Viral vector platform

AAV packaging

Adeno-associated virus (AAV) vectors support sustained transgene expression, particularly in non-dividing cells, and offer a favorable safety profile. FuBio provides AAV packaging services with a range of natural and engineered capsids, along with customizable promoter options for diverse research applications.

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What Are Adeno-Associated Viruses (AAVs)?

Adeno-associated viruses (AAVs) are small, non-enveloped viruses with a single-stranded DNA genome. First discovered in the 1960s, AAVs are among the smallest known viruses, measuring approximately 22 nm in diameter.

As their name suggests, AAVs were originally identified in association with adenoviruses. Wild-type AAVs depend on helper viruses, such as adenoviruses, for efficient replication in host cells. In recombinant AAV (rAAV) production, the essential helper functions are typically provided through helper plasmids rather than co-infection with live helper viruses.

Schematic of an AAV particle: an icosahedral capsid enclosing the single-stranded DNA genome, with the inverted terminal repeats and the viral cargo of about 2.7 kb labeled.

AAV Packaging and Production

The three plasmids are co-transfected into HEK293 producer cells, enabling vector genome replication, capsid assembly, and packaging of the vector genome into AAV capsids.

  • pAAV Cis Plasmid

    Contains the gene of interest (GOI), shRNA, or gRNA expression cassette flanked by two inverted terminal repeats (ITRs), which are essential for vector genome replication and packaging.

  • pAAV Rep/Cap Plasmid

    Provides the Rep proteins required for AAV genome replication and packaging, as well as the Cap proteins responsible for capsid formation and serotype determination.

  • pAd Helper Plasmid

    Supplies essential adenoviral helper functions, including E2A, E4, and VA RNA, to support efficient AAV production without the need for adenovirus co-infection.

Schematic overview of recombinant AAV production using a triple-plasmid transfection system: pAAV transgene, pAAV Rep/Cap and pAd helper plasmids are co-transfected into HEK293 packaging cells, followed by AAV harvest and purification to obtain encapsidated AAV particles carrying the transgene.
Figure. Schematic Overview of Recombinant AAV Production Using a Triple-Plasmid Transfection System

The three plasmids are co-transfected into HEK293 producer cells, enabling the replication of the vector genome, assembly of viral capsids, and encapsidation of the transgene. Following production, the rAAV particles are harvested and purified to remove cellular impurities and process-related contaminants.

Purified rAAV vectors undergo quality control testing to assess vector genome titer, purity, and other relevant quality attributes. Additional characterization, such as capsid integrity and full/empty capsid ratio analysis, may be available upon request to support specific research requirements.

AAV Packaging Service Advantages

  • Up to 1 × 10¹³
    gc/mL

    High-titer AAV production with titers of up to 1 × 10¹³ gc/mL, supporting efficient gene delivery and a wide range of research applications.

  • Multiple
    Serotype Options

    A broad selection of AAV serotypes to meet diverse tissue-targeting and research needs, with expert guidance for optimal serotype selection.

  • 50+
    Promoter Options

    50+ promoter options and flexible vector design solutions tailored to your specific research requirements.

  • Quality
    Control

    Comprehensive quality testing, including vector genome titer, purity, and safety-related assessments, to support consistent and reliable AAV production.

Applications of AAV Vectors

  1. 1

    Monogenic Disorders

    Treating inherited diseases caused by single-gene mutations, such as hemophilia.

  2. 2

    Neurological Disorders

    Enabling targeted gene delivery for neurological and neurodegenerative diseases.

  3. 3

    Rare Genetic Diseases

    Advancing gene therapies for rare disorders, including SMA and DMD.

  4. 4

    Ocular Diseases

    Restoring vision through targeted gene delivery to retinal cells.

  5. 5

    Cardiovascular and Muscular Diseases

    Supporting innovative gene therapies for heart and muscle disorders.

AAV Vector Features

Feature AAV
Gene Expression Long-term, stable expression
Immunogenicity Low
Payload Capacity ~4.7kb
Tropism (Targeting ability) High – tailored with different capsids
Best used for In-vivo studies, long-term expression
Serotype Selection Guide

Production and quality control

  • AAV vectors are produced using a triple-plasmid transfection system in HEK293 cells, consisting of a transfer plasmid, a serotype-specific Rep/Cap plasmid, and an adenoviral helper plasmid.
  • Following harvest and clarification, AAV vectors are purified using affinity chromatography and iodixanol density gradient ultracentrifugation, followed by buffer exchange into the final formulation.
  • Vector genome titers are determined by qPCR or digital PCR (dPCR) using assays targeting the ITR or transgene and reported in genome copies per milliliter (gc/mL). Each lot undergoes quality control testing for sterility, endotoxin, and mycoplasma.
  • Additional characterization, including full/empty capsid ratio and vector aggregation analysis, is available upon request for in vivo research applications.

Our Service Options

  • Standalone AAV Packaging

    For researchers with existing AAV transfer plasmids, we offer standalone AAV packaging using customer-supplied constructs, subject to compatibility review.

  • Vector Construction & AAV Packaging

    Our integrated service combines customized vector construction and AAV packaging, providing a streamlined solution from vector design to viral production.

Service Specifications

Specification Information
Vector Type ssAAV
Available Serotypes AAV1, AAV2, AAV6, AAV8, AAV9, AAV-DJ, AAV-PHP.eB; additional capsids on request
Vector Titer Up to 1 × 10¹³ gc/mL
Purification Affinity chromatography followed by iodixanol gradient ultracentrifugation
Quality Control Vector genome titer by qPCR/dPCR; purity, sterility, endotoxin and mycoplasma testing on every lot
Volume 50 μL
Turnaround Time 4-5 weeks from design to delivery
Application In vitro / In vivo Research

Note: AAV-PHP.eB transduction efficiency and CNS tropism are species- and strain-dependent. Serotype selection should be based on the target tissue, animal model, and route of administration.

Start Your AAV Packaging Project

Whether you need packaging for an existing transfer vector or a comprehensive vector construction and packaging solution, our team is ready to help you identify the right approach for your research.

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Experimental validation

The images below present AAV transduction and characterization data from the FuBio toolkit virus portfolio: capsid tropism across mouse brain regions, serotype comparison in dorsal root ganglia, and particle morphology by transmission electron microscopy.

Fluorescence images of AAV2/1 tropism in multiple mouse brain regions with quantified GFP-positive cells
Figure 1 · Tropism of AAV2/1 in multiple brain regions at 3 and 6 weeks post injection.
Fluorescence images of L3 dorsal root ganglia from AAV-injected mice across six serotypes, and a transmission electron microscopy image of purified AAV particles
Figure 2 · Representative images of L3 DRGs obtained from AAV-injected mice across the AAV2, AAV5, AAV6, AAV8, AAV9 and AAVrh10 serotypes (left), and AAV particles under transmission electron microscopy (right).

Resources

User Instructions

A general guide to transducing cultured mammalian cells using recombinant adeno-associated virus (rAAV) vectors. For detailed instructions, please refer to the product-specific protocol or contact our technical support team.

  1. 1

    Choose the Dose

    Optimal AAV dosage varies depending on the serotype, target cell type, and experimental conditions. Start with a range of vector doses and perform a dose-response study to determine the optimal conditions for your application.

  2. 2

    Thaw and Handle

    Thaw AAV stocks on ice and mix gently before use. Avoid vortexing and repeated freeze-thaw cycles. Use low-binding tubes and prepare single-use aliquots whenever possible to preserve vector stability and activity.

  3. 3

    Transduce

    Add diluted AAV vectors to target cells under appropriate culture conditions. Optimize cell density, vector dose, and incubation time based on the specific cell type and AAV serotype. Polybrene is generally not required for AAV transduction.

  4. 4

    Evaluate Transgene Expression

    Monitor transgene expression at appropriate time points following transduction. Expression kinetics vary depending on the vector design, serotype, and target cells. Evaluate transduction outcomes using fluorescence imaging, RT-qPCR, or protein-based assays, as appropriate.

  5. 5

    Plan for Long-Term Expression

    AAV vector genomes primarily remain episomal within transduced cells. In rapidly dividing cells, transgene expression may decrease over successive passages. Consider cell proliferation rates and experimental duration when planning long-term studies.

Important Note: These guidelines are intended for general in vitro research use. Optimal transduction conditions vary by AAV serotype, cell type, and experimental design. Follow product-specific protocols and institutional biosafety requirements.

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