AAV Capsid Tropisms
AAV Capsid Tropisms and Gene Therapies
Adeno-associated virus (AAV) vectors are widely used in gene therapy due to their efficient transduction, long-term gene expression, low immunogenicity, and favorable safety profile. It has been approved for human use, targeting different tissues due to the various tropisms of AAV.
| Drugs | Diseases | AAVs | Targets | Approval |
|---|---|---|---|---|
| Glybera | LPLD | AAV1 | Muscle | EMA 2012 |
| Luxturna | RPE65-LCA | AAV2 | Retina | FDA, 2017 |
| Zolgensma | SMA | AAV9 | Brain | FDA, 2019 |
| Upstaza | AADC | AAV2 | Brain | EMA, 2022 |
| Roctavian | Hemophia A | AAV5 | Liver | EMA,2022 |
| EtranaDez | Hemophia B | AAV5 | Liver | FDA, 2022 |
| ELEVIDYS | DMD | AAVrh74 | Muscle | FDA, 2023 |
AAV Receptors and Co-receptors
Adeno-associated virus (AAV) vectors are widely used in gene therapy due to their efficient transduction, long-term gene expression, low immunogenicity, and favorable safety profile. AAVs come in various serotypes, each with unique capsid proteins that determine their tropism—i.e., their ability to target specific cell types or tissues. To date, over 13 natural AAV serotypes, and more than 100 variants have been identified, many of which have gained regulatory approval. These serotypes differ in their tissue specificity and utilize diverse receptors and co-receptors for cellular entry.
- Primary Receptor:
- Heparan sulfate proteoglycans (HSPG) are the first identified primary receptor for AAV2.
- Co-Receptors for AAV2:
- FGFR1
- αVβ5 and α5β1 integrins
- HGFR
- Laminin receptor (LR)
- Other AAV Serotypes:
- AAV3, AAV6, AAV13: Also use HSPG as their primary receptor.
- AAV1: Binds to α2-3 N-linked sialic acid (SIA).
- AAV6: Binds to both α2-3 and α2-6 N-linked SIA and heparin. The amino acid K531 in AAV6 (absent in AAV1) enhances heparin binding and improves transduction.
- AAV4: Binds to α2-3 O-linked SIA.
- AAV5: Binds to α2-3 N-linked SIA.
- AAV7 and AAV8: Receptors remain unclear.
- AAV9: Binds to terminal N-linked galactose of SIA.
- AAVrh.10: Binds to sulfated N-acetyllactosamine (LacNAc) via a pocket on the capsid.
- Co-Receptors for Other Serotypes:
- AAV3: FGFR1, HGFR, and LR
- AAV5: PDGFR
- AAV6: EGFR
- AAV9: Putative integrin
- LR Functionality:
- Serves as a receptor for AAV8 and a co-receptor for AAV9.
Recent genome-wide screening studies identified novel host proteins facilitating AAV transduction, including the type I transmembrane protein KIAA0319L, which has been designated as the AAV receptor (AAVR). Another universal host protein, G protein-coupled receptor 108 (GPR108), plays a role in the transduction of several rAAV serotypes. Although knocking out AAVR and GPR108 in vivo reduces transduction, cell-surface binding is largely unaffected, suggesting that these host proteins primarily contribute to the transduction process post-attachment.
Table 1 Summary of natural AAV receptors and tissue specificity in humans [Wang et al., 2024].
| AAV serotype | Origin | Receptor for cellular attachment | Receptors for post-attachment | Tissue tropism in human | |
| Primary receptors | Co-receptors | ||||
| AAV1 | NHP | N-linked sialic acid | Unknown | AAVR, GPR108 | Skeletal muscle, CNS, airway, retina, pancreas |
| AAV2 | Human | HSPG | FGFR1, HGFR, LamR, CD9, Tetraspanin | AAVR, GPR108 | Retina, CNS |
| AAV3 | Human | HSPG | FGFR1, HGFR, LamR | AAVR, GPR108 | Liver |
| AAV4 | NHP | O-linked sialic acid | Unknown | GPR108 | Lung |
| AAV5 | Human | N-linked sialic acid | PDGFR | AAVR | Retina, CNS, kidney, pancreas, liver |
| AAV6 | Human | N-linked sialic acid | EGFR | AAVR, GPR108 | Airway, CNS |
| AAV7 | NHP | Unknown | Unknown | Unknown | Liver |
| AAV8 | NHP | Unknown | LamR | AAVR, GPR108 | Liver, CNS, retina |
| AAV9 | Human | Galactose | LamR | AAVR, GPR108 | Heart, CNS |
| AAVrh.8 | NHP | Unknown | Unknown | Unknown | CNS |
| AAVrh.10 | NHP | Unknown | Unknown | Unknown | CNS, skeletal muscle |
| AAVrh.74 | NHP | Unknown | Unknown | Unknown | Skeletal muscle |
AAV Serotypes and Their Tropism in C57B6 Mouse
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 Tropism in Different Brain Regions(B6C3 mouse)
The AAV9 is the best AAV serotypes with ability to crossing blood brain barrier (BBB) in B6C3 mice. It ranks in 1 for cerebellum, cortex hypothalamus, midbrain, striatum and hippocampus. AAV2 may have relative specific expression in Striatum.
* Ranking of RNA expression of 15 commonly used AAV serotypes in different regions of B6C3 brain after systematically injection of AAV Capsid Barcode Kit-CAG-EGFP.
Performance of Crossing BBB Capsids in B6C3 Mice
Among the tested AAV capsids, AAV9P31 demonstrated the highest efficiency in most brain tissues (cerebellum, cortex hypothalamus, midbrain, striatum and hippocampus), followed by AAV-F, AAV-MDV1A, AAV-PHP.C2, AAV-PHP.eB, AAV-PHP.B, and AAV-PHP.B3.
ATHENA-I Library was systematically injection into B6C3 mouse. All tissues were collected at 2 weeks post injection. All data shows RNA expression folds compared with AAV9.
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.
| AAV Capsids | AAV-Php.B | AAV-Php.eB | AAV.Cap-B10 | AAV.Cap-B22 |
Backbone | AAV9 | Php.B | Php.eB | Php.eB |
Engineering sites | VR-VIII | VR-VIII flanking substitutions | VR-IV | VR-IV |
Mouse | Yes | Yes | Yes | Yes |
NHP-Marmoset | NO | 4-fold | 4-fold | 12-fold |
NHP-Rhesus macaques | NO | NO | NO | NO |
Liver de-targeting | NO | Yes | Yes | NO |
AAV Capsid Barcode Kits for AAV Tropism Analysis
AAV Capsid Barcode Kits-Common
The AAV Capsid Barcode Kits-Common contain 15 common AAV serotypes, including AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAV11, AAV12, AAV13, AAVrh.74, AAV-DJ. Researchers can choose different reporter systems, such as AAV-CAG-EGFP, AAV-CMV-mCherry, and scAAV-CMV-mCherry.
AAV Capsid Barcode Kits-Tissue
In the tissue-specific AAV Capsid Barcode Kits, 9 selected tissue-targeting capsids are added into the AAV Capsid Barcode Kits-Common. with a total of 24 AAV capsids.
AAV Serotype Testing Kits
Our AAV Serotype Testing Kits include the 15 most commonly used AAV serotypes: AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAV12, AAV13, AAV-DJ, AAVrh.10, AAVrh.74, AAV2-RetroAAVrh.10, ,AAVrh.74, AAV2-Retro. Each kit contains vectors that carry either fluorescent proteins (EGFP, mCherry, TdTomato) or reporter luciferases (Fluc, Rluc, Cluc, Gluc), driven by a promoter (CAG, CMV, EF1α, or hSyn). Key Benefits:
- Cost-effective tools for testing AAV transduction efficiency.
- Assessing tissue-specific tropism related to different serotypes.
- Serving as a negative control to verify that observed biological effects stem from specific transgenes.
| Name | Price | Buy |
|---|
Custom AAV Capsid Barcode Kits
At AAVnerGene, we offer custom AAV capsid barcode kit services, allowing customers to tailor their kits to meet specific research needs. You can select your preferred AAV capsids, promoters, reporters, and other elements to create a personalized AAV capsid barcode kit. This flexibility enables you to design experiments that align precisely with your goals, whether for studying tissue tropism, transduction efficiency, or other applications. Let us help you build the perfect toolkit for your AAV research!
AAV Barcode Plasmids
AAV Barcode plasmids are used to generate AAV Capsid Barcode Kits. Each plasmid contains a unique DNA barcode, allowing for the identification and tracking of individual AAV capsid in the kits or libraries.
| Backbone | Product Name | SKU | Barcode Design | Request Plasmids |
| pAAVtri | pAAV-CAG-EGFP-BC-N25 | SA005001A | N25 (NNNNNNNNNNNNNNNNNNNNNNNNN) | pRCap+ mini-pHelper |
| pAAV-CAG-EGFP-BC-2XN12 | SA005001B | 2XN12(NNNNNNNNNNNNCGGAAATACGATGTCGGGANNNNNNNNNNNN | pRCap + mini-pHelper | |
| pAAVdual | pAAVdual-CMV-mCherry-BC-N25 | SA001006A | N25 (NNNNNNNNNNNNNNNNNNNNNNNNN) | pRcap |
| pAAVdual-sc-CMV-mCherry-BC-N25 | SA023006A | N25 (NNNNNNNNNNNNNNNNNNNNNNNNN) | pRcap |
AAV Tropism Analysis with Barcode-seq Technology
Barcode-seq technology involves tagging each AAV vector with a unique DNA barcode, allowing for precise tracking of individual viral particles within a mixed population. This technology is particularly useful for analyzing AAV tropism, as it enables high-throughput and quantitative assessment of vector distribution across different tissues. Here is a detailed overview of the process and its applications.
- Barcode Design and Library Preparation:
- Barcode Generation: Unique DNA sequences (barcodes) are synthesized and incorporated into the AAV vector genomes.
- Library Construction: A library of AAV vectors, each containing a different barcode, is prepared. This library can include various AAV serotypes or engineered capsids.
- In Vivo Administration:
- Injection: The AAV library is administered to animal models (e.g., mice, rats) via different routes, such as intravenous, intramuscular, or intrathecal injections.
- Tissue Collection:
- Harvesting: After a specified time period, tissues of interest (e.g., liver, muscle, brain) are collected from the treated animals.
- DNA Extraction: Genomic DNA containing AAV genomes is extracted from these tissues for subsequent analysis.
- RNA Extraction: Total RNA is extracted and covert to cDNA for NGS library preparation.
- Barcode Sequencing:
- Amplification: Barcodes are PCR-amplified from the extracted DNA or cDNA.
- High-Throughput Sequencing: The amplified barcodes are sequenced using next-generation sequencing (NGS) technologies.
- Data Analysis:
- Barcode Quantification: The abundance of each barcode in the different tissues is quantified based on sequencing data.
- Enrichment Calculation: Enrichment folds for each barcode are calculated by comparing their abundance in tissues relative to the input virus population.
AAV Capsid Tropism Analysis Services
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).