Adeno-Associated Virus Tet Inducible Gene Expression Vector

Overview

VectorBuilder’s adeno-associated virus (AAV) Tet inducible gene expression vector combines the highly versatile AAV vector system and the Tet inducible gene expression system to help you achieve AAV-mediated in vitro and in vivo delivery of tetracycline-inducible gene expression cassettes.

The Tet-On inducible system is a powerful tool to control the timing of expression of the gene(s) of interest (GOI) in mammalian cells. Our Tet-On inducible gene expression vectors are designed to achieve nearly complete silencing of a GOI in the absence of tetracycline and its analogs (e.g. doxycycline), and strong, rapid expression in response to the addition of tetracycline or one of its analogs (e.g. doxycycline). This is achieved through a multicomponent system which incorporates active silencing by the tTS protein in the absence of tetracycline and strong activation by the rtTA protein in the presence of tetracycline. In the absence of tetracycline, the tTS protein derived from the fusion of TetR (Tet repressor protein) and KRAB-AB (the transcriptional repressor domain of Kid-1 protein) binds to the tetracycline-responsive element (TRE) promoter, leading to the active suppression of gene transcription. The rtTA protein, on the other hand, derived from the fusion of a mutant Tet repressor and VP16 (the transcription activator domain of virion protein 16 of herpes simplex virus), binds to the TRE promoter to activate gene transcription only in the presence of tetracycline.

While our AAV Tet inducible gene expression vector includes an inducible gene expression cassette consisting of the TRE promoter driving the user-selected GOI, the TRE binding regulatory proteins tTS and rtTA have to be provided using a separate helper vector to achieve tetracycline induced gene expression in the presence of tetracycline, while minimizing leaky expression in the absence of tetracycline. 

The AAV Tet inducible gene expression vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with helper plasmids, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus. The tetracycline inducible gene expression cassette consisting of the TRE promoter driving the user-selected GOI is placed in-between the two ITRs, which is introduced into target cells along with the rest of viral genome. Gene expression can then be turned on in the presence of the rtTA regulatory protein and tetracycline.

AAV is effective in transducing many mammalian cell types, and, unlike adenovirus, has very low immunogenicity, being almost entirely nonpathogenic in vivo. This makes the AAV inducible gene expression vector the ideal viral vector system for achieving inducible gene expression in vivo.

The wild-type AAV genome is a linear single-stranded DNA (ssDNA) with two ITRs forming a hairpin structure on each end. It is therefore also known as ssAAV. In order to express genes on ssAAV vectors in host cells, the ssDNA genome needs to first be converted to double-stranded DNA (dsDNA) through two pathways: 1) synthesis of second-strand DNA by the DNA polymerase machinery of host cells using the existing ssDNA genome as the template and the 3' ITR as the priming site; 2) formation of intermolecular dsDNA between the plus- and minus-strand ssAAV genomes. The former pathway is the dominant one.

AAV genomic 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 can occur but is extremely rare. The lack of integration 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 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 earpancreas, 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 earliver, 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
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, AAV2.7m8
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 in Enzy. 507:229-54 (2012) Review of AAV virology and uses
Curr Opin Pharmacol. 24:59-67 (2015) AAV use in gene therapy, and serotype differences
Science. 268:1766-9 (1995) Development of rtTA.
J Gene Med. 1:4-12 (1999) Development of tTS.

Highlights

Our AAV Tet inducible gene expression vector when coexpressed with the Tet regulatory proteins tTS and rtTA can achieve nearly complete silencing of the GOI in the absence of tetracycline, and strong, rapid expression in response to the addition of tetracycline. The AAV Tet inducible gene expression vector is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient transduction of host cells, and high-level transgene expression. This viral vector can be packaged into virus using all known capsid serotypes, is capable of very high transduction efficiency, and presents low safety risk.

Advantages

High-level expression: The TRE promoter can drive very high levels of expression of the GOI in its induced state.

Safety: AAV is the safest viral vector system available. AAV 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 AAV vector can be packaged into high titer virus. When AAV virus is obtained through our virus packaging service, titer can reach >1013 genome copy 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 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: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.

Disadvantages

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: The use of viral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection. These demands can be alleviated by choosing our virus packaging 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 TRE promoter driving 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.

Regulatory element: Allows the user to add the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). WPRE enhances virus stability in packaging cells, leading to higher titer of packaged virus; enhances higher expression of transgenes.

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

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

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.