Photoreceptor Cells 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.

A photoreceptor cell is a specialized category of neuroepithelial cell found in the retina that is eligible of visual phototransduction. The great biological significance of photoreceptors is that they convert light (visible electromagnetic radiation) into signals that can facilitate biological procedures. To be more distinct, photoreceptor proteins in the cell absorb photons, triggering a difference in the cell's membrane potential.

Improvement of viral vectors eligible for transducing photoreceptors by less invasive procedures than a subretinal injection would give a major development in retinal gene therapy. Newly, photoreceptor transduction efficiency following intravitreal injection (IVT) has enhanced in rodent models through the aim of capsid-mutant AAV vectors; but stays limited in large animal models. The thickness of the inner limiting membrane (ILM) in big animals is thought to impair retinal penetration by AAV. 

Surface-exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were altered to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors including the smCBA promoter and mCherry cDNA were originally scored in vitro utilizing a cone photoreceptor cell line. Capsid mutants showing the increased transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). 

Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by measuring cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors including the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to evaluate whether productive expression exclusive to photoreceptors was attainable.

Production Process

Suitable serotypes for photoreceptor cells 

AAV2, 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.Foster, R.G.; Provencio, I.; Hudson, D.; Fiske, S.; Grip, W.; Menaker, M. (1991). "Circadian photoreception in the retinally degenerate mouse (rd/rd)". Journal of Comparative Physiology A. 169 (1): 39–50. 

2.Cideciyan AV, Hauswirth WW, Aleman TS, Kaushal S, Schwartz SB, et al. (2009) Human RPE65 gene therapy for Leber congenital amaurosis: persistence of early visual improvements and safety at 1 year. Hum Gene Ther 20: 999–1004.

3.Wright AF, Chakarova CF, Abd El-Aziz MM, Bhattacharya SS (2010) Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait. Nat Rev Genet 11: 273–284.