CNS Targeting AAV Production Services

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 utilized 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.

Mammalian central nervous system (CNS) is a complicated and precise connectivity of intertwining neurons nourished and assisted by glial cells- astrocytes, oligodendrocytes, and microglia. Smooth functioning of the CNS is produced by excitation and inhibition of neuronal firing/action potential i.e. relay of potential variation traveling between the cell body (soma) and its projections at the axonal terminus/ni.

Suitable Serotypes for CNS tissue

AAV1, AAV2, AAV4, AAV5, AAV8, AAV9

Disorders of the Central Nervous System

While timely excitation and inhibition of regional subpopulations of neurons controls motor, behavioral, hormonal, sensory and cognitive outcomes; unregulated neuronal activity and selective damage of neuronal or glial subgroups has been related with CNS disorders. Such disorders can happen from drug abuse, injuries, genetic, epigenetic and environmental factors.

Loss of functionality in affected cell types within the brain can often be associated to defects in single genes. For example, a range of neurological disorders happen from the inability of cells in the CNS to break down metabolic end products (e.g. lysosomal storage disorders (LSDs).

Therapeutic Approaches to CNS disorders

Gene therapy is being utilized for dealing with severely debilitating diseases by delivering healthy cargo of genetic data to afflicted cell/tissue types. Viral vector mediated gene therapy offers the ability to conduct efficient in vivo gene transfer of therapeutic transgenes organized to the CNS. In specific, Adeno-Associated Viruses (AAV) have emerged as promising tools for clinical gene transfer in a wide range of genetic diseases with neurological manifestations.

Recombinant Adeno-Associated Viral Vectors

Adeno-associated viruses (AAV) are non-enveloped, helper-dependent parvoviruses with an icosahedral capsid architecture ~25 nm in diameter. AAVs package ~4.7 kb genome flanked by ~145 bp inverted terminal repeats (ITRs) on the 5’ and 3’ ends.

Three developments have been instrumental in enabling the aim of AAV as a recombinant vector for gene transfer applications: a) the ability to pseudotype AAV vectors by utilizing AAV capsids of natural or synthetic origin ; b) cloning and characterization of adenoviral helper genes that are minimally necessary for generation of infectious AAV particles; and c) understanding that inverted terminal repeats (ITRs) are the only cis-acting molecular signature for profitable packaging of transgenes within an AAV capsids.

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

AAV Products

Human

•Over-Expression Products

•shRNA Products

•miRNA Products

Mouse

•Over-Expression Products

•shRNA Products

•miRNA Products

Rat

•Over-Expression Products

•shRNA Products

•miRNA Products

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.Simonato M, et al (2013) Progress in gene therapy for neurological disorders. Nat 

Rev Neurol 9(5): 277-291.

2.Gray SJ (2013) Gene therapy and neurodevelopmental disorders. 

Neuropharmacology 68: 136-142.

3.Agbandje-McKenna M & Kleinschmidt J (2011) AAV capsid structure and cell 

interactions. Methods Mol Biol 807: 47-92.

4.Lentz TB, Gray SJ & Samulski RJ (2012) Viral vectors for gene delivery to the 

central nervous system. Neurobiol Dis 48(2): 179-188.