Role of RPE65 and AAV vectors in LCA gene therapy

RPE65: role in the visual cycle

RPE65 is retinal pigment epithelium which is a key isomerase for
processing visual cycle or retinoid cycle. RPE65 is responsible for Commented [G1]: Inserted: s
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converting all-trans-retinyl ester to 11-cis-retinol so that the visual
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cycle after photoisomerization event can be done. Without RPE65, Commented [G9]: Deleted:ng
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retinyl ester will accumulate in RPE and the production of 11-cis
retinol will be reduced. The protein of RPE65 was 61 kDa in size and Commented [G3]: Inserted: be
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found in the smooth endoplasmic reticulum of the RPE cells. Based
on the research which was done in a rat, in embryonic day 18, the
formation of RPE65 is detectable and it will reach the peak twice Commented [G5]: Inserted: ,
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during the development of retinal. In the postnatal day 10 until 12,
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the developmental expression of RPE65 is coordinated with the
appearance of photoreceptor outer segment membranes and the
expression of some proteins.
There are two forms of RPE65; a soluble form (sRPE65) and a
membrane-bound form (mRPE65). The role of RPE65 was confined
to the binding and activation of all-trans-retinyl esters for preparing
by the isomerohydrolase in charge of the basic change of all-trans-to
11-cis-retinoids.
RPE65 is the isomerohydrolase because from the study it showed
that RPE65 protein associates with isomerohydrolase movement and
its enzymatic action can be reconstituted in vitro after transfection
with RPE65 cDNA. When a cDNA expression is conducted using
bovine RPE, there is high isomerohydrolase activity by isolated a
single clone, when it is sequenced, it proved to be RPE65. Commented [G11]: Inserted: ed

Despite the fact that RPE65 was initially distinguished just in RPE, it
is likewise communicated in both mammalian and amphibian
proficient and mammalian cone photoreceptors however not rods.
The role of RPE65 in a cone is still unknown but the mammalian Commented [G12]: Inserted: a

cones may use an alternate retinoid master cessing cycle

notwithstanding the conventional one utilized by rods, so the
retinoid regeneration in cones is likely still RPE65 dependent. At the
point when mice lacking RPE65 were crossed into the "cone-only", 11-
cis-retinal was imperceptible and retinyl esters and aggregate retinoids
were increased. Consistent with the nonattendance of color, retinal
affectability dropped by a factor of one thousand and cone OSs
degenerated.

Mutations in RPE65 causes 2% cases of recessive RP and 16% cases of

LCA. The retinas of LCA patients hold typical retinal lamina with a
perceptible photoreceptor layer into adulthood supporting the likelihood
that quality substitution treatment might be a practical treatment
alternative. Besides, numerous patients have a normal optic nerve, Commented [G13]: Inserted: a

anatomic structure, and both grey and white matter.

RPE is transduced by an assortment of infections, most recently is

lentivirus that used to move cDNA into transgene expression in the RPE
cells and keep of normal electroretinogram (ERG) function and cone
number. The gene therapy uses recombinant adeno-associated virus
(rAAV) as a vector. rAAV is a nonpathogenic parvovirus which contains
no viral coding regions and has low cytotoxicity. rAAV is the only viral
vector to transduce RPE and photoreceptors efficiently. The therapy
using that vector was done at different times; at embryonic day 14and
postnatal day 14 and 18. Treatment at all ages brought effective RPE Commented [G14]: Inserted: at
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transduction with exchanged RPE65 protein expression noticeable up to 7
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months post-infusion by immunohistochemistry. After the treatment, it
resulted in rhodopsin and retinyl-ester levels was normal, 11-cis-retinal
levels were increased, and retinal morphology was close to normal.
Treatment age affected treatment success. In a rat which treated at 17-26
months is less healed than a rat which treated at younger ages. Commented [G16]: Inserted: as
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Not only in a rat, the RPE65 gene replacement also success in a dog
model. rAAV was injected subretinally and resulted in significantly Commented [G19]: Inserted: a
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improved visual function, retinoid content, and visual behavior.