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Pharmaceutics 2022, 14, 137. https://doi.org/10.3390/pharmaceutics14010137 www.mdpi.com/journal/pharmaceutics
Review
Gene Therapy Using Nanocarriers for Pancreatic Ductal
Adenocarcinoma: Applications and Challenges in Cancer
Therapeutics
Eun-Jeong Won 1, Hyeji Park 2, Tae-Jong Yoon 1 and Young-Seok Cho 2,*
1 Laboratory of NanoPharmacy, College of Pharmacy, Research Institute of Pharmaceutical Science and
Technology (RIPST), Ajou University, 206 Worldcup-ro, Yeongtong-gu, Suwon 16499, Korea;
won4456@naver.com (E.-J.W.); tjyoon@ajou.ac.kr (T.-J.Y.)
2 Division of Gastroenterology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of
Medicine, The Catholic University of Korea, Seoul 06591, Korea; phj8637@naver.com
* Correspondence: yscho@catholic.ac.kr; Tel.: +82-1-2258-6021
Abstract: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide,
and its incidence is increasing. PDAC often shows resistance to several therapeutic modalities and
a higher recurrence rate after surgical treatment in the early localized stage. Combination chemo-
therapy in advanced pancreatic cancer has minimal impact on overall survival. RNA interference
(RNAi) is a promising tool for regulating target genes to achieve sequence-specific gene silencing.
Here, we summarize RNAi-based therapeutics using nanomedicine-based delivery systems that
are currently being tested in clinical trials and are being developed for the treatment of PDAC.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9
(Cas9) genome editing has been widely used for the development of cancer models as a genetic
screening tool for the identification and validation of therapeutic targets, as well as for potential
cancer therapeutics. This review discusses current advances in CRISPR/Cas9 technology and its
application to PDAC research. Continued progress in understanding the PDAC tumor microen-
vironment and nanomedicine-based gene therapy will improve the clinical outcomes of patients
with PDAC.
Keywords: pancreatic ductal adenocarcinoma; miRNA; siRNA; CRISPR/Cas9; nanocarrier
1. Introduction
Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer re-
lated deaths in the Unites States and the seventh leading cause worldwide. Its incidence
is increasing by 0.5% to 1.0% per year, and it is thought it will be the second leading
cause of cancer-related death by 2030 [1]. As its early diagnosis is very difficult, ap-
proximately 80% of patients present with locally advanced or metastatic disease [2].
PDAC often shows resistance to several therapeutic modalities and a higher recurrence
rate after surgical treatment [3]. Adjuvant chemotherapy with 5-fluorouracil, leucovorin,
irinotecan, and oxaliplatin (FOLFIRINOX), without bolus fluorouracil (modified
FOLFIRINOX) leads to a median overall survival (OS) of 54.4 months among patients
with resected pancreatic cancer [4]. However, combination chemotherapy such as
FOLFIRINOX or nab-paclitaxel plus gemcitabine in advanced pancreatic cancer has
minimal impact on OS in the range of weeks to months [5,6]. In addition, a number of
clinical trials have tested pathway-specific targeted therapeutic agents, such as vascular
endothelial growth factor inhibitors and multi-kinase inhibitors, alone or combined with
conventional chemotherapy in metastatic pancreatic cancer but failed to demonstrate
clinical meaningful benefits [7]. Although immune checkpoint inhibitors, such as cyto-
Citation: Won, E.-J.; Park, H.; Yoon,
T.-J.; Cho, Y.-S. Gene Therapy Using
Nanocarriers for Pancreatic Ductal
Adenocarcinoma: Applications and
Challenges in Cancer Therapeutics.
Pharmaceutics 2022, 14, 137.
https://doi.org/10.3390/
pharmaceutics14010137
Academic Editors: Elena
Mourelatou, Sophia Hatziantoniou,
Eleftheria Galatou and Gabriele
Grassi
Received: 20 November 2021
Accepted: 31 December 2021
Published: 6 January 2022
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and insti-
tutional affiliations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/license
s/by/4.0/).
Pharmaceutics 2022, 14, 137 2 of 16
toxic T lymphocyte protein 4 (CTLA4) and programmed cell death protein 1 (PD-1) have
shown promise for the treatment of multiple cancers, these agents demonstrate limited
responses for the treatment of patients with PDAC, probably due to the multiple im-
mune-regulatory pathways within the pancreatic tumor microenvironment (TME) [8].
At present, the initiating mechanisms of PDAC are relatively well understood. Most
PDACs develop from premalignant lesions called pancreatic intraepithelial neoplasias
(PanINs), which progress stepwise from low grade to high grade in types 1, 2, and 3.
Subsequently, they progress to invasive lesions with the accumulation of various genetic
alterations. Approximately 90% of PanINs of all grades have point mutations in the
KRAS oncogene (particularly within codon 12). In addition, mutational inactivation of
tumor suppressor genes, including cyclin-dependent kinase inhibitor 2A, tumor protein
53 (TP53), and SMAD family member 4 (SMAD4), are frequently detected in type 2 and 3
lesions. These findings suggest that KRAS mutations are associated with tumor initiation,
and subsequent gene mutations are a rate-limiting step for tumor progression [2,9].
However, the molecular mechanisms underlying metastatic spread still need to be clari-
fied. The TME of PDAC is heterogeneous and characterized by dense stroma, which
consists of proliferating myofibroblasts (pancreatic stellate cells), extracellular matrix
proteins and tumor vasculature, and inflammatory cells, including macrophages, mast
cells, plasma cells, and lymphocytes [10]. Extensive desmoplastic stroma and severe
hypovascularity of PDAC make it difficult to effectively deliver therapeutic agents to
PDAC cells and are associated with poor prognosis and increased tumor invasion and
metastatic spread [11]. A number of therapeutic agents to target or modify the TME are
currently being evaluated but have shown poor and contradictory results due to the
multi-faceted nature of tumor stroma. Genomic profiling has made novel therapeutics
feasible for a small subset of patients, but current approaches require further testing in
larger clinical trials [12].
Nanotechnology has led to significant advances in the diagnosis and treatment of
various malignancies. To successfully treat PDAC, it requires effective delivery of drugs
into the TME as well as tumor cells. Delivery using nanomaterial-based carrier systems
enhances the antitumor activity of conventional chemotherapeutic agents including
gemcitabine, fluorouracil, doxorubicin, and paclitaxel. The development and application
of these drugs for the treatment of pancreatic cancer have been comprehensively re-
viewed elsewhere [13]. In addition, there have been great advances in the development of
efficient delivery systems for gene therapy for the cancer treatment. Non-viral gene de-
livery systems are highly effective and are safer and easier to synthesize than delivery
systems using viral vectors. For successful gene therapy, a number of delivery systems
including liposomes, polymers, and inorganic nanoparticles have been developed and
investigated with cancer-targeting moieties [14].
In this review, we present RNA interference (RNAi)-based therapeutics using na-
nomedicine-based delivery systems that are currently being investigated in clinical trials
for the treatment of PDAC. Recently, we developed a novel therapeutic strategy that
targets KRAS and TP53 mutations at same time via liposome delivery of clustered regu-
larly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9
(Cas9), ribonucleoprotein (RNP) complexes, and single-guide RNA (sgRNA) to over-
come drug-resistance of PDAC, and this treatment significantly enhanced the anti-tumor
activity of gemcitabine [15]. In addition, current advances in the application of genome
editing technology to PDAC research are reviewed. Finally, we provide our perspective
on the development of gene therapy using nanotechnology for future clinical translation.
2. RNAi Therapy for Pancreatic Cancer
RNAi is a process that regulates target genes using double-stranded RNAs (dsR-
NAs), which bind to sequences complementary to a gene’s coding sequence, resulting in
the degradation of corresponding mRNAs and subsequent inhibition of translation to
proteins. It can be achieved using three major different types of RNAi molecules: small
Pharmaceutics 2022, 14, 137 3 of 16
interfering RNA (siRNA), microRNA (miRNA), and short hairpin RNA (shRNA). RNAi
is initiated by Dicer enzyme mediated processing of dsRNA to smaller fragments (~22
nucleotides) of siRNA, which are then incorporated into RNA-induced silencing com-
plexes [16]. Gene therapy using RNAi therapeutics is promising and improves precise
gene delivery to treat human diseases including various cancers. Patisiran is an siRNA
enveloped in lipid NPs, which inhibits the hepatic synthesis of transthyretin (TTR) and
improves several clinical manifestations of hereditary TTR-mediated amyloidosis (HTA)
[17]. It was approved in 2018 as the first RNAi-based therapy by the United States Food
and Drug Administration and European Union for the treatment of HTA in adults [18].
However, there are several limitations of RNAi-based therapies, including easy nucle-
ase-induce degradation within body fluids such as serum and inefficient delivery to de-
sired cells, tissues, and organs. To overcome these problems, various nanocarriers have
been developed, and these are highly attractive since they are safer and easier to synthe-
size than delivery systems using viral vectors. They fall into two categories: organic
complexes (lipid complexes, conjugated polymers, and cationic polymers) and inorganic
NPs (magnetic NPs, quantum dots, carbon nanotubes, and gold NPs).
2.1. RNAi Using miRNAs
miRNAs are 18–25 nucleotides in length and are endogenous non-coding RNA
molecules that bind, at least partially, to complementary mRNA sequences, subsequently
inducing target mRNA cleavage and degradation. Unlike siRNA, miRNAs can regulate
multiple mRNAs rather than just one. miRNAs play significant roles in the expression of
genes involved in cancer initiation, growth, progression, metastasis, drug resistance, and
therapeutic efficacy. In addition, those secreted by exosomes from cancer cells regulate
intercellular communication processes in the TME [19]. In PDAC, some miRNAs aber-
rantly express and regulate cancer initiation, progression, and invasion.
miR-21 functions as an oncogene and plays significant roles in cancer cell prolifera-
tion, differentiation, and survival in addition to cancer initiation and progression, which
suggests that it may be a promising therapeutic target for PDAC [20]. miR-21, miR-196a,
miR-196b, and let-7i are highly expressed and strongly associated with low survival in
patients with PDAC [21,22]. Researchers have developed anti-miR-21 oligonucleo-
tide-loaded tumor-penetrating complexes that consist of a C-terminal cell-penetrating
peptide iRGD and an N-terminal fatty acid group to enhance hydrophobic interactions
for the self-assembly of NPs (TPN-21). TPN-21 inhibits miR-21 expression and PDAC
growth in PDO and upregulates phosphatase and tensin homolog and programmed cell
death 4, resulting in strong suppression of tumor growth. Li et al. developed polyeth-
ylene glycol-polyethylenimine-magnetic iron oxide NPs conjugated with anti-CD44v6
single chain variable fragments for co-delivery of miR-21 antisense oligonucleotides and
gemcitabine into PDAC cells [23]. These NPs induce apoptosis and inhibit tumor growth
and metastasis in vitro and in vivo, suggesting synergistic anti-tumor effects of miR-21
gene silencing and chemotherapy in PDAC.
Xie et al. developed a local delivery system combining the silencing of miR-210,
KRASG12D, and blockade of C-X-C motif chemokine receptor 4 (CXCR4) with cholesterol-
modified polymeric CXCR4 antagonist (PCX) NPs for the co-delivery of anti-miR-210
and siKRASG12D [24]. Cholesterol is conjugated to improve the efficacy of enhanced per-
meability and retention (EPR)-independent delivery. PCX NPs block cancer-stroma in-
teractions, anti-miR-210 inactivates stroma-producing pancreatic stellate cells, and
siKRASG12D kills pancreatic cancer cells. In this study, NPs were delivered via intraperi-
toneal (IP) administration to an orthotopic PDAC mouse model as an effective
EPR-independent approach for targeting peritoneal tumors. There was nearly 15-fold
higher tumor accumulation of NPs with local IP delivery compared to intravenous (IV)
delivery.
Wu et al. revealed that miR-9 is positively associated with doxorubicin (DOX) sen-
sitivity and developed supramolecular NPs condensed with an arginine-based plectin-1
Pharmaceutics 2022, 14, 137 4 of 16
(PL-1)-targeting chimeric peptide to bind RNA and target PDAC. PL-1/miR-9 NPs sig-
nificantly enhanced DOX efficacy in PDAC cells and tumor growth from PDXs, which
suggests that miR-9/eIF5A2 might be a novel potential drug for the synergistic therapy of
PDAC [25].
miRNA-150 is a tumor suppressor that is downregulated in the majority of human
pancreatic cancer tissues, which suggests that restoration of miR-150 might be a thera-
peutic strategy for pancreatic cancer [26]. Arora et al. developed poly
(D,L-lactide-co-glycolide) (PLGA)-based nanoformulation (NF) for the delivery of miR-150
to pancreatic cancer cells, and treatment with it resulted in growth, clonogenicity, motility,
and invasion via significant downregulation of target gene mucin 4 in vitro [27].
Although several preclinical studies have demonstrated the efficacy of miRNA-based
gene therapy for the treatment of PDAC, many attempts at developing miRNA therapeu-
tics have not been successful in clinical trials [28]. In addition, clinical trials for miR-
NA-based therapeutics have not been performed for the treatment of PDAC to date.
2.2. RNAi Using siRNAs
siRNA is a short non-coding ds-RNA of 21–23 nucleotides, which can induce RNAi
silencing without eliciting non-specific interferon after introduction into the mammalian
cells. It has the potential to silence genes encoding proteins that cannot be controlled by
small molecules and programmable drugs [29]. Non-viral delivery systems using NPs
enhance accumulation in tumor cells via EPR resulting from several factors including
escape from the immune system, improved target to tumor and cell uptake, and pro-
longed circulation time [30]. Over the past two decades, a number of NP-based siRNA
therapeutics have been developed and are being investigated in clinical trials.[31] In this
section, recent advances in RNAi therapeutics using siRNA are described, focusing on
siRNAs currently in clinical trials (Table 1).
Table 1. Nanocarriers-based siRNA therapeutics in clinical trials for the treatment of pancreatic
cancer.
Therapeutic siRNAs
Indication
Target gene
/Protein
Delivery systems
Route of
administrations
Phase/Status
Reference
CALLA-01
Cancer, Solid tumors
RPM2
Cyclo-dextrin based
polymer
Systemc/IV
I/Terminated
[32]
Atu027
Pancreatic cancer
PKN3
Liposomes (Lipoplexes,
Cationic lipid)
Systemc/IV
II/Completed
[33,34]
siG12D LODER
Pancreatic cancer
KRASG12D
mutation
Polymer matrix (LODER
polymer)
Local/Surgical
implantation
II/Recruiting
[35]
TKM 080301
Solid tumors with liver
involvement
PLK-1
SNALP
Systemc/IV
II/Completed
[36]
iExosomes
Pancreatic cancer
KRASG12D
mutation
Mesenchymal Stromal
Cells-derived Exosomes
Systemc/IV
I/Recruiting
[37]
NBF 006
Non-small cell lung,
Colorectal, and Pancreatic
GSTP
Lipid nanoparticles
Systemc/IV
I/Recruiting
[38]
Abbreviation: GSTP, Glutathione-S-transferase; IV, intravenous; PKN2, protein kinase N3; PLK1,
polo-like kinase 1; RPM2, ribonuclease reductase M2; SNALP, Stable nucleic acid lipid particle.
Approximately 90% of PDAC patients have a KRAS mutation, which plays a major
role of PDAC initiation. Kamerkar et al. developed exosomes derived from normal fi-
broblast mesenchymal cells carrying siRNA or shRNA for silencing KRASG12D [37]. IP
injection of these engineered exosomes inhibited tumor growth and increased survival
compared to liposomes due to their CD47-mediated protection from phagocytosis by
macrophages and monocytes. A Phase I trial of patients with metastatic pancreatic cancer
with KRASG12D mutation is underway (NCT03608631). Glutathione-S-transferase
(GSTP) is a phase II detoxifying enzyme related to cell integrity maintenance, oxidative
Pharmaceutics 2022, 14, 137 5 of 16
stress, and protection against DNA damage. It is a regulator of proteins involved in RAS
signaling pathways and is highly expressed in several cancers with KRAS mutation in-
cluding lung, colorectal, and pancreatic cancers [39]. NBF-006, a lyophilized lipid NP,
consists of an ionizable, non-immunogenic, biodegradable lipid enveloping GSTP siR-
NA. Treatment with NBF-006 significantly inhibits tumor growth in KRAS mutant
non-small cell lung cancer (NSCLC) xenograft models and prolongs survival in surgically
implanted orthotopic lung tumor mice without toxicity [38]. It has recently entered a
Phase I trial for previously treated NSCLC with KRAS mutation and previously treated
progressive or metastatic NSCLC, pancreatic, and colorectal cancer (NCT03819387).
Khvalevsky et al. developed the first local prolonged siRNA delivery system (called
the Local Drug EluteR, or LODER), which is a miniature biodegradable polymeric matrix
that releases siRNA over a period of few months after administration to the tumors [40].
Treatment with LODER-encapsulated anti-KRASG12D siRNA (siG12D LODER) inhibits
cancer cell proliferation and epithelial-mesenchymal transition with significant decrease
in KRAS levels and inhibits orthotopic pancreatic tumor growth and prolonged mouse
survival in vivo. In a Phase 1/2a clinical trial that included 15 patients with inoperable
PDAC, treatment with standard of care chemotherapy following siG12D LODER inser-
tion into the tumor with a needle during an endoscopic ultrasound biopsy procedure
resulted in stable disease in 10 patients and a partial response in 2 patients with favorable
safety data (NCT01188785) [35]. In an ongoing trial that started in 2017, 80 patients with
unresectable or borderline resectable locally advanced pancreatic cancer have been as-
signed to receive repeated doses of 2.8 mg siG12D LODER with chemotherapy (gem-
citabine and nab paclitaxel or FOLFIRINOX) or chemotherapy alone (NCT01676259).
Ribonuclease reductase M2 subunit (RPM2) is correlated with biological behaviors of
tumor cells including proliferation, invasion, migration, cell cycle, and apoptosis and plays
an important role in tumorigenesis in several cancer types [41]. CALLA-01, a targeted NP
system, consists of cyclodextrin-containing polymer, polyethylene glycol (PEG), human
transferrin as a targeting ligand for binding transferrin receptors, and siRNA designed to
reduce expression of RPM2 for tumor inhibition and/or tumor size reduction. In 2008, it
entered the first human Phase I trial involving systemic administration of siRNA to pa-
tients with solid cancer including PDAC (NCT00689065). These NPs were successfully
delivered into cancer cells, and it was confirmed by cancer cells containing nanoparticles
in tumor biopsies from patients with melanoma after systemic administration. In addi-
tion, the expression of RPM2 mRNA and protein is reduced in patients who receive the
highest doses, which suggests that specific gene inhibition by an RNAi might be one
cancer treatment modality [42]. In a Phase Ia/Ib trial, CALLA-01 showed similar safety
profile in 24 patients with different cancers compared to animals [32].
Atu027 is an RNAi therapeutic formulation based on cationic lipids. It contains
neutral fusogenic, PEG-modified lipid components and siRNA molecules that specifi-
cally target protein kinase N3 in the vascular endothelium, a downstream effector of the
phosphoinositol-3-kinase signaling pathway [43]. Atu027 inhibits tumor growth and
lymph node metastasis in orthotopic mouse models for prostate and pancreatic cancer
mouse models and hematogenous metastasis in mouse models for spontaneous lung
cancer [43,44]. In the first human Phase I clinical trial of this compound (NCT01808638),
10 escalating doses of Atu027, as a single IV administration, followed by twice weekly
doses for a 28-day cycle, were given to 24 patients with advanced solid tumors. The
treatment was safe and resulted in tumor stabilization in 41% of patients. In addition,
most patients had a reduced soluble variant of vascular endothelial growth factor re-
ceptor-1, which suggests its potential as a biomarker [33]. In a Phase II trial, 23 patients
with metastatic pancreatic cancer received combination treatment of gemcitabine and
Atu027 (NCT01808638). The treatment was safe, and twice weekly Atu027 led to signifi-
cantly improved progression-free survival [34].
Polo-like kinase 1 (PLK1), an essential cell-cycle protein, plays multiple roles in mi-
tosis and cytokinesis. It is overexpressed in various types of cancer and negatively affects
Pharmaceutics 2022, 14, 137 6 of 16
patient outcome. In addition, inhibition of PLK1 expression induces mitotic arrest and
apoptosis, resulting in tumor growth inhibition [45]. Stable nucleic acid lipid particles
(SNALPs) are effective siRNA delivery system, and do not need active targeting moieties
due to passive disease-site targeting. In a preclinical liver tumor model,
SNALP-formulated PLK1 siRNA (TKM-080301) demonstrated potent antitumor activity
and induced no measurable immune reaction, minimizing potential nonspecific effects
[36]. A Phase I clinical trial of TKM-080301 was performed in patients with hepatic me-
tastasis from colorectal, pancreas, gastric, breast, and ovarian cancer, but it was termi-
nated without report of results (NCT01437007).
The unique TME of the PDAC make it difficult to deliver therapeutic agents to the
tumor cells and raises the need for the development of a novel therapy. Although the role
of hypoxia-inducible factor 1 (HIF1, including HIF1α and HIF1β) in PDAC development
mechanisms is not completely understood, HIF1α stabilization in the hypoxic TME is
associated with transcriptional activation of multiple signaling pathways involved in the
regulation of cell survival, tumor invasion, angiogenesis, and metabolism [46]. Zhao et al.
developed lipid-polymer hybrid NPs (LENPs), which consist of a single layer or bilayer
lipid shell around a polymeric core made from cationic ε-polylysine co-polymer (ENPs)
[47]. Negatively charged si-HIF1α is absorbed on the surface of ENPs and gemcitabine
encapsulated to the hydrophilic core. LENPs protected NP aggregation, have prolonged
lifetimes with a half-lifetime longer than 3 h, and improved drug release through an en-
hanced tumor vasculature effect within tumor tissues. Treatment with
LENP-Gem-si-HIF1α results in effective silencing of HIF1α both in vitro and in vivo as
well as significant synergistic tumor growth inhibition. Another hypoxia-associated ma-
jor transcription factor, HIF2α (also called endothelial PAS domain protein 1 [EPAS1]) in
pancreatic cancer, was targeted using NPs with siRNA loaded onto a polyethyl-
enimine-poly(lactide-coglycolide) (PLGA)/poloxamer [48]. Treatment inhibited PDAC
cell proliferation by apoptosis induction under hypoxic conditions in vitro, and signifi-
cantly inhibited microvascular formation and tumor growth in orthotopic PDAC mice.
The cytoskeleton, which included microtubules, is composed of α- and β-tubulin
heterodimers. Increased βIII-tubulin expression is associated with poor OS and drug re-
sistance in various types of cancer including pancreatic cancer, and silencing of
βIII-tubulin with shRNA leads to tumor growth inhibition and enhanced sensitivity to
chemotherapy in vitro and in vivo, which suggests the possibility of a novel therapeutic
target for PDAC [49]. The limitations of using highly charged cationic NPs as a delivery
system for siRNA include their toxicity and potential to interact with serum proteins in
the bloodstream. To overcome this problem, Teo et al. developed star polymers with
different lengths of cationic poly(dimethylaminoethyl methacrylate) sidearms and varied
amounts of poly(oligo(ethylene glycol) methyl ether methacrylate), which were highly
accumulated in orthotopic PDAC tumors in mice and had a silencing efficiency of 80% at
the gene and protein levels [50].
3. CRISPR-Cas Gene Editing
CRISPR functions as an immune system in E. coli [51,52]. Recently, the CRISPR systems
have been developed as tools in gene therapy and have shown good target gene specificity,
high editing efficiency, and research simplicity compared to zinc finger nuclease and tran-
scription activator-like effector nuclease [53,54]. CRISPR/Cas9 is an RNA-guided endonucle-
ase consisting of Cas9 protein and sgRNA with target sequence specificity for DNA cleavage.
DNA damage induced by Cas9 is repaired through a non-homologous end-joining DNA
repair pathway or homology-directed repair pathway (HDR), which are cellular DNA repair
mechanisms [55,56]. CRISPR variants have been developed based on this system. Base edit-
ing system (cysteine and adenine base editor) is composed of a single-strand DNA nickase
and deaminase, which can be applied for the treatment of diseases attributable to a point
mutation [57–59]. Cas13 (previously referred to as C2C2) edits RNA and reduces the risk for
DNA damage due to off-target effects [60,61]. The process of prime editing involves DNA
Pharmaceutics 2022, 14, 137 7 of 16
nickase and a reverse transcriptase enzyme, which generates new DNA by duplicating an
external RNA template [57,62]. It can lead to the formation of indels (insertions, deletions)
and base conversions without the DNA double-strand breaks or donor DNA templates.
However, it needs an appropriate delivery system for the treatment of cancer. Viral vectors
(retrovirus, adenovirus, adeno-associated virus vector) are popular in the gene therapy field
[63–65] but their clinical use is limited by several factors, such as the increased adaptive im-
mune response from neutralizing antibodies by the capsids of adeno-associated virus, the
potential immunogenicity, and gene mutations by their chromosomal insertion [66–70].
Therefore, an ideal vehicle would show target specificity, minimize off-target efficiency, and
retain non-immunogenicity.
CRISPR systems can be applied in PDAC gene therapy using three types of nanocarriers:
plasmid DNA (Cas9/sgRNA plasmid), in vitro transcribed Cas9 mRNA/sgRNA, and
pre-assembled ribonucleoprotein (RNP) complex (Figure 1). In general, the successful deliv-
ery of three forms requires cationic coatings to fully condense them [71]. Negatively charged
plasmid DNAs, mRNAs, and RNPs are coated with cationic molecules to form complexes
with structural stability. The complexes bind to cell membranes through electrostatic interac-
tions and are taken into cells via endocytosis. Typically, cationic lipid-based polymers (poly-
ethylenimine, PEI) and peptides (protamine) form positively charged particles that improve
the delivery efficiency and stability in vivo. Cationic lipid-based NPs have additional prop-
erties such as tumor targeting, co-encapsulation of chemotherapeutic agents, and others [72–
75]. PEI and protamine are also the most commonly used cationic molecules in vitro and in
vivo. They principally consist of high-density amine groups that interact with negatively
charged nucleic acids or RNPs. They enhance intracellular delivery efficiency and endosomal
escape at a lower pH due to the proton sponge effect [76,77]. In addition, inorganic materials,
such as gold (Au) NPs, are attractive for the delivery of RNPs and donor DNA into cells for
HDR repair. Au NPs are easily modified with thiol-terminated single-stranded DNA and
bind RNPs via non-specific electrostatic forces [78,79].
Figure 1. The cell delivery pathway of CRISPR/Cas9 encapsulated-nanoparticles. The CRISPR sys-
tems can be applied for gene therapy with non-viral carriers in three forms that include: plasmid
DNA (Cas9/sgRNA plasmid), in vitro transcribed Cas9 mRNA/sgRNA, and pre-assembled ribo-
nucleoprotein (RNP) complex. Such nanoparticles can take several forms, including PEGylated- or
cationic lipid-NPs (liposome), cell-mediated exosome, cationic polymers (polyethyleneimine, PEI;
protamine, PA), and cationic lipid- or polymer-coated gold NPs. The plasmid DNA, mRNA and
RNP of NPs may enter the cell via endocytosis and endosome escape, processing the RNP expres-
sion and gene editing.
Pharmaceutics 2022, 14, 137 8 of 16
3.1. Plasmid DNA (Cas9/sgRNA plasmid) Delivery
Although viral vectors such as adenoviruses and lentiviruses are efficient delivery
systems for CRISPR DNA, their immunogenicities make them potential carcinogens and
they have considerable molecular weights [80]. Furthermore, the CRISPR/Cas9 mRNA
and protein delivery remains challenging due to issues related to stability, encapsulation
efficiency, and their high cost of production. CRISPR/Cas9 plasmid delivery has been
used due to the low cost, storage stability, and the potential of prolonged expression.
However, plasmid delivery risks genomic integration, cell stress, and off-target effects
due to long-term transgene expression [81,82].
Plasmid DNA-encoded Cas9 nuclease and sgRNA-encapsulated lipid-based NPs
have been successfully studied in vivo for PDAC treatment. Li et al. reported a tu-
mor-targeted lipid-based CRISPR/Cas9 plasmid delivery system developed to suppress
HIF-1α (Figure 2a) [71]. They synthesized the R8-dGR peptide (Cys-RRRRRRRRdGR)
bound to integrin αvβ3 and neuropilin-1 in over-expressed tumor cells, and it enhanced
in vivo targeting of pancreatic cancer. The peptide was utilized to lower the charge den-
sity of DOTAP-based cationic liposome. In addition, this cell penetrating peptide en-
hanced cell internalization and transfection efficiency (Figure 2b). HIF-1α
over-expression in tumors is associated with aberrant p53 and upregulates angiogenesis
and metastasis-related signals such as vascular endothelial growth factor (VEGF) and
matrix metalloproteinase 9 (MMP9) [83,84]. Therefore, the Cas9-HIF-1α plasmid
DNA/protamine complex and paclitaxel (PTX) co-encapsulated liposome to promote the
anti-metastatic effects could suppress tumor growth in vivo (Figure 2c, d). Consequently,
R8-dGR-Lip/pHIF-1α and PTX downregulated HIF-1α and its downstream molecules
VEGF and MMP-9, leading to inhibition of metastasis to livers and lungs (Figure 2e, f).
Figure 2. R8-dGR peptide modified and loaded CRISPR/Cas9-HIF-1α and paclitaxel cationic lipo-
some for targeted pancreatic treatment. (a) Scheme of the structure of the R8-dGR-Lip/pHIF-1α
and paclitaxel (PTX) delivery in pancreatic cancer cells. (b) Cellular internalization of with or
without the R8-dGR peptide (Cys-RRRRRRRRdGR) on BxPC-3 cells. scale bar: 100 μm. (c) Tumor
volume of BxPC-3 xenograft models treated with different formulations (mean ± SD, n = 10, *p <
0.05). (d) In vivo downregulation of HIF-1α by pHIF-1α in tumor tissues. (e–f) MMP9 expression
in liver (e) and lung (f) of mice. Scale bar: 100 μm (adapted from Ref. [71]).
Pharmaceutics 2022, 14, 137 9 of 16
Exosomes are naturally released to nano-sized extracellular vesicles from all cells
and contain DNA, RNA, metabolites, cell-surface protein, and lipids depending on the
cell origin [85]. Unlike synthetic nanocarriers, exosomes originating from cells have the
advantage of biocompatibility, non-immunogenicity, and non-cytotoxicity [86]. In addi-
tion, they are contained in various plasma membranes and have enhanced half-live [87].
Therefore, they are suitable as carriers of internal cargo to recipient cells. Recently,
McAndrews et al. reported that exosomes could be successfully engineered to encapsu-
late CRISPR/Cas9 plasmid DNA and delivery it into tumor cells. The exosomes were de-
rived from bone marrow-derived MSCs and did not show repeated dose toxicity [86].
They suppressed the mutant KRASG12D oncogene, leading to inhibited tumor growth in a
syngeneic allograft model and orthotropic model (Figure 3) [88].
Figure 3. Exosome mediated treatment of CRISPR/Cas9 in as allograft and orthotropic model of
pancreatic cancer. (a,b) Tumor volume (a) and tumor weight (b) after exosomes-Cas9/KRASG12D
sgRNA intravenously administration in allograft models (KPC689 cells) every other day for 2
weeks (n = 8 mice in each group). (c) Cas9 mRNA expression levels in tumor tissues by quantita-
tive PCR (normalized to 18S, n = 5 mice per group). (d) Tumor growth by bioluminescent imaging
after exsomes-Cas9/KRASG12D sgRNA Intraperitoneal injection in orthotopic models
(KPC689-GFP-Luciferase cells) every other day for 3 weeks. (e) KRASG12D mRNA expression
levels at the end point in orthotropic tumor tissues (mean ± SD, *p < 0.05, **p < 0.01) (adapted from
Ref. [88]).
3.2. In Vitro Transcribed Cas9/sgRNA mRNA Delivery
CRISPR/Cas9 mRNA delivery has advantages over plasmid DNA. The mRNA is not
integrated into the target genome, which prevents off-target effects [65,89,90]. In addi-
tion, the mRNA leads to quick and transient Cas9 protein expression in the cytoplasm.
However, mRNAs are large in size, unstable, and susceptible to degradation during de-
livery [91].
Pharmaceutics 2022, 14, 137 10 of 16
3.3. Pre-Assembled Ribonucleoprotein (RNP) Complex Delivery
CRISPR/Cas9 RNP does not need to be translated into cells. It is directly transported
into the nucleus and edits the target gene. In addition, it has low toxicity, high gene-editing
efficiency, low off-target effects, and no chance of integration into the genome of recipient
cells [65,89,92,93]. The Cas9 protein and sgRNA can be simply mixed ex vivo for delivery to
cells. The Cas9 protein/sgRNA complex is negatively charged. The complex can be positively
charged by modification to facilitate cell membrane affinity and internalization [92,94–97]. In
addition, bio-reducible disulfide bonds of nanocarriers can be adjusted to improve degrada-
tion and endosomal escape [98]. In addition, the Cas9 protein can be modified to enhance the
encapsulation efficiency of NPs [99,100].
Zhao et al. studied CRISPR-Cas13a effector protein delivered to pancreatic cancer
cells using lipofectamine CRISPRMAX and found that the Cas13a-crRNA complex bound
to and cleaved target RNA and xenograft model by intratumoral injection (Figure 4a)
[101]. CRISPR-Cas13a system reduces mRNA expression in mammalian cells and shows
an improved efficacy and specificity over RNA interference [60,61]. The system reduced
the KRASG12D mRNA expression with up to 70% knockdown efficiency (Fig, 4b–d), lead-
ing to apoptosis and tumor growth inhibition (Figure 4e, f) [101].
Figure 4. CRISPR/Cas13a-lipofectamine CRISPRMAX delivery for KRAS mutated pancreatic cancer
treatment. (a) The scheme of Cas13 system differentiates wild type (WT, normal cells) and mutated
KRAS genes (cancer cells). (b) crRNA sequences of the crRNA19-target sequence in KRAS-G12D and
KRAS WT. (c) qRT-PCR analysis of KRAS-G12D mRNA expression after crRNA19-14 delivery into
KRAS-G12D mutated cells. (d) qRT-PCR analysis of KRAS-WT mRNA expression after crRNA19-14
delivery into KRAS-WT cells. (e) The tumor volumes of subcutaneous AsPC-1 xenograft after admin-
istration with repeated intratumoral injections of the Cas13a-lipofectamine for 21 days. (f) Staining for
apoptotic cells in tumor tissues (green fluorescence). Scale bar : 50 μm. (mean ± SD, *p < 0.05, **p < 0.01,
and ***p < 0.001) (adapted from Ref [101]).
Recently, our group has developed a tumor targeted-nanoliposome
(NL[Cas9/ABE]-Ab) that simultaneously encapsulates CRISPR/Cas9 and adenine-base editor
(ABE) proteins for dual-gene editing (Figure 5) [15]. The Cas9 and ABE proteins are modified
with his-tag protein to improve NL-encapsulation efficiency. In addition, we used
PEG-bound lipid for structure stability and increased receptor-mediated endocytosis. Gem-
citabine is used as first-line chemotherapy for PDAC but is limited in that most PDAC pa-
tients develop resistance to it [102]. The KRAS mutation and loss of P53 induces HIF-1α sta-
Pharmaceutics 2022, 14, 137 11 of 16
bilization, which is the master regulator of glucose metabolism and a major driver of gem-
citabine chemoresistance in PDAC [103,104]. Co-administration of NL(Cas9/ABE)-Ab and
gemcitabine markedly inhibits tumor proliferation in a PANC1 xenograft (Figure 5c).
NL(Cas9/ABE)-Ab inhibits KRAS and TP53 mutations, and regulates HIF-1α-related glycol-
ysis, which promotes gemcitabine sensitivity in vivo (Figure 5d-f). Therefore, the dual-gene
editing tool of the KRAS mutation and mutant TP53 might overcome drug-resistance in
PDAC [15].
Figure 5. Dual-gene editing nanoliposome system as an overcoming drug-resistance for PDAC. (a)
Graphical summary of NL(Cas9/ABE)-Ab composition. (b) Dynamic distributions of tumor targeting
efficiency by NL with or without Ab in vivo. Mice were injected intraperitoneally once with NL-Ab la-
beled with RITC. PANC1 xenograft tumor was marked as white circles. (c) The scheme of PANC1
xenografts co-administrated with NL(Cas9/ABE)-Ab and gemcitabine. The NL(Cas9/ABE)-Ab was in-
jected intraperitoneally once (red arrow) and then gemcitabine (50 mg/kg) was administered twice a
week for 4 weeks. Mean ± SEM, n = 3 per group, *p < 0.05, ***p < 0.001. (d) Immunofluorescence with
mutant P53 and KRAS on the tumor sections. Scale bar: 50 μm. (e) Immunostaining with HIF1a-related
glucose metabolism and gemcitabine transporter (GLUT1, TKT, CTPS and ENT1) on the tumor. Scale
bar: 50 μm. (f) Immunostaining with apoptotic cells (TUNEL assay and Ki67) by NL(Cas9/ABE)-Ab
and gemcitabine on the tumor. Scale bar: 100 μm (adapted from Ref. [15]).
Pharmaceutics 2022, 14, 137 12 of 16
4. Conclusions and Future Perspectives
Prerequisites for successful gene therapy include rapid, simple, and easy synthesis,
safety, and efficient delivery. Gene therapy using RNAi-based therapeutics and CRISPR
genome editing is promising and can improve precise gene delivery to tumor tissue.
Although there have been major advances in the development of gene therapy for the
PDAC treatment, there is still much room for improvement in pharmacokinetics, phar-
macodynamics, and methods to improve safety. RNAi-based therapeutics have several
barriers (e.g., systemic circulation and targeted delivery must be improved, off-target
effects must be minimized, etc.) and it is essential to further develop safe, biocompatible,
and biodegradable NP-based therapeutics for clinical application. EPR of nanoparti-
cle-based carriers make it easier to accumulate in the tumor cells. However, extracellular
and intracellular barriers in PDAC are hurdles to overcome. Nanoparticles with appro-
priate particle size and their chemical modifications and functionalization with targeting
ligands are necessary to enhance the uptakes into tumor cells and address other biologi-
cal issues such as intracellular trafficking and intracellular siRNA escape [105]. In addi-
tion, combination treatment with chemotherapy and RNAi-based therapeutics or a
strategy combining RNAi molecules and anticancer drugs would help overcome drug
resistance and TME barriers.
CRISPR/Cas9 genome editing has widely been used not only in the development of
cancer models and as a tool for the identification and validation of therapeutic targets but
also as a potential cancer therapeutic [106]. The field of cancer immunotherapy is the
most advanced clinical application. The first Phase I trial from China including 22 pa-
tients with NSLC used CRISPR-Cas9 PD-1-edited T cells from patient blood, and the
treatments were safe and showed some therapeutic efficacy (NCT02793856) [107]. Sever-
al clinical trials are ongoing to demonstrate the efficacy of more potent chimeric antigen
receptor T cells using CRISPR to knock out immune co-inhibitory pathways or signaling
molecules [108]. However, there are several huddles to cross before CRISPR/Cas9 ge-
nome editing can be used in clinical practice, including potential off-target effects and
safety issues. A novel therapeutic approach is required for efficient delivery, accurate
targeting of desired cells, efficient gene editing, and to minimize off-target effects and
immune responses. Although several clinical trials for gene therapy have not been suc-
cessful, the progress in understanding of the PDAC TME and nanomedicine-based gene
therapy can make it possible to improve the clinical outcomes of patients with PDAC.
Author Contributions: Conceptualization, E.-J.W. and Y.-S.C.; writing—original draft preparation,
E.-J.W., H.P., and Y.-S.C.; writing—review and editing, E.-J.W., H.P., T.-J.Y., and Y.-S.C.; supervi-
sion, T.-J.Y. and Y.-S.C. All authors discussed, edited, and approved the final version of the man-
uscript. All authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by a grant of the National Research Foundation of Korea
(grant number 2019R1F1A1058879).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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