RS-1 Gene Delivery to an Adult Rs1h Knockout Mouse Model Restores ERG b-Wave with Reversal of the Electronegative Waveform of X-Linked Retinoschisis.
OBJECTIVE
To create and evaluate a mouse model of human X-linked juvenile retinoschisis (XLRS) and then investigate whether supplementing with the retinoschisin protein by gene delivery can reverse the abnormal "electronegative" electroretinogram (ERG) retinal response.


METHODS
An X-linked retinoschisis mouse (Rs1h-KO) model was created by substituting a neomycin resistance cassette for exon 1 and 1.6 kb of intron 1 of Rs1h, the murine orthologue of the human RS-1 gene. RS protein was evaluated by immunohistochemistry and Western blot analysis with a polyclonal RS N-terminus antibody. Retinal function was evaluated by conventional, full-field flash ERG recordings. RS protein supplementation therapy was evaluated by gene transfer with an AAV(2/2)-CMV-Rs1h vector containing C57BL/6J Rs1h cDNA under the regulation of a CMV promoter, and ERG functional analysis was performed.


RESULTS
No RS protein was detected by Western blot analysis or immunohistochemistry in the Rs1h-KO mouse. Dark-adapted ERG responses showed an electronegative configuration, with b-wave reduction in both Rs1h(-/Y) and Rs1h-/- mice, typical of XLRS in humans. Histologic examination of Rs1h-KO mice showed disorganization of multiple retinal layers, including duplication and mislocalization of ganglion cells, laminar dissection through the inner plexiform layer, disorganization of the outer plexiform layer, loss of regularity of the outer nuclear layer, and shortening of the inner/outer segments with mislocalization of photoreceptor nuclei into this layer. After intraocular administration of AAV(2/2)-CMV-Rs1h, immunohistochemistry showed retinoschisin expression in all retinal layers of Rs1h(-/Y) mice, and ERG recordings showed reversal of the electronegative waveform and restoration of the normal positive b-wave.


CONCLUSIONS
The RS-KO mouse mimics structural features of human X-linked juvenile retinoschisis with dissection through, and disorganization of, multiple retinal layers. The Rs1h-KO functional deficit results in an electronegative ERG waveform that is characteristic of human retinoschisis disease and that implicates a synaptic transmission deficit in the absence of retinoschisin protein. Replacement therapy by supplementing normal Rs1h protein in the adult Rs1h-KO mouse restored the normal ERG configuration. This indicates that gene therapy is a viable strategy of therapeutic intervention even in the postdevelopmental adult stage of XLRS disease.
Zeng
Yong
Y
National Institute on Deafness and Other Communication Disorders, Bethesda, MD 20892, USA.
Takada
Yuichiro
Y
Kjellstrom
Sten
S
Hiriyanna
Kelaginamane
K
Tanikawa
Atsuhiro
A
Wawrousek
Eric
E
Smaoui
Nizar
N
Caruso
Rafael
R
Bush
Ronald A
RA
Sieving
Paul A
PA
D000818:Animals; D015815:Cell Adhesion Molecules; D002648:Child; D003623:Dark Adaptation; D004596:Electroretinography; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D012160:Retina / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D041441:Retinoschisis / Q000235:genetics / Q000473:pathology / Q000503:physiopathology*
D015815:Cell Adhesion Molecules; D005136:Eye Proteins; C491843:RS1 protein, mouse