RNA-based pharmacotherapy for tumors: From bench to clinic and back

Biomedicine & Pharmacotherapy 125 (2020) 109997

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

RNA-based pharmacotherapy for tumors: From bench to clinic and back Xiangping Liang

a,b,1

b,1

, Dongpei Li

c,

, Shuilong Leng **, Xiao Zhu

a,b,

T

*

a

Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical University, Zhanjiang, China Southern Marine Science and Engineering Guangdong Laboratory Zhanjiang, The Marine Medical Research Institute of Guangdong Zhanjiang (GDZJMMRI), Guangdong Medical University, Zhanjiang, China c Key Laboratory of Neurological Function and Health, School of Basic Medical Sciences, Affiliated Cancer Hospital and Institute of Guangzhou Medical University, Guangzhou, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: RNA therapy RNA drugs Cancer immunotherapy Clinical trials

RNA therapy is a treatment that regulates cell proteins and cures diseases by affecting the metabolism of mRNAs in cells, which has cut a figure in the studies on various incurable illnesses like hereditary diseases, tumors, etc. In this review, we introduced the discovery and development of RNA therapy and discussed its classification, mechanisms, advantages, and challenges. Moreover, we highlighted how RNA therapy works in killing tumor cells as well as what progresses it has made in related researches. And the development of RNA anti-tumor drugs and the clinical trial process were also included.

1. Introduction RNA therapy, a treatment based on RNA level, influences the metabolic process of messenger RNAs (mRNA) by using oligonucleotides combined with them by base pairing [1], which may include the splicing and mature process starting from precursor mRNAs (pre-mRNAs), transport, translation as well as degradation of mRNAs (Fig. 1). In recent 10 years, RNA therapy is highly active in many important fields and has become a research hotspot in immunotherapy. An increasing number of researchers have realized the potential of it and devote themselves to studying, especially when the first RNA interference (RNAi) drug Onpattro (patisiran) got approved by the Food and Drug Administration (FDA) in August 2018 [2,3]. With further studies in RNA therapy, scientists say that RNA-based drugs can inhibit a variety of genes in multiple cellular pathways. It is expected to be used to target multiple key sources of multi-gene diseases such as tumors, reducing drug resistance of tumor cells and effectively arresting the growth of advanced stage tumors. Now, the global prevalence rate of cancer is increasing, and the number of people dying from cancers is also rising year by year. In the face of cancer cells which lose growth inhibition, behave badly and seriously damage the body's structure and physiological functions, chemotherapy and surgeries are still far from the ideal effects. For a long time, scientists have treated tumors in a "general" way, ignoring the idiosyncrasies of them. However, advances in screening technology tell us that no tumors are

exactly the same at the mutation level, which has a decisive effect on tumor resistance and immune escape. Therefore, RNA therapy with high specificity, wide targets and good drug properties displays unique superiority in tumor therapeutics. In this study, the classification and application of RNA therapy were reviewed, with emphasis on the mechanisms of RNA anti-tumor therapy and the clinical research progress of RNA drugs. Additionally, we elaborated on the challenges of RNA therapy. 2. The classification of RNA drugs and therapeutics 2.1. ASO therapy Antisense oligonucleotides are small molecular drugs that specifically bind to their targeted mRNAs through the complementary basepairing rule, interfering with the steps of DNA unwinding, replication, transcription, and mRNA splicing, transport as well as translation (Fig. 1F, G), so as to regulate the growth and differentiation of cells. They are single-stranded DNAs or RNAs with a sequence length of 15–25 nucleotides. Because of weak hydrophilicity of single oligonucleotides, chemical modifications are usually required to improve their stability and drug potency. After chemical modification, the representative ASOs of the first, second and third generations are thiooligonucleotides, mixed skeleton oligonucleotides and polypeptide nucleic acids [4]. Their ability to be diffused in tissues and be absorbed by

⁎

Corresponding author at: Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical University, Zhanjiang, China. Corresponding author. E-mail addresses: [email protected] (S. Leng), [email protected] (X. Zhu). 1 Xiangping Liang and Dongpei Li contributed equally to this work. ⁎⁎

https://doi.org/10.1016/j.biopha.2020.109997 Received 5 December 2019; Received in revised form 2 February 2020; Accepted 6 February 2020 0753-3322/ © 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Biomedicine & Pharmacotherapy 125 (2020) 109997

X. Liang, et al.

Fig. 1. Mechanisms of RNA therapeutics. Five approaches to how RNA therapy treats diseases are shown in the figure. The first one is that anti-miRNA (A) complements the active chains of the aim miRNA, which weakens the gene silencing effect of endogenous miRNAs and then improves protein expression. Second, miRNA mimics (B) enhance the function of endogenous miRNAs and reduce intracellular protein expression. What’s more, block-miRNA agonists (C) are used to prevent RISC from binding to the specific mRNAs by "blocking" miRNA binding sites in order to increase the expression of relevant proteins. Usually, RNA therapy works by utilizing intracellular enzyme RNase H (G) or forming RNA-induced silencing complex (RISC) (D, E1, E2) to cut the targeted mRNA. In plant cells, miRNA bonds with the targeted mRNA completely to degrade it (E1) while in animal cells it cannot complementary to its target mRNA, which blocks ribosomes and reduces translation (E2). ASO is capable of regulating the splicing process of mRNA (F). It corrects wrong splicing by binding to aberrant mRNA splicing sites. Abbreviations: ASO antisense oligonucleotide; dsRNA double-stranded RNA; shRNA short hairpin RNA; pri-miRNA primary miRNA; pre-miRNA pre-cursor miRNA; TRBP Tar RNA binding protein; AGO argonaute; Pol II RNA polymerase II; siRISC siRNA-induced silencing complex; miRISC miRNA-induced silencing complex.

certain alternative spliceosome of proteins [6,7,9] in order to correct the wrong splicing, repair defective RNAs, restore production of some proteins or down-regulate expression of particular genes, finally achieving to treat diseases (Fig. 1F).

cells are enhanced, and they can directly bind to the targets after being injected into patients [5]. They work at targeted mRNAs by activating RNase H (Fig. 1G) [6,7], or inhibiting them from translation by preventing ribosomes binding through steric effect [8]. What's more, by closing splicing sites, ASOs can selectively promote expression of a 2

Biomedicine & Pharmacotherapy 125 (2020) 109997

X. Liang, et al.

2.2. RNA interference therapy

3. The manners of RNA therapeutics

As a mechanism that operates in cells to turn off gene expression when they detect infection or genetic abnormality, RNAi is triggered by double-stranded RNAs (dsRNA). Researchers explored it as a therapeutic to treat a lot of diseases. RNAi therapy utilizes natural molecular machinery of cells to efficiently knock down the expression of the concerned genes through the promotion of short interfering RNAs. There are diverse approaches to induce RNAi, including synthetic molecules, RNAi vectors and in vitro cleavage. Synthetic molecules include standard small interfering RNAs (siRNA) and specific RNAi sequences designed in programs developed by biological companies to meet customer needs [10]. RNAi vectors can be divided into small hairpin RNAs (shRNA) and micro RNAs (miRNAs) (Fig. 1E). In vitro, long dsRNA is mostly processed by Dicer (Fig. 1D). miRNA is a short endogenous non-coding RNA (ncRNA) that limits targeted gene expression by restricting mRNAs from transformation and promoting mRNAs to decay. First, in animal cells, miRNA is transcribed into a long primary miRNA in the nucleus. Then, a hairpin RNA with 60 ∼ 70 nucleotides processed by Drosha and its cofactor Pasha is a precursor miRNA. With the help of Exprotin-5 complex, it is transported out of the nucleus and cut into a mature miRNA by Dicer in the cytoplasm, with a length of about 22 bases. Eventually it is integrated into the RNA-induced silencing complex (RISC) to regulate expression based on complete or incomplete pairing with the 3’utr of the targeted mRNA (Fig. 1E). When miRNAs are not fully complementary to targeted mRNAs, they are used to suppress gene translation in mammals (Fig. 1E1). But they will lead targeted mRNAs to degrade if completely or almost completely complementary to their target sites, which is more common in plants (Fig. 1E2). miRNA also performs transcriptional regulation. It has been shown in recent studies that miRNA influences the CpG island methylation of gene promoters and directly regulates targeted genes at the transcriptional level [11–15]. Both siRNA and miRNA are cable of forming RISCs to silence targeted genes (Fig. 1D, E). The difference is that siRNA comes from the products after the cutting of a long dsRNA. It disrupts mRNAs before translation with 100 % complementarity so it has highly strict target specificity. miRNA, on the other hand, is from a single-stranded RNA. It folds itself to make so-called "stem-loops" [16], some small areas of a dsRNA. Thanks to imperfect base pairing, it is less limited so that it can affect hundreds of genes. Still, when miRNA acts as the lead chain of the targeted mRNA in RISC, it suppresses expression or reduces stability of the mRNA rather than lead to fracture of it on account of its incomplete match with the 3 'UTRs (Fig. 1E).

As is known to us, in cells, DNA is transcribed into mRNA which is translated into proteins, and proteins play an essential role in regulating life activities. RNA therapy works in the following ways (Fig. 1) to disturb certain parts of the process. a Degradation of mRNA complex. RNA drugs can use intracellular enzyme RNase H1 (Fig. 1G) or form RISCs (Fig. 1D, E) to cut targeted mRNAs to achieve the goal. b Regulating the splicing process of mRNA. RNA drugs inhibit or enhance specific RNA splicing via combination to mRNA splicing sites in order that they succeed in regulation of gene expression (Fig. 1F) [19]. c Regulating protein expression. Decoy transcription factors aiming at targeted mRNAs are introduced into cells to block translation, reduce production of disease-causing proteins, or up translation of synthetic mRNAs that produce therapeutic proteins. d miRNA mimics. miRNA mimics function similarly to natural miRNAs, which regulate critical developmental procedures and pathways to maintain cell recognition, enhancing the function of endogenous miRNAs and reducing intracellular proteins [20]. Besides, they can potentially promote cancer therapy by restoring disrupted miRNA maturation mechanisms and increasing expression of specific tumor suppressor miRNAs (Fig. 1B). e Anti-miRNA oligonucleotides. miRNA inhibitors are chemically modified ASOs that weaken the silencing effect of endogenous miRNAs by specifically binding to the active chains of them and increase protein expression (Fig. 1A) [21]. f miRNA competitive agonists. miRNA competitive agonists block RISCs from matching with targeted mRNAs by "shielding" miRNA binding sites on them, thereby up-regulating the expression of related proteins (Fig. 1C) [22]. 4. The mechanisms of RNA therapeutics in treating tumors 4.1. Inhibiting the proliferation of tumor cells The uncontrolled cell cycle is a key point for normal cells transforming into tumor cells. As a result, the first approach of RNA therapy to treat tumors is to find appropriate aims at the RNA level to inhibit the unlimited proliferation of tumor cells. Ali et al. discovered that some long non-coding RNAs (lncRNA) highly expressed during the cell division cycle in mice with lung cancer can be turned off by the lock nucleic acid-modified antisense oligonucleotides (LNA-ASO) injected, which contributed to a cure rate of 40–50 % of lung tumors in mice [23]. KRAS gene is one of the most ubiquitous mutated genes in human cancers. The protein encoded by it is a signaling molecule that triggers a series of molecular events to instruct cells to proliferate and differentiate. If KRAS gene mutates, it causes cells to divide out of control. Aimed at such a mutated “undruggable” KRAS gene, Pecot et al. sent a siRNA into mice suffered from cancers induced by KRAS mutation with a nanoliposomal delivery platform, DOPC, and KRAS oncogene was silenced successfully, making both two signaling molecules pERK and pMEK relevant to cancer cell proliferation and tumor growth decrease significantly, which restrained the growth of cancer cells in mice, but also effectively controlled their spread [24]. Kamerkar et al. further studied and confirmed that the delivery of siRNAs suppressing oncogenic KRAS by exosomes could be better than LNPs in treating pancreatic cancer mice, no matter in specific targeting or avoiding immune attack (Table 1) [25]. They both suggests that KRAS oncogene is no longer undruggable and many other genes like oncogenic KRAS will be figured out one by one through RNA therapy to yield highly specific molecular drugs. In addition to KRAS, there is another new target, a mutated RNA

2.3. mRNA therapy mRNA can carry genetic information from genes to protein synthetic factories to guide protein synthesis in cells. mRNA therapy is a treatment in which a specific mRNA is synthesized and injected into a patient's body so that the cells in vivo produce drugs themselves, forming various proteins to cure diseases. Weissman et al. suggested that mRNA therapy might be an alternative to DNA therapy in gene therapy [17]. Both have advantages. DNA therapy works in the long term, while mRNA therapy works in the short term. It may be more suitable for DNA therapy to deal with inherited diseases caused by genetic mutations while mRNA therapy can be emerged from other treatments in the rest of diseases. However, by contrast, mRNA is less stable than DNA and easy to be broken down by nucleases in the body, which is also possible to cause immune responses. After a long-time efforts, Kariko et al. have made a breakthrough in their quest for avoiding an immune response via modifying the nucleoside portion of the uracil ribose to produce a "pseudouracil" that escapes detection by the immune system [18]. Since then, mRNA therapy has developed rapidly and gained wide acceptance. 3

STP705

4

SV40 vectors carrying siRNA Bcr-Abl siRNA

Mesenchymal Stromal Cells-derived Exosomes with KRAS G12D siRNA NU-0129

siRNA-EphA2-DOPC

SXL01

siG12D LODER

Atu027

siRNA

AR V7 variant

siRNA siRNA

Bcr-Abl

siRNA

siRNA

Bcr-Abl

BCL2L12

Gliosarcoma

Chronic Myeloid Leukemia (CML) Chronic Myeloid Leukemia (CML)

KRAS G12D Mutation

siRNA

siRNA

KRAS

EphA2

siRNA

PKN3

siRNA

miRNA mimic

miRNA 193a3p

PLK1

RNAi

siRNA

siRNA

siRNA

MECHANISM

CTNNB1

TGF-β1 and COX-2

HSP90

HIF-2α

TARGET

Pancreatic Cancer

Adrenal Cortical Carcinoma (II), Neuroendocrine Tumor (II), Hepatocellular Carcinoma (II), Solid Tumors (I) Solid Tumors(I), Metastatic Pancreatic Cancer (II), Head and Neck Cancer (Hold) Pancreatic Ductal Adenocarcinoma, Pancreatic Cancer Metastatic castrationresistant prostate cancer (CRPC) Advanced Cancers

Hepatocellular Carcinoma (HCC), melanoma, TripleNegative Breast Cancer (TNBC), Non-Small Cell Lung Cancer (NSCLC)

INT-1B3

Clinical TKM-080301 (TKM-PLK1)

Cancer

DCR-BCAT

Nonmelanoma skin cancer (NMSC)

Cholangiocarcinoma (CCA)

si-PT-LODER

INDICATION(S)

Clear cell renal cell carcinoma (ccRCC) Prostate Cancer

Preclinical ARO-HIF2

THERAPEUTICS

Table 1 Preclinical Tests and Clinical Trials of Anti-tumor RNA Therapeutics.

Unmodified

Undisclosed

Undisclosed

Unknown

Undisclosed

Undisclosed

Undisclosed

2′-OMe

Undisclosed

Undisclosed

Undisclosed

Undisclosed

Undisclosed

PS, 2′-OMe, 2′-F, iB

MODIFICATION

Gold nanoparticle (SNA) Pseudoviral (SV40) particles Anionic liposome

Exosome

DOPC LNP

NA

LODER polymer (PLGA)

AtuPlex Technology

LNP

LNP

EnCore Lipid Nanoparticle

Histidine-Lysine Co-Polymer (HKP) peptide nanoparticles (PNP)

TRiM (RGDsiRNA conjugate) Polymeric matrix (LODER Polymer)

DELIVERY SYSTEM

i.v. or s.c.

Ex vivo transfection

i.v. infusion

i.v. infusion

i.v.

NA

Intratumor placement, Surgical implantation

i.v.

i.v.

i.v.

i.v.

NA

NCT00257647

NCT03020017

NCT03608631

NCT01591356

NCT02866916

NCT01188785 NCT01676259

NCT00938574 NCT01808638

NCT01262235 NCT01437007 NCT02191878

N/A

N/A

N/A

i.v. Topical administration

N/A

N/A

NCT ID

Intratumoral implantation

s.c.

ADMINIST-RATION ROUTE

Northwestern University Hadassah Medical Organization University of Duisburg-Essen

M.D. Anderson Cancer Center

M.D. Anderson Cancer Center

Institut Claudius Regaud

Silence Therapeutics GmbH Silenseed Ltd

Biopharma Arbutus Corporation

Dicerna Pharmaceuticals, Inc. InteRNA

Sirnaomics

Arrowhead Pharmaceuticals Silenseed Ltd

COMPANY

Phase 1

NS

Phase 1

Phase 1

Phase 1

Phase 1

Phase 1 Phase 2

Phase 1 Phase 1,2

Phase1,2 Phase1 Phase 1,2

Jensen et al. (2013) Kimchi-Sarfaty et al. (2002) Koldehoff et al. (2007), Scherr et al. (2003)

Duxbury et al. (2004), Landen Jr. et al. (2005), Nishimura et al. (2013) Kamerkar et al. (2017)

Aleku et al. (2008), Schultheis et al. (2018) Golan et al. (2013)

Demeure et al. (2016)

Ganesh et al. (2019), Ganesh et al. (2018)

Shemi and Khvalevsky, (2017) Zhou et al. (2017)

REF.

(continued on next page)

Preclinical

IND approved Regulatory Approval to Initiate Phase 2 study Preclinical

Preclinical

Preclinical

STAGE

X. Liang, et al.

Biomedicine & Pharmacotherapy 125 (2020) 109997

Solid Tumors, Multiple Myeloma, Non-Hodgkins Lymphoma, Pancreatic Neuroendocrine Tumors, Hepatocellular Carcinoma Solid Tumors

Cancer, Solid Tumor

Breast Cancer (III) Ovarian Cancer (III) Colorectal Cancer (II) metasta c melanoma (II) Ewing’s Sarcoma (II) metasta c Non-small Cell Lung Cancer (II), Solid Tumors(I)

Hematological Malignancies

DCR-MYC (DCR-M1711)

CALAA-01

Vigil™ vaccine (FANG, vigil, vigil EATC)

PSCT19 (MiHA-loaded PDL-silenced DC Vaccination)

5

Advanced and/or metastatic cancer Hepatocellular Carcinoma, liver cancer

pbi-shRNA STMN1 LP

MTL-CEBPA

Ewing's Sarcoma

pbi-shRNA™ EWS/FLI1 type 1 LPX

Lentivirus vector rHIV7shI-TAR-CCR5RZtransduced hematopoietic progenitor cells

CEBPA

STMN1

HIV-1 tat/rev (shI)-transactive response element (TAR), CCR5 ribozyme EWS/FLI1

saRNA

shRNA

Undisclosed

Unmodified

Unmodified

Unmodified

shRNA

shRNA

Unmodified

Cbl-b/DC cancer vaccine XPO1

Solid tumors which are metastatic or cannot be removed by surgery Chronic Lymphocytic Leukemia (CLL) AIDS-Related Lymphoma shRNA

Undisclosed

siRNA

LMP2, LMP7, and MECL1

Metastatic Melanoma, Absence of CNS Metastases

iPsiRNA (Proteasome siRNA and tumor antigen RNAtransfected DC) APN401 (siRNAtransfected PBMC)

Genetic: shRNA

Undisclosed

siRNA

CCR5

Unmodified

Undisclosed

Unmodified

Undisclosed

PS, 2′-OMe

Undisclosed

MODIFICATION

AIDS-Related Lymphoma

shRNA

siRNA

shRNA

Furin

PD-L1/PD-L2

siRNA

RRM2

siRNA

siRNA

MYC

VEGF, KSP

MECHANISM

TARGET

Lentivirus vector CCR5 shRNA

ALN-VSP02 (ASC-06)

INDICATION(S)

THERAPEUTICS

Table 1 (continued)

LNP

Lipoplex

Lipoplex

Lentivirus vector, Ex vivo transfection

Ex vivo siRNA electroporated PBMCs NA

Lentivirus vector, Ex vivo transfection Ex vivo transfection

Ex vivo transfection

Cyclodextrin NP (RONDEL) Ex vivo electroporation of shRNA

LNP (Dlin-DMA)

EnCore Lipid Nanoparticle

DELIVERY SYSTEM

i.v.

Intratumoral

NCT02716012

NCT01505153

NCT02736565

NCT00569985

i.v.

i.v. infusion

NCT02757586

NCT02166255 NCT03087591

i.v. infusion

NA

NCT00672542

i.d.

i.v. infusion

i.v.

NCT02797470

NCT02725489 NCT01867086 NCT01309230 NCT01551745 NCT03073525 NCT02346747 NCT01505166 NCT02574533 NCT01453361 NCT03842865 NCT02511132 NCT03495921 NCT01061840 NCT02639234 NCT02528682

i.d.

i.v.

NCT00882180 NCT01158079 NCT00689065

NCT0211056 NCT02314052

NCT ID

i.v.

i.v.

ADMINIST-RATION ROUTE

Mina Alpha Limited

Gradalis, Inc.

Gradalis, Inc.

Wake forest university health sciences Peking University People's Hospital City of Hope Medical Center

Scott Pruitt, Duke University

AIDS Malignancy Consortium

Radboud University

Alnylam Pharmaceuticals Calando Pharmaceuticals Gradalis, Inc.

Dicerna Pharmaceuticals, Inc

COMPANY

Phase 1

Phase 1

Phase 1

Phase 1

(continued on next page)

Barve et al. (2015), Rao et al. (2016) Phadke et al. (2011) Zhou et al. (2019)

DiGiusto et al. (2010), Li et al. (2005)

Loibner et al. (2018), Triozzi et al. (2015)

Phase 1 Phase 1 NS

Dannull et al. (2013)

Phase 1

Phase 1,2

2 3 1 2 1,2

Hobo et al. (2010), van der Waart et al. (2015) Walker et al. (2012)

Ghisoli et al. (2016), Ghisoli et al. (2015), Ghisoli et al. (2017), Nemunaitis et al. (2014), Oh et al. (2016), Senzer et al. (2012) Phase Phase Phase Phase Phase Phase Phase Phase Phase NS Phase Phase Phase Phase Phase

2 2 2 2 2 2 2 1 2

Tabernero et al. (2013) Davis et al. (2010)

Tolcher et al. (2015)

Phase 1 Phase 1,2

Phase 1 Phase 1 Phase 1

REF.

STAGE

X. Liang, et al.

Biomedicine & Pharmacotherapy 125 (2020) 109997

Cutaneous T-Cell Lymphoma (CTCL), Chronic Lymphocytic Leukemia (CLL), Diffuse Large B-Cell Lymphoma (DLBCL), Activated B-cell (ABC) Subtype, Adult T-Cell Leukemia/Lymphoma (ATLL) Hepatocellular Carcinoma (HCC), melanoma, TripleNegative Breast Cancer (TNBC), Non-Small Cell Lung Cancer (NSCLC) Melanoma

Solid Tumors, Melanoma

Melanoma, Colon Cancer, Gastrointestinal Cancer, Genitourinary Cancer, Hepatocellular Cancer

Colorectal Cancer (CRC), Non-Small Cell Lung Cancer (NSCLC), Pancreatic Adenocarcinoma Solid Tumors, lymphoma, Advanced Ovarian Carcinoma (Ph 2 cohort) Relapsed/Refractory Solid Tumor Malignancies, lymphoma Solid Tumors Prostate Cancer, Non-Small Cell Lung Cancer (NSCLC), Breast Cancer

Cobomarsen

mRNA-4157(Personalized cancer vaccine)

NCI-4650(Personalized cancer vaccine)

mRNA-5671

6

MEDI1191 Custirsen

mRNA-2752

mRNA-2416

ECI-006

INT-1B3

INDICATION(S)

THERAPEUTICS

Table 1 (continued)

Undisclosed

Undisclosed

Undisclosed

Undisclosed

miRNA mimic

mRNA vaccine

mRNA vaccine

mRNA vaccine

miRNA 193a3p

Undisclosed

mRNA

OX40 L, IL23, IL36γ (Triplet) IL12 Clusterin mRNA RNase H

Undisclosed

mRNA

OX40L

Undisclosed Mixed backbone

Undisclosed

mRNA vaccine

Five tumorassociated antigens (TAAs) Twenty tumorassociated antigens (TAAs) Twenty tumorassociated antigens (TAAs) KRAS

Undisclosed

LNA anti-miRNA

miRNA-155

MODIFICATION

MECHANISM

TARGET

LNP LNP

LNP

LNP

LNP

LNP

LNP

TriMix technology

LNP

LNP

DELIVERY SYSTEM

Intratumoral injection i.v.

Intratumoral injection

Intratumoral injection

i.m.

i.m.

i.m.

Intranodal injection

i.v.

i.v. or subcutaneous

ADMINIST-RATION ROUTE

NCT03946800 NCT01497470 NCT01578655 NCT00327340 NCT01083615 NCT01188187 NCT00138918 NCT00258388 NCT00054106 NCT01630733 NCT00138658 NCT00258375

NCT03739931

NCT03323398

NCT03948763

NCT03480152

NCT03313778 NCT03897881

NCT03394937

NA

NCT03837457 NCT02580552

NCT ID

ModernaTX, Inc. Achieve Life Sciences

ModernaTX, Inc.

ModernaTX, Inc.

ModernaTX, Inc.

National Cancer Institute

ModernaTX, Inc.

eTheRNA immunotherapies

InteRNA

miRagen Therapeutics, Inc.

COMPANY

Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase

1 1 3 2 3 3 2 2 1 3 1,2 2

Phase 1

Phase 1,2

Phase 1

Phase 1,2

Phase 1 Phase 2

Phase 1

N/A

Phase 2 Phase 1

STAGE

(continued on next page)

Beer et al. (2017), Chi et al. (2005), Chi et al. (2008), Saad et al. (2011), Laskin et al. (2012), Chia et al. (2009)

Seto et al. (2018)

REF.

X. Liang, et al.

Biomedicine & Pharmacotherapy 125 (2020) 109997

Advanced Solid Malignancies, Advanced adult Hepatocellular Carcinoma, Hepatocellular Carcinoma Metastatic Prostate Cancer

AZD9150

7

Ph + Chronic Myelogenous Leukemia, Acute Myeloid Leukemia (AML)

Undisclosed

miRNA mimic

mi-RNA 34a

Grb-2

RNase H

RNase H

STAT3

Androgen receptor

Mixed backbone

RNase H

Heat shock protein 27 (Hsp27)

Undisclosed

Undisclosed

Undisclosed

Undisclosed

MODIFICATION

MECHANISM

TARGET

LNP

LNP

LNP

LNP

LNP

DELIVERY SYSTEM

i.v.

i.v.

i.v.

i.v. infusion

i.v. or i.v. infusion

ADMINIST-RATION ROUTE

NCT02923986 NCT02781883 NCT01159028

NCT02862145 NCT01829971

NCT03300505

NCT01839604 NCT03421353 NCT03394144

NCT01454089

NCT02423590

NCT01829113

NCT01120470

NCT ID

Bio-Path Holdings, Inc.

University of Michigan Rogel Cancer Center Mirna Therapeutics, Inc.

British Columbia Cancer Agency SCRI Development Innovations, LLC Queen Mary University of London Achieve Life Sciences AstraZeneca

COMPANY

Phase 1,2 Phase 2 Phase 1

Phase 1,2 Phase 1

Phase 1,2

Phase 1 Phase 1,2 Phase 1

Phase 2

Phase 2

Phase 2

Phase 2

STAGE

Beg et al. (2017), Ling et al. (2017) Farooqi et al. (2016), Cortez et al. (2016), Adams et al. (2016), Zhao et al. (2017) Ohanian et al. (2018)

Reilley et al. (2018), Hong et al. (2015), Odate et al. (2017)

Chi et al. (2016), Dellis et al. (2016), Spigel et al. (2019), Hendriks et al. (2017)

REF.

Abbreviation: i.d., intradermal; i.v., intravenous; s.c. subcutaneous; N/A, not applicable; NA, not available; NS, not specified; 2′-F, 2′- fluoro substitution; 2′-OMe, 2′-methoxy group substitution; AIDS, acquired immunodeficiency syndrome; AR, androgen receptor; BCL2L12, B-cell lymphoma 2-like protein 12; Bcr-Abl, breakpoint cluster region gene-abelson murine leukemia viral oncogene homolog 1; Cbl-b, Cbl proto-oncogene B; CCR5, CeC chemokine receptor type 5; Cox-2, cyclooxygenase-2; CTNNB1, catenin beta-1 (β-catenin); DC, dendritic cell; DLin-DMA, 1,2-dilinoleyloxy-3-dimethylaminopropane; DOPC, 1,2-dioleoyl-sn-glycero-3phosphocholine; EphA2, EPH receptor A2 (ephrin type-A receptor 2); EWS/FLI1, the N-terminal transactivation domain of EWSR1 (EWS RNA binding protein 1) fuses with the C-terminal DNA binding domain of FLI1 (friend leukemia integration 1 transcription factor); GalNAc, N-Acetyl-D-galactosamine; HIF-2α, hypoxia-inducible factor-2 alpha; HIV, human immunodeficiency virus; HSP90, heat shock protein 90; iB, inverted base; KSP, kinesin spindle protein; LMP2, low molecular mass poylpeptides 2; LMP7, low molecular mass poylpeptides 7; LNP, lipid nanoparticle; LODER, Local Drug EluteR; MECL1, also known as PSMB10, proteasome subunit beta 10; MiHA, minor histocompatibility antigen; PBMC, peripheral blood mononuclear cell; PD-L1, programmed death-ligand 1; PD-L2, programmed death-ligand 2; PKN3, protein kinase N3; PLGA, poly(lacticco-glycolic acid); PLK1, polo-like kinase 1; PS, phosphorothioate linkage; RRM2, ribonucleoside-diphosphate reductase subunit M2; SNA, spherical nucleic acid; STMN1, stathmin 1; TGF-β1, transforming growth factor beta 1; TRiM, targeted RNAi molecule; VEGF, vascular endothelial growth factor; XPO1, exportin 1; i.m., intramuscular.

BP1001

MRX34

Melanoma, Primary Liver Cancer, Small Cell Lung Cancer (SCLC), Lymphoma, Renal Cell Carcinoma, NonSmall Cell Lung Cancer (NSCLC)

Prostate Cancer, Non-Small Cell Lung Cancer (NSCLC), Bladder Cancer

Apatorsen

AZD5312

INDICATION(S)

THERAPEUTICS

Table 1 (continued)

X. Liang, et al.

Biomedicine & Pharmacotherapy 125 (2020) 109997

Biomedicine & Pharmacotherapy 125 (2020) 109997

X. Liang, et al.

reduces the translation of RPS28 mRNA, blocks the 18S preribosomal RNA processing, and results in reduction of 40S ribosomal subunits [34]. Thus, it can induce apoptosis of tumor cells that grow and proliferate uncontrollably. Velagapudi and Costales et al. have chosen another approach of cancer cell pro-apoptosis, making use of the cell's RNA degradation system to awaken the body to kill tumor cells. In the study, they constructed a novel small molecule named ribonuclease-targeting chimeras (RIBOTAC), which bond with miRNA-96 largely expressed in TNBC and activated RNase L to eliminate miRNA-96. When it declined, the expression of FOXO1 gene increased, and the self-destruction system of tumor cells was awakened to stimulate the death of them [35,36]. In addition, Costales et al. found that targapremiR-210, a small molecule that can regulate the production of miR-210, was able to increase the occurrence of apoptosis in cells with TNBC through the hypoxia-inducible factor signaling pathway [37]. More related studies are still ongoing. Most of them indicate that RNA therapy is realizable to induce programmed death of tumor cells.

splicing factor named SF3B1, which can be seen in most uveal melanoma. Its mutation breaks in the normal splicing of RNAs, promoting cancers. Therefore, researchers created antisense oligonucleotides for mutant SF3B1 to cure mice with uveal melanoma. It was showed that the ASO drug did constrain cancer cell proliferation and make the whole tumor shrink [26]. In accordance with the above studies, RNA therapy is feasible and potential to interfere with tumor growth. 4.2. Preventing the metastasis of malignant tumor cells Many achievements are worth mentioning in the research of RNA therapy that prevents the metastasis of cancer cells along lymphatics and blood vessels [27–29]. Mendell et al. [30] confirmed overexpression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in human breast cancer by inhibiting MALAT1 in mice with lumen B breast carcinoma. And they reduced the carcinoma metastasis in the models via ASOs. Their findings demonstrated ASOs were helpful to treat MALAT1 featured breast cancers and also safe enough. Among all the breast cancers, the treatment of triple negative breast cancer (TNBC) is very tricky, and now there are a considerable number of researches on RNA therapy about it. Using beta 3 integrins as the target, Parvani et al. designed a siRNA and transported it in vivo through nanoparticles, and the in-situ tumor load of the treated mice was alleviated and the metastasis of cancer cells was significantly curbed [31]. Another study found that an ASO therapy designed for TROJAN strongly inhibited the progression of TNBC in humans [32]. This is because when analyzing the transcriptome of the entire human endogenous retrovirus genome, researchers discovered that TROJAN is overexpressed in patients suffering from TNBC, which can promote cancer cells reproduction and infiltration, usually associated with poor prognosis. Subsequently, it was further found that TROJAN could bind to ZMYND8, which encoded protein kinase C binding protein with the function of metastasis inhibition, increased the degradation of ZMYND8, and improved the ability of cancer cell metastasis. Generally speaking, TROJAN and beta 3 integrins are both expected to be new targets of RNA therapy in the treatment of TNBC, and they have a promising prospect. Furthermore, Zhao et al. developed bionic nanoparticles that were made up of cationic bovine serum albumin (CBSA) coupled with siS100A4 and exosome membrane coated nanoparticles, longing to handle the postoperative TNBC featuring high recurrence and metastasis. In contrast with CBSA/siS100A4@Liposome, CBSA/siS100A4@ Exosome had better affinity to the lung and showed excellent gene silencing effect, which inhibited malignancy to a remarkable extent. The results imply that CBSA/siS100A4@Exosome self-assembled nanoparticles have a bright future of prohibiting metastasis of postoperative breast cancer cells [33]. Compared with chemotherapy as well as radiotherapy that solely relieve symptoms, RNA therapy with target specificity can be regarded as a key way to cure cancers, which can efficiently stop cancer cells growth without harming normal cells.

4.4. Disrupting the expression of tumor cells Conde et al. [38] published a new study that they successfully delivered miRNAs into tumor cells by constructing a three-helix structure formed by the winding of three miRNA chains. After RNA enzyme splicing, the miRNA that inhibited tumors and the tumor-causing miRNA in the three-helix structure were simultaneously activated, which disturbed the expression of cancer cells and thus shrank the size of malignant tumors in mice. Such a three-helix miRNA not only delivers chain segments with high efficacy, but also finds a new means to shrink tumors and kill them. 4.5. Inhibiting the angiogenesis of tumor cells In the tumor microenvironment, vascular endothelial growth factors (VEGF) released by tumors play an important role in promoting tumor angiogenesis [39]. It has been found that downregulation of miR-29a/c in gastric cancer cells leads to an increasing expression and release of VEGF, thereby facilitating the proliferation of vascular cells. According to this finding, researchers transferred microvesicles (MV) carried miR29 a/c into the transplant tumor cells of mice, and finally the results showed angiogenesis and growth of the tumor in mice were significantly suppressed, proving the RNA therapy really could reduce VEGF expression to block the growth and metastasis of vascular cells as well as blood vessel formation [40]. This result will contribute to a novel anti-tumor therapeutic of using MVs to carry miRNAs. 4.6. Reconstructing the tumor microenvironment While exploring the cooperative effect between metabolic reprogramming and microenvironment reconstruction, Sang et al. made advantage of RNAi to suppress CamK-A in patient-derived xenograft (PDX) of TNBC model, which showed tumor proliferation, micro-vessels growth and macrophages recruitment were strongly inhibited, resulting in reshaping the tumor microenvironment [41]. This is due to the activation of the calcium signaling pathway by CamK-A under hypoxic tumor conditions, thereby inducing the activation of kappa-light-chainenhancer of activated B cells (NF-κB) [41,42]. It paves the way for further clinical application of CamK-A inhibitors.

4.3. Inducing the apoptosis of tumor cells One of the characteristics of tumor cells is that they resist cell death and do not undergo apoptosis as normal cells do when they reach a certain lifespan. Therefore, tumor cells increase sharply, and they spend energy crazily. If tumor cells can be induced to apoptosis, it's possible to make the tumor shrink or even disappear. LeuCAG3'tsRNA, which is found in high concentration in tumors and immortalized cells, enhances its own translation by binding at least two ribosomal protein mRNAs (RPS28 and RPS15). It has been suggested that the construction of anti- Leu3'tsLNA to inhibit Leu3'tsLNA based on the principle of RNAi can induce the apoptosis of rapidly dividing cells in vitro and in patient-derived primary hepatocellular carcinoma model mice. This is because the inhibition of Leu3'tsLNA

4.7. Reprogramming tomor cells Abu-hamad et al. explored a new anticancer RNA therapy with siRNA to silence the expression of mitochondrial gated protein VDAC1, which could greatly reduce the energy supply for cancer cells [43]. More surprisingly, it reprogrammed metabolic pathways of cancer cells to transform them into normal differentiated cells [44,45]. VDAC1 is 8

Biomedicine & Pharmacotherapy 125 (2020) 109997

X. Liang, et al.

but adaptive immunity and thus enhancing the efficacy of tumor immunotherapy [54]. What we learned from this study is that we can take it as a promoted strategy for tumor immune therapeutics to prohibit characteristic innate and adaptive checkpoints of cancer cells. PD-L1 has appeared in another study again. Zhou et al. made a corporation of doxorubicin (DOX) and PD-L1 siRNA through a nanodelivery system. They provides a brilliant idea for cancer treatment that it is combination of chemotherapy and immunotherapy, which generates more than working alone or simply pooling them together [55].

highly expressed in many solid tumors and hematomas, which is a hint to satisfy the needs of cancer cells for plenty of energy [46,47]. No matter in vitro or in mouse models of glioma, lung cancer and TNBC, this siRNA therapy successfully constrained the growth and angiogenesis of cancer cells and reduced their invasiveness [45,47–49]. 4.8. Decreasing drug resistance of tomor cells Cisplatin is a universal chemotherapeutic drug in antitumor treatment, whereas a part of tumors are now resistant to it. Li et al. optimized a self-assembled LNP system that delivered cisplatin prodrugs as well as siRNA at the same time, targeting the key component of the nucleotide excision repair (NER) pathway, the endonuclease xeroderma pigmentosum group F (XPF). They described that intrinsic NER function can be suppressed by siRNA, coupled with tumor cell DNAs being damaged by cisplatin prodrugs, which assured these specific LNPs promoted the antitumor activity of cisplatin and conquered the tumor cell resistance to it. It’s a new method to deal with cisplatin refractory tumors [50].

5.2. The development of tumor RNA vaccines Tumor vaccines not only prevent people from suffering tumors, but also activate human immune system to clean tumor cells and cure cancers. RNA tumor vaccines typically use mRNAs. Mostly mRNAs encode proteins that teach the immune system to recognize and target tumors, causing the body’s response to the tumors (Fig. 3). Clinical trials have shown that mRNAs directly delivered with LNPs produces a significant protective immune response, which also act as adjuvants to stimulate the innate immune response [56,57]. Several tumor-RNA vaccines are being tested. The application of single mRNA-4157 in resectable solid tumors and combined with antiPD-1 therapy, KEYTRUDA, in unresectable solid tumors has passed phase I clinical trial and is ready to enter phase Ⅱ (Table 1). The safety and immunogenicity of ECI-006 as adjunctive therapy in phase I clinical trials of resectable melanoma have been well documented (Table 1). mRNA-4650 is being tested in phase I/Ⅱ trials in patients with metastatic melanoma, gastric cancer, and urogenital cancer, each of which has at least one resectable site. The results indicate that mRNA -4650 is safe at all doses and no dose-limiting toxicity (DLT) or serious drugrelated side effects have been found (Table 1). The development of these RNA vaccines is encouraging, and we believe that they will certainly bring good news to cancer patients in the near future.

5. RNA therapeutics and Cancer immunotherapy Tumor immunotherapy plays its role by enhancing the activity of some components in the immune system or removing cancer cells’ inhibition of the immune system, such as immune checkpoint inhibitors, cancer vaccines, immune cell therapy and so on. It has an advantage over other traditional tumor therapies, because it can generate longlasting anti-tumor immune memory in patients, and has a long-term protective effect even after stopping treatment. But the current effectiveness is compromised by tumor immune escape. The immune escape refers that tumor cells flee from immune system recognition and attack by disguising themselves or changing their microenvironment [51]. RNA therapy can help immunotherapy to target exactly in the complex tumor microenvironment, thus reducing the escape of tumor cells [52]. Hence, the immunotherapy can work as well as possible.

5.3. Adjuvant cellular immunotherapy In 2017, researchers constructed c-Met-CAR T cells targeting c-Met, a molecule on the cell surface expressed in about 50 % of breast cancers via importing the chimeric antigen receptors (CAR) into the body by mRNA [58]. mRNA c-Met-CAR T cells were well tolerated by intratumor injection and could induce an inflammatory response to the tumors causing cancer cell necrosis when applied to metastatic breast cancers. This method improves the effectiveness of CAR-T cells in treating solid tumors. But what if CAR-T cells are equipped with tumor RNA vaccines? Someone has done this. A team wrapped CLDN6 RNA into liposomes for the reason that CLDN6 protein was overexpressed in solid tumors, and then introduced them into mice’s lymphatic system, driving expansion of claudin-CAR-T cells. In the light of the study data, the mice cured by the combination survived longer than the ones merely given CAR-T therapeutic [59]. The notion “CAR-T therapy is hard to treat solid tumors” has been reversed thoroughly due to the above researches, which have laid a solid foundation for its deeper exploration. We hope that with the assistance of RNA therapy, CAR-T therapy will be more powerful in coping with cancers.

5.1. The immune checkpoint inhibitors The self-delivering RNA (sd-rxRNA) interference technology is used to down-regulate immune checkpoints via destroying specific RNAs before they are translated into proteins (Fig. 2). sd-rxRNA compounds can target multiple immunosuppressive targets with high knockout efficiency without delivering technology. The new cancer immunotherapy invented by RXi Pharmaceuticals and the Karolinska Institute in Sweden combines advantages of the two most promising immunotherapies available, sd-rxRNAs and the adoptive cell transfer (ACT) therapy. The role of sd-rxRNAs in this process is to eliminate the expression of immunosuppressive receptors or proteins in therapeutic immune cells, so that the treated immune cells are not sensitive to the mechanism of tumor resistance and improve their ability to destroy tumor cells. These armed immune cells are then injected back into the patient's body to clear cancer cells (Fig. 2). A recently published study exhibited that sd-rxRNA targeting PD-1 can increase anti-tumor activity of tumor-infiltrating lymphocytes (TIL) against melanoma cells [53]. At present, other than sd-rxRNA targeting PD-1, there are sd-rxRNA compounds developed to target T cell immune-receptor with Ig and Immunoglobulin progenitor T cell immune receptor and other solid tumors. sd-rxRNA has great development potential and is a new powerful weapon for people to deal with frequently drug-resistant tumors. Lian et al. found another way to break down specific cancer cell checkpoints. They created EpCAM (epithelial cell adhesion molecule)targeted cationic liposomes (LPP-P4-Ep) because EpCAM was expressed commonly in tumor cells. LPP-P4-Ep was loaded with two kinds of siRNAs to knock down programmed death-ligand 1(PD-L1) and CD47 on the surface of cancer cells, helping to coordinate not only the innate

5.4. Regulating the release of immune modulators Moderna's investigational mRNA immune tumor therapy product mRNA-2752 allows simultaneous local delivery of mRNA encoding three immune modulators, including secreted cytokines IL23 and IL36 and membrane-bound T-cell co-stimulator OX40 L (Table 1). It induces and activates a wide range of immune responses in tumors injected and not injected at a distance, identifying and eradicating cancer cells and promoting tumor regression. Moreover, when combined with 9

Biomedicine & Pharmacotherapy 125 (2020) 109997

X. Liang, et al.

Fig. 2. Adoptive Cell Transfer (ACT) Therapy Improved by the sd-rxRNA Compound. T cells isolated from blood samples of cancer patients are divided into two types: those that can recognize tumor-specific antigens and those that cannot. Then culture T cells which don't recognize tumors and transform them into the ones that target precisely at tumor cells. sd-rxRNA compounds are used to eliminate the immunosuppressive receptors on the surface of T cells, making them insensitive to the drug resistance mechanisms of tumors, so as to boost the ability of T cells to kill tumor cells. After appropriate chemical modification and expansion, treated T cells are transferred back to the patients, which can attack the tumor cells quickly and accurately in vivo, thus achieving the goal of treating tumors. Abbreviation: sd-rxRNA, self-delivering RNA.

pathway. Through functional analysis, it was believed that ARLNC1 formed a positive feedback loop with androgen receptors to maintain the activation of androgen transcription. They finally proved that ASO therapy targeting ARLNC1 slowed the development of prostate cancer. The finding adds one more option for cancer patients, which could improve their cure rates.

checkpoint inhibitors, mRNA-2752 increased the complete remission rate in both immunosuppressive and immunocompromised tumor mice. 5.5. LncRNAs related Cancer immunotherapy Prostate cancer is closely relevant to the regulation of androgen secretion. Zhang et al. [60] discovered ARLNC1, a lncRNA with high specificity in prostate cancer related to androgen receptor signaling 10

Biomedicine & Pharmacotherapy 125 (2020) 109997

X. Liang, et al.

Fig. 3. Tumor RNA Vaccines. Tumor RNA vaccines are made to aim at tumor-specific antigens by analyzing a patient's tumor tissue. It is usually to use mRNA as the main component. The mRNA corresponding to tumor antigen can be directly transported into the body by liposomes, or presented by DCs, which can activate the immune system in vivo to produce an immune response and destroy tumor cells. Abbreviations: NGS, next-generation sequencing; DC, Dendritic cells.

of tumor treatment, can be more widely used. It targets characteristics of tumor cells at the RNA level, but without genetic changes, to cure cancers. Not only does it guarantee the specificity but avoid potential risks of gene therapy [63]. It's foreseeable that RNA therapy will become a possible direction of tumor therapy in the future.

5.6. miRNAs related cancer immunotherapy Another team is attempting to develop RNA therapy for acute myeloid leukemia (AML). Jiang et al. [61] found that miR-22 was downregulated in samples of all kinds of AML patients. They tried to use nanoparticles to deliver miR-22 into a model mouse of leukemia and discovered that increasing miR-22 expression can suppress specific CREB and MYC signaling pathways by aiming at three vital targets CRTC1, FLT3 and MYCBP, thereby delaying the development of AML. miRNAs related cancer immunotherapy also brings hopes to neuroblastoma patients. Neviani et al. loaded some exosomes derived from natural killer (NK) cells the cancer suppressor miR-186 and observed that these exosomes prohibited growth, proliferation and TGFbeta-dependent immune escape mechanisms of neuroblastoma, which was attributed to cytotoxicity of them against MYCN-amplified neuroblastoma [62]. The above progresses indicate that RNA therapy, as a new method

6. The advances in clinical trials of RNA therapeutics The undruggable RAS and MYC proteins in oncology have been long lack of small molecule drugs to target them [64], but now a growing number of experiments have shown that aiming at RNAs may create new drugs to direct at RAS, MYC or other sites (Table 1). Additionally, RNA therapy has the power to affect abnormal ncRNAs to modify changed protein profiles [65,66]. A diversity of small molecules can be bond to RNAs. Some of them reduce protein activity by blocking mRNA translation, while others increase protein activity by turning off inhibition of miRNA. Thus, they 11

Biomedicine & Pharmacotherapy 125 (2020) 109997

X. Liang, et al.

new ways to deliver therapeutic RNAs to cancer cells, but also reduce adverse side effects in normal tissues [79,80]. A year later, more new means of delivering siRNAs with greater safety have been discovered. Yalcin et al. modified albumin-sericin nanoparticles (Alb-Ser NPs) with poly-L-lysine (PLL), in an attempt to make them new hosts of target siRNAs, and added hyaluronic acid (HA) to the surface of Alb-Ser NPs. HA/PLL/Alb-Ser NPs, designed to aim at laryngeal cancer cell line, Hep2, were confirmed to be hopeful carriers in the research [81]. Kim et al. utilized peptide-targeted porous silicon nanoparticles as new carriers of siRNAs. Furthermore, a lipid coating for the nanoparticles to facilitate cytoplasmic membrane fusion was taken into consideration. This design has invented an independent uptake mechanism varied from common receptor-mediated endocytosis at the cellular level and has been tested its therapeutic effects at the animal level [82]. When it comes to the lung cancer, chimeric lipopepsomes (CLP) functionalized by selective cell penetrating peptides (CPP33) have made an immense improvement in treating it. CPP33-CLP encapsulated polo-like kinase 1 specific siRNA (siPLK1) tightly and sent it precisely into A549 human lung carcinoma in situ, which curbed on tumor growth, lengthening survival time of the mice bearing orthotopic A549 human lung carcinoma [83]. About a month later, Zheng et al. demonstrated that siRNA delivered by folate (FA)-exosomes successfully inhibited cancers because it avoided endosomal capture [84]. The study of this delivery mechanism will update the concept and arouse stronger interest in the use of FA as a targeting ligand in cancer therapy. Apart from these, numerous improvements have been made in anticancer ASO and mRNA drugs. Mou et al. supplied a novel approach of restoring sensitivity of cancer cells to chemotherapy, who created chemogenes with an antitumor drug integrated into ASOs. It referred to the results that chemogenes achieved to simplify delivery system, enhance druggability, and most critically, reverse chemoresistance [85]. As for delivering mRNA tumor vaccines, Lei et al. engineered some special liposomes, all of which shared the same structure: an unsaturated lipid tail, a dihydroimidazole linker, and cycloamine head groups. It was these liposomes that activated intracellular stimulators of the interferon gene (STING) pathway instead of Toll-like receptors to make antigen presenting cells mature, thus restricting systemic cytokines from expressing and facilitating mRNA vaccines antitumor effects [86]. A liposome-protamine lipoplex (CLPP) containing IVT mRNA that encoded survivin-T34A gene, named CLPP/mSur-T34A, is going to be a potential candidate in treating colon cancer [87]. Researches on the RNA therapy delivery system keep going on. Biodegradable ionized lipids have been used to reduce the toxicity of LNPs, but that's far from enough. Scientists still burry themselves into working out how to further improve the transmission and absorption of RNA drugs, decrease the cost, reduce the toxicity of carriers, and how to eventually establish production lines of RNA drugs.

become the focuses of researches on RNA drugs in laboratories, producing abundant RNA drugs for all types of tumors. Up to now, antitumor RNA drugs have got ideal achievements in clinical trials (Table 1), and the number of pharmaceutical companies who have joined in studying RNA anti-tumor therapy is rising. 7. The challenge of RNA therapeutics Although RNA therapy has many advantages in treating tumors, it faces many difficulties and challenges. These challenges are focused on three major areas, namely the pharmacokinetics, pharmacodynamics and drug production. 7.1. Pharmacokinetics The challenge for the development of in vivo applications of RNA therapy drugs is the potential for immune stimulation, lack of targeting specificity to the lesion area, avoidance of renal and reticuloendothelial tissue clearance, appropriate delivery vectors, and endosomal escape [67–69]. Overcome these challenges and improve the pharmacokinetics of RNA drugs to ensure that they are stable enough to reach target cells before they are degraded or excreted, and that they can successfully enter target cells and bind to intramolecular targets and exert their effects. Therefore, many efforts are paid to design RNA sequences through bioinformatics to avoid off-target effects. Besides, chemical modifications help to avoid degradation of nucleosidases and then reduce immunogenicity [22,70,71]. However, the bottlenecks of clinical application of RNA drugs are drug delivery system and connotation escape. 7.1.1. RNA therapy delivery system Lipid nanoparticles (LNPs) have been one of the most widely used delivery systems by now. Due to their good biocompatibility and the same structure as cell membrane, they can extend the time of drug action, reduce drug toxicity, and improve drug stability [72,73]. LNPs can also be extended to deliver mRNAs, making it possible to produce more therapeutic proteins [74]. But LNPs are often heterogeneous to a certain extent in terms of loading, particle composition, and properties, which makes them tough to establish a streamline in the clinical development process. On top of such, LNPs may become unstable and disintegrate, causing a toxic immune response after being transported into bodies. LNPs also accumulate in large numbers in the liver, preventing themselves from reaching other organs. Therefore, it is crucial to improve LNP system and develop new means of delivery. In 2014, Liu et al. used oligopeptide cyclo(RGDfK) (cRGD) to specifically bind to the endothelial membrane of tumor neovascularized cells to express the αvβ3 receptor, covalently coupling cRGD oligopeptide with the siRNA justice chain of VEGFR2, the silent vascular endothelial cell growth factor receptor, to obtain the crgd-sivegfr2 molecule. It does not require LNPs to specifically enter new vascular endothelial cells expressing αvβ3 receptors and silence the expression of target genes [75]. In 2016, a new type of RNA spherical nucleic acid (SNA) was successfully tested in humans [76], offering a new method to reduce the cleavage of RNA drugs by enzymes after entering cells. In 2017, researchers attached poly-A binding protein to the tail of the mRNA, delivering the mRNA with high efficiency, significantly increasing the effective loading of the mRNA and improving efficacy [77]. In the same year, Pi et al. used RNA nanotechnology to program natural extracellular vesicles (EVs) and control their direction, delivering vesicles to tumor cells. They succeeded in developing EVs that can target three types of cancers (prostate cancer, breast cancer and colon cancer) in animal models and contain therapeutic siRNA [78]. In 2018, researchers employed human red blood cells (RBC) to produce EVs for RNA therapy. It's proved that red blood cell extracellular vesicles (RBCEVs) are effective and safe. Moreover, coating RBCEVs with cancer-targeting peptides or antibodies cannot only further develop

7.1.2. RNA drug endosome escape Intracellular membrane is a major barrier to the application of RNA drugs [88]. As a result, fleeing from endosomes becomes a big challenge that RNA therapy must be confronted with and overcome [67]. When the carrier of the RNA drug reaches the target cell, the target cell endocytes it. Then the carrier and a part of membrane are assembled into endosomes, which next grow into mature lysosomes through various mechanisms. There are a great number of enzymes and acids in lysosomes, which will disrupt the drugs and inactivate them. Therefore, an effective RNA drug vector should facilitate the rapid escape of RNA drugs from endosomes, which is called endosome escape [89]. Several methods are listed below to realize endosomal escape. Make a membrane pore. For example, bee toxin, can change its own structure in the acidic environment of endosomes to bind to endosomal membrane, and form pores on it to release drugs [90,91]. Fuse membrane. Carriers modified by bacterial or viral peptides and proteins can escape by fusing phospholipid bilayer. Photochemical internalization technology. Light is capable of 12

Biomedicine & Pharmacotherapy 125 (2020) 109997

X. Liang, et al.

It is extremely hard, though, persistent efforts to develop RNA therapy have been paid off over these years. As many innovative technologies of drug delivery and target specificity have made marked progress, there is no doubt that more breakthroughs will be made.

activating photosensitive molecules on the endosomal membrane, making them produce reactive oxygen to destroy the membrane and release RNA drugs in it, such as adding porphyrin derivatives into the carriers. Reverse operation strategy and proton sponge effect are applied too. Despite these methods effectively enhance the probability of endosome escape, there still exist some problems such as high toxicity and low efficiency. The ideal method should be non-toxic, efficient, low cost and easy to produce. That's the goal pursued by every researcher

8. Conclusions and perspectives Small nucleic acid drugs represented by ASOs and siRNAs are launching a new wave in the pharmaceutical industry, while mRNA therapy has entered the fast lane as drug technology is developing, opening its golden age. To sum up, the spring of RNA therapy is coming, especially when the second RNAi drug and the first GalNAc conjugated RNA therapeutic agent, Givlaari, has received approval of FDA in November 2019 [101], which is taken as a landmark in advancement of precision genetic medicines. We can see that in the future there are both advantages and challenges ahead for RNA therapy. More teams devote themselves to RNA antitumor therapy, and more researches concentrate on this field. Particularly, in recent years, as the understanding of miRNAs has been gradually deepened, we are expected to develop "combination therapy" concentrated in one drug to treat multi-gene diseases such as tumors by making use of the characteristics of miRNAs against multiple targets in multiple disease-related pathways at one time. Developments in RNA antitumor therapy will continue, and we will overcome obstacles one by one towards the promising prospect of treating tumors precisely with RNA therapy. We believe that RNA therapy will bring a disruptive innovation to the field of cancer therapy and will be a great boon to countless cancer patients. But before we reach that goal, there is still a long way to go.

7.2. Pharmacodynamics In addition to the pharmacokinetic challenges, pharmaceutical preparations for RNA therapy must overcome pharmacokinetic-related challenges, including target specificity, off-target RNAi activity, and toxicity. A multitude of researches have been done to improve target specificity, including conjugating antibodies to siRNAs, binding the GalNAc to the end of precursor siRNAs to facilitate them to be accurately delivered towards the liver [92–94]. CRISPR-related Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNP) reconstituted in vitro using 5-Bromo-2′-deoxyuridine (BrdU) largely enhanced specificity and affinity of drugs to targets [95]. Toxicity is hard to be oversight. According to the causes, there are two kinds of RNA drug toxicity. One is pharmacodynamic based toxicity, which is caused by off-targeting and working on non-targeted genes. Another one is the result of drugs interacting with other molecules, such as proteins. They generate most undesirable effects, mainly including complement activation, immune stimulation, prolonged coagulation time, liver and kidney toxicity and lethal hemodynamic changes. The siRNA candidates conjugated by GalNAc have been shown in many clinical studies to be toxic to the liver. Zlatev et al. designed a nine-polymer of 5 locked nucleic acids (LNA) called REVERSIR that promoted the therapeutic effect of any long-acting GalNAc-siRNA and reduced the hepatotoxicity of the drugs [96]. Besides, some other researchers first confirmed that the off-target effect of siRNA [97–99] is actually the cause of its hepatotoxicity [100]. Because off-target drugs can be combined not only with targeted mRNAs by complete matching, but also other mRNAs by incomplete matching [97], leading to consequences similar to miRNA inhibition [99]. For this reason, the researchers deliberately added a nucleotide that could not be paired with any bases to their designed siRNA sequences according to different mechanisms by which siRNA and microRNA bind to mRNAs. While assuring the combination of the drug with its original target, it greatly reduced its chances of regulating the transcription of other mRNAs in the way that miRNAs acted. The result showed that this improvement achieved to lower the liver toxicity of siRNA drugs [100]. Although the risk of missed drug targets cannot be ruled out, the promoted siRNA strategy developed in this study may reduce the failure rate of siRNA therapy in clinical trials due to liver toxicity from 30 to 40 percent dropping to about 5 percent, which is a hard-won achievement to reduce the toxicity of RNA drugs.

Funding This work was supported partly by National Natural Science Foundation of China (81541153 and J18111211); Southern Science and Engineering Guangdong Laboratory Zhanjiang (ZJW-2019-07); Guangdong Provincial Science and Technology Department (2016A050503046, 2015A050502048 and 2016B030309002); The Public Service Platform of South China for R&D Marine Biomedicine Resources (GDMUK201808); Zhanjiang Science and Technology Plan (2017A06012); and “Group-type” Special Supporting Project for Educational Talents in Universities (4SG19057G). The funders had no role in the design of the study; the collection, analysis, and interpretation of the data; the writing of the manuscript; and the decision to submit the manuscript for publication. Declaration of Competing Interest The authors state that there is no conflict of interest. References [1] D. Bumcrot, M. Manoharan, V. Koteliansky, D.W. Sah, RNAi therapeutics: a potential new class of pharmaceutical drugs, Nat. Chem. Biol. 2 (12) (2006) 711–719. [2] H. Ledford, Gene-silencing technology gets first drug approval after 20-year wait, Nature 560 (7718) (2018) 291–292. [3] A. Mullard, FDA approves landmark RNAi drug, Nat. Rev. Drug Discov. 17 (9) (2018) 613. [4] C.F. Bennett, B.F. Baker, N. Pham, E. Swayze, R.S. Geary, Pharmacology of antisense drugs, Annu. Rev. Pharmacol. Toxicol. 57 (2017) 81–105. [5] S.T. Crooke, J.L. Witztum, C.F. Bennett, B.F. Baker, RNA-targeted therapeutics, Cell Metab. 29 (2) (2019) 501. [6] S.T. Crooke, Molecular mechanisms of antisense oligonucleotides, Nucleic Acid Ther. 27 (2) (2017) 70–77. [7] T.A. Vickers, S.T. Crooke, Antisense oligonucleotides capable of promoting specific target mRNA reduction via competing RNase H1-dependent and independent mechanisms, PLoS One 9 (10) (2014) e108625. [8] C. Boiziau, R. Kurfurst, C. Cazenave, V. Roig, N.T. Thuong, J.J. Toulme, Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism, Nucleic Acids Res. 19 (5) (1991) 1113–1119.

7.3. RNA therapy drug production It's one of the preconditions for widespread use of RNA therapy to solve the problem of drug production. As a qualified drug, it must first be produced in large quantities at a reasonable cost. The use of mRNA therapy, such as antibodies encoded by mRNAs, requires numerous mRNAs produced by a high level of pharmaceutical technology. However, even if we produce mRNAs in traditional technologies, they still cost much higher than traditional recombinant antibodies do. On top of the cost, the quantity and quality of mRNAs still restrict the application of mRNA therapy. In a word, it's essential to upgrade pharmaceutical technology which can attain the goal of high production, high purity, and the lowest cost. 13

Biomedicine & Pharmacotherapy 125 (2020) 109997

X. Liang, et al.

[9] D. Hodges, S.T. Crooke, Inhibition of splicing of wild-type and mutated luciferaseadenovirus pre-mRNAs by antisense oligonucleotides, Mol. Pharmacol. 48 (5) (1995) 905–918. [10] X. Zhu, J. Zhang, W. Fan, F. Wang, H. Yao, Z. Wang, S. Hou, Y. Tian, W. Fu, D. Xie, et al., The rs391957 variant cis-regulating oncogene GRP78 expression contributes to the risk of hepatocellular carcinoma, Carcinogenesis 34 (6) (2013) 1273–1280. [11] S. Wang, W. Wu, F.X. Claret, Mutual regulation of microRNAs and DNA methylation in human cancers, Epigenetics 12 (3) (2017) 187–197. [12] T. Wu, L. Qu, G. He, L. Tian, L. Li, H. Zhou, Q. Jin, J. Ren, Y. Wang, J. Wang, et al., Regulation of laryngeal squamous cell cancer progression by the lncRNA H19/ miR-148a-3p/DNMT1 axis, Oncotarget 7 (10) (2016) 11553–11566. [13] Z. Chen, S. Liu, L. Tian, M. Wu, F. Ai, W. Tang, L. Zhao, J. Ding, L. Zhang, A. Tang, miR-124 and miR-506 inhibit colorectal cancer progression by targeting DNMT3B and DNMT1, Oncotarget 6 (35) (2015) 38139–38150. [14] G. Roscigno, C. Quintavalle, E. Donnarumma, I. Puoti, A. Diaz-Lagares, M. Iaboni, D. Fiore, V. Russo, M. Todaro, G. Romano, et al., MiR-221 promotes stemness of breast cancer cells by targeting DNMT3b, Oncotarget 7 (1) (2016) 580–592. [15] Q. Lei, X. Liu, H. Fu, Y. Sun, L. Wang, G. Xu, W. Wang, Z. Yu, C. Liu, P. Li, et al., miR-101 reverses hypomethylation of the PRDM16 promoter to disrupt mitochondrial function in astrocytoma cells, Oncotarget 7 (4) (2016) 5007–5022. [16] K. Pinel, L.A. Diver, K. White, R.A. McDonald, A.H. Baker, Substantial dysregulation of miRNA passenger strands underlies the vascular response to injury, Cells 8 (2) (2019). [17] D. Weissman, K. Kariko, mRNA: fulfilling the promise of gene therapy, Mol. Ther. 23 (9) (2015) 1416–1417. [18] K. Kariko, M. Buckstein, H. Ni, D. Weissman, Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA, Immunity 23 (2) (2005) 165–175. [19] T.A. Vickers, H. Zhang, M.J. Graham, K.M. Lemonidis, C. Zhao, N.M. Dean, Modification of MyD88 mRNA splicing and inhibition of IL-1beta signaling in cell culture and in mice with a 2’-O-methoxyethyl-modified oligonucleotide, J. Immunol. 176 (6) (2006) 3652–3661. [20] R. Rupaimoole, F.J. Slack, MicroRNA therapeutics: towards a new era for the management of cancer and other diseases, Nat. Rev. Drug Discov. 16 (3) (2017) 203–222. [21] Y. Weng, H. Xiao, J. Zhang, X.J. Liang, Y. Huang, RNAi therapeutic and its innovative biotechnological evolution, Biotechnol. Adv. 37 (5) (2019) 801–825. [22] R.L. Setten, J.J. Rossi, S.P. Han, The current state and future directions of RNAibased therapeutics, Nat. Rev. Drug Discov. 18 (6) (2019) 421–446. [23] M.M. Ali, V.S. Akhade, S.T. Kosalai, S. Subhash, L. Statello, M. Meryet-Figuiere, J. Abrahamsson, T. Mondal, C. Kanduri, PAN-cancer analysis of S-phase enriched lncRNAs identifies oncogenic drivers and biomarkers, Nat. Commun. 9 (1) (2018) 883. [24] C.V. Pecot, S.Y. Wu, S. Bellister, J. Filant, R. Rupaimoole, T. Hisamatsu, R. Bhattacharya, A. Maharaj, S. Azam, C. Rodriguez-Aguayo, et al., Therapeutic silencing of KRAS using systemically delivered siRNAs, Mol. Cancer Ther. 13 (12) (2014) 2876–2885. [25] S. Kamerkar, V.S. LeBleu, H. Sugimoto, S. Yang, C.F. Ruivo, S.A. Melo, J.J. Lee, R. Kalluri, Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer, Nature 546 (7659) (2017) 498–503. [26] D. Inoue, G.L. Chew, B. Liu, B.C. Michel, J. Pangallo, A.R. D’Avino, T. Hitchman, K. North, S.C. Lee, L. Bitner, et al., Spliceosomal disruption of the non-canonical BAF complex in cancer, Nature 574 (7778) (2019) 432–436. [27] W. Ngamcherdtrakul, W. Yantasee, siRNA therapeutics for breast cancer: recent efforts in targeting metastasis, drug resistance, and immune evasion, Transl. Res. 214 (2019) 105–120. [28] X. Zhu, H. Luo, Y. Xu, Transcriptome analysis reveals an important candidate gene involved in both nodal metastasis and prognosis in lung adenocarcinoma, Cell Biosci. 9 (2019) 92. [29] B. Guo, D. Li, L. Du, X. Zhu, piRNAs: biogenesis and their potential roles in cancer, Cancer Metastasis Rev. (2020). [30] J.T. Mendell, Targeting a long noncoding RNA in breast cancer, N. Engl. J. Med. 374 (23) (2016) 2287–2289. [31] J.G. Parvani, M.D. Gujrati, M.A. Mack, W.P. Schiemann, Z.R. Lu, Silencing beta3 integrin by targeted ECO/siRNA nanoparticles inhibits EMT and metastasis of triple-negative breast cancer, Cancer Res. 75 (11) (2015) 2316–2325. [32] X. Jin, X.E. Xu, Y.Z. Jiang, Y.R. Liu, W. Sun, Y.J. Guo, Y.X. Ren, W.J. Zuo, X. Hu, S.L. Huang, et al., The endogenous retrovirus-derived long noncoding RNA TROJAN promotes triple-negative breast cancer progression via ZMYND8 degradation, Sci. Adv. 5 (3) (2019) eaat9820. [33] L. Zhao, C. Gu, Y. Gan, L. Shao, H. Chen, H. Zhu, Exosome-mediated siRNA delivery to suppress postoperative breast cancer metastasis, J. Control. Release 318 (2019) 1–15. [34] H.K. Kim, G. Fuchs, S. Wang, W. Wei, Y. Zhang, H. Park, B. Roy-Chaudhuri, P. Li, J. Xu, K. Chu, et al., A transfer-RNA-derived small RNA regulates ribosome biogenesis, Nature 552 (7683) (2017) 57–62. [35] S.P. Velagapudi, M.D. Cameron, C.L. Haga, L.H. Rosenberg, M. Lafitte, D.R. Duckett, D.G. Phinney, M.D. Disney, Design of a small molecule against an oncogenic noncoding RNA, Proc Natl Acad Sci U S A 113 (21) (2016) 5898–5903. [36] M.G. Costales, Y. Matsumoto, S.P. Velagapudi, M.D. Disney, Small molecule targeted recruitment of a nuclease to RNA, J. Am. Chem. Soc. 140 (22) (2018) 6741–6744. [37] M.G. Costales, C.L. Haga, S.P. Velagapudi, J.L. Childs-Disney, D.G. Phinney, M.D. Disney, Small molecule inhibition of microRNA-210 reprograms an oncogenic hypoxic circuit, J. Am. Chem. Soc. 139 (9) (2017) 3446–3455. [38] J. Conde, N. Oliva, M. Atilano, H.S. Song, N. Artzi, Self-assembled RNA-triple-helix

[39] [40]

[41]

[42] [43]

[44]

[45]

[46]

[47]

[48]

[49] [50]

[51]

[52] [53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

14

hydrogel scaffold for microRNA modulation in the tumour microenvironment, Nat. Mater. 15 (3) (2016) 353–363. J. Liu, D. Li, H. Luo, X. Zhu, Circular RNAs: the star molecules in cancer, Mol. Aspects Med. 70 (2019) 141–152. H. Zhang, M. Bai, T. Deng, R. Liu, X. Wang, Y. Qu, J. Duan, L. Zhang, T. Ning, S. Ge, et al., Cell-derived microvesicles mediate the delivery of miR-29a/c to suppress angiogenesis in gastric carcinoma, Cancer Lett. 375 (2) (2016) 331–339. L.J. Sang, H.Q. Ju, G.P. Liu, T. Tian, G.L. Ma, Y.X. Lu, Z.X. Liu, R.L. Pan, R.H. Li, H.L. Piao, et al., LncRNA CamK-A regulates Ca(2+)-Signaling-Mediated tumor microenvironment remodeling, Mol. Cell 72 (1) (2018) 71–83 e77. J. Luo, L.F. Langer, J. Liu, A novel role of LncRNA in regulating tumor metabolism and angiogenesis under hypoxia, Cancer Commun. 39 (1) (2019) 2. S. Abu-Hamad, S. Sivan, V. Shoshan-Barmatz, The expression level of the voltagedependent anion channel controls life and death of the cell, Proc Natl Acad Sci U S A 103 (15) (2006) 5787–5792. T. Arif, Z. Amsalem, V. Shoshan-Barmatz, Metabolic reprograming via silencing of mitochondrial VDAC1 expression encourages differentiation of cancer cells, Mol. Ther. Nucleic Acids 17 (2019) 24–37. T. Arif, A. Paul, Y. Krelin, A. Shteinfer-Kuzmine, V. Shoshan-Barmatz, Mitochondrial VDAC1 silencing leads to metabolic rewiring and the reprogramming of tumour cells into advanced differentiated states, Cancers (Basel) 10 (12) (2018). V. Shoshan-Barmatz, D. Ben-Hail, L. Admoni, Y. Krelin, S.S. Tripathi, The mitochondrial voltage-dependent anion channel 1 in tumor cells, Biochim. Biophys. Acta 1848 (10 Pt B) (2015) 2547–2575. T. Arif, L. Vasilkovsky, Y. Refaely, A. Konson, V. Shoshan-Barmatz, Silencing VDAC1 expression by siRNA inhibits Cancer cell proliferation and tumor growth in vivo, Mol. Ther. Nucleic Acids 3 (2014) e159. T. Arif, Y. Krelin, I. Nakdimon, D. Benharroch, A. Paul, D. Dadon-Klein, V. Shoshan-Barmatz, VDAC1 is a molecular target in glioblastoma, with its depletion leading to reprogrammed metabolism and reversed oncogenic properties, Neuro Oncol 19 (7) (2017) 951–964. Z. Tang, D. Li, S. Hou, X. Zhu, The cancer exosomes: clinical implications, applications and challenges, Int. J. Cancer (2019). C. Li, T. Li, L. Huang, M. Yang, G. Zhu, Self-assembled lipid nanoparticles for ratiometric codelivery of cisplatin and siRNA targeting XPF to combat drug resistance in lung Cancer, Chem. Asian J. 14 (9) (2019) 1570–1576. R. Ajina, D.J. Zahavi, Y.W. Zhang, L.M. Weiner, Overcoming malignant cell-based mechanisms of resistance to immune checkpoint blockade antibodies, Semin. Cancer Biol. (2019). S. Tan, D. Li, X. Zhu, Cancer immunotherapy: pros, cons and beyond, Biomed. Pharmacother. 124 (2020) 109821. M.A. Ligtenberg, Y. Pico de Coana, T. Shmushkovich, Y. Yoshimoto, I. Truxova, Y. Yang, M. Betancur-Boissel, A.V. Eliseev, A.D. Wolfson, R. Kiessling, Self-delivering RNAi targeting PD-1 improves tumor-specific t cell functionality for adoptive cell therapy of malignant melanoma, Mol. Ther. 26 (6) (2018) 1482–1493. S. Lian, R. Xie, Y. Ye, X. Xie, S. Li, Y. Lu, B. Li, Y. Cheng, V.L. Katanaev, L. Jia, Simultaneous blocking of CD47 and PD-L1 increases innate and adaptive cancer immune responses and cytokine release, EBioMedicine 42 (2019) 281–295. Y.J. Zhou, W.J. Wan, Y. Tong, M.T. Chen, D.D. Wang, Y. Wang, B.G. You, Y. Liu, X.N. Zhang, Stimuli-responsive nanoparticles for the codelivery of chemotherapeutic agents doxorubicin and siPD-L1 to enhance the antitumor effect, J Biomed Mater Res B Appl Biomater (2019). J. Lutz, S. Lazzaro, M. Habbeddine, K.E. Schmidt, P. Baumhof, B.L. Mui, Y.K. Tam, T.D. Madden, M.J. Hope, R. Heidenreich, et al., Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines, Npj Vaccines 2 (2017) 29. G. Lindgren, S. Ols, F. Liang, E.A. Thompson, A. Lin, F. Hellgren, K. Bahl, S. John, O. Yuzhakov, K.J. Hassett, et al., Induction of robust B cell responses after influenza mRNA vaccination is accompanied by circulating hemagglutinin-specific ICOS+ PD-1+ CXCR3+ t follicular helper cells, Front. Immunol. 8 (2017) 1539. J. Tchou, Y. Zhao, B.L. Levine, P.J. Zhang, M.M. Davis, J.J. Melenhorst, I. Kulikovskaya, A.L. Brennan, X. Liu, S.F. Lacey, et al., Safety and efficacy of intratumoral injections of chimeric antigen receptor (CAR) t cells in metastatic breast cancer, Cancer Immunol. Res. 5 (12) (2017) 1152–1161. K. Reinhard, B. Rengstl, P. Oehm, K. Michel, A. Billmeier, N. Hayduk, O. Klein, K. Kuna, Y. Ouchan, S. Woll, et al., An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors, Science (2020). Y. Zhang, S. Pitchiaya, M. Cieslik, Y.S. Niknafs, J.C. Tien, Y. Hosono, M.K. Iyer, S. Yazdani, S. Subramaniam, S.K. Shukla, et al., Analysis of the androgen receptorregulated lncRNA landscape identifies a role for ARLNC1 in prostate cancer progression, Nat. Genet. 50 (6) (2018) 814–824. X. Jiang, C. Hu, S. Arnovitz, J. Bugno, M. Yu, Z. Zuo, P. Chen, H. Huang, B. Ulrich, S. Gurbuxani, et al., miR-22 has a potent anti-tumour role with therapeutic potential in acute myeloid leukaemia, Nat. Commun. 7 (2016) 11452. P. Neviani, P.M. Wise, M. Murtadha, C.W. Liu, C.H. Wu, A.Y. Jong, R.C. Seeger, M. Fabbri, Natural killer-derived exosomal miR-186 inhibits neuroblastoma growth and immune escape mechanisms, Cancer Res. 79 (6) (2019) 1151–1164. R. Martier, J.M. Liefhebber, A. Garcia-Osta, J. Miniarikova, M. Cuadrado-Tejedor, M. Espelosin, S. Ursua, H. Petry, S.J. van Deventer, M.M. Evers, et al., Targeting RNA-Mediated toxicity in C9orf72 ALS and/or FTD by RNAi-Based gene therapy, Mol. Ther. Nucleic Acids 16 (2019) 26–37. S. Slovak, T. Evan-Salem, Y. Cohen, Self-assembly of a hexameric aggregate of a lipophilic calix[4]pyrrole-resorcinarene hybrid in solution: a diffusion NMR study, Org. Lett. 12 (21) (2010) 4864–4867.

Biomedicine & Pharmacotherapy 125 (2020) 109997

X. Liang, et al.

[85] Q. Mou, Y. Ma, F. Ding, X. Gao, D. Yan, X. Zhu, C. Zhang, Two-in-One chemogene assembled from drug-integrated antisense oligonucleotides to reverse chemoresistance, J. Am. Chem. Soc. 141 (17) (2019) 6955–6966. [86] L. Miao, L. Li, Y. Huang, D. Delcassian, J. Chahal, J. Han, Y. Shi, K. Sadtler, W. Gao, J. Lin, et al., Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation, Nat. Biotechnol. 37 (10) (2019) 1174–1185. [87] X. Zhang, K. Men, Y. Zhang, R. Zhang, L. Yang, X. Duan, Local and systemic delivery of mRNA encoding survivin-T34A by lipoplex for efficient colon cancer gene therapy, Int. J. Nanomedicine 14 (2019) 2733–2751. [88] P. Lonn, A.D. Kacsinta, X.S. Cui, A.S. Hamil, M. Kaulich, K. Gogoi, S.F. Dowdy, Enhancing endosomal escape for intracellular delivery of macromolecular biologic therapeutics, Sci. Rep. 6 (2016) 32301. [89] M. Dominska, D.M. Dykxhoorn, Breaking down the barriers: siRNA delivery and endosome escape, J. Cell. Sci. 123 (Pt 8) (2010) 1183–1189. [90] S. Maher, L. Feighery, D.J. Brayden, S. McClean, Melittin as an epithelial permeability enhancer I: investigation of its mechanism of action in Caco-2 monolayers, Pharm. Res. 24 (7) (2007) 1336–1345. [91] G. van den Bogaart, J.V. Guzman, J.T. Mika, B. Poolman, On the mechanism of pore formation by melittin, J. Biol. Chem. 283 (49) (2008) 33854–33857. [92] S. Matsuda, K. Keiser, J.K. Nair, K. Charisse, R.M. Manoharan, P. Kretschmer, C.G. Peng, A. VKi, P. Kandasamy, J.L. Willoughby, et al., siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes, ACS Chem. Biol. 10 (5) (2015) 1181–1187. [93] K.G. Rajeev, J.K. Nair, M. Jayaraman, K. Charisse, N. Taneja, J. O’Shea, J.L. Willoughby, K. Yucius, T. Nguyen, S. Shulga-Morskaya, et al., Hepatocytespecific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo, Chembiochem 16 (6) (2015) 903–908. [94] J.K. Nair, J.L. Willoughby, A. Chan, K. Charisse, M.R. Alam, Q. Wang, M. Hoekstra, P. Kandasamy, A.V. Kel’in, S. Milstein, et al., Multivalent N-acetylgalactosamineconjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing, J. Am. Chem. Soc. 136 (49) (2014) 16958–16961. [95] A. Lapinaite, J.A. Doudna, J.H.D. Cate, Programmable RNA recognition using a CRISPR-associated argonaute, Proc. Natl. Acad. Sci. U. S. A. 115 (13) (2018) 3368–3373. [96] I. Zlatev, A. Castoreno, C.R. Brown, J. Qin, S. Waldron, M.K. Schlegel, R. Degaonkar, S. Shulga-Morskaya, H. Xu, S. Gupta, et al., Reversal of siRNAmediated gene silencing in vivo, Nat. Biotechnol. 36 (6) (2018) 509–511. [97] A.L. Jackson, S.R. Bartz, J. Schelter, S.V. Kobayashi, J. Burchard, M. Mao, B. Li, G. Cavet, P.S. Linsley, Expression profiling reveals off-target gene regulation by RNAi, Nat. Biotechnol. 21 (6) (2003) 635–637. [98] A. Birmingham, E.M. Anderson, A. Reynolds, D. Ilsley-Tyree, D. Leake, Y. Fedorov, S. Baskerville, E. Maksimova, K. Robinson, J. Karpilow, et al., 3’ UTR seed matches, but not overall identity, are associated with RNAi off-targets, Nat. Methods 3 (3) (2006) 199–204. [99] A.L. Jackson, J. Burchard, J. Schelter, B.N. Chau, M. Cleary, L. Lim, P.S. Linsley, Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity, RNA 12 (7) (2006) 1179–1187. [100] M.M. Janas, M.K. Schlegel, C.E. Harbison, V.O. Yilmaz, Y. Jiang, R. Parmar, I. Zlatev, A. Castoreno, H. Xu, S. Shulga-Morskaya, et al., Selection of GalNAcconjugated siRNAs with limited off-target-driven rat hepatotoxicity, Nat. Commun. 9 (1) (2018) 723. [101] P.R. de Paula Brandao, S.S. Titze-de-Almeida, R. Titze-de-Almeida, Leading RNA interference therapeutics part 2: silencing delta-aminolevulinic acid synthase 1, with a focus on givosiran, Mol. Diagn. Ther. (2019).

[65] A. Mullard, Small molecules against RNA targets attract big backers, Nat. Rev. Drug Discov. 16 (12) (2017) 813–815. [66] X. Tang, Y. Huang, J. Lei, H. Luo, X. Zhu, The single-cell sequencing: new developments and medical applications, Cell Biosci. 9 (2019) 53. [67] S.F. Dowdy, Overcoming cellular barriers for RNA therapeutics, Nat. Biotechnol. 35 (3) (2017) 222–229. [68] R. Titze-de-Almeida, C. David, S.S. Titze-de-Almeida, The race of 10 synthetic RNAi-Based drugs to the pharmaceutical market, Pharm. Res. 34 (7) (2017) 1339–1363. [69] K. Li, H. Luo, L. Huang, H. Luo, X. Zhu, Microsatellite instability: a review of what the oncologist should know, Cancer Cell Int. 20 (2020) 16. [70] X. Zhu, S. Gao, H. Luo, W. Fan, S. Guo, H. Yao, S. Leng, Z. Xu, T. Tao, X. Liu, et al., 9q33.3, a stress-related chromosome region, contributes to reducing lung squamous cell carcinoma risk, J. Thorac. Oncol. 9 (7) (2014) 1041–1047. [71] J. Tang, Z. Xu, L. Huang, H. Luo, X. Zhu, Transcriptional regulation in model organisms: recent progress and clinical implications, Open Biol. 9 (11) (2019) 190183. [72] L.M. Ickenstein, P. Garidel, Lipid-based nanoparticle formulations for small molecules and RNA drugs, Expert Opin. Drug Deliv. 16 (11) (2019) 1205–1226. [73] X. Zhu, M.C.M. Lin, W. Fan, L. Tian, J. Wang, S.S. Ng, M. Wang, H. Kung, D. Li, An intronic polymorphism in GRP78 improves chemotherapeutic prediction in nonsmall cell lung cancer, Chest 141 (6) (2012) 1466–1472. [74] J.A. Kulkarni, P.R. Cullis, R. van der Meel, Lipid nanoparticles enabling gene therapies: from concepts to clinical utility, Nucleic Acid Ther. 28 (3) (2018) 146–157. [75] X. Liu, W. Wang, D. Samarsky, L. Liu, Q. Xu, W. Zhang, G. Zhu, P. Wu, X. Zuo, H. Deng, et al., Tumor-targeted in vivo gene silencing via systemic delivery of cRGD-conjugated siRNA, Nucleic Acids Res. 42 (18) (2014) 11805–11817. [76] H. Nemati, M.H. Ghahramani, R. Faridi-Majidi, B. Izadi, G. Bahrami, S.H. Madani, G. Tavoosidana, Using siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation in psoriasis, J. Control. Release 268 (2017) 259–268. [77] J. Li, Y. He, W. Wang, C. Wu, C. Hong, P.T. Hammond, Polyamine-mediated stoichiometric assembly of ribonucleoproteins for enhanced mRNA delivery, Angew. Chem. Int. Ed. Engl. 56 (44) (2017) 13709–13712. [78] F. Pi, D.W. Binzel, T.J. Lee, Z. Li, M. Sun, P. Rychahou, H. Li, F. Haque, S. Wang, C.M. Croce, et al., Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression, Nat. Nanotechnol. 13 (1) (2018) 82–89. [79] W.M. Usman, T.C. Pham, Y.Y. Kwok, L.T. Vu, V. Ma, B. Peng, Y.S. Chan, L. Wei, S.M. Chin, A. Azad, et al., Efficient RNA drug delivery using red blood cell extracellular vesicles, Nat. Commun. 9 (1) (2018) 2359. [80] C.T. Pham, X. Zhang, A. Lam, M.T. Le, Red blood cell extracellular vesicles as robust carriers of RNA-based therapeutics, Cell Stress. 2 (9) (2018) 239–241. [81] E. Yalcin, G. Kara, E. Celik, F.A. Pinarli, G. Saylam, C. Sucularli, S. Ozturk, E. Yilmaz, O. Bayir, M.H. Korkmaz, et al., Preparation and characterization of novel albumin-sericin nanoparticles as siRNA delivery vehicle for laryngeal cancer treatment, Prep. Biochem. Biotechnol. 49 (7) (2019) 659–670. [82] B. Kim, S. Sun, J.A. Varner, S.B. Howell, E. Ruoslahti, M.J. Sailor, Securing the payload, finding the cell, and avoiding the endosome: peptide-targeted, fusogenic porous silicon nanoparticles for delivery of siRNA, Adv Mater 31 (35) (2019) e1902952. [83] M. Qiu, J. Ouyang, Y. Wei, J. Zhang, Q. Lan, C. Deng, Z. Zhong, Selective cell penetrating peptide-functionalized envelope-type chimeric lipopepsomes boost systemic RNAi therapy for lung tumors, Adv. Healthc. Mater. 8 (16) (2019) e1900500. [84] Z. Zheng, Z. Li, C. Xu, B. Guo, P. Guo, Folate-displaying exosome mediated cytosolic delivery of siRNA avoiding endosome trapping, J. Control. Release 311–312 (2019) 43–49.

15