May 2005
Volume 46, Issue 13
ARVO Annual Meeting Abstract  |   May 2005
Genes Associated With Adeno–Associated Virus (AAV2) Transduction Enhancement in the Human Trabecular Meshwork (HTM)
Author Affiliations & Notes
  • T. Borras
    Ophthalmology, University of North Carolina, Chapel Hill, NC
  • S.S. Chisolm
    Ophthalmology, University of North Carolina, Chapel Hill, NC
  • J.S. Bartlett
    Pediatrics, Ohio State University, Columbus, OH
  • A.M. Eaton
    Ophthalmology, University of North Carolina, Chapel Hill, NC
  • W. Xue
    Ophthalmology, University of North Carolina, Chapel Hill, NC
  • Footnotes
    Commercial Relationships  T. Borras, None; S.S. Chisolm, None; J.S. Bartlett, None; A.M. Eaton, None; W. Xue, None.
  • Footnotes
    Support  EY13126, EY11906, Research to Prevent Blindness; SCC was a Holderness scholar
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1155. doi:
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      T. Borras, S.S. Chisolm, J.S. Bartlett, A.M. Eaton, W. Xue; Genes Associated With Adeno–Associated Virus (AAV2) Transduction Enhancement in the Human Trabecular Meshwork (HTM) . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1155.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: AAV2 vectors offer great promise for gene therapy due to their long–term gene expression and low immune response. We have shown that HTM is refractory to AAV2 but transduction can be drastically enhanced by co–infecting AAV+Adenovirus (Ad) (viral particles 1/0.05). Here we investigate mechanisms of AAV transduction in the HTM using a pseudotyped AAV and microarray analysis. Methods: An AAV packaging plasmid encoding capsid proteins with an inserted RGD peptide was constructed by PCR–based mutagenesis. AAV.GFP viruses were generated by triple transfection of the packaging plasmids with a plasmid carrying the CMV–eGFP cassette and ITRs plus Ad helper plasmid. Our Ads: AdNull (carrying no foreign gene), AdhTIG3 (encoding MYOC) and Ad.LacZ (encoding B–gal) have been described. Primary HTM cells were infected with AAV.GFP, pseudotyped AAV.RGD.GFP (moi 5–10) or co–infected with each of the AAV.GFP+Ad combinations. GFP mRNA and DNA were measured by RQ–PCR and normalized with 18S RNA and genomic DNA respectively. Total RNA was processed and hybridized to Affymetrix U95Av2 GeneChips (n=5) at the UNC core facility. Comparisons of co–infected cells (AAV.GFP+AdNull, AAV.GFP+AdhTIG3 and AAV.GPF+Ad.LacZ) vs. cells infected with AAV.GFP alone, and that of AAV.GFP alone vs mock–infected cells, were analyzed using Affymetrix software. Results: While AAV transduction levels (GFP mRNA) increased 25–300X in the AAV+Ad co–infected cells, AAV DNA levels inside HTM cells were the same in all conditions. DNA from AAV.RGD.GFP was 2.5X higher than AAV but, likewise, yielded no GFP transduction. Infection with AAV.GFP alone induced cellular expression changes higher than 1.4–fold in just 0.7% of the 12,626 genes while AAV+Ads co–infections induced the same change in 8.8.% (+Ad.LacZ), 7.3% (+AdhTIG3) and 3.9% (+AdNull) of the cellular genes. Among the 25 most upregulated genes in all three enhancement co–infections were, as expected, genes involved in inflammatory and stress responses, such IL–6 and heat shock 70 kDa protein; surprisingly, this list also included the CAR receptor and four TM preferred genes: carbonic anhydrase II, secretogranin II, tenascin and versican. Conclusions: The inability of AAV gene transfer to HTM is not due to viral cell entry failure. Adenoviral vectors elicit a considerably higher TM cellular expression response than AAV. Identification of genes associated with AAV transduction enhancement could help circumvent the use of Ads and obtain a long–term, low immunogenic gene delivery to the TM.

Keywords: gene transfer/gene therapy • gene microarray • trabecular meshwork 

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