Milagros Sergio, de Erenchun Pablo Ramírez-Ruiz, Guembe Maite, Carte Beatriz, Méndez Miriam, Uribarri Ander, Aldabe Rafael
DNA and RNA Medicine Division, CIMA Universidad de Navarra, 31008, Pamplona, Spain.
J Biol Eng. 2024 May 14;18(1):34. doi: 10.1186/s13036-024-00430-7.
Several treatments for genetic diseases utilizing recombinant adeno-associated viruses (AAVs) have recently gained approval. However, the development of a greater number of therapeutic AAVs is constrained by certain limitations. While extensive efforts have concentrated on screening AAV genetic libraries, an alternative strategy involves modifying the AAV capsid by attaching various moieties. The capsid of AAV plays a pivotal role in transducing target cells and evading immune responses, making modifications a key avenue for engineering improved variants.
In our study, we replaced specific AAV9 capsid residues with an unnatural amino acid bearing a bioorthogonal group, identifying four positions with no adverse impact on production. Utilizing click chemistry, we attached varying proportions of Cy5.5 to these positions, allowing us to assess the impact of these modifications on AAV9 infectivity in cultured cells. Our findings reveal that both the position and degree of capsid modification significantly affect AAV transduction. While higher amounts of attached molecules lead to an increased number of AAV genomes within cells, this does not positively impact transgene expression. Conversely, a negative impact on transgene expression is observed when the AAV capsid is highly modified, with the degree of this effect associated with the modified residue.
Careful control of both the degree and specific position of capsid modifications is crucial for optimizing transduction efficiency and minimizing undesired effects on transgene expression. These results underscore the importance of precision in AAV capsid modification to achieve optimal transduction efficiency while mitigating potential drawbacks on transgene expression.
最近，几种利用重组腺相关病毒（AAV）治疗遗传疾病的方法已获得批准。然而，更多治疗性AAV的开发受到某些限制。虽然大量工作集中在筛选AAV基因文库上，但另一种策略是通过连接各种基团来修饰AAV衣壳。AAV的衣壳在转导靶细胞和逃避免疫反应中起关键作用，因此修饰是构建改良变体的关键途径。
在我们的研究中，我们用带有生物正交基团的非天然氨基酸取代了特定的AAV9衣壳残基，确定了四个对生产没有不利影响的位置。利用点击化学，我们将不同比例的Cy5.5连接到这些位置，从而能够评估这些修饰对培养细胞中AAV9感染性的影响。我们的研究结果表明，衣壳修饰的位置和程度都会显著影响AAV的转导。虽然附着分子的量增加会导致细胞内AAV基因组数量增加，但这对转基因表达没有积极影响。相反，当AAV衣壳高度修饰时，会观察到对转基因表达有负面影响，这种影响的程度与修饰的残基有关。
仔细控制衣壳修饰的程度和特定位置对于优化转导效率和最小化对转基因表达的不良影响至关重要。这些结果强调了在AAV衣壳修饰中精确性的重要性，以实现最佳转导效率，同时减轻对转基因表达的潜在不利影响。
