Engineered AAV13 variants with enhanced transduction and confined spread.

Precise targeting of specific regions within the central nervous system (CNS) is crucial for both scientific research and gene therapy in the context of brain diseases. Adeno-associated virus 13 (AAV13) is known for its restricted diffusion range within the CNS, making it an ideal choice for precise labeling and administration within small brain regions. However, AAV13 mediates relatively low expression of target genes. Here, we introduced specifically engineered modifications to the AAV13 capsid protein to enhance its transduction efficiency. We first constructed AAV13-YF by mutating tyrosine to phenylalanine on the surface of the AAV13 capsid. We then inserted the 7m8 peptide, known to enhance cell transduction, into positions 587/588 and 585/586 of the AAV13 capsid, resulting in two distinct variants named AAV13-587-7m8 and AAV13-585-7m8, respectively. We found that AAV13-YF exhibited superior in vitro infectivity in HEK293T cells compared to AAV13, while AAV13-587-7m8 and AAV13-585-7m8 showed enhanced CNS infection capabilities in C57BL/6 mice, with AAV13-587-7m8 infection retaining a limited spread range. These modified AAV13 variants hold promising potential for applications in gene therapy and neuroscience research.

AAV11 enables efficient retrograde targeting of projection neurons and enhances astrocyte-directed transduction.

Viral tracers that enable efficient retrograde labeling of projection neurons are powerful vehicles for structural and functional dissections of the neural circuit and for the treatment of brain diseases. Currently, some recombinant adeno-associated viruses (rAAVs) based on capsid engineering are widely used for retrograde tracing, but display undesirable brain area selectivity due to inefficient retrograde transduction in certain neural connections. Here we developed an easily editable toolkit to produce high titer AAV11 and demonstrated that it exhibits potent and stringent retrograde labeling of projection neurons in adult male wild-type or Cre transgenic mice. AAV11 can function as a powerful retrograde viral tracer complementary to AAV2-retro in multiple neural connections. In combination with fiber photometry, AAV11 can be used to monitor neuronal activities in the functional network by retrograde delivering calcium-sensitive indicator under the control of a neuron-specific promoter or the Cre-lox system. Furthermore, we showed that GfaABC1D promoter embedding AAV11 is superior to AAV8 and AAV5 in astrocytic tropism in vivo, combined with bidirectional multi-vector axoastrocytic labeling, AAV11 can be used to study neuron-astrocyte connection. Finally, we showed that AAV11 allows for analyzing circuit connectivity difference in the brains of the Alzheimer's disease and control mice. These properties make AAV11 a promising tool for mapping and manipulating neural circuits and for gene therapy of some neurological and neurodegenerative disorders.

Computer-Aided Directed Evolution Generates Novel AAV Variants with High Transduction Efficiency.

Adeno-associated viruses (AAVs) have become safe and effective tools for therapeutic in vivo gene drug delivery. Among many AAV serotypes, AAV2 is the most well-characterized. Although many studies have been carried out on the engineering of the capsid VR-VIII region, few attempts have been made in the VR-IV region. Here, we targeted amino acid positions 442-469 of the VR-IV region and established an engineering paradigm of computer-aided directed evolution, based on training samples from previous datasets, to obtain a viral vector library with high diversity (~95,089). We further examined two variants selected from the library. The transduction efficiency of these two novel AAV variants, AAV2.A1 and AAV2.A2, in the central nervous system was 10-15 times higher than that of AAV2. This finding provides new vehicles for delivering gene drugs to the brain.

AAV13 Enables Precise Targeting of Local Neural Populations.

As powerful tools for local gene delivery, adeno-associated viruses (AAVs) are widely used for neural circuit studies and therapeutical purposes. However, most of them have the characteristics of large diffusion range and retrograde labeling, which may result in off-target transduction during in vivo application. Here, in order to achieve precise gene delivery, we screened AAV serotypes that have not been commonly used as gene vectors and found that AAV13 can precisely transduce local neurons in the brain, with a smaller diffusion range than AAV2 and rigorous anterograde labeling. Then, AAV13-based single-viral and dual-viral strategies for sparse labeling of local neurons in the brains of C57BL/6 or Cre transgenic mice were developed. Additionally, through the neurobehavioral test in the ventral tegmental area, we demonstrated that AAV13 was validated for functional monitoring by means of carrying Cre recombinase to drive the expression of Cre-dependent calcium-sensitive indicator. In summary, our study provides AAV13-based toolkits for precise local gene delivery, which can be used for in situ small nuclei targeting, sparse labeling and functional monitoring.