RPE (Retinal Pigment Epithelium) 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 employed in our vector cloning services. As an outcome, we have a growing base of highly satisfied clients who come back to us time after time for their AAV vector cloning and AAV packaging requirements.

The pigmented layer of retina or retinal pigment epithelium (RPE) is the pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells, and is firmly connected to the underlying choroid and overlying retinal visual cells. The RPE has various functions namely, light absorption, epithelial transport, spatial ion buffering, visual cycle, phagocytosis, secretion and immune modulation. Research has revealed that the RPE is where macular degeneration begins.

Retinal pigment epithelial (RPE) cells are an important component of the human eye because they not only mediate and regulate the transfer of fluids and solutes but also safeguard the retina against photooxidative injury and renew photoreceptor cells through phagocytosis. Nevertheless, their function necessitates cumulative exposure to the sun arising in UV damage, which may direct to the improvement of age-related macular degeneration (AMD). Various studies have indicated that UVB induces direct DNA damage and oxidative stress in RPE cells by improving ROS and dysregulating endogenous antioxidants. Activation of various signaling pathways attached to inflammation, cell cycle arrest, and intrinsic apoptosis was reported as well. 

Adeno-associated virus (AAV) is emerging as a favorable vector for gene therapy. Adeno-associated viral (AAV) vectors have been utilized to express various different proteins in the eye. Adeno-associated virus-based vectors are eligible to efficiently transduce and stably persist in RPE cells.

Production Process

Suitable serotypes for RPE

AAV1, AAV2, AAV4,AAV5,AAV8

1.Subcloning (Optional)

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

2.Large-scale Production (Optional)

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

3.Large scale Transfection

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

4.Purification

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

5.Titer Determination 

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

Workflow

Genome-wide 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.Cassin, B. & Solomon, S. (2001). Dictionary of eye terminology. Gainesville, Fla: Triad Pub. Co.  

2.Strauss O (2005) "The retinal pigment epithelium in visual function". Physiol Rev 85:845–81.

3.C. E. Willoughby, D. Ponzin, S. Ferrari, A. Lobo, K. Landau, and Y. Omidi, “Anatomy and physiology of the human eye: effects of mucopolysaccharidoses disease on structure and function – a review,” Clinical & Experimental Ophthalmology, vol. 38, pp. 2–11, 2010.