Articles
https://doi.org/10.1038/s41587-022-01334-x
1
Alnylam Pharmaceuticals, Cambridge, MA, USA. 2
These authors contributed equally: Kirk M. Brown, Jayaprakash K. Nair, Maja M. Janas.
✉e-mail: mmaier@alnylam.com; vjadhav@alnylam.com
R
NA interference (RNAi) therapeutics use an endogenous
mechanism whereby short interfering RNAs (siRNAs) direct
the RNA-induced silencing complex (RISC) to sequence
matchedtargettranscriptsforknockdown1
.Bothlipidnanoparticles
and N-acetylgalactosamine (GalNAc) conjugates are clinically validated
and approved delivery strategies for liver targets2–8
. Building
on nearly 2 decades of siRNA design and chemistry optimiza-
tion9–12
, we demonstrate here that, with suitable delivery solutions,
the RNAi pathway can be harnessed in extrahepatic tissues, such as
the central nervous system (CNS), eye and lung. Multiple CNS diseases,
representing some of the highest unmet medical needs and
greatest therapeutic challenges, have been associated with dominant
gain-of-function mutations, making them suitable candidates
for an RNAi-based silencing approach. As such, chemically modified
siRNAs have demonstrated potent and sustained silencing
in rodents and non-human primates (NHPs); however, using an
invasive intracerebroventricular (ICV) administration approach13
that is not suitable for repeated dosing in humans. Furthermore,
technologies enabling siRNA delivery across the blood–brain barrier
following a less challenging systemic administration are similarly
being explored14–17
, which are, however, still in early stages of
discovery. In the eye, intravitreal (IVT) dosing of siRNAs has been
evaluated in late-stage clinical studies, with few safety concerns,
but did not advance further due to lack of efficacy18
. Recently, the
Coronavirus Disease 2019 (COVID-19) pandemic has highlighted
the importance of optimizing siRNA delivery to the lung for the
treatment of emergent viral respiratory diseases. Although earlier
programs have already shown potential clinical benefits of
siRNA-based therapeutics in the lung19
, 2′-O-hexadecyl (C16)
conjugates demonstrate enhanced delivery and siRNA uptake into
the alveolar and bronchiolar epithelium. Taken together, this work
highlights that the combination of a C16 lipophilic modification
with our fully chemically modified, metabolically stable siRNAs
achieves efficient delivery to the CNS, eye and lung, resulting in
a robust and durable gene silencing in rodents and NHPs, with
a favorable safety profile. We think that these advances have the
potential to generate multiple candidates for investigating clinical
safety and efficacy in humans.
Results
Optimization of the siRNA conjugate design. Lipophilic moieties
represent one of the earliest approaches to improve cellular uptake
and delivery of antisense oligonucleotides (ASOs) and siRNAs to
the liver and various other organ systems20
, including the CNS21–23
.
We reasoned that, by carefully optimizing the lipophilicity of chemically
modified siRNAs, we could enhance intracellular delivery
without compromising broad biodistribution, potency and safety.
We used the 2′ position of the ribose sugar backbone to introduce
Expanding RNAi therapeutics to extrahepatic
tissues with lipophilic conjugates
Kirk M. Brown1,2
, Jayaprakash K. Nair1,2
, Maja M. Janas1,2
, Yesseinia I. Anglero-Rodriguez   1
,
Lan T. H. Dang1
, Haiyan Peng1
, Christopher S. Theile1
, Elena Castellanos-Rizaldos1
, Christopher Brown1
,
Donald Foster1
, Jeffrey Kurz1
, Jeffrey Allen1
, Rajanikanth Maganti1
, Jing Li   1
, Shigeo Matsuda1
,
Matthew Stricos1
, Tyler Chickering1
, Michelle Jung1
, Kelly Wassarman1
, Jeff Rollins1
, Lauren Woods1
,
Alex Kelin1
, Dale C. Guenther1
, Melissa W. Mobley1
, John Petrulis1
, Robin McDougall1
, Timothy Racie1
,
Jessica Bombardier1
, Diana Cha1
, Saket Agarwal   1
, Lei Johnson1
, Yongfeng Jiang1
, Scott Lentini1
,
Jason Gilbert1
, Tuyen Nguyen1
, Samantha Chigas1
, Sarah LeBlanc1
, Urjana Poreci1
, Anne Kasper1
,
Arlin B. Rogers1
, Saeho Chong1
, Wendell Davis1
, Jessica E. Sutherland1
, Adam Castoreno1
,
Stuart Milstein1
, Mark K. Schlegel   1
, Ivan Zlatev   1
, Klaus Charisse1
, Mark Keating1
,
Muthiah Manoharan   1
, Kevin Fitzgerald1
, Jing-Tao Wu1
, Martin A. Maier   1 ✉ and Vasant Jadhav   1 ✉
Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions
are needed to target additional organs. Here we show that conjugation of 2′-O-hexadecyl (C16) to siRNAs enables safe,
potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad
cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS
regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration
to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of
an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of
Alzheimer’s disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs
has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.
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ArticlesNatureBiotechnology
the lipophilic moiety, as it provides many options for the positioning
of the lipophile within the siRNA duplex.
The lipophile was introduced at the N6 (position 6 from the 5′
end) of the sense strand, and the effect of its chain length was evaluated
in the rat CNS using a superoxide dismutase 1 (Sod1)-targeting
siRNA (Supplementary Table 1). The RNAi activity of siRNA conjugates
in the CNS generally increased with lipid chain length, with
C16 providing optimal activity in the rat spinal cord and brain at
2 weeks after an intrathecal (IT) dose (Fig. 1a). To determine the
positional effect, C16 was walked across both strands of the siRNA
and screened via transfection in RPE-J cells (Fig.1b). The C16 conjugation
was well-tolerated across both strands, with potency similar
to the unconjugated siRNA, except for a few positions known to be
sensitive to sterically demanding 2′ modifications9
. A subset of the
C16-siRNAs was selected for in vivo evaluation in rats via a single
0.9-mg IT dose. Most tested positions provided activity equivalent
to the sense strand N6 after 1 month (Fig. 1c), confirming positional
tolerance of C16 modification. In vitro–in vivo discrepancy was
observed with the antisense strand N16 conjugate, potentially due
to a metabolic liability in vivo that affected the duration. The sense
strand N6 was chosen to maintain functional RISC activity and to
reduce the risk of exonuclease-mediated cleavage of the conjugate
from termini.
Next, we confirmed that known siRNA design elements, such
as the strategic placement of 2′-fluoro and 2′-O-methyl chemical
modifications to increase potency9
, or glycol nucleic acid (GNA) in
the antisense seed region to increase specificity10,24
, retained similar
benefits in the CNS (Fig. 1d). Furthermore, we assessed the relative
importance of C16 and/or 5′-(E)-vinylphosphonate (VP), a 5′
phosphate mimic placed at the 5′ end of the antisense strand to promote
RISC loading25
, on potency (Fig. 1e). Although the addition
of either C16 or VP produced a robust mRNA knockdown in the
spinal cord (up to 80%), only a modest knockdown in the brain (up
to 50%) was observed. The siRNA combining both VP and C16 had
the best activity across CNS regions, with up to 90% and 75% mRNA
knockdown in the spinal cord and brain, respectively. There was no
activity in the rat striatum, liver and kidney at the tested dose level
and time point. Consistent with the functional knockdown data
across CNS regions, the C16-siRNA demonstrated superior brain
biodistribution compared to its unconjugated version after a single
IT injection in rats (Fig. 1f).
C16 conjugation enables broad and efficient siRNA delivery
to the rodent CNS. To understand C16-siRNA uptake and RNAi
activity across the major CNS cell types, we designed siRNAs
against cell-type-specific targets uniquely expressed in neurons
(Map2), astrocytes (Gfap), microglia (Iba1) or oligodendrocytes
(Mbp) and the endothelium/perivascular macrophages (Pecam1)
(Supplementary Table 1). After an IT dose in rats, immunohistochemistry
(IHC) to detect siRNAs revealed widespread drug
distribution in neurons and glial cells (Fig. 2a). Dual IHC confirmed
cell-specific uptake in neurons, astrocytes and microglial cells
(Fig. 2a), whereas siRNA accumulation in oligodendrocytes was
below the IHC limit of detection (Fig. 2a). Robust knockdown was
observed in neurons, astrocytes and microglia (Fig. 2b), whereas it
was less effective in oligodendrocytes, consistent with lower siRNA
exposure. Vascular endothelium and perivascular macrophages did
not show functional RNAi activity (Fig. 2b). Overall, these data
indicate that C16-siRNAs are productively taken up by most CNS
cell types of therapeutic relevance.
Knockdown of Sod1 mRNA was dose dependent in the spinal
cord and the brain (Fig. 2c). The duration of knockdown was
assessed in a 6-month study in rats after a single 0.9-mg IT dose
(Fig. 2d). In the lower spinal cord, the maximal level of target knockdown
was observed as early as 1 week post-dose (earliest time point
tested), with sustained silencing out to 6 months. Brain regions
analyzed showed >75% silencing for at least 3 months with partial
recovery out to 6 months, except for the temporal cortex where Sod1
expression started to recover at 2 months post-dose. One additional
cohort of rats received multiple doses of 0.3 mg at approximately
monthly intervals for up to five doses, with the last group killed at
2 weeks post-final-dose (4.5 months after the first dose) (Fig. 2e).
Silencing appeared to be additive, with similar Sod1 knockdown in
the frontal cortex, cerebellum and cervical spinal cord at 1 month
after three 0.3 mg doses versus a single 0.9 mg dose at the same time
point (Fig. 2d). Knockdown appeared to recover in the thoracic
spinal cord after 3 months in the multi-dose rat IT study (Fig. 2e),
which is inconsistent with the 6-month single-dose rat IT data with
the same C16-siRNA (Fig. 2d), and the other CNS regions of the
same experiment, and may be due to assay or technical variability.
Dose-dependent silencing in the frontal cortex correlated with
drug exposure (Fig. 2f), with the siRNA half-life for the 0.9 mg
group calculated to be 3–4 months. Frontal cortex exposure was
generally linear with increasing dose at all time points. Evidence of
accumulation was observed in this tissue after multiple doses, with
the siRNA exposure after the third monthly 0.3 mg dose equivalent
to a single 0.9 mg dose, with additive accumulation observed
after the fourth and fifth doses. The dose-exposure relationship of
the Sod1 C16-siRNA could be characterized across all dose levels
explored with a first-order pharmacokinetic model.
C16-siRNAs are potent and durable in NHP CNS. To understand
whether the C16 and VP siRNA modifications that enable robust
CNS activity in the rat would translate to NHPs, we used an amyloid
precursor protein (APP)-targeting siRNA (Supplementary
Table 1). After a single IT dose of 60 mg, siRNA lacking C16 as
well as VP had minimal activity in the spinal cord and no activity
in the brain at 3 months post-dose (Fig. 3a). Consistent with the
rat data, siRNA containing both VP and C16 had the best activity
across CNS regions, with up to 70% APP knockdown in the spinal
Fig. 1 | Optimization of the siRNA design for CNS delivery in the rat. a, 5′ Sod1-targeting siRNA was conjugated to lipids via 2′-O position at N6 nucleotide
of the sense strand and administered as a single IT bolus injection to rats at 0.9 mg (on the basis of siRNA sequence I; Supplementary Table 1). Spinal
cord and brain regions were collected 2 weeks post-dose for Sod1 mRNA knockdown measurement by RT–qPCR. n = 4 animals per group. b, Single
C16 modification walk across sense strand and antisense strand of siRNA and transfected into RPE-J cells for potency assessment via RT–qPCR. n = 3
biologically independent samples. c, C16 at various positions of Sod1-targeting siRNA was evaluated after a single IT bolus injection to rats at 0.9 mg for
knockdown in spinal cord and brain regions by RT–qPCR. n = 3 animals per group. d, Partially or fully modified (ESC, ESC+) 5′-VP-modified Sod1-targeting
siRNA (Supplementary Table 1) was conjugated to C16 at N6 of the sense strand and administered as a single IT bolus injection to rats at 0.9 mg. Spinal
cord and brain regions were collected 2 weeks post-dose for Sod1 mRNA knockdown measurement by RT–qPCR. n = 3 animals per group for aCSF and
partially modified; n = 5 animals per group for ESC; n = 2 animals per group for ESC+. e, Sod1-targeting siRNA was modified with VP at the 5′ end of the
antisense strand, with 2′-O-C16 at N6 of the sense strand or with both and administered as a single IT bolus injection to rats at 0.9 mg. Spinal cord, brain
regions and peripheral organs were collected at 1 month post-dose for Sod1 mRNA knockdown measurement by RT–qPCR. n = 3 animals per group. All
error bars represent standard deviation. f, Unconjugated or C16-modified Sod1-targeting siRNA was administered as a single IT bolus injection to rats at
0.9 mg, and siRNA biodistribution was assessed in whole brain at 24 h post-dose using IHC with anti-siRNA antibody. Three animals were analyzed per
group with similar results.
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Articles NatureBiotechnology
aCSF C10 C12 C14 C16 C18
0
25
50
75
100
125 Rat IT, 0.9 mg, day 14
ProportionofSod1mRNA
remainingrelativetoaCSF(%)
Lumbar spinal cord
Thoracic spinal cord
Cervical spinal cord
Cerebellum
Brainstem
Temporal cortex
Frontal cortex
Hippocampus
SS1
SS2
SS3
SS4
SS5
SS6
SS7
SS8
SS9
SS10
SS11
SS12
SS13
SS14
SS15
SS16
SS17
SS18
SS19
SS20
SS21
AS2
AS3
AS4
AS5
AS6
AS7
AS8
AS9
AS10
AS11
AS12
AS13
AS14
AS15
AS16
AS17
AS18
AS19
AS20
AS21
AS22
AS23
NoC16
0
25
50
75
100
125 RPE-J cells, 24 hours
ProportionofSod1mRNA
remainingrelativetoaCSF(%)
0.1 nM
10 nM
aC
SF
SS1
SS2
SS5
SS6
SS7
SS10
SS11
SS16
SS17
SS21
AS5
AS15
AS16
0
25
50
75
100
125 Rat IT, 0.9 mg, day 28
ProportionofSod1mRNA
remainingrelativetoaCSF(%)
aCSF ESC ESC+ Partially
modified
0
25
50
75
100
125 Rat IT, 0.9 mg, day 14ProportionofSod1mRNA
remainingrelativetoaCSF(%)
Thoracicspinalcord
Cerebellum
Frontalcortex
0
25
50
75
100
125
150
175 Rat IT, 0.9 mg, day 28
ProportionofSod1mRNA
remainingrelativetoaCSF(%)
Lumbar spinal cord
Thoracic spinal cord
Cervical spinal cord
Brainstem
Cerebellum
Hippocampus
Temporal cortex
Frontal cortex
Striatum
Liver
Kidney
C16 + + – –
aCSF
VP + – + –
Unconjugated siRNA C16-siRNA 2 mm
24 h 24 h
a
b
c d
e
f
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ArticlesNatureBiotechnology
a
b
d e
c
f
Neuron Astrocyte Microglia
Green: Map2
Purple: siRNA
Cortex
Purple: siRNA
Oligodendrocyte
Yellow: Gfap
Purple: siRNA
Yellow: Iba1
Purple: siRNA
Yellow: Mbp
Purple: Duplex
Oligodendrocytes
(note linear array)
Astrocyte
200 µm 15 µm 20 µm 10 µm 100 µm
aC
SF
G
fap
C
16-siR
N
A
(day
14)
Iba1
C
16-siR
N
A
(day
32)
M
ap2
C
16-siR
N
A
(day
28)
M
bp
C
16-siR
N
A
(day
14)
Pecam
1
C
16-siR
N
A
(day
14)
0
25
50
75
100
125
150
Rat IT, 0.9 mg
ProportionoftargetmRNA
remainingrelativetoaCSF(%)
Thoracic spinal cord
Cervical spinal cord
Cerebellum
Brainstem
Lumbar spinal cord
aCSF 0.07 mg 0.3 mg 0.9 mg
0
25
50
75
100
125
Rat IT, day 28
ProportionofSod1mRNA
remainingrelativetoaCSF(%)
Thoracic spinal cord
Cerebellum
Frontal cortex
0
25
50
75
100
125
150
175
Rat IT, 0.9 mg
Days post-dose
ProportionofSod1mRNA
remainingrelativetoaCSF(%)
Lumbar spinal cord
Thoracic spinal cord
Cervical spinal cord
Cerebellum
Frontal cortex
Temporal cortex
Hipppocampus
Liver
Kidney7 28 42 56 70 84 98 126 168
0
25
50
75
100
125
150
175
Rat IT, 0.3 mg QMx5
Days post-dose
ProportionofSod1mRNA
remainingrelativetoaCSF(%)
Lumbar spinal cord
Thoracic spinal cord
Cervical spinal cord
Cerebellum
Frontal cortex
Temporal cortex
Hipppocampus
Liver
Kidney
7 28 42 56 70 84 98 126
0 28 56 84 112 140 168
0.1
1
10
100
siRNAconcentration(µgg–1
)
Rat IT, frontal cortex
0.07 mg (single)
Days post-dose
0.3 mg (single)
0.9 mg (single)
0.3 mg (QMx5)
Fig. 2 | Characterization of the C16-siRNA in rat CNS. a, siRNA IHC demonstrating robust neuronal and glial cell drug accumulation (magenta) in cerebral
cortex (left panel). Dual IHC for the detection of siRNA and cell-type-specific targets in neurons (Map2), astrocytes (Gfap) and microglia (Iba1). siRNA
signal in oligodendrocytes (Mbp) was below the IHC detection limit. Three animals were analyzed per group with similar results. b, C16-siRNAs targeting
CNS cell-type-specific transcripts were administered as a single IT bolus injection to rats at 0.9 mg. Spinal cord and brain regions were collected at 2 weeks
post-dose (Gfap, Pecam1 and Mbp) or 1 month post-dose (Map2 and Iba1) for target mRNA knockdown measurement by RT–qPCR. n = 3 animals per
group for aCSF, Gfap, Iba1 and Mbp; n = 6 animals per group for Map2; n = 2 animals per group for Pecam1. c, C16-siRNA targeting Sod1 was administered
as a single IT bolus injection to rats at 0.07 mg, 0.3 mg or 0.9 mg. Spinal cord and brain regions were collected at 1 month post-dose for target mRNA
knockdown measurement by RT–qPCR. n = 3 animals per group. d, C16-siRNA targeting Sod1 was administered as a single IT bolus injection to rats
at 0.9 mg. Spinal cord, brain regions and peripheral organs were collected at indicated days post-dose, out to 6 months, for target mRNA knockdown
measurement by RT–qPCR. n = 3 animals per group. e, C16-siRNA targeting Sod1 was administered as a monthly IT bolus injection to rats at 0.3 mg for a
total of up to five injections over 4 months. Spinal cord, brain regions and peripheral organs were collected at indicated days, out to 4.5 months, for target
mRNA knockdown measurement by RT–qPCR. n = 3 animals per group. f, Sod1 C16-siRNA was administered as single IT injections of 0.07 mg, 0.3 mg or
0.9 mg or monthly 0.3 mg injections for a total of up to five injections over 4 months. Frontal cortex concentrations show dose linearity in the single-dose
escalation study in rats and additive exposure in the multi-dose arm. The straightforward pharmacokinetics of C16-siRNA in rat CNS are well-characterized
by a first-order pharmacokinetic model (solid lines). n = 3 animals per group. All error bars represent standard deviation.
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Articles NatureBiotechnology
cord and up to 80% in the brain, including 25% knockdown in the
striatum. There was no activity in NHP liver or kidney at 3 months
post-60 mg IT dose.
Duration of silencing in the NHPs was evaluated by analyzing
serially collected cerebrospinal fluid (CSF) samples using soluble
APPα (sAPPα) and soluble APPβ (sAPPβ) protein biomarker assays
(Fig. 3b). Nadir of knockdown was observed at 1 week post-dose
(the earliest time point tested), with sustained >75% APP silencing
for approximately 2.5 months, 50% at 4.5 months and approaching
full recovery at 9 months in most animals.
The relationship of APP silencing in CNS tissues to exposure was
evaluated in samples from animals in Fig. 3a,b as well as a third
cohort of animals that was killed at approximately 4.5 months after
a single 45 mg dose of C16-siRNA. The exposures in the frontal cortex
and thoracic spinal cord ranged from <1 µg g−1
to approximately
9 µg g−1
in these animals (Fig. 3c) and largely reflected the variability
in IT administration, as has been previously noted for ASOs26
.
Fortuitously, the variability in dosing allowed us to observe a robust
exposure-response relationship across brain regions analyzed.
To evaluate the tolerability and safety in NHPs, histopathological
evaluations of brain, spinal cord and dorsal root ganglia
were performed after terminal necropsy procedures at 3 months,
6 months or 9 months after a single IT dose of up to 60 mg using
the APP-targeting siRNAs (Supplementary Table 1). APP siRNAs,
with or without VP and/or N6 C16 conjugation, were well-tolerated,
and there were no test item-related microscopic findings in the
a
b c
0
25
50
75
100
125
150
175 NHP IT, 60 mg, day 85
ProportionofAPPmRNA
remainingrelativetoaCSF(%)
Lumbar spinal cord
Thoracic spinal cord
Cervical spinal cord
Brainstem
Cerebellum
Hippocampus
Temporal cortex
Frontal cortex
Striatum
Liver
Kidney
C16 + + – –
aCSF
VP + – + –
0
25
50
75
100
125 NHP IT, 60 mg
Days post-dose
ProportionofsAPPremaining
relativetopre-dose(%)
sAPPα
sAPPβ
1 15 43 71 105 129 163 193 224 255 0 2 4 6 8 10
0
25
50
75
100
125
NHP IT, 45 or 60 mg, day 85 or 129
siRNA concentration (µg g–1
)
ProportionofAPPmRNAremaining
relativetoaCSF(%)
Striatum
Prefrontal cortex
Thoracic spinal cord
Fig. 3 | Translation of C16-siRNA activity in NHP CNS. a, APP-targeting siRNA (compounds VII, VIII, IX and X; Supplementary Table 1) was administered as
a single IT bolus injection to cynomolgus monkeys at 60 mg, and, 3 months post-dose, tissue samples were analyzed for APP mRNA levels by RT–qPCR after
85 days. n = 5 animals per group for aCSF; n = 2 animals per group for +VP/+C16; n = 4 animals per group for −VP/+C16; n = 1 animal per group for +VP/−
C16; n = 3 animals per group for −VP/−C16. b, C16-siRNA targeting APP was administered as a single IT bolus injection to three cynomolgus monkeys at
60 mg. CSF was collected at indicated days post-dose, out to 9 months, for sAPPα and sAPPβ measurement relative to pre-dose. n = 3 animals per group.
c, C16-siRNA concentrations were evaluated by mass spectrometry from CNS tissue samples obtained from animals from a and b and from a third group of
animals (n = 3) killed at 4.5 months after a 45 mg nominal dose. All error bars represent standard deviation.
Fig. 4 | Efficacy of APP silencing in the CVN mouse model. a, Human APP-targeting siRNA XVIII (Supplementary Table 1) reduced APP mRNA and sAPPα
protein. aCSF, n = 6 per group; siRNA XVIII, n = 3 per group. b, Single 120 µg ICV bolus dose showed ~75% reduction of APP mRNA at 30 days and >50%
reduction at 60 days post-dose. Day 30 and Day 180, n = 4 per group; Day 60, n = 1 per group; Day 90, n = 11 per group. c, Overview of the experimental
design and disease progression in the CVN mice. Animals were dosed pre-symptomatically and assessed by IHC for changes in deposition of AB40 (e,f)
and inflammation (IBA1) (e,g) within the cortex and hippocampus 3 months or 6 months post-dose. d, After 3 months, a reduction of ~25% and ~50% of
APP mRNA was observed in the cortex and hippocampus, respectively, which corresponded to a ~50% reduction in sAPPα protein. aCSF, n = 3 per group;
siRNA XVIII, n = 4 per group. f, Tissue AB40 deposits assessed by IHC. aCSF, n = 2 per group at 6 months; n = 4 for the remaining groups. g, Tissue IBA1
levels assessed by IHC and qPCR (Iba1). aCSF, n = 2 per group at 6 months; n = 4 for the remaining groups. Simple linear regression was used to compare
the slopes. *P < 0.05 (P = 0.0237 in the siRNA XVIII group). h, Glutamate and N-acetylaspartate levels as measured by 1
H-MRS at 12 months of age
(6 months post-dose) show normalization of glutamate levels in the siRNA-treated group. WT aCSF, n = 9 per group; n = 8 per group for the remaining
groups. All error bars represent standard error. *P < 0.05. Unpaired t-test assuming equal variance was used. CR, creatine i, siRNA-treated animals show
normalization of total distance traveled and rearing frequency. WT aCSF, n = 9 per group; n = 8 per group for the remaining groups. All error bars represent
standard error. *P < 0.05, **P < 0.005. Unpaired t-test assuming equal variance was used unless indicated otherwise. NS, not significant.
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ArticlesNatureBiotechnology
a
c
e
h
i
f
g
d
b Ventral cortex mRNA
Single dose
120 µg by ICV
Single dose
60 µg by ICV
aC
SFsiR
N
A
XVIII
0
100
200
300
400
APP
(Normalized to Xpnpep1)
Proportionofmessage
remainingrelativeto
aCSF(%)
30 days post-dose
aC
SFsiR
N
A
XVIII
0
50
100
150
200
sAPPα
CSFsAPPα(pg/ml)
30 days post-dose
30
60
90
180
0
50
100
150
APP
%messageremaining
(relativetoaCSF)
Time (days) post-dose
siRNA XVIII
Ventral cortex mRNA Hippocampus mRNA
Glutamate N-acetylaspartate
Ventral cortex mRNA CSF protein
9 months
CSF protein
Cortex IHC
Cortex IHC
Hippocampus IHC
Hippocampus IHC
Single dose
60 µg by ICV
3 6 9 12Age (months)
4
DiseaseSingle dose
120 µg by
ICV
IHC - AB40, Iba1
RNA and protein
Metabolite
open field test
siRNA XVIII
siRNA XVIII
aC
SFsiR
N
A
XVIII0
50
100
150
APP
(Normalized to Xpnpep1)Proportionofmessage
remainingrelativeto
aCSF(%)
90 days post-dose
aC
SFsiR
N
A
XVIII
0
50
100
150
APP
(Normalized to Xpnpep1)
Proportionofmessage
remainingrelativeto
aCSF(%)
90 days post-dose
aC
SFsiR
N
A
XVIII
0
0.1
0.2
0.3
sAPPα
CSFsAPPα(pg/ml)
90 days post-dose
AB40 + IBA1 AB40 IBA1
AB40 IBA1
aCSF
siRNA XVIII
AB40 + IBA1
100 um
aC
SF
siR
N
A
XVIII
aC
SF
siR
N
A
XVIII
0
0.01
0.02
0.03 AB40
Stainingpercentage
ofregionarea(%)
6 months 9 months
aC
SF
siR
N
A
XVIII
aC
SF
siR
N
A
XVIII
0
0.05
0.10
0.15
0.20 AB40
Stainingpercentage
ofregionarea(%)
6 months 9 months
5 6 7 8 9 10
0
1
2
3
4
Age (months)
Meanintensity(%)
IBA1
aCSF
siRNA XVIII
P = 0.0237
*
5 6 7 8 9 10
0
2
4
6
8
Age (months)
Meanintensity(%)
IBA1
aCSF
siRNA XVIII
P = 0.0268
*
aC
SF
siR
N
A
XVIII
0
20
40
60
80
100
Relativeexpression
Iba1
W
T
aC
SF
C
VN
aC
SF
C
VN
siR
N
A
XVIII
2.0
2.5
3.0
3.5
4.0
1
H-MRSGLU/CRratio
GLU/CR
W
T
aC
SF
C
VN
aC
SF
C
VN
siR
N
A
XVIII
1.5
2.0
2.5
3.0
3.5
1
H-MRSGLU/CRratio
NAA/CR
*
W
T
aC
SF
C
VN
aC
SF
C
VN
siR
N
A
XVIII
0
5,000
10,000
15,000
20,000
Distance traveled
Distance(cm)
NS
W
T
aC
SF
C
VN
aC
SF
C
VN
siR
N
A
XVIII
0
500
1,000
1,500
Rearing frequency
Meanrearing
frequency(events)
NS
W
T
aC
SF
C
VN
aC
SF
C
VN
siR
N
A
XVIII
0
20
40
60
80
Velocity
Velocity(cms–1
)
*
**
*
*
*
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Articles NatureBiotechnology
examined brain, spinal cord, dorsal root ganglia and liver sections
(Extended Data Figs. 1–3). Additionally, conjugation of the C16
ligand to fully chemically modified siRNAs did not induce cytokine
release in a human whole blood assay (Supplementary Table 2).
PreclinicalefficacyofC16-siRNAinamousemodelofAlzheimer’s
disease. To evaluate the preclinical efficacy of C16-siRNAs, we used
the Tg-hAPPSwDI
/mNos2−/−
(CVN) transgenic mouse expressing
human APP harboring the Swedish K670N/M671L, Dutch E693Q
and Iowa D694N mutations crossed to a Nos2−/−
background27
,
which accumulates Aβ in the brain parenchyma and vasculature28
.
Using an APP targeting C16-siRNA, XVIII (Supplementary Table
1), we demonstrated that a single 60 µg ICV administration reduced
APP mRNA and sAPPα protein levels by 50% in the ventral cortex
(VC) and CSF (Fig. 4a). A higher dose at 120 μg showed a ~75%
reduction of APP mRNA at 30 days and >50% reduction at 60 days,
finally normalizing at 90 days in the VC (Fig. 4b,d). When dosed
at 3 months of age (Fig. 4c), hippocampal APP mRNA and CSF
sAPPα protein levels remained reduced at ~40–50% after 90 days
(Fig. 4d). We further asked whether knockdown of APP is sufficient
to reduce pathology in the CVN mice and divided the study
into two categories: the first group, dosed pre-symptomatically,
was assessed for Aβ40 (AB40) deposits as well as inflammation;
the second group, dosed post-symptomatically, was evaluated for
changes in metabolites as well as behavior (Fig. 4c). Knockdown of
APP lasted for 3 months in the VC (Fig. 4b); however, AB40 deposits
were reduced by ~50% even after 6 months (Fig. 4e, f), albeit not
significantly due to the small sample size (n = 2–4). In this model,
ionized calcium-binding adaptor molecule 1 (Iba1) is upregulated
at 4 months and continues to elevate at 9 months, concomitant
with increases in inflammation when neurodegeneration occurs29
.
The siRNA XVIII-treated group showed a significantly slower rate
of increase in IBA1 IHC intensity in the cortex and hippocampus
(Fig. 4g). In addition, the expression of Iba1 mRNA was reduced by
20–30% in siRNA XVIII-treated animals after 6 months (Fig. 4g).
To determine whether deceases in Aβ deposition and inflammation
can be associated with restoration of neuronal function, we used
proton magnetic resonance spectroscopy30
(1
H-MRS) to measure
metabolites, such as glutamate (Glu) and N-acetylaspartate (NAA).
These metabolites have been found to be reduced in patients with
Alzheimerʼs disease, are reflective of neuronal integrity and are predictors
of cognitive deficits31,32
. Here, we noted a significant reduction
in both Glu and NAA levels in the CVN mice at 12 months of
age compared with wild-type (WT) (Fig. 4h). Knockdown of APP
in the CVN mice normalized Glu levels to that of the WT; however,
it had little effect on NAA levels (Fig. 4h). In the open field test,
we observed that the CVN mice showed a measurable increase in
total distance traveled and an increase in rearing frequency (Fig. 4i).
Treatment with siRNA XVIII normalized both measures to levels
seen in the WT (Fig. 4i). Velocity measures in all groups were not
significantly different from each other (Fig. 4i).
C16 siRNA shows potent and durable activity in the eye. To
assess potency and durability in NHP eyes, we used a transthyretin
(TTR)-targeting C16-siRNA with VP (Supplementary Table 1). A
single IVT dose of this conjugate showed dose-dependent lowering
of TTR protein in the aqueous humor, with >80% sustained knockdown
for 6 months at 100 μg per eye or 300 μg per eye (Fig. 5a).
In the retinal pigmented epithelium (RPE), dose-dependent TTR
mRNA lowering was observed, with >95% knockdown at a dose
of 100 μg per eye (Fig. 5b). TTR protein knockdown in RPE cells
was further confirmed by TTR IHC at 6 months post-dose (Fig. 5c).
a
c
b
d
0
25
50
75
100
125
150 NHP IVT, aqueous humor
Days post-dose
ProportionofTTRprotein
remainingrelativetoPBS(%)
28 84 168
PBS
PBS
3
µg
30
µg
100
µg
300
µg
0
25
50
75
100
125
150 NHP IVT, day 168, RPE
ProportionofTTRmRNA
remainingrelativetoPBS(%)
0
25
50
75
100
125
150
175
NHP IVT, aqueous humor
Days post-dose
ProportionofTTRprotein
remainingrelativetoPBS(%)
VP/C16 - 10 µg
VP/No C16 - 10 µg
28 56
VP/No C16 - 30 µg
84
PBS
C16-siRNAPBS
Purple: TTR Purple: TTR
3 µg
30 µg
100 µg
300 µg
Fig. 5 | Translation of C16-siRNA activity in NHP eye. a, C16-siRNA targeting TTR (compound XI; Supplementary Table 1) was administered via IVT
injection to cynomolgus monkeys at various doses. Aqueous humor was collected for secreted TTR protein measurement relative to PBS-dosed animals.
n = 4 animals per group. b, TTR knockdown via RNAi was further confirmed on day 168 by TTR mRNA normalized to housekeeping GAPDH mRNA relative
to PBS-dosed animals in micro-dissected RPE. n = 4 animals per group. c, TTR IHC confirmed protein knockdown in RPE, along with knockdown of secreted
protein adherent to photoceptor cells above. d, Unconjugated TTR-targeting siRNA modified with VP at the 5′ end of the antisense strand was dosed IVT
in cynomolgus monkeys at 10 μg per eye and 30 μg per eye and compared with a dose of 10 μg per eye of the C16-conjugated version. Aqueous humor was
collected at indicated days post-dose, out to 3 months, for secreted TTR protein measurement relative to PBS-dosed animals. n = 4 animals per group. All
error bars represent standard deviation.
Nature Biotechnology | VOL 40 | October 2022 | 1500–1508 | www.nature.com/naturebiotechnology1506
ArticlesNatureBiotechnology
The unconjugated siRNAs (without C16) showed no knockdown at
a dose of 10 μg per eye and a modest 33% knockdown at a dose of
30 μg per eye compared with 81% knockdown observed for the C16
conjugate at a dose of 10 μg per eye (Fig. 5d).
C16 siRNA shows potent and durable siRNA activity in the
lung. C16-siRNA distribution in the mouse lung tissue was evaluated
after intranasal (IN) administration using IHC (Fig. 6a,b and
Supplementary Table 1). siRNA distributed throughout the whole
lung, with the greatest accumulation near the mainstem bronchi
(Fig. 6a). Closer evaluation showed robust bronchiolar and alveolar
uptake (Fig. 6b). The siRNA distribution correlated well with RNAi
activity assessed by in situ hybridization (ISH) of the Sod1 mRNA
(Fig. 6c). We were not able to assess activity in the alveoli because
Sod1 is expressed only in the large airways. The RNAi activity was
dose dependent as measured by Sod1 mRNA from whole lung tissue
(Fig. 6e) and was sustained out to 2 months post-dose, with 57%
mRNA lowering at 10 mg kg−1
via IN administration (Fig. 6d). The
single intravenous (IV) injection at 30 mg kg−1
reduced Sod1 mRNA
by 42%(Fig. 6e), indicating a clear benefit of IN administration for
delivery of C16-siRNAs to the lungs.
Discussion
Systematic efforts to optimize RNAi therapeutics for potent, durable
and safe silencing of disease-associated gene transcripts in the
liver9,10,33
provided the framework for designing siRNAs for extrahepatic
applications. Lipophilic ligands, primarily cholesterol, conjugated
via a linker to the 5′ or 3′ ends of the siRNA, have been widely
used to facilitate systemic and local siRNA delivery into extrahepatic
tissues23,34–37
, including the eye38
, lung35
and CNS21,39,40
, albeit
with limited distribution beyond the injection site and, in certain
cases, an unacceptable safety profile41
. Here we report, to our knowledge
for the first time, a systematic evaluation of lipophilic siRNA
conjugates combined with additional design features to arrive at an
optimal conjugate design that enables widespread siRNA distribution
in the spinal cord and brain after administration into rodents
and NHPs via a clinically feasible IT dosing route, with a favorable
safety profile. Notably, we provide a preclinical proof-of-concept
study whereby administration of a C16-siRNA targeting APP in a
mouse model of Alzheimerʼs disease produced a sufficiently potent
and durable knockdown in the CNS to both alter the physiological
deficits, such as Aβ deposition and inflammation, and normalize
behavioral deficits. This program has already advanced into clinical
a b
1 mm
Mainstem
bronchus
Accessory
Cranial
Middle
Caudal
Test article
Right lung
100 um 200 um 50 um
Bronchus Bronchiole Alveoli
0 1 3 10 28 56
0
25
50
75
100
125
150
Days post-dose
ProportionofSod1mRNA
remainingrelativetoPBS(%)
PBS
10 mg kg–1
Mouse IN
PBS0.3
m
g
kg–1
1
m
g
kg–1
3
m
g
kg–1
10m
g
kg–1
30
m
g
kg–1
0
25
50
75
100
125
150 Mouse IN, day 10
ProportionofSod1mRNA
remainingrelativetoPBS(%)
IN IV
e
d
1 mm
Control
BronchusBronchiole
Sod1 siRNA
c
Fig. 6 | C16-siRNA distribution and activity in mouse lung. a,b, C16-siRNAs were administered in mice intranasally at 10 mg kg−1
(Sod1) (a) or 30 mg kg−1
(Traf6) (b) (Supplementary Table 1). Lungs were collected on day 10 or day 28 post-dose, respectively, for siRNA IHC. siRNA, magenta; hematoxylin
counterstain, blue. Three animals were analyzed per group with similar results. c, Sod1 mRNA knockdown was visualized by ISH on day 10 after 10 mg kg−1
IN dose of Sod1-targeting C16-siRNA. Sod1 mRNA, brown; hematoxylin counterstain, blue. Three animals were analyzed per group with similar results.
d, RT–qPCR of Sod1 mRNA in whole lung measured on day 10 after 0.3, 1, 3 or 10 mg kg−1
single IN dose or 30 mg kg−1
single IV dose of Sod1-targeting
C16-siRNA. n = 3 animals per group. All error bars represent standard deviation. e, RT–qPCR of Sod1 mRNA in whole lung measured at multiple time points
after 10 mg kg−1
IN dose of Sod1-targeting C16-siRNA. n = 3 animals per group.
Nature Biotechnology | VOL 40 | October 2022 | 1500–1508 | www.nature.com/naturebiotechnology 1507
Articles NatureBiotechnology
development for the treatment of early onset Alzheimerʼs disease
and cerebral amyloid angiopathy.
The same C16 conjugation strategy also enables broad siRNA
distribution and durable activity in the eye and lung after local
delivery. Combined with the durability of siRNAs, the addition of
the C16 conjugation has the potential for therapeutic silencing of
target genes outside the liver with infrequent dosing. The precise
mechanisms whereby C16 promotes intracellular delivery is currently
under investigation; however, we hypothesize that it promotes
moderate affinity interactions with the extracellular matrix
and various cell membrane components, thus allowing for binding
and uptake into various cell types.
Online content
Any methods, additional references, Nature Research reporting
summaries, source data, extended data, supplementary information,
acknowledgements, peer review information; details of
author contributions and competing interests; and statements of
data and code availability are available at https://doi.org/10.1038/
s41587-022-01334-x.
Received: 28 September 2021; Accepted: 22 April 2022;
Published online: 2 June 2022
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ArticlesNatureBiotechnology
Methods
Care and use of laboratory animals. All studies were conducted using protocols
consistent with local, state and federal regulations, as applicable, and approved
by the Institutional Animal Care and Use Committees (IACUCs) at Alnylam
Pharmaceuticals, Charles River Laboratories (CRL) or LabCorp (formerly
Covance) as applicable. Animal studies in Figs. 1a,d,e and 2b were conducted
in 8–10-week-old female Sprague Dawley rats and approved by the IACUC at
Alnylam Pharmaceuticals. Animal studies in Figs. 1c,f and 2c–f were conducted
in 8–10-week-old male Sprague Dawley rats and approved by the IACUC at CRL
(Figs. 1c and 2c–f) or Alnylam Pharmaceuticals (Fig. 1f). Animal studies in Fig. 4
and in Extended Data figures were conducted in 2–4-year-old male cynomolgus
macaques and approved by the IACUC at CRL. Animal studies in Fig. 5 were
conducted in 2–5-year-old female cynomolgus macaques and approved by the
IACUC at CRL. Animal studies in Fig. 5 were conducted in 8–10-week-old female
C57BL/6 mice and approved by the IACUC at Alnylam Pharmaceuticals. Animal
numbers are provided in figure legends.
Oligonucleotide synthesis. All oligonucleotides were synthesized on an MerMade
192 or MerMade 12 synthesizer according to previously published protocols24,42
.
5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-fluoro- and 5′-O-(4,4′-dimethoxytrityl)-
2′-O-methyl-3′-O-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite monomers of
uridine, 4-N-acetylcytidine, 6-N-benzoyladenosine and 2-N-isobutyrylguanosine
were purchased commercially, and (S)-GNA phosphoramidites were synthesized
according to previously published protocols43–45
. 2′-O-C16 phosphoramidites
(WO2019217459) were used at a concentration of 100–150 mM in acetonitrile
with no other changes to synthetic protocols. Phosphorothioate linkages were
introduced by sulfurization of phosphite linkages using 0.1 M 3-((N,N-dimethylaminomethylidene)amino)-3H-1,
2, 4-dithiazole-5-thione (DDTT) in pyridine.
After completion of the solid-phase synthesis, the solid support was incubated
in a sealed container with aqueous ammonium hydroxide (28–30%) with added
5% diethylamine by volume, with shaking overnight at 35 °C, after a procedure
optimized for 5′-(E)-vinyl phosphonate oligonucleotide deprotection46
. The
oligonucleotide was filtered to remove the support with 5× volume of water and
analyzed by liquid chromatography–mass spectrometry (LC-MS) and ion exchange
(IEX) high-performance liquid chromatography (HPLC).
After de-protection and crude quality confirmation, IEX HPLC purification
was performed. Purification buffer A consisted of 20 mM sodium phosphate and
15% acetonitrile, pH 8.5. Buffer B was the same composition with an additional
1 M sodium bromide. TSKgel Super Q-5PW (20) IEX resin (Tosoh, 0018546) was
used for purification, and a general purification gradient of 15% to 48% in about
20 column volumes was applied. Fractions were analyzed by IEX analysis using a
Dionex DNAPac PA200 IEX analytical column, 4 mm × 250 mm (Thermo Fisher
Scientific, 063000) at room temperature. Buffer A consisted of 20 mM sodium
phosphate and 15% acetonitrile, pH 12. Buffer B was identical with an additional
1 M sodium bromide. A gradient of 30% to 50% over 12 minutes with a flow rate of
1 ml min−1
was used to analyze fractions. Fractions with >85% purity were pooled,
concentrated and desalted over size exclusion columns (GE Healthcare, 17-5087-
01) with a flow rate of 10 ml min−1
. All oligonucleotides were purified and desalted
and further annealed to form siRNA duplexes as previously described12
. All siRNAs
were synthesized by Alnylam Pharmaceuticals.
The identities and purities of all oligonucleotides were confirmed using
electrospray ionization (ESI)-LC–MS and IEX HPLC, respectively.
In vitro screening. Rat RPE-J cells (American Type Culture Collection, CRL-
2240) were transfected by adding 0.1 µl of RNAiMAX (Invitrogen, 13778) diluted
in 4.9 µl of Opti-MEM to 5 µl of siRNA in a 384-well plate. After a 15-minute
room temperature incubation, 40 µl of media containing ~5 × 103
cells was
added to the wells. Cells were incubated for 24 h, and RNA was isolated using an
automated protocol on a BioTek EL406 platform using Dynabeads mRNA DIRECT
Purification Kit (Invitrogen, 61012) according to the manufacturer’s instructions.
RNA was eluted with 150 µl of elution buffer.
Complementary DNA (cDNA) synthesis was performed using High-Capacity
cDNA Reverse Transcription Kit (Applied Biosystems, 4368813) according
to the manufacturer’s instructions. Next, 2 µl of cDNA was added to a master
mix containing 0.5 µl of rat Gapdh TaqMan probe (Thermo Fisher Scientific,
4352338E), 0.5 µl of rat Sod1 TaqMan probe (Thermo Fisher Scientific,
Rn00566938_m1) and 5 µl of LightCycler 480 probe master mix (Roche,
04887301001) per well in a 384-well plate (Roche, 04887301001). Quantitative PCR
with reverse transcription (RT–qPCR) was performed in a LightCycler 480 Real
Time PCR system (Roche). qPCR data were analyzed using the ΔΔCt method.
Rat IT studies. siRNAs formulated at up to 30 mg ml−1
in artificial cerebrospinal
fluid (aCSF) were administered as 30-µl IT injections by lumbar puncture in the
dorsal region of the spine between the L3–L5 vertebral space to male Sprague
Dawley rats. At a minimum of 30 minutes before surgery, rats were subcutaneously
administered 1 mg kg−1
of meloxicam and 0.1 mg kg−1
of buprenorphine. After
anesthesia with isoflurane, rats were placed on a warm heating pad and treated
with eye lubricant; the IT injection site was shaved and disinfected; and an incision
was made to expose the spinal column. siRNA was administered with an insulin
syringe. Proper placement of the needle was confirmed via CSF backflow in the
hub of the needle. Once siRNA administration was completed, gentle constant
pressure on the plunger was maintained for 30 seconds; the incision was sutured
and secured with tissue glue; and the rats were placed in sternal recumbency on a
heating pad until recovery.
NHP IT studies. siRNAs formulated at 30 mg ml−1
in aCSF were administered as
2-ml IT injections over 3 minutes by lumbar puncture in the dorsal region of the
spine between the L2–L3, L3–L4, L4–L5 or L5–L6 vertebral space of cynomolgus
monkeys. Anesthesia was induced with dexmedetomidine and ketamine and was
maintained with supplemental isoflurane as needed. Proper placement of the
needle was confirmed via CSF backflow in the hub of the needle. A total volume
of 1 ml of CSF was removed before dosing, and 0.3-ml aCSF flush was performed
after siRNA administration. After completion of the flush, the syringe and needle
were removed; pressure was applied to the injection site for at least 30 seconds;
and the animals were allowed to recover. Dexmedetomidine was reversed with
atipamezole as necessary. Serial CSF collections were performed from cisterna
magna after procedural anesthesia induced with dexmedetomidine and ketamine
and maintained with supplemental isoflurane as needed.
NHP IVT studies. A topical antibiotic (tobramycin) was applied to both eyes twice
on the day before and twice on the day after IVT injection to cynomolgus monkeys.
The animals received an intramuscular injection of a sedative cocktail (ketamine
5 mg kg−1
; dexmetedomidine 0.01 mg kg−1
) followed by isoflurane/oxygen mix
through a mask, if deemed necessary to maintain anesthesia. Either 1× PBS or siRNA
formulated in 1× PBS was administered in both eyes by IVT injection using a 1-ml
syringe and a 30-gauge, ½-inch needle. The dose volume administered was 50 μl per
eye. IVT injections were performed by a board-certified veterinary ophthalmologist.
After completion of the dosing procedure, animals received an intramuscular
injection of 0.1 mg kg−1
of atipamezole, a reversal agent for dexmetedomidine, as
considered necessary. The conjunctivae were flushed with diluted benzalkonium
chloride (Zephiran). Mydriatic drops were applied to each eye as needed.
Mouse IN studies. Female C57BL/6 mice (8–10 weeks of age) were dosed via IN
instillation (50 μl total, 25 μl per nostril) at the indicated siRNA concentration
diluted in 1× PBS and were killed at the indicated day.
CVN mouse studies. Male and female CVN mice were bred at CRL Germany and
genotyped, housed, dosed and analyzed (IHC, 1
H-MRS and open field test) at CRL
Finland, as previously reported, under regular temperature, humidity and light/
dark cycle conditions27,28,47
. Homozygous Tg-hAPPSwDI
/mNos2−/−
(CVN) mice were
produced by crossing mice expressing the vasculotropic Swedish K670N/M671L,
Dutch E693Q and Iowa D694N human APP mutations under control of the Thy-1
promoter48
with mNos2−/−
(B6 129PNos2 tau1Lau/J) mice49
. The study was divided
into two phases. In the first phase, 15 CVN mice were dosed by ICV at 3 months of
age, with terminal tissue sampling at 6 months of age and 9 months of age. In the
second phase, 40 CVN mice and 20 WT mice were divided into three experimental
groups, with terminal tissue sampling at 9 months of age and 12 months of age.
Metabolic analyses using 1
H-MRS were at 12 months of age, respectively, as
previously described28
. Open field test measurements were performed at ~9 months
of age. Activity chambers (Med Associates, 27 × 27 × 20.3 cm) were equipped
with infrared beams. Mice were placed in the center of the chamber, and their
behaviors were recorded for 30 minutes. The following parameters were recorded
and analyzed: distance traveled, number of vertical rearings and average velocity.
IHC was performed as described27,29
using anti-amyloid-beta antibody WO2
(Sigma-Aldrich, MABN10, lot 3557956, 1:500) and anti-IBA1 antibody (Wako
Pure Chemicals, 01-1941, lot LEP3218, 1:200). All data are presented as mean ±
standard deviation or standard error of the mean, and differences were statistically
significant at the P < 0.05 level. Statistical analyses were performed using the
PRISM statistical program (GraphPad Software).
Mouse ICV infusion. ICV administration was performed using stereotaxically
guided Hamilton syringe and infusion system (Harvard Apparatus). A single
5-µl unilateral injection was performed in the right lateral ventricle at the
following coordinates: AP = −0.5 mm (posterior from bregma), ML = +1.0 mm
and DV = −1.75 mm, using standard aseptic surgical procedures. After midline
skin incision on the scalp, a burr hole was generated at selected coordinates using
stereotaxic coordinates and a dental drill. Thereafter, a small puncture in the dura
was made at the epicenter of the burr hole, and the needle was lowered to specified
depth to reach the lateral ventricle. A Hamilton syringe with a 28-gauge blunt
needle was filled with a volume slightly greater than 5 µl. Administration volume of
the test articles was at 5 µl and infusion rate at 1 µl min−1
(over 5 minutes). After the
infusion, the needle was left in place for a stabilization period of 5 minutes before
withdrawal from the ventricle. Withdrawal is performed slowly and paused for
30 seconds when the needle tip is at the cortical area.
RNA extraction and RT–qPCR. For the extraction of RNA, powdered tissues
(~10 mg) were resuspended in 700 µl of QIAzol and homogenized by vigorous
pipetting. Alternatively, two 5-mm steel grinding balls were added to each sample,
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Articles NatureBiotechnology
followed by homogenization at 25 per second for 1 minute at 4 °C using TissueLyser
II (Qiagen, 85300). Samples were incubated at room temperature for 5 minutes,
followed by the addition of 140 µl of chloroform. Samples were mixed by shaking,
followed by a 10-minute incubation at room temperature. Samples were spun at
6,000g for 15 minutes at 4 °C; the supernatant was transferred to a new tube; and
1.5 volumes of 100% ethanol was added. Samples were then purified using an
miRNeasy Kit (Qiagen, 217061) according to the manufacturer’s instructions. The
RNA was eluted from miRNeasy columns with 50–60 µl of RNAse-free water and
quantified on a NanoDrop (Thermo Fisher Scientific).
cDNA synthesis was performed using High-Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, 4368813) according to the manufacturer’s
instructions. qPCR reactions were performed using gene-specific TaqMan assays
for rat Sod1 (Thermo Fisher Scientific, Rn00566938_m1), rat Ppib (Thermo
Fisher Scientific, Rn03302274_m1), mouse Sod1 (Integrated DNA Technologies,
Mm.PT.58.12368303), mouse Gapdh (Thermo Fisher Scientific, Mm99999915_g1),
NHP APP (Thermo Fisher Scientific, Mf01552283_m1), NHP TTR (Thermo
Fisher Scientific, Mf02799963_m1), NHP PPIB (Thermo Fisher Scientific,
Mf02802985_m1) or NHP GAPDH (forward primer: 5′-GCATCCTGGGCTACAC
TGA-3′, reverse primer: 5′-TGGGTGTCGCTGTTGAAGTC-3′, probe: 5′HEX-CC
AGGTGGTCTCCTCC-3′BHQ-1), mouse Iba1 (Thermo Fisher Scientific,
Mm00479862), mouse Xpnpep1 (Thermo Fisher Scientific, Mm00460029) and
human APP (Thermo Fisher Scientific, Hs00169098). qPCR reactions were
performed in a Roche LightCycler 480 using LightCycler 480 Probes Master Mix
(Roche, 04707494001). qPCR data were analyzed using the ΔΔCt method.
Protein analyses. For sAPP protein analysis in NHP CSF, a V-PLEX panel for
sAPPα and sAPPβ (Meso Scale Discovery, K15120E) was used according to
the manufacturer’s instructions. Plates were incubated at room temperature
for ~10 minutes before reading on a MSD SECTOR Imager Instrument
(Meso Scale Discovery).
For TTR protein analysis in NHP aqueous humor, an in-house sandwich ELISA
was developed. Then, 96-well Nunc MaxiSorp plates (Invitrogen, 44-2404-21) were
coated overnight at 4 °C with rabbit anti-human TTR pAb (Dako, A0002) at a final
concentration of 5.63 µg ml−1
prepared in 50 mM carbonate/bicarbonate buffer. The
next day, plates were washed 5× with 300 µl of Tris buffer containing 0.05% Tween
20 (TBS-T) on a BioTek plate washer. Plates were blocked with 150 µl per well of
1× Power Block (Biogenex Laboratories, HK0855K) for 2 h at room temperature
and washed 5× with 300 µl of TBS-T. An in-house cynomolgus TTR standard was
used to prepare the highest standard at 59.5 ng ml−1
, followed by a 2.5-fold dilution
series in 1× Power Block to generate an 8-point standard curve. Aqueous humor
samples were tested at a 1:600 dilution in 1× Power Block. Standards and samples
were added to the plate, 100 µl per well, in duplicate and incubated for 2 h at room
temperature with shaking at 600 r.p.m. Plates were washed 5× with 300 µl of TBS-T,
followed by the addition of secondary antibody, anti-hTTR pAb (Abcam, ab9015),
at a final concentration of 4 ug ml−1
prepared in 1× Power Block. Plates were
incubated for 1 hour at room temperature with shaking at 600 r.p.m. Plates were
washed 5× with 300 µl of TBS-T, and the detection antibody, donkey anti-sheep
pAb alkaline phosphatase, was applied at a final concentration of 1.4 µg ml−1
in
1× Power Block (Sigma-Aldrich, A5187). Plates were incubated for 1 hour at
room temperature with shaking at 600 r.p.m. Plates were washed 5× with 300 µl of
TBS-T, followed by the addition of substrate, 1.0 mg ml−1
of pNPP (Sigma-Aldrich,
N2770). Plates were incubated in the dark at room temperature for ~20 minutes
before the addition of 1 M sodium hydroxide to stop the reaction. Absorbance was
read at 405 nm on an M5 SpectraMax (Molecular Devices). For data analysis, the
blank optical density (OD) units were subtracted from all wells, and the OD units
for each calibrator were plotted against the calibrator concentrations and fit with
a 4-parameter logistical fit. The concentrations of each replicate of unknowns and
the back-calculated concentrations of the standards were interpolated to determine
the ng ml−1
of TTR.
Quantitation of siRNA. Quantitation of siRNAs was performed by liquid
chromatography coupled with high-resolution mass spectrometry (LC–HRMS)
as described previously50,51
. The samples extracted using a Clarity OTX 96-well
plate (Phenomenex) were analyzed by LC–HRMS. The mobile phases used
were as follows: mobile phase A: H2O/HFIP/DIEA (100:1:0.1, v:v:v) with 10 µM
EDTA; mobile phase B: H2O/ACN/HFIP/DIEA (35:65:0.75:0.0375, v:v:v:v) with
10 µM EDTA. The column used was a DNAPac RP column (4 µm, 50 × 2.1 mm,
Thermo Fisher Scientific). Column temperature was set at 90 °C, and flow rate
was 0.2–0.3 ml min−1
. The gradient started with 10% B, progressed to 50% B over
2.5 minutes and then increased to 100% B in 0.2 minutes and maintained for
0.8 minutes; the column was then re-equilibrated with 10% B for 1.5 minutes. A
Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific) in combination
with an Accela Open Autosampler (Thermo Fisher Scientific) and a Q Exactive
mass spectrometer (Thermo Fisher Scientific) was used for the LC–HRMS
analysis. The oligonucleotides were analyzed in negative ionization mode. The
mass spectrometer was set at parallel reaction monitoring mode.
Histopathology, IHC and ISH. All animals were killed in accordance with AVMA
Guidelines for the Euthanasia of Animals. Tissues were collected52
and fixed in
10% neutral-buffered formalin, processed routinely and stained with hematoxylin
and eosin (H&E) as described previously10
. H&E-based microscopic findings
were recorded in the Pristima version 7.0.0 system (Xybion) and graded on a
scale of 1–5 (1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe) by a
board-certified veterinary pathologist.
Luxol fast blue/cresyl-echt violet (LFB/CEV) stain for myelin was performed
on spinal cord, brainstem and cerebellum. In brief, de-paraffinized sections were
dehydrated through three changes in xylene, 2 minutes each, and rehydrated
through two changes of 100% and 95% ethanol, 2 minutes each. Sections were
stained with 0.1 % LFB (StatLab) either for 1 hour at 60 °C or overnight at
room temperature, followed by 3–5 rinses in 95% ethanol, distilled water and
differentiation in 0.05% lithium carbonate for 3–5 seconds. Next, sections were
rinsed in distilled water, followed by counterstaining with CEV for 10 minutes,
dehydrated through graded alcohols and xylene and routinely coverslipped.
Decreases in LFB staining intensity and increases in IBA1 staining indicative
of microgliosis were semi-quantitatively scored on a scale of 1–4 (1 = minimal,
2 = mild, 3 = moderate, 4 = marked).
IHC and ISH were performed on the Discovery Ultra automated instrument
(Ventana Medical Systems) using manufacturer-provided reagents and protocols.
For IHC, antibodies against the following targets were used: IBA1 1:1,600
(019-19741, Fujifilm Wako), GFAP 1:4,000 (Z0334, Agilent-Dako), CD31 1:75
(ab23874, Abcam), MAP2 1:1,000 (ab5392, Abcam), TTR 1:800 (prealbumin,
SC-8104, Santa Cruz Biotechnology) and in-house anti-siRNA rabbit polyclonal
antibody ab19151 at 1:12,000. The anti-siRNA antibody was isolated from
rabbits dosed with Keyhole limpet hemocyanin (KLH)-conjugated fully modified
siRNA mixed with Freund’s adjuvant. Iba1, Gfap and Pecam1 were detected with
anti-rabbit NP multimer, followed by anti-NP AP multimer and Discovery Yellow
or Disciovery Purple AP Chromagen (all Ventana Medical Systems). MAP2 was
detected with goat anti-chicken antibody (6100-08, Southern Biotech), followed
by anti-goat HRP secondary antibody and Discovery Green HRP Chromogen
(both Ventana Medical Systems). TTR was detected by anti-goat HRP secondary
antibody, followed by Discovery Purple HRP Chromogen (both Ventana Medical
Systems). siRNA was detected with anti-rabbit HRP secondary antibody, followed
by Discovery Purple HRP Chromogen (Ventana Medical Systems). For ISH,
sections were hybridized with a Sod1 mRNA probe (Advanced Cell Diagnostics,
428589), followed by detection with RNAScope DAB amplification kit (Advanced
Cell Diagnostics, 323200) and visualization with mRNA DAB Chromogen
(Ventana Medical Systems).
Human whole blood cytokine release assay. Whole blood from four healthy
human donors was collected into 10-ml sodium heparin Vacutainer tubes (Becton
Dickinson), diluted 1:1 (v:v) in 0.9% saline (Baxter International) and added to
96-well tissue culture plates at a volume of 180 μl per well. siRNAs were transfected
within 2 h of collection at a final concentration of 300 nM using 8 ug ml−1
of
DOTAP. After overnight incubation, plasma was collected for cytokine multiplex
analysis (IL-1β, IL-1RA, IL-6, IL-8, G-CSF, IP-10, MIP1α and TNF-α) using
Bio-Plex Pro assay kits (Bio-Rad).
Reporting summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.
Data availability
All datasets generated and analyzed in the study are provided within the
manuscript files. Source data are provided with this paper.
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Acknowledgements
The authors thank Alnylam’s Medicinal Chemistry Team, Medium Scale Synthesis Team,
High Throughput Synthesis Team and Duplex Annealing Team for siRNA synthesis.
They also thank A. Bisbe, P. Miller and R. Malone for siRNA purification; C. Wong,
S. Maczko, M. Mendez, I. Duarte and D. Rooney for in vivo study support; A. Guan
for tissue processing; J. Varao for sample management; B. Fitzsimmons for tissue drug
concentration analysis; B. Carito, P. Gedman, C.-E. Stephany and K. Bittner for histology
support; Alnylam’s Research Analytical Team for metabolic stability studies; and the
research team at CRL Finland, especially J. Puolivali and F. Khan.
Author contributions
K.M.B., J.K.N., M.M.J., S.M., M.K., C.B, D.F., K.F., M.A.M. and V.J. conceived the
projects. K.M.B., J.K.N., M.M.J., S.M., Y.I.A.-R., L.T.H.D., H.P., C.S.T., E.C.-R., C.B., D.F.,
J.K., J.A., A.C., M.K.S., I.Z., M.K., M.A.M. and V.J. contributed to experimental design.
L.T.H.D., H.P., C.S.T., E.C.-R., C.B., D.F., J.K., J.A., R.M., J.L., S.M., M.S., T.C., M.J., K.W.,
J.R., L.W., A.K., D.G., M.W.M., J.P., R.M., T.R., J.B., D.C., S.A., L.J., Y.J., S.L., J.G., T.N.,
S.C., S.L.B, U.P., A.K., A.B.R. and S.C. contributed experimentally. R.M., J.E.S. and W.D.
provided non-human primate evaluations. C.S.T., T.C., K.W., J.R. and L.W. synthesized
the duplexes. K.M.B., J.K.N., M.M.J., Y.I.A.-R., L.T.H.D., M.M., K.F., J.-T.W., M.A.M. and
V.J. wrote the manuscript.
Competing interests
All authors are, or were during the time this work was conducted, employees of Alnylam
Pharmaceuticals, with salary and stock options.
Additional information
Extended data is available for this paper at https://doi.org/10.1038/s41587-022-01334-x.
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41587-022-01334-x.
Correspondence and requests for materials should be addressed to Martin A. Maier
or Vasant Jadhav.
Peer review information Nature Biotechnology thanks the anonymous reviewers for their
contribution to the peer review of this work.
Reprints and permissions information is available at www.nature.com/reprints.
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Articles NatureBiotechnology
Extended Data Fig. 1 | Histopathological evaluation of the nonhuman primate (NHP) spinal cord at three months following a single IT dose of 60 mg
C16-siRNA. Representative H&E (a), LFB (b) and IBA-1 (c) staining of NHP thoracic spinal cord. Microscopic findings in the spinal cord were limited to
procedure-related changes (that is, IT bolus administration in lumbar region). These consisted of minimal to mild degeneration of nerve fibers with dilated
axonal sheaths and few digestion chambers containing gitter cells (arrowheads) that were localized to the white matter of spinal cord (cervical, thoracic,
and lumbar segments). Limited to procedure-affected areas, there were minimal decreases in LFB staining intensity (arrows) and a minimal increase in
the number of IBA-1 (brown chromogen) positive microglia (open arrows). Three animals were analyzed per group with similar results. GM = grey matter;
WM = white matter.
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ArticlesNatureBiotechnology
Extended Data Fig. 2 | Histopathological evaluation of the nonhuman primate (NHP) brain at three months following a single IT dose of 60 mg
C16-siRNA. Representative H&E (a), LFB (b) and IBA-1 (c) staining of NHP brainstem (medulla oblongata region adjacent to aqueduct and underlying
pons). Microscopic findings in the brain were limited to procedure-related changes (that is, CSF collection from cisterna magna). These consisted of
minimal to mild degeneration of nerve fibers with dilated axonal sheaths and few digestion chambers containing gitter cells (arrowheads) that were
localized to the peripheral white matter areas. Limited to procedure-affected areas, there were minimal decreases in LFB staining intensity (arrows) and a
minimal increase in the number of IBA-1 (purple chromogen) positive microglia (open arrows). Three animals were analyzed per group with similar results.
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Articles NatureBiotechnology
Extended Data Fig. 3 | Histopathological evaluation of the nonhuman primate (NHP) liver at three months following a single IT dose of 60 mg
C16-siRNA. Representative H&E staining of NHP liver (H = hepatocyte; S = sinusoid; KC = Kupffer cell). There were no remarkable microscopic findings.
Three animals were analyzed per group with similar results.
Nature Biotechnology | www.nature.com/naturebiotechnology
natureresearch Corresponding author(s): Martin A. Maier,Vasant Jadhav
Last updated by author(s): _April 19, 2022
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Laboratory animals Mouse (C57BL/6, female, 8-10 weeks), Rat (Sprague Dawley, male, 8-10 weeks), Monkey (Cynomolgus, male and female, 1.5-3
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Ethics oversight All studies were conducted using protocols consistent with local, state and federal regulations, as applicable, and approved by the
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