Integrated AAV Offerings: From Cutting-Edge Products to Advanced Custom Services

AAV Capsid Barcode Kits Based on the ATHENA I Platform

Streamlining AAV Capsid Optimization with Innovative Screening Kits

AAV vectors are powerful tools for gene delivery and gene therapy, with hundreds of natural AAV serotypes and an increasing number of engineered variants available for targeting specific cell types and tissues. Identifying the optimal capsid for each target is crucial for effective gene therapies. However, traditional methods for comparing AAV variants often involve testing them individually side-by-side, which can be labor-intensive, difficult to control, and impractical when handling hundreds of AAV vectors in a laboratory setting. This limitation reduces the ability to find better AAV variants and optimize gene delivery.

To address this challenge, AAVnergene offers two innovative solutions: AAV Capsid Barcode Kits and AAV Serotype Testing Kits, designed to help researchers efficiently compare and select the best AAV serotypes and variants for their specific applications.

AAV Capsid Barcode Kits are built on the ATHENA-I platform and utilize Barcode-Seq technology along with next-generation sequencing (NGS) to systematically evaluate a group of AAV serotypes or variants. In these kits, each AAV capsid variant is assigned three different DNA barcodes (BCs). By comparing the DNA or RNA levels of these barcodes using NGS, researchers can determine which AAV capsid variant performs best for their specific research or therapeutic needs. This high-throughput approach overcomes the limitations of traditional methods, allowing for the efficient screening and optimization of AAV capsids.

On the other hand, AAV Serotype Testing Kits provide a quicker method to identify a specific subset of AAV serotypes by measuring the expression levels of fluorescent proteins (EGFP, mCherry, TdTomato) or luciferases (Gluc, Rluc, Fluc, Cluc). This approach is particularly useful when researchers have a clear understanding of the target cell lines or tissues and seek a streamlined way to narrow down their choices to a few optimal AAV serotypes.

Together, these solutions enable researchers to effectively explore a wide range of AAV capsids and variants, enhancing the precision and efficiency of gene delivery systems and accelerating the development of next-generation gene therapies.
 

Pre-made Collections of AAV capsids Barcode Kits

AAV Capsid Barcode Kit - Common: This kit includes 15 widely used AAV serotypes such as AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAV12, AAV13, AAVrh.10, AAVrh.74, AAV-DJ, and AAV2-Retro. It is ideal for broad-spectrum AAV testing and screening applications. AAV

Capsid Barcode Kit - Tissues: In addition to the 15 common AAV serotypes, this kit contains 9 additional tissue-targeting AAV variants, totaling 24 capsids. It is specifically designed for researchers focusing on tissue-specific applications and targeting.​

​Pre-made Collections of AAV Serotype Testing Kits

AAV Serotype Testing Kits provide a quicker method to identify a specific subset of AAV serotypes by measuring the expression levels of fluorescent proteins (EGFP, mCherry, TdTomato) or luciferases (Gluc, Rluc, Fluc, Cluc). This approach is particularly useful when researchers have a clear understanding of the target cell lines or tissues and seek a streamlined way to narrow down their choices to a few optimal AAV serotypes.

In this category, 15 most used AAV serotypes (AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAV12, AAV13, AAV-DJ, AAVrh.10, AAVrh.74, and AAV2-Retro) are included in each kit. The vectors carry fluorescent protein (EGFP, mCherry, TdTomato) or reporter luciferase (Fluc, Rluc, Cluc, Gluc), which is driven by a promoter (CAG, CMV, EF1a or hSyn). The kits are pretended to provide useful tools for customers, with minimum cost, in testing AAV transduction efficiency, testing tissue specific tropism related to serotypes, and using as negative control to examine whether the observed biological effects come from specific transgenes. We also provide custom AAV Capsid Kits developing services.

Custom AAV Capsid Barcode Library Design

Select Your Promoter: Choose from a range of promoters such as CMV, CAG, EF1a, Syn, TTR, CaMKII, hAAT, or other options to drive the expression of your gene of interest.
Select Your Reporter: Select a reporter gene that can be easily detected or quantified to assess the transduction efficiency or tropism of AAV capsids. Commonly used reporter genes include EGFP, mCherry, TdTomato, Gluc, Fluc, Rluc, Cluc, AAT, LacZ, among others.
Select Your Capsid: Choose from a diverse range of AAV serotypes or engineered variants such as AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and others. Consider factors such as tissue specificity, receptor binding, and immunogenicity for your library.
Contact Us: Reach out our team at [email protected] to discuss your specific requirements and initiate the custom library creation process.
Make Barcoded Reporter Library: Insert random barcodes between reporters and PolyA to create a barcoded library. Miniprep of plasmids and sequencing are used to confirm the barcode in each plasmid.
Produce AAV Capsid Library: Each capsid is matched to one unique barcoded plasmid. AAV vectors are produced separately, then pooled and purified together. qPCR primers are designed according to the barcodes.
​Perform Experiments: Conduct screening experiments in vitro using cell lines, primary cells, or organoids, or in vivo using mouse and non-human primate (NHP) models. Collect Data: Use qPCR, NGS, and data analysis to evaluate the performance and efficiency of the AAV capsid variants.

Custom AAV Capsid Selection Kits

AAVnerGene’s ​ATHENA I platform

​ATHENA I platform offers a comprehensive service for capsid library design and production, as well as one round of screening using next-generation sequencing (NGS). This screening can be performed both in vitro (e.g., cell lines, primary cells, organoids) and in vivo (e.g., mouse and NHP models). For specific screening needs, we recommend discussing them with AAVnerGene’s technical support team to ensure the best results.
Potential models: 
In vitro: cell lines, primary cells, organoids
In vivo: mouse and non-human primate (NHP) 

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scRNA-AAVseq for AAV Tropism Analysis

Analyzing AAV tropism in different tissues is challenging due to the complexity of cell types and the diversity of hundreds of AAV serotypes and variants. To address these challenges, Biohippo, in collaboration with AAVnerGene, has developed a novel pipeline called scRNA-AAVseq (single-cell RNA-AAV barcode sequencing). This advanced technique allows for in vivo characterization of barcoded AAV pools at an unprecedented single-cell resolution, enabling a deep and detailed understanding of AAV tropism across various tissues and cell types.

Overview of the scRNA-AAVseq Workflow

Injection of Barcoded AAV Variants:
Multiple barcoded AAVs are pooled and injected into the target organism, such as mice, via retro-orbital injection. This enables the distribution of different AAV variants across multiple tissues and cell types.

Tissue Dissociation and Single-Cell Isolation:
After sufficient expression time, tissues (e.g., cortex) are harvested and dissociated into single-cell suspensions. This step involves tissue dissociation, preparation of single-cell suspensions, and droplet-based sorting to isolate single cells.

Library Preparation and Sequencing:
Single-cell libraries are prepared using droplet-based sequencing platforms such as Illumina NGS. Both cell transcriptomes and viral variant transcripts are processed for sequencing.
The transcriptome aliquots undergo the standard library construction protocol, further amplified using AAV barcode-specific primers to enrich AAV capsids. These amplified viral transcripts are then sequenced separately to generate NGS libraries.

Data Processing and Analysis:
The single-cell transcriptome analysis is performed using CeleScope 2.0 and annotated with in-house software. The sequencing reads from amplified viral transcripts are used to quantify the abundance of each viral barcode associated with each cell barcode and unique molecular identifier (UMI).
The most abundant viral barcode for each cell barcode and UMI is assumed to be the correct viral barcode, which is then used to construct a variant lookup table for further analysis.



​Application: AAV Tropism Analysis in PBMCs

In a specific example, the ATHENA-I library, containing 463 AAVs, was used to infect Peripheral Blood Mononuclear Cells (PBMCs) at a multiplicity of infection (MOI) of 2e5 vg/cell. Samples were collected 5 days post-infection for detailed analysis.

Subgrouping of Major Cell Types: Major cell types (e.g., CD4+ T cells, CD8+ T cells, B cells, monocytes, NK cells, etc.) were identified following scRNA-seq analysis.
Overlap Between Cell Types and AAVs: Visualization of the overlap between cell types and AAV variants helped identify which AAV serotypes preferentially target specific cell types.
AAV Positive Rates in Each Cell Type: The proportion of AAV-positive cells within each cell type was calculated to determine AAV transduction efficiency.
Log2 RNA Fold Changes Across Different AAVs: Differential expression analysis was conducted to compare AAV-mediated transcriptional changes across different AAVs relative to AAV6.
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