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. 2017 Jun 12;8:132–143. doi: 10.1016/j.omtn.2017.06.005

Therapeutic miRNA and siRNA: Moving from Bench to Clinic as Next Generation Medicine

Chiranjib Chakraborty 1,2,4,∗∗, Ashish Ranjan Sharma 2,4, Garima Sharma 2, C George Priya Doss 3, Sang-Soo Lee 2,∗
PMCID: PMC5496203  PMID: 28918016

Abstract

In the past few years, therapeutic microRNA (miRNA) and small interfering RNA (siRNA) are some of the most important biopharmaceuticals that are in commercial space as future medicines. This review summarizes the patents of miRNA- and siRNA-based new drugs, and also provides a snapshot about significant biopharmaceutical companies that are investing for the therapeutic development of miRNA and siRNA molecules. An insightful view about individual siRNA and miRNA drugs has been depicted with their present status, which is gaining attention in the therapeutic landscape. The efforts of the biopharmaceuticals are discussed with the status of their preclinical and/or clinical trials. Here, some of the setbacks have been highlighted during the biopharmaceutical development of miRNA and siRNA as individual therapeutics. Finally, a snapshot is illustrated about pharmacokinetics, pharmacodynamics with absorption, distribution, metabolism, and excretion (ADME), which is the fundamental development process of these therapeutics, as well as the delivery system for miRNA- and siRNA-based drugs.

Keywords: miRNA, siRNA, drug development

Introduction

Victor Ambros1 and his groups from Harvard University led to a new milestone in research by discovering microRNAs (miRNAs) in 1993. The first miRNA gene discovered was lin-4, which contains sequences complementary to a repeated sequence element in the 3′ UTR of the lin-14 mRNA from Caenorhabditis elegans.1 Next, lin-4 was considered a worm genetics discovery. However, it was not until the discovery of a second miRNA, let-7, that miRNAs were found to be highly conserved among all groups of animals, including humans.2, 3 This discovery augmented the research and development of numerous miRNAs in C. elegans, Drosophila, and human cell lines, especially in HeLa cells. The increased research on miRNAs leads to studies linking miRNA dysregulation and human disease; such a study was published in 2002. In this work, Calin et al.4 describe two human miRNA genes, mir-15a and mir-16-1, that are downregulated or frequently deleted chronic lymphocytic leukemia (CLL) diseases. Presently, thousands of miRNAs have been found in plants and animals including humans. In 2011, Kozomara and Griffiths-Jones5 recorded that 18,226 miRNAs have been noted in animals, plants, and viruses, and that 1,921 miRNAs were encrypted in the human genome. Significantly, the world’s first miRNA therapeutic, miravirsen, a short locked nucleic acid (LNA) for miR-122, is currently moving toward the market. This drug is currently in phase II clinical trials for the treatment of hepatitis C virus (HCV) infection.6

Small interfering RNA (siRNA) is a potent tool for target-specific gene silencing through RNAi. Gene silencing by RNAi was first observed in 1998 by Craig Mello and his co-researchers in C. elegans.7 For this work, Mello and Fire received the Nobel Prize in 2006.8 Now, Dr. Craig Mello is one of the founders of RXi Pharmaceuticals, a company that develops RNAi compounds. The most important idea behind RXi Pharmaceuticals Corporation is the unique ability of their compounds to be “self-delivering,” meaning that no additional delivery vehicles are needed for specific targeting.9 Currently, “Big Pharma” companies are watching the clinical trial trends for RNAi therapeutics.

To date, approximately 20 clinical trials have been initiated using miRNA- and siRNA-based therapeutics. These drugs are running platforms driven by four leading RNA-therapeutic companies. Only one miRNA therapeutic, the compound SPC3649 (miravirsen), which is an inhibitor of miR-122 developed by Santaris Pharma from Denmark, is entered in a clinical trial. Several other miRNA therapeutics are in the preclinical stage and aiming to enter clinical trials. In contrast, several siRNA-based therapeutics have been introduced into clinical trials. Some researchers believe that miRNAs fall into the category of RNAi-based therapeutics.10 The biogenesis and mechanism of action of miRNAs are quite similar to siRNAs with respect to post-transcriptional gene silencing.11, 12 It has been noted that miRNAs are endogenous short RNAs that combine with Argonaute proteins to regulate gene expression. At the translational level, these two categories of small RNAs are important for the gene regulatory landscape in the present scientific world. miRNAs are known as regulators of endogenous genes.13 In contrast, siRNAs help to maintain genome stability. Both are single-stranded forms and were found to associate with effector associations14 that are recognized as RNA-induced silencing complexes (RISCs).15 However, there are differences between miRNA and siRNA modes of action, because both of these ribonucleic acids act differently. miRNAs mostly use eight nucleotides from their 5′ end to identify target mRNA sequences and utilize their inhibitory activities on the translation process. In contrast, siRNAs use approximately their full lengths to recognize target sequences and mediate cleavage of the target mRNA.16

In this paper, we examine the patent landscape of therapeutic miRNAs and siRNAs. We present insights regarding significant siRNAs and miRNAs in the therapeutic landscape and their useful therapeutic modalities. We also focus on their parent biopharmaceutical companies and the status of their preclinical and/or clinical trials. Some of the bottlenecks involved have been highlighted in the discussion. Finally, we also tried to elucidate the fundamental views on pharmacokinetics, pharmacodynamics, and efficient delivery systems for therapeutic RNAs.

Patent Landscape for Therapeutic miRNAs and siRNAs

The potential importance of patent rights is increasing day by day in the area of innovation. Patent rights are not limited to pharmaceuticals, as it has become a common practice for other innovations as well. It has been noted that newly developed modern biology techniques, such as monoclonal antibodies and rDNA techniques, are subject to patent rights.17 Additionally, biological drug candidates and small-molecule drugs have also been subject to patent rights. This instance resulted in the transfer of academic technologies to industries. The government has provided licenses to industry for several biomolecules. Presently, innovation and patents play a crucial role in biopharmaceuticals, as well as for the biopharmaceutical industry.18 Over the past decade, the number of patents issued to biopharmaceutical companies has increased substantially. Therefore, obtaining patent rights for small RNA-based therapeutics is a significant area. However, the market value that each company can derive from the supporting technologies also depends on their ability to protect the patents with well-licensed purposes. The companies have to keep them as trade secrets to get a competitive advantage in areas that are curtailed for miRNA or RNAi technological success. Laitala-Leinonen19 has reviewed recent patent applications regarding miRNA biology20 and described the patent search through the Delphion patent database, where 1,661 American miRNA-related patent papers were found, as documented by International Patent Classification (IPC) codes. miRNA-related patent documents cover approximately 60 IPC code categories. Nearly 50% of USA patent papers were categorized as relating to some type of medicinal preparation consisting of pharmaceutical compositions that encompass miRNA-modulating compounds or the methods related to miRNA-modulating activity for the treatment of diseases. The researchers also describe a large number of patent filings related to methods for treating cancer, significantly more than any other illness or disorder. In the case of other diseases, less than 10% of the patents were issued for each other disorder. The authors also show that the first miRNA-based patent was published in Europe in 2008.21 However, we found from the Google Scholar database that a patent was granted for miRNA analysis (US 20070092882 A1); this is an American patent. It has been noted that the initial patents for specific miRNAs were filed by several advanced research institutes, universities, or pharmaceutical companies throughout the globe, including in Europe (Max Planck Society for the Advancement of Science, Munich, Germany); the United States (University of Massachusetts, Massachusetts Institute of Technology, and Rockefeller University, New York, NY); Asia (College of Medicine, Pochon Cha University Industry-Academic Cooperation Foundation, Gyeonggi-do, South Korea and Council of Scientific & Industrial Research, India); Western Asia (Rosetta Genomics, Rehovot, Israel), etc. Methods for the functional analysis of miRNA, as well as anti-miRNAs with improved activities and efficacies, have also been patented by companies and universities such as Regulus Therapeutics, Stanford University, etc.21 The US Patent and Trademark Office’s (USPTO’s) Utility Guidelines were published in 2001 for the controversy related to the patenting of gene sequences developed in the late 1990s. The goal of the USPTO’s Utility Guidelines was to propagate patent requirements and satisfy the requirements.22, 23 We performed a search for the total number of patents with the terms “microRNA” and “siRNA” from the USA patent search database (http://patft.uspto.gov/) and European Patent Office database (http://www.epo.org/searching-for-patents.html), and found that more patents have been granted for siRNA compared with miRNAs in both databases (Figure 1). However, we found a difference in the trends between the number of US and European patent filings. We have also performed a search regarding the number of patents with the terms “microRNA” and “siRNA” with different diseases, such as cancer, viral infections, inflammatory disorders, cardiovascular disorders, neurological disorders, ocular disorders, and metabolic disorders. In the US patent database, we found that the highest number of patents was granted for “siRNA and cancer” (6,560 patents), whereas the lowest number of patents was granted for “miRNA and ocular disorder” (17 patent). These filings reveal that there is a problem with miRNA therapeutics with respect to ocular disease; this may be because of problems with nucleic acid delivery systems in eye-related diseases.

Figure 1.

Figure 1

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miRNA and siRNA Patents

(A) Total number of keyword searches (“microRNA” and “siRNA”) performed in both the US patent search database and the European patent office database. (B) Number of patents in “microRNA” and “siRNA” in case of the different diseases. (C) Number of patents in “microRNA” and “siRNA” in event of the different type of cancers.

We performed an analysis on the US patents for different cancers and found that the highest number of patents was granted for “siRNA and breast cancer” (3,284 patents). These findings demonstrate that this is an important research area. In contrast, the lowest number of patents was granted for “miRNA and renal cancer” (109 patents). These searches may assist us in identifying intellectual properties (IPs) that may help us better understand the real potential of miRNA- and siRNA-based products for market uniqueness. Table 1 shows a summary of several important US and European patents related to miRNA and siRNA. This table denotes several important properties of the patents, including the patent application number, content of the patent statement, inventors, and applicant institute.

Table 1.

Some Relevant Issued Patents Related to MicroRNA and siRNA

Patent No. Patent Type Application No. Publication Date Content of the Patent Statement Inventors Applicant
CA2404890 C USA PCT/US2001/010188 November 19, 2013 RNA sequence-specific mediators of RNAi Thomas Tuschl et al. Whitehead Institute for Biomedical Research and seven more
EP 1309726 B1 European EP20010922870 December 2, 2009 RNA sequence-specific mediators of RNAi Thomas Tuschl et al. Whitehead Institute for Biomedical Research and three more
EP1550719 B1 European EP20050002454 December 24, 2008 double-stranded RNA (dsRNA) for inhibition of the expression of a defined gene Roland Kreutzer and Stefan Limmer Alnylam Europe AG
US7056704 B2 USA US 10/832,432 June 6, 2006 RNAi mediating small RNA molecules Thomas Tuschl et al. Max-Planck-Gesellschaft zur Foerderung der Wissenschaften E.V.
US 7750144 B2 USA US 10/912,440 July 6, 2010 mediates silencing of a target gene; lessening the base pair strength between the 5′ end of the first strand and the 3′ end of the second strand of the duplex as compared with the base pair strength Phillip D. Zamore et al. University of Massachusetts
EP1633890 B1 European EP20040753972 October 20, 2010 methods and compositions for enhancing the efficacy and specificity of RNAi Phillip D. Zamore et al. University of Massachusetts
EP1309726 B1 European EP20010922870 December 2, 2009 RNA sequence-specific mediators of RNAi Thomas Tuschl et al. Whitehead Institute for Biomedical Research and three more
US7825230 B2 USA US 11/545,280 November 2, 2010 human microRNA targets in HIV genome and a method of identification thereof Samir Kumar Brahmachari et al. Council of Scientific & Industrial Research
US7683036 B2 USA US 10/909,125 March 23, 2010 oligomeric compounds and compositions for use in modulation of small non-coding RNAs Christine Esau et al. Regulus Therapeutics
US7582744 B2 USA US 11/200,703 September 1, 2009 chemically modified oligonucleotides Muthiah Manoharan et al. Alnylam Pharmaceuticals
US 7592441 B2 USA US 11/418,875 September 22, 2009 miRNA for diagnosis, prognosis, and treatment of liver cancer Itzhak Bentwich et al. Rosetta Genomics
US7642348 B2 USA US 11/429,720 January 5, 2010 miRNA for diagnosis, prognosis, and treatment of prostate cancer; linear amplification and labeling for hybridization techniques like Luminex and microarray analysis Itzhak Bentwich et al. Rosetta Genomics
US 7825229 B2 USA US 11/418,870 November 2, 2010 miRNAs; diagnosis, prognosis, and treatments; drug screening; linear amplification and labeling for hybridization techniques like Luminex and microarray analysis; gene expression inhibition Itzhak Bentwich et al. Rosetta Genomics
US7635563 B2 USA US 11/171,175 December 22, 2009 method for identifying miRNA expression (microarrays; RT-PCR) H. Robert Horvitz et al. Massachusetts Institute of Technology
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Biopharmaceutical Industry and Therapeutic miRNAs and siRNAs

The biotechnology industry is currently flourishing. It has been noted that US biotechnology industry revenues have increased from $20 billion in 1996 to $70.1 billion in 2008. In contrast, biotechnology research and development (R&D) expenditures in industry increased from $10.8 billion to $30.4 billion.24 The biopharmaceutical industry plays a major role in the US biotechnology industry. Biopharmaceutical industries are investing in the development of therapeutic miRNAs and siRNAs. Several biopharmaceutical industries, such as Alnylam Pharmaceuticals, Rosetta Genomics, and Regulus Therapeutics, were established during the last 25 years (Figure 2). These biopharma companies are investing in the development of miRNA- and siRNA-based therapeutic molecules. However, there is a challenge for small biotechnology companies because there is some financial volatility in this area.25, 26 Big Pharma is using small companies to develop molecules for R&D to clinical trials. Big Pharma is investing in this new area to enter into the market with new therapeutic miRNA and siRNA molecules as early as possible.

Figure 2.

Figure 2

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List of Biopharmaceutical Companies Involved in miRNA and siRNA Therapeutics

Different significant biopharmaceutical companies with their year of establishment since 1983 to 2017 that are involved in the development of the therapeutic miRNA and siRNA molecules.

Significant siRNA Therapeutics in Clinical Trials and Their Biopharmaceutical Companies

It is well established that siRNAs are therapeutic for gene silencing.27 Therefore, nucleic-acid-based biopharmaceuticals are entering the market with the help of new biopharmaceutical companies. It has been noted that more than 14 RNAi therapeutic programs have entered clinical trials in the past decade (Figure 3, top left). The NIH in the United States has developed a database regarding the molecules that have completed clinical trials or are in ongoing clinical trials (https://www.clinicaltrials.gov). Seven out of 14 RNAi therapeutics are for localized and topical use (4 for the eye, 2 for the respiratory tract, and 1 for the skin). Approximately 1,500 patients and healthy volunteers have been enrolled in RNAi clinical programs that use siRNA therapeutics.28 We will discuss some of the important therapeutic siRNAs with ongoing clinical trials below. Snapshots of these therapeutics are presented in Table 2.

Figure 3.

Figure 3

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Status of Various miRNA and siRNA Therapeutics under Clinical Trials

(Top left panel) Different siRNA molecules that are in a clinical trial and their status of clinical trial. (Top right panel) Different miRNAs molecules that are in preclinical and/or clinical trial and their status of clinical trial. (Bottom panel) Different important issues related to ADME during development of miRNA- and siRNA-based therapeutics molecules.

Table 2.

Significant Therapeutics siRNAs That Are in the Development Phase, Their Indication, and Their Biopharmaceutical Company

Therapeutic siRNAs Indication Target Pharmaceutical Company Remarks
ALN-RSV01 treatment of respiratory syncytial virus (RSV) infection during lung transplantation RSV nucleocapsid Alnylam Pharmaceuticals phase IIb clinical trial
ALN-TTR02 treatment of transthyretin-mediated amyloidosis transthyretin (TTR) Alnylam Pharmaceuticals phase III APOLLO study
PF-04523655 (formerly known as RTP-801i) treatment of age-related macular degeneration and diabetic macular edema HIF-1-responsive gene, RTP801 Quark Pharmaceuticals phase II wet AMD with 0.5 mg of Lucentis given by intravitreal injection with 3 mg of PF-04523655 given every 2 weeks from week 4
QPI-1002 for the avoidance of AKI following primary cardiovascular surgery as well as for the prophylaxis of delayed graft function (DGF) following deceased donor renal transplantation p53 Quark Pharmaceuticals granted Orphan drug designation; in phase II study with randomized 1:1 to receive 10 mg/kg single-bolus intravenous (i.v.) dose of QPI-1002
Excellair for the treatment of inflammatory disorders like asthma spleen tyrosine kinase (Syk) gene Zabecor Pharmaceuticals phase II clinical trial for asthma
ALN-VSP for the treatment of liver cancer VEGF gene, kinesin spindle (KSP) protein gene Alnylam Pharmaceuticals completed phase I extension study
CALAA-01 to inhibit tumor and cancer therapy M2 subunit of ribonucleotide reductase (RRM2) gene Calando Pharmaceuticals phase 1b clinical trial; delivery system is into a nanoparticle <100 nm in diameter
Atu-027 for the treatment of advanced solid tumors protein kinase N3 gene Silence Therapeutics phase I clinical trial
PF-655 (formerly REDD14NP and RTP801i) for the treatment of age-related macular degeneration RTP801 gene Quark Pharm phase II clinical trial
QPI-1007 treatment of nonarteritic anterior ischemic optic neuropathy caspase-2 gene Quark Pharm
AGN211745 treatment of age-related macular degeneration VEGFRI gene Sirna Therapeutics phase II clinical trial with 164 subjects
ApoB SNALP for the treatment of hypercholesterolemia apolipoprotein B gene Tekmira Pharmaceuticals phase I clinical trial concluded
RXI-109 for the treatment of fibrosis or scarring of the skin at a post-surgical wound site or the prevention of dermal scarring for the management of proliferative vitro retinopathy (PVR) and other ocular disorders connective tissue growth factor (CTGF) gene RXi Pharmaceuticals phase 1 clinical trial
SYL040012 ocular hypertension β2-adrenergic receptor gene Sirna Therapeutics phase II trial completed
Bevasiranib treatment of AMD or diabetic macular edema VEGF gene OPKO Health phase III clinical trial, bevasiranib’s clinical trial was terminated
AGN21174 age-related macular degeneration VEGFR1 gene Allergan terminated in phase II
SPC2996 chronic lymphocytic leukemia Bcl-2 gene Santaris Pharma completed phase II trial
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ALN-RSV01 and ALN-TTR from Alnylam Pharmaceuticals

Alnylam Pharmaceuticals has strategic alliances with two pharmaceutical companies (Cubist Pharmaceuticals and Kyowa Hakko Kirin) and has initiated siRNA therapeutic (molecular name: ALN-RSV01) human clinical trials for the treatment of respiratory syncytial virus (RSV) infection during lung transplantation. ALN-RSV01 is a naked and unchanged siRNA that targets the conserved N protein in the RSV genome. This molecule completed its phase IIb trial. The results documented the safety and tolerability of inhaled ALN-RSV01 in naturally infected patients (http://www.alnylam.com/capella/presentations/complete-results-of-our-aln-rsv01-Phase-IIb-study/). The safety and tolerability study involved 101 healthy adults (65 active, 36 placebo-controlled) with doses ranging up to 150 mg as a single dose with five daily doses.29

ALN-AAT is another molecule developed against alpha-1-antitrypsin (AAT)-mediated liver diseases that is currently in phase 1/2 of clinical trials. AAT insufficiency is frequently assessed as liver disease in patients.30 This pharmaceutical company is developing another siRNA-based therapeutic called ALN-TTR. ALN-TTR is a targeting molecule for transthyretin-mediated amyloidosis for polyneuropathy. This molecule is in clinical trials sponsored by Alnylam Pharmaceuticals. The licensed program includes ALN-TTR02, which is in a phase III APOLLO clinical trial. Alnylam will retain rights to develop a program in the United States, Europe, and the rest of the world.31

PF-04523655 from Quark Pharmaceuticals

Quark Pharmaceuticals made strategic alliances with Pfizer for the further development of PF-04523655. Pfizer invested $145 million in the formulation of the compound. The companies have completed a phase I dose-related study for this molecule in human subjects with choroidal neovascularization (CNV), as well as minor age-related macular degeneration (wet AMD).32 They are currently performing phase II clinical trial as an open-label multi-center CNV study. These companies are also conducting a clinical trial for a different indication for diabetic macular edema. In this clinical trial, they are performing a multi-center, randomized, comparator study evaluating the efficacy and safety of the molecule versus laser treatments in subjects with diabetic macular edema.33

QPI-1002 and Quark Pharmaceuticals

QPI-1002, a synthetic siRNA, can inhibit the expression of the pro-apoptotic protein p53. This drug is being developed to prevent acute kidney injury (AKI) following primary cardiovascular surgery, as well as for prophylaxis of the delayed graft function (DGF) after deceased donor renal transplantation.34 It has been noted that AKI is a clinically overwhelming disease that leads to approximately 5% of hospital admissions, and within 30 days, the mortality rate has been recorded to be more than 50% after the onset of AKI after surgery.35 DGF is also one of the most universal complications in the immediate period after renal transplantation, affecting 25%–40% of deceased donor renal transplant patients.36 It is currently in a phase II clinical trial by Quark Pharmaceuticals. The QPI-1002 molecule has been granted orphan drug designation for prophylaxis of DGF in kidney transplantation by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA). However, in August 2010, Quark signed an agreement with Novartis for the exclusive license of this molecule for all indications.

Excellair and Zabecor Pharmaceuticals

Excellair is a designed siRNA that has anti-inflammatory properties. This siRNA targets and silences the Syk kinase gene, a critical gene linked to inflammation. This drug has shown potential and security in preclinical studies for the management of asthma and other inflammatory disorders.37 This molecule has completed a phase II clinical trial.

ALN-VSP and Alnylam Pharmaceuticals

The molecule ALN-VSP comprises two siRNAs. This molecule is a systemically delivered RNAi therapeutic that targets two genes, VEGF and KSP. It is used for the management of advanced solid liver tumors (mainly primary and secondary liver cancers).38 Lipid nanoparticles (LNPs) can efficiently deliver siRNAs to cellular targets. Alnylam Pharmaceuticals, an important RNAi therapeutics company, has completed a phase I clinical trial with this drug.

ALN-RSV is currently one of the most advanced siRNA programs in the world that uses an original siRNA formulated in a saline environment to target the RSV N gene. It completed a phase I trial (two intranasal and one inhalational) to prove its safety and tolerability at doses up to 3 mg/kg. A double-blind, randomized, placebo-controlled study using 88 patients was shown to reduce the occurrence of upper respiratory tract infection with RSV upon intranasal ALN-RSV treatment, thus showing its safety and efficacy.29

CALAA-01 and Calando Pharmaceuticals

The targeted therapeutic molecule CALAA-01 is a tumor inhibitor that targets the M2 subunit of ribonucleotide reductase (RRM2). RRM2 protein is involved in DNA replication and is an essential protein for completing cell division. It has been noted that RRM2 regulates Bcl-2 in different cancers, especially head, neck, and lung cancers, and plays an active role in tumor progression. Therefore, RRM2 is a potential target for cancer therapy.39 An anti-RRM2 siRNA sequence was developed that exhibits significant anti-proliferative activity in human, mouse, rat, and monkey cancer cells.40 Nanoparticles containing anti-RRM2 siRNA sequence were evaluated in vivo for targeted delivery. The nanoparticles contain a synthetic delivery arrangement that uses a linear, cyclodextrin-containing polycation, transferrin, and siRNA.41 The Investigational New Drug (IND) application was submitted to the US FDA, and Calando Pharmaceuticals received approval in 2008 to initiate a phase I clinical trial for this siRNA-based drug in patients with solid tumors. The provisional phase I clinical trial study data were published in 2010.42, 43 This siRNA therapeutic is currently a significant drug in cancer programs.

Atu-027 and Silence Therapeutics

Atu027 contains siRNA combined with a lipoplex delivery system and displays RNAi-mediated suppression of protein kinase N3 (PKN3) gene expression in vascular endothelial cells. Silence Therapeutics is conducting a human phase I study for therapeutic Atu027 in patients with advanced cancer.44 The PKN3 target gene is a critical factor for cancer progression and metastasis. A phase Ib/IIa study for Atu027 in combination with gemcitabine was completed after the lead-in safety period.45

ApoB SNALP and Tekmira Pharmaceuticals

Apolipoprotein B (ApoB) stable nucleic acid lipid particles (SNALPs) are designed for the treatment of elevated low-density lipoprotein (LDL) cholesterol or “bad” cholesterol (hypercholesterolemia). ApoB-specific siRNAs were encoded in SNALPs.46 Tekmira Pharmaceuticals initiated a phase 1 clinical trial by enrolling 23 adult volunteers with mild hypercholesterolemia. Among them, 17 were exposed to ApoB SNALP, while the rest received a placebo.28 They further concluded the program in 2010 and mentioned that ApoB SNALP were well tolerated among the majority of patients with no liver toxicity; however, in some cases, immune stimulation was observed.

RXI-109 and RXi Pharmaceuticals

RXI-109 is an siRNA (self-delivering RNAi compound [sd-rxRNA]) that acquired FDA clearance to start clinical trials. Its phase I clinical trial started in June 2012 for the treatment of fibrosis or scarring of the skin at post-surgical wound sites or for dermal scarring prevention.47 It is used to manage proliferative vitro retinopathy (PVR) and other ocular disorders.48

Setbacks to siRNA Therapeutics Development

There has been a delay in clinical trials with siRNA therapeutics, because two clinical trials have been withdrawn because of poor performance.

Bevasiranib and OPKO Health

Bevasiranib is a so-called siRNA that can control vascular endothelial growth factor (VEGF) gene expression; this molecule is a naked siRNA. OPKO Health is a multi-national pharmaceutical company that has selected bevasiranib for the treatment of AMD or diabetic macular edema.49, 50

At first, it was expected that the drug would enter the market very quickly. The bevasiranib phase III clinical trial was terminated in 2009 because of poor performance. In the initial phase III trial called the COBALT study, a combination therapy study was examined. This phase III trial was also canceled because of unsatisfactory performance. However, further phase III clinical studies with other regimens using bevasiranib are in preparation.

AGN21174 and Allergan

During the development of AGN211745, a clinical study program was conducted by Allergan in collaboration with Sirna Therapeutics. The molecule targets VEGF receptor I (VEGFRI) for treating AMD.51 Unfortunately, AGN211745 was also terminated in phase II for those specific indications.

After reports from the independent data monitoring committee, these two studies were terminated ahead of time before reaching their endpoints. Even though the safety of the molecules was acceptable, the exiting of these molecules from their clinical trials was a major setback for the siRNA-based therapeutic industry.

Significant miRNA Therapeutics in Preclinical and/or Clinical Trials and Their Biopharmaceutical Companies

miRNA-based therapeutics have entered in the past few years and are currently entering clinical trials (Figure 3, top right). Newer companies have established miRNA therapeutics for particular indications. These companies include miRagen Therapeutics, Regulus Therapeutics, and Mirna Therapeutics. Snapshots of these therapeutics are presented in Table 3.

Table 3.

Significant Therapeutics miRNA that Are in the Development Phase, Their Indication, and Their Biopharmaceutical Company

Therapeutic miRNAs Indication Biopharmaceutical Company Remarks
Miravirsen the indication of the hepatitis C virus (HCV) infection Santaris Pharma phase IIa clinical trial
MRX34 for the treatment of a variety of cancers such as colon cancer, non-small-cell lung cancer (NSCLC), hepatocellular carcinoma, cervical cancer, ovarian cancer, etc. Mirna Therapeutics phase 1 clinical trial halted because of immune responses
RG-101 for the treatment of HCV Regulus Therapeutics an owned GalNAc-conjugated anti-miR
RG-012 for the treatment of Alport syndrome Regulus Therapeutics in the pipeline to initiate clinical trial phase II
MGN-1374 for the treatment of post-myocardial infarction remodeling miRagen Therapeutics targets miR-15 and miR-195; it is in the preclinical stage
MGN-2677 for the treatment of vascular disease miRagen Therapeutics targets miR-143/145; it is in the pipeline
MGN-4220 for the treatment of cardiac fibrosis miRagen Therapeutics targets miR-29; it is in the pipeline
MGN-4893 for the treatment of disorders like abnormal red blood cell production such as polycythemia vera miRagen Therapeutics targets miR-451; it is in the pipeline
MGN-5804 for the treatment of cardiometabolic disease miRagen Therapeutics targets miR-378; it is in the pipeline
MGN-6114 for the treatment of peripheral arterial disease miRagen Therapeutics targets miR-92; it is in the pipeline
MGN-9103 for the treatment of chronic heart failure miRagen Therapeutics targets miR-208; it is in the pipeline
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Miravirsen and Santaris Pharma

Miravirsen is an LNA-modified DNA phosphorothioate antisense oligonucleotide (ASO) that inhibits miR-122.52, 53 This is the first miRNA-targeted drug to enter phase II clinical trials to understand its safety and tolerability in the patients; this drug is sponsored by Santaris Pharma (Roche Innovation Centre). The clinical trials were conducted for its use against HCV infection. In addition to the United States, the phase IIa clinical trial is also being undertaken in several other countries, including the Netherlands, Germany, Poland, Romania, and Slovakia. The study is investigating miravirsen in combination with telaprevir and ribavirin in null responders to pegylated interferon and ribavirin.

MRX34 and miRNA Therapeutics

Several miRNAs can perform as tumor-suppressor genes in human cancers, including miR-34.54, 55 MRX34 delivers an imitation miRNA to miR-34 that performs as a tumor suppressor. This miRNA is lost or repressed in patient tumors.56 This drug can be used with an extensive variety of cancers, such as colon cancer, non-small-cell lung cancer (NSCLC), hepatocellular carcinoma, cervical cancer, and ovarian cancer. Mirna Therapeutics is the biopharmaceutical company that is performing the MRX34 phase 1 clinical trial on liver-based cancers. This clinical study was halted in 2016 because of multiple immune-related severe adverse events (SAEs).

RG-101, RG-012, and Regulus Therapeutics

It has been noted that miRNA-122 is a liver-related miRNA.57 It has also been shown that miR-122 is an important target for HCV infection therapy.58 Regulus Therapeutics is developing RG-101, an N-acetylgalactosamine (GalNAc)-conjugated anti-miR that targets miRNA-122 to treat HCV.

miR-21 plays an important role in fibrogenic diseases in different organs, including the kidneys. Therefore, anti-miRNA-21 oligonucleotides can prevent Alport nephropathy.59 RG-012, an anti-miR targeting miRNA-21, is a Regulus Therapeutics molecule in the clinical trial pipeline for the treatment of Alport syndrome. This disease is a life-threatening genetic kidney disease that does not currently have an approved therapy.60

Anti-miRs and miRagen Therapeutics

miRagen Therapeutics is a USA-based biopharmaceutical company that is developing innovative RNA-targeting therapies with a particular emphasis on miRNAs for unmet human health needs. miRagen Therapeutics is developing several miRNA-based therapeutics, including MGN-1374, MGN-2677, MGN-4220, MGN-4893, MGN-5804, MGN-6114, MGN-8107, and MGN-9103.

MGN-9103 was suggested to play useful roles in diabetes and obesity. Obesity, type 2 diabetes, and heart failure are associated with aberrant cardiac metabolism. It has been noted that the cardiac-specific miR-208 target gene MED13 provides resistance to high-fat-diet-induced obesity and produces insulin sensitivity and glucose tolerance in mice.61 MGN-9103 is an LNA-modified ASOs.62, 63 DNA-like nucleotides lacking contiguous stretch of DNA are also called ASOs.64 Such ASOs are capable of blocking gene expression by binding to RNA and are also known as “steric blockers.” miR-208 is used for the treatment of persistent heart failure and is located in an alphaMHC gene intron. It is a heart-specific miRNA that is required for cardiac hypertrophy, fibrosis, and myosin switching.63, 65

Another miRagen Therapeutics lead drug is MGN-4893, which targets miR-451. miR-451 is required for red blood cell expansion. Inhibition of miR-451 in mice, using anti-miR-451, blocked erythrocyte differentiation, which was helpful for the treatment of disorders relevant to abnormal red blood cell production.66, 67, 68 Another significant miRagen Therapeutics drug is MGN-1374, an LNA-modified ASO for myocardial infarction. It is an 8-mer LNA oligonucleotide that targets the miR-15 family seed region.

ADME for Therapeutic miRNAs and siRNAs

ADME denotes the absorption, distribution, metabolism, and excretion of a new chemical entity (NCE) molecule.69, 70 Any new pharmaceutical compound that is administered in the human body undergoes pharmacokinetics and pharmacodynamics. Pharmacokinetics illustrates how the body affects a particular drug after taking it through the method of absorption and distribution. The ADME of drugs occurs through drug-metabolizing enzymes, as well as different drug transporters expressed in various tissues such as the small intestine, liver, and kidney. Drug-metabolizing enzymes, such as cytochrome P450 (CYP or P450) isoforms, play crucial roles in drug metabolism.71 Drug transporters, including ABC and SLC transporters, also play key roles in drug absorption, distribution, and excretion.72 However, several important issues related to ADME have been observed by biopharmaceutical companies during the development of miRNA- and siRNA-based therapeutics (Figure 3, bottom).

In the majority of the cases, polyanionic molecules are used to develop anti-miRs because they are extremely water-soluble. Another important point is that these molecules have small molecular mass ranging from 2 to 6 kDa. These miRNA-based NCEs are weak candidates for oral administration, and their intestinal absorption is low.73 Even with the application of oral enhancers, intestinal absorption of anti-miRs is not up to the mark. Therefore, anti-miR oligonucleotides are currently administered using a parenteral route. The two types of parenteral administration routes that are currently being used are intravenous and subcutaneous injections, as well as infusions.20, 74 It is currently easier to make an anti-miR aqueous solution because of its water solubility. However, more studies focused on ADME intravenous and subcutaneous injections, as well as infusions, are necessary. A better delivery system that can provide long biological half-lives is required; thus, more research is needed in these directions. With longer biological half-lives, it may be possible to reduce the frequency of injections so that anti-miRs can be administered more frequently.

Levin75 described that LNA-modified DNA/phosphorothioate (PS) oligonucleotide backbone modifications improve the pharmacokinetic properties of antisense LNA-modified DNA/PS oligonucleotides. Different studies have reported efficient LNA-modified anti-miRs that harbor complete PS backbones. miR-122 silencing uses a high-affinity 15-nt LNA/DNA PS oligonucleotide.76, 77 Saline-formulated LNA-anti-miR-122 delivery uses intraperitoneal (i.p.) or intravenous (i.v.) injection methods to treat mice. This compound has been efficiently taken up by the liver.78, 79

In other study, Elmén et al.76, 77 used single LNA-anti-miR i.p. injections with doses ranging from 1 to 200 mg/kg in a dose-dependent study to show decreased serum cholesterol in mice. In the same survey, administration of PBS-formulated LNA-anti-miR to African green monkeys with doses ranging from 1 to 10 mg/kg shows accumulation of the LNA-anti-miR compound in the liver.76, 77 The same research group also showed that PCSK9 LNA ASOs induced a significant reduction in LDL cholesterol in nonhuman primates.80 These results show that i.p. injections of LNA-anti-miR compounds were well accepted in both mice and primates. Therefore, the parenteral route of administration for miRNA-based new chemical entities (NCEs) may provide better pharmacokinetics and pharmacodynamics.

Efficient Delivery Systems for Therapeutic miRNAs and siRNAs Can Reduce the Effective Dose

Delivering a therapeutic miRNA and siRNA to its target tissue is a challenging task. The drug molecule enters the cell through the membrane and eventually comes into the cytoplasm.81 RNA-based therapeutics show poor pharmacological properties, such as off-targeting, low serum stability, and innate immune responses, which cause significant challenges for clinical applications.82, 83

Studies have shown that local or topical delivery (eye, skin) of therapeutic RNAs displayed better bioavailability in target tissues than non-target tumor tissues.

Different delivery systems are used for better bioavailability, including PEGylated liposomes, lipidoids, and biodegradable polymers. Vesicles with diameters between 50 and 500 nm have been used to deliver therapeutic miRNAs and siRNAs. These vesicles prevent the drugs from being filtered by the kidneys and improve intracellular delivery.81, 82, 84

Liposomes contain lipid bilayers with an aqueous core that can contain therapeutic miRNA and siRNA molecules.16 Lipoplexes are a liposome-based delivery system that contains cationic lipids; this delivery system allows negatively charged therapeutic miRNA and siRNA molecules to traverse the lipid bilayer. The negatively charged miRNA and siRNA molecules and their hydrophilicity are balanced by the cationic lipids.81, 85, 86

The resulting net positive charge facilitates the liposomes to bind to anionic cell surface molecules. The composition of these lipid particles is modified to promote fusion with cytoplasmic, nuclear, and endosomal membranes to support endosomal release within the cell. This can also solve the problem of pH sensitivity for the therapeutic molecules. In the end, liposomes intermingled with anionic phospholipids in the endosome produce non-bilayer structures that disrupt the endosomal membrane and liberate the miRNA and siRNA molecules.

It is extremely important to develop new in vivo delivery systems for miRNA and siRNA molecules that can target specific cells and tissues. Liposomal encapsulation technology has improved the half-life of these therapeutic RNAs in human blood. Diverse miRNA and siRNA molecule carriers have been suggested and developed differently that may have better stabilities and efficiencies with therapeutic RNA delivery systems. However, pharmaceutical companies need to apply these delivery systems to promote more efficient delivery of commercial therapeutic RNAs.

Future Prospects

RNA-based therapeutics has demonstrated great promise for the treatment of different diseases and is still evolving. The main obstruction linked with the clinical application of RNA-based therapeutics targeting strategies is determining how to accurately deliver the therapeutic agents into the targeted cells. Recent efficient delivery systems such as nanoparticle-based delivery systems may reduce doses, which will be beneficial for treatment of different diseases including cancer. Engineered nanoparticles are specially used for delivery to specific cells, which will help to achieve this goal. It will also help the co-delivery approach of RNA-based therapeutics with anticancer drugs. In addition, synchronized delivery of RNA-based therapeutics and chemotherapy agents to the tumor cells is highly challenging. RNA-based therapeutics combined with conventional chemotherapy agents might offer a new approach to treat malignant tumors in the near future and will ultimately help to bring the RNA-based therapeutics amalgamation therapy to the clinic for the treatment of patients.

Conclusions

The discovery of therapeutic miRNA and siRNA is considered one of the most exhilarating and significant therapeutic breakthroughs in pharmaceutical development, IP rights (IPRs), and business points of view. The development of therapeutic miRNAs and siRNAs is progressing at a quick pace. However, pharmaceutical companies working with therapeutic miRNAs and siRNAs are somewhat different from those focusing on NCE molecules. This is primarily due to the technical differences between these two kinds of molecules. RNA-based drug molecules are more similar to traditional gene therapy. Soon, there will be no specific applications or efficacy guidelines for miRNAs and siRNAs, which will allow researchers and companies to utilize this important technology to solve real-world problems.

It is likely that there may be product failure during the development of drug molecules, which can be attributed to a number of factors including safety, efficacy, target selection, and delivery technologies. These and other factors, such as clinical trial design and commercial considerations, will require optimization to produce successful drugs.

Nevertheless, in the near future, RNA-based therapeutics will overcome these obstacles, and therapeutic miRNAs and siRNAs will enter into the clinic as next generation drugs. This group of therapeutics definitely has the potential to contribute significantly to the future of medicine.

Author Contributions

C.C. and S.-S.L. designed the manuscript, and C.C. and A.R.S. wrote the manuscript. G.S. assisted in the design of the manuscript and prepared the figures. C.G.P.D. and S.-S.L. edited the manuscript.

Conflicts of Interest

The authors declare that they have no competing interests.

Acknowledgments

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) under the Ministry of Education (grant NRF-2014R1A1A4A03009388) and by a grant of the Korea Health Technology R&D project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant HI12C1265). We acknowledge our colleagues for useful discussion and comments on this manuscript.

Contributor Information

Chiranjib Chakraborty, Email: drchiranjib@yahoo.com.

Sang-Soo Lee, Email: 123sslee@gmail.com.

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