Engineered tissue arrays to streamline deimmunized DMD gene therapy vectors

工程组织阵列可简化去免疫 DMD 基因治疗载体

基本信息

批准号：
10724882
负责人：
Guy Leary Odom
金额：
$ 41.11万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10724882
关键词：
3-Dimensional Actins Activities of Daily Living Address Affinity Aftercare Age Months Amino Acid Sequence Amino Acids Antigen Presentation Antigen-Presenting Cells Binding Biological Assay Body Weight Cardiac Cell Separation Child Clinical Clinical Trials Collection Complex Computer software Cytotoxic T-Lymphocytes Data Databases Disease Progression Dose Duchenne muscular dystrophy Dystrophin Echocardiography Engineering Epitopes Evaluation Excision Exclusion Exclusion Criteria Exons Fiber Future Gene Transduction Agent Gene therapy trial Generations Genetic Diseases Glycoproteins Hand Strength Haplotypes Head Helper-Inducer T-Lymphocyte Hindlimb Human Immune Immune response Immunity Immunologics Individual Magnetism Major Histocompatibility Complex Measures Modeling Muscle Muscle Fibers Muscular Atrophy Mutation Outcome Patients Peptides Performance Phase Phase I Clinical Trials Phenotype Progressive Disease Proteins Proteomics Protocols documentation Rattus Recombinant adeno-associated virus (rAAV)Relaxation Residual state Resolution Respiratory Diaphragm Role Running Selection Criteria Serious Adverse Event Signal Transduction Spectrin Striated Muscles Structure System T cell response T-Cell Activation T-Cell Receptor T-Lymphocyte T-Lymphocyte Epitopes Testing Therapeutic Effect Time Tissue Engineering Tissue Microarray Update Validation Variant Viral Vector adeno-associated viral vector cell mediated immune response clinically relevant data integration de-immunization delivery vehicle design disease phenotype early phase clinical trial gene therapy gene therapy clinical trial genomic data immunogenic immunogenicity in vivo induced pluripotent stem cell innovation intravenous administration mdx mouse mechanical properties micro-dystrophin mini-dystrophin miniaturize muscle engineering muscular system novel pre-clinical predicting response predictive modeling predictive tools prevent screening skeletal symposium three dimensional cell culture treadmill vector

项目摘要

A major driver for immunogenicity is the presence of T-cell epitopes within a protein sequence that can activate helper T cells, resulting in neutralization of the therapeutic effect. For DMD gene therapy, the possibility of T-cell responses to pre-existing (from revertant fibers) &/or post-treatment dystrophin epitopes has recently emerged as a serious treatment issue. In the past year 5 DMD patients treated with AAV-microdystrophin (µDys) have developed T cell mediated immune responses against dystrophin. Based on immunological data, in all cases the antigenic region was highly suggested to be encoded by exons 8-11.1 Additionally, to being observed in patients deleted for this region (updates provided at MDA and ASGCT 2022 scientific conferences). A key component of our approach takes advantage of the fact that a single amino acid can anchor a novel peptide for a preferred binding state for antigen presenting cells’ via its major histocompatibility complex (MHC) and subsequent T cell receptor (TCR). It is the TCR recognition which leads to downstream signaling cascades and immune sequelae. Alternatively, conservatively modified residue(s), can disfavor antigen presentation, preventing dystrophin- specific immunity. Thus, avoidance of a specific immune response can be achieved if key epitope anchor residue(s) necessary for antigen presentation are modified to disrupt MHC recognition. Here, our proposal addresses the fundamental role of immunogenicity against dystrophin that has been found in DMD patients. This proposal integrates deimmunization protocols and structure-based protein redesign via Rosetta-software, with streamlined functional testing via our magnetic tissue array (Mantarray) platform-allowing for daily 3D DMD contractile performance assessment in-a-dish. In turn, dystrophin epitopes can be predicted & ranked according to their antigenicity by correlating their amino acid sequence content with immunological databases. Known and predicted epitopes will be targeted for redesign to reduce or eliminate putative MHC-binding peptides and subsequent T cell activation. Our deimmunization approach will build on previous efforts while focusing on exons 8-11 of dystrophin, & integrating data as it becomes available through any DMD clinical trial alliance that unfolds. The exon 8-11 region, now part of Pfizers’ updated patient exclusion criteria, encodes a portion of the actin- binding domain, extending through spectrin-like repeat 1 (note: we have already deimmunized much of exons 6- 8). Importantly, restricting deimmunization efforts to exons 8-11 of dystrophin would be beneficial for all µDys designs currently in clinical trials. In summary, we propose to deliver deimmunized µDys vectors to human DMD 3D engineered muscle tissues and measure contractile force(s) via our Mantarray system in an effort to screen for highly functional leading candidates with reduced immunogenicity. To fully validate our 3D muscle system as a predictive model we will test optimized candidates(s) via rAAV vectors driven by highly muscle specific regulatory cassettes for their capacity to ameliorate disease progression in the DMDmdx rat model of DMD. These results will provide necessary preclinical outcome metrics required to advance towards Phase 1 clinical trials.

免疫原性的一个主要驱动因素是蛋白质序列中存在 T 细胞表位，可以激活辅助性T细胞，导致治疗效果中和对于DMD基因治疗，T细胞的可能性。最近出现了对预先存在的（来自回复纤维）和/或治疗后肌营养不良蛋白表位的反应作为一个严重的治疗问题，在过去的一年中，有 5 名 DMD 患者接受了 AAV-微肌营养不良蛋白 (μDys) 治疗。根据免疫学数据，在所有情况下都产生了 T 细胞介导的针对肌营养不良蛋白的免疫反应。强烈建议抗原区域另外由外显子 8-11.1 编码，以在患者中观察到已删除该区域（MDA 和 ASGCT 2022 科学会议上提供的更新）。我们的方法利用了这样一个事实，即单个氨基酸可以锚定一种新的肽以获得优选的抗原呈递细胞通过其主要组织相容性复合体 (MHC) 和随后的 T 细胞的结合状态 TCR 识别导致下游信号级联反应和免疫后遗症。或者，保守修饰的残基可能不利于抗原呈递，从而防止肌营养不良蛋白- 因此，如果关键表位锚定，则可以避免特异性免疫反应。抗原呈递所必需的残基被修饰以破坏 MHC 识别。在此，我们的建议是。解决了在 DMD 患者中发现的抗肌营养不良蛋白免疫原性的基本作用。该提案通过 Rosetta 软件整合了去免疫协议和基于结构的蛋白质重新设计，通过我们的磁性组织阵列 (Mantarray) 平台简化功能测试 - 允许日常 3D DMD 反过来，可以根据培养皿中的收缩性能评估来预测抗肌萎缩蛋白表位并对其进行排名。通过将其氨基酸序列内容与已知和免疫学数据库相关联来确定其抗原性。预测的表位将被重新设计，以减少或消除假定的 MHC 结合肽，我们的去免疫方法将建立在之前的努力的基础上，同时关注外显子。 8-11 的肌营养不良蛋白，并整合通过任何开展的 DMD 临床试验联盟获得的数据。外显子 8-11 区域现已成为辉瑞公司更新的患者排除标准的一部分，编码了肌动蛋白的一部分结合域，延伸至血影蛋白样重复序列 1（注：我们已经对大部分外显子 6- 进行了去免疫处理） 8) 重要的是，限制抗肌萎缩蛋白外显子 8-11 的去免疫工作对所有 µDys 都是有益的。总之，我们建议向人类 DMD 提供去免疫的 µDys 载体。 3D 工程肌肉组织并通过我们的 Mantarray 系统测量收缩力，以筛选用于具有降低免疫原性的高功能领先候选者，以充分验证我们的 3D 肌肉系统。我们将通过由高度肌肉特异性驱动的 rAAV 载体测试优化的候选模型的预测模型调节盒能够改善 DMDmdx 大鼠模型中的疾病进展。结果将提供推进一期临床试验所需的必要的临床前结果指标。