Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle

Article abstract of DOI:10.1093/nar/gkz354

Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma C_max and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.

Full text of DOI:10.1093/nar/gkz354

Published online 25 May 2019
Nucleic Acids Research, 2019, Vol. 47, No. 12 6029–6044
doi: 10.1093/nar/gkz354
Fatty acid conjugation enhances potency of antisense
oligonucleotides in muscle
Thazha P. Prakash1,*, Adam E. Mullick2, Richard G. Lee2, Jinghua Yu1, Steve T. Yeh2,
Audrey Low2, Alfred E. Chappell1, Michael E. Østergaard1, Sue Murray2, Hans J. Gaus1, Eric
1
E. Swayze1 and Punit P. Seth
1Ionis Pharmaceuticals, Medicinal Chemistry, USA and 2Antisense Drug Discovery, 2855 Gazelle Ct., Carlsbad, CA
92010, USA
Received March 22, 2019; Revised April 19, 2019; Editorial Decision April 25, 2019; Accepted April 26, 2019
ABSTRACT
of clinical development (3). Conjugation of tri-antennary
N-acetyl galactosamine enhanced ASO uptake into hepa-
tocytes through ASGR-mediated internalization, improv-
ing hepatocyte potency 10- to 60-fold (4). Recent stud-
ies showed that conjugation of ASOs to a ligand of the
glucagon like peptide-1 receptor (GLP1R) improved ASO
uptake into pancreatic ß-cells and enhanced potency >50-
fold (5). This was remarkable since pancreatic ß-cells are
known to be refractory to ASO uptake (6) and this opens
therapeutic opportunities to treat diseases affecting the pan-
creas such as diabetes. To further expand the utility of an-
tisense technology it is important to improve ASO potency
in additional tissues beyond the liver and ß-cells. Therefore,
delivery approaches which enhance ASO potency in extra-
hepatic tissues will be beneficial.
ASOs show robust gene silencing in extrahepatic tissues
such as muscle after systemic administration but higher
doses are required (6). Development of Ionis DMPK-2.5Rx
was recently discontinued because of inadequate therapeu-
tic benefit in short-term clinical trials in patients with my-
otonic dystrophy type 1 (DM1) (7). We envisage that more
potent ASOs with improved muscle delivery technologies
would ameliorate poor efficacy of this drug in patients.
Previous work showed that cholesterol conjugated ASOs
show increased activity and cellular association through
LDL receptor mediated endocytosis (8,9). Wolfrum et al.
conjugated cholesterol and variety of lipids to siRNA and
demonstrated that long-chain fatty acids and cholesterol
can facilitate siRNA uptake into cells for effective gene si-
lencing in mice (10). This group also showed that efficient
and selective uptake of siRNA conjugates depends on inter-
action with lipoprotein particles, lipoprotein receptors and
other cell-surface receptors (10). There are additional exam-
ples where lipid conjugation improved potency of siRNA
and ASOs in cells (11) and in mice (12–15). Cholesterol con-
jugated siRNA also inhibited myostatin mRNA expression
in skeletal muscle after systemic administration (16).
Enhancing the functional uptake of antisense
oligonucleotide (ASO) in the muscle will be beneficial
for developing ASO therapeutics targeting genes ex-
pressed in the muscle. We hypothesized that improv-
ing albumin binding will facilitate traversal of ASO
from the blood compartment to the interstitium of
the muscle tissues to enhance ASO functional up-
take. We synthesized structurally diverse saturated
and unsaturated fatty acid conjugated ASOs with a
range of hydrophobicity. The binding affinity of ASO
fatty acid conjugates to plasma proteins improved
with fatty acid chain length and highest binding affin-
ity was observed with ASO conjugates containing
fatty acid chain length from 16 to 22 carbons. The de-
gree of unsaturation or conformation of double bond
appears to have no influence on protein binding or
activity of ASO fatty acid conjugates. Activity of fatty
acid ASO conjugates correlated with the affinity to
albumin and the tightest albumin binder exhibited
the highest activity improvement in muscle. Palmitic
acid conjugation increases ASO plasma Cmax and im-
proved delivery of ASO to interstitial space of mouse
muscle. Conjugation of palmitic acid improved po-
tency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to
7-fold) in mouse muscle. Our approach provides a
foundation for developing more effective therapeutic
ASOs for muscle disorders.
INTRODUCTION
Nucleic acid-based therapeutics represent a distinct drug-
discovery platform with the ability to target genes linked
to disease that are considered undruggable by classic
small molecule approaches (1–3). Several nucleic-acid based
drugs are currently approved for clinical use or in late stage
Skeletal and cardiac muscle cells rely heavily on the ox-
idation of long-chain fatty acids for contractile work (17).
*To whom correspondence should be addressed. Tel: +1 760 603 2590; Fax: +1 760 603 2600; Email: tprakash@ionisph.com
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C
The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

6030 Nucleic Acids Research, 2019, Vol. 47, No. 12
Fatty acids are transported to muscle tissue via the blood
either complexed to albumin or covalently bound in tri-
acylglycerols forming the neutral lipid core of circulating
triglyceride-rich lipoproteins such as chylomicrons or very
low-density lipoproteins (17). The capillary endothelium
represents one of the first barriers fatty acids have to tra-
verse on their way from the vascular compartment to skele-
tal and cardiac muscle cells (17). The mechanism responsi-
ble for transmembrane movement of fatty acids is incom-
pletely understood, however recent studies have revealed
that interaction of the albumin-fatty acid complex with the
endothelial membrane may facilitate fatty acid uptake (17).
Serum albumin is a transport protein for endogenous
fatty acids. Albumin is the most abundant plasma protein
in human blood (35–50 g/l, Molecular weight 66.5 kDa)
(18) and it is synthesized in the liver and released into the
vascular space (19). Albumin interacts with multiple cellu-
lar receptors such as glycoprotein Gp60, Gp30 and Gp18a,
the Megalin/Cubilin complex, and the neonatal Fc recep-
tor (FcRn) (20). The interaction with these receptors are
responsible for albumin’s recycling, transcytosis and ex-
tended half-life (∼19 days) in circulation (20). Albumin con-
tains multiple hydrophobic pockets that bind fatty acids and
steroids as well as different drugs (20). Palmitic acid conju-
gated glucagon-like type-1 (GLP-1) agonist liraglutide (21)
and Myristic acid conjugated detemir (22) also utilize the
fatty-acid binding properties of albumin to improve their
pharmacology.
ASOs with PS linkages are known to bind serum albumin
with dissociation constants in the low micromolar range
(23). We hypothesized that fatty acid conjugation will im-
prove the binding of PS ASO with serum albumin and fa-
cilitate ASO transport across the continuous capillary en-
dothelial in the skeletal and cardiac muscle (24). In this
report, we describe results from our detailed investigation
of using fatty acid conjugation to enhance ASO activity in
muscle. We show that conjugation of palmitic acid enhances
affinity of PS ASOs for serum albumin and that these con-
jugates show enhanced potency in muscle tissues. We also
report the detailed SAR of fatty acid-ASO conjugates to
identify the optimal fatty acid for enhancing ASO potency.
F
F
F
F
F
O
O
Pfp-TFA, Solvent, rt
R
R
OH
O
3, 15-21
4, 22-28
5'
3'
O
O
H2N
P
O
OH
5, 7, 70, 73, 76
sodium tetraborate, pH 8.5, TEA, DMSO, rt
5'
O
3'
R
O
O
N
P
H
O
OH
2, 6, 8-14, 69, 72, 75
3, 4, 2 6
,
,
R:
69, 72, 75
15, 22, 8
R:
R:
R:
16, 23, 9
17, 24, 10
18, 25, 11
19, 26, 12
R:
R:
20, 27, 13
21,28, 14
R:
R:
Scheme 1. Synthesis of 5ꢁ-fatty acid conjugated ASO 2, 6, 8–14, 69,
72 and 75; ASO 5: 5ꢁ-TmCAGkmCkAk TdTd mCd TdAdAdTdAd
GdmCd AkGkmCk 3ꢁ, ASO 7: GkmCk AkTdTd mCd TdAdAdTdAd
GdmCd AkGkmCk, ASO 70: 5ꢁ- TdmCdAdAkGkGkAdTd AdTd
GdGdAdAdmCdmCd AkAkAk-3ꢁ, ASO 73: 5ꢁ-AkmCkAk AdTd AdAd
AdTdAd mCd mCdGd AkGkGk-3ꢁ, ASO 76: 5ꢁ-mCkmCk mCk Td TdTdAd
TdTd Gd mCdAdGdmCkAkmCk, k: cEt BNA, d: DNA, mC: 5-methyl
cytidine, Backbone all PS, underline PO.
General method for the synthesis of fatty acid ASO conju-
gates 2, 6, 8–14, 29, 32–43, 69, 72, 75
MATERIALS AND METHODS
To a solution of 5ꢁ-hexylamino ASOs 5, 7, 70, 73, 76
(Schemes 1 and 2) in 0.1 M sodium tetraborate buffer, pH
8.5 (2 mM) a solution of fatty acid PFP ester 4, 22–28, 31,
56–67 (3–9 mole equivalent, Scheme 1 and 2) dissolved in
DMSO (40 mM), acetonitrile (20 mM) and triethylamine
was added and the reaction mixture was stirred at room
temperature for 3–18 h. The reaction mixture was diluted
with water and purified by HPLC on a strong anion ex-
change column (GE Healthcare Bioscience, Source 30Q, 30
␮m, 2.54 × 8 cm, A = 100 mM ammonium acetate in 30%
aqueous CH3CN, B = 1.5 M NaBr in A, 0–60% of B in
60 min, flow 14 ml min−1). HPLC fractions containing full
length ASO conjugates (analyzed by LC MS) were pooled
together and diluted three fold volume with water and de-
salted by HPLC on a reverse phase column to yield the 5ꢁ-
fatty acid conjugated ASOs 2, 6, 8–14, 29, 32–43, 69, 72,
75 in an isolated yield of 50–78%. The ASOs were charac-
terized by ion-pair-HPLC–MS analysis with Agilent 1100
MSD system (Supporting Information).
General method for the synthesis of fatty acid pentafluo-
rophenyl esters 4, 22–28, 31, 56–67
Fatty acids 3, 15–21, 30, 44–55 (1 mmol, Schemes 1 and
2), TEA (2.25 ml, 16 mmol) were dissolved in DCM (1
ml/mmol) and pentafluorophenyl trifluoroacetate (4 mmol)
was added. Stirred the reaction mixture at room tem-
perature for 1 h. The reaction mixture was diluted with
dichloromethane (6 ml/mmol) and washed with aqueous
saturated NaHCO3 (5 ml/mmol) solution and 1 N NaHSO4
(5 ml/mmol) solution. The organic layer was separated,
dried (Na2SO4), filtered and concentrated under reduced
pressure. The crude product was purified by silica gel col-
umn chromatography and eluted with solvents (See Sup-
porting Information) to yield 4, 22–28, 31, 56–67 in 70–82%
isolated yield. All the compounds were characterized by 1H
and 13C NMR spectroscopic analysis (Supporting Informa-
tion).

Nucleic Acids Research, 2019, Vol. 47, No. 12 6031
a clear solution. To this solution, tetrazole (1.15 g, 16.40
mmol) and 1-methylimidazole (0.41 ml, 5.13 mmol) were
added and the reaction mixture was cooled in an ice bath
(Note: solution became cloudy). 2-Cyanoethyl-N,N,N’,N’-
tetraisopropylphosphorodiamidite (9.78 ml, 30.8 mmol)
was added and the reaction mixture was removed from
the ice bath and stirred at room temperature for 12 h.
The reaction mixture was then diluted with ethyl acetate
(50 ml), washed with saturated NaHCO3 solution (50 ml)
followed by brine (50 ml), and then dried (Na2SO4), fil-
tered and concentrated under reduce pressure. The residue
obtained was purified by silica gel column chromatogra-
phy and eluted with 30% ethyl acetate in hexanes contain-
ing 1% triethylamine to yield 77 (10.7 g, 93%). 31P NMR
(121MHz, CDCl3) ␦: 147.49; HRMS (ESI) m/z calcd for
C33H66N3O3P [M + H]+ 584.4915, found 584.4943.
F
F
F
F
F
O
O
Pfp-TFA, Solvent, rt
R
R
OH
O
30, 44-55
31, 56-67
5'
3'
O
O
H2N
P
O
OH
7
sodium tetraborate, pH 8.5, TEA, DMSO, rt
O
5'
3'
R
O
O
N
H
P
O
OH
29, 32-43
29, 30, 31
32, 44, 56
R:
R:
Fluorescence polarization assay
Fluorescence polarization experiments were performed us-
ing ALEXA647-labeled ASOs. Measurements were per-
formed in 1× phosphate-buffered saline (PBS). The assay
was setup in 96-well Costar plates (black flat-bottomed
non-binding) purchased from Corning, NY, USA. Bind-
ing was evaluated by adding ALEXA 647-labeled ASOs to
yield 2 nM concentration to each well containing 100 ␮l of
protein from sub nM to low mM concentration. Readings
were taken using the Tecan (Baldwin Park, CA, USA) In-
finiteM1000 Pro instrument (␭ex = 635 nm, ␭em = 675 nm).
Using polarized excitation and emission filters, the instru-
ment measures fluorescence perpendicular to the excitation
plane (the ‘P-channel’) and fluorescence that is parallel to
the excitation plane (the ‘S-channel’), and then it calculates
FP in millipolarization units (mP) as follows: mP = [(S – P *
G)/(S + P × G)] × 1000. The ‘G-factor’ is measured by the
instrument as a correction for any bias toward the P chan-
nel. Polarization values of each ALEXA 647-labeled ASO
in 1× PBS at 2 nM concentration were subtracted from
each measurement. Kd values were calculated with Graph-
Pad Prism 5 software (GraphPad Software, La Jolla, CA,
USA) using non-linear regression for curve fit assuming one
binding site.
33, 45, 57
34, 46, 58
R:
R:
35, 47, 59
36, 48, 60
37, 49, 61
38, 50, 62
39, 51, 63
40, 52, 64
R:
R:
R:
R:
R:
R:
41, 53, 65
42, 54, 66
43, 55, 67
R:
R:
R:
Scheme 2. Synthesisof 5’-unsaturated fatty acid conjugated ASO 29, 32-
43.
N-(6-hydroxyhexyl)palmitamide 78
To a solution of palmitic acid 3 (10.0 g, 39.0 mmol) and
triethylamine (16.3 ml, 117.0 mmol) in dichloromethane
(800 ml) pentafluorophenyl trifluoroacetate (10.1 ml, 58.5
mmol) was added dropwise at room temperature. To this 6-
aminohexanol 79 (5.48 g, 46.8 mmol) and dichloromethane
(200 ml) were added. The reaction mixture was stirred at
room temperature for 12 h. Compound 78 (11.6 g, 84%)
precipitated from the solution and collected by filtration.
1H NMR (300MHz, CDCl3) ␦: 5.42 (br s, 1H), 3.65 (t, J =
6.3 Hz, 2H), 3.29-3.23 (m, 2H), 2.16 (t, J = 6.3 Hz, 2H),
1.70–1.46 (m, 6H), 1.45–1.19 (m, 28H), 0.89 (t, J = 6.3 Hz,
3H); 13C NMR (75MHz, CDCl3) ␦: 173.11, 62.71, 39.24,
36.95, 32.56, 31.91, 29.70, 29.68, 29.64, 29.61, 29.49, 29.35,
29.32, 26.49, 25.82, 25.27, 22.68, 14.09. LRMS (ESI) m/z
calcd for C22H46NO2 [M + H]+ 356.6, found 356.3.
Animal treatment
Animal experiments were conducted in accordance with the
American Association for the Accreditation of Laboratory
Animal Care guidelines and were approved by the Animal
Welfare Committee (Cold Spring Harbor Laboratory’s In-
stitutional Animal Care and Use Committee guidelines).
The animals were housed in micro-isolator cages on a con-
stant 12 h light–dark cycle with controlled temperature and
humidity and were given access to food and water ad libi-
tum. Blood was collected by cardiac puncture exsanguina-
tion with K2-EDTA (Becton Dickinson Franklin Lakes, NJ,
USA) and plasma separated by centrifugation at 10 000 rcf
for 4 min at 4◦C. Plasma transaminases were measured us-
ing a Beckman Coulter AU480 analyzer. Tissues were col-
lected, weighed, flash frozen on liquid nitrogen and stored
at −60◦C. Reduction of target mRNA expression was deter-
mined by real time RT-PCR using StepOne RT–PCR ma-
chines (Applied Biosystems). Briefly, RNA was extracted
2-Cyanoethyl (6-palmitamidohexyl) diisopropylphospho-
ramidite 77
To compound 78 (7.3 g, 20.5 mmol) DMF (50 ml) and THF
(40 ml) were added. The mixture was heated at 50◦C to get

6032 Nucleic Acids Research, 2019, Vol. 47, No. 12
primer: 5ꢁ-CACGAAT GAGG TCCTG AGCTT-3ꢁ and 5ꢁ
fluorescein and 3ꢁ TAMRA probe 5ꢁ-AACACTTGT CG
CTG CCGCTGGC. Target RNA levels were normalized
from ∼50 to 100 mg tissue from each mouse using Pure-
Link Pro 96 Total RNA Purification Kit (LifeTechnologies,
Carlsbad, CA, USA) and mRNA was measured by qRT-
PCR using Express One-Step SuperMix qRT-PCR Kit (Life
Technologies, Carlsbad, CA, USA). Primers and probes for
the PCR reactions were obtained from Integrated DNA
technologies (IDT). The assay is based on a target-specific
probe labeled with a fluorescent reporter and quencher dyes
at opposite ends. The probe is hydrolyzed through the 5ꢁ-
exonuclease activity of Taq DNA polymerase, leading to
an increasing fluorescence emission of the reporter dye that
can be detected during the reaction. Target RNA levels
were normalized to cyclophilin mRNA expression or total
mRNA as measured by Ribogreen.
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C
to total RNA (Ribogreen assessment).
Caveolin-3 (Cav3) mouse protocol
ASOs 74–75 administrated subcutaneously to 6- to
8-week old male C57BL/6 mice at 1.68, 5 and 15
␮mol/kg once a week for two weeks. Heart and quadri-
ceps were harvested after 4 days after the last dose.
Cav3 mRNA was quantitated using Invitrogen Express
One-Step qRT-PCR kit using Cav3 forward primer: 5ꢁ-
CATCAAGGACATTCACTGCAAG-3ꢁ, reverse primer:
5ꢁ-CTCCGCAATCACGTCTTCA-3ꢁ and probe 5ꢁ-
AACCGCGACCCCAAGAACATCA-3ꢁ with 5ꢁ fluores-
cein and 3ꢁ TAMRA. Target RNA levels were normalized
Malat-1 mouse protocol
Malat-1 ASOs 1–2, 6, 8–14, 29 and 32–43 subcutaneously
administrated to 8-week-old male C57BL/6 mice (Jack-
son Laboratories) at various doses. RNA was extracted
using Invitrogen PureLink Pro 96 Total RNA purifica-
tion kit after 5 days of last dose, mice were sacrificed
for mouse heart, quadriceps, liver and kidney Malat-
1 RNA quantification. PCR was done using Invitro-
gen Express One-Step qRT-PCR kit using primer probe
set MALAT1 5ꢁ-TGGGTTAGAGAAGGCGTGTACTG-
3ꢁ for the forward primer, 5ꢁ-TCAGCGGCAACTGGG
AAA-3ꢁ for the reverse primer, and 5ꢁ- CGTTGGCAC-
GACACC TTCAGGGACT-3ꢁ for the probe. Target RNA
levels were normalized to cyclophilin mRNA expression.
The sequences for the primers and probe used for mouse
Cyclophilin A are 5ꢁ-TCGCCGCTTGCTGCA-3ꢁ for the
forward primer, 5ꢁ-ATCGGCCGTGATGTCGA-3ꢁ and 5ꢁ-
CCATGGTCAACCCCACCG TGTTCX-3ꢁ for the probe
with 5ꢁ fluorescein and 3ꢁ TAMRA.
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C
to total RNA (Ribogreen assessment).
ED50 determination
ED50 values were determined with GraphPad Prism 5 soft-
ware. The log dose of ASOs were plotted against mRNA
level relative to untreated controls. The curves obtained
were fitted using a four-parameter fit with variable slope and
constraining bottom = 0 and top = 1.
Early distribution mouse protocol
Twelve-week-old male C57BL/6 mice were administered
7.5 ␮mol/ kg ASO subcutaneously and sacrificed (n = 2/
ASO) at 0.5, 1, 2, 4, 8 and 24 h. Plasma was collected by
cardiac puncture on K2-EDTA. Systemic tissues including
heart were collected at necropsy and immediately frozen on
dry ice. Concentration of ASOs in heart were determined
using the protocol describe below. Immunohistochemistry
analysis was done following the protocol reported in the lit-
erature (6).
CD36 mouse protocol
CD36 ASOs 68–69 intravenous administrated to six to
8-week-old female C57BL/6 mice (Jackson Laborato-
ries) at 1, 3 and 9 ␮mol/kg once a week for 3 weeks.
Mice liver, kidney, heart and quadriceps were harvested
5 days after the last dose for CD36 mRNA quantita-
tion using Invitrogen Express One-Step qRT-PCR kit.
The sequences for the primers and probe used for mouse
CD36 are 5ꢁ-TCCAGCCAATGCCTTTGC-3ꢁ for the for-
ward primer, 5ꢁ-GAGATTACTTTTTCAGTGCAGAA-3ꢁ
for the reverse primer, and 5ꢁ-TCACCCCTCCAGAA
TCCAGACAACCAT-3ꢁ for the probe with 5ꢁ fluorescein
and 3ꢁ TAMRA. Target RNA levels were normalized to to-
Extraction and quantitation of ASO in tissues and plasma by
LCMS
ASO was extracted from plasma and tissues using pre-
viously described methods using phenol/chloroform fol-
lowed by solid phase extraction using phenyl-functionalized
support (25). Tissues were minced and 50–200-mg or 50–
100 ␮l plasma samples were homogenized in 500 ␮l ho-
mogenization buffer (0.5% NP40 substitute (Sigma-Aldrich
St. Louis, MO, USA) in Tris-buffered saline, pH 8) with
Lysing Matrix D beads (MPBio Santa Ana, CA, USA)
on a ball mill homogenizer (Retsch Haan, Germany) at
30 Hz for 45 s. Standard curves of each ASO were estab-
lished using 500 ␮l aliquots of control tissue homogenate
(50–200-mg tissue or 50–100 ␮l plasma/ml homogenization
buffer). A 27-mer, fully PS, MOE/DNA oligonucleotide
was added as an internal standard (IS) to all standard
curves and study samples. Samples and curves were ex-
tracted with phenol/chloroform followed by solid-phase
extraction (SPE) of the resulting aqueous extract using
phenyl-functionalized silica sorbent (Biotage, Upsalla, Swe-
den). Eluate from SPE was dried down using a warm forced-
air (nitrogen) evaporator and reconstituted in 100–200 ␮l
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C
tal RNA (Ribogreen assessment).
DMPK mouse protocol
ASOs administrated subcutaneously to 6-week-old
male Balb/c mice at ASO 71 at 1.84, 3.68 and 7.36
␮mol/kg and ASO 72 at 0.85, 1.70 and 3.40 ␮mol/kg
once a week for 3.5 weeks. Heart and quadriceps
were harvested after 2 days after the last dose. DMPK
mRNA was quantitated using Invitrogen Express One-
Step qRT-PCR kit using DMPK forward primer: 5ꢁ-
GACATATGCCAAGATTGTGCACTAC-3ꢁ,
reverse

Nucleic Acids Research, 2019, Vol. 47, No. 12 6033
100 ␮M EDTA. Extracts were then analyzed and ASO con-
centration determined by LC–MS using a method simi-
lar to that described by Gaus et al. (26). Briefly, separa-
tion was accomplished using an 1100 HPLC–MS system
(Agilent Technologies, Wilmington, DE, USA) consisting
of a quaternary pump, UV detector, a column oven, an
autosampler and a single quadrupole mass spectrometer.
Samples were injected on an X-bridge OST C18 column (2.1
× 50 mm, 2.5-␮m particles; Waters, Milford, MA, USA)
equipped with a SecurityGuard C18 guard column (Phe-
nomenex, Torrance, CA, USA). The columns were main-
tained at 55◦C. Tributylammonium acetate buffer (5 mM)
and acetonitrile were used as the mobile phase at a flow
rate of 0.3 ml/min. Acetonitrile was increased as a gradi-
ent from 20 to 70% over 11 min. Mass measurements were
made online using a single quadrupole mass spectrometer
scanning 1000–2100 m/z in the negative ionization mode.
Molecular masses were determined using the ChemStation
analysis package (Agilent, Santa Clara, CA, USA). Man-
ual evaluation was performed by comparing a table of cal-
culated m/z values corresponding to potential metabolites
with the peaks present in a given spectrum. Peak areas from
extracted ion chromatograms were determined for ASOs
and IS and a trendline established using the calibration
standards, plotting concentration of ASO against the ratio
of the peak areas ASO/IS. Concentration of ASOs in study
samples were determined using established trendlines and
reported as ␮g ASO/g tissue.
A
B
RESULTS
Palmitoyl conjugation improves plasma protein binding of cEt
BNA ASO
Figure 1. Characterizing the effect of palmitoyl conjugation on binding to
selected plasma protein of Gapmer ASOs. (A) Binding curves showing dif-
ferences between binding of unconjugated Malat-1 ASO 1 and palmitic
acid conjugated ASO 2. (B) Binding constants for ASO 1 and 2 to se-
lected plasma proteins; NB: no binding; HSA: human serum albumin;
HDL: high-density lipoprotein; LDL: low-density lipoprotein; HRG: his-
tidine rich glycoprotein; ASO sequence blue: cEt BNA, black: DNA, C:
5-methylcytidine, backbone all PS; underline PO.
Phosphorothioate (PS) ASOs are known to bind plasma,
cell surface and intra-cellular proteins which facilitate dis-
tribution, cellular uptake and intracellular trafficking to pe-
ripheral tissues (27). Plasma protein bound ASOs circulate
transiently in the blood compartment and partition onto
cell surface proteins and enter cells by endocytosis (28).
Palmitic acid is known to bind to albumin and conjugation
of palmitic acid to PS ASO is expected to improve albumin
binding. To characterize the effect of palmitic acid conju-
gation on ASO binding to plasma proteins we synthesized
5ꢁ-plamitic acid conjugated 3–10–3 cEt BNA Gapmer ASO
1 (Figure 1) targeting the metastasis associated lung ade-
nocarcinoma transcript 1 (Malat-1) (29). Malat-1 is an evo-
lutionary conserved noncoding RNA gene highly expressed
in many tissues (6). Synthesis of 5ꢁ-palmitic acid conjugated
ASO 2 (Figure 1) is described in Scheme 1. The palmitoyl
ASO 2 was fully characterized by ion-pair LC–MS analysis
(Supporting Information).
To characterize the interaction of palmitoyl ASO 2 with
albumin, we determined the binding constants using a flu-
orescence polarization assay (30). In addition, we also an-
alyzed binding affinity of palmitic acid conjugates to HDL
(high-density lipoproteins), LDL (low-density lipoproteins)
and HRG (histidine rich glycoprotein). Palmitoyl conjuga-
tion improved the binding affinity of PS ASOs to albumin
>150-fold (Figure 1). Interestingly, palmitic acid conjuga-
tion also enhanced affinity of the ASO to HDL and LDL
while no change in affinity was observed for HRG (Figure
1).
Palmitoyl conjugation improves potency of cEt BNA Malat-1
ASO 3–6-fold in muscle
We next examined the effect of palmitoyl conjugation on
potency of a 3–10–3 cEt BNA ASO 2 (Figure 2) targeting
Malat-1 RNA to understand the broader effect of palmi-
toyl conjugation. The potency of ASO 1 and 2 to inhibit
Malat-1 RNA was evaluated in mice. Mice (C57BL/6, n =
4/group) were injected subcutaneously with 0.4, 1.2, and 3.6
␮mol/kg of ASOs 1 or 2 for 3 weeks. Five days following
the last injection, mice were sacrificed and heart, quadri-
ceps, livers and kidney were homogenized and analyzed for
Malat-1 RNA expression. The 5ꢁ-palmitoyl Malat-1 ASO 2
showed improved potency relative to unconjugated ASO 1
in quadriceps (3-fold, Figure 2) and heart (>6 fold, Figure
2). However, no potency improvement was observed in liver
and kidney. Malat-1 ASOs 1 and 2 were well tolerated with
no elevations in plasma transaminases or organ weights.

6034 Nucleic Acids Research, 2019, Vol. 47, No. 12
Figure 2. Palmitic acid conjugation enhances potency of Malat-1 ASO in mouse heart and quadriceps. Mice (C57BL/6, n = 4/group) were injected
subcutaneously Malat-1 ASO 1 and 5ꢁ-palmitoyl conjugated Malat-1 ASO 2 at 0.4, 1.2 and 3.6 ␮mol/kg once a week for 3 weeks for a total of three doses
and sacrificed after 5 days. (A) Malat-1 RNA expression analyzed in mice heart, quadriceps, liver and kidney using qRT-PCR. All data are expressed as
mean standard deviation. (B) ED50 (␮mol/kg/week) for reducing Malat-1 RNA in mouse heart, quadriceps and liver.
The tissue concentration of Malat-1 ASOs (1,2) in heart,
quadriceps and liver of treated mice were quantitated (Table
1) (31,32). Palmitoyl conjugation improved accumulation of
Malat-1 ASO in heart (2–4-fold, Table 1) and quadriceps
(2-fold, Table 1) relative to unconjugated ASO 1. This data
suggest that improved potency observed for palmitoyl ASO
2 is due to higher uptake of ASO in these tissues. To de-
termine the metabolic fate of the 5ꢁ-pamitoyl ASO in heart,
quadriceps and liver tissues from the mice treated with ASO
2 were homogenized and the ASO and metabolites were ex-
tracted and identified by LCMS. Interestingly, very little in-
tact ASO 2 was extracted from tissues. The major metabo-
lite isolated from the tissues corresponds to unconjugated
ASO (data not shown) suggesting that the 5ꢁ-palmitoyl was
metabolized liberating free ASO in the heart, quadriceps
and liver.
4-fold from 222 30 ␮g•h/mL to 915 153 ␮g•h/ml (Fig-
ure 3A). Similarly, Cmax in heart increased nearly two-fold
from 67- to 119 ␮g/g and AUC more than doubled from 682
41 ␮g•h/g to 1676 118 ␮g•h/g (Figure 3B) for uncon-
jugated and palmitate-conjugated ASO respectively. Histo-
logical examination indicates that much of the ASO accu-
mulated in heart (Figure 3C) at early time points appears
diffuse or concentrated along intercellular boundaries sug-
gesting it primarily resides in the interstitial space, however,
by 24 h, the ASO appears in punctate foci, likely occupying
intracellular vesicles. This increase in ASO exposure in the
heart correlates with a 6-fold increase in potency of palmi-
toyl ASO 2 compared to unconjugated ASO 1 in the heart.
Linker SAR to identify optimal linker strategy for palmitoyl
conjugate
We next studied the early distribution pharmacokinet-
ics of ASO 1 and palmitic acid conjugate ASO 2 in mice.
Mice (C57BL/6) were administered 7.5 ␮mol/kg ASOs 1
or 2 subcutaneously and sacrificed at 0.5, 1, 2, 4, 8, and 24
h. Plasma and heart tissues were was collected and ASO
concentration was analyzed by LCMS (25,26). Conjugation
with palmitic acid increased plasma Cmax of ASO nearly 3-
fold, from 74 ␮g/ml for unconjugated ASO to 215 ␮g/ml
for palmitate-conjugated ASO and increased plasma AUC
In our initial linker strategy, we attached the palmitoyl moi-
ety using a phosphodiester d(TCA) linker which is metab-
olized in tissues to release the ASO. To further probe the
importance of the PO d(TCA) linker moiety on potency,
we evaluated ASO conjugate 6 where the fatty acid was di-
rectly attached to the ASO using a phosphodiester-linked
hexylamino linker (Figure 4). 5ꢁ-Hexylamino Malat-1 ASO
7 (Scheme 1) with a PO linker was synthesized according to
the reported procedure (33). The 5ꢁ-hexylamino ASO 7 was

Nucleic Acids Research, 2019, Vol. 47, No. 12 6035
Table 1. Concentration of Malat-1 ASO 1 and 5ꢁ-palmitoyl Malat-1 ASO 2 in the heart, quadriceps and liver
ASO No
Heart tissue concentration (␮g/g)
Quadriceps tissue concentration (␮g/g)
Liver tissue concentration (␮g/g)
0.4 ␮mol
1.2 ␮mol
3.6 ␮mol
kg−1
0.4 ␮mol
1.2 ␮mol
3.6 ␮mol
0.4 ␮mol
1.2 ␮mol
3.6 ␮mol
kg−1
kg−1
kg−1
kg−1
kg−1
kg−1
kg−1
kg−1
1
2
0.12 0.1
0.40 0.1
0.53 0.1
2.13 0.9
2.7
6.53 2.9
1.2
-
-
0.53 0.53
0.80 0.4
1.67 0.33
2.80 1.13
2.76 0.07
4.82 1.72
8.28 1.03
19.31 3.45
23.10 2.07
43.45 8.62
A
B
Plasma
Heart
300
200
100
0
1
2
150
1
2
100
50
0
0.1
1
10
100
0.1
1
10
100
Time (h)
Time (h)
C
Heart ASO IHC
ASO 1
ASO 2
1h
2h
4h
8h
24
Figure 3. Pharmacokinetics of Malat-1 ASO 1 and 5ꢁ-palmitoyl conjugated Malat-1 ASO 2; mice plasma total ASO concentration (A) and mice heart
tissue total ASO concentration (B) after 0.5, 1, 2, 4, 8 and 24 h of subcutaneous administration of ASOs 1–2 (7.5 ␮mol kg−1); (C) ASO distribution in
mice heart tissue after 1, 2, 4, 8 and 24 h of subcutaneous administration ASOs 1–2 (7.5 ␮mol kg−1) analyzed for ASOs by immunohistochemistry (IHC)
using a PS-oligonucleotide antibody. Shown is a representative example of one of the four animals per group.
Fatty acid structure activity relation (SAR) to identify op-
timal fatty acid strategy for enhancing potency of ASO in
muscle and heart
reacted with pentafluorophenyl palmitate 4 to yield ASO
6 and was fully characterized by ion-pair LC–MS analysis
(Supporting Information).
Mice (C57BL/6, n = 4/group) were injected subcuta-
neously with 0.4, 1.2 and 3.6 ␮mol/kg of ASOs 1, 2 and 6
for three weeks. Mice were sacrificed after 5 days and heart
and liver Malat-1 RNA expression were analyzed. ASO 2
with a PO d(TCA) (Figure 4) linker and ASO 6 with a PO
linker (Figure 4) showed similar ASO activity in heart and
liver. This data suggests that the PO d(TCA) linker between
the fatty acid and the ASO is not required and just a PO
linkage is sufficient. According to this data we selected the
PO linked design chemistry for further characterization of
fatty acid conjugated ASOs.
Binding of fatty acids with varying chain length to albumin
showed that primary association constant increased with
chain length (34). Moreover, the number of high-affinity
sites also increased with chain length, octanoate (C8) and
decanoate (C10) bind to one site while longer chain fatty
acid such as laurate (C12) and myristate (C14) bind to two
sites (34). To investigate the effect of fatty acid hydropho-
bicity on the functional uptake of ASO we studied in vivo
activity and plasma protein binding of eight fatty acid con-
jugates (Figure 5) with varying chain length.
We synthesized fatty acid conjugated ASOs 8–14 target-
ing Malat-1 RNA employing the similar solution phase

6036 Nucleic Acids Research, 2019, Vol. 47, No. 12
fully characterized by ion-pair LC–MS analysis (Support-
ing Information).
We examined the plasma protein binding of ASOs 8–
14 using a reported fluorescence polarization (FP) com-
petition binding assay (35) which measures the change in
FP upon displacement of a 5ꢁ-palmitoyl ASO fluorophore
tracer from human albumin, LDL or HDL. Interestingly,
all fatty acids, regardless of chain length, improved plasma
protein binding of ASO to albumin, LDL and HDL (Figure
5) relative to unconjugated ASO 1. Albumin binding affini-
ties of ASO fatty acid conjugates (8-11) with chain length
C8–C14 was 2–5-fold less than ASO conjugated with chain
length C16-C22 (6, 12-14). A similar trend was observed for
the binding affinities of fatty acid conjugates 6 and 8–14 to
LDL and HDL. ASO conjugates with fatty acids containing
longer chain lengths of C12 to C22 (6, 10-14) exhibited im-
proved binding to HDL and LDL proteins relative to ASO
8–9 containing fatty acid chain length C8 to C10.
We next tested the activity of ASOs 8–14 (Figure 5) for
inhibiting Malat-1 RNA expression in mouse quadriceps
and heart. For comparison we also evaluated the activity
of unconjugated Malat-1 ASO 1 and 5ꢁ-palmitoyl conju-
gated ASO 6. Mice (C57BL/6, n = 4/group) were injected
subcutaneously with 3.6 ␮mol/kg of ASOs 1, 6, 8–14 for
three weeks. Mice were sacrificed 5 days post-injection and
Malat-1 RNA expression was analyzed from quadriceps
and heart (Figure 5). ASOs conjugated to fatty acids with
chain length C8 (8, Figure 5) and C10 (9, Figure 5) and
unconjugated ASO 1 showed similar potency in quadriceps
and heart. Interestingly, in the quadriceps and heart ASOs
10–14 conjugated to fatty acids with chain length from C12
to C22 were more active than ASO 1 (Figure 5). ASO conju-
gated to fatty acids with chain length C16 to C20 (6, 12–13,
Figure 5) showed the greatest quadriceps and heart activity
improvements. The ASO conjugates 6, 12–14 with longer
fatty acid chain conjugates C16–C22 showed the highest
plasma protein binding affinities. In general, albumin bind-
ing affinity appeared correlated with the activity of fatty
acid conjugates in skeletal and cardiac muscle. The ASO
conjugates 6, 12–14 with longer fatty acid chain conjugates
C16–C22 showed the highest plasma protein binding affin-
ity and in vivo muscle activity.
Figure 4. Potency of palmitic acid conjugated Malat-1 ASO with and with-
out PO d(TCA) linker is similar in mouse heart and liver. Mice (C57BL/6,
n = 4/group) were injected subcutaneously ASO 1 at 0.4, 1.2, 3.6, 10.8 and
ASO 2 with PO d(TCA), 6 without PO d(TCA) at 0.4, 1.2 and 3.6 ␮mol/kg
for 3 weeks for a total of 3 doses then sacrificed after 5 days. Malat-1 RNA
expression analyzed in mice heart and liver by qRT-PCR. All data are ex-
pressed as mean standard deviation. ED50 (␮mol/kg/wk) for reducing
Malat-1 RNA in mouse heart and ASO sequence: blue = cEt BNA, black
= DNA, C = 5-methylcytidine, backbone all PS; o = PO, X = palmitoyl.
SAR of unsaturated fatty acid ASO Conjugates
conjugation method used for palmitoyl ASO 6 (Scheme 1).
All the ASOs contain similar PO linker design as in ASO 6
without any DNA linker. Fatty acids octanoic acid 15, de-
canoic acid 16, dodecanoic acid 17, myristic acid 18, stearic
acid 19, eicosanoic acid 20 and docosanoic acid 21 (Scheme
1) were treated with pentafluorophenyl trifluroacetate in ap-
propriate solvents in the presence of triethylamine at room
temperature afforded corresponding pentafluorophenyl es-
ters 22–28 (Scheme 1, Supporting Information). A solution
of pentafluorophenyl esters 22–28 in appropriate solvent
were added to a solution of 5ꢁ-hexylamino Malat-1 ASO 7
(Scheme 1) in sodium tetraborate buffer (pH 8.5) and the
resulting solution was stirred at room temperature for 5–
18 h followed by HPLC purification to provide the fatty
acid conjugates ASO 8–14 (Figure 5) with varying fatty acid
chain length C8-C22. Fatty acid ASO conjugates 8–14 were
We also investigated the effect of conjugation of unsatu-
rated fatty acid on muscle ASO potency. We hypothesized
that unsaturated fatty acids will assume a different struc-
ture than saturated fatty acid leading to significant differ-
ences in the protein binding characteristics of unsaturated
fatty acid and saturated fatty acid ASO conjugates. To test
this hypothesis, we synthesized ASO 29 (Figure 6, Scheme
2) which contained an oleic acid conjugation.
ASO 29 (Figure 6) activity in mouse liver and heart was
examined and compared with 5ꢁ-palmitoyl ASO 2 and un-
conjugated ASO 1. Mice (C57BL/6, n = 4/group) were in-
jected with 0.2, 0.6 and 1.8 ␮mol/kg of ASOs 1, 2 and 29 for
three weeks. Mice were sacrificed after 5 days and Malat-1
RNA expression was analyzed from liver and heart (Fig-
ure 6). 5ꢁ-Palmitoyl ASO 2 and 5ꢁ-oleoyl ASO 29 showed
similar potency in liver and heart (Figure 6). To further in-

Nucleic Acids Research, 2019, Vol. 47, No. 12 6037
A
B
C
Figure 5. (A) Structures of ASO fatty acid conjugates 6, 8–14; (B) protein binding affinity of fatty acid ASO conjugates 6, 8–14; (C) Malat-1 RNA expression
in mice heart and quadriceps after subcutaneous administration of ASO 6, 8–14 at 3.6 ␮mol/kg once a week for three weeks for a total of three doses;
ASO sequence: X = lipid, blue: cEt BNA, black: DNA, C: 5-methylcytidine, Backbone all PS, o: PO.
vestigate the effect of unsaturated bond number, position,
and conformation we synthesized a series of naturally oc-
curring unsaturated fatty acid ASO conjugates and studied
their activity in skeletal and cardiac muscle (Figure 7). In
addition, we also characterized the plasma protein binding
profile (Figure 7) of these conjugates.
We first investigated the cis and trans and positional iso-
mers of fatty acid with one double bond. Myristolenoyl
ASO 32 (Figure 7A, C14, ␻ 5Z), Palmitoleyoyl ASO 33
(Figure 7A, C16, ␻ 7Z) and sapienoyl ASO 34 (Figure 7A,
C16, ␻ 10Z) with cis double bond at ␻ 5, 7 and 10 were
synthesized (Scheme 2). To understand the effects on activ-
ity of fatty acid chain length and the degree of unsaturation
we synthesized nervonoyl ASO 35 (Figure 7A, C16, ␻ 9Z)
with chain length C24 and a cis double bond at ␻ 9. To ex-
amine the effect of double bond conformation on ASO ac-
tivity we synthesized three fatty acid ASO conjugates 36–37
(Figure 7A) with a trans double bond at ␻ 7 and 9 respec-
tively as well as linoelaidoyl ASO 38 (Figure 7A, C18, ␻ 6E,
9E) containing two trans double bond at ␻ 6 and 9. Fur-
thermore, we also characterized the effect of conjugation of
fatty acids containing 2, 3, 4 and 6 cis double bonds con-
taining fatty acids on the activity and plasma protein bind-
ing of ASO. Linoleoyl ASO 39 (Figure 7A, C18, ␻ 6Z,9Z),
octadecatrienoyl ASO 40 (Figure 7A, C18, ␻ 3Z, 6Z,9Z), ␥-
linolenoyl ASO 41 (Figure 7A, C18, ␻ 6Z,9Z,12Z), arachi-
donyl ASO 42 (Figure 7A, C20, ␻ 6Z,9Z,12Z, 15Z) and
DHA ASO 43 (Figure 7A, C22, ␻ 3Z,6Z,9Z,12Z, 15Z,

6038 Nucleic Acids Research, 2019, Vol. 47, No. 12
Liver
100
Heart
100
50
0
50
0
2
29
29
1
1
2
Dose µmole kg-1
Dose µmol kg-1
ED50 Liver
µmol kg-1
0.2
ED50 Heart
µmol kg-1
>1.8
ASO No.
Sequence (5’ to 3’)
X =
1
2
29
GCATTCTAATAGCAGC
none
X-TCAGCATTCTAATAGCAGC
X-TCAGCATTCTAATAGCAGC
5’-Palmitoyl
5’-Oleoyl
0.1
0.9
0.1
1.1
Figure 6. 5ꢁ-Oleoyl ASO 29 and 5ꢁ-palmitoyl Malat-1 ASO 2 exhibited similar potency in mice heart and liver. Mice (C57BL/6, n = 4/group) were injected
subcutaneously with Malat-1 ASO 1, 5ꢁ-plamitoyl Malat-1 ASO 2 and 5ꢁ-oleyol Malat-1 ASO 29 at 0.2, 0.6 and 1.8 ␮mol/kg once a week for 3 weeks,
sacrificed after 5 days. (A) Malat-1 RNA expression analyzed in mice heart and liver by qRT-PCR. All data are expressed as mean standard deviation. (B)
ED50 (␮mol/kg/week) for reducing Malat-1 RNA in mouse heart and liver. ASO sequence: X = lipid, blue: cEt BNA, black: DNA, C: 5-methylcytidine,
Backbone all PS, Underline PO. (C) structure of 5ꢁ-oleic acid conjugated Malat-1 ASO 29.
18Z) were synthesized. ASOs 32–43 were synthesized us-
ing methods (Scheme 2) similar to the synthesis methods of
other fatty acid conjugates described in this report.
weeks. Mice were sacrificed after 5 days and Malat-1 RNA
expression was analyzed from quadriceps and heart (Fig-
ure 7C). All fatty acid conjugates ASOs showed improved
activity in quadriceps and heart compared to unconjugated
ASO 1 (Figure 7C). However, 5ꢁ-palmitoyl ASO 6 exhibited
improved activity compared to all other unsaturated fatty
acid conjugated ASOs examined in the study (Figure 7C).
It appears that geometry or number of unsaturation do not
significantly influence the functional uptake of unsaturated
fatty acid conjugates. ASOs 32–35 containing fatty acids
with cis double bond at different position and ASOs 36–38
containing trans double bonds showed similar activity. Sim-
ilarly, ASO conjugates 39–43 containing two to six cis dou-
ble bonds also showed similar activity in muscle. Consistent
with saturated fatty acid conjugates, plasma protein bind-
ing affinity correlated with the skeletal and cardiac muscle
activity of unsaturated fatty acid ASO conjugates
Binding of unsaturated fatty acid ASO conjugates 32–43
to human albumin, LDL and HDL were measured using a
competition binding assay (Figure 7B) (35). Comparable to
saturated fatty acid ASO conjugates, all unsaturated fatty
acid ASO conjugates bound to albumin, LDL and HDL
(Figure 7B) with higher affinity than unconjugated ASO
1. Interestingly, palmitoyl ASO conjugate 6 showed tighter
binding to albumin, LDL and HDL relative to all unsat-
urated fatty acids ASO conjugates 32–43 assessed in this
study. Consistent with previous observation, binding affin-
ity of unsaturated fatty acid conjugates with shorter chain
length (ASO 32, C14 ␻ 5Z) was less than longer chain un-
saturated fatty acids. Double bond confirmation and po-
sition appears to have minimum effect of the plasma pro-
tein binding of ASO conjugates. ASOs 33–35 containing cis
double bond at different position and ASOs conjugates 36–
38 with trans double bonds at different positions showed
similar plasma protein binding affinity (Figure 7B). Fatty
acid conjugates containing different degrees of unsaturation
exhibited interesting albumin binding affinity trends. ASO
conjugate 43 with six cis unsaturated double bonds bound
to albumin (Ki 5 ␮M) at a lower affinity compared to ASOs
32–35 and 39–42 (Ki 1.7–2 ␮M) with one to four cis double
bonds.
Palmitoyl conjugation improves potency of cEt BNA CD36
ASO 3–7 fold in heart and muscle
Next, we examined the effects of palmitic acid conjuga-
tion on the potency of ASO targeting CD36 mRNA in
mouse heart and quadriceps. CD36 ASO 68 (Figure 8)
is a fully PS modified cEt BNA Gapmer ASO targeting
CD36 mRNA, which is expressed in both murine liver and
extra-hepatic tissues (36). Activity of ASOs targeting mouse
CD36 mRNA have been previously characterized (36). To
study the effect of palmitic acid modification on activity of
CD36 ASO we synthesized 5ꢁ-palmitoyl conjugated CD36
ASO 69 (Figure 8) using the method described in Scheme 1.
Mice (C57BL/6, n = 4/group) were injected intra-
venously with 1, 3 and 9 ␮mol/kg of ASOs 68 or 69 for
To assess the effect of structural variation of fatty acid
on activity, we evaluated the activity of ASO conjugates
32–43 for inhibiting Malat-1 RNA expression in mice and
compared it to 5ꢁ-palmitoyl ASO 6 and unconjugated ASO
1. Mice (C57BL/6, n = 4/group) were subcutaneously in-
jected with 3.6 ␮mol/kg of ASOs 1, 6 and 32–43 for three

Nucleic Acids Research, 2019, Vol. 47, No. 12 6039
Figure 7. (A) Structure of ASO conjugates 32–43; (B) protein binding affinity of lipid conjugates ASOs 32–43; (C) Malat-1 RNA expression from hearts
and quadriceps of ASOs 32–43 administered mice.; ASO sequence: X = lipid, blue: cEt BNA, black: DNA, C: 5-methylcytidine, Backbone all PS, o: PO.

6040 Nucleic Acids Research, 2019, Vol. 47, No. 12
Figure 8. Palmitic acid conjugation enhances potency of CD36 ASO in mice heart, quadriceps and, liver. Mice (C57BL/6, n = 4/group) were injected
intravenously CD36 ASO 68, 5ꢁ-plamitoyl CD36 ASO 68 at 1, 3 and 9 ␮mol/kg once a week for 3 weeks, sacrificed after 5 days. (A) CD36 mRNA expression
analyzed in mice hearts quadriceps, liver and kidney by qRT-PCR. All data are expressed as mean standard deviation. (B) ED50 (␮mol/kg/week) for
reducing CD36 mRNA in mouse heart, quadriceps, liver and kidney. ASO sequence: X = lipid, blue: cEt BNA, black: DNA, C: 5-methylcytidine, backbone
all PS, underline PO.
3 weeks. Mice were sacrificed 5 days post-injection and,
heart, quadriceps, liver and kidney were homogenized and
analyzed for reduction of CD36 mRNA. Palmitoyl ASO
69 showed more than 7-fold enhanced potency in quadri-
ceps and heart relative to the parent ASO 68 (Figure 8). In
liver, potency improvement was only 3-fold whereas in kid-
ney similar potency was exhibited with unconjugated ASO
and palmitic acid conjugated ASO. All ASOs were well tol-
erated with no elevations in plasma transaminases or organ
weights (data not shown).
histology (38). However, in the clinic the DMPK ASO ex-
hibited limited therapeutic benefit (7), indicating an urgent
need for improved potency. In this report we evaluated the
potency of a previously characterized (37) DMPK ASO 71
(Figure 9) in muscle and show that palmitoyl conjugation
improves the potency of DMPK ASO in mice quadriceps
and heart (Figure 9).
Mice (n = 4/group) were injected subcutaneously with
DMPK ASOs 71 at 1.84, 3.68 and 7.36 ␮mol/kg and 72
at 0.85, 1.70 and 3.40 ␮mol/kg per week for 4 weeks. Mice
were sacrificed 2 days following their last dose and quadri-
ceps and heart DMPK mRNA expression was analyzed
(Figure 9). Palmitoyl modification led to a 3–4-fold im-
provement of DMPK ASO 72 activity in quadriceps and
heart relative to unconjugated ASO 71 (Figure 9).
Palmitoyl conjugation enhances potency of ASO targeting
DMPK mRNA in mice
Myotonic dystrophy type 1 (DM1) is a neuromuscular dis-
eases caused by inherited CTG repeat expansion in the gene
encoding DM Protein Kinase (DMPK) (7). The CTG re-
peats in the gene are transcribed into mRNA which cause
hairpins to form and bind with high affinity to the mus-
cle blind-like (MBNL) family of proteins. This complex is
sequestered and prevents them from performing their nor-
mal function (7). In preclinical studies, antisense oligonu-
cleotides targeted to DMPK mRNA efficiently reduced
mRNA in different skeletal muscle (37,38) which led to im-
provements in body weight, muscle strength and muscle
Palmitoyl conjugation enhances potency of ASO targeting
Cav3 mRNA more than 5-fold in mice skeletal and cardiac
muscle
Caveolin 3 (Cav3) is a caveolin family member protein ex-
pressed in cell types that act as a scaffolding protein for
the organization and concentration of certain caveolin-
interacting molecules. Mutations in this gene is attributed to
lead to Limb-Girdle muscular dystrophy type-1C (LGMD-

Nucleic Acids Research, 2019, Vol. 47, No. 12 6041
Figure 9. Palmitic acid conjugation enhances potency of DMPK ASO in mice heart and quadriceps. Mice (Bal/c, n = 4/group) were administered subcu-
taneously DMPK ASO 71 at 1.84, 3.68 and 7.36 and 5ꢁ-palmitoyl DMPK ASO 72 at 0.85, 1.70, 3.40, ␮mol/kg once a week for 3.5 weeks, sacrificed after 2
days. (A) DMPK mRNA expression analyzed in mice heart and quadriceps by qRT-PCR. All data are expressed as mean standard deviation. (B) ED50
(␮mol/kg/week) for reducing DMPK mRNA in mouse heart and quadriceps. ASO sequence: X = lipid, blue: cEt BNA, black: DNA, C: 5-methylcytidine,
backbone all PS, o = PO.
1C), hyperCKemia, or rippling muscle disease (RMD) (39).
It has been reported that Cav3 expression in mice is primar-
ily in heart and skeletal muscle (40). We designed antisense
oligonucleotide 74 (Figure 10) targeting Cav3 mRNA and
demonstrated modest activity in quadriceps and heart. To
evaluate the effect of palmitic acid conjugation on potency
of Cav3 ASO we synthesized 5ꢁ-palmitoyl conjugated Cav 3
ASO 75 (Figure 10, Scheme 1) and studied activity in mice.
We then evaluated the potency of ASOs 74–75 to in-
hibit Cav3 mRNA in mice quadriceps and heart. Mice (n
= 4/group) were injected subcutaneously with ASOs 74 or
75 at 1.68, 5 and 15 ␮mol/kg per week for two weeks. Mice
were sacrificed 4 days following the final dose and Cav3
mRNA expression was analyzed from quadriceps and heart
(Figure 10). Palmitoyl Cav3 ASO 75 exhibited improved po-
tency in quadriceps (5-fold) and in heart (> 4-fold) com-
pared to unconjugated ASO 74 (Figure 10). This data fur-
ther confirms that that palmitoyl conjugation is a worth-
while approach to improve the potency of clinically relevant
ASO in mice.
a solid phase DNA synthesizer using phosphoramidite 77
using standard protocol in good yield (29).
DISCUSSION
Antisense oligonucleotide therapeutics is a maturing drug
discovery platform with six ASO drugs approved for clinical
use (3,41,42), and over 40 more in clinical development (3).
Given the predominant distribution of ASOs to the liver, it
is not surprising that the majority of systemically adminis-
trated ASOs in clinical use or development target the liver-
expressed genes. Thus, delivery strategies will be essential
to define a new class of RNA therapeutics that target extra-
hepatic sites such as muscle. We demonstrate that fatty acid
conjugation can improve potency of RNase H ASOs 3 to7
fold for suppressing gene expression in skeletal and cardiac
muscles.
Fatty acids constitute a fundamental source of energy
production (43). The majority of fatty acids in the plasma
are bound to serum albumin and there are seven binding
sites for long-chain non-esterified fatty acids on the albu-
min (44). Albumin is one of the most abundant proteins in
plasma and provides the transport of fatty acids, drugs, ions
and other metabolites (45). Albumin interacts with multi-
ple receptors such as glycoprotein Gp60, Gp30 and Gp18,
(SPARC), the Megalin/Cublin complex, and the neonatal
Fc receptor (FcRn) (20). Albumin’s interaction with these
receptors is responsible for its recycling and cellular transcy-
tosis (20). This property makes albumin an attractive ‘self’
drug delivery molecules to transit across tissues and cellu-
lar barriers (20). Transport from circulation to muscle cells
ASO must pass the endothelium, the interstitial space, and
the plasma membrane (Figure 11). We hypothesized that
conjugation of fatty acid will increase the albumin bind-
ing affinity of ASOs enhancing their ability to cross the
endothelia barrier and improve its functional uptake into
Solid phase synthesis of 5ꢀ-palmitic acid conjugated ASOs
We developed a solid phase DNA synthesis method
for convenient synthesis of 5ꢁ-palmitic acid conjugated
ASOs. First, we developed a 6-palmitamidohexyl phospho-
ramidite 77 (Scheme 3) for the incorporation of palmitic
acid modification using a solid phase DNA synthesis
method. Phosphoramidite 77 was synthesized accord-
ing to the method described in Scheme 3. In brief, 6-
palmitamidohexanol 78 (84%) was generated from palmitic
acid, Pfp-TFA, triethylamine and 6-aminohexanol 79 in
dichloromethane in a one pot synthesis. Compound 78 was
subsequently phosphitylated to afford phsophoramidite 77
(93%). 5ꢁ-Pamitic acid conjugated ASO were synthesized on

6042 Nucleic Acids Research, 2019, Vol. 47, No. 12
Figure 10. Activity of Cav3 ASO 74 and 5ꢁ-palmitoyl Cav3 ASO 75 in mice heart and quadriceps; Mice (C57BL/6, n = 4/group) were injected subcuta-
neously Cav3 ASO 74, 5ꢁ-palmitoyl Cav3 ASO 75 at 1.68, 5 and 15 ␮mol/kg once a week for 2 weeks, sacrificed after 4 days. (A) Cav3 mRNA expression
analyzed in mice heart and quadriceps by qRT-PCR. All data are expressed as mean standard deviation. (B) ED50 (␮mol/kg/week) for reducing Cav3
mRNA in mouse heart and quadriceps. ASO sequence: X = lipid, blue: cEt BNA, black: DNA, C: 5-methylcytidine, backbone all PS, o = PO.
binding affinity 370–480 ␮M (46). A recent report suggests
that fatty acid modified gapmer ASOs can self-assemble
into constructs that offer improved tissue distribution (47).
As expected, palmitoyl conjugation improved binding
affinity of ASO to human serum albumin (Figure 1) >150-
fold. While, palmitic acid modification enhanced binding
affinity of ASO to other most prominent plasma proteins
such as HDL, and LDL but binding affinity to HRG re-
mained unchanged (Figure 1). We next studied the effect
of palmitic acid conjugation on potency of Malat-1 ASOs
in mice quadriceps, heart, liver and kidney. Interestingly,
palmitic acid conjugation improved the potency of Malat-1
ASO more than five-fold in heart and quadriceps (Figure
2) after systemic administration. In kidney unconjugated
and palmitoyl conjugated Malat-1ASOs exhibited similar
potency. In addition, palmitoyl conjugation improved tissue
uptake of Malat-1 ASO in heart and quadriceps (Table 1)
thus providing a rationale for the enhanced potency. Inter-
estingly, palmitic acid conjugation increased plasma Cmax
of ASO and plasma AUC relative to unconjugated ASO.
Histological examination indicates that much of the ASO
accumulated in heart at early time points appears diffuse
or concentrated along intercellular boundaries suggesting
it primarily resides in the interstitial space, however, by 24
h, the ASO appears in punctate foci, likely occupying intra-
cellular vesicles. These data provide initial evidence for im-
proved delivery of ASO when conjugated to palmitic acid
in the muscle interstitial space compared to unconjugated
ASO.
3
, Pfp-TFA,CH2Cl2,N(Et)3, rt, 84%
NH2
HO
R
79
H
N
O
O
78
: R = H
Phosphitylation
93%
O
CN
P
77
: R =
N(iPr)2
Scheme 3. Synthesis of 6-palmitamidohexyl phosphoramidite 77; Pfp-
TFA: pentaflurophenyl trifluoroacetate.
One of the principal functions of human serum albumin
is to transport fatty acids. The primary association con-
stant of albumin to fatty acids is a function of chain length
and degree of unsaturation (34), where the primary associa-
tion constant and number of high affinity sites increase with
fatty acid chain length (34). We characterized the effect of
fatty acid chain length and degree of unsaturation on the al-
Figure 11. Schematic representation of the likely route taken by lipid con-
jugated ASO from the capillary to cytosol in muscle. AlbLA: albumin
bound lipid ASO; AlbR: albumin receptor.
muscles (Figure 11). Previous studies suggested that uncon-
jugated PS ASOs bind to human serum albumin with low

Nucleic Acids Research, 2019, Vol. 47, No. 12 6043
bumin binding affinity of fatty acid conjugated ASOs. Bind-
ing affinity increased with chain length and the optimum
affinity was observed in ASO conjugates with C16-C18 fatty
acid chain length. Double bond unsaturation number or
stereochemistry had little effect on albumin binding affin-
ity of the ASO conjugate. Fatty acid conjugates with one
to four unsaturated double bonds in cis conformation and
with one or two trans double bond exhibited similar albu-
min binding. Interestingly, ASO conjugates containing C16
to C18 saturated or unsaturated fatty acids exhibited sim-
ilar activity in mice skeletal and cardiac muscle. Taken to-
gether, our data suggest that palmitoyl (C16 saturated) and
oleoyl (C18, ␻ 9Z) conjugation provided maximum potency
enhancement for ASOs in skeletal and cardiac muscle.
We also demonstrate that potency improvement observed
for palmitoyl conjugation in mouse skeletal and cardiac
muscle is not sequence or target specific. Palmitic acid con-
jugation improved potency (3 to 7 fold) of ASOs targeting
CD36, DMPK and Cav3 mRNA in mouse muscle com-
pared to corresponding unconjugated ASOs. The unconju-
gated DMPK ASO was evaluated in clinical trials for the
treatment of myotonic dystrophy type 1.
Our data demonstrate that altering the interaction of PS
ASO with specific plasma proteins could modulate the tis-
sue distribution of PS ASO. Conjugation of palmitic acid, a
known albumin ligand, enhanced the albumin affinity of PS
ASO. Albumin is actively transported across the capillary
endothelium via caveolin-1 mediated transcytosis and sig-
nificant amounts (60%) of total albumin is associated with
the interstitial space in muscle (48). We hypothesize fatty
acid conjugation enhances albumin binding thereby facili-
tating ASO movement across the vascular wall into the in-
terstitium. Additional work is needed to understand more
completely this process, as well as whether myocyte delivery
itself is also enhanced by fatty acid interactions on plasma
membrane receptors.
In conclusion, we report a detailed structure-activity re-
lationship of fatty acid conjugated ASOs for harnessing
albumin-based delivery into extrahepatic tissues. Activity
and binding to plasma proteins of ASO fatty acid conju-
gates containing varying fatty acid chain length, degree of
unsaturation and cis trans isomers were studied. The bind-
ing affinity of ASO to plasma proteins improved with fatty
acid chain length with the highest binding affinity observed
with fatty acid chain length from 16 to 18 carbons. Degree
of unsaturation or conformation of double bond appears
to have no influence on protein binding of ASO fatty acid
conjugates. Activity of fatty acid ASO conjugates correlate
with the affinity to albumin and the tightest albumin binder
exhibited highest activity improvement in muscle. Palmitic
and oleic acid appears to be the optimum fatty acid struc-
ture for improving functional uptake of ASO into skeletal
and cardiac muscle. To access the site of action in muscle,
ASO must traverse the vascular wall and enter the intersti-
tium to have access to the myocyte. Our data suggest that
fatty acid conjugation enables the ASO to bind tightly to al-
bumin thereby facilitating ASO delivery to the interstitium
leading to enhanced myocyte uptake and ASO activity. Fur-
ther improvement in effectiveness of fatty acid ASO conju-
gates could be achieved by combining with ligands which
target cell-surface receptors in muscle tissues. Strategy de-
scribed in this report provides a foundation for designing
more effective therapeutic ASOs for targeting muscle tissues
to develop treatments for muscle related diseases.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank Tracy Reigle for the help in preparing Figure 11
and Amy Dan, Rick Carty, Chris Watson, Mark Andrade
for their contribution to this work.
FUNDING
Funding for open access charge: Ionis Pharmaceuticals Inc.
Conflict of interest statement. None declared.
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