April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Impact of Single Amino Acid Modifications of Adeno-Associated Virus (AAV) Capsid and Viral Dose on Transduction Patterns After Transuterine Subretinal Delivery of AAV Vectors to the Fetal Retina
Author Affiliations & Notes
  • D. C. Chung
    FM Kirby Ctr Molecular Ophth, Scheie Eye Institute/Univ. of Pennsylvania, Philadelphia, Pennsylvania
  • V. Lee
    FM Kirby Ctr Molecular Ophth, Scheie Eye Institute/Univ. of Pennsylvania, Philadelphia, Pennsylvania
  • L. H. Vandenberghe
    Dept. of Pathology and Lab Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
  • D. Cheng
    FM Kirby Ctr Molecular Ophth, Scheie Eye Institute/Univ. of Pennsylvania, Philadelphia, Pennsylvania
  • Z. Wei
    FM Kirby Ctr Molecular Ophth, Scheie Eye Institute/Univ. of Pennsylvania, Philadelphia, Pennsylvania
  • J. M. Wilson
    Dept. of Pathology and Lab Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
  • J. Bennett
    FM Kirby Ctr Molecular Ophth, Scheie Eye Institute/Univ. of Pennsylvania, Philadelphia, Pennsylvania
  • Footnotes
    Commercial Relationships  D.C. Chung, None; V. Lee, None; L.H. Vandenberghe, None; D. Cheng, None; Z. Wei, None; J.M. Wilson, None; J. Bennett, None.
  • Footnotes
    Support  : NIH Grants K08 EY017024, RO1 EY10820, P30-DK-47747-10, Foundation Fighting Blindness, Research to Prevent Blindness, the Paul and Evanina Mackall Foundation Trust, Hope for Vision and the F.M. Kirby
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 3019. doi:
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      D. C. Chung, V. Lee, L. H. Vandenberghe, D. Cheng, Z. Wei, J. M. Wilson, J. Bennett; Impact of Single Amino Acid Modifications of Adeno-Associated Virus (AAV) Capsid and Viral Dose on Transduction Patterns After Transuterine Subretinal Delivery of AAV Vectors to the Fetal Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3019.

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

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Abstract

Purpose: : To compare transduction patterns after transuterine subretinal delivery of transgenes through novel AAVs with wildtype or modified capsids to the fetal retina in mice. It is proposed that viral tropism and transduction characteristics could be altered by single nucleotide viral capsid modifications or viral dosage. Optimization of retinal transduction properties could lead to increased efficacy in treatments designed for early onset retinal degenerative diseases.

Methods: : Wild-type C57Bl6 mice underwent timed pregnancies to determine the date of conception. At embryonic day 14.5-15.5 (E14.5-15.5), transuterine injections were conducted to place a wildtype or modified AAV2 vector carrying the enhanced green fluorescent protein marker gene driven by the CMV promoter into the embryonic subretinal space. Adeno-associated viral vectors tested were AAV2/6, AAV2/6.1, AAV2/6.1.2, AAV2/6.2. Vectors were injected at a high dose (2.8E9gc - 2.4E10gc) and low dose (5.5E8gc). Following successful gestation and delivery, pups were maintained for an additional 28 days. Animals were then euthanized, eyes enucleated, cryoprotected and sectioned. Retinal sections were examined on a fluorescent microscope for GFP expression.

Results: : The cellular specificity and efficiency of transduction exhibited slight differences in expression patterns after high titer injections. At high titer, photoreceptor transduction was seen with all vectors used, however transduction with AAV2/6.2 was significantly reduced, as measured by eGFP expression. At low titer, all vectors showed expression in retinal pigment epithelial cells only. All injected mice developed normally and without apparent toxicity secondary to the transuterine injection. The majority of dams were able to rear subsequent litters without difficulty.

Conclusions: : Viral titer and AAV capsid modification can alter tropism and expression efficiency after transuterine subretinal delivery to the fetal retina. Such differences could be exploited to optimize gene targeting to the fetal retina and influence therapies for congenital or pediatric retinal degenerative diseases.

Keywords: gene transfer/gene therapy • retinal degenerations: cell biology • retinal development 
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