Kaczmarek et al. Genome Medicine (2017) 9:60
`DOI 10.1186/s13073-017-0450-0
`
`R E V I E W
`Advances in the delivery of RNA
`therapeutics: from concept to clinical reality
`James C. Kaczmarek1,2†, Piotr S. Kowalski2† and Daniel G. Anderson1,2,3,4*
`
`Open Access
`
`Abstract
`
`The rapid expansion of the available genomic data continues to greatly impact biomedical science and medicine.
`Fulfilling the clinical potential of genetic discoveries requires the development of therapeutics that can specifically
`modulate the expression of disease-relevant genes. RNA-based drugs, including short interfering RNAs and antisense
`oligonucleotides, are particularly promising examples of this newer class of biologics. For over two decades, researchers
`have been trying to overcome major challenges for utilizing such RNAs in a therapeutic context, including intracellular
`delivery, stability, and immune response activation. This research is finally beginning to bear fruit as the first RNA drugs
`gain FDA approval and more advance to the final phases of clinical trials. Furthermore, the recent advent of CRISPR,
`an RNA-guided gene-editing technology, as well as new strides in the delivery of messenger RNA transcribed in vitro,
`have triggered a major expansion of the RNA-therapeutics field. In this review, we discuss the challenges for clinical
`translation of RNA-based therapeutics, with an emphasis on recent advances in delivery technologies, and present an
`overview of the applications of RNA-based drugs for modulation of gene/protein expression and genome editing that
`are currently being investigated both in the laboratory as well as in the clinic.
`Keywords: Antisense oligonucleotide, Clinical trial, CRISPR, Gene editing, Gene therapy, Messenger RNA delivery, mRNA
`vaccine, RNA nanoparticle, Short interfering RNA delivery
`
`Background
`Fourteen years after the completion of the human gen-
`ome project, our understanding of human genomics
`continues to develop at an unprecedented rate. Thanks
`to advances in next-generation sequencing technology,
`scientists have been able to identify the genetic roots of
`many common diseases [1]. Diseases such as cancer [2],
`Parkinson’s [3], rheumatoid arthritis [4], and Alzheimer’s
`[5] have all had many of their genetic components re-
`vealed, bringing us closer than ever to ‘personalized
`medicine’ [6]. Thus far, this knowledge has been well
`adapted for diagnostic use—but has not yet been fully
`translated to pharmaceutical
`interventions addressing
`the genetic defects underlying diseases. Currently, the
`two major structural classes of FDA-approved drugs are
`small molecules and proteins [7]. Small-molecule drugs,
`
`* Correspondence: dgander@mit.edu
`†Equal contributors
`1Department of Chemical Engineering, Massachusetts Institute of
`Technology, Cambridge, Massachusetts 02139, USA
`2David H. Koch Institute for Integrative Cancer Research, Massachusetts
`Institute of Technology, Cambridge, Massachusetts 02139, USA
`Full list of author information is available at the end of the article
`
`which consist predominantly of hydrophobic organic
`compounds, typically act by deactivating or inhibiting
`target proteins through competitive binding. However,
`the proteins that might possess such binding pockets
`have been estimated to account for only 2–5% of the
`protein-coding human genome [8]. Protein-based drugs
`(e.g., antibodies), by contrast, can bind with high specifi-
`city to a variety of targets or be used to replace mutated
`or missing proteins (e.g., delivering insulin for diabetes).
`However, the size and stability of proteins limit their
`utility towards many potential disease targets [7]. Thus,
`true realization of the therapeutic potential of personal-
`ized genomics requires treatments beyond those offered
`by current small-molecule and protein therapies.
`In summary, both protein and small-molecule drugs
`are limited in that they cannot target every disease-
`relevant protein or gene. The mRNA and DNA precur-
`sors of proteins, however, are promising therapeutically
`in that they can be specifically targeted via Watson–
`Crick base pairing and,
`in the case of gene editing,
`which aims to permanently change the host’s DNA, rep-
`resent an avenue to cure a genetic defect as opposed to
`
`© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
`International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
`reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
`the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
`(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
`
`Alnylam Exh. 1029
`
`

`

`Kaczmarek et al. Genome Medicine (2017) 9:60
`
`Page 2 of 16
`
`just treating it. Over the past few decades, RNA drugs
`have emerged as candidates to address diseases at the
`gene and RNA levels. Although it has been known since
`1990 that nucleic acids can be used to modulate protein
`production in vivo [9], therapeutic RNA delivery has
`been limited by a number of
`factors. Naked, single-
`stranded RNA is prone to nuclease degradation, can
`activate the immune system, and is too large and nega-
`tively charged to passively cross the cell membrane—and
`must, therefore, be provided with additional means of
`cellular entry and escape from endosomes, which trans-
`port extracellular nanoparticles into the cytoplasm [10].
`As such, the nucleic acid delivery field has centered on
`the design of delivery methods and materials that will
`transport RNA drugs to the site of
`interest. In this
`review, we provide an overview of the current status of
`advances in RNA and RNA–protein therapy, with an
`emphasis on materials that have been developed for
`RNA delivery and applications of RNA-based drugs for
`the modulation of gene/protein expression and gene
`editing.
`
`Delivery materials and chemical modifications for
`RNA
`Delivery materials
`Broadly speaking, RNA delivery can be mediated by viral
`and non-viral vectors. For viral RNA delivery, there has
`been a great deal of
`interest
`in engineering adeno-
`associated viruses to carry nucleic acid cargo [11]—how-
`ever, this section will focus mainly on the development
`of non-viral materials (Table 1). Of the non-viral RNA
`delivery vehicles, nanoparticles are perhaps the most
`studied. Nanoparticle encapsulation of RNA physically
`protects nucleic acids from degradation and, depending
`on the specific chemistry, can aid in cellular uptake and
`endosomal escape. Given their high degree of chemical
`flexibility, polymers are commonly used materials for
`nanoparticle-based delivery [12]. Typically, cationic poly-
`mers are used to electrostatically condense the nega-
`tively charged RNA into nanoparticles (Fig. 1a) [13].
`
`Table 1 Comparison of clinically relevant RNA delivery platforms
`Delivery vehicle Type of RNA in clinical trials Advantages
`Naked RNA
`siRNA, ASO, mRNA
`No additional materials or
`synthesis required
`
`Nanoparticle
`
`siRNA, ASO, mRNA
`
`Increased half life
`Protection from nucleases
`Aids in endocytosis and
`endosomal escape
`
`Conjugate
`
`siRNA, ASO
`
`Defined chemical structure
`Ability to target specific receptors
`Limited toxicity due to lack of
`excipient materials
`ASO antisense oligonucleotide, siRNA short interfering RNA
`
`These positively charged groups often consist of amines
`that become protonated at physiological pH (pKa ~7.4),
`thought to lead to an ion imbalance that results in endo-
`somal rupture [14, 15], although this so-called ‘proton
`sponge’ hypothesis has yet
`to be rigorously demon-
`strated for various materials [16]. Regardless of the exact
`mechanism by which polymers aid in RNA delivery,
`commercially available amine-containing polymers were
`some of the earliest non-viral materials adopted for
`nucleic acid delivery. Synthetic polymers such as poly-L-
`lysine [17], polyamidoamine [18], and polyethyleneimine
`[19], as well as naturally occurring polymers such as
`chitosan [20], have all been applied to RNA delivery,
`with varying levels of success. In addition, some investi-
`gators have synthesized polymers specifically for nucleic
`acid delivery. Poly(β-amino esters),
`in particular, have
`gained widespread use in DNA delivery owing to their
`ease of synthesis and biodegradability [21], but have also
`proved to be capable of effecting delivery of short inter-
`fering RNA (siRNA) [22–24] and mRNA [25].
`Lipids and lipid-like materials represent the second
`major class of nanoparticle-based delivery vehicles for
`RNA. As with polymers, cationic lipids are often used to
`electrostatically bind the nucleic acid. Many laboratories,
`however, have started utilizing ionizable lipids, which are
`lipids that are positively charged only at acidic pH. This
`ionizable behavior
`is
`thought
`to enhance efficacy
`through helping with endosomal escape [26] and redu-
`cing toxicity [27] as compared with particles that remain
`cationic at physiological pH. Lipids are also capable of
`self-assembly into well-ordered nanoparticle structures,
`known as lipoplexes (Fig. 1b), driven by a combination
`of electrostatic interactions with RNA and hydrophobic
`interactions [28, 29]. Optimizing the formulation of lipid
`nanoparticles (LNPs) by addition of other hydrophobic
`moieties, such as cholesterol and PEG-lipid, in addition
`to an ionizable/cationic lipid, enhances nanoparticle
`stability and can significantly enhance efficacy of RNA
`delivery [30]. However, similarly to polymers,
`it was
`found that ionizable lipid structure is the main factor
`
`Disadvantages
`Prone to degradation
`Immunogenic
`Difficulty entering cell
`Poor circulation half-life
`
`References
`[63–65, 73–78, 101, 103, 114, 115]
`
`Elevated risk of toxicity with
`introducing excipient materials
`
`[12–37, 58–60, 82–85, 106–108,
`110–113, 131, 145, 156–159]
`
`High doses required
`Dependent on chemical
`modifications for RNA stability
`
`[38–43, 62]
`
`

`

`Kaczmarek et al. Genome Medicine (2017) 9:60
`
`Page 3 of 16
`
`Cholesterol
`
`PEG
`
`O
`
`–
`O
`
`O
`P
`O
`
`N
`
`OH
`
`O
`
`Base
`
`O
`
`Base
`
`O
`
`Base
`
`a
`
`c
`
`b
`
`RNA cargo
`
`Cationic polymer
`
`RNA cargo
`
`Cationic/ionizable lipid
`
`Phospholipid
`
`O
`
`HN
`
`O
`
`O
`
`O
`
`NH
`
`O
`
`O
`
`HN
`
`NH2
`N
`
`O
`
`NH
`
`HN
`
`HN
`
`O
`
`NH
`
`O
`
`HN
`
`O
`
`OH
`
`OH
`
`HO
`
`AcHN
`
`O
`
`AcHN
`HO
`
`OH
`
`OH
`
`OH
`
`O
`
`OH
`
`O
`
`HO
`
`AcHN
`
`d
`
`O
`
`HN
`
`NH
`
`Br
`
`O
`
`NH
`
`O
`HH
`H
`–
`H
`S
`
`H
`O
`
`O P O
`
`Base
`
`O
`
`H
`H
`O
`
`H
`H
`H
`
`O
`
`O
`
`H
`OH
`
`H
`
`H
`OH
`
`O
`
`O
`O
`
`N
`
`O
`
`O
`
`O
`
`H
`
`H
`OH
`
`H
`
`O
`
`H
`
`N
`
`O O
`
`Base
`
`O
`
`O
`
`H
`
`H
`OH
`
`H
`
`O
`
`H
`
`O
`
`H
`
`O
`
`H
`
`H
`
`H
`
`H
`
`O
`
`H
`
`O
`
`H
`
`H
`
`O
`
`H
`
`O
`H H
`HH
`Base
`
`H
`
`O
`
`HN
`
`O
`H H
`H
`H
`H
`O
`
`Pseudouridine
`
`5-Bromo-uridine
`
`5-methylcytidine
`
`2’-Deoxy
`
`2’-OMe
`
`Amide-3 linkage
`
`Thioate linkage
`
`Fig. 1 Common delivery modalities for RNA. a Schematic depicting polymeric nanoparticles comprising RNA and cationic polymer. b Schematic
`depicting lipid nanoparticles containing RNA, a cationic/ionizable lipid, and other hydrophobic moieties (such as cholesterol) commonly used
`in nanoparticle formulation. c Chemical structure of a tertiary conjugate between N-acetylgalactosamine (GalNAc) and RNA that is currently
`in clinical trials [38]. d Examples of base, sugar, and linker modifications that have been utilized to deliver nucleic acids (modified chemistry
`highlighted in blue)
`
`the nanoparticle. As such, one
`affecting efficacy of
`laboratory has pioneered the use of semi-automated
`high-throughput synthesis methods to create libraries of
`chemically diverse lipids and lipid-like materials for
`RNA delivery [31–35], resulting in highly potent nano-
`particles capable of delivering a variety of RNA types to
`both the liver [32, 36, 37] and the lung [33] following
`systemic delivery in vivo.
`As an alternative to nanoparticles, a more conceptually
`straightforward and chemically well-defined means of
`delivery is to directly conjugate a bioactive ligand to the
`RNA that will allow it to enter the cell of
`interest.
`Perhaps the most clinically advanced example of this
`
`technique is the conjugation of N-acetylgalactosamine
`(GalNAc; Fig. 1c), which targets the asialoglycoprotein
`receptor on hepatocytes, to siRNA [38]. Unlike many
`nanoparticles, which are given intravenously, GalNAc
`conjugates are typically dosed subcutaneously and have
`shown an ability to rapidly enter systemic circulation and
`target the liver [39]. Other conjugates, such as cholesterol
`[40], vitamin E [41], antibodies [42], and cell-penetrating
`peptides [43], have been explored in the past, although
`none but the specialized triantennary GalNAc–siRNA
`conjugate has gained any clinical traction (Table 2), sug-
`gesting the need for additional work on the design of con-
`jugates for efficient delivery of nucleic acids.
`
`

`

`Kaczmarek et al. Genome Medicine (2017) 9:60
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`Page 4 of 16
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`I
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`III
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`NCT02243124
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`NCT02144051
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`NCT02900027
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`NCT00100672
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`NCT02564614
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`NCT02406833
`
`NCT01960348
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`NCT02610296
`NCT02610283
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`II/III
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`NCT02341560
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`II
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`II
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`II
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`II
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`II
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`I/II
`/II
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`II
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`/II
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`I/II
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`I/II
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`I/II
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`I/II
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`I
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`I
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`I
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`I
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`I
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`Phase
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`NCT02250612
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`NCT02455999
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`NCT01445899
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`NCT03060577
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`NCT01676259
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`NCT01262235
`NCT02191878
`NCT01437007
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`NCT02314052
`NCT02110563
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`NCT02949830
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`NCT02352493
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`NCT02554773
`
`NCT02706886
`
`NCT02956317
`
`NCT02795325
`
`NCT02227459
`NCT01858935,
`
`NCT00938574
`
`Myelodysplasticsyndrome
`
`Intravenousinfusion
`
`Prostatecancer
`
`Intravenousinfusion
`
`NCT02623699
`
`Amyotrophiclateralsclerosis
`
`Intrathecalinjection
`
`Elevatedtriglycerides
`
`Subcutaneousinjection
`
`Advancedcancer
`
`Intravenousinfusion
`
`Livercancer
`
`Glaucoma
`
`Intravitrealinjection
`
`Familialamyloidpolyneuropathy
`
`Intravenousinfusion
`
`transplantrecipients
`Delayedgraftfunctioninkidney
`Preventionofacutekidneyinjury
`
`opticneuropathy
`Acutenonarteriticanteriorischemic
`
`Glaucoma
`
`Dryeyesyndrome
`
`Intravenousinfusion
`
`Intravitrealinjection
`
`Eyedrops
`
`Eyedrops
`
`Diabeticmacularedema
`
`Intravitrealinjection
`
`Hypercholesterolemia
`
`Subcutaneousinjection
`
`Pancreaticcancer
`
`Localimplantation
`
`Adrenocorticalcarcinoma
`Livercancer
`Livercancer
`
`Intravenousinfusion
`Intravenousinfusion
`Liverinjection
`
`Solidcancer
`
`Intravenousinfusion
`
`Acuteintermittentporphyria
`
`Subcutaneousinjection
`
`Paroxysmalnocturnalhemoglobinuria
`
`Subcutaneousinjection
`
`SeverehemophiliaAorB
`
`Subcutaneousinjection
`
`Primaryhyperoxaluriatype1
`
`Subcutaneousinjection
`
`Hypertrophicscarring
`
`Intradermalinjection
`
`Primaryhyperoxaluriatype1
`
`Intravenousinfusion
`
`Liverfibrosis
`
`Solidcancer
`
`NCT00716014
`
`NCT01591356
`identifier
`ClinicalTrials.gov
`
`Pachyonychiacongenita
`
`Intralesionalinjection
`
`Solidcancer
`
`Disease
`
`Administrationmethod
`
`Naked(modified)
`
`Naked(modified)
`
`Naked(modified)
`
`Conjugate(GalNAc)
`
`Lipidnanoparticle
`
`Intravenousinfusion
`
`Naked(modified)
`
`Naked(modified)
`
`Lipidnanoparticle
`
`Naked(modified)
`
`Naked(modified)
`
`Naked(modified)
`
`Naked(modified)
`
`Naked(modified)
`
`Conjugate(GalNAc)
`
`Degradablepolymer
`
`Lipidnanoparticle
`
`Lipidnanoparticle
`
`Conjugate(GalNAc)
`
`p53
`
`Androgenreceptor
`
`SOD1
`
`ApoCIII
`
`C-raf
`
`HIF1A
`TGF-β2
`TTR
`
`p53
`
`Caspase2
`β-2adrenergicreceptor
`TRPV1
`
`RTP801
`
`PCSK9
`
`KRASG12D
`
`PLK1
`
`MYC
`
`ALAS-1
`
`Conjugate(GalNAc)
`
`ComplementcomponentC5
`
`Conjugate(GalNAc)
`
`Conjugate(GalNAc)
`
`Polymernanoparticle
`
`Lipidnanoparticle
`
`Plasmaantithrombin
`
`siRNA
`
`Glycolateoxidase
`TGF-1βandCox-2
`Glycolateoxidase
`
`Intravenousinfusion
`
`Lipidnanoparticle
`
`Intravenousinfusion
`
`Lipidnanoparticle
`
`Naked(unmodified)
`
`Intravenousinfusion
`
`Lipidnanoparticle
`
`HSP47
`
`PKN3
`
`K6a
`
`EphA2
`
`Deliveryvehicle
`
`Genetic/proteintarget
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`Cenersen
`
`AZD5312
`
`BIIB067(IONIS-SOD1Rx)
`
`AKCEA-APOCIII-LRx
`
`LErafAON-ETU
`
`EZN-2968(RO7070179)
`
`ISTH0036
`
`siRNA
`
`Patisiran(ALN-TTR02)
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`siRNA
`
`QPI-1002
`
`QPI-1007
`
`Bamosiran(SYL040012)
`
`SYL1001
`
`PF-655
`
`Inclisiran(ALN-PCSSC)
`
`siG12D-LODER
`
`TKM080301
`
`DCR-MYC
`
`ALN-AS1
`
`ALN-CC5
`
`Fitusiran(ALN-AT3SC)
`
`ALN-GO1
`
`STP705
`
`DCR-PH1
`
`ND-L02-s0201
`
`Atu027
`
`TD101
`
`siRNA-EphA2-DOPC
`
`Name
`Table2CurrentclinicaltrialsinvolvingRNAdelivery
`
`Treatment
`
`

`

`Kaczmarek et al. Genome Medicine (2017) 9:60
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`Page 5 of 16
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`I
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`I
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`III
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`II/III
`III
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`III
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`III
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`ND
`
`NCT02238756
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`NCT02241135
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`NCT02316457
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`NCT02035956
`NCT02316457
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`NCT02410733
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`NCT01630733
`NCT01578655
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`NCT01737398
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`NCT02527343
`NCT02658175
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`NCT02525523
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`NCT02255552
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`II/III
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`NCT02947867
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`II
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`II
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`II
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`II
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`II
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`I/II
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`NCT00780052
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`NCT02079688
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`NCT01743768
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`NCT00822861
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`NCT02824003
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`NCT02981602
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`NCT01829113
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`NCT03070782
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`NCT02667483
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`NCT00882869
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`NCT02129439
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`NCT02532764
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`NCT01563302
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`NCT02709850
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`NCT02519036
`
`NCT01713361
`NCT02553889
`
`NCT02781883
`
`InfluenzaA
`
`Intramuscularinjection
`
`RSV,HIV,rabies
`
`Intramuscularinjection
`
`Rabies
`
`Intramuscularinjection
`
`Triplenegativebreastcancer
`
`Intravenousinfusion
`
`Melanoma
`Triplenegativebreastcancer
`
`Intra-nodal
`
`Melanoma
`
`Intravenousinfusion
`
`Nonsmallcelllungcancer
`Prostatecancer
`
`Intravenousinfusion
`
`Familialamyloidpolyneuropathy
`
`Subcutaneousinjection
`
`Familialpartiallipodystrophy
`Familialchylomicronemiasyndrome
`
`Subcutaneousinjection
`
`Pouchitis
`
`Enema
`
`Duchennemusculardystrophy
`
`Intramuscularinjection
`
`Neovascularglaucoma
`
`Eyedrops
`
`Clottingdisorders
`
`Subcutaneousinjection
`
`Myeloidleukemia
`
`Liquidcancer
`
`Intravenousinfusion
`
`Atopicdermatitis
`
`Topical
`
`Asthma
`
`Nebulization(inhaled)
`
`Allergen-inducedasthma
`
`Nebulization(inhaled)
`
`Type2diabetes
`
`Subcutaneousinjection
`
`HepatitisBinfection
`
`Subcutaneousinjection
`
`Solidcancer
`
`Intravenousinfusion
`
`Hyperlipoproteinemia(a)
`
`Subcutaneousinjection
`
`Duchennemusculardystrophy
`
`Subcutaneousinjection
`
`Solidcancer
`
`Intravenousinfusion
`
`Ulcerativecolitis
`
`Enema
`
`Cysticfibrosis
`
`Nebulization(inhaled)
`
`Solidcancer
`
`Intravenousinfusion
`
`hypercholesterolemia
`Elevatedtriglycerides/familial
`
`Huntington'sdisease
`
`Intrathecalinjection
`
`ND
`
`Hemagglutinin7(H7)protein
`
`NakedmRNA
`
`NakedmRNA
`mRNA–Lipoplex
`
`RNA-basedadjuvant
`
`Rabiesvirusglycoprotein
`
`Tumor-associatedantigens
`
`NakedmRNA
`mRNA–Lipoplex
`
`Patient-specifictumorantigens
`
`Tumor-associatedantigens
`
`Naked(modified)
`
`Naked(modified)
`
`Naked(modified)
`
`Naked(modified)
`
`Clusterin
`
`TTR
`
`ApoCIII
`
`ICAM-1
`
`Naked(modified)
`
`Dystrophin(exonskipping)
`
`Naked(modified)
`
`Naked(modified)
`
`Intravenousinfusion
`
`Lipidnanoparticle
`
`Naked(modified)
`
`Naked(modified)
`
`Naked(modified)
`
`Naked(modified)
`
`Naked(modified)
`
`Naked(modified)
`
`Naked(modified)
`
`Conjugate(GalNAc)
`
`IRS-1
`
`FactorXI
`
`Grb2
`
`C-myb
`
`GATA-3
`
`GATA-3
`
`andGM-CSF
`CCR3,β-chainofIL-3,IL-5,
`GCGR
`
`HBVsurfaceantigen
`
`Hsp27
`
`ApoA
`
`ASO
`
`Naked(modified)
`
`Dystrophin(exonskipping)
`
`Naked(modified)
`
`Naked(modified)
`
`XIAP
`
`GATA-3
`
`Naked(modified)
`
`CFTR(causesbaseinsertion)
`
`Naked(modified)
`
`Subcutaneousinjection
`
`Conjugate(GalNAc)
`
`Naked(modified)
`
`STAT3
`
`ANGPTL3
`
`Huntingtin
`
`mRNA
`
`mRNA
`
`mRNA
`
`mRNA
`
`mRNA-1851
`
`CV8102
`
`CV7201
`
`TNBC-MERIT
`
`IVACmutanome/warehousemRNA
`
`mRNA
`
`Lipo-MERIT
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`ASO
`
`Custirsen(OGX-011)
`
`IONIS-TTRRx
`
`Volanesorsen
`
`Alicaforsen
`
`Eteplirsen(AVI-4658)
`
`Aganirsen(GS-101)
`
`IONIS-FXIRx
`
`Prexigebersen(BP1001)
`
`G4460
`
`SB011
`
`SB010
`
`ASM8
`
`IONIS-GCGRRx
`
`IONIS-HBVRx
`
`Apatorsen(OGX-427)
`
`AKCEA-APO(a)-LRx
`
`DS-5141b
`
`AEG35156
`
`SB012
`
`QR-010
`
`AZD9150
`
`IONISANGPTL3-LRx
`
`IONIS-HTTRx
`
`Table2CurrentclinicaltrialsinvolvingRNAdelivery(Continued)
`
`

`

`Kaczmarek et al. Genome Medicine (2017) 9:60
`
`Page 6 of 16
`
`III
`II
`
`II
`
`II
`
`I
`II
`
`I
`II
`
`I/II
`
`I/II
`
`I/II
`
`I
`
`I
`
`I
`
`NCT01582672
`NCT00678119
`NCT01482949
`
`NCT02888756
`
`NCT02662634
`
`NCT01686334
`NCT01995708
`
`NCT02707900
`NCT01069809,
`
`NCT01197625
`
`NCT00831467
`
`NCT03014089
`
`NCT02935712
`
`ND
`
`ND
`
`Renalcellcarcinoma
`
`therapy
`Autologousdendriticcell
`
`HIVinfections
`
`Intranodalroute
`
`Non-smallcelllungcancer
`
`therapy
`Autologousdendriticcell
`
`Acutemyeloidleukemia
`Multiplemyeloma
`
`therapy
`Autologousdendriticcell
`
`HIVinfections
`
`Prostatecancerpatients
`
`Prostatecancer
`
`therapy
`Autologousdendriticcell
`
`therapy
`Autologousdendriticcell
`
`ND
`
`NakedmRNA
`
`NakedmRNA
`
`Tumor-specificantigens
`
`Tumor-associatedantigens
`
`Zika
`
`Intramuscularinjection
`
`Lipidnanoparticle
`
`Viralantigenicproteins
`
`Cardiovasculardisease
`
`Intradermal
`
`Naked(modified)
`
`Undisclosed
`
`Intramuscularinjection
`
`InfluenzaA
`
`Intramuscularinjection
`
`ND
`
`ND
`
`VEGF-A
`
`Vaccine
`
`protein
`Hemagglutinin10(H10)
`
`mRNA
`
`mRNA
`
`mRNA
`
`mRNA
`
`mRNA
`
`mRNA
`
`CV9103
`
`mRNA-1325
`
`mRNAAZD-8601
`
`mRNAMRK-1777
`
`mRNA-1440
`
`ASOantisenseoligonucleotide,mRNAmessengerRNA,siRNAshortinterferingRNA,NDnotdisclosed
`
`NakedmRNA
`
`NakedmRNA
`
`Tumor-specificantigens
`
`HIVtargetantigens
`
`mRNA
`
`mRNA
`
`AGS-003
`
`iHIVARNA-01
`
`NakedmRNA
`
`Tumor-specificantigens
`
`mRNA
`
`AGS-003-LNG
`
`NakedmRNA
`
`CT7,MAGE-A3,andWT1
`
`mRNA
`
`NakedmRNA
`
`Vaccine
`
`mRNA
`
`AGS-004
`
`Table2CurrentclinicaltrialsinvolvingRNAdelivery(Continued)
`
`

`

`Kaczmarek et al. Genome Medicine (2017) 9:60
`
`Page 7 of 16
`
`RNA modifications
`Equally important for effective nucleic acid delivery are
`chemical modifications made to the RNA itself, which
`can impart degradation resistance to the RNA [44] and
`render them unrecognizable by the immune system [45].
`This is true of both conjugate delivery systems, which
`leave the RNA exposed immediately upon injection, as
`well as nanoparticulate delivery systems, which must at
`some point expose the RNA to intracellular immune re-
`ceptors. RNAs can be modified by means of chemical al-
`terations to the ribose sugar (of particular importance is
`the 2′ position [45, 46]), the phosphate linkage and the
`[47–50]. RNAs delivered
`individual bases
`(Fig. 1d)
`through nanoparticles, discussed later, are also typically
`modified in order to avoid recognition by endosomally
`expressed pattern recognition receptors [51]. With few
`exceptions, modified RNAs are the gold standard in clin-
`ical trials (Table 2). The degree to which the RNA can
`be modified and still retain its potency depends, to a
`large extent, on the nature of the nucleic acid and its
`mechanism of action. For instance, short RNAs such as
`siRNAs, which rely on the relatively robust RNA-
`induced silencing complex (RISC) [52], can typically be
`heavily modified. By contrast, large mRNAs, which must
`be effectively translated by ribosomes, are more sensitive
`to modifications and utilize naturally occurring RNA
`modification such as pseudouridine and 5-methylcytidine
`substitution [53]. Indeed, recent studies have shown that
`base modification of mRNA can actually decrease potency
`in certain situations [54], whereas chemical modification in
`siRNAs is almost ubiquitously applied for in vivo use [55].
`
`Applications of RNA-based gene/protein
`modulation
`Protein downregulation—siRNA, ASOs, and microRNA
`In simplistic terms, disease-relevant proteins can be al-
`tered in one of two ways: upregulated or downregulated.
`The use of RNAs to selectively downregulate proteins
`experienced a paradigm shift following the discovery of
`siRNA by Fire and colleagues [56]. Short interfering
`RNAs are typically 21–23 base-pairs in length and can
`selectively bind and degrade complementary mRNA
`through the RISC (Fig. 2) [57]. After almost two decades
`of research, siRNA-based therapies represent one of the
`more clinically advanced platforms for RNA drugs.
`Alnylam Pharmaceuticals,
`in particular, has
`several
`siRNA drugs in clinical trials. Their most advanced drug,
`also one of the most advanced siRNA therapeutics, pati-
`siran, is a LNP containing siRNA against mutant trans-
`thyretin for the treatment of transthyretin amyloidosis
`[58]. Patisiran is currently in phase III of clinical trials
`[59], having shown significant dose-dependent knock-
`down, with minimal adverse events,
`in phase II trials
`[60], and other companies have also invested in the use
`
`of lipoplex-based siRNA drugs (Table 2). Increasingly,
`however, Alnylam and others have reported significant
`progress with the GalNAc
`conjugate
`technology
`(Table 2). Despite Alnylam’s recent decision to discon-
`tinue development of revusiran, a GalNAc–siRNA con-
`jugate drug that also treats transthyretin amyloidosis
`[61], the company has several more GalNAc conjugates
`in its pipeline that utilize a newer ‘enhanced stabilization
`chemistry’ [62] that could address the issues that led to
`the removal of revusiran from clinical trials [61]. Sur-
`prisingly, some of the current clinical trials utilize naked,
`albeit chemically modified, siRNAs. Almost all of these
`naked siRNAs are delivered locally (Table 2), reducing
`the risk of RNA degradation and systemic immune acti-
`vation compared with that associated with systemic
`delivery. An intriguing use of naked siRNA is Silenseed’s
`siG12D LODER, which encapsulates siRNA targeted
`against the KRAS oncoprotein in an implantable and
`degradable polymeric matrix for the treatment of pan-
`creatic cancer [63, 64]. However, there is concern that
`the positive effects of such treatments might in some
`cases be mediated by the induction of non-specific and
`immunological mechanisms such as siRNA binding to
`toll-like receptors [65].
`Despite its significant presence in clinical trials, siRNA
`is not the only, or even the first, RNA drug to be investi-
`gated for protein knockdown at the clinical stage. The
`first RNA drugs widely used in clinical trials were anti-
`sense oligonucleotides (ASOs). Like siRNA, ASOs are
`designed to block protein translation through Watson–
`Crick base-pairing with the target mRNA [66] and can
`be modified to improve stability [67]. The ASOs, how-
`ever,
`inhibit protein production through a variety of
`mechanisms, such as sterically blocking ribosome at-
`tachment or eliciting RNase-H activation [68]. They can
`also promote exon skipping (a form of RNA splicing
`which leaves out faulty exons), which allows for the dele-
`tion of faulty sequences within proteins [69], and,
`in
`some cases, can even lead to protein upregulation, which
`could be used therapeutically in diseases where certain
`genes are repressed [70]. An additional utility of ASOs is
`their ability to enter cells without the use of a transfec-
`tion reagent, although this uptake does not always lead
`to therapeutic action [71]. Four ASOs have been clinic-
`ally approved, all of which are chemically modified and
`used without a delivery vehicle, representing the only
`RNA drugs for protein modulation to be cleared by the
`FDA so far. The most recent, Spinraza (nusinersen), is
`injected intrathecally to treat spinal muscular atrophy
`[72]. It joined Exondys 51 (eteplirsen), an intravenously
`infused ASO for treatment of Duchenne muscular dys-
`trophy [73], Vitravene (fomivirsen), an intravitreally
`injected ASO indicated for the treatment of ocular cyto-
`megalovirus [74], and Kynamro (mipomersen), which is
`
`

`

`Kaczmarek et al. Genome Medicine (2017) 9:60
`
`Page 8 of 16
`
`ASO
`
`siRNA
`
`I.
`
`II.
`
`III.
`
`IV.
`
`sgRNA
`
`mRNA
`
`AAAAA
`
`III
`
`II
`
`Ribosome
`
`Protein expression
`
`AAAAA
`
`RISC
`
`AAAAA
`
`Cas9
`
`IV
`
`+
`
`CRISPR-Cas9
`
`Cleaved
`DNA
`
`Protein
`knockout
`
`I
`
`Rnase H
`
`AAAAA
`
`Cleaved
`mRNA
`
`Protein
`knockdown
`
`Fig. 2 Regulation of gene and protein expression using RNA. Once delivered into the cells, RNA macromolecules can utilize diverse intracellular
`mechanisms to control gene and protein expression. (I) Hybridization of antisense oligonucleotides (ASOs) to a target mRNA can result in specific
`inhibition of gene expression by induction of RNase H endonuclease activity, which cleaves the mRNA–ASO heteroduplex. (II) Short interfering
`RNA (siRNA) is recognized by the RNA-induced silencing complex (RISC), which, guided by an antisense strand of the siRNA, specifically binds and
`cleaves target mRNA. (III) In vitro transcribed mRNA utilizes the protein synthesis machinery of host cells to translate the encoded genetic information
`into a protein. Ribosome subunits are recruited to mRNA together with a cap and poly(A)-binding proteins, forming a translation initiation complex.
`(IV) In the CRISPR–Cas9 system, co-delivery of a single guide RNA (sgRNA) together with the mRNA encoding the Cas9 DNA endonuclease allows
`site-specific cleavage of double-stranded DNA, leading to the knockout of a target gene and its product. CRISPR, clustered regularly interspaced short
`palindromic repeats
`
`injected subcutaneously and targets mRNA encoding
`apolipoprotein B for the treatment of hypercholesterol-
`emia [75, 76]. There are still several ASOs in clinical
`trials, the majority of which are delivered without a
`vehicle (Table 2). Of particular interest are studies by
`Ionis Pharmaceuticals utilizing a GalNAc–ASO conju-
`gate similar to that developed by Alnylam to deliver
`siRNA. Optimism from such approvals and clinical stud-
`ies has also led researchers to continue investigation of
`ASOs to treat diseases such as amyotrophic lateral scler-
`osis (ALS) [77] and spinocerebellar ataxia [78].
`An emerging, albeit less clinically advanced, RNA-
`based platform for protein knockdown is microRNA
`(miRNA). Endogenous microRNAs
`are non-coding
`RNAs that act as key regulators for a variety of cellular
`pathways, and are often downregulated in diseases [79].
`Thus, exogenous microRNAs, or microRNA mimics,
`delivered therapeutically could be used to knockdown
`several proteins simultaneously, which is particularly
`useful in diseases such as cancer where having a single
`disease-relevant target is rare [80]. It is also worth not-
`ing that a rare subset of microRNAs is thought
`to
`enhance protein production, and that targeting of gene-
`
`suppressing microRNAs using ASOs could also be used
`to increase protein production [81]. The majority of
`current clinical trials involving microRNA are screens to
`investigate microRNA involvement in certain diseases,
`although there are several ongoing animal studies utiliz-
`ing microRNA delivery. Examples include the use of
`LNPs to treat a mouse model of colorectal cancer [82],
`and polymeric nanoparticles to deliver microRNA to
`the heart to treat fibrosis [83]. The first microRNA
`mimic therapy to enter clinical trials was MRX-34—a
`liposomal-encapsulated microRNA mimic from Mirna
`Therapeutics meant to treat a variety of cancers [84].
`However, the company terminated the study earlier in
`2017 after reports of several
`immune-related severe
`adverse events [85]. The fact that the adverse events
`were immunological
`in character further highlights
`the importance of RNA modification for clinical ap-
`plications, as such modifications remain one of the
`most important means of evading immune detection
`for RNA drugs. Chemical modification of miRNA
`mimics in particular, however, might prove challen-
`ging owing to the complex nature of miRNA-induced
`gene regulation [86].
`
`

`

`Kaczmarek et al. Genome Medicine (2017) 9:60
`
`Page 9 of 16
`
`Protein overexpression—mRNA
`Expression of disease-relevant proteins can be achieved
`by intracellular delivery of plasmid DNA (pDNA) or
`messenger RNA (mRNA). Application of DNA or
`mRNA as protein intermediate enables expression of
`virtually any desired protein inside the host cells and
`tissues. This approach can address formulation and
`delivery
`challenges
`encountered with protein-based
`drugs, especially those aimed at intracellular targets [87].
`mRNA-based therapeutics in particular offer several
`advantages over pDNA,
`including rapid and transient
`protein production, no risk of insertional mutagenesis,
`and greater efficacy of non-viral delivery by virtue of
`mRNA cytoplasmic activity (Fig. 2) [88]. Since the first
`pre-clinical studies in the 1990s, mRNA technology has
`greatly developed and now holds
`the potential
`to
`revolutionize vaccination, protein-replacement therapies,
`and treatment of genetic diseases, consequently gaining
`a considerable level of interest among the scientific com-
`munity and biotech industry [53].
`The delivery of mRNA therapeutics has been facilitated
`by significant progress in maximizing the translation and
`stability of mRNA, preventing its immune-stimulatory
`activity and the development of in vivo delivery technolo-
`gies, some of which are discussed below. The 5′ cap and
`3′ poly(A) tail are the main contributors to efficient trans-
`lation and prolonged half-life of mature eukaryotic
`mRNAs. Incorporation of cap analogs such as ARCA
`(anti-reverse cap analogs) and poly(A) tail of 120–150 bp
`into in vitro transcribed (IVT) mRNAs has markedly
`improved expression of
`the encoded proteins and
`mRNA stability [89, 90]. New types of cap analogs,
`such as 1,2-dithiodiphosphate-modified caps, with re-
`sistance against RNA decapping complex, can further
`improve
`the
`efficiency of RNA translation [91].
`Replacing rare codons within mRNA protein-coding
`sequences with synonymous
`frequently occurring
`codons, so-called codon optimization, also facilitates
`better efficacy of protein synthesis and limits mRNA
`destabilization by rare codons, thus preventing accel-
`erated degradation of the transcript [92, 93]. Similarly,
`en