Limbal Approach-subretinal Injektion af virale vektorer til genterapi i mus retinale pigmentepitel
Summary
Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.
Abstract
The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.
Introduction
I oftalmologi har genterapi opstået som behandlingsmodalitet i monogeniske nedarvede retinopatier. Der er nedarvede retinopatier forbundet med gener i retinal pigment epitel (RPE), herunder Leber medfødt amurosis 1,2, retinitis pigmentosa 3, og choroideremia 4. Inden for genterapi forskning ekspanderer i både prækliniske og kliniske forsøg med virale vektorer, såsom adeno-associeret virus (AAV), lentivirus (LV) og adenovirus (Ad) 5. Forskellige virale vektorer har forskellige tropisme i nethinden. For en sikker og effektiv genterapi bør virale vektorer nøje udvalgt efter målceller og målgener.
Ruten for genafgivelse er også vigtige for effektiv genafgivelse til målceller, således skal det omhyggeligt valgt som godt. De to mest almindelige metoder til intraokulær afgivelse af virale vektorer subretginal injektion og intravitreal injektion 6. Sidstnævnte, intravitreal injektion, har været meget anvendt til lægemiddelafgivelse til behandling choroidal neovaskularisering i våd aldersrelateret maculadegeneration (AMD) og makulært ødem i diabetisk retinopati 7. Intravitreal rute giver eksponering af virale vektorer til glaslegemet og indre nethinde, men diffusionen af vektorerne til ydre nethinde er begrænset. På den anden side, den subretinal rute giver direkte levering af virale vektorer til potentielle rum mellem retina og RPE, inducere en lokaliseret bleb. Derfor er subretinal injektion i øjeblikket betragtes som en mere effektiv vej til at målrette fotoreceptorceller og RPE. Med hensyn til kirurgisk tilgang, pars plana er valgt som et sikkert område til intravitreal injektion for at undgå beskadigelse af nethinden hos humane patienter. Ved blot at ændre denne tilgang til mus, kunne vi tilføre virale vektorer subretinalt eller intravireally via limbal tilgang.
I denne videoartiklen viser vi en nem og bekvem metode til subretinal injektion af virale vektorer i mus RPE. Efter en enkelt punktering ved posteriort for limbus med en 30 G 1/2 nål, er en 33 G stump nål udstyret mikroliter sprøjte indsat i subretinarummet via limbale indstikssted. De virale vektorer af 1,5-2 ul volumen injiceres til den potentielle rum mellem retina og RPE inducerende subretinal bleb'er. Denne procedure kan udføres under direkte visualisering under anvendelse af kirurgisk mikroskop. Gentagne praksis vil garantere reproducerbare resultater selv uden direkte visualisering af bleb formation. Dette vil hjælpe forskerne til at udføre nøjagtige og tidsbesparende eksperimenter for subretinal genafgivelse i mus RPE.
Protocol
Representative Results
Discussion
I denne video artiklen, beskrev vi den limbale-tilgang subretinal injektionsteknik i detaljer med de repræsentative resultater af RPE / choroidea / sclera fladskærms-mount. Dette er en nem og bekvem teknik til subretinal injektion af virale vektorer i RPE. Direkte visualisering af bleb dannelse under injektionen er et vigtigt skridt for præcis levering for begyndere. Der er nogle subretinal injektionsteknik indført i Journal of visualiseret Forsøg 8-10. Subretinal rum er det muligt rum mellem retina og R…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Denne undersøgelse blev støttet af Seoul National University Research Grant (800-20140542), Pioneer Research Program for NRF / MEST (2012-0009544), og Bio-Signal Analysis Technology Innovation Program for NRF / MEST (2009-0090895), og meddelelse af NRF / MEST (2015M3A9E6028949).
Materials
| TWEEZERS DUMONT #5 11cm DUMOSTAR 0.1 x 0.06 mm TIPS | WPI | 500233 | |
| VANNAS Scissors S/S, 105mm | WPI | 555583S | |
| 33G Blunt needle | WPI | NF33BL-2 | |
| NanoFil Syringe, 10 microliter | WPI | NANOFIL | |
| RPE-KIT | WPI | RPE-KIT | For easy one hand injection |
| 30Gx1/2 (0.3mmx 13mm) BD PrecisionGlideTM Needle | BD | 305107 | Initial puncture for subretinal injection |
| Microscope Cover Glasses (No. 1 3 mm diameter) | Warner Instruments | 64-0720 (CS-3R) | |
| Leica operating microscope | Leica | LM M80 | |
| Fluoresecein microscope | Nikon | Eclipse 80i | |
| Lentivirus | Thermo scientific | TMO.LV-Ctr | Used to dilute vectors |
| PBS | Gibco | 10010-015 | Used to dilute vectors |
| Troperin (Phenylephrin 0.5%-Tropicamide 0.5%) | Hanmi | For dilation | |
| Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5% (Sterile) | Bausch&Lomb | For topical anesthesia | |
| Healon GV OVD | Abbott Medical Optics Inc. | ||
| Zoletil 50 (tiletamine hypochloride and zolazepam hypochloride) | Virbac | For general anesthesia | |
| Rompun® injection (Xylazine HCl) | Bayer | For general anesthesia |
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