The tyrosine capsid mutations on retrograde adeno-associated virus accelerates gene transduction efficiency

Abstract

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.

AAV13 Enables Precise Targeting of Local Neural Populations

“…AAV2-7m8 was developed by inserting the heptamer peptide (LGETTRP) into loop IV of AAV2, allowing enhanced retinal targeting over its parental serotype AAV2 [38,39]. While 7m8 insertion Replacing surface-exposed tyrosines of AAV2 and AAV2 mutant capsid with phenylalanine (YF mutation) to reduce virion degradation has been demonstrated as an effective means of improving transgene expression efficiency [36,37]. AAV2-7m8 was developed by inserting the heptamer peptide (LGETTRP) into loop IV of AAV2, allowing enhanced retinal targeting over its parental serotype AAV2 [38,39].…”

“…Replacing surface-exposed tyrosines of AAV2 and AAV2 mutant capsid with phenylalanine (YF mutation) to reduce virion degradation has been demonstrated as an effective means of improving transgene expression efficiency [ 36 , 37 ]. AAV2-7m8 was developed by inserting the heptamer peptide (LGETTRP) into loop IV of AAV2, allowing enhanced retinal targeting over its parental serotype AAV2 [ 38 , 39 ].…”

The circadian clock in the piriform cortex intrinsically tunes daily changes of odor-evoked neural activity

“…Adeno-associated virus injection. We performed AAVs injection as previously described 52,53 . In brief, mice 6-8 weeks of age were stereotaxically injected.…”

Chemical approaches to probe and engineer AAV vectors

“…In addition, recent advances in mass-spectrometry-based platforms have also revealed the presence of many novel post-translational modifications on the AAV capsid residues across multiple serotypes. 18,19 However, the distribution of these modifications is quite heterogeneous and, except for a few well-documented cases such as phosphorylation mediated capsid degradation, [20][21][22] and their physiological significance remains poorly understood.…”