ATHENA-I Capsid Evaluation Platform

The ATHENA-I AAV Capsid Platform is a comprehensive library of over 1,000 distinct AAV capsids, each linked to three unique DNA barcodes for assessment using Barcode-seq technology. By analyzing the DNA or RNA levels of these barcodes, researchers can identify the AAV capsid variant with the highest efficiency for their specific application.

Highlights

  • Semi-High-Throughput Screening: Enables rapid and efficient comparison of hundreds of AAV variants simultaneously, significantly reducing the time required for capsid evaluation.

  • Unique Barcoding System: Each AAV capsid is assigned three unique DNA barcodes, allowing precise tracking and identification of individual variants during screening.

  • Dual Application: Supports both in vivo and in vitro testing, providing comprehensive insights into capsid performance across different biological contexts.

  • Streamlined Workflow: Integrates plasmid preparation, co-transfection, AAV purification, and titration into a seamless process, ensuring consistency and reliability.

  • Scalability: Suitable for small-scale research as well as large-scale therapeutic development, offering flexibility to meet diverse experimental needs.

  • Enhanced Precision: Facilitates the identification of capsids with improved targeting, reduced immunogenicity, and higher transduction efficiency.

  • Pooling Capability: Allows selected AAV variants to be pooled for targeted experiments, optimizing resource utilization and experimental efficiency.

  • Accelerated Discovery: Empowers researchers to quickly identify the most effective capsids for specific cells, tissues, or therapeutic applications, advancing the development of next-generation gene therapies.

Purpose of ATHENA-I

AAV vectors are highly effective tools for gene delivery and therapy. With numerous natural and engineered AAV serotypes available, identifying the optimal capsids for targeting specific cells and tissues is crucial for successful gene therapy. Additionally, researchers often generate hundreds of novel capsids through multiple rounds of evolution during capsid engineering. However, individually testing these AAV variants is both time-intensive and laborious. To address this challenge, AAVnerGene has developed the ATHENA-I AAV Capsid Platform, which enables rapid, semi-high-throughput comparison of AAV variants in both in vivo and in vitro environments. This innovative platform streamlines the evaluation process, accelerating the discovery of the most effective capsids for therapeutic applications.

Design of ATHENA-I

In the ATHENA-I, each capsid variant is linked to three unique DNA barcodes, all carrying the same reporter gene. This reduces variability and improves accuracy in high-throughput screening. For example, The AAV Capsid Barcode Kit-CAG-EGFP includes AAV genomes with a CAG promoter driving EGFP expression, followed by the WPRE enhancer and bGH PolyA signal. The DNA barcode is placed between the WPRE and bGH PolyA. The CAG promoter ensures strong expression across cell types, while the EGFP reporter gene helps identify and isolate successfully transduced cells. WPRE and bGH PolyA enhance EGFP expression and stabilize mRNA.

Production of ATHENA-I

In the ATHENA-I system, each AAV capsid is uniquely identified by three distinct DNA barcodes. The production process involves the following steps:

  • Plasmid Preparation: The selected pRCap plasmid (encoding the AAV capsid) is paired with three AAV barcode plasmids (each containing a unique DNA barcode).

  • Co-Transfection: The pRCap plasmid and the three barcode plasmids are co-transfected into HEK 293T cells using a standardized transfection protocol to produce AAV particles.

  • AAV Purification and Titration: The produced AAVs are purified using advanced purification techniques to ensure high-quality yields. Each AAV variant is individually titrated to determine its concentration and ensure consistency.

  • Pooling for Injection: Selected AAV variants can be pooled together based on experimental requirements before being prepared for in vitro or in vivo injections.

This streamlined process ensures precise tracking of AAV capsids through their unique barcodes, enabling efficient screening and characterization of capsid libraries for gene therapy applications.

Capsid Evaluation with ATHENA-I

Next-generation sequencing (NGS) is used to analyze barcode data. Researchers can assess the effectiveness of different capsids for specific target cells using unique DNA or RNA barcodes. This enables the simultaneous evaluation of hundreds of AAV vectors in both in vivo (living organisms) and in vitro (cell culture) experiments.

Example: AAV Capsid Barcode Kits-Common

The AAV Capsid Barcode Kit-CAG-EGFP was systematically injected into C57B6 mice, and all tissues were collected two weeks post-injection. DNA and RNA were extracted from the tissues to prepare NGS libraries. The data reflects the enrichment fold of barcodes (BCs) in each tissue relative to the input viruses, as determined through NGS analysis.

AAV tissue tropisms in mice Results:

  • Overall RNA: AAV9, AAVrh.10.
  • Overall DNA: AAV-DJ, AAVrh.74.
  • Liver: RNA: AAV9>AAVrh.10>AAV8; DNA, AAV-DJ>AAVrh.74>AAV8
  • Lung: RNA: AAV9>AAV11>AAVrh.10; DNA, AAV11>AAVrh.74=AAV8=AAVrh.10
  • Brain(BBB): RNA, AAV9>AAVrh.10>AAV8; DNA, AAV9>AAVrh.10=AAV8=AAV11
  • Heart RNA: AAVDJ>AAV9>AAVrh.10; DNA, AAV11>AAVrh.74>AAV-DJ
  • Kidney RNA: AAV9>AAVrh.10>AAV8; DNA, AAV-DJ>AAV8>AAV3B
AAV Capsid Barcode Kit-data

Notes: 

1. AAVnerGene provides custom AAV capsid barcoded kit construction and production services. Customers can design their own expression cassettes and DNA barcode strategies, and add any AAV capsid into the libraries.

2. AAVnerGene has a capsid collection over 1000 different serotypes and variants.

3. We also provide AAV capsid optimization services based on the ATHENA-I platform.

AAV Capsid Barcode Kits are known AAV capsid selection kits build on our ATHENA-I platform. They are used to systematically evaluate a group of AAV serotypes or variants by using Barcode-Seq technology and next generation sequencing (NGS). In AAV Capsid Barcode Kits, each capsid variant was assigned to three different DNA barcodes (BCs). By comparing DNA or RNA levels of BCs with NGS, researchers can determine which AAV capsid variant is the most efficient for their specific application. 

  • AAV Capsid Barcode Kit-Common: It contains 15 common used AAV serotypes, AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAV12, AAV13, AAVrh.10, AAVrh.74, AAV-DJ, and AAV2-Retro.
  • AAV Capsid Barcode Kit-Tissues: Besides the 15 common AAV serotypes, it also contain 9 reported tissue-targeting AAV variants, with a total of 24 capsids.
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Custom AAV Capsid Barcode Kits

AAVnerGene offers end-to-end services for the custom construction and production of AAV Capsid Barcode Kits. Customers can tailor their kits by selecting their preferred promoter, reporter gene, barcode design, and capsid variants, ensuring fully customized solutions to meet specific experimental needs. Customization Options:

  • Select Your Capsids: Choose from a diverse set of capsids representing various serotypes, tropisms, and transduction efficiencies. Options include well-characterized AAV serotypes, newly engineered capsids, or variants with specific properties.
  • Choose Your Promoter: Select the optimal promoter to ensure your gene of interest is expressed in the right cells, at the desired level, and for the required duration.
  • Select Your Reporter System: Pick a reporter gene or cassette for easy detection and quantification of transduction efficiency and tropism. Common options include:
    • Fluorescent proteins: GFP, mCherry, etc.
    • Enzymatic reporters: Luciferase (Cluc, Fluc, Gluc, Rluc), β-galactosidase, etc.
  • Design Your DNA Barcode Strategy: Incorporate unique DNA barcodes to track and identify individual capsid variants. Barcodes are typically placed in non-coding regions and designed for compatibility with downstream sequencing or detection methods.
    • Barcode Design Options: N25, 2xN12
    • Number of Barcodes per Capsid: We recommend using 3 barcodes per capsid to balance cost and experimental accuracy.
  • Determine Production Strategy and Scale: Plan the production strategy and scale based on the number of capsid variants, desired yields, and intended applications.
    • Individual Purification: 1e12 vg, 2e12 vg, 5e12 vg, 1e13 vg, etc.
    • Pooled Purification: 10 cm dish, 15 cm dish, 850 cm² roller bottle, etc.

Unlike custom AAV Capsid Barcode Kits, the AAV Capsid Barcode Library includes a larger number of AAV capsid variants, and the library is purified and provided as pooled particles.  The production process involves the following steps:

  • Plasmid Preparation: The selected pRCap plasmid (encoding the AAV capsid) is paired with requested AAV barcode plasmids (each containing a unique DNA barcode).

  • Co-Transfection: The paired plasmids are co-transfected into HEK 293T/HEK 293 or our HEK 293one cells using our PEIone transfection reagent in our standard protocol to produce AAV particles.

  • AAV Purification and Titration: The produced AAVs are pooled and purified using advanced purification methods to ensure high-quality yields. The final AAV capsid barcode library is then titrated to determine its concentration and ensure batch-to-batch consistency.

  • Pooling for Injection: The pool AAV vectors can be directly for in vitro or in vivo injections.

The production cost is based on the production scales and requested barcode number per capsid. 

Production ScalePrice/CapsidBulk Order DiscountTurnaround  Time
Barcodes/Capsid345More10~20, 10%;
21-40, 15%;
41~70, 20%;
71-100, 25%;
>100, request		3-4 weeks
25 ml culture or 15-cm dish$400$450$500Quote
100 ml culture or 850-cm2 roller bottle$1,000$1,100$1,200Quote
AAV Capsid Barcode Library Production
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AAV barcode reporter plasmids are essential for generating AAV Capsid Barcode Kits. Each plasmid contains a unique barcode strategically positioned between the WPRE enhancer and the PolyA sequence. At AAVnerGene, we offer a diverse range of AAV barcode plasmids featuring various backbones, promoters, reporters, and barcodes to meet your specific research needs. Researchers can order sets of 100 AAV barcode plasmids, each containing unique barcode, directly from AAVnerGene.

AAV Barcode Plasmids
BackboneProduct NameSKUBarcode DesignRequest Plasmids
pAAVtripAAV-CAG-EGFP-BC-N25SA005001AN25 (NNNNNNNNNNNNNNNNNNNNNNNNN)pRCap+  mini-pHelper
pAAV-CAG-EGFP-BC-2XN12SA005001B2XN12(NNNNNNNNNNNNCGGAAATACGATGTCGGGANNNNNNNNNNNNpRCap + mini-pHelper
pAAVdualpAAVdual-CMV-mCherry-BC-N25SA001006AN25 (NNNNNNNNNNNNNNNNNNNNNNNNN)pRcap
pAAVdual-sc-CMV-mCherry-BC-N25SA023006AN25 (NNNNNNNNNNNNNNNNNNNNNNNNN)pRcap

AAVnerGene’s 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). The screening can be performed in vitro using cell lines, primary cells and organoids, as well as in vivo using mouse and non-human primate (NHP) models. For specific screening needs, it is recommended to discuss with AAVnerGene’s technical support team to ensure the best results. Contact us to request a quote (customer@aavnergene.com).

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Potential models: 

  • In vitro: cell lines, primary cells, IPS cells, organoids
  • In vivo: mouse, rat, and non-human primate (NHP) 

Targeting tissues:

AAV Biodistribution Analysis in different tissues

The complexity of cell types in different tissues, as well as the presence of hundreds of AAV serotypes/variants, make it very difficult to analyze AAV tropism with traditional methods. To fully leverage the output of these large screening paradigms across multiple targets, we have developed a scRNA-AAVseq (single cell RNA-AAV barcode sequencing) pipeline for in vivo characterization of barcoded AAV pools at unprecedented resolution. In this workflow, multiple barcoded AAVs are injected into mice followed by tissue dissociation, single-cell library construction  and sequencing with multiplex Illumina NGS. The cell transcriptome aliquots undergoing the standard library construction protocol was further amplified using AAV bar-code-specific primer to enrich AAV capsids. The amplified viral transcripts are then sequenced as separate NGS libraries. The single cell transcriptome analysis is performed using CeleScope 2.0 and annotated using our in-house program. In parallel, reads from the amplified viral transcripts are used to count 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, and is used to construct a variant lookup table.

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Picture is from Brown et al., 2021. Deep Parallel Characterization of AAV Tropism and AAV-Mediated Transcriptional Changes via Single-Cell RNA Sequencing.

scRNA-seq for AAV Tropism Analysis in PBMCs

The ATHENA-I library containing 463 AAVs, was used to infect PBMCs at an MOI of 2e5 vg/cell. Samples were collected 5 days post-infection. A. Subgrouping of major cell types following scRNA-seq analysis. B. Overlap between cell types and AAVs. C. AAV positive rates in each cell type. D. Log2 RNA fold changes compared to AAV6 across different AAVs.

We conducted a systematic comparison of commonly used AAV serotypes by injecting them into C57BL/6 mice using our AAV Capsid Barcode Kit-CAG-EGFP. At two weeks post-injection, tissues were collected and analyzed. The data, derived from next-generation sequencing (NGS), reveal the enrichment folds of barcodes (BCs) in each tissue relative to the input viral pool. This approach provides a comprehensive and quantitative assessment of the tissue tropism and transduction efficiency of each AAV serotype, offering valuable insights for selecting the optimal vector for specific applications. The results showed that:

  • Overall:
    • AT RNA Level: The AAV serotype with the best overall performance in different tissues is AAV9, followed by AAVrh.10.
    • AT DNA Level: The two AAV serotypes with the best overall performance are AAV-DJ and AAVrh.74.
  • Tissues:
    • Liver: RNA, AAV9>AAVrh.10>AAV8; DNA, AAV-DJ>AAVrh.74>AAV8
    • Lung: RNA, AAV9>AAV11>AAVrh.10; DNA, AAV11>AAVrh.74=AAV8=AAVrh.10
    • Brain(BBB): RNA, AAV9>AAVrh.10>AAV8; DNA, AAV9>AAVrh.10=AAV8=AAV11
    • Heart: RNA, AAVDJ>AAV9>AAVrh.10; DNA, AAV11>AAVrh.74>AAV-DJ
    • Kidney: RNA, AAV9>AAVrh.10>AAV8; DNA, AAV-DJ>AAV8>AAV3B
AAV Capsid Library(I)-Common