ALN-TTR, an RNAi Therapeutic for the Treatment of Transthyretin-Mediated Amyloidosis
Rene Alvarez, Ph.D.
TTR-Mediated Amyloidosis (ATTR) ProgramALN-TTR
RNAi to treat significant orphan disease
Transthyretin (TTR)-mediated amyloidosis (ATTR)» Caused by mutation in TTR gene
» Amyloid deposits in nerves and heart
– Familial Amyloid Polyneuropathy (FAP)
– Familial Amyloid Cardiomyopathy (FAC)
» ~50,000 patients with significant morbidity and mortality
Clinical pathology » Typical onset ~40 to >60 yr
» Fatal within 5-15 years
» Loss of autonomic function, painful neuropathy
» Congestive heart failure
Liver transplant current standard-of-care for
subset of FAP patients
Initiate Phase I trial H1, 2010
22
RNA Interference
mRNA
Target-specific
mRNA degradation
dsRNAdicer
Cleavage
Strand separation
Complementary pairing
Cleavage
(A)n
(A)n
Natural Process of RNAi
Synthetic siRNA
Reduced protein
expression
RNA-induced
silencing complex
3
Systemic RNAi DeliveryLipid Nanoparticles
Unformulated siRNAs are rapidly cleared from circulation Lipid nanoparticle formulations (SNALP) of siRNAs prolong half-life and enable hepatic deliverySystemic administration of SNALP-formulated siRNAs results in» Dose-dependent mRNA and protein
suppression of hepatocyte-expressed disease targets
» Suppression maintained for 2 to 4 weeks» Has been demonstrated in multiple species
(rodents, non-human primate) for multiple targets (apoB, TTR, Factor VII, PCSK9)*
4
*Zimmerman et al., Nature, 2006; Akinc et al., Nature Biotech, 2008; Frank-Kamenetsky et al., PNAS, 2008
ATTR Program Rationale
Effective delivery of siRNA to hepatocytes with
current LNP platform
» Chemically modified TTR siRNA
» Formulated in SNALP for systemic delivery
Hepatocytes primary site of TTR expression
» Mutant and wild-type TTR proteins pathogenic
– Liver transplant can stabilize or improve V30M FAP patients
– However, cardiac disease accelerates in other ATTR
patients due to increased production of wild-type TTR
» Production of both wild-type and mutant TTR
ideally targeted
Target well validated with human genetics
» ~90% FAP caused by V30M mutation
» FAC caused primarily by V122I mutation
5
Transthyretin crystal structure
ALN-TTR siRNA Selection
6
>100 Mutations identified in TTR gene
ALN-TTR targets region of TTR mRNA common to wild-type and all
known mutant forms of TTR
Asp18Glu
Asp18Gly
Ala25Ser
Ala25Thr
Arg34Thr
Ala36Pro
Trp41Leu
Gly47Ala
Gly47Arg
Gly47Glu
Gly47Val
Ser50Arg
Glu54Gln
Glu54Gly
Leu58Arg
Leu58His
Thr60Ala
Phe64Leu
Phe64Ser
Tyr69His
Tyr69Ile
Val71Ala
Asp74His
Ile84Asn
Ile84Ser
His90Asn
Gln92Lys
Gly101Ser
Arg104Cys
Arg104His
Ala109Thr
Ala109Val
Ala109Ser
Ser112Ile
Tyr116Ser
Thr119Met
Gly6Ser
Leu12Pro
Val20Ile
Pro24Ser
Val28Met
Phe33Cys
Phe33Ile
Phe33Leu
Phe33Val
Lys35Asn
Asp38Ala
Glu42Asp
Glu42Gly
Ala45Asp
Ala45Ser
Ala45Thr
Thr49Ala
Thr49Pro
Glu51Gly
Gly53Glu
Leu55Arg
Leu55Pro
Leu55Gln
Thr59Lys
Glu61Lys
Ile68Leu
Lys70Asn
Ile73Val
Ser77Phe
Ser77Thy
Glu89Gln
Glu89Lys
Ala97Gly
Ala97Ser
Pro102Arg
Ile107Val
Leu111Met
Tyr114Cys
Tyr114His
Ala120Ser
Pro125Ser
Val122Ala
Val122IleVal122 (-)Cys10Arg
Ser23Asn Phe44Ser
Ser52Pro
Ala91Ser Thr118Met
Val30Ala
Val30Gly
Val30Leu
Val30 Met
5’ UTR
3’ UTR
Durable Suppression of TTR In Vivo
Durable reduction of TTR mRNA with rodent TTR siRNA analog Single i.v. infusion of rodent TTR analog or control siRNA; 1 mg/kg dose
Liver mRNA levels measured on Days 3, 8, 11, 15, 19, 22, 25 and 29 post-dose
7
** p < 0.01, *** p < 0.001(one-way ANOVA, Bonferroni post- hoc test)
** *
***
** **
0 1 3 8 11 15 19 22 25 290.0
0.2
0.4
0.6
0.8
1.0
1.2
Days after administration
Rela
tive T
TR
mR
NA
Level
(no
rmalized
to
day 0
)
Control siRNA
Rodent TTR analog
ALN-TTR Reduces TTR mRNA Non-Human Primates
ALN-TTR shows dose dependent silencing of TTR mRNASingle i.v. infusion of ALN-TTR or control siRNALiver mRNA levels measured 48 hr post-dose
8
ALN-TTR
(mg/kg)
1.0Control siRNA
(mg/kg)
3 0. 3 3.0
*** ***
***
47% 62%
82%TT
R/G
AP
DH
mR
NA
(Rela
tive
to
Con
tro
l)
0.0
0.2
0.4
0.6
0.8
1.0ED50 ~ 0.3 mg/kg
*** p< 0.001 (one-way ANOVA, Dunn’s post-hoc test)
ALN-TTR
0.3 mg/kg
1 mg/kg
3 mg/kg
Day 0 1 2 3 4 5 7 10 14
Durability of Reduction of Wild-Type TTR In Vivoin NHP
9
Control siRNA 3 mg/kg
ALN-TTR Silences Mutant Human TTRV30M TTR Transgenic Mouse Model
ALN-TTR silences human V30M TTR mRNA and suppresses mutant protein levelsSingle i.v. dose of ALN-TTR or control siRNA
Liver mRNA and serum TTR levels measured 48 hr post-dose
10
ED50 ~ 0.15 mg/kg
Liver mRNA
TT
R/G
AP
DH
mR
NA
(rela
tive t
o c
on
tro
l siR
NA
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
3
Control
siRNA
(mg/kg)
0.03
***
0.3
ALN-TTR
(mg/kg)
***
3
Serum Protein
TT
R s
eru
m l
evels
(rela
tive t
o c
on
tro
l siR
NA
)
ALN-TTR
(mg/kg)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0.03
***
0.3
***
33
Control
siRNA
(mg/kg)
*** p< 0.001 (one-way ANOVA,
Dunn’s post-hoc test)
*** p< 0.001 (one-way ANOVA,
Dunn’s post-hoc test)
ALN-TTR Silencing is DurableV30M TTR Transgenic Mouse Model
ALN-TTR efficacy is both rapid and durable
Single i.v. bolus of ALN-TTR or control siRNA; 1 mg/kg
Liver mRNA and serum protein levels measured on Days 3, 8, 15 and 22 post-dose
11
TT
R/G
AP
DH
mR
NA
(rel
ativ
e to
Co
ntr
ol
siR
NA
)
Control
siRNA
Day 3
TT
R S
eru
m P
rote
in
(rel
ativ
e to
co
ntr
ol
siR
NA
)
ALN-TTR ALN-TTR
Day 8 Day 15 Day 22
Liver mRNA
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6Serum Protein
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Control
siRNA
Day 3 Day 8 Day 15 Day 22
Therapeutic Efficacy for ALN-TTR
V30M TTR Transgenic Mouse Model
ALN-TTR blocks pathogenic accumulation of mutant human TTR in peripheral tissuesMulti-dose i.v. bolus of ALN-TTR or control siRNA, 3 mg/kg (d0, 14, 28)Quantitation of TTR deposition by immunohistochemistry on day 56
12
0
5
10
15
20
25
30
Mea
n T
TR
IHC
In
ten
sity
Esophagus Colon Sciatic
nerve
Dorsal root
ganglionStomach
Control siRNAALN-TTR
Collaboration with M. Saraiva
8 weeks
ALN-TTR
or control siRNA
16 weeks
Therapeutic Efficacy for ALN-TTR
V30M TTR Transgenic Mouse Model
13
Marked reduction of mutant TTR in tissues associated with human diseaseMutant V30M TTR quantified with immunohistochemistry
ALN-TTR
IntestineStomachSciatic NerveDorsal Root Ganglion
control
siRNA
ALN-TTR Therapeutic Efficacy V30M TTR Transgenic Mouse Model
ALN-TTR promotes regression of pathogenic mutant human TTR deposits in peripheral tissues>90% Regression of existing V30M hTTR tissue deposits
Multi-dose IV bolus of ALN-TTR01 or control siRNA, 3 mg/kg (week 0, 2, 4, 6, 8, 10)
Quantitation of TTR deposition by immunohistochemistry (week 11)
14
21 months
ALN-TTR
or control siRNA
24 months
ALN-TTR01
Control siRNA
0
10
20
30
40
50
60
70
Sciatic
nerve
Dorsal root
ganglion
Esophagus Colon Stomach
Rela
tive T
TR
Tis
su
e L
evels
Control siRNAALN-TTR
Dorsal Root Ganglion
100%
98.8%
98.5%
97%
98.8%
TTR-Mediated Amyloidosis (ATTR) Program Summary
We have identified a lead siRNA targeting wild-type and all mutant
forms of TTR with an IC50 of 3 pM in hepatocyte cell lines
ALN-TTR reduced TTR mRNA levels (ED50 ~ 0.3 mg/kg) in non-
human primate, with significant reduction of TTR serum protein levels
and durability of suppression >14 days post-single administration
ALN-TTR reduced mutant TTR mRNA and plasma protein levels >
90% in the hTTR V30M transgenic mouse model, with suppression
lasting > 21 days post-single administration
ALN-TTR, when administered to hTTR V30M transgenic mice,
prevents TTR deposition (young mice) and leads to regression (old
mice) of pre-existing TTR deposits in key target tissues, including:
dorsal root ganglia, sciatic nerve, stomach, and intestines
These findings demonstrate the potential therapeutic benefit of an
RNAi therapeutic targeting TTR for the treatment of TTR-mediated
amyloidosis (ATTR)
15
TTR TeamProgram Advisors and Extended Team
Victor KotelianskiDave KonysStu PollardStu MilsteinGreg HinkleRichard DuncanTuyen NguyenGang WangAkin AkincMartin MaierMara BrobergWill CantleyKlaus Charisse Jason CostiganPei GeMartin Maier Rachel MeyersKathleen Mills Jonathan O'SheaChang PengBill QuerbesJeffrey Rollins Sarfarz ShaikhChristopher SherrillMarjie SolomonSergey Shulga-MorskoyMark TracyLigang ZhangAbby CapobiancoFrances WongMark TracyTony RossomandoChunhua WangJeff KopaczCaroline Stark-BeerRebecca Howard
16
Core TeamRene Alvarez (Program Leader)
Dinah Sah (Program Leader)
Rena Denoncourt (Program Manager)
Christina Gamba-Vitalo
Jeff Cehelsky
Renta Hutabarat
Jared Gollob
Klaus Charisse
Lubo Nechev
Matthias Kretschmer
Mano Manoharan
Sara Nochur
Lauri Binne
Akshay Vaishnaw
Rick Falzone
David Watkins
Research TeamTodd Borland
Qingmin Chen
Saraiva Lab, Tekmira