Theranostics 2023, Vol.

13, Issue 11 3497

Ivyspring
International Publisher
Theranostics
2023; 13(11): 3497-3508. doi: 10.7150/thno.82228
Research Paper

Lipid nanoparticles-loaded with toxin mRNA represents

a new strategy for the treatment of solid tumors
Yasmin Granot-Matok1,2,3,4, Assaf Ezra1,2,3,4, Srinivas Ramishetti1,2,3,4, Preeti Sharma1,2,3,4, Gonna Somu
Naidu1,2,3,4, Itai Benhar2,4,5, and Dan Peer1,2,3,4,
1. Laboratory of Precision NanoMedicine, Shmunis School for Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv University,
Tel Aviv 69978, Israel.
2. Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv 69978, Israel.
3. Department of Materials Sciences and Engineering, Iby and Aladar Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel.
4. Cancer Biology Research Center, Tel Aviv University, Tel Aviv 69978, Israel.
5. Laboratory of Antibody Engineering, Shmunis School for Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv University,
Tel Aviv 69978, Israel.

 Corresponding author: Email: [email protected].

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/).
See http://ivyspring.com/terms for full terms and conditions.

Received: 2022.12.28; Accepted: 2023.05.20; Published: 2023.06.12

Abstract
Background and rationale: Cancer therapy have evolved remarkably over the past decade, providing
new strategies to inhibit cancer cell growth using immune modulation, with or without gene therapy.
Specifically, suicide gene therapies and immunotoxins have been investigated for the treatment of tumors
by direct cancer cell cytotoxicity. Recent advances in mRNA delivery also demonstrated the potential of
mRNA-based vaccines and immune-modulators for cancer therapeutics by utilizing nanocarriers for
mRNA delivery.
Methods: We designed a bacterial toxin-encoding modified mRNA, delivered by lipid nanoparticles into
a B16-melanoma mouse model.
Results: We showed that local administration of LNPs entrapping a modified mRNA that encodes for a
bacterial toxin, induced significant anti-tumor effects and improved overall survival of treated mice.
Conclusions: We propose mmRNA-loaded LNPs as a new class of anti-tumoral, toxin-based therapy.
Keywords: lipid nanoparticles, mRNA, cancer therapy, gene therapy, suicide-gene therapy, immunotoxins

Introduction
mRNA therapeutics have gained great interest in Toll-like receptors mediated RNA recognition [1,4–7].
the last years, being developed to treat a wide range Because of such limitations, DNA was preferred over
of diseases including rare genetic diseases, mRNA for gene therapy development until recent
neurodegenerative diseases, inflammation, cancer years [8–13]. Specifically, viral vectors and naked or
and infectious diseases. Like DNA molecules, mRNA plasmid DNA were mostly investigated for suicide
molecules have the potential to express any protein of gene therapy purposes, using intracellular expression
interest in diseased cells, using the cell’s own protein of a toxic protein or an enzyme that converts a
synthesis machinery [1–4]. non-toxic compound to a cytotoxic molecule to induce
Unlike DNA, mRNA lacks the risk of insertional cancer cell death [8,14–19].
mutagenesis since nucleus entrance is not required for Another biological class of drugs aiming to
its functionality. However, mRNA is less stable than induce tumor cell death are recombinant or
DNA and susceptible to both extracellular and conjugated immunotoxins, which are antibody-toxin
intracellular degradation, caused by either unspecific chimeric proteins [20–24]. Moxetumomab pasudotox
RNase activity or immunogenicity triggered by (Lumoxiti), for instance, is an anti-CD22 Fv murine

https://www.thno.org
Theranostics 2023, Vol. 13, Issue 11 3498

antibody fused to PE38, a 38 kDa truncated form of purchased from TriLink Biotechnologies, having a
pseudomonas exotoxin A (PE) that was approved by 5'-methoxy uridine modification and a Cap1
the U.S. Food and Drug Administration for the structure. Cholesterol, DSPC (1,2- distearoyl-sn-
treatment of relapsed or refractory hairy cell leukemia glycero-3-phosphocholine), polyethylene glycol
[25–27]. The PE domain used for immunotoxins is a (PEG)– DMG (1,2-dimyristoyl-rac-glycerol), and
NAD+-diphthamide ADP-ribosyltransferase that DSPE (1,2-distearoyl-snglycero-3-phosphoethanol-
targets and inactivates eukaryotic translation elonga- amine)–PEG were purchased from Avanti Polar
tion factor 2 (eEF2), leading to apoptosis [24,28–32]. Lipids Inc. Briefly, one volume of lipid mixture
The conjugation of this domain to a specific antibody (ionizable lipid, DSPC, cholesterol, DMG-PEG, and
against cancer-related receptor, is intended to enhance DSPE-PEG at 50:10.5:38:1.4:0.1 molar ratio) in ethanol
drug specificity and lower the risk of adverse effects and three volumes of designated mmRNA (1:10 molar
[20,31,33]. ratio RNA to ionizable lipid) in a citrate buffer, pH 4.5
Although those immunotoxins have reached the were injected into a NanoAssemblr microfluidic
clinic, they have been shown to have inherent mixing device (Precision Nanosystems Inc.) at a
characteristics causing resistance [33]. Their combined flow rate of 12 mL min−1. The formed LNPs
mechanism of action includes target receptor binding, were dialyzed twice against PBS (pH 7.4) for 16 h to
internalization, intracellular toxin processing and remove ethanol.
trafficking [22,27,30,32-33]. All these processes are
prone to resistance such as decreased cell-surface Size distribution
antigen presentation, impaired processing of the toxin mmRNA-LNPs size distribution and ζ potential
or toxin cleavage in the lysosome [33]. were determined by dynamic light scattering using a
To overcome some of the anti-immunotoxins Malvern Nano ZS ζ sizer (Malvern Instruments). For
resistance mechanisms, while avoiding potential risks size measurements, LNPs were diluted 1:20 in PBS.
of DNA-based suicide gene therapy, we suggest an For ζ potential measurements, LNPs were diluted
mRNA-based therapy as an alternative. Herein, we 1:200 in double distilled water.
used modified mRNA encoding the pseudomonas
Transmission electron microscopy
exotoxin A domain, a toxic domain originally
produced by the bacteria pseudomonas aeruginosa. A drop of an aqueous solution containing LNPs
We entrapped in-vitro transcribed modified mRNA was placed on a carbon-coated copper grid, air dried
(mmRNA) encoding for this toxin in lipid nano- and analyzed using a JEOL 1200 EX transmission
particles (LNPs), and intratumorally administered electron microscope.
them to B16-melanoma tumor-bearing mice. We LNP quantification and encapsulation
hypothesize that such platform represents an imp-
To quantify the RNA in LNPs and to determine
roved approach for toxin-based anti-tumor therapy,
the RNA encapsulation efficiency, the Quant-iT
with better safety profile and low immunogenicity
RiboGreen RNA assay (Life Technologies) was used
while maintaining high potency.
as previously described [36]. Briefly, 2 µL of LNPs or
Methods dilutions of ribosomal RNA at known concentrations
were diluted in a final volume of 100 µL of TE buffer
Cell culture growth and maintenance (10 mM tris-HCl, pH 7.4 and 20 mM EDTA) in the
Monolayers of B16F10.9 (murine skin melanoma) presence or absence of 0.5% Triton X-100 (Sigma-
cells were grown in T-75 flasks. The cells were Aldrich) in a 96-well fluorescence plate (Costar,
cultured in Dulbecco's modified Eagle's medium Corning). The plate was incubated for 10 min at 40°C
(DMEM) containing 10% fetal bovine serum, to allow particles to become permeabilized before
penicillin (100 U per mL), streptomycin (0.1 mg/mL), adding 99 µL of TE buffer and 1 µL of RiboGreen
nystatin (12.5 U per mL) and L-glutamine (2 mM). All reagent to each well. Plates were shaken at room
cells were routinely checked every 2 months for temperature for 5 min, and fluorescence (excitation
mycoplasma contamination using the EZ-PCR wavelength of 485 nm and emission was measured
Mycoplasma Test Kit (Biological Industries, Israel). using a plate reader (BioTek Industries) according to
the manufacturer’s protocol.
LNP preparation
DLin-MC3-DMA (MC3) and EA-PIP were
LNP transfection
synthesized according to a previously described Cells were counted using trypan blue (Biological
method [34-35]. Firefly Luciferase, EGFP and PE Industries), and 105 cells/well were placed in 24-well
mmRNA (custom- synthesized, sequence shown in tissue culture plates (Greiner Bio-One, Germany) with
supplementary data) modified mRNA were 0.5 mL of growing medium, or 104 cells/well were

https://www.thno.org
Theranostics 2023, Vol. 13, Issue 11 3499

placed in 96-well tissue culture plates (Greiner In-vivo biodistribution study and expression
Bio-One, Germany) with 100 µl growing medium. kinetics
MC3 or EA-PIP LNPs were added to the wells at RNA Eight to ten weeks old female C57BL/6 mice
amounts of 0.06 to 1 mg/ mL. Cells were incubated (Envigo, Rehovot, Israel) were inoculated
with the LNPs under standard culture conditions for subcutaneously to the right flank with 105 B16F10.9
up to 48 h. Then, cells were washed three times, cells suspended in 50 µL HBSS. 10 days post
incubated in fresh culture medium, and were taken inoculation, mice were intratumorally (I.T.) injected
for functional Luciferase assay system (Promega), with mmFluc-LNPs (mRNA dose: 0.15 mg/Kg). At 6
XTT cell proliferation assay (Biological Industries) or h post-injection, mice were intraperitoneally injected
FACS analysis for EGFP expression or stained with with D-Luciferin (150 mg/Kg) and major organs were
APC Annexin V (BioLegend) and propidium iodide harvested for imaging using IVIS (PerkinElmer Inc).
(SigmaAldrich) according to the manufucturers' For mmRNA expression kinetics of intratumorally-
recommendations and then analyzed by FACS injected mmFluc LNPs, mice were imaged every 24 h
(Cytoflex, Beckman Coulter, USA. 2X105 cells per as described above.
FACS measurement).
mmRNA translation inhibition by mmPE LNPs
B16F10.9 tumor-bearing mice
B16F10.9 were seeded at 3×105 cells/well and
Eight to thirteen weeks old female C57BL/6 mice cultured as described above. 24 h after seeding, cells
(Envigo, Rehovot, Israel) were injected subcuta- were treated with mmPE LNPs and mmFluc LNPs as
neously to the right flank with 105 B16F10.9 cells control at the indicated concentrations. 2 h post
suspended in 50 µL HBSS. For in-vivo imaging treatment, cells were transfected with 0.25 µg/mL
experiments, we used B16F10.9 cells stably trans- EGFP mRNA (TriLink, USA) via messenger max
duced by a lentivirus to express mCherry and Firefly transfection reagent (Invitrogen, USA) according to
luciferase reporter proteins. Treatment start point was the manufacturers’ recommendations. 12 h post EGFP
determined when the tumor volumes reached 40 to 50 transfection, cells were trypsinized, resuspended in
mm3, or for imaging experiments, when labeled PBS/1%FBS and 104 cells were measured by flow
tumors reached luminescence of at least 4×107 p/s, 8– cytometry (Cytoflex, Beckman Coulter, USA).
12 days post tumor inoculation. The tumor
dimensions were measured using Caliper, and tumor In-vivo transfection of tumoral B16F10.9 cells
volume was calculated as: (width)2×length/2. For by I.T. injection of LNPs
imaging experiments, we imaged mice using the Eight to ten weeks-old female C57BL/6 mice
in-vivo imaging system (IVIS, PerkinElmer). To (Envigo, Rehovot, Israel) were inoculated subcutane-
observe luminescence, we intraperitoneally injected ously to the right flank with 105 B16F10.9-mCherry-
mice with 200 µL of 0.15 mg/mL XenoLight Luc labeled cells suspended in 50 µL HBSS. Ten days
D-Luciferin Potassium Salt (PerkinElmer) reconsti- post inoculation, mice were I.T. injected with
tuted in PBS, and the mice were imaged 5 min post mmEGFP-LNPs (mRNA dose: 0.15 mg/Kg). 24 h post
injection. The mice were randomly separated into injection, tumors were dissected, and tumor single
three groups (n = 6 per group): PBS, mmFluc and cells were extracted according to the manufacturer’s
mmPE. mmLNPs were intratumorally injected at a recommendations (mouse tumor dissociation kit,
dose of 0.15 mg/Kg every 2-3 days. Milteny, USA). 105 cells were analyzed by flow
cytometry (Cytoflex, Beckman Coulter, USA).
Histology
All histological sectioning, staining and Statistical analysis
histological evaluation were done by Dr. Zohar Statistical analysis for comparing two
Gavish, Patho-Logica, Ness-Ziona, Israel. Histological experimental groups was performed using two-sided
evaluation and scoring were performed by Dr. Student’s t tests. Kaplan-Meier curves were used to
Emmanuel Loeb, a Veterinary Pathologist. analyze survival. A value of P < 0.05 was considered
statistically significant. Analyses were performed
Animal experiments
with Prism 7 (GraphPad Software). Differences are
All animal protocols were approved by the Tel labeled n.s. for not significant, * for P ≤ 0.05, ** for
Aviv University Institutional Animal Care and Usage P ≤ 0.01, *** for P ≤ 0.001, and **** for P ≤ 0.0001.
Committee and in accordance with current regula- Pre-established criteria for the removal of animals
tions and standards of the Israel Ministry of Health. from the experiment were based on animal health,
Mice were randomly divided at the beginning of each behavior, and well-being as required by the ethical
experiment. guidelines.

https://www.thno.org
Theranostics 2023, Vol. 13, Issue 11 3500

Figure 1. Schematic and microscopic representation of toxin encoding mmRNA-loaded lipid nanoparticles. A. Self-assembly of lipid mixture and mRNA
molecules in acidic buffer composes mRNA-LNPs. mRNA is encoding for the pseudomonas exotoxin A (PE) toxin, which is delivered by the LNPs to cancer cells (C). The
delivered mmPE is then translated by target cells into PE toxin (D) that induces apoptosis (E). B. Representative TEM image of Firefly Luciferase mmRNA-loaded LNPs. Bar scale
– 200 nm.

ion and delivery while maintaining minimal toxicity,

Results we encapsulated in-vitro transcribed mmRNA in
Toxin-encoding mmRNA LNPs design and LNPs composed of a novel ionizable lipid that was
characterization developed by our group, named EA-PIP [35,45]. We
compared EA-PIP (Figure 2B) with the clinically
We utilized in-vitro transcribed mRNA, into
approved, well-studied ionizable lipid Dlin-MC3-
which chemically modified nucleotides were incor-
DMA (MC3) (Figure 2A). We showed that while both
porated, shortly termed modified mRNA (mmRNA),
EA-PIP LNPs and MC3 LNPs had an average size
encoding for either Firefly Luciferase, EGFP or PE, to
distribution below 100 nm and a similar
optimize LNPs formulation. Modified bases incorpo-
encapsulation efficiency, they differed in their ζ
ration into the in-vitro transcribed (IVT) mRNA is a
potential, which was more consistent and closer to
well-investigated method to enhance protein expres-
neutral in EA-PIP LNPs, while MC3 LNPs were
sion and lower mRNA immunogenicity [7,37-38].
marginally negative (Figure 2C-E).
LNPs were synthesized using the NanoAssemblr®
To evaluate their functionality, we examined
microfluidic mixing system (Precision Nanosystems
mmRNA-LNPs loaded with either Firefly Luciferase
Inc., Vancouver, Canada), in which mmRNA mole-
or EGFP mmRNA on different cancer cell lines, from
cules interact with ionizable lipids in acidic conditions
both human and murine origin. Like MC3 LNPs,
and self-assembled with other components to form
EA-PIP LNPs did not exhibit any significant
highly uniform nanoparticles (Figure 1). This control-
cytotoxicity to cells when loaded with reporter
led electrostatic interaction between the mRNA
mmRNA molecules (Figure S1). However, they
molecules and the ionizable lipids, allows an efficient
demonstrated improved in-vitro expression of
encapsulation of the mRNA payload on the one hand,
delivered mmRNA over MC3 LNPs, in agreement
while avoiding the positive charge of the traditional
with our previous publication [46]. We observed this
cationic lipids, which is known to enhance toxicity
dose-dependent effect for both mmFluc-LNPs and
[3,39–44]. We characterized the size distribution and ζ
mmEGFP-LNPs, as reflected by increasing lumines-
potential of the mmRNA-LNPs using dynamic light
cence and fluorescence intensities, respectively
scattering (DLS) measurements. To assess mmRNA
(Figure 2F-H, S2, S7). In mmEGFP LNPs- treated cells,
encapsulation efficiency, we used the Quant-it™
we could show that in high mmRNA concentrations
Ribogreen assay as previously reported [36].
(0.5 and 1 µg/ mL), there was only one population of
To facilitate optimal mmRNA encapsulation in
cells expressing EGFP (Figure 2F-I).
lipid nanoparticles, allowing both mmRNA protect-

https://www.thno.org
Theranostics 2023, Vol. 13, Issue 11 3501

Pseudomonas exotoxin A mmRNA (mmPE) an in-vitro protein translation measurement, using a

encapsulated in LNPs induces high rate of reporter gene mmRNA. We pre-incubated B16F10.9
cancer cell apoptosis and attenuates protein melanoma cells with either mmPE LNPs or mmFluc
translation in-vitro LNPs for 2 h, and then transfected them with EGFP
Next, we encapsulated mmRNA encoding for mmRNA using a commercial transfection reagent. We
the pseudomonas exotoxin A domain III, which is the demonstrated EGFP expression inhibition in cells
catalytic domain of the PE toxin, in LNPs. We pre-treated with mmPE-LNPs compared to cells
hypothesized that encoding the active domain only, pre-treated with mmFluc LNPs and this effect was
without the binding and translocation domains of the dose-dependent (Figures 3B, S6).
original toxin, will allow potent intracellular effect Intratumorally administered mmPE-LNPs
upon mRNA translation, while reducing the risk of lead to intratumoral apoptosis and tumor
side-off effects in case of toxin release from target growth inhibition in a B16-melanoma mouse
cells. We assessed these mmPE LNPs’ ability to model
induce cancer cell death in-vitro. We incubated
To test mmPE-LNPs efficacy in-vivo, we intratu-
B16F10.9 and other cancer cell lines with increasing
morally injected B16-melanoma tumor-bearing mice
amounts of encapsulated PE mmRNA (mmPE) and
with either mmFluc-LNPs or mmPE-LNPs (0.15
demonstrated a significant reduction in cancer cell
mg/Kg) or PBS as a negative control (n = 6 mice /
viability 48 h post treatment (Figures 3A, S3). We also
group). The treatment regimen included four intratu-
confirmed that this cytotoxicity was due to apoptosis,
moral doses, with a gap of 2-3 days between injections
using a PI-Annexin-V staining and FACS analysis.
and final analysis 72 h post the last injection (Figure
B16F10.9 cells treated with mmPE-LNPs had an
5A). These experiment settings and treatment
increasing fraction of apoptotic cells starting from 24 h
protocol corresponds mmFluc LNPs expression
post treatment to a massive rate of ~90% late-
kinetics upon intratumoral injection showed in a
apoptotic cells 48 h post treatment (Figures 3C-D, S8).
different experiment (Figure S4 D&E). Firefly
To further validate that our mmPE-LNPs
luciferase expression caused high luminescence in the
mechanism of action correlates the original PE toxin
tumor 24 h post intratumoral administration, with a
mechanism [24,28], and that the encoded mmPE
decay in the signal over time up to 96 h post
triggers protein translation inhibition, we performed
administration (Figure S4).

Figure 2. Physicochemical characterization and in-vitro expression of MC3-mmRNA-LNPs and EA-PIP-mmRNA-LNPs. A-B. Chemical structures of MC3 (A)
and EA-PIP (B) ionizable lipids. C-D. LNPs' size and zeta potential measurements by dynamic light scattering (DLS). E. mmRNA encapsulation efficiency as reflected in a
RiboGreen-based assay, allowing mmRNA concentration calculation, according to the absorbance of an RNA-binding dye. F. Firefly luciferase expression in B16F10.9 cells 48 h
post incubation with mmFluc-LNPs composed of either MC3 or EA-PIP lipids. H-G. EGFP expression level in B16F10.9 cells 48 h post incubation with mmEGFP-LNPs composed
of either MC3 (H) or EA-PIP (I) lipids. Multiple paired t-tests statistical analysis was performed by Prism GraphPad, *p ≤ 0.05, ****P < 0.0001.

https://www.thno.org
Theranostics 2023, Vol. 13, Issue 11 3502

Figure 3. mmPE-LNPs therapeutic effect in-vitro: cancer cell viability reduction via apoptosis and protein translation inhibition. A. B16F10.9 cell viability rate
48 h post treatment with mmPE-LNPs. Cells were incubated with increasing doses of mmPE-LNPs, composed of either MC3 or EA-PIP lipids. 48 h post treatment, the viability
rate of treated cells was exceptionally low (~10%) in all tested conditions. B. B16F10.9 cells pre-treated with mmPE-LNPs, had lower expression levels of transfected EGFP
mmRNA compared to cells pre-treated with mmFluc LNPs, suggesting that mmPE LNPs inhibit protein translation. C. PI-Annexin assay for determination of necrosis and
apoptosis rates 3 h, 24 h and 48 h post treatment. D. Graphical representation of PI-Annexin-V-stained cells according to their viability state: live, necrotic, early apoptotic or late
apoptotic at all tested timepoints, indicating mmPE-LNPs caused significant apoptosis 48 h post treatment.

Figure 4. Intratumorally-injected, repeated doses of mmPE-LNPs caused in-vivo apoptosis in tumor cells. Representative images of mice tumors IHC stained for
either caspase-3 (A-C) or TUNEL (E-G), yellow arrows mark stained cells. Left panel (A, E) represents PBS-treated mice, middle panel (B, E) represents mmFluc-LNPs treated
mice and right panel (C, G) represents mmPE-LNPs treated mice (0.15 mg/Kg, 50 µl, four doses). All specimens (N = 3) were classified by a veterinary pathologist according to
the following: Grade 0 = no positive reaction at all, Grade 1= Only few cells are positive (< 5 cells per a X20 field), Grade 2 = Very mild positive stain (5-15 cells per a X20 field),
Grade 3 = Mild positive stain (15-25 cells per a X20 field), Grade 4= Moderate positive stain (25-50 cells per a X20 field), Grade 5 = Marked positive stain (> 50 cells per a X20
field). D&H are graphical representations of the average scoring of caspase-3 and TUNEL positive cells, respectively.

https://www.thno.org
Theranostics 2023, Vol. 13, Issue 11 3503

Figure 5. Intratumorally-injected mmPE-LNPs effect on tumor growth in a B16F10.9 mouse model. A. Experiment settings and timeline. Mice were
subcutaneously inoculated with B16F10.9 melanoma cells and were treated with four intratumoral, repeated doses of either PBS, mmFluc LNPs or mmPE LNPs (0.15 mg/Kg, 50
µl), starting 12 days post tumor inoculation. B. Average percentage of mice's weight compared to first day of treatment, representing weight loss rate. C. Ex-vivo tumors at
experiment endpoint, visually demonstrating tumor sizes. D. Average tumor volume of mice from first day of treatments [n = 6 mice / group. Student's t-test statistical analysis
of every pair of groups was performed using Prism GraphPad, *p ≤0.05.

Mice receiving mmPE-LNPs had smaller tumor concerns when developing a potent anti-cancer
volumes in all tested timepoints, and significantly therapy. In addition to monitoring mice weight
lower tumor volumes at the experiment endpoint in-vivo, indicating there is no substantial adverse
compared to the control group, as reflected by both effect that caused weight loss, we also performed a
tumor volumes measured in the whole animal, and biodistribution test and demonstrated minimal
ex-vivo tumor sizes (Figure 5C-D). We also did not mmRNA expression in the main blood-filtrating
observe a significant reduction in the average weight organs upon mmFluc LNPs I.T. injection (Figure
of the treated mice compared to the control mice, S4A-C).
suggesting there was no substantial systemic toxicity We have evaluated general toxicity by liver and
of the treatment (Figure 5B). spleen histology, liver enzyme levels and pro-
To strengthen these observations and validate inflammatory cytokines measurement in the blood.
the mechanism of action of the toxin encoded by the H&E staining of liver and spleen tissues of mice
therapeutic mmRNA, PE, which is known to induce receiving four doses of either PBS, mmFluc LNPs or
protein synthesis arrest leading to apoptosis [24,28], mmPE LNPs (0.15 mg/Kg mmRNA) indicated that
we harvested tumors from the mice at the final day of there were no significant changes in tested tissues
the experiment and analyzed them using immuno- compared to healthy mice, three days post last
histochemistry staining. We employed caspase-3 injection (Figure 6A-H). Additionally, a single,
staining to detect early apoptotic changes, and intratumoral dose of mmPE LNPs (0.5 mg/Kg) did
TUNEL assay to detect late apoptosis reflected by not affect serum liver enzymes levels, as compared to
DNA fragmentation. We observed higher staining for untreated mice 24 h post administration (Figure 6I).
both caspase-3 and TUNEL in the mmPE LNPs- Furthermore, we could not observe any detectable
treated group compared to the control groups, with a levels of either IL-6 or TNF-α in mice’s serum 2 h and
significantly higher scoring of TUNEL staining in 24 h post mmPE LNPs intratumoral injection (data not
mmPE LNPs group compared to the controls, shown).
indicating late apoptosis in the treated tumors at the
experiment endpoint (Figure 4). Repeated doses of mmPE-LNPs inhibited
tumor growth and increased overall survival
Safety profile of intratumorally administered rate
mmPE LNPs Next, we aimed to evaluate the effect of our
Systemic toxicity and side-off effects are main treatment on mice survival rate and determine tumor

https://www.thno.org
Theranostics 2023, Vol. 13, Issue 11 3504

growth inhibition in a quantitative manner. For that, lower tumor volumes at days 20, 22 and 25 post tumor
we utilized mCherry-Luciferase-labeled B16F10.9 cells inoculation compared to the control group (Figure
for tumor inoculation. Mice were intratumorally 8B&D). We could also show a significant increase in
injected with PBS, mmFluc LNPs or mmPE LNPs, the survival rate of mmPE-LNPs treated mice,
every 2-3 days. Each mouse was treated until reaching compared to the control mice, with no considerable
an ethical sacrificing criterion of tumor volume of average weight loss (Figure 7B&E).
1500 mm3, and then was sacrificed. Overall, the Facilitating the same mCherry-Luciferase-
experiment lasted 35 days from tumor inoculation, labeled tumor model, we were able to demonstrate
with 10 injection timepoints (Figure 7A). The tumors' cancer cells-specific transfection in-vivo. We displayed
mCherry and Luciferase intensities were tracked expression of mmEGFP delivered intratumorally by
using in-vivo imaging system (IVIS) every 2-3 days our LNPs. We observed that 44-60% of the mCherry-
until the experiment endpoint. We could exhibit labeled cells expressed EGFP 24 h following a single
tumor growth inhibition, reflected in either tumor size mmEGFP LNPs intratumoral injection. Delivery
measured with Caliper, mCherry signal and specificity was excellent, as a great part of the
Luciferase signal in the mmPE-LNPs treated group EGFP-expressing cells in-vivo were mCherry positive,
compared to the control groups (Figures 7C&D, 8). indicating most LNPs have reached cancer cells
Mice receiving mmPE-LNPs had reduced tumor (Figures 7F, S5).
volumes in all tested timepoints, and significantly

Figure 6. Safety profile analysis of mmPE-LNPs. A-H. Histology of liver (A-D) and spleen (E-H) samples (hematoxylin and eosin staining, representative images) of either
healthy (A,E), PBS-injected (B,F), mmFluc-LNPs-injected (C,G) or mmPE-LNPs-injected mice (D,H), after four intratumoral injections, 0.15 mg/Kg, 50 µl. I. Liver enzymes of mice
intratumorally injected with either mmFluc LNPs or mmPE LNPs, compared to untreated mice, 24 h post injection (0.15 mg/Kg, 50 µl), indicating no significant increase has
occurred in the treatment group.

https://www.thno.org
Theranostics 2023, Vol. 13, Issue 11 3505

Figure 7. Intratumorally injected mmPE-LNPs caused tumor volume reduction, cancer-cell specific expression and better survival rate, with minimal
systemic toxicity. A. Experiment timeline and settings. B. Average percentage of mice's weight compared to first day of treatment, representing weight loss rate. No significant
weight loss in the treated group (mmPE, repeated doses, 0.15 mg/Kg, 50 µl) compared to the control groups (PBS and mmFluc) was observed. C. Average tumor volume of all
groups. [n = 6 mice / group. Statistical analysis was done using student's t-test for every pair of groups, performed by Prism GraphPad, *p ≤0.05]. D. Tumor volume of each
individual mouse until reaching sacrificing criteria (tumor volume ≥ 1500 mm3). E. Kaplan-Meier plot representing survival rate of all groups., showing significant positive effect of
mmPE-LNPs on mice's survival rate. [Log-rank (Mantel-Cox) test was used for curve comparison using Prism GraphPad, *p ≤0.05, **p ≤0.01.]. F. Representative FACS analysis
of mCherry-labeled tumor cells of mice receiving a single IT dose of mmEGFP-LNPs, 24 h post injection. A substantial fraction of almost 60% out of the mCherry-labeled cells also
co-expressed EGFP (FITC), demonstrating a high delivery rate. Additionally, nearly all EGFP expressing cells were also mCherry positive, representing extremely high specificity.

Figure 8. Intratumorally-injected mmPE-LNPs inhibited B16-melanoma growth, as reflected by lower luminescence and fluorescence signals of labelled
tumors. A&C. Representative IVIS images showing mCherry-luc labelled B16F10.9 tumor progression as reflected by either mCherry (A) or Firefly-luciferase (C) signals starting
48 h post the first injection (PBS, mmFluc LNPs or mmPE LNPs, 0.15 mg/Kg, 50 µl). Mice reaching a threshold tumor size of 1500 mm3 were sacrificed and therefore had no
imaging in later timepoints. Mice have reached larger tumors sooner in the control groups compared to the mmPE-LNPs treated groups. B&D. Average flux of mCherry (B) or
Firefly Luciferase (D) signals from tumors of all groups over time showing that tumor signals were significantly lower in the treatment group for both Firefly luciferase and
mCherry reporters at days 20, 22 and 25 post tumor inoculation, compared to the control groups. [Statistical analysis was done using student's t-test for every pair of groups,
performed by Prism GraphPad, *p ≤0.05.]

https://www.thno.org
Theranostics 2023, Vol. 13, Issue 11 3506

immunotoxin currently under clinical evaluation

Discussion showed differentiated effect on tumors originated
LNPs are rapidly emerging in recent years as from different mesothelin expressing cell lines, and
vehicles for mRNA delivery and were shown to be a the researchers claimed that the target receptor
flexible and quickly scalable platform for this purpose shedding might be the reason [23]. Low receptor
[47–50]. Their clinical application for inducing tumor expression on target cells can also decrease efficacy, as
cell death is emerging as a novel, promising field. The seen for the immunotoxin BL22 that was tested in CLL
mainstream of such developing therapies nowadays patients [32]. Some immunotoxins can still bind
are immunotherapies, including cancer vaccines normal cells and lead to vascular leak syndrome, in
employing mRNA encoding for tumor associated which endothelial cells are damaged, so fluid from
antigens [51–54], mRNA expressing monoclonal circulation leaks into the tissues and can lead to
antibodies against such antigens [55], and immuno- edema, a fall in serum proteins, hypotension, and
modulating cytokines such as IL-12 and TGF-β- vascular collapse [20]. In addition, mAb conjugation
encoding mRNA [56–59]. may reduce the anti-cancerous toxic effect of the toxin.
However, there is a need for alternative The conjugation of F1G4 mAb to Abrin toxin, for
treatments in tumors that do not express an identified example, lowered the cancer cell death rate in-vitro,
unique antigen, or tumors which are resistant or compared to the free toxin [21]. We assume that
unresponsive to immunological treatments. Expres- intracellular synthesis of the intact, catalytically-active
sing a cytotoxic gene by the tumor cells represents a toxic domain, can overcome such limitations.
more straightforward approach, which could be mmRNA delivery into tumor cells allows such
useful in such indications. In this respect, mmRNA- intracellular synthesis, due to the universality of the
entrapped in LNPs has the potential to expend the genetic code of mRNA molecules. In an interesting
therapeutic index of the expressed lethal protein, as study that might address some of those challenges,
the toxic element is not found in the bloodstream, and the investigators showed that immunotoxin
it is produced at the site of mmRNA internalization. expression and secretion by human primary T cells
To our knowledge, similar studies aiming to transfected with immunotoxin-encoding mRNA,
intratumorally express a toxin or a cytotoxic protein caused cancer cell death when co-cultured with
made use of either cationic lipid-based formulations, ovarian cancer cells. However, the cytotoxic effect was
or a protamine-lipid formulations [60–62]. While mild and in-vivo evaluation was not presented [63].
ionizable lipid- based lipid nanoparticles have Here we demonstrated the concept of utilizing
close-to neutral surface charge, positively charged mmRNA encapsulated in LNPs, as a feasible type of
nanoparticles are known to have an increased in-vivo suicide-gene therapy, for the treatment of solid
surface protein corona formation, accelerated tumors. We showed that mmRNA encoding for the
elimination by the reticuloendothelial system (RES), pseudomonas exotoxin A domain III encapsulated in
non-specific uptake and high immunogenicity [3,39– LNPs, caused a significant anti-tumoral effect, both
44]. The delivery of toxin-encoding mRNA entrapped in-vitro and in-vivo. This treatment not only inhibited
in lipid nanoparticles is more clinically- relevant and tumor growth, but also enhanced the overall survival
preferable when immune stimulation is unnecessary. of the treated animals. Furthermore, we could show
In addition, we suggest LNP-mediated mmRNA that cancer cell death was due to apoptosis (Figures 3,
delivery to tumor cells as an alternative to immuno- 4). We also demonstrated that the molecular basis for
toxins, which require receptor binding, intracellular cell apoptosis corresponds the known mechanism of
processing and trafficking for functionality. Tumor action of pseudomonas exotoxin A (Figures 4B, S6).
cells can alter these processes to acquire resistance to Additionally, although its high efficacy, our treatment
the treatment [27,33]. Like previously stated regard- did not cause any significant systemic toxicity, as
ing mRNA-based vaccines, immunotoxins provide reflected by animal weight, H&E staining of the liver
targeting to cancers expressing known cell-surface and spleen sections, liver enzymes levels and the
antigens, and so are irrelevant for other types of major cytokines evaluated (Figures 5B, 6, 7B).
cancer. Moreover, there is no clinically- approved Moreover, tumor single cell analysis revealed high
immunotoxin-based therapy for solid tumors efficacy of our treatment, as a single I.T. dose resulted
indications to date. This may be due to low in 44-60% transfection of cancer cells (mCherry
distribution and penetration of large molecules into positive). Specificity was also high as negligible
solid tumors [27]. percent of EGFP-expressing cells were mCherry
Furthermore, receptor shedding from the negative, meaning that the vast majority of the
cellular membrane can hamper immunotoxins detected EGFP signal was expressed by
internalization. For instance, a mesothelin-targeted mCherry-labeled-cancer cells (Figures 7F, S5).

https://www.thno.org
Theranostics 2023, Vol. 13, Issue 11 3507

One important drawback reported is the time Writing the manuscript: Y.G-M, I.B., A.E and
limitation of the therapeutic window effect, as we D.P.
observed a tumor relapse in late stages of the repeated
dosing experiment (Figures 7C, 8). One explanation is Competing Interests
that B16 melanoma models are known to be overly D.P. declares the following competing financial
aggressive with a very rapid growth rate. Therefore, interest(s): D.P. receives licensing fees (to patents on
intratumoral injections have an inherent barrier in which he was an inventor) from, invested in, consults
reaching massive portion of the tumor cells, in late (or on scientific advisory boards or boards of
stages of tumor growth. We cannot rule out antibody directors) for, lectured (and received a fee) or
production against the PE toxin as the reason for conducts sponsored research at TAU for the following
tumor relapse. However, this explanation is less entities: ART Biosciences, BioNtech SE, Eleven
feasible due to the immunosuppressive activity of the Therapeutics, Kernal Biologics, Merck, Newphase
PE toxin [64]. Developing technologies for inducing Ltd., NeoVac Ltd., RiboX Therapeutics, Roche,
targeted, tumor-specific mRNA expression upon SirTLabs Corporation, Teva Pharmaceuticals Inc.
systemic administration has the potential to overcome All other authors declare no competing financial
this obstacle, as the mmRNA-LNPs will reach the interests.
tumor more homogenously via blood circulation. We
hope to further investigate into this aspect and References
demonstrate improved therapeutic effect in more 1. Granot Y, Peer D. Delivering the right message: Challenges and opportunities
in lipid nanoparticles-mediated modified mRNA therapeutics—An innate
tumor models in the future. immune system standpoint. Semin Immunol. 2017; 34: 68-77.
In conclusion, using toxin-encoding mmRNA 2. Meyer RA, Neshat SY, Green JJ, Santos JL, Tuesca AD. Targeting strategies for
mRNA delivery. Mater Today Adv. 2022; 14: 11-20.
entrapped in LNPs, can overcome some of the above 3. Rahman MM, Zhou N, Huang J. An Overview on the Development of
discussed limitations of the current immunotoxin- mRNA-Based Vaccines and Their Formulation Strategies for Improved
Antigen Expression In Vivo. Vaccines. 2021; 9: 244–244.
based anti-cancer therapies, with an additional 4. Sahin U, Karikó K, Türeci Ö. mRNA-based therapeutics — developing a new
advantage of a safer payload delivery to tumor cells. class of drugs. Nat Rev Drug Discov 2014; 13: 759–80.
5. Beck JD, Reidenbach D, Salomon N, et al. mRNA therapeutics in cancer
This platform may represent a new class of treatment immunotherapy. Mol Cancer. 2021; 20: 1–24.
that can address an unmet clinical need for solid 6. Weng Y, Li C, Yang T, et al. The challenge and prospect of mRNA therapeutics
landscape. Biotechnol Adv. 2020; 40: 107534.
tumors therapy. 7. Guan S, Rosenecker J. Nanotechnologies in delivery of mRNA therapeutics
using nonviral vector-based delivery systems. Gene Ther. 2017; 24: 133–43.
8. Youn H, Chung J-K. Modified mRNA as an alternative to plasmid DNA
Abbreviations (pDNA) for transcript replacement and vaccination therapy. Expert Opin Biol
Ther. 2015; 15: 1337–48.
MC3: DLin-MC3-DMA; LNPs: Lipid nanoparti- 9. Mizrahi A, Czerniak A, Levy T, et al. Development of targeted therapy for
cles; mmRNA: modified mRNA; PI: propidium ovarian cancer mediated by a plasmid expressing diphtheria toxin under the
control of H19 regulatory sequences. J Transl Med 2009; 7: 69.
iodide; FACS: Fluorescence-activated cell sorting; 10. Gofrit ON, Benjamin S, Halachmi S, et al. DNA Based Therapy with
TEM: transmission electron microscopy; PE: pseudo- Diphtheria Toxin-A BC-819: A Phase 2b Marker Lesion Trial in Patients with
Intermediate Risk Nonmuscle Invasive Bladder Cancer. J Urol. 2014; 191:
monas exotoxin; mAB: monoclonal antibody; I.T.: 1697–702.
intratumorally. 11. Amit D, Tamir S, Birman T, Gofrit ON, Hochberg A. Development of targeted
therapy for bladder cancer mediated by a double promoter plasmid
expressing diphtheria toxin under the control of IGF2-P3 and IGF2-P4
Supplementary Material regulatory sequences. Int J Clin Exp Med. 2011; 4: 91–102.
12. Moradian C, Rahbarizadeh F. PE38-based gene therapy of HER2-positive
Supplementary figures. breast cancer stem cells via VHH-redirected polyamidoamine dendrimers. Sci
Rep. 2021; 11: 15517.
https://www.thno.org/v13p3497s1.pdf 13. Shapira S, Boustanai I, Kazanov D, Ben Shimon M, Fokra A, Arber N.
Innovative dual system approach for selective eradication of cancer cells using
Acknowledgements viral-based delivery of natural bacterial toxin-antitoxin system. Oncogene.
2021; 40: 4967–79.
14. Vago R, Collico V, Zuppone S, Prosperi D, Colombo M.
Funding Nanoparticle-mediated delivery of suicide genes in cancer therapy. Pharmacol
Res. 2016; 111: 619–41.
This work was supported in part by the Shmunis 15. Ardini M, Vago R, Fabbrini MS, Ippoliti R. From Immunotoxins to Suicide
Toxin Delivery Approaches: Is There a Clinical Opportunity? Toxins. 2022; 14:
Family Foundation awarded to D.P. 579–579.
16. Karjoo Z, Chen X, Hatefi A. Progress and problems with the use of suicide
Graphical illustrations genes for targeted cancer therapy. Adv Drug Deliv Rev. 2016; 99: 113–28.
17. Duarte S, Carle G, Faneca H, Lima MCP de, Pierrefite-Carle V. Suicide gene
All graphical illustrations were created with therapy in cancer: Where do we stand now? Cancer Lett. 2012; 324: 160–70.
BioRender.com. 18. Yahya EB, Alqadhi AM. Recent trends in cancer therapy: A review on the
current state of gene delivery. Life Sci. 2021; 269: 119087–119087.
19. Sun W, Shi Q, Zhang H, et al. Advances in the Techniques and Methodologies
Author contributions of Cancer Gene Therapy. Discov Med. 2019; 27: 45–55.
20. Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ. Immunotoxin treatment of
Conceptualization: Y.G-M, I.B. and D.P. cancer. Annu Rev Med. 2007; 58: 221–37.
Experimentation: Y.G-M, S.R., A.E. 21. Gadadhar S, Karande AA. Abrin Immunotoxin: Targeted Cytotoxicity and
Intracellular Trafficking Pathway. PLoS One. 2013; 8 (3): e58304.
22. Kreitman RJ. Immunotoxins for targeted cancer therapy. AAPS J. 2006; 8:
E532–51.

https://www.thno.org
Theranostics 2023, Vol. 13, Issue 11 3508
23. Cerise A, Bera TK, Liu X, Wei J, Pastan I. Anti-Mesothelin Recombinant TGF-β receptor ii potentiates antitumor immunity. Oncotarget. 2014; 5: 10100–
Immunotoxin Therapy for Colorectal Cancer. Clin Colorectal Cancer. 2019; 18: 13.
192-199. 57. Lei S, Zhang X, Men K, et al. Efficient Colorectal Cancer Gene Therapy with
24. Pastan I. Targeted therapy of cancer with recombinant immunotoxins. IL-15 mRNA Nanoformulation. Mol Pharm. 2020; 17: 3378–91.
Biochim Biophys Acta Rev Cancer. 1997; 1333(2): C1-6. 58. Men K, Huang R, Zhang X, et al. Delivery of interleukin-22 binding protein
25. Nobre CF, Newman MJ, DeLisa A, Newman P. Moxetumomab (IL-22BP) gene by cationic micelle for colon cancer gene therapy. RSC Adv. 8:
pasudotox-tdfk for relapsed/refractory hairy cell leukemia: a review of 16537–48.
clinical considerations. Cancer Chemother Pharmacol. 2019; 84: 255–63. 59. Hewitt SL, Bailey D, Zielinski J, et al. Intratumoral IL12 mRNA Therapy
26. Dhillon S. Moxetumomab Pasudotox: First Global Approval. Drugs. 2018; 78: Promotes TH1 Transformation of the Tumor Microenvironment. Clin Cancer
1763–7. Res. 2020; 26: 6284–98.
27. Kim JS, Jun SY, Kim YS. Critical Issues in the Development of Immunotoxins 60. Men K, Zhang R, Zhang X, et al. Delivery of modified mRNA encoding
for Anticancer Therapy. J Pharm Sci. 2020; 109: 104–15. vesicular stomatitis virus matrix protein for colon cancer gene therapy. RSC
28. Michalska M, Wolf P. Pseudomonas Exotoxin A: optimized by evolution for Adv. 2018; 8: 12104–15.
effective killing. Front Microbiol. 2015; 6: 963–963. 61. Wang Y, Su HH, Yang Y, et al. Systemic delivery of modified mRNA encoding
29. Hwang J, Fitzgerald DJ, Adhya S, Pastan I. Functional domains of herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy. Mol
Pseudomonas exotoxin identified by deletion analysis of the gene expressed in Ther. 2013; 21: 358–67.
E. coli. Cell. 1987; 48: 129–36. 62. Lin Y-X, Wang Y, Ding J, et al. Reactivation of the tumor suppressor PTEN by
30. Weldon JE, Pastan I. A guide to taming a toxin--recombinant immunotoxins mRNA nanoparticles enhances antitumor immunity in preclinical models. Sci
constructed from Pseudomonas exotoxin A for the treatment of cancer. FEBS J. Transl Med. 2021; 13: eaba9772.
2011; 278: 4683–700. 63. Eggers R, Philippi A, Altmeyer MO, Breinig F, Schmitt MJ. Primary T cells for
31. Ibrahim AA. Current status of immunotoxins application in cancer patient’s mRNA-mediated immunotoxin delivery. Gene Ther. 2018; 25: 47–53.
treatment. Int J Hosp Res. 2021; 10 (3). 64. Holt PS, Misfeldt ML. Variables which affect suppression of the immune
32. Havaei SM, Aucoin MG, Jahanian-Najafabadi A. Pseudomonas response induced by Pseudomonas aeruginosa exotoxin A. Infect Immun.
Exotoxin-Based Immunotoxins: Over Three Decades of Efforts on Targeting 1986; 52: 96–100.
Cancer Cells With the Toxin. Front Oncol. 2021; 11: 5158–5158.
33. Dieffenbach M, Pastan I. Mechanisms of Resistance to Immunotoxins
Containing Pseudomonas Exotoxin A in Cancer Therapy. Biomolecules. 2020;
10: 979.
34. Singh MS, Peer D. RNA nanomedicines: the next generation drugs? Curr Opin
Biotechnol. 2016; 39: 28–34.
35. Kampel L, Goldsmith M, Ramishetti S, et al. Therapeutic inhibitory RNA in
head and neck cancer via functional targeted lipid nanoparticles. J
Control Release. 2021; 337: 378–89.
36. Veiga N, Goldsmith M, Granot Y, et al. Cell specific delivery of modified
mRNA expressing therapeutic proteins to leukocytes. Nat Commun. 2018; 9:
4493.
37. Gómez-Aguado I, Rodríguez-Castejón J, Beraza-Millor M, Rodríguez-Gascón
A, del Pozo-Rodríguez A, Solinís MÁ. mRNA delivery technologies: Toward
clinical translation. Int Rev Cell Mol Biol. 2022; 372: 207–93.
38. Li B, Luo X, Dong Y. Effects of Chemically Modified Messenger RNA on
Protein Expression. Bioconjugate Chem. 2016; 27: 849–53.
39. Liu C, Zhang L, Zhu W, et al. Barriers and Strategies of Cationic Liposomes for
Cancer Gene Therapy. Mol Ther Methods Clin Dev. 2020; 18: 751–64.
40. Tan Y, Huang L. Overcoming the Inflammatory Toxicity of Cationic Gene
Vectors. J Drug Target. 2002; 10: 153–60.
41. Lv H, Zhang S, Wang B, Cui S, Yan J. Toxicity of cationic lipids and cationic
polymers in gene delivery. J Control Release. 2006; 114: 100–9.
42. Christensen D, Korsholm KS, Rosenkrands I, Lindenstrøm T, Andersen P,
Agger EM. Cationic liposomes as vaccine adjuvants. Expert Rev Vaccines.
2007; 6: 785–96.
43. Lonez C, Vandenbranden M, Ruysschaert J-M. Cationic lipids activate
intracellular signaling pathways. Adv Drug Deliv Rev. 2012; 64: 1749–58.
44. Ruseska I, Fresacher K, Petschacher C, Zimmer A. Use of Protamine in
Nanopharmaceuticals—A Review. Nanomaterials. 2021; 11: 1508–1508.
45. Singh MS, Ramishetti S, Landesman-Milo D, et al. Therapeutic Gene Silencing
Using Targeted Lipid Nanoparticles in Metastatic Ovarian Cancer. Small.
2021; 17: 2100287–2100287.
46. Ramishetti S, Hazan-Halevy I, Palakuri R, et al. A Combinatorial Library of
Lipid Nanoparticles for RNA Delivery to Leukocytes. Adv Mater. 2020; 32:
e1906128.
47. Yang W, Cao J, Cheng H, et al. Nanoformulations targeting immune cells for
cancer therapy: mRNA therapeutics. Bioact Mater. 2023; 23: 438–70.
48. Dobrowolski C, Paunovska K, Hatit MZC, Lokugamage MP, Dahlman JE.
Therapeutic RNA Delivery for COVID and Other Diseases. Adv Healthc
Mater. 2021; 10 (15): e2002022.
49. Park KS, Sun X, Aikins ME, Moon JJ. Non-viral COVID-19 vaccine delivery
systems. Adv Drug Deliv Rev. 2021; 169: 137–51.
50. Liang F, Lindgren G, Lin A, et al. Efficient Targeting and Activation of
Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine
Administration in Rhesus Macaques. Mol Ther. 2017; 25: 2635–47.
51. Grunwitz C, Kranz LM. mRNA Cancer Vaccines-Messages that Prevail. Curr
Top Microbiol Immunol. 2017; 405: 145–64.
52. Mockey M, Bourseau E, Chandrashekhar V, et al. mRNA-based cancer
vaccine: prevention of B16 melanoma progression and metastasis by systemic
injection of MART1 mRNA histidylated lipopolyplexes. Cancer Gene Ther.
2007; 14: 802–14.
53. Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines - a new era in
vaccinology. Nat Rev Drug Discov. 2018; 17: 261–79.
54. Lorentzen CL, Haanen JB, Met Ö, Svane IM. Clinical advances and ongoing
trials of mRNA vaccines for cancer treatment. Lancet Oncol. 2022; 23: e450–8.
55. Rybakova Y, Kowalski PS, Huang Y, et al. mRNA Delivery for Therapeutic
Anti-HER2 Antibody Expression In Vivo. Mol Ther. 2019; 27: 1415–23.
56. Van der Jeught K, Joe PT, Bialkowski L, et al. Intratumoral administration of
mRNA encoding a fusokine consisting of IFN-β and the ectodomain of the

https://www.thno.org