Liver Targeting AAV Production Service

Seattle Genova offers outstanding quality AAV packaging services to benefit your AAV-based gene therapy experiments. We have improved a series of proprietary technologies and reagents that have greatly improved recombinant AAV production protocols in terms of titer, purity, potency and consistency, particularly for the AAV vector systems used in our vector cloning services. As an outcome, we have a growing base of highly satisfied customers who come back to us time after time for their AAV vector cloning and AAV packaging requirements.

Recent clinical successes have intensified interest in utilizing adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological purposes and involvement in a vast range of genetic diseases. Therefore, several inherited diseases could be effectively treated by targeting the liver utilizing gene transfer approaches. The challenges related to liver-directed gene therapy are efficient targeting of hepatocytes, stability of the vector genome, and persistently increased level expression. Numerous of these obstacles can be overcome with adeno-associated viral (AAV) gene transfer vectors. 

The first AAV gene transfer -vector formulated for in vivo use was based on the AAV2 serotype. AAV2 has a wide tropism and transduces many cell types, comprising hepatocytes, relatively efficiently in vivo. The capsid protein suggests the serological profile and at least 12 primate AAV serotypes have already been defined. Importantly, pseudotyping a recombinant AAV vector with various capsid proteins can dramatically change the tropism. Both AAV8 and AAV9 have higher affinities for hepatocytes when distinguished from AAV2.

Production Process

Subcloning (Optional)

We begin by subcloning the gene of interest (GOI), shRNA or gRNA into an associated pAAV cis-plasmid.

Large-scale Production (Optional)

Large-scale preparation of the pAAV cis-plasmid and complimentary plasmids utilizing Qiagen Endo-free Mega Prep kits.

Large scale Transfection

Large-scale transfection of engaged plasmids into 40x15cm plates of HEK293 cells.

Purification

Harvest the AAV production cells and purify the AAVs through a sequence of CsCl centrifugations.

Titer Determination 

Find the titer of the viral stock (in genome copy number per ml, or GC/ml) through quantitative real-time PCR.

Workflow 

Key Features and Benefits

•Ready to utilize for in vitro and in vivo applications

•High titer: improved transduction efficiency

•Transduction of dividing and non-dividing cells

•Expert Technical Support 

•Quality and user friendly 

References

1.Mount JD, Herzog RW, Tillson DM, et al.. Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy. Blood 2002;99:2670–2676.

2.Snyder RO, Miao C, Meuse L, et al.. Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nat Med 1999;5:64–70.

3.Nathwani AC, Reiss UM, Tuddenham EG, et al.. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 2014;371:1994–2004