Dataset Information

Quantitative 3D tracing of gene-delivery viral vectors in human cells and animal tissues.

ABSTRACT: Trafficking through a variety of cellular structures and organelles is essential for the interaction between gene-delivery vectors (i.e., adeno-associated virus (AAV) and liposomes) and host cells/tissues. Here, we present a method of computer-assisted quantitative 3D biodistribution microscopy that samples the whole population of fluorescently-labeled vectors and document their trafficking routes. Using AAV as a working model, we first experimentally defined numerical parameters for the singularity of Cy5-labeled particles by combining confocal microscopy and atomic force microscopy (AFM). We then developed a robust approach that integrates single-particle fluorescence imaging with 3D deconvolution and isosurface rendering to quantitate viral distribution and trafficking in human cells as well as animal tissues at the single-particle level. Using this quantitative method, we uncovered an as yet uncharacterized rate-limiting step during viral cell entry, while delineating nuclear accumulation of virions during the first 8 hours postinfection. Further, our studies revealed for the first time that following intramuscular injection, AAV spread progressively across muscle tissues through endomysium between myofibers instead of traversing through target cells. Such 3D resolution and quantitative dissection of vector-host interactions at the subcellular level should significantly improve our ability to resolve trafficking mechanisms of gene-delivery particles and facilitate the development of enhanced viral vectors.

SUBMITTER: Xiao PJ

PROVIDER: S-EPMC3277235 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Quantitative 3D tracing of gene-delivery viral vectors in human cells and animal tissues.

Xiao Ping-Jie PJ Li Chengwen C Neumann Aaron A Samulski R Jude RJ

Molecular therapy : the journal of the American Society of Gene Therapy 20111122 2

Trafficking through a variety of cellular structures and organelles is essential for the interaction between gene-delivery vectors (i.e., adeno-associated virus (AAV) and liposomes) and host cells/tissues. Here, we present a method of computer-assisted quantitative 3D biodistribution microscopy that samples the whole population of fluorescently-labeled vectors and document their trafficking routes. Using AAV as a working model, we first experimentally defined numerical parameters for the singula ...[more]

PMID: 22108857

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Project description:Limbal stem cell (LSC) transplantation is a promising treatment for ocular surface diseases especially LSC deficiency. Genetic engineering represents an attractive strategy to increase the potential for success in LSC transplantations either by correcting autologous diseased LSCs or by decreasing the immunogenicity of allogeneic LSCs. Therefore, two popular viral vectors, adeno-associated viral (AAV) vector and lentiviral (LV) vector, were compared for gene delivery in human LSCs. Transduction efficiency was evaluated by flow cytometry, quantitation of viral genomes, and fluorescence microscopy after introducing eight self-complementary AAV serotypes or LV carrying a green fluorescent protein (GFP) cassette to fresh limbal epithelial cells, cultivated LSC colonies, or after corneal intrastromal injection into human explant tissue. For fresh limbal epithelial cells, AAV6 showed the highest transduction efficiency, followed by LV and AAV4 at 24?h after vector incubation, which did not directly correlate with internalized genome copy number. The colony formation efficiency, as well as colony size over time, showed no significant differences among AAV serotypes, LV, and nontreated controls. The percentage of GFP+ colonies at 14 days post-seeding was significantly higher in the LV group, which plateaued at 50% GFP+ upon serial passages. Interestingly, AAV6-treated colonies initially showed a variegated transduction phenotype with no GFP+ colonies in serial passages. Quantitative polymerase chain reaction and AAV6 capsid staining revealed that transduction was restricted to differentiated cells of LSC colonies at a post-entry step. Following central intrastromal injection of human corneas, both LV and AAV6 transduced the stroma and endothelial cells, and AAV6 also transduced cells of the epithelia. However, no transduction was observed in derived LSC colonies. The collective results demonstrate the effectiveness of LV for stable human LSC genetic engineering and an unreported phenomenon of AAV6 transduction restriction in multipotent cells derived from the human limbus.

Project description:Over the last two decades, enormous effort has been focused on developing virus-based gene delivery vectors to target the respiratory airway epithelium as a potential treatment for cystic fibrosis (CF) lung disease. However, amongst other problems, the efficiency of gene delivery to the differentiated airway epithelial cells of the lung has been too low for clinical benefit. Although not a target for CF therapy, the nasal epithelium exhibits cellular morphology and composition similar to that of the lower airways, thus representing an accessible and relevant tissue target for evaluating novel and improved gene delivery vectors. We previously reported that replication-competent human parainfluenza virus (PIV)-based vectors efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to sufficient numbers of cultured CF airway epithelial cells to completely correct the bioelectric function of CF cells to normal levels, resulting in restoration of mucus transport. Here, using an in vitro model of rhesus airway epithelium, we demonstrate that PIV mediates efficient gene transfer in rhesus epithelium as in the human counterpart. Naive rhesus macaques were inoculated intranasally with a PIV vector expressing rhesus macaque ?-fetoprotein (rhAFP), and expression was monitored longitudinally. rhAFP was detected in nasal lavage fluid and in serum samples, indicating that PIV-mediated gene transfer was effective and that rhAFP was secreted into both mucosal and serosal compartments. Although expression was transient, lasting up to 10 days, it paralleled virus replication, suggesting that as PIV was cleared, rhAFP expression was lost. No adverse reactions or signs of discomfort were noted, and only mild, transient elevations of a small number of inflammatory cytokines were measured at the peak of virus replication. In summary, rhAFP proved suitable for monitoring in vivo gene delivery over time, and PIV vectors appear to be promising airway-specific gene transfer vehicles that warrant further development.

Dataset Information

Quantitative 3D tracing of gene-delivery viral vectors in human cells and animal tissues.

Publications

Quantitative 3D tracing of gene-delivery viral vectors in human cells and animal tissues.

Similar Datasets

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