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. 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 |
AAV-PHP.eB |
CNS |
AAV-PHP.S |
PNS |
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 |
CNS |
AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-PHP.eB |
PNS |
AAV-PHP.S |
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.