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Introduction to AAV

Adeno-associated viruses (AAVs), from the parvovirus family, are small viruses (22 nm) that are replication-defective, non-enveloped, and with a genome of single stranded DNA.

AAVs are non-pathogenic, as 80-90% of humans are sero-positive with AAV2.

In contrast to adenoviruses, most people treated with AAV will not build an immune response to remove the virus and the cells that have been successfully transduced by it.

Gene therapy vectors using AAV can infect both dividing and non-dividing cells and persist in an extrachromosomal state without integrating into the genome of the host cell.

The AAV genome is built of single-stranded DNA, either positive- or negative-sensed. The total genome size that is able to be packaged is about 4.7 to 4.9 kilobase long. The genome comprises 145 nucleotide-long inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsids proteins, VP1, VP2, and VP3, which interact together to form a capsid of an icosahedral symmetry.
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The characteristics of AAVs have allowed the scientific community to establish methods for the production of recombinant AAV (rAAV) vectors. The only sequences required in cis next to the therapeutic gene (or gene of interest) are the ITRs. The structural (cap) and packaging (rep) genes can be delivered in trans. rAAVs are now widely used in both basic science research (single gene studies) and disease treatments (gene therapy).
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As many as 11 naturally-occurring AAV serotypes have been discovered (with many pseudotypes being generated by several different research groups). All of the known serotypes can infect cells from multiple diverse tissue types. Tissue specificity is determined by the capsid serotype, where serotype 2 (AAV2) has been the most extensively examined so far.
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BAC-to-AAV

World’s Most Efficient Large-Scale AAV Manufacturing System, in GLP and Pre-GMP

Virovek has developed a patented BAC-to-AAV technology that utilizes the baculovirus expression system to produce AAV vectors in insect cells under serum-free condition.

Available in GLP grade, as well as in seed stock and pre-GMP preparation tailored to customer requirement.

 

Recognized as the world’s most powerful and efficient AAV production platform, Virovek has the capability to generate over 3e+16vg of AAV vectors with a single production run, unmatched by any other AAV production system.

Alternatively, check out here for our AAVs in HEK293 cells.

Three Key Steps to Generating
Your Custom-Made AAV Vector

Sub-clone your gene of interest (GOI) into Virovek’s AAV shuttle vector

We will design the best cloning strategy, sub-clone your GOI into our AAV shuttle vector, and perform sequence analysis to verify the integrity of your GOI.

Generate baculovirus containing your GOI

We will then generate bacmid DNA containing your GOI, purify the bacmid DNA, transfect Sf9 insect cells to produce recombinant baculovirus containing your GOI, and then amplify the recombinant baculovirus.

Produce AAV vectors containing your GOI

Finally, we will double infect Sf9 cells using the amplified recombinant baculovirus containing your GOI and another recombinant baculovirus containing the Rep-Cap genes of your choice to produce recombinant AAV vectors containing your GOI, purify the recombinant AAV vectors, perform qPCR to determine the viral titer, and perform SDS-PAGE to verify the purity of AAV vectors.

Three Key Steps to Generating Your Custom-Made AAV Vector

Sub-clone your gene of interest (GOI) into Virovek’s AAV shuttle vector

MORE

We will design the best cloning strategy, sub-clone your GOI into our AAV shuttle vector, and perform sequence analysis to verify the integrity of your GOI.

 

Generate baculovirus containing your GOI

MORE
We will then generate bacmid DNA containing your GOI, purify the bacmid DNA, transfect Sf9 insect cells to produce recombinant baculovirus containing your GOI, and then amplify the recombinant baculovirus.

 

Produce AAV vectors containing your GOI

MORE
Finally, we will double infect Sf9 cells using the amplified recombinant baculovirus containing your GOI and another recombinant baculovirus containing the Rep-Cap genes of your choice to produce recombinant AAV vectors containing your GOI, purify the recombinant AAV vectors, and perform qPCR to determine the viral titer SDS-PAGE to verify the purity of AAV vectors.

AAV Production Timeline

The whole AAV production process takes approximately 6-8 weeks to complete. (see table below for timeline).

Clone gene of interest (GOI) into Virovek’s AAV shuttle plasmid

1~2 WEEKS

Generation of Bacmid + Purification of Bacmid DNA

1~2 WEEKS

Transfection of Sf9 cells to generate baculovirus

1~2 WEEKS

Amplifying baculovirus and titration

1 WEEK

AAV Production and CsCl purification

1 WEEK

Desalting, filter sterilization, and AAV titration

2 DAYS

Patented Toxin-Based
Cell Ablation Technology, available for licensing

Virovek has developed the world’s first and only manufacturing system to produce viral vectors harboring toxin genes. This patented technology also utilizes the same baculovirus expression system to produce AAV vectors in insect cells under serum-free condition.

The toxin genes are deactivated during the production process, and only reactivated once the AAV-toxin vectors are produced. Using promoter-specific and/or capsid-specific AAaV vectors harboring toxin genes, this cell ablation technology is able to hone in on and destroy specific subsets of cells within a larger tissue sample or organ.

As a result, this toxin-based cell ablation technology can be used to study the functionality of particular types of cells, as well as for therapeutic purposes by targeting diseased cells.

The world’s first to produce viral vectors harboring toxin genes. Using promoter-specific and/or capsid-specific AAV vectors harboring toxin genes. Cancer cell killing effects of these AAV-toxin vectors in vitro.

We have successfully demonstrated the cancer cell killing effects of these AAV-toxin vectors in vitro. In vivo testings of these AAV-toxin vectors in different animal cancer models are now underway.

All of our proprietary technologies are available for non-exclusive licensing. Please inquire for details.

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Safety & Storage

AAV vectors are considered risk group 1 agents (not associated with disease in healthy adult humans) according to the National Institutes of Health.

Our recombinant AAV vectors can be handled in a Biosafety Level (BSL-1) environment.
Short-term storage

AAV vectors are stable at room temperature and 4°C. You may elect to have us ship your vectors on Ice Packs at 4°C.

Long-term storage

Keep AAV vectors at -80°C. Do NOT store at -20°C. Research suggests that long-term storage at -20°C is not necessarily effective, and may result in decreased transduction efficiency of the virus when it is removed from storage, thawed, and used. You may elect to have us ship your vectors on Dry Ice at -80°C.

Additional AAV handling

Our AAV vectors are not considered Hazardous Materials by Federal OSHA or CalOSHA, and are not legally required to come with a Safety Data Sheet (SDS). However, we would like to provide relevant information on recommended handling practices and safety. This data sheet may be helpful if you anticipate difficulties with product identification in international shipping requirements, or within your organization. Please click on the title below to view the document.

AAV Storage and Handling Information

Our AAV vectors are not considered Hazardous Materials by Federal OSHA or CalOSHA, and are not legally required to come with a Safety Data Sheet (SDS). However, we would like to provide relevant information on recommended handling practices and safety. This data sheet may be helpful if you anticipate difficulties with product identification in international shipping requirements, or within your organization. Please click on the title below to view the document.

AAV Vector Storage and Handling Information
Virovek AAV Safety Data Sheet

Partners & Customers

Virovek has collaborated with over 100 world-class universities, non-profit institutions and industry pioneers to deliver gene therapy applications for tomorrow.
Genentech
Mount Sinai
UCSF
Charles river
BioMarin Pharmaceutical
Arup laboratories
https://www.nih.gov/
https://www.mit.edu/
https://www.tenayatherapeutics.com/
http://med.stanford.edu/
https://burke.weill.cornell.edu/
https://www.ku.dk/english/
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AAV Serotype

Retina

Heart

Liver

Lung

CNS/Brain

Skeletal Muscle

Other

AAV1

X

X

AAV2

X

X

AAV2-Retro

X

AAV3A, 3B

X

AAV4

X

X

AAV5

X

X

Immune system

AAV6

X

X

X

Immune system, pancreas

AAV7

X

X

X

AAV8

X

X

X

AAV9

X

X

AAVShH10

X

AAVrh10

X

X

X

AAV7m8

X

AAVDJ

X

X

X

Kidney, cervix, ovary, skin, fibroblast

AAVphp.b

X

AAVphp.eb

X

AAV10

X

X

X

X

kidney, uterus

AAV11

X

X

X

kidney, spleen, stomach

Serotype 2 generally tranduces cells more effectively in vitro than other serotypes

All other serotypes transduce cells more effectively in vivo