Lecture 10 Gene Therapy

Sclera

Covers and protects most of the eye.

Cornea

Transparent protective layer on the front of the eye.

Iris

Controls the amount of light that enters the eye by dilating and contracting.

Pupil

The hole through which light enters the eye.

Ciliary body

Produces humors and controls the shape of the lens to focus light on the fovea.

Fovea

Patch of retina responsible for visual acuity.

Choroid

Contains blood vessels to nourish the retina.

Neural retina

Made up of photoreceptor cells.

Retinal pigmented epithelium (RPE)

Thin layer behind the neural retina that nourishes rods and cones and absorbs stray light.

Optic nerve

Transmits nerve impulses from the photoreceptor cells to the brain.

Optic disc

Where the optic nerve exits the eye; a blind spot with no photoreceptor cells.

Rods

Photoreceptors for low light vision found in the periphery of the neural retina.

Cones

Photoreceptors for bright color vision concentrated in the fovea.

Visual cycle process

Opsin is struck by photon, 11-cis-retinal isomerizes to all-trans-retinol, triggering a signal to the brain.

All-trans-retinal gets recycled back into 11-cis-retinal by RPE65 and reused

LCA Type 2

Leber Congenital Amaurosis caused by loss of function of RPE65 leading to early vision loss / complete blindness by 3rd decade.

Luxturna gene therapy steps

Build gene construct, replace AAV genome with cDNA, add enhancer-promoter, grow viral particles

Tissue culture - grow 10^10 viral particles (not patient’s cells)

Inject purified viral particles carrying construct into patient close to target cells, AAV genome construct creates circular episome which is replicated by helper virus proteins (Adenovirus)

Luxturna effects in cells

Creates circular episome that replicates and produces normal protein to replace the defective one (with help of Adenovirus).

Factors for Luxturna success

Non-proliferative, minimal side effects, rod cell preservation if treated early.
Caused by LOF RPE65 - introducing functional gene is affective

Heterozygous PAX6 mutation effects

Leads to aniridia, small/missing iris, opaque cornea and/or lens, under-developed fovea and optic nerve.

Who benefits from Ataluren

Individuals with missense mutations or premature stop codons.

Hypothesis for Ataluren action

It makes ribosomes ignore premature stop codons, reading UGA as Trp, Arg, or Cys.