AAV Capsid Evolution Services
AAV Capsid Evolution
AAV capsid evolution is a process that involves modifying the capsid proteins of AAV to create novel variants with optimized characteristics. By introducing genetic diversity into the capsid's variable regions (VRs), researchers can generate libraries of AAV variants. These libraries are then subjected to iterative rounds of selection and screening to identify capsids with desired traits. Starting with a highly diverse library of up to 1e8 (100 million) capsid variants, the evolution process systematically narrows down the candidates to identify the single best-performing capsid. AAV capsid evolution enables the creation of customized vectors tailored to specific therapeutic needs. By leveraging advanced technologies such as next-generation sequencing (NGS), high-throughput screening, and bioinformatics, researchers can rapidly identify and optimize capsid variants with superior performance.
AAV Capsid Evolution Services
AVnerGene offers AAV capsid evolution services through its ATHENA-II and ATHENA-I Platform. Those platforms allow for the high-throughput screening of AAV capsid libraries to identify the most efficient capsids for specific cell types or tissues. The ATHENA platform uses advanced techniques such as next-generation sequencing (NGS) to analyze the capsids and select the most promising candidates. AAVnerGene can also use directed evolution strategies to improve AAV capsid properties, such as tissue tropism and transduction efficiency, to better meet the needs of specific gene therapy applications.
Our standard evolution process involves four rounds of enrichment and verification.
- Round-I, the top 1e5 variants from a high-complexity initial library are corrected.
- Round-II, the selection is narrowed down to the top 100 variants.
- Round-III, the selected 100 capsids are recorded and verified using next-generation sequencing (NGS) to identify the top 10 candidates.
- Round-IV, the top 10 candidates are individually tested to determine their performance and suitability for further development.
This iterative process ensures the identification of highly optimized AAV capsids for specific gene therapy applications.
Round-I: From >1e8 to 1e5 variants
Library Generation: The process begins with the creation of a diverse library of AAV capsid variants. Our Premade AAV Capsid Libraries offer a wide range of unique capsids with extensive sequence diversity. If these libraries do not meet your specific needs, we also provide custom AAV capsid library construction and production services to tailor the library to your requirements.
Transduction: The viral library is used to transduce target cells or tissues of interest. Key factors to consider during this step include:
Dosage: The amount of viral particles used for transduction.
Injection Route: The method of delivery (e.g., intravenous, intramuscular, or tissue-specific injection).
Selection: The selection process is tailored to the specific application and desired outcome.
In Vivo: Capsids are selected based on their ability to target and transduce specific tissues or cell types in living organisms.
In Vitro: Capsids are screened in cell culture systems to identify variants with enhanced properties, such as improved transduction efficiency or tissue specificity.
- Recovery: After transduction, the transduced cells or tissues are harvested, and DNA or RNA is extracted to recover the capsid sequences for further analysis.This step is critical for identifying the most effective capsid variants based on their ability to target specific tissues (tropism) and drive transgene expression (expression).
DNA Recovery: Used to assess AAV tropism, as it reveals which capsid variants successfully delivered their genetic cargo to the target cells or tissues.
RNA Recovery: Used to evaluate AAV expression, as it indicates whether the delivered transgene is being actively transcribed in the target cells.
- Sequencing: The enriched or amplified capsid sequences are characterized using next-generation sequencing (NGS). This step identifies the specific capsid variants present in the selected population.
Analysis: Bioinformatic analysis is performed to evaluate the diversity, frequency, and characteristics of the identified capsids. Top 1e5 variants will be sleeted from next round evolution.
Round-II: From 1e5 to 100 variants
The process for Round-II is similar to Round-I, with the exception of library generation. In this round, the top 1e5 variants identified from Round-I are synthesized as an oligo pool and cloned into the same variable region as the initial library. In the Round-II library, each variant is present in equal amounts, and the requested library complexity is relatively lower (1e7, providing 100-fold coverage of the 1e5 variants). This focused approach allows for more efficient screening and enrichment of the most promising capsid variants, advancing the evolution process toward identifying highly optimized candidates. This step will generate top 100 variants for next round evolution.
Round-III: From 100 to 10 variants
Unlike Round-I and Round-II, this step employs the ATHENA-I platform to evaluate the top 100 capsids. In this process, the AAV helper plasmid containing distinct peptide sequences is generated individually for each capsid variant. Each capsid is assigned 3 unique barcodes to ensure precise tracking and identification following DNA/RNA recovery. For more details, please refer to our AAV Evaluation Services. This step is designed to identify the top 10 variants, which will advance to the next round of evolution for further refinement and optimization. Single-cell level comparison can be performed using the scRNA-AAVseq technology. This advanced approach enables the analysis of AAV capsid performance at an unprecedented resolution, allowing researchers to assess transduction efficiency, tropism, and transgene expression in individual cells. By leveraging this technology, it is possible to identify the most effective capsid variants with high precision, accelerating the development of optimized AAV vectors for targeted gene therapy applications.
Round-IV: Individual testing
In this step, the top 10 capsids are individually packaged with a reporter gene and transduced into the target cells or tissues. Standard assays, such as EGFP expression and qPCR analysis, are used to evaluate and identify the best-performing capsids. These assessments provide critical insights into transduction efficiency, tropism, and overall functionality, enabling the selection of the most effective capsid variants.
AAV Capsid Evolution Models
AAVnerGene provides comprehensive AAV capsid screening services to support the development of optimized gene therapy vectors. Our services include both in vitro and in vivo models, enabling thorough evaluation of capsid performance under controlled and physiologically relevant conditions.
In Vitro Models:
We utilize a variety of cell-based systems to assess and evolution AAV capsids in a controlled environment. These include:
Human and Animal Cell Lines: For high-throughput screening and initial capsid evaluation.
Primary Cells: To study capsid performance in more physiologically relevant cell types.
iPSC-Derived Cells: For disease-specific modeling and personalized therapy development.
Organoids: To evaluate capsid tropism and transduction efficiency in 3D tissue-like structures.
In Vivo Models:
To assess capsid performance in complex biological systems, we screen AAV capsids using a range of animal models, including:
Small Animals: Mice and rats for initial in vivo testing and proof-of-concept studies.
Non-Human Primates (NHPs): For translational studies and immune response evaluation.
Large Animals: Pigs, dogs, and other large animals to model human physiology and disease more closely.
AAV Cross-species Issues
Animal models play a critical role in screening AAVs designed to target specific tissues. Recent studies have highlighted a key finding: AAVs can perform differently across species and animal strains, underscoring the importance of cross-species testing [Tabebordba et al., 2021; Gonzalez et al., 2022]. For instance, AAV-PHP.B displayed the ability to cross the blood-brain barrier (BBB) in specific mouse strains but not in non-human primates (NHPs) [Hordeaux et al., 2018; Matsuzaki et al., 2019]. To identify variants that are more likely to be effective in humans, it is essential to conduct variant screening in a range of animal models and human-based systems, ensuring the selection of AAVs with translatable performance. The directed evolution of a random peptide insertion library of AAV9 (VR-VIII) led to the development of the AAV-PHP family, with AAV-PHP.B showing significantly enhanced BBB-crossing capabilities in mice. Subsequent engineering of AAV-PHP.B resulted in the creation of AAV-PHP.eB, which retains the same peptide insertion but incorporates additional flanking substitutions. AAV-PHP.eB demonstrated efficient central nervous system (CNS) transduction in mice, achieving 55–76% neuron transduction depending on the region—more than 2.5 times that of AAV-PHP.B. Further modifications led to liver de-targeted variants of AAV-PHP.eB, including AAV.CAP-B10 and AAV.CAP-B22. AAV.CAP-B10 maintained CNS targeting in mice while exhibiting reduced transduction of peripheral organs and a 50-fold decrease in liver tropism compared to AAV-PHP.eB, and over 100-fold compared to AAV9. This variant showed specific neuronal targeting within the CNS, with fewer transduced astrocytes and oligodendrocytes compared to AAV-PHP.eB. Notably, AAV.CAP-B10 achieved broad and robust transgene expression in the CNS of adult marmosets, with a 4-fold increase in CNS transduction over AAV9 and a 17-fold reduction in liver expression compared to AAV9. However, when administered as a pool in infant rhesus macaques, AAV.CAP-B10 produced only slightly higher CNS enrichment compared to AAV9. AAV.CAP-B22, another variant, demonstrated even higher CNS transduction in marmosets, achieving a 12-fold increase compared to AAV9. However, it also showed greater astrocyte transduction than both AAV9 and AAV.CAP-B10. While AAV.CAP-B22 showed similar liver tropism to AAV9, it did not effectively translate in newborn rhesus macaques.
Summary
AAVnerGene offers comprehensive AAV capsid screening services both in vivo and in vitro. These screening services are designed to support AAV-based research and therapeutic development, offering insights into vector performance and AAV evolution, and suitability for different applications. Given the complexity of the evolution processes, we encourage you to contact us for assistance. Our team is ready to provide guidance and support to ensure the success of your AAV capsid optimization and gene therapy projects.
| Model | Tissue | Evolution Process | Turnaround | Price |
| Cell line iPSC Organoid Mouse Rat NHP Others | Brain Liver Lung Heart Muscle Retina Others | Round-I Round-II Round-III Round-IV | TBD | Request a Quote |