presentation #: OR-77 Date/Time: Friday, October 27, 11:00-13:00 CEST Session: 11a: Gene editing: Towards clinical trials The study was funded and conducted through a collaborative research and development agreement with the National Heart, Lung, and Blood Institute (NHLBI), part of the National Institutes of Health. Disclaimer: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Study Summary: “Development of an ex vivo precision gene engineered B cell medicine that produces highly active and sustained levels of acid sphingomyelinase for the treatment of Niemann-Pick disease” Niemann-Pick disease (NPD) is an autosomal recessive lysosomal storage disease caused by acid sphingomyelinase (ASM) deficiency. Sphingomyelin accumulates in multiple organs leading to organ failure, and in the severest form causes death early in life. Currently, the only treatment which is approved in NPB, a subset of NPD, is an enzyme replacement therapy (ERT) requiring an intensive dose escalation phase followed by Q2W maintenance intravenous infusions. This study evaluated the potential of engineering B cells to produce therapeutic levels of ASM, thus offering a potential new treatment for NPD. In this study, primary human B cells were expanded, engineered by CRISPR/Cas9 genome editing with an AAV-delivered template for homology-directed repair (HDR) to insert a constitutive promoter followed by the ASM gene, SMPD1, into a safe harbor locus, which showed up to 30% HDR. BCMs were subsequently expanded and differentiated in vitro. Engineered BCMs secreted ASM up to ~150 ng/1e6 cells/24hr as measured by ELISA. HAP1 SMPD1 knockout cells exposed to supernatant from SMPD1-engineered BCMs restored ASM activity. Liquid chromatography–mass spectrometry analysis demonstrated that HAP1 SMPD1 knockout cells treated with supernatant from SMPD1-engineered BCMs mitigated the accumulation of lyso-sphingomyelin and that BCM-produced ASM was approximately 10-fold more active than recombinant ASM made from CHO cells. Details for the poster presentation of this study are as follows: Title: “Development of an ex vivo precision gene engineered B cell medicine that produces highly active and sustained levels of acid sphingomyelinase for the treatment of Niemann-Pick disease” Lead Author: Monika Musial-Siwek, Ph.D., Director Protein Science, Be Biopharma Co-Presenters: Timothy Mullen, Ph.D., Senior Scientist, Be Biopharma Anja Hohmann, Ph.D., Senior Director, Cell Engineering, Be Biopharma Poster #: P579 Poster Session: October 25, 17:00 to 18:15, and October 26 from 20:30 to 21:30 CEST About B Cells – A New Class of Cellular Medicines Imagine what could “Be?” In nature, a single B cell engrafts in the bone marrow and can produce thousands of proteins per second at constant levels over decades. B cells are nature’s exquisite medicine factories, manufacturing proteins to fight disease and maintain health. Unleashing the power of B cells is driving a new class of cellular medicines – Engineered B Cell Medicines (BCMs). BCMs have the potential to be durable, allogeneic, redosable and administered without toxic conditioning. The promise of BCMs could transform therapeutic biologics with broad application — across protein classes, patient populations and therapeutic areas. About Be Biopharma Be Biopharma (“Be Bio”) is pioneering Engineered B Cell Medicines (BCMs) to dramatically improve the lives of patients who are living with cancer, rare diseases and other serious conditions. With eyes locked on the patient, our team of purpose-driven scientists, technologists, manufacturing experts and business builders collaborate to create a bold new class of cell therapies. Be Bio was founded in October 2020 by Longwood Fund and B cell engineering pioneers David Rawlings, M.D., and Richard James, Ph.D., from Seattle Children’s Research Institute. Be Bio is backed by ARCH Venture Partners, Atlas Venture, RA Capital Management, Alta Partners, Longwood Fund, Bristol Myers Squibb, Takeda Ventures, Seattle Children’s Research Institute and others. Since our founding, Be Bio’s investors have committed over $180 million to enable the Company to re-imagine medicine based on the power of B cell therapy. For more information, please visit us at Be.Bio and our LinkedIn page. 1 Landsverk et al (2017) J Exp Med 2 Hibi and Dosch (1986) J Immunol; Eyer et al (2017) Nat Biotech

皱皮木瓜（Chaenomeles speciosa），又名贴梗海棠，为蔷薇科木瓜海棠属落叶灌木。皱皮木瓜花朵艳红，贴生枝干之上，鲜艳夺目。陆游“千点猩红蜀海棠，谁怜雨里作啼妆” 皱皮木瓜（Chaenomeles speciosa），又名贴梗海棠，为蔷薇科木瓜海棠属落叶灌木。皱皮木瓜花朵艳红，贴生枝干之上，鲜艳夺目。陆游“千点猩红蜀海棠，谁怜雨里作啼妆”中所指便是贴梗海棠。除观赏价值外，皱皮木瓜还兼具食用价值与药用价值，其干果富含齐墩果酸和熊果酸，具有抗菌抗炎、抗病毒、抗氧化等作用。构建皱皮木瓜高质量基因组，解析皱皮木瓜药用成分生物合成途径，可为调控其重要药用成分的生物合成、提高药用木瓜品种提供新见解、新途径。 近日，华中农大药用植物学梅之南教授团队联合南京农业大学、中南民族大学、国药集团中联药业有限公司、武汉碳码生物科技有限公司以及北卡罗莱纳州立大学等单位科研人员在园艺一区（1/36）Horticulture Research上联合发表题为“A Telomere-to-telomere Reference Genome Provides Genetic Insight into the Pentacyclic Triterpenoid Biosynthesis in Chaenomeles speciosa”的研究论文。该研究通过HiFi和Hi-C测序，构建了首个皱皮木瓜T2T参考基因组，并结合比较基因组解析皱皮木瓜的三萜生物合成机制，为了解蔷薇科植物驯化和植物进化提供了宝贵的资源。 皱皮木瓜为二倍体植物，原产于中国，广泛栽培于世界各地。基于基因组survey，研究团队评估了皱皮木瓜基因组大小约为650.4 Mb。随后进行PacBio HiFi和Hi-C测序，利用Hifiasm组装出两套单体型，Hap1和Hap2。Hap1的基因组大小为592.84 Mb，包含230个contigs (N50为25.57 Mb)，125个scaffold (N50为36.12 Mb)，GC含量为37.41%。Hap2基因组大小为608.28 Mb，包含97个contigs (N50为29.34 Mb)， 42个scaffold (N50为36.92 Mb)， GC含量为37.25%。Hap1和Hap2，在基因组大小和基因数量方面具有相似性，同时具有良好的共线性。此外，两个单倍型基因组之间的单核苷酸多态性(SNP)、插入-缺失(InDels)和结构变异(SV)存在明显差异，为了解皱皮木瓜遗传多样性提供了大量信息来源。基于Hap1和Hap2，研究团队组装了T2T水平的皱皮木瓜基因组。其基因组全长650.4Mb，contig N50为35.5Mb。其中，632.3 Mb被锚定在17条染色体上，有12条染色体仅有1条contig组成，在11条染色体两端鉴定到端粒，17条染色体上均鉴定到着丝粒。BUSCO评估结果、LAI评分、二三代数据比对结果均证实了组装基因组良好的完整性和准确性。 图1 皱皮木瓜的基因组调查与组装 研究团队使用从头注释和同源注释的方法来鉴定重复序列，49.5%的皱皮木瓜基因组序列被预测为重复序列，约312.9 Mb。序列分析表明，重复序列主要由TE组成。其中，LTR最为丰富，占整个基因组的36.3%，包括Gypsy元件(22.7%)、Copia元件(7.3%)和其它序列。 基于从头注释、同源注释和转录组注释，共在皱皮木瓜中基因组中预测到45515个高置信度的蛋白编码基因，其中93%的基因都能得到功能注释。共鉴定到685个tRNA、4205个rRNA、274个snRNA和739个snoRNA。 表1 皱皮木瓜基因组组装数据统计 研究团队选择11个物种与皱皮木瓜进行比较基因组学分析。基因家族聚类分析表明11个物种中普遍存在6958个基因家族，皱皮木瓜存在5658个特有基因家族。GO富集分析表明这些特有基因主要参与辅因子代谢、辅酶代谢和蛋氨酸代谢过程。KEGG富集分析表明，物种特有基因主要参与碳代谢、丙酮酸代谢、半乳糖代谢和萜类物质生物合成。系统进化发育分析表明，皱皮木瓜和苹果、杜梨、西洋梨被聚类在一起，它们可能由同一祖先进化而来。皱皮木瓜和苹果的分化时间约为10.0个百万年前，苹果族与桃亚属的祖先在约10.92个百万年前分化，蔷薇科物种在约98.81个百万年前分化。基因家族收缩扩张分析表明，皱皮木瓜有320个基因家族发生扩张，与苹果扩张基因家族数量相似；有158个基因家族发生收缩，约为苹果收缩基因家族的50%。皱皮木瓜收缩的基因家族主要富集于脂质转运、信号转导过程等，扩张的基因家族与单萜类、二苯乙烯庚烷类、姜辣素、亚油酸、类黄酮、倍半萜类和三萜类的生物合成有关。 六种蔷薇科物种的系统发育分析表明，西洋梨、苹果、皱皮木瓜同属一个分支，约46个百万年前从同一祖先分化而来。这三个物种共同经历了一次四倍化事件，导致它们的染色体数量加倍，达到16条。WGD分析显示，Ks分布在0.2~0.3处出现了显著峰值，这一事件可能与西洋梨、苹果、皱皮木瓜中近期发生的四倍化事件有关。 图2 皱皮木瓜基因组同源聚类和系统发育分析 图3 皱皮木瓜的WGD特征、共线性和染色体进化 齐墩果酸和熊果酸是羟基五环三萜烯酸，《中国药典》中以齐墩果酸和熊果酸含量作为木瓜中药材质量控制的重要指标。植物五环三萜类化合物主要由甲羟戊酸途径和戊糖磷酸途径合成。本研究将皱皮木瓜与其它七个物种中涉及甲羟戊酸途径的四个基因家族进行比较，发现皱皮木瓜基因组中存在11个β-香树素合酶基因，其中10个基因分布在9号染色体上的两个基因簇中。基因簇1的长度从12,157,281 bp到12,428,418 bp，包含8个β-香树素合酶基因，9个其它基因和64个LTR反转录转座子。基因簇2的长度从31,206,028 bp到31,327,645 bp，包括2个β-香树素合酶基因，5个其它基因和29个LTR反转录转座子。 基因家族的鉴定进一步揭示了2个角鲨烯合酶的成员、12个角鲨烯2,3-氧化环化酶的成员、11个β-香树素合酶的成员和9个β-香树脂醇28-单加氧酶的成员。与其它蔷薇科物种相比，角鲨烯合酶、β -amyrin合酶和β-香树脂醇28-单加氧酶基因家族在皱皮木瓜基因组中显著扩张，这可能是该物种具有高含量的齐墩果酸和熊果酸的原因。 图4 皱皮木瓜中齐墩果酸、熊果酸的生物合成途径和β-香树素合酶基因簇 本研究通过HiFi和Hi-C测序构建了首个木瓜属植物皱皮木瓜的T2T基因组，通过比较基因组分析确定了皱皮木瓜扩张和收缩的基因家族与植物代谢或生物过程的关联，鉴定了与齐墩果酸和熊果酸生物合成途径相关的基因家族，为了解植物五环三萜类物质的生物合成提供了宝贵的资源，为了解蔷薇科植物驯化提供了新的见解。

本文转载自“小桔灯网”自2012年CRISPR-Cas9技术问世以来，基因编辑便驶入了快车道，取得了一系列新突破。如果将CRISPR-Cas9比作能够破坏目标基因的分子剪刀，那么base editor（碱基编辑器）可以称为分子铅笔，因其能对单个核苷酸进行替换。2019年开发的prime editor则具有更强大的功能，能够对基因组进行搜索和替换，堪称分子世界的“文字处理器”。开发这些技术的最终目标是修复人类基因中的有害突变。16,000多个小的缺失变异与人类疾病存在因果关系，理论上可以通过插入缺失的序列来修复。囊性纤维化就是一个很好的例子，70%的病例由三核苷酸缺失而引起。为了实现临床应用，人们需要一种技术来准确、高效且安全地插入序列，避免出现意外的结果。prime editing系统虽然在治疗囊性纤维化等遗传病上表现出巨大潜力，但目前仍不清楚哪些因素决定了编辑效率。英国Wellcome Sanger研究院和爱沙尼亚塔尔图大学的研究人员近日在Nature Biotechnology杂志上发文称，他们开发出一种新工具，可预测将基因编辑的DNA序列成功插入基因组的几率。影响插入效率的多个因素 在这项研究中，研究人员试图系统评估插入序列的长度和组成、细胞系、目标位点以及prime editor的不同版本如何影响插入效率。为此，他们共设计了3,604条pegRNA，编码切口位点上游的插入物，这些插入物的长度从1nt到69nt不等，且GC含量不同。他们将序列插入两个细胞系（HEK293T和HAP1细胞）中，靶向四个目标位点（HEK3、EMX1、FANCF和CLYBL）。一周后，他们对这些细胞进行基因组测序，观察编辑是否成功。评估插入效率的整体策略详见图1。图1 prime插入效率的高通量测定由于插入率的变化跨越了三个数量级，于是研究人员试图了解相关的特征，首先是插入物的长度。他们在HEK293T细胞中发现了两个特点：3和4nt序列的插入率高于其他序列；15-21nt序列的插入率高于周围的序列。不过在HAP1细胞中，1-4 nt短序列的插入率并没有高于较长序列。他们将其归因于错配修复（MMR）系统，因为HEK293T细胞在MMR上存在部分缺陷。敲除错配修复基因MLH1的HAP1细胞也证明了这一点，表明MMR系统阻碍了短序列的插入。之后，他们分析了prime editing的不同步骤如何影响序列的插入率。他们发现，若pegRNA中带有四个或更多的连续腺嘌呤，则插入率会大大下降。此外，prime editing中的另一个重要步骤是带有5’ flap（包含野生型序列）和3’ flap（包含插入物）的中间产物之间达到平衡，而5’ flap核酸酶FEN1及3’ flap核酸酶TREX1和TREX2介导了这种平衡。他们发现，3’ flap核酸酶TREX1和TREX2抑制了较长序列的插入。同时，插入序列的核苷酸组成和二级结构也会影响插入率。研究人员发现，prime editing系统对胞嘧啶有着明显的偏好。插入序列中每增加1%的胞嘧啶，则插入率平均增加2.2%。相反，腺嘌呤和胸腺嘧啶的百分比则降低了各个位点的插入率。此外，他们还发现，结构强度更高的序列能够被更有效地插入。在此过程中，他们使用了Twist Bioscience提供的寡核苷酸池。据Twist介绍，他们独特的硅基DNA合成平台能够在单次运行中生成超过一百万条寡核苷酸，对数量几乎没有限制，而且寡核苷酸池精准均一，让人们对实验结果充满信心。此外，实验中使用的多个载体和基因片段也是由Twist Bioscience提供的。预测不同序列的插入率 在了解影响插入率的多个因素后，研究人员接下来想预测不同序列在同一位点的插入效率。他们采用了机器学习的方法，挑选出10个特征来训练数据，包括插入序列的长度、组成、pegRNA二级结构和MMR等。这种称为MinsePIE的方法能够很好地预测测试数据的插入效率，相关性达到0.68（图2）。在现有数据上进行训练后，他们在新数据上测试MinsePIE模型，发现它能够准确预测多个插入序列的成功率。之后，他们还对预测的序列进行实验测试。与预测插入率较低的变体相比，预测插入率较高的密码子变体确实表现出较高的插入率，这凸显了MinsePIE模型在密码子优化方面的优势。研究人员认为，这种计算模型可以帮助人们选择出最有效的序列写入基因组中。图2 预测prime插入效率最后，对于如何提高prime editing系统的插入效率，研究人员提出了几点建议。他们建议选择胞嘧啶含量高且容易形成二级结构的序列。对于使用U6启动子的pegRNA，尽量避免腺嘌呤的插入。对于小于14nt的序列，暂时抑制MMR或敲除MLH1将极大提高插入效率。总的来说，这项工作增加了人们对短序列插入效率的理解，有望实现复杂的基因组工程和纠正各种致病突变。责编|文竞择校对|文竞择End参考资料：[1]Koeppel, J., Weller, J., Peets, E.M. et al. Prediction of prime editing insertion efficiencies using sequence features and DNA repair determinants. Nat Biotechnol (2023). https://doi-org.libproxy1.nus.edu.sg/10.1038/s41587-023-01678-y  往期精选 围观历史性突破！不需卵子和精子就可以合成人类胚胎 热文 富豪用儿子血浆“回春”科学吗？Science：血液牛磺酸缺乏驱动衰老 热文 世界首例！35岁的她，通过机器人操刀移植的子宫，成功诞下宝宝 热文‍为了避免心脏骤停，53岁的他成为“第一个”植入开创性除颤器的心脏病患者 热文 首次披露！175家生物制药公司的薪资数据：薪资中位数达20万美元 点击“阅读原文”，了解更多~