Long%term*functional*delivery*of*the*human*L%opsin*cDNA*via*intravitreal*administration*

of*an*AAV*vector*in*Mongolian*gerbilsDiana%Cepeda,%Jim%Ver Hoeve,%Ming%Ni,%Julio%Nieves,%Edward%Yeh,%Annahita Keravala,%Angelica%Phillips%and%Mehdi%GasmiAdverum Biotechnologies2Inc.,2Menlo2Park,2CA,2USA

#*5696

Background Purpose

Study*Design*and*Methods

Results*&*Conclusion

Disclosures* Contact*Information

• Blue%cone%monochromacy%(BCM)%is%a%rare%XGlinked%visual%

disorder%that%affects%of%1/100,000%individuals,%with%a%high%

unmet%medical%need%for%treatment.

• BCM%causes%severe%color%vision%deficiency,%poor%visual%acuity,%

nystagmus%and%photophobia,%resulting%from%the%deficiency%in%

the%longGwave%(L)%and%mediumGwave%(M)%opsin%pigments%in%

cone%photoreceptor%cells%in%the%retina.%

• AAVGmediated%gene%therapy%using%intravitreal%(IVT)%

administration%is%one%of%the%most%promising%and%minimally%

invasive%delivery%systems%to%treat%retinal%diseases.

• AAV.7m8%is%a%variant%of%AAV2%discovered%by%directed%evolution%

in%the%retina.%It%contains%a%10%amino%acid%insertion%in%Loop%IV%of%

AAV2%VP3%(Dalkara et2al,%2013).

• AAV.7m8%efficiently%transduces%photoreceptors%when%delivered%

by%IVT%injection.

• The%regulatory%cassette%MNTC%was%engineered%to%drive%efficient%

and%specific%gene%expression%in%LG and%MGcone%photoreceptors.

• MNTC%confers%more%pronounced%and%abundant%reporter%

expression%in%cones%in%nonhuman%primate%retina%transduced%by%

IVT%injection,%as%compared%to%the%wellGcharacterized%coneG

specific%PR2.1%promoter.

Fovea Mid%periphery Periphery

Rho*L/M%opsin GFP DAPI10x 20x

L/M%Opsin*GS GFP DAPI Calbindin L/M%Opsin*GFP DAPI10x

In#vivo#Fundus#Imaging

Immunofluorescence#staining#of#NHP#retina#tissue#transduced#with#AAV.7m8>

MNTC>GFP

LCR IntronUTRMTNC KOZs OPSIN pAlg

AUG

MP U U

MNTC#Cassette

Robust*Cone%Specific*Expression*Mediated*by*IVT*Administration*of*

AAV.7m8%MNTC%GFP*in*Nonhuman*Primate*Retina

OD OS

AAV.7m8%MNTC

AAV.7m8%PR2.1

OD OS

12%weeks

Dose:%5E11%vg/eye

• Adverum%is%developing%ADVMG062%as%an%intravitreal%gene%

therapy%vector%to%treat%blue%cone%monochromacy.

• ADVMG062%is%an%AAV.7m8%capsid%vector%containing%human%LG

opsin%(OPN1LW)%cDNA%under%the%control%of%the%pMNTC cassette%

to%drive%coneGspecific%gene%expression.%

• The%aim%of%the%study%was%to%assess%whether%IVT%delivery%of%

AAV.7m8GMTNCGLOpsin%gene%(γpeak =%560%nm)%in%the%Mongolian%

gerbil%could%translate%into%a%functional%ERG%readout.

Hypothetical*ADVM%062*Treated*Gerbil

4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

W ave le n g th 3(nm )

Absorption

Gerbil%blue/green%opsin

ADVMG062%LGopsin

• Mongolian%gerbils%were%used%as%a%model%for%BCM%in%that%their%

retina%contains%11G14%%cone%cells%with%peak%light%absorbance%in%

the%short%and%medium%wave%(UVGblue/green)%spectral%regions.%

Gerbils%lack%longGwave%(red)%sensitive%cone%visual%pigment.

• Animals%were%injected%IVT%with%a%single%3E11%vg/5%μl dose%of%

ADVMG062,%or%formulation%buffer%in%both%eyes%(n=4%animals).%

• Ocular%examinations%were%performed%before%dosing%and%

throughout%the%study.

• Functional%expression%of%human%LGopsin%was%evaluated%using%

fullGfield%color%electroretinogram%(cERG)%with%light%emitting%

diodes%of%different%wavelengths,%at%different%intensities%and%

frequencies,%for%over%1.5%years.%Exposure%to%green%light%prior%to%

and%during%cERG%measurements%was%used%to%reduce%MGcone%

contribution to%long%wavelength%light%responses.

• Histopathology,%immunofluorescence%staining%and%mRNA%

expression%were%performed%on%eye%tissues%and%brain%at%

termination,%90%weeks%postGtreatment.%

cERG ParametersBlue*

(440nm)

Green*

(513*nm)

Red*

(630*nm)

Far*Red*

(680*nm)

Flash Intensity 0.1%cd.s/m2 0.5%cd.s/m2 3.0%cd.s/m2 1.0%cd.s/m2

Flash*Frequency 1 Hz 1%Hz 1%Hz 1 Hz

Green Background 30%cd.s/m2 30%cd.s/m2 30%cd.s/m2 30%cd.s/m2

• IVT%injection%of%ADVMG062%was%well%tolerated%based%on%bright%field%ocular%exams%and%histopathology%of%ocular%tissue.

• Increased%cERG responses%to%red%and%farGred%light%%were%observed%from%week*14 and%up%to%week*90 following%%ADVMG062%IVT%

administration.

Conclusion*and*Future*Directions

cERG Recordings*in*Green%Light*Adapted*Gerbil*Eyes

Top:#Representative2cERGs tracings2of2

naïve2control,2vehicleCtreated2and2

ADVMC062Cdosed2gerbils,2at2862weeks2

postCdosing.2Arrows2point2to2the2coneC

driven2bCwave2elicited2by2red2and2farCred2

light2sources2observed2in2ADVMC062C

treated2gerbils,2which2is2not2observed2in2

control2eyes.2

Bottom:#Scatter2and2bar2plots2of2the2

mean2bCwave2amplitude2from2a2total2of2

82eyes2per2group.2Error2bars:2SEM.2

Significance2***:2p<0.0012(nonC

parametric2WilcoxonCMannC

Whitney test).

• Our%data%indicates%that%ADVMG062%produces%functional,*long%term LGopsin%expression%following%single intravitreal*delivery%in%

Mongolian%gerbil%eyes.%

• Future%validation%studies%in%a%mouse%model%of%BCM%will%be%performed%to%investigate%the%functional%state%of%photoreceptors%

that%do%not%express%opsin.

• Electrophysiology%and%behavioral%studies%in%nonGhuman%primates%will%aim%at%determining%if%the%ERG%signal%elicited%by%the%

longGwave%light%sources%is%transmitted%to%the%visual%cortex.%

Blue*(440nm) Green*(513nm) Red*(630nm) Far%Red*(680nm)

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Mean%bGwave%

amplitude

Naive

Vehicle

ADVM%062

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ve

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le

ADVM

-062

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plitu

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***

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***

L%Opsin*protein*is*expressed*in*cone*cells*of*retinas*treated*with*ADVM%062,*and*is*not*detected*in*brain*tissue

Vehicle

L%opsin*

PNA

DAPI*

ADVM%062 ADVM%062,*zoomed

A.#Immunofluorescence2labeling2of2gerbil2retinal2tissue2sections2902weeks2post2IVT2dosing.2CoClabeling2of2LCopsin2with2peanut2agglutinin2lectin2(PNA)2confirms2ADVMC

0622expression2in2cone2photoreceptors.2B. RTCPCR2of2human2LCopsin2and2gerbil2GAPDH2in2retina2and2brain2samples.

A. B.

Naïve

Vehicle

ADVM

G062

Retina Brain

Naïve

Vehicle

ADVM

G062

L%opsin

GAPDH

DC,%JN,%EY,%AP,%and%MG%are%employed%by%Adverum Biotechnologies,%Inc. dcepeda@adverum.com