Department of Infectious Diseases/Virology, Section Viral Vector Technologies, Medical Faculty, University of Heidelberg, BioQuant, Center for Integrative Infectious Diseases (CIID), Heidelberg, Germany.
German Center for Infection Research (DZIF) and German Center for Cardiovascular Research (DZHK), Partner Site Heidelberg, Heidelberg, Germany.
Hum Gene Ther. 2023 May;34(9-10):350-364. doi: 10.1089/hum.2023.057.
The ability to specifically, safely, and efficiently transfer therapeutic payloads to the striated musculature via a minimally invasive delivery route remains one of the most important but also most ambitious aims in human gene therapy. Over the past two decades, a flurry of groups have harnessed recombinant adeno-associated viruses (AAVs) for this purpose, carrying cargoes that were packaged either in one of the various wild-type capsids or in a synthetic protein shell derived by molecular bioengineering. In this study, we provide an overview over the most commonly used techniques for the enrichment of muscle-specific (myotropic) AAV capsids, typically starting off with the genetic diversification of one or more extant wild-type sequences, followed by the stratification of the ensuing capsid libraries in different muscle types in small or large animals. These techniques include the shuffling of multiple parental capsid genes, peptide display in exposed capsid loops, mutagenesis of individual capsid residues, creation of chimeras between two viral parents, or combinations thereof. Moreover, we highlight alternative experimental or bioinformatic strategies such as ancestral reconstruction or rational design, all of which have already been employed successfully to derive synthetic AAV capsids or vectors with unprecedented efficiency and/or specificity in the musculature. Most recently, these efforts have culminated in the isolation of unique clades of myotropic vectors called AAVMYO or MyoAAV that have in common the display of the amino acid motif RGD (arginine-glycine-aspartate) on the capsid surface and that exhibit the highest transduction rate in striated muscles of mice or nonhuman primates reported to date. Finally, we note essential looming improvements that will facilitate and accelerate clinical translation of these latest generations of myotropic AAVs, including the identification and utilization of capsid selection or validation schemes that promise optimal translation in humans, and continued efforts to enhance patient safety by minimizing hepatic off-targeting.
通过微创输送途径将治疗性有效载荷特异性、安全且有效地递送至横纹肌仍然是人类基因治疗中最重要但也是最具挑战性的目标之一。在过去的二十年中，许多研究小组利用重组腺相关病毒（AAV）实现了这一目标，携带的货物要么包装在各种野生型衣壳中，要么包装在通过分子生物工程衍生的合成蛋白壳中。在本研究中，我们概述了最常用于富集肌肉特异性（肌向性）AAV 衣壳的常用技术，这些技术通常从一个或多个现有野生型序列的遗传多样化开始，然后在小动物或大动物中不同肌肉类型中对随后的衣壳文库进行分层。这些技术包括多个亲本衣壳基因的改组、暴露衣壳环中的肽展示、单个衣壳残基的诱变、两个病毒亲本之间嵌合体的创建，或它们的组合。此外，我们还强调了替代的实验或生物信息学策略，如祖先重建或合理设计，所有这些策略都已成功用于衍生具有前所未有的效率和/或肌肉特异性的合成 AAV 衣壳或载体。最近，这些努力的结果是分离出了称为 AAVMYO 或 MyoAAV 的独特肌向性载体的分支，这些载体的共同点是在衣壳表面展示氨基酸基序 RGD（精氨酸-甘氨酸-天冬氨酸），并且在报道的迄今为止的小鼠或非人类灵长类动物的横纹肌中转导率最高。最后，我们注意到即将出现的重要改进，这将促进和加速这些最新一代肌向性 AAV 的临床转化，包括鉴定和利用有望在人类中实现最佳转化的衣壳选择或验证方案，以及通过最小化肝脏靶向非特异性来继续努力提高患者安全性。
