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AAV Capsid Evolution & Screening Service

OverviewCapsid Engineering PlatformProprietary AAV Capsid LibraryContact Us

With over a decade of experience in gene therapy vector development, Creative Biogene offers advanced AAV capsid engineering services aimed at providing efficient and precise solutions for AAV optimization. By integrating rational design and directed evolution technologies, our platform serves research institutions and pharmaceutical companies seeking high-performance AAV vectors. Combining high-throughput screening, AI-driven prediction, and multi-omics analysis, we are dedicated to developing engineered AAV vectors with enhanced tissue specificity, low immunogenicity, and increased packaging capacity to facilitate the translation of gene therapy from bench to bedside.

Overview

Gene therapy has emerged as a transformative approach in modern medicine, offering new paradigms for treating genetic disorders, cancer, and neurodegenerative diseases by correcting or replacing defective genes. Adeno-associated virus (AAV) vectors stand out among the various gene delivery systems due to their safety profile, low immunogenicity, and long-term gene expression. However, native AAV vectors face several limitations, such as limited tropism, restricted packaging capacity, and pre-existing immunity, which hinder broader clinical application. Using rational design and directed evolution, Capsid engineering technologies have been developed to overcome these barriers by modifying the viral capsid to enhance its biological properties.

AAV Capsid Structure and Function

The AAV capsid adopts a T=1 icosahedral symmetry, composed of 60 VP proteins (VP1, VP2, and VP3). VP3 forms the capsid core, while VP1 and VP2 participate in nuclear entry and genome release via their extended N-terminal regions. Key structural features include:

Fivefold Channels: Gateways for DNA packaging and release.
Threefold Protrusions: Host receptor-binding sites determining tissue tropism.
Twofold Depressions: Structurally weaker zones, possibly involved in immune evasion.

The capsid's surface consists of β-barrel cores and inter-strand loops (e.g., GH loop), forming variable regions (VRs I–IX) that significantly differ among AAV serotypes and influence receptor binding, tropism, and antigenicity.

Limitations of Native AAV

Limited Tropism: Poor transduction efficiency in certain tissues like the brain and heart.
Pre-existing Immunity: High prevalence of neutralizing antibodies against common serotypes (e.g., AAV2, AAV9).
Restricted Packaging Capacity: ~4.7 kb, which limits the delivery of large genes or complex regulatory elements.
Capsid Instability: Certain mutations may disrupt capsid assembly or infectivity.

Creative Biogene AAV Capsid Engineering Platform

Creative Biogene offers an integrated service platform for AAV capsid engineering, covering library design and construction, high-throughput screening, in vivo biodistribution analysis, mechanistic studies, and GMP-grade production. Our goal is to help clients develop safer and more efficient AAV-based gene therapy vectors.

Engineering of AAV Capsid Genes

Library Construction

Rational Design

Peptide Insertion
Receptor Retargeting
Site-Directed Mutagenesis

Directed Evolution

Error-Prone PCR
DNA Shuffling
Random Peptide Display
Semi-Random Loop Replacement
Visualization-Guided Family Shuffling

Computational Design and Evolution

Ancestral Sequence Reconstruction
Structure-Guided SCHEMA Algorithm
Machine Learning-Based Prediction

Variants Screening

In Vitro Cell Models

In Vivo Rodent Models

Clinically Relevant Models

Expanded Packaging Capacity
Neutralizing Antibody Evasion
Modulation of Tissue Tropism

Technical Advantages

1. Multi-dimensional Technology Integration

Our AAV capsid engineering platform combines rational design, directed evolution, and high-throughput screening to optimize vector performance. Structural modeling and machine learning guide targeted modifications, while large capsid libraries (>10⁹ variants) undergo in vitro/in vivo selection to enrich efficient candidates. Barcode-seq enables pooled screening and NGS-based identification of top-performing variants.

2. Comprehensive Workflow

We offer AAV capsid engineering across 12 serotypes with targeted peptide insertion to ensure diversity and functionality. High-quality AAVs are produced via a triple plasmid system and purified by ultracentrifugation and affinity chromatography. Validation includes in vitro transduction, immunogenicity (ELISA), in vivo targeting, and long-term expression tracking.

3. Core Parameter Optimization

Capsids are optimized for enhanced tissue tropism, immune evasion through epitope mutation, and expanded packaging capacity (up to 5.2 kb), enabling efficient and specific gene delivery.

Service Workflow

Creative Biogene provides a comprehensive AAV directed evolution service, covering the entire process from project design to final AAV production. This service enables the development of optimized AAV capsids with improved transduction efficiency, tissue specificity, and therapeutic potential. The workflow includes:

Technical Process

Our services are designed to ensure smooth, efficient, and reliable results. We support every step of the cell line development process:

1Project Consultation & Strategy Design
We start by understanding your specific needs-target tissue, delivery route, and performance criteria-and design a tailored evolution strategy, including library type and screening method.

2Library Construction
AAV capsid libraries are generated using random mutagenesis, DNA shuffling, or peptide insertion. Library diversity and quality are validated by sequencing.

3Library Packaging & QC
The AAV libraries are packaged and assessed for titer and quality to ensure robust diversity and infectivity.

4In Vitro/In Vivo Screening
Directed evolution is performed through 2–5 rounds of selection in cell lines or animal models, enriching for high-performing variants under defined conditions.

5Variant Recovery & Analysis
Enriched variants are recovered and analyzed by sequencing to identify capsids with superior properties.

6Lead Candidate Validation
Top capsids are individually produced and tested for transduction efficiency and tissue specificity in relevant models.

7Large-Scale AAV Production
Final candidates are produced at scale with high purity and titer, ready for downstream research or preclinical applications.

Applications

1. Rare Disease Therapies

Neurological Disorders: Engineering BBB-penetrant AAVs (e.g., PHP.eB) for Rett syndrome, SMA, and more.
Ophthalmic Diseases: Intravitreal AAV vectors targeting retinal layers and RPE for treating inherited retinal diseases.

2. Oncology and Chronic Disease

Cancer Therapy: AAVs delivering immune-regulatory genes to tumor microenvironments to enhance immunotherapy.
Metabolic Disorders: Liver-targeting AAVs for gene replacement in hemophilia and familial hypercholesterolemia.

3. Vaccine Development

Infectious Disease Prevention: AAV-based delivery of viral antigens (e.g., SARS-CoV-2 spike protein) for inducing durable humoral and cellular immunity.

Proprietary AAV Capsid Library

Creative Biogene holds nearly 100 plasmids encoding AAV variants with peptide insertions, as cataloged by Buning et al. These resources are readily available for direct AAV vector packaging and further development.

AAV2 VP3 (I-587) Peptide Insert

AAV2 VP3 (I-588) Peptide Insert

Non-AAV2 VP3 Peptide Insert

Name	Target Cell Type	Insert
AAV-I-587	β1-integrin positive tumor cells	QAGTFALRGDNPQG
AAV-588NGR	CD13-positive tumor cells	NGRAHA
AAV-MO7A	Tumor cells	RGDAVGV
AAV-MO7T	Tumor cells	RGDTPTS
AAV-MecA	Tumor cells	GENQARS
AAV-MecB	Tumor cells	RSNAVVP
rRGD587	αv-integrin positive tumor cells	CDCRGDCFC
AAV-C4	Tumor cells	PRGTNGP
AAV-D10	Tumor cells	SRGATTT
AAV-SIG	Endothelial cells	SIGYPLP
AAV-MTP	Endothelial cells	MTPFPTSNEANL
AAV-QPE	Endothelial cells	QPEHSST
AAV-VNT	Endothelial cells	VNTANST
AAV-CNH	Endothelial cells	CNHRYMQMC
AAV-CAP	Endothelial cells	CAPGPSKSG
AAV-EYH	Smooth muscle cells	EYHHYNK
AAV587MTP	Skeletal muscle cells	ASSLNIA
AAV-r3.45	Neuronal stem cells	TQVGQKT
AAV2-LSS	CNS	LPSSLQK
AAV2-PFG	CNS	WPFYGTP
AAV2-PPS	CNS	DSPAHPS
AAV2-TLH	CNS	GWTLHNK
AAV2-GMN	CNS	GMNAFRA
AAV2-7m8	Retinal cell types	LGETTRP
AAV-Kera1	Keratinocytes	RGDTATL
AAV-Kera2	Keratinocytes	PRGDLAP
AAV-Kera3	Keratinocytes	RGDQQSL
AAV-588Myc	None	EQLSISEEDL
AAV2-Z34C	Adaptor	Z34C
AAV2.N587_R588insBAP	Adaptor	GLNDIFEAQKIEWHE
AAV2Ald13	Adaptor	LCTPSRAALLTGR
DMD4	Vaccine	QVSHWVSGLAEGSFG
DMD6	Vaccine	LSHTSGRVEGSVSLL

Name	Target Cell Type	Insert Sequence
A588-RGD4C	αv integrin-positive tumor cells	CDCRGDCFC
A588-RGD4CGLS	αv integrin-positive tumor cells	CDCRGDCFC
AAV-VTAGRAP	Tumor cells	VTAGRAP
AAV-APVTRPA	Tumor cells	APVTRPA
AAV-DLSNLTR	Tumor cells	DLSNLTR
AAV-NQVGSWS	Tumor cells	NQVGSWS
AAV-EARVRPP	Tumor cells	EARVRPP
AAV-NSVSLYT	Tumor cells (CML)	NSVSLYT
AAV-LS1	Tumor cells (CML), CD34+ cells	NDVRSAN*
AAV-LS2	Tumor cells (CML), CD34+ cells	NESRVLS
AAV-LS3	Tumor cells (CML), CD34+ cells	NRTWEQQ
AAV-LS4	Tumor cells (CML), CD34+ cells	NSVQSSW
AAV-RGDLGLS	Tumor cells	RGDLGLS
AAV-RGDMSRE	Tumor cells	RGDMSRE
AAV-ESGLSQS	Tumor cells	ESGLSQS
AAV-EYRDSSG	Tumor cells	EYRDSSG
AAV-DLGSARA	Tumor cells	DLGSARA
AAV-NDVRSAN	Tumor cells	NDVRSAN*
AAV-GPQGKNS	Tumor cells	GPQGKNS
AAV-NSSRDLG	Endothelial cells	NSSRDLG
AAV-NDVRAVS	Endothelial cells	NDVRAVS#
AAV-NDVRSAN	Endothelial cells	NDVRSAN*
AAV-NDVRAVS	Endothelial cells	NDVRAVS#
AAV-PRSTSDP	Lung (maybe endothelial cells)	PRSTSDP
AAV-DIIRA	Endothelial cells	DIIRA
AAV-SYENV	Endothelial cells	SYENVASRRPEG
AAV-PENSV	Endothelial cells	PENSVRRYGLEE
AAV-LSLAS	Endothelial cells	LSLASNRPTATS
AAV-NDVWN	Endothelial cells	NDVWNRDNSSKRGGTTEAS
AAV-NRTYS	Endothelial cells	NRTYSSTSNSTSRSEWDNS
rAAV2-ESGHGYF	Pulmonary endothelial cells	ESGHGYF
AAV-GQHPRPG	Cardiomyoblasts	GQHPRPG+
AAV-PSVSPRP	Cardiomyoblasts	PSVSPRP
AAV2-VNSTRLP	Cardiomyoblasts	VNSTRLP
AAV-GQHPR	Cardiomyoblasts	GQHPRPG+
AAV-LSPVR	Cardiomyoblasts	LSPVRPG
AAV-MSSDP	Cardiomyoblasts	MSSDPRRPPRDG
AAV-GARPS	Cardiomyoblasts	GARPSEVTTRPG
AAV-GNEVL	Cardiomyoblasts	GNEVLGTKPRAP
AAV-KMRPG	Cardiomyoblasts	KMRPGAMGTTGEGTRVTRE
AAV588MTP	Skeletal muscle	ASSLNIA

Serotype	Position	Name	Target Cell Type	Insert
AAV1	I-590	BAP-AAV1	Scavidin-displaying BT4C (rat glioma)	GLNDIFEAQKIEWHE
AAV1	I-590	BAP-AAV1	Endothelial cells	GLNDIFEAQKIEWHE + CDCRGDCFC
AAV1	I-590	AAV1-RGD	Tumor cells, endothelial cells	CDCRGDCFC
AAV1	I-590	AAV1-RGD/BAP (90/10) (mosaic capsid)	Tumor cells, endothelial cells	CDCRGDCFC and GLNDIFEAQKIEWHE
AAV1	I-590	Tet1c-AAV1 (mosaic capsid)	Tetanus toxin GT1b receptor positive cells	HLNILSTLWKYR
AAV1	I-590a	AAV1.9-3-SKAGRSP	Fibroblast	SKAGRSP
AAV3	I-586	BAP-AAV3	Tumor cells	GLNDIFEAQKIEWHE
AAV4	I-586	BAP-AAV4	Tumor cells	GLNDIFEAQKIEWHE
AAV5	I-575	BAP-AAV4	Tumor cells	GLNDIFEAQKIEWHE
AAV5	I-575	AAV5-7m8	Not successful	LGETTRP80
AAV6	I-585	AAV6-RGD	Tumor cells	RGD
AAV6	I-585 + Y705-731F + T492V	AAV6-RGD-Y705-731F+T492V	Tumor cells	RGD
AAV6	I-585 + Y705-731F + T492V + K531E	AAV6-RGD-Y705-731F+T492V+K531E	Tumor cells	RGD
AAV8	I-585c	AAV2/8-BP2	ON-bipolar cells	PERTAMSLP
AAV8	I-590	AAV8-PRSTSDP	Not successful	PRSTSDP135
AAV8	I-590	AAV8-ESGLSOS	Tumor cells	ESGLSOS135
AAV8	I-590	AAV8-VNSTRLP	Not successful	VNSTRLP138
AAV8	I-590	AAV8-ASSLNIA	Heart (weakly improved transduction)	ASSLNIA122
AAV8	I-590d	AAV8-PSVSPRP	Not successful	PSVSPRP138
AAV8	I-590d	AAV8-GQHPRPG	Heart (weakly improved transduction)	GQHPRPG86
AAV8	I-590d	AAV8-SEGLKNL	Liver	SEGLKNL
AAV8	I-590	AAV8-7m8	Not successful	LGETTRP80
AAV9	I-589	AAV-SLRSPPS	Endothelial cells, smooth muscle cells	SLRSPPS
AAV9	I-589	AAV-RGDLRVS	Endothelial cells, smooth muscle cells	RGDLRVS
AAV9	I-589d	AAV9-NDVRAVS	Endothelial cells	NDVRAVS82
AAV9	I-589d	AAV9-PRSTSDP	Not successful	PRSTSDP135
AAV9	I-589d	AAV9-ESGLSOS	Tumor cells (weak targeting)	ESGLSOS135
AAV9	I-588	AAV-PHP.B	CNS	TLAVPFK
AAV9	I-588	AAV-PHP.A	CNS	YTLSQGW
AAV9	I-588	AAV9-7m8	Retinal cells	LGETTRP80
AAV9P1	Not disclosed	AAV9P1	Neuronal progenitor cells	RGDLGLS

Contact Us

Ready to advance your AAV vector development with high-performance capsid engineering? Contact Creative Biogene today to explore how our AAV capsid-directed evolution service can accelerate your gene therapy pipeline.

FAQ

Q: Why are positions I-587 and I-588 on AAV2 frequently used for peptide insertion?

A: Positions I-587 and I-588 are located near the threefold protrusions on the AAV2 capsid surface, which are tolerant to insertions without disrupting capsid assembly or genome packaging. Studies have shown that up to 34-amino-acid-long peptides can be inserted here, enabling redirection of viral tropism and functional enhancement of vector properties.

Q: What are the key structural features of the AAV capsid relevant to engineering?

A: The AAV capsid is a T=1 icosahedral structure assembled from 60 VP subunits (mostly VP3), featuring:

Threefold protrusions, which mediate receptor binding.
Fivefold channels, essential for genome packaging and capsid assembly.
Variable regions (VRs) in surface-exposed loops, which differ among serotypes and are primary targets for immune recognition and retargeting efforts.

Understanding these structures guides rational design and functional customization of AAV vectors.

Q: What additional support do you offer beyond vector design?

A: Creative Biogene and its partners offer end-to-end services including:

AAV vector design and cloning
High-titer virus packaging (research- to GMP-grade)
In vitro and in vivo transduction assays
Molecular validation (qPCR, ELISA, western blot)
Capsid performance comparison
Regulatory consulting for IND submission

Q: Can I order a ready-to-use engineered AAV vector?

A: Yes. A wide library of engineered AAV vectors is available as off-the-shelf products. Custom orders are also welcome, and timelines are optimized for fast project turnover, typically within 1-4 weeks depending on complexity.

References:

Lee EJ, Guenther CM, Suh J. Adeno-Associated Virus (AAV) Vectors: Rational Design Strategies for Capsid Engineering. Curr Opin Biomed Eng. 2018 Sep;7:58-63.
Ku CA, Pennesi ME. Retinal Gene Therapy: Current Progress and Future Prospects. Expert Rev Ophthalmol. 2015 Jun;10(3):281-299.
Büning H, Srivastava A. Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors. Mol Ther Methods Clin Dev. 2019;12:248-265.

* For research use only. Not intended for any clinical use.

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