scholarly journals Efficient transduction of green fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters

Gene Therapy ◽  
1997 ◽  
Vol 4 (1) ◽  
pp. 16-24 ◽  
Author(s):  
AL Peel ◽  
S Zolotukhin ◽  
GW Schrimsher ◽  
N Muzyczka ◽  
PJ Reier
Keyword(s):  
Spinal Cord ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Type ◽  
Adeno Associated Virus ◽  
Spinal Cord Neurons ◽  
Virus Vectors ◽  
Cell Type Specific ◽  
Green Fluorescent

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.

Delayed accumulation of activated macrophages and inhibition of remyelination after spinal cord injury in an adult rodent model

2008 ◽  
Vol 8 (1) ◽  
pp. 58-66 ◽  
Author(s):  
Masaaki Imai ◽  
Masahiko Watanabe ◽  
Kaori Suyama ◽  
Takahiro Osada ◽  
Daisuke Sakai ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Bone Marrow ◽  
Spinal Cord Injury ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Activated Macrophages ◽  
Cord Injury ◽  
Myelin Debris ◽  
Chemotactic Factors ◽  
Green Fluorescent

Object Inhibition of remyelination is part of the complex problem of persistent dysfunction after spinal cord injury (SCI), and residual myelin debris may be a factor that inhibits remyelination. Phagocytosis by microglial cells and by macrophages that migrate from blood vessels plays a major role in the clearance of myelin debris. The object of this study was to investigate the mechanisms underlying the failure of significant remyelination after SCI. Methods The authors investigated macrophage recruitment and related factors in rats by comparing a contusion model (representing contusive SCI with residual myelin debris and failure of remyelination) with a model consisting of chemical demyelination by lysophosphatidylcholine (representing multiple sclerosis with early clearance of myelin debris and remyelination). The origin of infiltrating macrophages was investigated using mice transplanted with bone marrow cells from green fluorescent protein–transfected mice. The changes in levels of residual myelin debris and the infiltration of activated macrophages in demyelinated lesions were investigated by immunostaining at 2, 4, and 7 days postinjury. To investigate various factors that might be involved, the authors also investigated gene expression of macrophage chemotactic factors and adhesion factors. Results Activated macrophages coexpressing green fluorescent protein constituted the major cell population in the lesions, indicating that the macrophages in both models were mainly derived from the bone marrow, and that very few were derived from the intrinsic microglia. Immunostaining showed that in the contusion model, myelin debris persisted for a long period, and the infiltration of macrophages was significantly delayed. Among the chemotactic factors, the levels of monocyte chemoattractant protein–1 and granulocyte–macrophage colony-stimulating factor were lower in the contusion model at 2 and 4 days postinjury. Conclusions The results suggest that the delayed infiltration of activated macrophages is related to persistence of myelin debris after contusive SCI, resulting in the inhibition of remyelination.

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
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