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
Nai
ve
Vehic
le
ADVM
-062
0
100
200
300
Am
plitu
de (µ
V)
n.s.
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ADVM
-062
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V) n.s.
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-062
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Am
plitu
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V)
***
Nai
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-062
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150200300
Am
plitu
de (µ
V)
***
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. [email protected]
