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

• 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
• 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

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.

免疫原性的主要驱动力是蛋白质序列中的T细胞表位的存在 辅助T细胞，导致治疗作用中和。对于DMD基因疗法，T细胞的可能性 最近出现了对预先存在的（来自恢复纤维）和/或治疗后肌营养不良蛋白表位的反应 作为严重的治疗问题。在过去的一年中 开发了T细胞介导的抗肌营养不良蛋白的免疫血液。基于免疫数据，在所有情况下 强烈建议在患者中观察到抗原区域由外显子编码8-11.1。 为该地区删除（在MDA和ASGCT 2022科学会议上提供的更新）。一个关键组件 我们的方法利用了一个事实，即单个氨基酸可以锚定新型肽的首选肽 通过其主要的组织相容性复合物（MHC）和随后的T细胞的结合态呈现抗原的结合状态 受体（TCR）。这是TCR识别，导致下游信号级联和免疫后遗症。 另外，保守修改的居住地可以不利于抗原表现，防止肌营养不良蛋白 特定的免疫力。那就是，如果关键的表位锚锚可以避免特定的免疫反应 修改抗原表现所需的居住地以破坏MHC识别。在这里，我们的建议 解决了在DMD患者中发现的免疫原性对肌营养不良蛋白的基本作用。这 提案通过Rosetta-Software重新设计了集成的Demmunization协议和基于结构的蛋白质，并带有 通过我们的磁性组织阵列（Mantarray）平台进行简化的功能测试，可用于每日3D DMD 收缩绩效评估在一个境内。反过 通过将其氨基酸序列含量与免疫数据库相关联，它们的抗原性。已知和 预测的表位将被针对重新设计，以减少或消除推定的MHC结合肽和 随后的T细胞激活。我们的Deimmunization方法将基于以前的努力，同时专注于外显子 8-11的肌营养不良蛋白和整合数据，可以通过任何展开的DMD临床试验联盟获得。 外显子8-11地区现在是Pfizers更新的患者排除标准的一部分，编码了肌动蛋白的一部分 结合结构域，通过光谱蛋白样重复1延伸（注意：我们已经将大部分外显子6-- 8）。重要的是，限制对外显子8-11肌营养不良蛋白的限制对所有µDys都是有益的 目前在临床试验中的设计。总而言之，我们建议将Deimmunized µDys向量传递到人DMD 3D工程的肌肉组织并通过我们的壁炉架系统测量收缩力 对于高度功能的领先候选人，免疫原性降低。充分验证我们的3D肌肉系统 一个预测模型，我们将通过由高度肌肉特异性驱动的RAAV矢量测试优化的候选者 在DMD的DMDMDX大鼠模型中改善疾病进展的能力。这些 结果将提供前进到1阶段临床试验所需的必要临床前结果指标。