AAV Capsids for NHPs

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

NHP Models for Researches

Non-human primates (NHPs) are indeed valuable models for AAV capsid evolution and evaluation due to their close similarities to humans in terms of tissue structure, immune response, and cognitive function. Below is a list of the commonly used monkey models in AAV research:

  • Rhesus Macaque (Macaca mulatta)
    • Widely used due to their genetic, physiological, and immunological similarity to humans.
    • Frequently employed in studies of neurological diseases, retinal disorders, and liver-targeted gene therapies.
  • Cynomolgus Macaque (Macaca fascicularis)
    • Another popular model, particularly in toxicology and immunogenicity studies.
    • Often used for evaluating AAV capsid tropism and safety in liver, muscle, and CNS-targeted therapies.
  • Marmoset (Callithrix jacchus)
    • Smaller size and shorter lifespan make them cost-effective for certain studies.
    • Used in neuroscience research and retinal gene therapy due to their well-characterized visual system.
  • African Green Monkey (Chlorocebus sabaeus)
    • Known for their use in vaccine development and infectious disease research.
    • Gaining traction in AAV studies due to their relevance in immunogenicity assessments.
  • Pig-tailed Macaque (Macaca nemestrina)
    • Used in neurological and retinal studies because of their larger brain size compared to other macaques.
    • Valuable for evaluating AAV delivery to the central nervous system (CNS).
  • Squirrel Monkey (Saimiri spp.)
    • Smaller NHP model used in neuroscience and retinal research.
    • Suitable for studies requiring a smaller primate model with similar ocular and neural anatomy to humans.

These NHP models provide critical preclinical data on AAV capsid performance, helping researchers optimize tissue targeting, safety profiles, and therapeutic efficacy before advancing to human clinical trials. The choice of model depends on the specific research goals, such as the target tissue, disease type, and the need for immunological or neurological relevance.

AAV Capsid Biodistribution Data in NHPs

We have characterized approximately 500 AAV transduction properties across various tissues in both non-human primate (NHP) and mouse models using our advanced ATHENA-I platform. Researchers interested in accessing or collaborating on this rich dataset are encouraged to contact us to explore potential partnerships. Additionally, we warmly welcome gene therapy companies to invest in our ongoing efforts to develop tailored AAV capsids, especially those optimized and validated using NHP models. Collaborating with us offers a unique opportunity to accelerate capsid innovation with robust preclinical data in relevant species, ultimately enhancing the success of gene therapy programs.

AAV Biodistribution Analysis in different tissues

AAV Capsid Evolution Platform at AAVnerGene-ATHENA

At AAVnerGene, we developed ATHENA AAV screening platforms, which allow users to quickly evaluate, evolve, and create AAV serotypes or variants tailored for specific therapeutic applications.

  • ATHENA-I platform is used to systematically evaluate different AAV serotypes or variants by using Barcode-Seq technology.  
  • ATHENA-II platform is designed to evolve novel AAV capsid with tissue-specific tropism from high complexity random peptide insert library.
  • ATHENA-III is a rational DNA shuffling library used to create novel hybrid AAV capsids.

By combining the three sub-platforms with AI, the ATHENA platform can efficiently identify, evolve and create the best AAV capsids for specific applications, potentially improving the effectiveness of gene therapy and reducing costs.

ATHENA AAV Capsid Engineering platform

AAV-ShD: Crossing BBB AAV Capsids in NHP-Cynomolgus Macaques

Using the ATHENA-I platform, we systematically evaluated a range of AAV capsids for their ability to cross the blood-brain barrier (BBB) in Cynomolgus Macaques. Among all the tested capsids, AAV9P801 demonstrated the best performance, achieving superior BBB penetration and CNS transduction. Additionally, we developed a novel AAV variant, AAV-ShD, which showed remarkable improvements in gene delivery efficiency. Specifically, AAV-ShD exhibited over 380-fold higher DNA levels and 8-fold higher RNA levels in the CNS compared to the natural AAV9 serotype. These results highlight the potential of AAV-ShD as a highly efficient vector for CNS-targeted gene therapy in primates.

Retina Targeting AAV Capsids-AAV-Eye

Gene therapy holds great promise for the treatment of eye diseases. Adeno-associated virus (AAV) serotypes, such as AAV2, AAV5, and AAV8, are widely used for retinal gene delivery. AAV2 is predominantly utilized for intravitreal injection (IVT), while AAV5 and AAV8 are often used for subretinal injection (SRI). Due to its minimally invasive nature and the ability to be performed in an outpatient setting, IVT represents a safe and promising approach for ocular gene therapy. IVT delivers AAV vectors into the vitreous space, primarily targeting the ganglion cell layer (GCL) and other inner retinal cells. However, the inner limiting membrane (ILM) poses a significant barrier, restricting the penetration of natural AAV serotypes into deeper retinal layers beyond the GCL.

To overcome these limitations, engineered AAV2 variants such as AAV2-YFs, AAV2-7M8, and AAV-4D-R100 have been developed through directed evolution, enabling improved delivery to the outer retina. However, significant challenges remain when translating these advancements to large-animal retinas, which differ substantially from rodent retinas. Large-animal retinas, such as those in dogs and non-human primates (NHPs), feature specialized high-acuity regions (e.g., the area centralis in dogs and the fovea in primates), a thicker vitreous, and a more robust ILM. Consequently, AAV2 may not be the optimal serotype for retina-targeting capsid engineering in NHPs using the IVT route.

To identify potential AAV capsids for further engineering, we systematically evaluated commonly used AAV serotypes (AAV2, AAV5, AAV6, AAV8, AAV9, and AAVrh.74) and a series of AAV2 variants (AAV2-Y444F-Y500F, AAV2-Y444F-A493V-Y500F, AAV2-GL, AAV2-NN, and AAV2-7M8) in the whole retina of Rhesus Macaque using barcode technology. The results showed that among the commonly used serotypes, AAV2 exhibited the highest DNA levels in the retina, followed by AAV5, AAV9, AAV8, and AAV6. AAV2-YFs slightly increased DNA levels. AAV2 variants selected from large-animal models (AAV2-7M8) demonstrated a ~10-fold increase in DNA levels compared with AAV2. However, AAV2 variants evolved using mouse models (AAV2-NN and AAV2-GL) showed either comparable or reduced DNA levels.

At the RNA level, AAV6 exhibited an ~8-fold increase in expression compared to AAV2. Notably, AAV2-7M8 demonstrated a several-hundred-fold increase in RNA expression relative to AAV2. Moreover, we identified a novel AAV capsid, designated AAV(Eye), which achieved a 3.4-fold increase in DNA levels and a 47-fold increase in RNA expression compared to AAV2. Additionally, AAV(Eye) demonstrated greater specificity in the retina compared to AAV2-7M8. Importantly, AAV(Eye) also exhibited a ~12-fold increase in RNA expression in Cynomolgus Monkeys compared to AAV2. Using single-cell molecular sequencing, we aim to identify the specific target cells for this capsid.

In summary, AAV(Eye) demonstrates strong potential as a template for future AAV capsid engineering efforts, providing an efficient platform for targeted retinal gene delivery especially in NHP models. Researchers are invited to collaborate on the ecodevelopment of AAV(Eye), with the goal of further enhancing its transduction efficiency across various non-human primate (NHP) models. By working together, we aim to optimize this AAV variant for improved gene delivery to ocular tissues, advancing its potential for treating eye-related disorders. This collaborative effort will leverage shared expertise and resources to accelerate the development of next-generation AAV vectors for translational research and therapeutic applications.

AAV(Eye): A Promising Template for AAV Capsid Engineering in NHP Retinas

[1]Tabebordbar M, Lagerborg KA, Stanton A, King EM, Ye S, Tellez L, et al. Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell 2021;184:4919-4938.e22.

[2]Gonzalez TJ, Simon KE, Blondel LO, Fanous MM, Roger AL, Maysonet MS, et al. Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing. Nat Commun 2022;13:5947.

[3]Hordeaux J, Wang Q, Katz N, Buza EL, Bell P, Wilson JM. The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J Mice. Mol Ther 2018;26:664-8. 

[4]Matsuzaki Y, Tanaka M, Hakoda S, Masuda T, Miyata R, Konno A, et al. Neurotropic Properties of AAV-PHP.B Are Shared among Diverse Inbred Strains of Mice. Mol Ther 2019;27:700–4. 

[5]Goertsen D, Flytzanis NC, Goeden N, Chuapoco MR, Cummins A, Chen Y, et al. AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset. Nat Neurosci 2022;25:106–15. 

[6]Chuapoco MR, Flytzanis NC, Goeden N, Octeau JC, Roxas KM, Chan KY, Scherrer J, Winchester J, Blackburn RJ, Campos LJ, Man KNM, Sun J, Chen X, Lefevre A, Singh VP, Arokiaraj CM, Shaya TF, Vendemiatti J, Jang MJ, Mich J, Bishaw Y, Gore B, Omstead V, Taskin N, Weed N, Ting J, Miller CT, Deverman BE, Pickel J, Tian L, Fox AS, Gradinaru V. Intravenous functional gene transfer throughout the brain of non-human primates using AAV. Nat Nanotechnol. 2023 Oct;18(10):1241-1251. 

[7]Chen X, Ravindra Kumar S, Adams CD, Yang D, Wang T, Wolfe DA, Arokiaraj CM, Ngo V, Campos LJ, Griffiths JA, Ichiki T, Mazmanian SK, Osborne PB, Keast JR, Miller CT, Fox AS, Chiu IM, Gradinaru V. Engineered AAVs for non invasive gene delivery to rodent and non-human primate nervous systems. Neuron. 2022 Jul 20;110(14):2242-2257.e6.