Angewandte
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penetration and cellular uptake is more challenging.
CRISPR/Cas editing using viruses,[2b,c,3] membrane deforma-
tion,[4] ribonucleoprotein complex delivery,[5] and hydrody-
namic injection[2d] are functional, but have limitations that
could hinder in vivo therapeutic use in the clinic, including
persistent expression of Cas9 and off-target editing.
epoxides and acrylates to append 6 to 18 carbon alkyl tails and
alcohol/ester groups to enhance ZAL-RNA interactions
(Figures S1–S4). To verify that ZNPs could generally bind
and deliver RNA, the 72-member library was first screened
for siRNA delivery to HeLa cells that stably expressed firefly
luciferase (HeLa-Luc; Figure S5). This allowed structural
identification of key amine cores, including ZA1, ZA3, and
ZA6. Interestingly, epoxide-based ZALs (ZAx-Epn), were
also more active than acrylate-based ZALs (ZAx-Acn; Fig-
ure S6). With lead compounds in hand, we focused on the
delivery of sgRNAs and Cas9 mRNA. Both temporally staged
and simultaneous co-delivery enabled fully exogenous gene
editing.
ZALs were evaluated for their ability to deliver CRISPR/
Cas9 components using a stable cell line expressing both Cas9
and luciferase (HeLa-Luc-Cas9). A single HeLa-Luc-Cas9
cell clone was isolated following Cas9 lentiviral transduction
of HeLa-Luc cells (Figure S7). sgRNAs against luciferase
were designed and generated according to previously
reported methods targeting the first third of the gene
(Table S1)[12] and evaluated by pDNA transfection (Fig-
ure S8). The most active sgRNA against luciferase (sgLuc5,
henceforth sgLuc) as well as control sgRNAs were synthe-
sized by in vitro transcription. Next, lead ZNPs were loaded
with sgLuc and evaluated for delivery to HeLa-Luc-Cas9
cells. Luciferase and viability[13] were measured after 48 hours
(h) relative to untreated cells. As anticipated from the
chemical design combining cationic and zwitterionic func-
tionalities, ZNPs do not require inclusion of helper phospho-
lipids (Figure 3A).
Among the lead ZALs, ZA3-Ep10 was found to be the
most efficacious for delivery of sgLuc (Figure S9). Editing of
luciferase DNA resulted in a dose-dependent decrease in
luciferase expression (Figure 3B). We verified CRISPR/Cas
editing using the Surveyor nuclease assay,[14] which can detect
indels (Figure 1C). Given that sgRNAs require loading into
Cas9 nucleases in cells and trafficking to the nucleus to
perform sequence-guided editing, we wanted to understand
the kinetics of this process, particularly in comparison to
RNAi-mediated gene silencing. siLuc-mediated mRNA deg-
radation is a fast process, where expression decreased by 40%
within the first 4 h. Luciferase was silenced by 92% by 20 h
and remained low for about 3 days. Thereafter, the protein
expression steadily increased and reached baseline level
6 days after transfection (Figure 1B and Figure S10 (early
time points)). In contrast, sgLuc-mediated DNA editing was
kinetically slower, possibly because of the requirements to
load into Cas9 and survey the DNA for PAMs. It took 20 h for
luciferase expression to decrease by 40%, ultimately going
down by 95% after 2 days and remaining there indefinitely.
The low luciferase expression (5%) persisted throughout the
duration of the assay (9 days) because of the permanent
genomic change, even after multiple rounds of cellular
division, suggesting that edited cells grew at the same rate
of non-edited cells (Figure 1B, Figure S11).
Although great advances have been made in the delivery
of short RNAs (siRNA, miRNA; ꢀ 22 base pairs (bp) in
length) by lipid nanoparticles (LNPs),[6] the ideal chemical
and formulation composition is largely unknown for longer
RNA cargo (mRNA, sgRNA). Highly effective LNPs are
composed of a cationic lipid, zwitterionic phospholipid,
cholesterol, and lipid poly(ethylene glycol) (PEG). Cationic
lipids bind RNAs at low pH during mixing, and promote
intracellular release as the pH decreases during endosomal
maturation.[7] Computational modeling has shown that phos-
pholipids function by solubilizing small RNAs inside of
aqueous pockets within multi-component LNPs.[8] High
cationic lipid density may thus minimize phospholipid-
stabilizing interactions with longer RNAs in LNPs. Cationic
lipids also take up space within LNPs and could hinder
inclusion of organized long RNAs at pH 7.4. Recent reports
on mRNA delivery using alternative helper phospholipids
(for example, DOPE) further suggests that associated solu-
bilizing forces may improve NP construction.[9] We therefore
hypothesized that combining the chemical and structural roles
of zwitterionic[10] and cationic lipids[9,11] into a single lipid
compound might improve delivery of longer RNAs by
increasing molecular interactions within the LNP.
Zwitterionic amino lipids (ZALs) were rationally synthe-
sized to contain a zwitterionic sulfobetaine head group, an
amine-rich linker region, and assorted hydrophobic tails
(Figure 2). A zwitterionic electrophilic precursor (SBAm)
was prepared by the ring-opening reaction of 2-(dimethyl-
amino)ethyl acrylamide with 1,3-propanesultone, which was
easily isolated by in situ precipitation in acetone. Conjugate
addition of different polyamines to SBAm afforded a series of
zwitterionic amines that could be reacted with hydrophobic
Figure 2. ZALs were designed to increase molecular interactions with
longer RNAs by combining the chemical and structural roles of
zwitterionic lipids and cationic lipids into a single lipid compound.
High-efficiency reactions provided access to a library of unique charge
unbalanced lipids.
Having demonstrated that ZA3-Ep10 ZNPs could effec-
tively deliver sgRNAs ( ꢀ 100 nt), we next examined their
ability to deliver even longer mRNA (1,000 to 4,500 nt). We
delivered mCherry mRNA ( ꢀ 1,000 nt) or luciferase mRNA
2
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Angew. Chem. Int. Ed. 2016, 55, 1 – 6
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