Lecture 10 Gene Therapy

  1. What are the parts of the eye and what do they do?

    • Sclera - covers and protects most of eye

    • Cornea - transparent protective layer on the front

    • Iris - dilates and contracts controlling the amount of light that enters eye

    • Pupil - hole through which light enter eye

    • Ciliary body - produces humors and also controls shape of lens to allow focus of light on fovea

    • Fovea - patch of retina responsible for visual acuity

    • Choroid - contains blood vessels to nourish retina

    • neural retina - made up of photoreceptor cells

    • Retinal pigmented epithelium (RPE) - thin layer behind neural retina, nourishes rods and cones, absorbs stray light

    • Optic nerve - transmits nerve impulses from the photoreceptor cells to brain

    • Optic disc - where the optic nerve exits the eye, blindspot where there are no photoreceptor cells

  2. What are the differences between rods and cones and why do we need both?

    • rods - low light vision, in periphery of neural retina

      • rhodopsin

      • multiple rods to each bipolar cell

    • cones - bright colour vision, concentrated in fovea

      • photopsin

      • visual acuity

      • 3 varieties

      • one cone to each bipolar cell

    • detect different aspects of light

  3. What happens during the visual cycle?

    • opsin struck by photon, 11-cis-retinal isomerizes to all-trans-retinol

    • all-trans-retinol diffuses away from Opsin, conformation change sends signal to brain

    • all-trans-retinal gts recycled back into 11-cis-retinal by RPE65 and re-used

  4. Describe LCA Type 2. What gene is affected and what are the challenges of this disorder?

    • LCA 2 - Leber Congenital Amaurosis

      • early vision loss

      • complete blindness by 3rd decade

      • rods more strongly affected than cones

    • LOF of RPE65

  5. What are the steps for making the Luxturna gene therapy?

    Build gene construct

    • replace AAV genome with cDNA of human gene

    • add strong enhancer-promoter combo

    • add ITRs that flank elements above

    Tissue culture - grow 10^10 viral particles

    Inject purified viral particles carrying construct into patient

    • close to target cells

    • AAV genome construct creates circular episome

    • episome is replicated by helper virus replication proteins (Adenovirus)

  6. What happens to Luxturna inside the patient’s cells?

    • AAV genome construct creates circular episome

    • episome is replicated by helper virus replication proteins (Adenovirus)

    • normal protein is made to replace defective protein

  7. What are the factors that made Luxturna a success story?

    • non-proliferative

    • patients are willing to go through treatment

    • no serious side effects in human trials

    • caused by LOF RPE65 - introducing functional gene is effective

    • if treated early, can preserve rod cells

  8. Describe the effects of heterozygous PAX6 mutation on the eye.

    • leads to aniridia

      • small/missing iris

      • opaque cornea and/or lens

      • under-developed fovea and optic nerve

  9. Who might benefit from taking Ataluren

    • those with missense mutations

      • premature stop codon

  10. What is the hypothesis for how Ataluren works?

    • makes ribosomes “ignore” premature stop codon by reading UGA as Trp, Arg, or Cys