Adeno-Associated Virus Encoding Green Fluorescent Protein as a Label for Retinal Pigment Epithelium

2003 ◽  
Vol 44 (2) ◽  
pp. 772 ◽  
Author(s):  
Kathleen A. Hansen ◽  
Ilene K. Sugino ◽  
Fumihiko Yagi ◽  
Hao Wang ◽  
Itsuro Tsukahara ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Retinal Pigment Epithelium ◽  
Fluorescent Protein ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Adeno Associated Virus ◽  
Green Fluorescent

scholarly journals Novel Adeno-Associated Virus Serotypes Efficiently Transduce Murine Photoreceptors

2007 ◽  
Vol 81 (20) ◽  
pp. 11372-11380 ◽  
Author(s):  
Mariacarmela Allocca ◽  
Claudio Mussolino ◽  
Maria Garcia-Hoyos ◽  
Daniela Sanges ◽  
Carolina Iodice ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Ex Vivo ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Therapeutic Success ◽  
Adeno Associated Virus ◽  
Quantitative Manner ◽  
Green Fluorescent ◽  
Rod And Cone Photoreceptors

ABSTRACT Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed in photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct retinal pigment epithelium (RPE) defects in animal models, approaches for the correction of photoreceptor-specific diseases are less efficient. We evaluated the ability of novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, and AAV2hu.29R) in combination with constitutive or photoreceptor-specific promoters to improve photoreceptor transduction, a limiting step in photoreceptor rescue. Based on a qualitative analysis, all AAV serotypes tested efficiently transduce the RPE as well as rod and cone photoreceptors after subretinal administration in mice. Interestingly, AAV2/9 efficiently transduces Müller cells. To compare photoreceptor transduction from different AAVs and promoters in both a qualitative and quantitative manner, we designed a strategy based on the use of a bicistronic construct expressing both enhanced green fluorescent protein and luciferase. We found that AAV2/8 and AAV2/7 mediate six- to eightfold higher levels of in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient AAV serotype for photoreceptor targeting. In addition, following subretinal administration of AAV, the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested. Finally, we show that AAV2/7, AAV2/8, and AAV2/9 outperform AAV2/5 following ex vivo transduction of retinal progenitor cells differentiated into photoreceptors. We conclude that AAV2/7 or AAV2/8 and the rhodopsin promoter provide the highest levels of photoreceptor transduction both in and ex vivo and that this may overcome the limitation to therapeutic success observed so far in models of inherited severe photoreceptor diseases.

scholarly journals Gene Transfer in Rodent Nervous Tissue Following Hindlimb Intramuscular Delivery of Recombinant Adeno-Associated Virus Serotypes AAV2/6, AAV2/8, and AAV2/9

2019 ◽  
Vol 14 ◽  
pp. 117906951988902 ◽  
Author(s):  
Asad Jan ◽  
Mette Richner ◽  
Christian B Vægter ◽  
Jens R Nyengaard ◽  
Poul H Jensen
Keyword(s):  
Nervous System ◽  
Viral Load ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Adeno Associated Virus ◽  
Green Fluorescent ◽  
Virus Delivery ◽  
Intramuscular Delivery

Recombinant adeno-associated virus (rAAV) vectors have emerged as the safe vehicles of choice for long-term gene transfer in mammalian nervous system. Recombinant adeno-associated virus–mediated localized gene transfer in adult nervous system following direct inoculation, that is, intracerebral or intrathecal, is well documented. However, recombinant adeno-associated virus delivery in defined neuronal populations in adult animals using less-invasive methods as well as avoiding ectopic gene expression following systemic inoculation remain challenging. Harnessing the capability of some recombinant adeno-associated virus serotypes for retrograde transduction may potentially address such limitations (Note: The term retrograde transduction in this manuscript refers to the uptake of injected recombinant adeno-associated virus particles at nerve terminals, retrograde transport, and subsequent transduction of nerve cell soma). In some studies, recombinant adeno-associated virus serotypes 2/6, 2/8, and 2/9 have been shown to exhibit transduction of connected neuroanatomical tracts in adult animals following lower limb intramuscular recombinant adeno-associated virus delivery in a pattern suggestive of retrograde transduction. However, an extensive side-by-side comparison of these serotypes following intramuscular delivery regarding tissue viral load, and the effect of promoter on transgene expression, has not been performed. Hence, we delivered recombinant adeno-associated virus serotypes 2/6, 2/8, or 2/9 encoding enhanced green fluorescent protein (eGFP), under the control of either cytomegalovirus (CMV) or human synapsin (hSyn) promoter, via a single unilateral hindlimb intramuscular injection in the bicep femoris of adult C57BL/6J mice. Four weeks post injection, we quantified viral load and transgene (enhanced green fluorescent protein) expression in muscle and related nervous tissues. Our data show that the select recombinant adeno-associated virus serotypes transduce sciatic nerve and groups of neurons in the dorsal root ganglia on the injected side, indicating that the intramuscular recombinant adeno-associated virus delivery is useful for achieving gene transfer in local neuroanatomical tracts. We also observed sparse recombinant adeno-associated virus viral delivery or eGFP transduction in lumbar spinal cord and a noticeable lack thereof in brain. Therefore, further improvements in recombinant adeno-associated virus design are warranted to achieve efficient widespread retrograde transduction following intramuscular and possibly other peripheral routes of delivery.

scholarly journals Green Fluorescent Protein-Tagged Adeno-Associated Virus Particles Allow the Study of Cytosolic and Nuclear Trafficking

2005 ◽  
Vol 79 (18) ◽  
pp. 11776-11787 ◽  
Author(s):  
Kerstin Lux ◽  
Nico Goerlitz ◽  
Stefanie Schlemminger ◽  
Luca Perabo ◽  
Daniela Goldnau ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Helper Virus ◽  
Nuclear Entry ◽  
Adeno Associated Virus ◽  
Prolonged Incubation ◽  
Viral Capsids ◽  
Green Fluorescent ◽  
Perinuclear Area

ABSTRACT To allow the direct visualization of viral trafficking, we genetically incorporated enhanced green fluorescent protein (GFP) into the adeno-associated virus (AAV) capsid by replacement of wild-type VP2 by GFP-VP2 fusion proteins. High-titer virus progeny was obtained and used to elucidate the process of nuclear entry. In the absence of adenovirus 5 (Ad5), nuclear translocation of AAV capsids was a slow and inefficient process: at 2 h and 4 h postinfection (p.i.), GFP-VP2-AAV particles were found in the perinuclear area and in nuclear invaginations but not within the nucleus. In Ad5-coinfected cells, isolated GFP-VP2-AAV particles were already detectable in the nucleus at 2 h p.i., suggesting that Ad5 enhanced the nuclear translocation of AAV capsids. The number of cells displaying viral capsids within the nucleus increased slightly over time, independently of helper virus levels, but the majority of the AAV capsids remained in the perinuclear area under all conditions analyzed. In contrast, independently of helper virus and with 10 times less virions per cell already observed at 2 h p.i., viral genomes were visible within the nucleus. Under these conditions and even with prolonged incubation times (up to 11 h p.i.), no intact viral capsids were detectable within the nucleus. In summary, the results show that GFP-tagged AAV particles can be used to study the cellular trafficking and nuclear entry of AAV. Moreover, our findings argue against an efficient nuclear entry mechanism of intact AAV capsids and favor the occurrence of viral uncoating before or during nuclear entry.

Semiquantitative Methods for GFP Immunohistochemistry and In Situ Hybridization to Evaluate AAV Transduction of Mouse Retinal Cells Following Subretinal Injection

2020 ◽  
pp. 019262332096480
Author(s):  
Gennadiy Bondarenko ◽  
Steven D. Sorden ◽  
Brian J. Christian ◽  
Sharron Webster ◽  
Alok K. Sharma
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent Protein ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Photoreceptor Layer ◽  
Rna In Situ Hybridization ◽  
Semiquantitative Evaluation ◽  
Green Fluorescent ◽  
Subretinal Injection

The goal of this study was to develop methods for the evaluation of green fluorescent protein (GFP) and GFP transcript biodistribution in paraformaldehyde-fixed paraffin-embedded (PFPE) eye sections to assess the effectiveness of Adeno-associated virus (AAV) gene delivery in an experimental ocular toxicity study. Female C57BL/6NTac mice were administered AAV2-enhancedGFP vector once via subretinal injection. One group also received anti-inflammatory therapy (meloxicam). Immunohistochemistry (IHC) and RNA in situ hybridization (ISH) for GFP were performed on PFPE serial eye sections and evaluated using semiquantitative methods. On day 43, GFP labeling in both IHC and ISH sections was greatest in the retinal pigment epithelium, compared with other retinal layers in which expression was negative to moderate. Despite the presence of IHC GFP labeling in the photoreceptor layer (PRL) in some animals, only low numbers of transduced cells were detected by ISH in the PRL. Simultaneous analysis of IHC and ISH may be needed for comprehensive assessment of gene transduction and protein biodistribution. This study demonstrates approaches for semiquantitative evaluation of IHC and ISH that allow interpretation and reporting of GFP expression in toxicity studies.

scholarly journals Comparative Analysis of Gene Transfer to Human and Rat Retinal Pigment Epithelium Cell Line by a Combinatorial Use of Recombinant Adeno- Associated Virus and Ultrasound or/and Microbubbles

2009 ◽  
Vol 9 (3) ◽  
pp. 174-181 ◽  
Author(s):  
Xiao-Zhi Zheng ◽  
Hong-Li Li ◽  
Lian-Fang Du ◽  
Hui-Ping Wang ◽  
Qing Gu
Keyword(s):  
Retinal Pigment Epithelium ◽  
Gene Transfer ◽  
Cell Line ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelium Cell ◽  
Blue Staining ◽  
Adeno Associated Virus ◽  
Epithelium Cell

Ultrasound-targeted microbubble destruction has been utilized to deliver a drug/gene into cells in both in vitro and in vivo studies. This work was performed to investigate the feasibility of gene transfer to human retinal pigment epithelium cell line(ARPE-19) and rat retinal pigment epithelium cell line(RPE-J) by a combinatorial use of recombinant adeno-associated virus (rAAV) and ultrasound (US) or/and mi-crobubbles (MBs) and compare the difference between them. Different doses of serotype 2 rAAV encoding a enhanced green fluorescent protein (rAAV2-EGFP) gene and MBs was administered to ARPE-19 and RPE-J cells under different US conditions. Transfection efficiency and cell viability were assessed by fluorescence microscopy, flow cytometry (FCM) analysis, trypan blue staining. The results indicated that US and MBs could respectively improve rAAV2mediated gene transfer to RPE-J cells, but neither US nor MBs could do so in ARPE- 19 cells. US plus MBs could significantly enhance rAAV2-mediated gene transfer to ARPE-19 cells, however, the same effects were not seen in RPE-J cells. These findings demonstrated it is not always coincident that US, MBs and US plus MBs exert the similar effects on gene transfer in vitro RPE cells. So, it is necessary to choose appropriate RPE cell line for the study of US or/and MBs-mediated rAAV gene transfer in retinal gene therapy.

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