Transcript AAV-mediated Gene Therapy Restores Cone Function In A Rat With

AAV-mediated Gene Therapy Restores Cone Function In A Rat With An M-cone Opsin Deficiency,
A Model For Blue Cone Monochromacy
Zuoming
1Clinical
1
Zhang ,
Jijing
2
Pang ,
Feng
1
Xia ,
Qun
1
Guo ,
Li
1
Li ,
Jing
1
An ,
Lei
1
Zhang ,
William W.
2
Hauswirth ,
Shaowei
1
Yang ,
Zhenfeng
1
Li .
Aerospace Medicine, Fourth Military Medical University, Xi'an, China;
2Department
of Ophthalmology, University of Florida, Gainesville, FL.
Purpose: Using an AAV vector targeting human L-cone opsin or rat M-cone opsin expression to cones, we aimed to test gene therapy in a
naturally occurring m-cone opsin mutant middle-wavelength opsin cone dysfunction, MCD) rat model.
Methods: Abnormal cone response phenotype male Sprague-Dawley (SD) rats (outbreed strain) were analyzed to identify the inherited trait
and the causative mutation. To develop a gene therapy, two AAV vectors were constructed. The first was a serotype 5 AAV with the human
red opsin promoter (PR2.1) driving expression of a human L-opsin cDNA (hROps). The second was a serotype 8 AAV containing a Y-F mutation
at capsid position 733 (AAV8-733) with the same promoter driving a rat M-opsin cDNA. One microliter of each vector containing 1010 vector
genomes was subretinally injected into one eye of a cohort of 30 mutant rats, respectively at postnatal day 14 (P14). At 2 months posttreatment the therapeutic effect of vector expression of the two opsin cDNAs was tested by full field photopic and flicker ERG analyses.
Results: The rat with abnormal cone function has an X-linked recessive trait and the causative mutation is in the Opn1mw gene. There was
no cone response or flicker ERG under standard intensity flashes in untreated rats, a phenotype that was stably maintained for 16
generations over 7 years. Histologically, there was no obvious change in retinal thickness, structure or the number of M-cones. After
subretinal injection of the AAV5 vector expressing the human L-opsin in rat cones, there was no obviously change in cone single flash or
flicker ERG at 2 months post-treatment. In contrast, the AAV8 (733) vector encoding the rat M-opsin yielded robust cone function rescue.
Fig 2 Inherit trait in MCD rat
It was showed a (X-linked
recessive trait
Fig 4 Retinal cone densities and thickness at
different month in wild and MCD rat
Bar denotes 10 μm in length.
Fig 3 Identification of the rat mcd mutation locus
In the rats, the nucleotide conversion G-to-T indicated
by the box occurred at the invariant splicing acceptor
site AG of intron 4.
Fig 5 Expression of Opn1mw transcripts and
M-opsin protein in the retina of wild rat (SD)
and MCD rats.
Fig 1 Scotopic and phototic ERG in wild and MCD rat
Fig 6 ERGs after the AAV8 (733) vector encoding treatment in MCD rat
Uper trace was right eye (treated eye)
down trace was left eye (un treated eye)
Conclusions: Since there is no L-cone opsin in the rat, this rat strain lacking M-cone function can be considered a model for combined L
and M cone function loss in humans, a condition termed Blue Cone Monochromacy. Replacement gene therapy using a subretinally
delivered AAV vector restores cone function in this model when using the homologous rat M-opsin cDNA but not when using the human
L-opsin cDNA perhaps due to incompatibility of the human opsin with one or more components of the rat phototransduction complex.
Reference
1. Gu YH, et al. A naturally occurring rat model of X-linked cone dysfunction. Invest Ophthalmol Vis Sci. 2003;44(12):5321-6.
2. Xie B, et al. A novel middle-wavelength opsin (M-opsin) null-mutation in the retinal cone dysfunction rat. Exp Eye Res. 2010;91(1):26-33.
3. Pang JJ,et al. Achromatopsia as a potential candidate for gene therapy.Adv Exp Med Biol, 2010;664:639-46.