α No SNA
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Spherical Nucleic Acids: Oral Delivery and Efficacy in TNBS-induced IBD Model Bart R. Anderson , SubbaRao Nallagatla, Richard S. Kang, Matthias Schroff, and Ekambar R. Kandimalla Exicure, Inc., 8045 Lamon Avenue, Skokie, IL 60077 INTRODUCTION Spherical nucleic acids (SNAs) are a novel class of therapeutic molecules consisting of densely packed oligonucleotides arranged radially around a spherical nanoparticle core. As a consequence of their 3-dimensional structure, SNAs have increased cellular uptake compared with the same oligonucleotide sequence in the conventional linear format. SNA delivery and activity have been previously demonstrated in various tissues including topical administration to the skin, aerosol delivery to the lung, and intracerebral ventricular injection to the central nervous system. Here we assessed the delivery, biological activity, and disease-modifying activity of an antisense SNA targeted to murine TNFα mRNA (anti-mTNFα SNA) in gastrointestinal (GI) tissues. SNA structure Figure 2. Effect of anti-TNFα SNA on body weight following TNBS-induction of IBD in mice. Body weight of mice following IBD induction and treatment with vehicle or anti-mTNFα SNA. Mean + SEM is shown. ** P-value < 0.01 vs Vehicle on each D1-D7, 2-way ANOVA with Holm-Sidak multiple test correction. Anti-mTNFα SNA Treatment Improves Body Weight 0 2 4 6 15 16 17 18 19 Study day Body weight (g) Naive Vehicle anti-mTNF SNA, 200 μg anti-mTNF SNAs, 1000 μg 7 ** Figure 3. Survival of TNBS-induced IBD mice following anti-mTNFα SNA treatment. Survival curves of mice following IBD induction and treatment with vehicle or anti-mTNFα SNA. Anti-mTNFα SNA Treatment Improves Survival 0 2 4 6 8 0 50 100 Study day Survival (%) Naive Vehicle anti-mTNF SNA, 200 μg anti-mTNF SNA, 1000 μg A B Anti-mTNFα SNA Treatment Reduces TNFα mRNA in Colon Tissues Figure 6. TNFα mRNA knockdown by anti-mTNFα SNA. The proximal and distal colons of mice were collected on study day 7, washed with PBS, and flash frozen. Total mRNA was extracted from homogenized tissues and cDNA was generated for qRT-PCR analysis. The levels of TNF mRNA were quantified and normalized to GAPDH using the 2 -ΔΔCt method. Mean + SEM of n=4 (day 4) or n=5 – 12 (day 7 surviving animals) is shown. 4 7 0 200 400 600 Proximal Colon Study day TNF mRNA (% Naive) Naive Vehicle anti-mTNF SNA, 1000 g 4 7 0 200 400 600 Distal Colon Study day TNF mRNA (% Naive) Naive Vehicle anti-mTNF SNA, 1000 g Figure 5. IBD gross pathology following anti-mTNFα SNA treatment. Gross pathology scores in mice following IBD induction and treatment with vehicle or anti-mTNFα SNA. Mean + SEM is shown. *** P-value < 0.001 vs Vehicle, 1-way ANOVA with Holm-Sidak multiple test correction. Anti-mTNFα SNA Treatment Reduces Gross Pathology in the Proximal Colon 0 1 2 3 4 Gross pathology score Naive Vehicle anti-mTNF SNA, 200 μg anti-mTNF SNA, 1000 μg *** Anti-mTNFα SNA Treatment Significantly Improves IBD Clinical Scores Figure 4. Effect of anti-mTNFα SNA on IBD clinical score. IBD clinical scores in mice following IBD induction and treatment with vehicle or anti-mTNFα SNA. Mean +SEM is shown. 2-way ANOVA with Holm-Sidak multiple test correction P-values vs Vehicle: * < 0.05, *** < 0.001, **** < 0.0001. 0 2 4 6 Study day Clinical score Naive Vehicle anti-mTNF SNA, 200 μg anti-mTNF SNA, 1000 μg 7 0 2 4 6 8 * * **** *** * ** **** **** **** **** **** **** TNBS-induced Inflammatory Bowel Disease (IBD) Model Description On study day 0, inflammatory bowel disease was induced in female BALB/C mice aged 6 – 7 weeks by intrarectal (i.r.) administration of 2,4,6-Trinitrobenzenesulfonic acid (TNBS), except mice in the naïve group, in n=12 animals per group. On day 1, prior to treatment with test articles, animals were randomized into groups by clinical score to give similar means and distributions of clinical scores across groups. On days 1 – 4, vehicle only or anti-TNFα SNA was administered once daily by oral gavage at a volume of 200 μL per mouse in 0.1 M NaHCO 3 pH 9.0. Body weight, survival, and clinical score were assessed daily. On day 7 the surviving animals were sacrificed. Gross pathology assessment was performed on the proximal colon. GI tissues were flash frozen and subsequently the TNFα mRNA levels were assessed by qRT-PCR. From naïve, vehicle, and 1000 μg/dose SNA groups an additional n=4 parallel animals were removed on day 4 for qRT-PCR assessment of TNFα mRNA levels in GI tissues. Mice found dead in cage (FDC) were represented through the remainder of the study using the last measured body weight, the maximal clinical score of 11, and the maximum gross pathology score of 5. Mice terminated for ethical considerations were represented with the last measured body weight, last measured clinical score, or the maximum gross pathology score of 5. RESULTS Anti-mTNFα SNA is Distributed Throughout the GI Tract With Increasing Accumulation in Distal Segments Over Time Figure 1. Biodistribution in GI tissues of orally administered SNA Fluorescently-labeled SNA (Cy5-SNA) was administered by oral gavage in a volume of 200 μL per mouse in 0.1 M NaHCO 3 pH 9.0 to female C57BL/6 mice aged 6 – 7 weeks, n=2 per treatment group. At 2 hours or 10 hours following administration, GI tissues were collected, washed, and imaged using Cryofluorescence Tomography (CFT) and fluorescence microscopy. In (A) and (B), P.Cecum and D.Cecum are the proximal and distal portions of the Cecum, respectively. Similarly, D.Colon (1) and D.Colon (2) are two most distal portions of the Colon. (A) Schematic of experimental outline (B) CFT imaging of SNA distribution in GI tissues. Each row is an individual animal. (C)Fluorescence microscopy imaging of SNA distribution in GI tissues. Representative images from one animal in each group are shown. B C A CONCLUSIONS Orally delivered SNAs: Accumulate throughout the GI tract in mice Show target mRNA knockdown Produce clinical improvement in the TNBS-induced IBD mouse model, including increased survival, increased body weight, reduced IBD clinical score, and reduced gross pathology These data support the therapeutic potential of orally delivered SNAs for treating GI diseases www.exicuretx.com Jejunum Ileum Duodenum Proximal Distal P.Cecum D.Cecum M.Colon D.Colon (1) D.Colon (2) No SNA 2 hours 10 hours DAPI Cy5-SNA Jejunum Ileum Duodenum Proximal Distal 1200 8 Normalized Corrected Counts P.Cecum D.Cecum M.Colon D.Colon (1) D.Colon (2) No SNA 2 hours 10 hours
Transcript of α No SNA
Spherical Nucleic Acids: Oral Delivery and Efficacy in TNBS-induced IBD ModelBart R. Anderson, SubbaRao Nallagatla, Richard S. Kang, Matthias Schroff, and Ekambar R. Kandimalla
Exicure, Inc., 8045 Lamon Avenue, Skokie, IL 60077
INTRODUCTION
Spherical nucleic acids (SNAs) are a novel class of therapeutic molecules
consisting of densely packed oligonucleotides arranged radially around a spherical
nanoparticle core. As a consequence of their 3-dimensional structure, SNAs have
increased cellular uptake compared with the same oligonucleotide sequence in the
conventional linear format.
SNA delivery and activity have been previously demonstrated in various
tissues including topical administration to the skin, aerosol delivery to the lung, and
intracerebral ventricular injection to the central nervous system. Here we assessed
the delivery, biological activity, and disease-modifying activity of an antisense SNA
targeted to murine TNFα mRNA (anti-mTNFα SNA) in gastrointestinal (GI) tissues. SNA structure
Figure 2. Effect of anti-TNFα SNA on body weight following TNBS-induction of IBD in
mice.
Body weight of mice following IBD induction and treatment with vehicle or anti-mTNFα SNA.
Mean + SEM is shown. ** P-value < 0.01 vs Vehicle on each D1-D7, 2-way ANOVA with
Holm-Sidak multiple test correction.
Anti-mTNFα SNA Treatment Improves Body Weight
0 2 4 6
1 5
1 6
1 7
1 8
1 9
S tu d y d a y
Bo
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igh
t (g
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N aive
V e h ic le
a n ti-m T N F S N A , 2 0 0 µ g
a n ti-m T N F S N A s , 1 0 0 0 µ g
7
**
Figure 3. Survival of TNBS-induced IBD mice following anti-mTNFα SNA treatment.
Survival curves of mice following IBD induction and treatment with vehicle or anti-mTNFα SNA.
Anti-mTNFα SNA Treatment Improves Survival
0 2 4 6 8
0
5 0
1 0 0
S tu d y d a y
Su
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V e h ic le
a n ti-m T N F S N A , 2 0 0 µ g
a n ti-m T N F S N A , 1 0 0 0 µ g
A
B
Anti-mTNFα SNA Treatment Reduces TNFα mRNA in Colon
Tissues
Figure 6. TNFα mRNA knockdown by anti-mTNFα SNA.
The proximal and distal colons of mice were collected on study day 7, washed with PBS, and
flash frozen. Total mRNA was extracted from homogenized tissues and cDNA was generated
for qRT-PCR analysis. The levels of TNF mRNA were quantified and normalized to GAPDH
using the 2-ΔΔCt method. Mean + SEM of n=4 (day 4) or n=5 – 12 (day 7 surviving animals) is
shown.
4 7
0
2 0 0
4 0 0
6 0 0
P r o x im a l C o lo n
S tu d y d a y
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)
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a n ti-m T N F S N A , 1 0 0 0 g
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6 0 0
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S tu d y d a y
TN
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mR
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(%
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N a iv e
V e h ic le
a n ti-m T N F S N A , 1 0 0 0 g
Figure 5. IBD gross pathology following anti-mTNFα SNA treatment.
Gross pathology scores in mice following IBD induction and treatment with vehicle or anti-mTNFα SNA. Mean +
SEM is shown. *** P-value < 0.001 vs Vehicle, 1-way ANOVA with Holm-Sidak multiple test correction.
Anti-mTNFα SNA Treatment Reduces Gross Pathology in the Proximal
Colon
0
1
2
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4
Gro
ss
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co
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a n ti-m T N F S N A , 2 0 0 µ g
a n ti-m T N F S N A , 1 0 0 0 µ g
***
Anti-mTNFα SNA Treatment Significantly Improves IBD Clinical Scores
Figure 4. Effect of anti-mTNFα SNA on IBD clinical score.
IBD clinical scores in mice following IBD induction and treatment with vehicle or anti-mTNFα SNA. Mean +SEM
is shown. 2-way ANOVA with Holm-Sidak multiple test correction P-values vs Vehicle: * < 0.05, *** < 0.001, ****
< 0.0001.
0 2 4 6
S tu d y d a y
Cli
nic
al
sc
ore N aive
V e h ic le
a n ti-m T N F S N A , 2 0 0 µ g
a n ti-m T N F S N A , 1 0 0 0 µ g
7
0
2
4
6
8
* *****
***
*
** **** **** **** **** **** ****
TNBS-induced Inflammatory Bowel Disease (IBD) Model
Description
On study day 0, inflammatory bowel disease was induced in female BALB/C mice aged
6 – 7 weeks by intrarectal (i.r.) administration of 2,4,6-Trinitrobenzenesulfonic acid (TNBS),
except mice in the naïve group, in n=12 animals per group. On day 1, prior to treatment with
test articles, animals were randomized into groups by clinical score to give similar means and
distributions of clinical scores across groups. On days 1 – 4, vehicle only or anti-TNFα SNA
was administered once daily by oral gavage at a volume of 200 μL per mouse in 0.1 M
NaHCO3 pH 9.0. Body weight, survival, and clinical score were assessed daily. On day 7 the
surviving animals were sacrificed. Gross pathology assessment was performed on the
proximal colon. GI tissues were flash frozen and subsequently the TNFα mRNA levels were
assessed by qRT-PCR.
From naïve, vehicle, and 1000 μg/dose SNA groups an additional n=4 parallel animals
were removed on day 4 for qRT-PCR assessment of TNFα mRNA levels in GI tissues.
Mice found dead in cage (FDC) were represented through the remainder of the study
using the last measured body weight, the maximal clinical score of 11, and the maximum
gross pathology score of 5. Mice terminated for ethical considerations were represented with
the last measured body weight, last measured clinical score, or the maximum gross
pathology score of 5.
RESULTS
Anti-mTNFα SNA is Distributed Throughout the GI Tract With Increasing Accumulation in
Distal Segments Over Time
Figure 1. Biodistribution in GI tissues of orally
administered SNA
Fluorescently-labeled SNA (Cy5-SNA) was administered
by oral gavage in a volume of 200 μL per mouse in 0.1 M
NaHCO3 pH 9.0 to female C57BL/6 mice aged 6 – 7
weeks, n=2 per treatment group. At 2 hours or 10 hours
following administration, GI tissues were collected,
washed, and imaged using Cryofluorescence Tomography
(CFT) and fluorescence microscopy. In (A) and (B),
P.Cecum and D.Cecum are the proximal and distal
portions of the Cecum, respectively. Similarly, D.Colon (1)
and D.Colon (2) are two most distal portions of the Colon.
(A) Schematic of experimental outline
(B) CFT imaging of SNA distribution in GI tissues. Each
row is an individual animal.
(C)Fluorescence microscopy imaging of SNA distribution
in GI tissues. Representative images from one animal
in each group are shown.
B
C
A
CONCLUSIONS
Orally delivered SNAs:
Accumulate throughout the GI tract in mice
Show target mRNA knockdown
Produce clinical improvement in the TNBS-induced IBD mouse model,
including increased survival, increased body weight, reduced IBD clinical
score, and reduced gross pathology
These data support the therapeutic potential of orally delivered SNAs for
treating GI diseases
www.exicuretx.com
Jejunum IleumDuodenum
Proximal Distal
P.Cecum D.Cecum M.Colon D.Colon (1) D.Colon (2)
No SNA
2 hours
10 hours
DAPI
Cy5-SNA
Jejunum IleumDuodenum
Proximal Distal
1200
8
No
rma
lize
d C
orr
ecte
d C
ou
nts
P.Cecum D.Cecum M.Colon D.Colon (1) D.Colon (2)
No SNA
2 hours
10 hours
