Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Mol Brain. 2022 Aug 8;15(1):70. doi: 10.1186/s13041-022-00957-0.
Adeno-associated virus (AAV) vector is a critical tool for gene delivery through its durable transgene expression and safety profile. Among many serotypes, AAV2-retro is typically utilized for dissecting neural circuits with its retrograde functionality. However, this vector requires a relatively long-term incubation period (over 2 weeks) to obtain enough gene expression levels presumably due to low efficiency in gene transduction. Here, we aimed to enhance transgene expression efficiency of AAV2-retro vectors by substituting multiple tyrosine residues with phenylalanines (YF mutations) in the virus capsid, which is previously reported to improve the transduction efficiency of AAV2-infected cells by evading host cell responses. We found that AAV2-retro with YF mutations (AAV2-retroYF)-mediated transgene expression was significantly enhanced in the primary culture of murine cortical neurons at 1 week after application, comparable to that of the conventional AAV2-retro at 2 week after application. Moreover, transgene expressions in the retrogradely labeled neurons mediated by AAV2-retroYF were significantly increased both in the cortico-cortical circuits and in the subcortical circuits in vivo, while the retrograde functionality of AAV2-retroYF was equally effective as that of AAV2-retro. Our data indicate that YF mutations boost AAV2-retro-mediated retrograde gene transduction in vivo and suggest that the AAV2-retroYF should be useful for efficient targeting of the projection-defined neurons, which is suited to applications for dissecting neural circuits during development as well as future clinical applications.
腺相关病毒 (AAV) 载体是通过其持久的转基因表达和安全特性进行基因传递的重要工具。在许多血清型中，AAV2-retro 通常因其逆行功能而用于剖析神经回路。然而，由于基因转导效率低，该载体需要相对较长的孵育期（超过 2 周）才能获得足够的基因表达水平。在这里，我们旨在通过在病毒衣壳中的多个酪氨酸残基上取代苯丙氨酸（YF 突变）来增强 AAV2-retro 载体的转基因表达效率，这先前已被报道可通过逃避宿主细胞反应来提高 AAV2 感染细胞的转导效率。我们发现，在应用后 1 周，AAV2-retro 中的 YF 突变（AAV2-retroYF）介导的转基因表达在鼠皮质神经元的原代培养物中显著增强，与应用后 2 周的常规 AAV2-retro 相当。此外，AAV2-retroYF 介导的 retrogradely 标记神经元中的转基因表达在体内的皮质间和皮质下回路中均显著增加，而 AAV2-retroYF 的逆行功能与 AAV2-retro 同样有效。我们的数据表明，YF 突变可增强 AAV2-retro 介导的体内逆行基因转导，并表明 AAV2-retroYF 应该可用于有效地靶向投射定义的神经元，这适合于在发育过程中剖析神经回路以及未来的临床应用。
