Cone Targeted Gene Therapy: Animal Models

Rialto Beach, WA 2007 Which cone disease?

Targets only cones

Genetics are well established

Animal model mimics human disease

Clear clinical assay for therapy Cone genes causing Phototransduction foveal function loss

3 genes causing Achromatopsia

CNGA3 CNGB3

GNAT2

J Med Genet 2002;39:656–660 Cone genes causing Phototransduction foveal function loss

3 genes causing Achromatopsia

CNGA3 CNGB3

GNAT2

X-linked color blindness

J Med Genet 2002;39:656–660 Cone genes causing Phototransduction foveal function loss

3 genes causing Achromatopsia

CNGA3 CNGB3

GNAT2

X-linked color blindness Blue Cone Monochromacy

J Med Genet 2002;39:656–660 Cone genes causing Phototransduction foveal function loss

3 genes causing Achromatopsia

CNGA3 CNGB3

GNAT2

X-linked color blindness Blue Cone Monochromacy

J Med Genet 2002;39:656–660 Mouse model for GNAT2 Achromatopsia

Alexander, J, Umino, Y , Chang, B , Min, S , Li, Q, Timmers, A , Hawes, N, Everhart, D, Barlow, R, Pang, P and Hauswirth, WW “Restoration of Cone Function in a Mouse Model of Achromatopsia” Nature Medicine. 13:685-687 (2007).

AAV5-PR2.1-mGnat2 Treatment effects Is useful vision restored? Cone ERG ~100% of normal Cone structure ~normal Mouse model for GNAT2 Achromatopsia

AAV5-PR2.1-mGnat2 Treatment effects

Cone ERG ~100% of normal Cone structure ~normal

Visual Acuity 0.4

0.3

0.2

Visual Acuity Visual 0.1 (cycles / degree) / (cycles

0.0 Untreated Treated Wild Type Cpfl3 Eyes Cpfl3 Eyes Eyes

Nature Medicine (2007). Visual Acuity 0.4

0.3

0.2

Visual Acuity Visual 0.1 (cycles / degree) / (cycles

0.0 Untreated Treated Wild Type Cpfl3 Eyes Cpfl3 Eyes Eyes

Nature Medicine (2007). Can other genetic forms of Achromatopsia and other species with Achromatopsia be treated? Phototransduction Achromatopsia Genes

CNGA3 CNGB3

X-linked color blindness Blue Cone Monochromacy

J Med Genet 2002;39:656–660 J Med Genet 2002;39:656–660 Dog model for CNGB3 Achromatopsia

Komáromy AM, Alexander JJ, Rowlan JS, Garcia MM, Chiodo VA, Kaya A, Tanaka JC, Acland GM, Hauswirth WW, Aguirre GD. “Gene therapy rescues cone function in congenital achromatopsia” Hum Mol Genet. 19:2581-2593 (2010).

AAV5-PR2.1-hCngb3 Treatment effects Cone ERG ~10% of normal, but 50% in Tx area Cone structure ~normal in Tx area > 3 year persistence Dog model for CNGB3 Achromatopsia

AAV5-PR2.1-hCngb3 Treatment effects Cone ERG ~10%Behavioral of normal, Effects? but 50% in Tx area Cone structure ~normal in Tx area > 3 year persistence Visually guided maze test for dogs (n=6)

Dog Movie

Hum Mol Genet (2010). Visually guided maze test for dogs (n=6)

10 Transit Time (sec) Time Transit

0 Normal noTx Tx Hum Mol Genet (2010). Visually guided maze test for dogs (n=6)

10 Transit Time (sec) Time Transit

0 Normal noTx Tx Hum Mol Genet (2010). Cone genes causing Phototransduction foveal function loss

3 genes causing Achromatopsia

CNGA3 CNGB3

GNAT2

X-linked color blindness Blue Cone Monochromacy

J Med Genet 2002;39:656–660 Sheep model for CNGA3 Achromatopsia

Edward Averbukh; Ron Ofri; Elisha Gootwine; Raaya Ezra-Elia; Hen H. Honig; Alexander Rosov ; Esther Yamin; Alexey Obolensky; William W. Hauswirth; Eyal Banin ARVO (2013)

AAV5-PR2.1-hCnga3

Treatment effects Cone ERG ~30% of normal, but ~100% in Tx area Cone structure ? Sheep model for CNGA3 Achromatopsia

Edward Averbukh; Ron Ofri; Elisha Gootwine; Raaya Ezra-Elia; Hen H. Honig; Alexander Rosov ; Esther Yamin; Alexey Obolensky; William W. Hauswirth; Eyal Banin ARVO (2013)

AAV5-PR2.1-hCnga3

Treatment effects Behavioral Effects? Cone ERG ~30% of normal, but ~100% in Tx area Cone structure ? Visually guided maze test at 6 Mos. postTx

N = 6

ARVO (2013) Visually guided maze test at 6 Mos. postTx

N = 6

ARVO (2013) Visually guided maze test at 6 Mos. postTx

N = 6

ARVO (2013) Cone genes causing Phototransduction foveal blindness

3 genes causing Achromatopsia

CNGA3 CNGB3

GNAT2

X-linked color blindness Blue Cone Monochromacy

J Med Genet 2002;39:656–660 Mouse Model of BCM The thyroid hormone receptor KO mouse (THrβ KO) has no detectible M- opsin, no recordable M-cone response to red light and a normal S-cone function. It is therefore a mouse model of BCM. AAV8-IRBP/GNAT2-rat M-opsin M-cone function (photopic red ERG)

Normal mouse Treated THrβ KO mouse Untreated THrβ KO mouse 70uV 25uV 5uV 36% of normal 7% of normal Are M-cones restored in treated THb KO mice? M-opsin cones are Red S-opsin cones are Green

No vector Vector No vector Vector

No vector Vector THrb KO mouse treatment effect – retinal whole mounts

Normal

45% of cones M-cones are red have M-opsin All cones are Green

Tx THrb KO THrb KO

24% of cones 0% of cones have M-opsin Have M-opsin Cone genes causing Phototransduction foveal blindness

3 genes causing Achromatopsia

CNGA3 CNGB3

GNAT2

XX--linkedlinked colorcolor blindnessblindness Blue Cone Monochromacy

J Med Genet 2002;39:656–660 Primate model of human red color blindness

Dalton

Jay and Maureen Neitz, Med. Coll. Wisc. Color Vision Testing Apparatus

Cambridge Colour Test

The color vision threshold defines how saturated each hue must be to distinguish it from gray Gene Therapy for X-Linked Color Blindness (Protanopy)

Mancuso, K, Hauswirth, WW, Li, Q, Connor, TB, Kuchenbecker,JA, Mauck, MC, Neitz, J and Neitz, M “Gene therapy for red-green colour blindness in adult primates” Nature, 461:784-787 (2009). PMID: 19759534

AAV5-PR2.1-hR-Ops

Treatment effect Gain of ~normal red light ERG response (but only in treated area) Squirrel Monkey #1 Thresholds

490nm (protan confusion l)

550nm (normal response l)

Nature (2009) Squirrel Monkey #1 Thresholds

490nm (protan confusion l) Cortical “learning”?

550nm (normal response l)

GeneAny othertherapy example treated of RPE65 a slow LCA response patients only start tousing retinal the gene treated therapy? part of their retina after ~one year. Nature (2009) Why develop a treatment for red color blindness?

AAV5-PR2.1-hR-Ops

Blue Cone Monochromacy patients are missing both R-Ops and G-Ops. They have clinical symptoms very similar to Achromats. Cone Targeted Gene Therapy Species Disease Gene Cone ERG Vision Mouse Achromo. gnat2 normal ~normal Achromo. cnga3 90% normal ~normal Achromo. cngb3 50% normal ?? Dog Achromo. cngb3 10% normal ~normal Sheep Achromo. cnga3 30% normal ~normal Rat BCM M-opsin 20% normal ?? Mouse BCM TRb2 36% normal ?? Monkey Protanopia L-opsin ~normal ~normal

Rialto Beach, WA 2007 ConeWhat’s Targeting next AAV forVectors cone targeted gene therapy? Qui-Hong Li Mouse Cone Therapy Vince Chiodo John Alexander LucyRecently Glushakova funded NEI R24 forWentao B3 Achm. Deng IND/clin. trial Bo Chang, Norm Hawes (JAX Labs)  Corporate support for B3 andBob A3 Barlow Achm. (Syracuse) to parallel R24 Monkey Cone Therapy (Med. Coll. Wisconsin) Jay Neitz Maureen Neitz Dog Cone Therapy Katherine Mancuso Andras Komaromy (Penn) Gus Aguirre (Penn) Greg Acland (Cornell) John Alexander

Rialto Beach, WA 2007 * Light from Astronomic star Adaptive Optics Retinal Adaptive Optics A New Adaptive Optics Technology Split Detector AO

Conventional AO

J. Carroll, 2014, pers. Comm. Conventional AO Split-detector AO

Control

ACHM

J. Carroll, 2014, pers. Comm. Achromatopsia Patients OCT Images JC10089 Conventional AO JC10069

KC10088

JC10028

JC10089

J. Carroll, 2014, pers. Comm. Split Detector AO Supported by

NEI FFB MVRF RPB Achroma Corp. BCM Families Fdn. Thanks Gerstein Fund Overstreet Fund

Mac Stone Suwannee River Florida, 2010