Engineered tissue arrays to streamline deimmunized DMD gene therapy vectors

免疫原性的主要驱动因素是蛋白质序列中存在T细胞表位，其可以激活免疫原性。 辅助性T细胞，导致治疗效果的中和。对于DMD基因治疗，T细胞 对预先存在的（来自回复纤维）和/或治疗后的肌营养不良蛋白表位的反应最近已经出现 作为一个严重的治疗问题。在过去的一年中，5名接受AAV-微肌营养不良蛋白（µDys）治疗的DMD患者， 发展了针对肌营养不良蛋白的T细胞介导的免疫应答。根据免疫学数据，在所有情况下， 抗原区高度提示由外显子8- 11编码。1此外，在患者中观察到， 删除了该地区的数据（在MDA和ASGCT 2022科学会议上提供了更新）。的关键组成部分 我们的方法利用了这样一个事实，即单个氨基酸可以锚新的肽， 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 受体（TCR）。正是TCR识别导致下游信号级联和免疫后遗症。 或者，保守修饰的残基可以不利于抗原呈递，防止肌营养不良蛋白- 特异性免疫因此，如果关键表位锚结合到免疫应答中， 修饰抗原呈递所必需的残基以破坏MHC识别。在这里，我们的建议 阐述了在DMD患者中发现的抗肌营养不良蛋白的免疫原性的基本作用。这 该提案通过Rosetta软件整合了去免疫方案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）平台进行简化的功能测试-允许进行日常3D DMD 在培养皿中评估收缩性能。反过来，肌营养不良蛋白表位可以根据 通过将它们的氨基酸序列含量与免疫学数据库相关联来确定它们的抗原性。已知和 预测的表位将被靶向重新设计以减少或消除推定的MHC结合肽， 随后T细胞活化。我们的去免疫方法将建立在以前的努力，同时侧重于外显子 8-11和整合数据，因为它可以通过任何DMD临床试验联盟展开。 外显子8-11区域，现在是辉瑞公司更新的患者排除标准的一部分，编码肌动蛋白的一部分， 结合结构域，延伸通过血影蛋白样重复1（注：我们已经去免疫的外显子6- 8）。重要的是，将去免疫努力限制在肌营养不良蛋白的外显子8-11将有利于所有µDys 目前在临床试验中的设计。总之，我们建议将去免疫化的µDys载体递送至人DMD 3D工程肌肉组织，并通过我们的Mantarray系统测量收缩力，以筛选 用于具有降低的免疫原性的高功能性领先候选物。为了充分验证我们的3D肌肉系统， 预测模型，我们将通过高度肌肉特异性驱动的rAAV载体测试优化的候选物， 调节盒改善DMD的DMDmdx大鼠模型中的疾病进展的能力。这些 结果将提供必要的临床前结果指标，以推进1期临床试验。

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.