June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Different transduction efficiencies between single- and double-stranded Adeno-associated viruses (ssAAV & dsAAV) in the human trabecular meshwork extend across seven serotypes
Author Affiliations & Notes
  • Laura Rodriguez Estevez
    Ophthalmology, Univ of NC at Chapel Hill, Chapel Hill, North Carolina, United States
  • Priyadarsini Asokan
    Ophthalmology, Univ of NC at Chapel Hill, Chapel Hill, North Carolina, United States
  • Terete Borras
    Ophthalmology, Univ of NC at Chapel Hill, Chapel Hill, North Carolina, United States
    Gene Therapy Center, Univ of NC at Chapel Hill, Chapel Hill, North Carolina, United States
  • Footnotes
    Commercial Relationships   Laura Rodriguez Estevez, None; Priyadarsini Asokan, None; Terete Borras, None
  • Footnotes
    Support  EY011906; EY026220
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3496. doi:
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      Laura Rodriguez Estevez, Priyadarsini Asokan, Terete Borras; Different transduction efficiencies between single- and double-stranded Adeno-associated viruses (ssAAV & dsAAV) in the human trabecular meshwork extend across seven serotypes. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3496.

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

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Abstract

Purpose : Our long term goal is to increase transgene transduction in the TM to lower dose and toxicity in gene therapy. Previously, we demonstrated that ssAAV2 viral transduction in the human TM was very inefficient while that of the ds form (scAAV2) was very high. Our goal here was to investigate whether this ss/ds characteristic extended to serotypes 1, 2.5, 5, 6, 8 & 9, to determine the rank of their efficiency, and to explore whether a capsid mutated scAAV2 virus could significantly enhance gene transduction in the HTM cells.

Methods : A primary HTM cell line from a single individual was used at passage 3-5. AAV- & scAAV.GFP high titer stocks were made at the UNC vector core. A tyrosine mutated AAV2 capsid (Y444F) was generated by synthesizing a 767bp DNA fragment containing a single nucleotide change in codon TAC (Y) to TTC (F), and swapping it for the corresponding wild-type in the pXR2 plasmid (BsiWI-XcmI sites). Viral infections were performed at moi 100-1000 pfu on subconfluent cells. Quantification of intracellular viral DNA (24h) and RNA (72h) was conducted on total cell DNA and cDNA by TaqMan PCR using a GFP probe. Single-copy RPPH1 and 18S probes were used for respective normalizations. Quantification of fluorescence (transduction) was conducted on images captured on a monochrome camera (DP70) at the same exposure and threshold using the Metamorph software (Olympus).

Results : In all serotypes, there was a high difference in transduction (cDNA fold-changes and fluorescence intensity units) between each scAAV and its corresponding AAV. In contrast, the number of intracellular AAV viral genomes was higher than the scAAV in serotypes 2.5, 1, 5 & 2 (11-6 fold), and just slightly lower in serotypes 9, 6 & 8 (0.31-0.65 fold). The highest transduction efficiency of the scAAV serotypes ranked 2, 6 & 5 followed by a distant, negligible transduction of 9, 8, 1 & 2.5. The capsid mutated scAAV2.Y444F virus showed a large transduction enhancement compared to scAAV2 (14.2-fold).

Conclusions : In the human TM, all seven serotypes showed a large transduction difference between the AAV’s ds and ss forms. The serotype 2 capsid mutation Y444F conferred a marked transduction enhancement to the human TM. Determining the optimal conditions for the human TM transduction is key for the development of gene therapy of glaucoma.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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