June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Co-expression of human L- or M-opsin restores M-Cone Function in the Opn1mw Knock-out mouse, a model for blue cone monochromacy (BCM)
Author Affiliations & Notes
  • Wentao Deng
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Ji-Jing Pang
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Jie Li
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Ping Zhu
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Wolfgang Baehr
    University of Utah, Salt Lake City, Utah, United States
  • W. Clay Smith
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • William W Hauswirth
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Footnotes
    Commercial Relationships   Wentao Deng, None; Ji-Jing Pang, None; Jie Li, None; Ping Zhu, None; Wolfgang Baehr, None; W. Clay Smith, None; William Hauswirth, AGTC (P), AGTC (F), AGTC (C)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4477. doi:
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      Wentao Deng, Ji-Jing Pang, Jie Li, Ping Zhu, Wolfgang Baehr, W. Clay Smith, William W Hauswirth; Co-expression of human L- or M-opsin restores M-Cone Function in the Opn1mw Knock-out mouse, a model for blue cone monochromacy (BCM). Invest. Ophthalmol. Vis. Sci. 2017;58(8):4477.

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

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Abstract

Purpose : BCM is an X-linked congenital disorder characterized by loss of both L- and M- cone function. Here we tested gene therapy for BCM using an M-cone knock out mouse model by AAV-mediated expression of either or both human M- and L-opsin.

Methods : Since human L- and M-opsin are highly homologous there is no antibody that can distinguish between them. We generated AAV5 vectors expressing either C-terminal myc tagged L-opsin-myc cDNA or C-terminal HA tagged M-opsin-HA cDNA, both driven by the cone-specific PR2.1 promoter. Vectors were delivered either individually or as a mixture containing equal numbers of vector genomes of each. 1 μL of vector(s) was injected subretinally into one eye of one month old M-opsin Knockout (Opn1mw-/-) mice, while the contralateral eyes served as controls. M-cone function was assessed by photopic ERG under middle wavelength (510nm) flash conditions. In retinal sections human L- and M-opsin expression were analyzed by myc and HA antibodies respectively, and their cellular localizations analyzed by peanut agglutinin (PNA) and anti-human L/M-opsin and anti-mouse S-opsin.

Results : Retinal functional analysis of Opn1mw-/- mice treated with either vector individually or as an equal mixture showed significant ERG rescue compared to untreated controls. In eyes treated with individual vectors, expression of myc, HA and M/L-opsin were detected and co-localized correspondingly. In the dorsal retina where no mouse M-opsin is expressed, human M- or/and L-opsin expression co-localized with PNA labeled cone sheaths. In the ventral retina, where mouse S-opsin is present in cone outer segments, they co-localized with endogenous S-opsin. In retinas treated with the vector mixture, a majority of the cones co-express both M- and L-opsin as seen by HA and Myc co-localization. We also note that individual cones can contain different ratios of human M- and L-opsin.

Conclusions : We demonstrate that C-terminal tagging human opsins does not interfere either function or cellular localization. Therefore, these tags can be employed to distinguish L- from M-opsin in the same retina. Functional and structural analyses in Opn1mw-/- retinas confirm that either human opsin or their mixture can restore both significant M-cone function and cone outer segments. These observations provide preclinical proof-of-principle justifying gene therapy treatment of human BCM.

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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