Oligo(serine ester) Charge-Altering Releasable Transporters: Organocatalytic Ring-Opening Polymerization and their Use for in Vitro and in Vivo mRNA Delivery

Article abstract of DOI:10.1021/jacs.9b03154

RNA technology is transforming life science research and medicine, but many applications are limited by the accessibility, cost, efficacy, and tolerability of delivery systems. Here we report the first members of a new class of dynamic RNA delivery vector

Full text of DOI:10.1021/jacs.9b03154

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Communication
Oligo(serine ester) Charge-Altering Releasable Transporters:
Organocatalytic Ring-Opening Polymerization and
their Use for In Vitro and In Vivo mRNA Delivery
Nancy L. Benner, Rebecca L. McClellan, Christopher R. Turlington,
Ole A. W. Haabeth, Robert M. Waymouth, and Paul A. Wender
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b03154 • Publication Date (Web): 14 May 2019
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Oligo(serine ester) Charge-Altering Releasable Transporters: Organo-
catalytic Ring-Opening Polymerization and their Use for In Vitro and In
Vivo mRNA Delivery
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†
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Nancy L. Benner, Rebecca L. McClellan, Christopher R. Turlington, Ole A. W. Haabeth, Robert
*
,†
*,†,‡
M. Waymouth, Paul A. Wender
†
Department of Chemistry, Stanford University, Stanford, California 94305 (USA)
‡ Department of Chemical and Systems Biology, Stanford University, Stanford, California 94305 (USA)
§
9
Department of Medicine, Division of Oncology, Stanford Cancer Institute, Stanford University, Stanford, California
4305 (USA)
Supporting Information Placeholder
prompted the search for improved delivery systems with par-
ticular emphasis on degradable vectors.
ABSTRACT: RNA technology is transforming life science re-
search and medicine, however, many applications are limited
by the accessibility, cost, efficacy, and tolerability of delivery
systems. Here we report the first members of a new class of dy-
namic RNA delivery vectors, oligo(serine ester)-based charge-
altering releasable transporters (Ser-CARTs). Composed of li-
pid-containing oligocarbonates and cationic oligo(serine es-
ters), Ser-CARTs are readily prepared (one flask) by a mild
ring-opening polymerization using thiourea anions and, upon
simple mixing with mRNA, readily form complexes which de-
grade to neutral serine-based products, efficiently releasing
their mRNA cargo. mRNA/Ser-CART transfection efficiencies of
>95% are achieved in vitro. Intramuscular or intravenous (IV)
injections of mRNA/Ser-CARTs into living mice result in in vivo
expression of a luciferase reporter protein with spleen locali-
zation observed after IV injection.
17-22
We recently reported a new class of synthetic biodegradable
gene delivery materials, dubbed Charge-Altering Releasable
Transporters (CARTs, e.g. 1).23 These first generation CARTs 1
are amphipathic diblock co-oligomers, consisting of a lipophilic
oligocarbonate sequence followed by a cationic morpholinone-
derived α-amino ester backbone. These transporters operate
through an unprecedented mechanism, with the cationic ol-
igo(α-amino ester) block serving to electrostatically complex
the anionic nucleic acid cargo and subsequently undergoing an
irreversible rearrangement to neutral small molecules (e.g., 2),
resulting in cargo release (Scheme 1A). While morpholinone-
based CARTs 1 are effective for mRNA and plasmid delivery in
many cell lines, including T-lymphocytes, these first generation
transporters represent only one subclass of a potentially broad
and unexplored platform of charge-altering vectors for gene
23-27
delivery.
Here, we report the synthesis and evaluation of a new class
of charge-altering vectors, based on oligo(serine esters), Ser-
CARTs 3. Differing from CARTs with oligocationic backbones,
Ser-CARTs incorporate a charge-altering side-chain amine to
complex the mRNA cargo and produce neutral serine-based by-
products upon degradation, resulting in mRNA release
Messenger RNA (mRNA) is advancing fundamental research
and medicine due to its ability to induce the transient catalytic
expression of target proteins in vitro, in vivo, and ex vivo. mRNA
applications include protein replacement therapy, gene edit-
1
ing, vaccination, and cancer immunotherapy. However, the
challenge of developing synthetically accessible, affordable,
safe, and effective delivery vectors that extracellularly protect
and intracellularly release mRNA have hampered applications,
(
Scheme 1B). In addition to their biocompatibility and expected
2
8-30
rearrangement into peptides 4,
oligo(serine esters) were
selected for study over other degradable amine-functionalized
2-3
driving demand for improved delivery systems. Current de-
1
8, 31
polyesters
due to their activating α-amino ester motif.
livery strategies focus on mechanical methods, and viral and
Studies suggest that the rapid rearrangement of morpholinone-
derived oligo(α-amino esters) is partially due to the activation
of a backbone ammonium group positioned alpha to the ester
4
-6
nonviral vectors. Mechanical methods that temporarily ren-
der the cellular membrane permeable are limited to accessible
tissues and ex vivo techniques, often suffer from poor cell via-
2
3
repeating unit. In this study, we synthesize and characterize
the degradation of side-chain ammonium-containing oligo(ser-
7
bility, and encounter scalability challenges. Viral vectors offer
broader administration options but are coupled with cost and
3
2
ine esters), structural isomers of oligo(serine amides) 4. We
further demonstrate that Ser-CARTs are readily formed (one
flask) and efficiently deliver mRNA in cultured cells and live
mice.
8-9
immunogenicity concerns and cargo size limitations. These
restrictions have stimulated interest in nonviral vectors, typi-
cally lipid nanoparticles and cationic polymers that form elec-
7
, 10-16
trostatic complexes with polyanionic nucleic acids.
De-
spite advances in nonviral vectors, challenges with accessibil-
ity, formulation, efficacy, tolerability and targetability, have
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KH (1 eq.)
TU (3.5 eq.)
NHTrt
O
O
RO
O
H
Toluene (0.5-1.5M)
RT, 4 h
TrtHN
O
5
n
a-9a
Serine
lactone
20-50 eq.)
(
1% TFA/DCM
RT, 45 min
CF3
S
NH3TFA
RO
O
F3C
N
N
H
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0
O
TU
n
5
b-9b
OH
ROH:
OH
PyOH
BnOH
Figure 1. Poly(serine ester) synthesis.
Scheme 1. mRNA release mechanism of (A) morpholinone-de-
2
3
rived CARTs 1 and (B) Ser-CARTs 3. Both systems utilize ac-
tivated α-amino esters, with oligo(serine esters) rearranging to
neutral serine-based products via O-N acyl shifts.
Table 1. Poly(serine ester) characterization.
DPc,e
ROH
[M] [M]/
Conv
M
(kD
a)
n
Ð
To study Ser-CARTs, we developed a polymerization method
that avoids the control issues and harsh conditions previously
a
ROH] (%)b
[
d
30, 33-37
reported for oligo(serine ester) synthesis.
Our proce-
dure benefits from the commercial availability of the N-trityl-
L-serine lactone monomer (serine lactone) and an organocata-
5
a
a
BnOH
BnOH
1.0
0.5
1.5
1.0
1.0
50
50
50
20
50
>95% 62
16.7 1.17
12.6 1.24
15.6 1.20
6
89%
93%
47
61
38-43
lytic ring-opening polymerization (OROP)
strategy, utiliz-
ing a thiourea anion catalyst recently developed for the OROP
7a BnOH
BnOH
9a PyOH
4
4
of lactones and cyclic carbonates (Fig. 1). Specifically, we
found that the OROP of serine lactone with 1-(3,5-bis-trifluoro-
methyl-phenyl)-3-cyclohexyl-thiourea (TU) and potassium hy-
dride (KH) in the presence of an alcohol initiator proceeds at
RT in hours to generate trityl-protected poly(serine esters) 5a-
8
a
>95% 20
7.7
1.21
>95% 49
15.8 1.21
Polymerizations run for 4h at RT in toluene. [a]Serine lactone
concentration (molar). Determined by [b]NMR, NMR end-
group analysis after dialysis, and gel permeation chromatog-
raphy. Degree of polymerization.
[c]
[
d]
9a with predictable molecular weights (M
n
= 7 - 17 kDa) and
[
e]
narrow dispersities (Đ = 1.11 - 1.24) avoiding multimodal dis-
tributions previously reported (Table 1, Supporting Infor-
3
5-36
mation Table S1).
removed using 1% TFA to yield cationic poly(serine esters) 5b-
b with no significant decrease in molecular weight, as deter-
After polymer isolation, trityl groups are
As studies indicate that the ring-opening of β-lactones can
4
8
occur by two mechanisms, we performed the stoichiometric
ring-opening of serine lactone with one equivalent of benzyl al-
cohol using the KH/TU catalyst. Analysis of the resulting prod-
uct by HMBC NMR indicates that ring-opening proceeded
through acylation of benzyl alcohol by the lactone to generate
the alkoxy-terminated benzyl serine ester, rather than by nu-
9
mined by end-group analysis (Fig. S1). This facile controlled
polymerization of serine lactone is noteworthy, as prior re-
ports suggested that β-lactone OROP was inefficient.4
5-47
48
cleophilic attack at the β-carbon to generate the carboxylate
(
Fig. S2).
Having developed an effective poly(serine ester) synthesis,
we next investigated whether they would rearrange at biologi-
cally relevant pH regimes in the absence of an mRNA cargo.
While uncomplexed poly(serine ester) 9b (DP 47) is stable un-
der its generation conditions, at pH 7.4 it begins degrading in
minutes, producing in hours the known rearrangement prod-
2
8-29
uct, oligo(serine amide) 4,
and also a previously unre-
ported product, dimerized serine diketopiperazine (DKP, 10)
as confirmed by NMR and LC-MS (Fig. 2A, Supporting Infor-
mation). Analysis of 9b degradation at pH 7.4 reveals that the
DKP yield increases over 24 hours resulting in a final yield of
55% DKP and 45% oligo(serine amides) of various lengths (Fig.
2
B). While prior reports indicated that the
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A
O
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b
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-membered
O
O
H
O
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NH2
O-to-N acyl shift
H
60
NH2
N
OH
RO
O
O
RO
O
5
0
n-2
n-2
NH3
NH2
NH2
O
OH
OH
a
40
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10
0
Poly(serine ester)
6-membered
5-membered
O-to-N acyl shift
9b
O
O
O-to-N acyl shift
a
b
+
HN
OH
HO
NH
O
O
HO
O
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O
H
+
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OH
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H
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RO
HO
N
OH
RO
O
HO
NH
n-2
H
n-2
O
NH2
NH3
0
400
800
1200
1600
Oligo(serine amide)
4
DKP
10
Time (min)
Figure 2. A) Proposed rearrangement of poly(serine esters) B) Time-dependent DKP 10 yield when 9b subjected to pH 7.4 buffered
O at RT (■, ●, ▲).
D
2
Figure 3. Ser-CART 13b-16b syntheses.
aqueous degradation of poly(serine esters) generates poly(ser-
ine amides) by a series of O-to-N acyl shifts,
covered DKP as a significant serine-based byproduct.
fluorescent protein (EGFP) and analyzed by flow cytometry the
total fluorescence and percentage of cells transfected. Notably,
mRNA/Ser-CART polyplexes are produced by simple mixing of
mRNA with Ser-CARTs. To optimize the in vitro formulation, we
screened charge ratios of 5:1 to 100:1 (cation:anion (+/-)) us-
ing dodecyl-Ser-CART 13b formulated with EGFP mRNA for de-
livery into HeLa cells. The highest fluorescence was observed
at a 50:1(+/-) charge ratio in serum-free conditions and the in-
tracellular EGFP expression was confirmed by confocal and flu-
orescence microscopy (Fig. S4-6). Using this charge ratio, we
compared EGFP mRNA delivery using Ser-CART 13b to com-
mercial transfection reagent Lipofectamine 2000 (L2000), as a
positive control, and to naked EGFP mRNA. Significantly, 13b-
mediated EGFP mRNA delivery resulted in highly efficient
2
8-29
our studies un-
We propose that the degradation of poly(serine esters) to
generate DKP 10 follows a charge-altering mechanism (Fig. 2A)
related to that proposed for the degradation of morpholinone-
based poly(α-amino esters) (Fig. S3) but now involving a pri-
2
3, 28-29
mary side-chain amine.
For pH values at which some of
the pendant ammonium groups are deprotonated, nucleophilic
attack of the resultant primary amine on an adjacent H-bond
activated ester carbonyl (5-membered O-N acyl shift) would
generate an amide and contract the polymer backbone, posi-
tioning the proximal amine for a 6-membered O-N acyl shift to
liberate DKP. Importantly, formation of the expected oligo(ser-
ine amide) 4 and the newly observed DKP 10 provide charge-
altering transformations from cationic α-amino esters to neu-
tral β-hydroxyamides critical for CART-mediated mRNA com-
(
>95%) transfection in multiple cell lines (HeLa, CHO-K1, Raw-
Blue), markedly outperforming L2000 (55-71% transfection)
(Fig. 4A). The transfection efficiency of 13b is consistent with
that of morpholinone-based CARTs 1, highlighting the im-
portance of the charge-altering block for mRNA delivery (Fig.
S7). Additionally, greater fluorescence was observed with 13b-
mediated EGFP mRNA delivery than with L2000-mediated de-
livery in HeLa cells (Fig. 4B). Ser-CART 14b also exhibited
23
plexation, delivery, and release.
Having shown that uncomplexed poly(serine esters) de-
grade to neutral products, we explored the use of Ser-CARTs
for polyanion complexation and delivery, focusing on mRNA.
1
0, 24, 49-
As lipid domains are vital in polyanion delivery vehicles,
>
95% transfection for EGFP mRNA delivery in HeLa cells, sug-
5
3
we generated a series of amphiphilic diblock co-oligomers
gesting that the initiator does not significantly influence trans-
fection, as pyrene butanol was used for 13b and benzyl alcohol
for 14b (Fig. 4C). In contrast to dodecyl-Ser-CARTs 13b and
4b, lower transfection levels were observed with more lipid-
rich 15b. Oleyl-Ser-CART 16b also resulted in >95% transfec-
tion. Ser-CARTs retain these high transfection efficiencies
when stored (0 C) under nitrogen (Fig. S8). Notably, formula-
tion of EGFP mRNA with poly(serine ester)47 9b or DKP 10 re-
sulted in negligible fluorescence (Fig. S9). Importantly, DKP 10
was non-toxic at concentrations up to 500 μM in HeLa cells (Fig.
S10).
comprised of a dodecyl-(C12) or oleyl-(C18) modified oligocar-
bonate sequence and a cationic oligo(serine ester) sequence
from carbonate monomer 11 or 12 and serine lactone, respec-
tively, using our thiourea anion catalyst system. Co-oligomers
1
13a-16a (R’=dodecyl: n=10, m=15; n=10, m=17; n=19, m=17,
R’=oleyl: n=15, m=38) were synthesized by a straightforward
three-component, step economical (one-flask) procedure using
alcohol initiators (Fig. 3, Table S2). Deprotection of 13a-16a
with 1% TFA afforded cationic Ser-CARTs 13b-16b.
o
To assess the efficacy of Ser-CARTs for mRNA delivery and
expression, we investigated the transfection of cultured cells
using Ser-CARTs complexed with mRNA encoding green
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were used to study the time-dependent surface charge. When
A
B
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**
**
**
added to RNAse-free water, mRNA/Ser-CARTs were initially
positive (mV, 13b: 37 ± 6; 16b: 52 ± 10) but over 1 hour be-
came negative (mV, 13b: -20 ± 7; 16b: -15 ± 5), consistent with
rearrangement of the cationic oligo(serine ester) block to neu-
tral products (Fig. S11).
1
00
Ser-CART 13b mRNA
L2000
Untreated
80
6
4
2
0
0
0
0
Encouraged by these in vitro studies, we explored the in vivo
utility of Ser-CARTs for mRNA delivery using two different
modes of administration in female BALB/c mice. Luciferase
(
fLuc)-coding mRNA was chosen as a model reporter gene
HeLa
CHO-K1
RAW
3
4
0
10
10
since luciferase expression can be quantitatively monitored in
Untreated
L2000
Naked mRNA
Ser-CART 1 43 b
GFP Fluorescence
55-56
0
1
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9
0
real-time in living mice.
After confirming fLuc mRNA deliv-
ery in vitro (Fig. S12), fLuc mRNA/13b polyplexes were admin-
istered via intramuscular (IM) injection into mice at a 10:1 or
50:1(+/-) ratio, and expression visualized after 7 hours by bio-
C
D
**
Bright field
Fluorescence Overlay
100
1.2
**
3
7°C
25
1
.8
.6
luminescence imaging (Fig. 4E). Both conditions resulted in
8
6
4
2
0
0
0
0
0
4°C
0
0
protein expression, however, mice treated at the lower
10:1(+/-) ratio resulted in enhanced luciferase expression (Fig.
S13). Using the 10:1(+/-) ratio, fLuc mRNA/14b or 16b poly-
plexes administered to mice via intravenous (IV) tail vein injec-
tion resulted in luciferase expression localized in the spleen, a
target organ for several therapeutic indications (Fig. 4F, S14).
Importantly, mRNA/Ser-CARTs formulated at the 10:1(+/-) ra-
tio resulted in improved cell viability (78-87% relative to un-
treated HeLa cells) compared to 50:1(+/-) in vitro (Fig. S15).
0.4
0.2
0
d
b
b
b
b
ted
13b
A
e
t
e
1
3
1
4
1
5
6
1
N
a
a
T
tre
R
T
R
A
t
r
e
R
R
T
R
T
R
T
y5-m
C
n
A
A
A
A
n
U
er-C er-C er-C er-C
U
r
-
C
S
S
S
S
d
e
S
k
a
N
E
F
IM
IV
In conclusion, mRNA delivery with the readily synthesized
(one flask) Ser-CARTs results in efficient transfection and high
protein expression in vitro and in vivo. Further benefit of Ser-
CARTs over first-generation CARTs comes from the degrada-
tion of the oligo(serine ester) block at biological pH into serine
peptides (oligo(serine amides) and DKP), commercial availa-
bility of the monomer, and smaller size of the polyplexes. The
accessibility, tunability, effectiveness, and organ selectivity of
mRNA/Ser-CART polyplexes bode well for their use in biomed-
ical research and therapeutic applications. Furthermore, this
study establishes the generality of charge-altering architec-
tures for polyanion delivery. We are currently exploring Ser-
CARTs for targeting, co-transfections and clinical indications
with an initial emphasis on vaccination and immunotherapy.
Ser-CART
3b (10:1)
Ser-CART
13b (50:1)
Ser-CART Ser-CART
14b (10:1) 16b (10:1)
Naked
mRNA
1
5
.0e6 1.0e7 1.5e7 2.0e7 2.5e7
1.0e6
2.0e6
3
3.0
.0
e
6
e6 4.0e6
5
.0e6
1.5e7
2.5e7
1.0e6 2.0e6
4.0e6
Figure 4. Delivery of mRNA/Ser-CARTs in vitro and in vivo.
mRNA/Ser-CARTs formulated at a 50:1(+/-) ratio unless spec-
ified. A) Percent transfection of HeLa, CHO-K1, or Raw-Blue
RAW) cells using L2000 or EGFP mRNA/13b. B) Histogram of
HeLa cells treated with naked EGFP mRNA, L2000, or
mRNA/13b. C) Percent transfection of HeLa cells treated with
EGFP mRNA/13b-16b. D) Cy5-labeled-mRNA/13b uptake
into HeLa cells after 1h incubation at 4°C or 37°C. Representa-
tive bioluminescence images of mice 7h post E) IM injection
with fLuc mRNA/13b at a 10:1(+/-) (left, n=4) or 50:1(+/-)
right, n=2) ratio, or F) IV injection with fLuc mRNA/14b (left,
n=4), fLuc mRNA/16b (middle, n=3), both at a 10:1(+/-) ratio,
or naked fLuc mRNA (right, n=1). Average of ≥3 experiments.
Error bars represent ±SD. **p<0.0004, *p<0.05
ASSOCIATED CONTENT
a
(
Supporting Information
Experimental details, including representative spectra and bi-
ological assays, available in Supporting Information (PDF).
a
a
AUTHOR INFORMATION
(
Corresponding Authors
* Robert M. Waymouth: waymouth@stanford.edu
* Paul A. Wender: wenderp@stanford.edu
a
We next explored the temperature-dependent uptake of Ser-
CARTs. HeLa cells treated with Cy5-labeled-mRNA/13b at 4°C
resulted in 70% reduction in Cy5-fluorescence relative to cells
incubated at 37°C, indicating mainly endocytic uptake of the
Author Contributions
z
These authors contributed equally.
Notes
5
4
polyplexes as 4°C incubation inhibits endocytosis (Fig. 4D).
The authors declare no competing financial interests.
Analysis of the EGFP mRNA/Ser-CART polyplexes by dy-
namic light scattering indicated a hydrodynamic diameter of
190 nm (dodecyl-based 13b: ~154 nm; oleyl-based 16b:
174 nm), significantly smaller than morpholinone-based
ACKNOWLEDGMENT
<
~
This work was supported by NSF CHE-1607092 (R.M.W.), NSF
CHE848280 and NIH-CA031845 grants (P.A.W.), the Child
23
CARTs (~250 nm) (Table S3). Zeta potential measurements
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Journal of the American Chemical Society
Health Research Institute at Stanford University and the Stan-
ford SPARK Translational Research Program (R.M.W, P.A.W).
R.L.M. was supported by an Abbott Laboratories Stanford Grad-
uate Fellowship. C.R.T acknowledges support from the National
Institute of Biomedical Imaging And Bioengineering of the NIH
under Award Number F32EB021161. Flow cytometry data was
collected in the Stanford Shared FACS Facility using NIH S10
Shared Instrument Grant S10RR027431-01. We gratefully
acknowledge: Dr. Timothy Blake, Dr. Colin McKinlay and Prof.
Ronald Levy for helpful discussion, B.K. Marshall for fluores-
cence microscopy, Prof. Lynette Cegelski for use of tissue-cul-
ture equipment, and Prof. Richard Zare for use of the Malvern
Zetasizer.
Stable Polyplexes with Plasmids and Promote Liver-Targeted Delivery.
Biomacromolecules 2016, 17 (3), 830-840.
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Zu, Y., Transfecting the hard-to-transfect lymphoma/leukemia cells
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