Ability Of Adeno Associated Virus Biology Essay

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The ability of adeno-associated virus (AAV) serotypes 1-9 in addition to nineteen novel vectors isolated from various tissues, to transduce mouse and human ciliated airway epithelium (HAE) are concerned. Vectors expressing α-1-antitrypsin (AAT) and β-galactosidase are co-instilled into the mouse lung.

First step the DNA build and vector production, the AAV vectors are flanked with AAV2 inverted terminal repeats (ITRs) contained either (i) a LacZ gene fused to a nucleus localization sequence at the N-terminus (nLacZ); (ii) produce green fluorescence protein gene (iii) an hAAT gene under the transcriptional control of the cytomegalovirus-enhanced chicken-β-actin or the cytomegalovirus promoter. Then AAV isolates from exacting residues called singletons that are known by structure-function analyses. After that Novel capsid sequences transferred from a PCR isolate into TOPO cloning vectors (Invitrogen, Carlsbad, CA) and thus cloned into an AAV packaging make by partial restriction digestion at the first internal cap XhoI site on the 5′ end and, depending on the direction in the TOPO plasmid, a PmeI or EcoRV blunt end digestion.

Second step, the model animal ( Mice C57BL/6) are anesthetized using an intraperitoneal injection of ketamine/xylazine, and are inoculated by intranasal or intratracheal way with 1011 genome copies of AAV vector in a final volume of 50 µl PBS and the control-treated animals received PBS only

For nLacZ gene expression in the processed lungs. Transduction efficiency is estimated by examining 20 high-power (-100) fields from two cryosections spaced 200 µm apart and data presented as nLacZ expressing cells per - 100 fields. Assessment of gene transfer to the nasal airway epithelium was performed and is presented as the total number of LacZ positive cells in the respiratory epithelium which are quantitated in three standard cross-sections of nasal airway. For hAAT detection, serum samples are collected and the concentration is determined by ELISA. For Green Fluorescence Protein (GFP) expression, each image was analyzed using ImageJ software. For each brightness value between 60 (which is above background) and 255 (the maximum value), the number of concerned pixels is determined. The number of pixels was then multiplied with their brightness value and the products added to give a final value for GFP expression

Fresh donor human tracheobronchial tissues are obtained and airway epithelial cells are isolated. Freshly isolated airway epithelial cells are seeded at 250,000 per cm2 on permeable membrane supports (Millicells PICM 01250, 0.4 µm pore size, Millipore, Billerica, MA), and allowed to coming together, upon which the cultures are maintained at an air-liquid interface to permit differentiation into pseudostratified ciliated airway epithelium. The HAE cultures are maintained at an air-liquid interface for 4-6 weeks before injection with AAV vectors

For viral inoculation, the apical surfaces of HAE cultures are washed with PBS, then are inoculated with 1011 genome copies (MOI = 200,000) of candidate AAVGFP vectors in 100 µl. All AAV vector candidates expressed GFP using the cytomegalovirus promoter. To identify transduced epithelial cell types, HAE cultures are fixed in 4% paraformaldehyde and cilia immuno-labelled with a β-tubulin IV specific monoclonal antibody (Sigma, St. Louis, MO) and fluorescent XZ confocal images obtained with a Zeiss 510 Meta Laser Scanning Confocal Microscope

A mixture of AAV vectors expressing hAAT and nLacZ are administered to mice (n = 15) by intratracheal instillation. Twenty-eight days later, mice are killed and sera analyzed for hAAT concentration and lung tissue evaluated for nLacZ expression. Based on hAAT serum concentrations, the 19 candidate vectors, counting the well established serotypes AAV1, 2, 5, 6, 7, 8, and 9, are segregated into three groups of expression levels (high, medium, and low). The high-expressors group conferring hAAT concentrations in serum >1,500 ng hAAT/ml serum are serotypes AAV1, 6, 9, and novel vectors rh.8, rh.10, rh.20, rh.46, rh.64R1, hu.48R3, and cy.5R4 (Figure: 3). In the medium-expressors group with 500-1,500 ng hAAT/ml serum are serotypes AAV7, 8 and novel vectors rh.2R, rh.32.33, rh.64R2, hu.13, hu.32, hu.37, and pi.2. Of note is the observation that both high and medium expressors all generated significantly higher hAAT levels than produced by AAV2 or AAV5 (P < 0.05, analysis of variance, Student-Newman-Keuls test, n = 15). In the low-expressors group (<500 ng/ml hAAT) including AAV2 and AAV5, the novel vectors rh.39, rh.43, hu.11, hu.29R, and hu.51 produced very low levels of hAAT which approximated the detection sensitivity of the hAAT ELISA [16, 17].

Of all the vectors tested rh.64R1, AAV5 and AAV6 are the most efficient. The high transduction observed in mouse was reproduced in HAE cell cultures for both rh.64R1 and AAV6 but not for AAV5. While AAV6 was the most efficient vector in mouse and HAE are also tested the transduction efficiencies of the AAV6 singleton vectors (AAV6 variants with targeted mutations) in these models. Of these, AAV6.2 transduced mouse airway epithelium and HAE with greater efficiency than all other AAV vectors tested. Then, demonstrated that AAV6.2 exhibits improved transduction efficiency compared to previously reported AAVs in mouse airways and in culture models of human airway epithelium and that this vector requires further development for preclinical and clinical testing [16].

Many viral gene therapy vectors, adeno-associated virus (AAV)-based vectors hold great promise for efficiently targeting airway epithelium in vivo. However, efforts to replace a functional copy of the CF gene (CFTR) to the lungs of CF patients using AAV2-based vectors have proven a significant challenge [16].

Furthermore, the AAVs have the ability to infect and express in non dividing cells and they create a risk of mutagenesis due to random insertion into multiple sites [14].

Future outlook

The generation of animal models in CF was of great benefit in such the detailed study of the disease could be carried out in models than is possible in humans. The models allowed the identification of the CFTR gene and the environmental factors that influence the severity of the disease. The animal models generated are essential in testing the pharmacological strategies for modifying the severity of the disease and furthermore, they are useful for determining an effective vector to correct the mutation in gene therapy [14].

However, since cystic fibrosis is a complex disease affecting many organs, there is no single model that represents all its features hence the generation of many models. Furthermore, the mouse models have a shorter life span and may not have a chance to develop all problems shown by human [12].