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Chimeric Baculovirus-AAV Gene Expression Vector


The adeno-associated virus (AAV) vector system is a popular and versatile tool for in vitro and in vivo gene delivery. AAVs have emerged as one of the most effective vehicles for gene therapy due to their ability to transduce a wide variety of mammalian cell types and their low immunogenicity in humans. The chimeric baculovirus-AAV vector offers a highly efficient tool for the large-scale production of recombinant AAV vectors in insect cells, thereby making it an attractive candidate for pre-clinical and clinical stage gene therapy applications.

Baculovirus-based recombinant AAVs are produced by co-infecting insect cells with two recombinant baculoviruses, specifically the first one expressing the gene of interest (GOI) flanked by the AAV inverted terminal repeats (ITRs) and a second helper baculovirus expressing the AAV rep and cap genes. For generating the two recombinant baculoviruses, the expression cassette for each baculovirus is first cloned into a baculovirus transfer vector. The entire expression cassette, along with a gentamicin resistance gene, is flanked by the Tn7 transposon terminal elements, Tn7L and Tn7R. This vector is then transformed into E. coli carrying the bacmid shuttle vector and a helper plasmid. The bacmid is essentially a large plasmid containing the baculovirus genome modified to carry a lacZ gene and an attTn7 docking site inserted in the lacZ coding region. The helper plasmid expresses the Tn7 transposase. The transposase would then mediate the transposition of the region flanked by Tn7R and Tn7L on the baculovirus transfer vector, which contains the expression cassette and gentamicin resistance, into the attTn7 docking site of the bacmid. Colonies containing recombinant bacmids can then be identified by gentamicin selection and blue/white screening (non-recombinant colonies are blue due to lacZ expression whereas recombinant colonies are white due to disruption of lacZ by transposon insertion). The purified bacmid DNA is then transfected into insect cells to generate live recombinant baculovirus.

Our chimeric baculovirus-AAV gene expression vector is used for expressing a user-selected GOI flanked by the AAV ITRs. Recombinant baculovirus generated from this vector can then be co-infected along with the helper baculovirus expressing the AAV rep and cap genes into insect cells to produce baculovirus-based recombinant AAV particles. When recombinant AAV is added to target cells, the single-stranded linear DNA genome is delivered into cells, where it is converted by the host cell DNA polymerase machinery into double-stranded DNA. AAV vector DNA forms episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers can remain for the life of the host cells. In dividing cells, AAV DNA is lost through the dilution effect of cell division, because the episomal DNA does not replicate alongside host cell DNA. Random integration of AAV DNA into the host genome is extremely rare. This is desirable in many gene therapy settings where the potential oncogenic effect of vector integration can pose a significant concern.

A major practical advantage of AAV is that in most cases AAV can be handled in biosafety level 1 (BSL1) facilities. This is due to AAV being inherently replication-deficient, producing little or no inflammation, and causing no known human disease.

Many strains of AAV have been identified in nature. They are divided into different serotypes based on different antigenicity of the capsid protein on the viral surface. Different serotypes can render the virus with different tissue tropism (i.e. tissue specificity of infection). When our AAV vectors are packaged into virus, different serotypes can be conferred to the virus by using different capsid proteins for the packaging. The serotypes currently offered by us for packaging our chimeric baculovirus-AAV vector systems include - serotypes 1, 2, 6, 8 and 9. During cloning, ITRs from AAV2 are used, as this is common practice in the field and does not impact specificity. Packaging helper plasmids include a Rep/Cap plasmid, containing the replication genes from AAV2 and the capsid proteins for a chosen serotype to determine tropism. The table below lists different AAV serotypes and their tissue tropism.

List by Serotype
List by Tissue Type

Serotype Tissue tropism
AAV1 Smooth muscle, skeletal muscle, CNS, brain, lung, retina, inner ear, pancreas, heart, liver
AAV2 Smooth muscle, CNS, brain, liver, pancreas, kidney, retina, inner ear, testes
AAV3 Smooth muscle, liver, lung
AAV4 CNS, retina, lung, kidney, heart
AAV5 Smooth muscle, CNS, brain, lung, retina, heart
AAV6 Smooth muscle, heart, lung, pancreas, adipose, liver
AAV6.2 Lung, liver, inner ear
AAV7 Smooth muscle, retina, CNS, brain, liver
AAV8 Smooth muscle, CNS, brain, retina, inner ear, liver, pancreas, heart, kidney, adipose
AAV9 Smooth muscle, skeletal muscle, lung, liver, heart, pancreas, CNS, retina, inner ear, testes, kidney, adipose
AAVrh10 Smooth muscle, lung, liver, heart, pancreas, CNS, retina, kidney
AAV-DJ Liver, heart, kidney, spleen
AAV-DJ/8 Liver, brain, spleen, kidney
AAV2-retro Spinal nerves
AAV2-QuadYF Endothelial cell, retina
AAV2.7m8 Retina, inner ear
Tissue type Recommended AAV serotypes
Smooth muscle AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10
Skeletal muscle AAV1, AAV9
Brain AAV1, AAV2, AAV5, AAV7, AAV8, AAV-DJ/8
Retina AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2-QuadYF, AAV2.7m8
Inner ear AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8
Lung AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAVrh10
Liver AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
Pancreas AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh10
Heart AAV1, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV-DJ
Kidney AAV2, AAV4, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
Adipose AAV6, AAV8, AAV9
Testes AAV2, AAV9
Spleen AAV-DJ, AAV-DJ/8
Spinal nerves AAV2-retro
Endothelial cells AAV2-QuadYF

For further information about this vector system, please refer to the papers below.

References Topic
Methods Mol Bio. 1937:91 (2019) AAV production using baculovirus expression vector system
Mol Ther. 17:1888 (2009) Using a simplified baculovirus-AAV expression vector system yields high-titer rAAV stocks
Mol Ther. 12:1217 (2005) Production of pseudotyped rAAV vectors using a modified baculovirus expression system


The chimeric baculovirus-AAV gene expression vector system enables efficient and large-scale production of recombinant AAV in insect cells. Our vector is optimized for high copy number replication in E. coli, high-titer packaging of recombinant baculovirus and presents low safety risk.


Scalability and efficiency: Insect cell lines such as Sf9 can be grown in high-density suspension cultures using large-scale bioreactors which allows baculovirus-based AAV to be produced with increased efficiency and at larger scales than compared to the conventional approach of producing AAV from adherent cell cultures.

Safety: Baculovirus cannot replicate outside of insect cells and are nonpathogenic to mammals and plants. Therefore, our expression system can be used in insect cell lines under minimal biosafety conditions. Moreover, insect cells can be grown under serum-free conditions which eliminates the chances of any animal-derived proteins being present, thus further enhancing the biosafety of this system. Lastly, recombinant AAV produced using this system is inherently replication-deficient and is not known to cause any human diseases.

Low risk of host genome disruption: Upon transduction into host cells, AAV vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.

High viral titer: Our chimeric baculovirus-AAV vector can be used for producing high-titer recombinant AAV. When AAV is obtained through our AAV packaging service (by baculovirus infection in Sf9 insect cells), titer can reach >1013 genome copies per ml (GC/ml).

Broad tropism: A wide range of cell and tissue types from commonly used mammalian species such as human, mouse and rat can be readily transduced with our AAV vector system when it is packaged into the appropriate serotype. But some cell types may be difficult to transduce, depending on the serotype used (see disadvantages below).

Effectiveness in vitro and in vivo: Recombinant AAV is often used to transduce cells in live animals, but it can also be used effectively in vitro.


Small cargo space: AAV has the smallest cargo capacity of any of our viral vector systems. AAV can accommodate a maximum of 4.7 kb of sequence between the ITRs, which leaves ~4.2 kb cargo space for user's DNA of interest.

Difficulty transducing certain cell types: Our AAV vector system can transduce many different cell types including non-dividing cells when packaged into the appropriate serotype. However, different AAV serotypes have tropism for different cell types, and certain cell types may be hard to transduce by any serotype.

Technical complexity: Recombinant AAV production using the chimeric baculovirus-AAV expression system requires multiple steps, including cloning the GOI into the baculovirus transfer vector, generating recombinant bacmid from the transfer vector, transfecting the bacmid into insect cells and subsequently infecting insect cells with recombinant baculovirus. These procedures are technically demanding and time consuming relative to conventional triple transfection-based AAV production. These demands can be alleviated by choosing our AAV packaging (by baculovirus infection in Sf9 insect cells) services when ordering your vector.

Key components

5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.

Promoter: The promoter that drives your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.

Tn7L: Tn7 transposon left terminal element. It is recognized by Tn7 transposase. DNA flanked by Tn7R and Tn7L can be transposed by Tn7 transposase into attTn7 docking sites.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Tn7R: Tn7 transposon right terminal element. It is recognized by Tn7 transposase. DNA flanked by Tn7R and Tn7L can be transposed by Tn7 transposase into attTn7 docking sites.

Gentamicin: Gentamicin resistance gene. It allows for drug selection of E. coli carrying recombinant bacmids.

Representative vector design

VB ID Vector name Descriptions
VB010000-9306ded pBV-ITR-CMV>EGFP-ITR CMV-driven EGFP-expressing baculovirus transfer plasmid, including a gentamicin resistance gene and Tn7 transposon terminal elements.
VB231214-1628epu pBV-ITR-CAG>hSMN1[NM_000344.4]:WPRE-ITR A baculovirus transfer plasmid expressing CAG-driven human survival motor neuron gene SMN1, which is associated with SMA, with a WPRE regulatory element.
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