1. Ryan Smith October 20, 2008

2.  Rare inherited eye disease (1 in 80,000)  Symptoms first occur in early infancy ◦ Irregular behavior, nystagmus  Progresses through time resulting in total blindness by 3 rd or 4 th decade of life  No treatment (until now?...)  Described by Theodore Leber in 19 th Century  Amaurosis – Vision loss without any lesions  Congenital – Not acquired, present at birth 2

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4. Cell Virus DNA of Interest Nucleus Insert DNA into genome Functional Protein 4

5.  Common human virus (80%)  Not known to cause a disease ◦ Very mild immune response  Advantages ◦ Integrates into the same place in the genome nearly every time ◦ Elicits no clear cytotoxic response ◦ Actually shown to fight cancer 5

6.  Disadvantages ◦ Cannot hold a lot of DNA in the head ◦ Efficiently packing it can require removing the viral DNA  This may cause it to not integrate in the proper location 6

7.  Identified three subjects (1 male, 2 female) age 19 to 26 with LCA2 and profound vision loss.  Put a cDNA of RPE65 gene with a chicken beta actin promoter into an AAV vector.  Injected AAV2.hRPE65v2 into subretinal tissue under general anesthesia. ◦ Only in one eye (the worse based on objective and subjective measurement) – Right in all three ◦ Other used as a control 7

8.  Patients’ vision and health evaluated before and after surgery  Subjective Vision tests: ◦ Pupillary light reflex ◦ Nystagmus testing  Objective Vision tests: ◦ Visual Acuity tests ◦ Goldmann visual-field examination ◦ Ability to navigate an obstacle course 8

9.  Injection caused localized retina detachment  Reattached 14 hours after surgery  All patients showed unremarkable retinas after surgery ◦ Patient 2  developed outer lamellar cyst in fovea  Also developed a macular hole 9

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11.  Vector DNA only found in tear of patient 1 on day 1 after surgery  Vector was not observed to spread through the body  No humoral or cell-mediated immune response in any of the patients  Neutralizing antibody titers to AAV2 capsid observed in Patient 2, but went away over time 11

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14.  All patients showed an increased light sensitivity  The injected eye was approximately three times as sensitive to light than at baseline  The injected eye drove the light response in both eyes  In each situation the less functional eye became better than the previously better functioning eye 14

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17.  Patient 1 ◦ HM -> 20/1050 (P<0.001)  Patient 2 ◦ HM -> 20/710 (P<0.001)  Patient 3 ◦ 20/640 -> 20/290 (P=0.002)  Visual Field size increased  Nystagmus was reduced  Ability to navigate obstacle course improved (for patient 2) 17

18.  All patients showed improvement for 6 weeks, then improvement slowed (did not go away)  Reduction in nystagmus may account for improvement in uninjected eye  No apparent local or systemic effects of AAV 18

19.  No inflammation or acute retinal toxicity  Could be contraction of a preexisting membrane stimulated by the surgery ◦ Could be caused by the surgery  No reported vision loss by the patient ◦ Definitely would cause vision loss to normally functioning retina 19

20.  These three patients will continue to be monitored  This was first of three experiment groups involving 9 people ◦ First group got a small dose ◦ Second group gets a medium dose ◦ Third group gets a large dose  Possible improved response if patients are treated in childhood 20

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