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mRNA transcript therapy

Expert Review of Vaccines

October 2014
14(2)

DOI:10.1586/14760584.2015.973859

Authors:

Drew Weissman

University of Pennsylvania

mRNA is the central molecule of all forms of life. It is generally accepted that current life on Earth descended from an RNA world. mRNA, after its first therapeutic description in 1992, has recently come into increased focus as a method to deliver genetic information. The recent solution to the two main difficulties in using mRNA as a therapeutic, immune stimulation and potency, has provided the basis for a wide range of applications. While mRNA-based cancer immunotherapies have been in clinical trials for a few years, novel approaches; including, in vivo delivery of mRNA to replace or supplement proteins, mRNA-based generation of pluripotent stem cells, or genome engineering using mRNA-encoded meganucleases are beginning to be realized. This review presents the current state of mRNA drug technologies and potential applications, as well as discussing the challenges and prospects in mRNA development and drug discovery.

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mRNA transcript therapy

Expert Rev. Vaccines Early online, 1–17 (2014)

Drew Weissman

Department of Medicine, University of

Pennsylvania, Philadelphia, PA 19104,

USA

Tel.: +1 215 573 8491

Dreww@upenn.edu

mRNA is the central molecule of all forms of life. It is generally accepted that current life on

Earth descended from an RNA world. mRNA, after its first therapeutic description in 1992, has

recently come into increased focus as a method to deliver genetic information. The recent

solution to the two main difficulties in using mRNA as a therapeutic, immune stimulation and

potency, has provided the basis for a wide range of applications. While mRNA-based cancer

immunotherapies have been in clinical trials for a few years, novel approaches; including, in vivo

delivery of mRNA to replace or supplement proteins, mRNA-based generation of pluripotent

stem cells, or genome engineering using mRNA-encoded meganucleases are beginning to be

realized. This review presents the current state of mRNA drug technologies and potential

applications, as well as discussing the challenges and prospects in mRNA development and

drug discovery.

KEYWORDS:modified nucleoside • mRNA • PKR • pseudouridine • purification • therapeutic protein • vaccine

The first use of mRNA encoding a potentially

therapeutic protein delivered in vivo occurred in

1990 when Wolff et al. demonstrated the

expression of reporter proteins after direct injec-

tion of mRNA and occurred with the first

description of the in vivo delivery of plasmid

DNA [1]. Interestingly, mRNAs were used ther-

apeutically in only a single follow-up with injec-

tion into the brain and amelioration of disease

symptoms [2]. In the mean time, the field

focused on plasmid and DNA based technolo-

gies and, then, viral vector delivery systems.

In considering the advantages and disadvan-

tages of mRNA-based therapy, there are several

conceptual advantages compared to other

nucleic acid-based approaches. Unlike DNA-

based therapy, mRNA does not have the risk

of integration into the chromosomes [3–7],

which can lead to insertional mutagenesis with

potentially disastrous results [8,9]. mRNA deliv-

ered therapeutically only results in transient

translation that can be controlled by both

changes in the UTRs or coding sequence and

is completely degraded through physiologic

pathways [10–13]. This is considered both an

advantage and a disadvantage depending on

therapeutic needs. In principle, mRNA-based

therapies appear to be much safer than DNA

or viral and are applicable to a broad spectrum

of disorders both acute and chronic.

The use of mRNA in the field of cancer vac-

cination has experienced the greatest amount of

preclinical investigation and has achieved

multiple stages of clinical testing. With the

advent of new discoveries that have both

increased the amount of protein produced per

delivered mRNA, through improvements in

mRNA structure and delivery, and the reduc-

tion of intrinsic immunogenicity of mRNA

[14–24], new approaches to replace proteins in

cardiology, oncology, endocrinology and the

treatment of genetic disorders, such as cystic

fibrosis or hemophilia [25,26], are underway.

These therapeutic approaches are still very early

in development and require both basic science

and pharmaceutical development of nearly every

step from synthesis to delivery of mRNA.

In this review, we attempt to offer a summary

on the current state of therapeutic mRNA tech-

nology, including vaccines against epidemic and

pandemic infections, protein replacement for

specific disease therapy and genetic deficiency,

and gene editing. The strengths and the upcom-

ing challenges that will likely impact on the

progress of this new class of drug will also

be highlighted.

mRNA pharmacology

At the present time, therapeutic mRNA is made

through the process of in vitro transcription,

where a linearized plasmid, PCR product or

synthesized DNA contains a promoter that is

recognized by a phage polymerase, such as T7,

T3 or SP6. In an in vitro reaction, multiple cop-

ies of mRNA are made from the DNA. The

DNA to be transcribed, in addition to

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containing the coding sequence of the protein of interest, can

contain optimized and specialized 5’and 3’UTRs and extended

lengths of A:T to produce a defined mRNA. In addition to the

cognate nucleotides, modified nucleoside triphosphates and vari-

ous versions of 5’caps can be included in the transcription reac-

tion. The DNA template is digested with DNases to terminate

transcription, and the mRNA is purified by conventional meth-

ods of nucleic acid isolation. The mRNA is engineered to resem-

ble fully processed mature mRNA molecules, as they would be

found in the cytoplasm of eukaryotic cells. Additional posttran-

scriptional modifications can be added, such as the addition of a

5’cap and 2-O-methylation of the cap or extension of the poly

(A) tail. The resulting product should optimally contain a 5’cap,

a coding sequence or ORF that encodes the protein of interest

and starts and stops with the appropriate codons, which can be

flanked by UTRs that can contain additional instructions for

translating the coding region.

While DNA and RNA are both protein-encoding molecules

that utilize cellular systems to produce the protein of interest,

there are many differences in the use of mRNA versus DNA.

The first involves the site of initial activity, which is the

nucleus for plasmid DNA and the cytoplasm for mRNA. This

represents a major difference between DNA and RNA, as

DNA requires nuclear envelope breakdown during cell division

to reach the nucleus and produce encoded protein, while

mRNA only needs to reach the cytoplasm for translation.

For mRNA to be translated into protein, it must survive in

the extracellular space that contains high levels of ubiquitous

RNases; it must reach or be targeted to the cells of interest for

translation, and finally, it must cross the cell membrane. The

cell membrane hinders passive diffusion of large negatively

charged mRNA molecules. Although, it has been demonstrated

that eukaryotic cells can actively engulf naked mRNA through

a receptor mediated mechanism, in most cell types, the rate of

uptake with transfer to the cytoplasm is extremely low [27,28],

and naked mRNA has a very short half-life in tissues and fluids

containing high levels of RNase activity. The delivery of

mRNA to cells both in vitro and in vivo can be greatly

increased by the use of complexing agents that protect the

mRNA from degradation by RNases, increase cellular uptake

and can perform targeting to specific tissues and cell types.

The route for complexed and naked nucleic acid uptake by

cells is via endosomes [29]. The mRNA must then escape from

the endosome and reach a translationally competent region of the

cytoplasm. Once in the cytoplasm, the pharmacology of mRNA

is subject to the same complex cellular mechanisms that regulate

the stability and translation of native mRNAs [30,31], with the pos-

sible exception that the bound proteins will at least initially differ

between mRNAs that have been processed and exited the nucleus

versus being in vitro transcribed. The roles that these proteins

play in cytoplasmic regulation of translation and mRNA degra-

dation are unknown. The encoded protein that is translated from

the in vitro transcribed mRNA undergoes posttranslational modi-

fication as directed by both the host that the mRNA is injected

into and the cell type that is translating the mRNA. The half-

lives of both the mRNA and the protein are critical factors for

the pharmacokinetics of delivered mRNA therapeutics and both

can be easily modulated to increase or decrease the duration of

protein activity.

Therapeutically, mRNA may be directly conveyed into a

patient’s cells, ex vivo, which are then administered back to the

patient. This allows a more precise control of elements of mRNA

pharmacology that have not been solved for in vivo delivery,

including specific targeting of a cell type for protein expression

and control over the amount of expression. Alternatively, the

future of mRNA therapeutics will rely upon mRNA used in a

suitable formulation with direct in vivo administration. Advances

in cell or tissue targeting are needed to make this possible.

Factors modulating the translation & stability of mRNA

Significant advances have been made in modifying the 5’cap, 5’-

and 3’-UTRs, the coding sequence and the poly(A) tail to

increase the translational efficiency and cytoplasmic stability of

mRNA. The optimization of translation can take on many forms

depending on the therapeutic needs and can include very rapid

initial production of the protein and sustained duration of trans-

lation or high peak translation followed by rapid reduction of

protein production. At present, protein production can be modu-

lated from minutes to nearly a week [23,31,32]. The field of modu-

lating mRNA pharmacology will advance as more therapeutic

mRNAs are described, but, at present, there is much space for

advances and deeper understanding. Further research into

mRNA binding factors and their respective mRNA binding sites

will unlock continued chances to engineer mRNA for diverse

pharmacokinetic activities.

In vivo mRNA delivery

In vitro transcribed mRNA used for immunization has been in

numerous clinical trials. Clinical trials using uncomplexed

(naked) or protamine-complexed mRNA in vaccines with deliv-

ery either by the intradermal route [33–37] or with direct injec-

tion into lymph nodes [38–40] are currently ongoing. The

delivery of naked mRNA seems to be satisfactory to produce

potent immune responses; it is likely to not be sufficient for

other clinical applications where higher amounts of protein will

be required and the delivery to a variety of cell types is needed.

The in vivo delivery of therapeutic mRNA for nonimmuniza-

tion purposes is currently the greatest hurdle and challenge.

Depending on the therapeutic goal of the delivered mRNA,

delivery can take on many different objectives; including, target-

ing specific cell types; delivery of high level protein production to

as many cells as possible without regard to cell type; delivery to a

majority of a particular cell type, such as bone marrow stem cells,

with or without regard to delivery to other cell types; and delivery

to specific organs, such as liver, brain, heart, and so on.

Therapeutic mRNAs can be divided between extracellular

and intracellular site of action of delivered encoded proteins.

The pharmacokinetics of each are very different. Systemic cir-

culation of an effector protein, such as observed with many

hormones and cytokines, is much easier to achieve, especially if

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any producer cell is qualified to make the appropriate func-

tional protein. The targeting of a specific cell type in vivo is

much more difficult and approaches that attempt this are still

early in development. This is further compounded if the major-

ity or that entire cell type needs to receive the mRNA for a

complete therapeutic effect.

Basic science studies in both mice and primates have demon-

strated that delivery of mRNA by the intraperotineal, subcuta-

neous and intramuscular routes achieves elevated plasma levels

for protein hormones, including erythropoietin (EPO) ([41] and

[DREW WEISSMAN,UNPUBLISHED DATA]). Lipid nanoparticles (LNPs), origi-

nally called stable nucleic acid lipid particles [42,43], have been

used in clinical trials for the delivery of siRNA to the liver

(reviewed in [44]). Liver cells are reached by many types of lipo-

somal and polymer delivery systems, and the liver serves as the

production site for many secreted systemic proteins; thus, liver

targeting could be an efficient method for producing high

amounts of recombinant proteins with mRNA delivery [45].

There are many other in vivo delivery systems using a variety

of approaches including lipids, polymers, sugars and den-

drimers being developed for nucleic acids including mRNA.

The results of these studies will direct future approaches to use

mRNA therapeutically.

The delivery of ‘intracellular acting’proteins to correct defec-

tive or deficient proteins with mRNA is a greater challenge,

especially when the fraction of cells that need to be restored is

high, in order to have a clinical result. The first determinations

for such approaches needs to consider both the percent of tar-

get cells that have to produce the encoded protein and the

amount of protein that needs to be present per cell for ade-

quate function/activity. Unlike in vitro transfection of cells that

can typically achieve over 80% and often close to 95% effi-

ciency, except for certain cell types that are highly resistant to

most chemical transfection methods, such as lymphocytes,

in vivo delivery is highly variable and poorly studied. At pres-

ent, the most work has been performed with LNPs and the

delivery of siRNA, including human clinical trials. It has been

reported that with such a delivery, over 80% of all cell types in

the liver can have siRNA within them [46]. For most delivery

systems with mRNA, especially when used as a vaccine, the

percent and type of cell that takes up the mRNA and produces

the protein is rarely identified and the measure for efficiency is

the resultant immune response.

5‘ cap

Eukaryotic mRNAs have a 7-methylguanosine (m7G) cap at

the 5’end of the mRNA that is appended during the transcrip-

tion process through a 5´-5´-triphosphate (ppp) bridge

(m7GpppNpNp…). The 5’cap is critical for efficient transla-

tion through its binding to eIF4E (reviewed in [47]). Three

decapping enzymes, Dcp1, Dcp2, and DcpS, also bind the 5’

cap and are involved in the regulation of mRNA decay [48].

There are 2 main approaches to adding a 5’cap to in vitro syn-

thesized mRNA. The first uses recombinant Vaccinia virus-

derived enzymes, one of which adds the m

GpppN cap, and

the second adds a 2-O-methyl group to the penultimate nucle-

otide (m

Gppp 2’-O-methyl-NpNp…) [49,50]. The Vaccinia

virus enzymes make a 5’cap-1 that is identical to the cap struc-

ture that is most frequently found in eukaryotic mRNAs.

A second more commonly used approach is to include a syn-

thetic cap analog in the in vitro transcription reaction. There

are advantages and disadvantages to both approaches, adding

the cap after transcription requires a second reaction, while

including a cap analog during transcription results in a decrease

in the total amount of mRNA made and a fraction of the

mRNA produced will not contain a cap analog. This is because

the cap analog competes with GTP in the transcription reac-

tion and a balance must be struck between the total amount of

mRNA produced and the percent that is capped by varying the

ratio of GTP to cap analog.

Current cap analogs comprise 3 basic families: m7GpppG cap

analog; antireverse cap analogs (ARCA), 3´-O-Me-m7GpppG

and modified antireverse cap analogs. Initial mRNA research was

done using mRNAs produced with m7 cap analog (GpppG) [1,51]

and continues to be used in most mRNA clinical trials. Unfortu-

nately, a significant amount of m7GpppG cap analog used dur-

ing in vitro transcription is incorporated in an opposite

orientation [52] resulting in inefficient recognition by translational

machinery. To combat this, ARCAs were developed [53,54].

mRNAs constructed with ARCA-caps exhibit superior transla-

tional efficiency in a variety of cell types compared to standard

cap analog [55–57]. A new class of ARCA caps has been established

that contains a phosphorothioate bond [58]. This results in resis-

tance to decapping by the Dcp2 enzyme leading to an extension

of the half-life of the mRNA [59]. Phosphorothioate containing

cap analog mRNA encoding an antigen when given to mice

resulted in a potent antigen-specific response that was greater

than that observed for mRNA with standard ARCA-mRNA [24].

Untranslated regions

Eukaryotic cells use the UTRs in mRNAs to regulate transla-

tion; multiple different approaches are used, including

upstream ORFs, sequences that promote degradation, modula-

tory protein binding sequences and micro RNA sites [60–63].

The description of these mechanisms is beyond the scope of

this review. For the purpose of therapeutic mRNA, most appli-

cations desire UTRs that lead to the highest level of translation,

although some applications may seek very fast translation or

short durations of protein translation.

Many mRNAs in clinical trials and in use for basic science

research utilize the 3’UTRs derived from a- and b-globin

mRNAs. Multiple sequence elements have been observed to

increase the translation and stability of mRNAs that harbor

them [64–66]. Studies have also identified multiple cellular and

viral 5’and 3’UTRs that enhance the translational efficiency

and stability of mRNAs they are appended to. 5’UTRs found

in numerous orthopoxvirus mRNAs have been demonstrated to

inhibit both cap removal and 3’to 5’exonuclease degradation

(reviewed in [6]). Depending on the therapeutic need, mRNA

with limited lengths of protein production may be needed.

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This can be achieved by incorporating sequence elements, such

as AU-rich sequences, into 3’UTRs, which results in rapid

mRNA degradation and associated short interval of protein

expression [67]. New approaches to develop and optimize UTR

function using techniques such as Systematic Evolution of

Ligands by Exponential enrichment [68,69] are currently being

used. The results of these studies will be important in defining

methods to improve UTR function and in the creation of

improved mRNA therapeutics.

Coding sequence

The first element of the mRNA coding sequence is the signal

sequence/peptide, which is a short (5–30 amino acids) peptide

found at the N-terminus of most newly synthesized proteins

that will enter the secretory pathway. In the construction of

mRNA encoding therapeutic proteins, the signal sequence can

be the natural version found in the cellular mRNA or it can be

engineered, which is typically used when the fate of the desired

proteins needs to be redirected, such as membrane bound to

secreted. In the case of mRNA encoding vaccine immunogens,

the desired processing of the immunogen by antigen presenting

cells helps to determine the type of signal sequence, in order to

obtain the correct immune response. As part of proteasome

processing, peptides that can be loaded onto MHC class I mol-

ecules are generated that can then be presented to CD8

T cells. The loading of MHC class II molecules to generate a

CD4

T cell response to a vaccine immunogen can be pro-

moted by modifying the mRNA with an endosome targeting

sequence [70–72].

Codon optimization serves several purposes. It replaces rarely

used codons with more abundant and frequently used codons

based on tRNA preferences of the host, but it also removes

RNA secondary structures, potential splice motifs, and other

sequences that may interfere with efficient transcription, nuclear

export and translation of an mRNA. The role of codon optimi-

zation to avoid rare and seldom used tRNAs in mRNA-based

therapies has been minimally studied. Mammalian codons usu-

ally have G or C in their third degenerative position, and such

sequences are expressed more efficiently than those in which

the codons end with A or T [73]. Typically, codon optimiza-

tions are performed by comparing protein production from

DNA plasmids carrying the wild-type sequence to the opti-

mized sequence [73]. In this setting, it is difficult to sort out if

the increased protein expression from the codon-optimized

construct is caused by enhanced transcription, increased RNA

stability, or augmented translation. However, studies that have

directly compared codon optimization that optimizes tRNA use

and removes RNA secondary structure, potential splice motifs

and other wild-type sequences in mRNA have found small

(~1.6 to threefold) increases in the protein produced [74,75].

CureVac GMBH, the first company to perform Phase II clini-

cal trials of mRNA-based vaccination for cancer, has identified

and patented the replacement of all third position Us with G

or C. Preliminary studies in our lab demonstrated an approxi-

mately fivefold increase in protein produced. This approach to

codon optimization is mechanistically different from other

studies [74,75] and only seeks to reduce third position Us and

replace them with Gs or Cs, which likely accounts for the dif-

ference in enhancement to translation.

While the increase in protein production by certain types of

codon-optimization is significant, there are potential difficulties

that could be encountered. Certain proteins require slow trans-

lation for their proper folding and modification and the pres-

ence of rare codons allows this to happen [76]. Responses to

hidden and unexpected epitopes have been identified in a num-

ber of cancers and in HIV infection [SHAW,GEORGE M, PERS.

COMM.] [77–82]. Some of these occur due to translation in different

frames caused by ribosomal frame shifting or alternate start site

utilization. Codon optimization of vaccine immunogens would

not contain these epitopes.

Poly(A) tail

The poly(A)-binding protein bound to the poly(A) tail interacts

with the eIF4F complex bound to the cap as part of the transla-

tion initiation complex. The poly(A) tail regulates the transla-

tional efficiency and stability of mRNA in synergy with the 5’

cap and with various other determinants [83].In vitro synthesized

mRNA can either have an encoded poly(A) tail in the template

vector or as a separate reaction after transcription where the poly

(A) tail is added by recombinant poly(A) polymerase. A downside

that precludes clinical use of enzymatic polyadenylation is that

each preparation of RNA contains a mixture of RNA species

with different lengths of poly(A) tail, which is unacceptable to

the US FDA. Typical lengths of encoded poly(A) tail range

around 120–150 nucleotides [23,56,84].

mRNA immunogenicity

There are more innate immune receptors that recognize RNA

than any other foreign or self-molecule, indicating the impor-

tance of RNA in the immune system. RNA, including in vitro

transcribed, induces innate immune responses through the activa-

tion of pattern recognition receptors, whose function is to iden-

tify and respond to pathogenic RNAs (FIGURE 1). Innate immune

recognition of RNA has been extensively reviewed [85–93].

RNA sensor activation can have both direct and indirect effects

on translation. In vitro transcribed RNA activates protein kinase

R and 2’-5’-oligoadenylate synthetase that lead to a direct inhibi-

tion of protein synthesis [14,20,21]. Activation of other cytoplasmic

and endosomal RNA sensors, which include TLR3, TLR7,

TLR8, RIG-I, MDA5, NOD2, IFIT-2, DDX60, DHX9,

DDX3, the DDX1-DDX21-DHX36 complex, HMGB proteins,

and LRRFIP1 (reviewed in [94]) can result in the release of Type I

interferon that activate interferon-inducible genes including pro-

tein kinase R, RNase L, 2’-5’-oligoadenylate synthetase and

others that when activated, directly inhibit translation (FIGURE 1).

The negative effects of RNA sensor activation has been

highlighted for mRNA vaccination where lipid-complexed

mRNA encoding HIV gag induced Type I interferon that led to

an inhibition in translation of the mRNA and a reduced immune

response [95].

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The intrinsic immunogenicity of in vitro transcribed mRNA

is viewed as advantageous in vaccine therapeutics due to its

adjuvant activity [96–98] that results in potent antigen-specific

humoral and cellular immune responses [99,100]. RNA has a dis-

tinctive pattern of immune stimulation, but this can be par-

tially modulated by altering the characteristics (type and size)

of the particle used to deliver it [22,101,102].

mRNA was first used therapeutically with in vivo administra-

tion to treat a disease in 1992 [2], but was not used again outside

of vaccination until the source of its immunogenicity and meth-

ods to overcome it were discovered. The observation that natu-

rally occurring RNAs differed in their immune activating

potential and the level of immune activation correlated with the

amount of modified nucleosides contained in the RNA led to the

finding that naturally occurring modified nucleosides such as

pseudouridine, 5-methyluridine, 2-thiouridine, 5-methylcytidine

(m5C), and N6-methyladenosine suppressed RNAs immune-

stimulatory effect by avoiding activation of TLR3, TLR7 and

TLR8 [103]. This was followed by the identification of other RNA

sensors, including RIG-I [18,19], protein kinase R [20,21],2’-5’-oli-

goadenylate synthetase and RNase L [14], similarly not being

activated by RNA containing nucleoside modifications. Double-

stranded RNA contaminants are formed during phage polymer-

ase transcription [104–107], and the presence of pseudouridine

and/or m5C did not obviate their immunogenicity. The dsRNA

contaminants were eliminated using an HPLC purification

method that resulted in mRNA that did not have any immune-

stimulatory activity [22]. The absence of the activation of RNA

sensors led to mRNA that was translated at much greater levels

(> 1000-fold) in vivo without inducing proinflammatory

cytokines, Type I interferons or adverse events [22,41] and led to

the current resurgence of nonvaccine mRNA therapeutics.

Preclinical & clinical applications

The main major therapeutic areas that mRNA or nucleoside

modified mRNA are currently being explored are the fields of

regenerative medicine, immunotherapy, vaccines and protein

replacement (FIGURE 2).

Vaccines

The first preclinical application of mRNA was used to induce a

protective immune response and employed direct injection of

carcinoembryonic antigen encoding mRNA [108]. Direct addi-

tion of mRNA derived from tumor cells or encoding tumor

specific antigens to dendritic cells (DCs) ex vivo followed by

administration of DCs back to the host, followed this first

demonstration of efficacy and led to extensive development

[109–112] leading to clinical trials (reviewed in [5,113]). Multiple

enhancements to this approach to therapy were attempted,

including the addition of mRNAs encoding costimulators

and cytokines [55,114–117]. Some spectacular results have been

reported, including the use of dendritic cells electroporated

with melanoma-associated antigen fused to a HLA-class II

targeting signal (DC-LAMP), adjuvanted with mRNAs

encoding CD40 ligand, a constitutively active Toll-like

receptor 4, and CD70 (TriMix [118]), in treated melanoma

patients where antitumor activity with durable disease con-

trol was observed [119]. Tumor-derived mRNA loaded DCs

have entered a Phase III clinical trial for patients with

advanced renal cell cancer [120].

Signals to IVT RNA

????????XXXX XXX

RNase LOASPKR

Murine

IFIT-2

LRRFIP1HMGB1, 3DDX3

DHX9

IPS1

DDX60NOD2MDA5RIG-ITLR8TLR7TLR3

TRIF MYD88

MAVS

Type I IFNs

Inhibits viruses

unable to 2-O-

methylate caps

Inhibits protein

translation

2′-5′ linked

oligoadenylates

- Rnase L

elF2α

β-catenin

-IRF3

Promote signaling

through TLRs

and RIG-I

Proinflammatory

cytokines and

type I IFNs

DDX1-

DDX21-

DHX36

complex

X†

Figure 1. Innate immune receptors demonstrated to respond to RNA. In vitro transcribed mRNA and other forms of

single-stranded and double-stranded RNA are recognized by numerous innate immune receptors. Whether signaling is initiated by in vitro

transcribed RNA is indicated. Signaling occurs through different pathways, as indicated, and results in inflammation associated with Type

I interferon, activation of cascades of transcriptional programs, proinflammatory cytokines and chemokines or inhibition of

translational machinery.

†

Indicates DREW WEISSMAN [UNPUBLISHED DATA].

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Direct injection of mRNA encoding an antigen has many

advantages over ex vivo manipulations, which led to multiple

investigations using direct injection of mRNA with different

strategies, including a variety of complexing agents and gene

gun delivery (reviewed in [7]). CureVac (Tu

¨bingen, Germany)

took the lead early on to develop direct intradermal injection

of mRNA with and without protamine complexing [33,34,101].

They have completed multiple clinical trials including ones

directed at prostate and non-small cell lung cancer [121] and

continue to develop this approach for other types of cancer.

BioNTech (Mainz, Germany) took an alternative approach

that has entered a clinical trial in melanoma patients [40]. They

used direct injection of uncomplexed mRNA into lymph

nodes. The mRNAs were optimized for efficient and high level

translation. Their studies demonstrated selective uptake by local

DCs with RNA induced DC maturation and effective presenta-

tion to T cells [38,122–124]. They have also begun to personalize

mRNA antigen vaccination therapy by massive parallel

sequencing of tumor cells with identification of tumor antigens

and construction of multi-epitope polypeptides containing each

patient’s tumor antigens and identified mutations [125–127].

Testing this approach has been initiated. Recently, the Univer-

sity Hospital Brussels in collaboration with the Vrije University

Brussels have started a clinical trial where hepatocellular

Protein replacement

Direct injection

Ex vivo treatment

Transposases

CRISPR/Cas9

TALE nuclease

Zinc-finger nuclease

Preclinical

Direct injection

Ex vivo treatment

Clinical

Direct injection

Ex vivo treatment

T cells: CAR-mesothelin [132]

Preclinical

Ex vivo treatment

HIV [79,133,134]

Direct injection

RSV [135,136], influenza [135,137,138], TB-associated Hsp65 [139], SV40 large T [140]

Clinical

Ex vivo treatment

iPS cell generating [146-151]

Injection of mRNA encoding allergens [144,145]

Allergy

Reprogramming of cells

DCs: HIV antigens [141-143]

Infectious disease vaccines

DCs: pancreatic adenocarcinoma [122,123], prostate cancer [124,125], melanoma [126-130], colon cancer [131]

renal cell carcinoma [12], melanoma [14]

T cells: CAR-Her-2/neo [117], CAR-mesothelin [118,119], CAR-CD19 [120,121]

DCs: MUC1[114], iLRP [115], survivin[116]

CEA [110], NY-ESO [111], Trp2, tyrosinase [112], gp100 [113]

Cancer Immunotherapy

knock out in rats [108], gene editing in zebrafish [109]

transgenic rat [104], mutated mice [105,106], zebrafish [43,107] editing

gene editing in mice [102], zebrafish [103]

gene editing in Drosophila [97], mouse embryo [98,99], HT1080 cells, mouse liver in vivo [100], HeLa cells [101]

Genome editing

MSCs with Slex, P-selectin glycoprotein ligand-1, IL-10 [95], DCs with IL-4 [96]

VEGF-A [90], BAX [91], Vasopressin [2], EPO [15,92], HSV1-TK, [93]SP-B [92], Foxp3 [94]

Figure 2. Fields of therapeutic applications of mRNA. Clinical and preclinical development of mRNA therapeutics, whether delivered

by ex vivo treatment of cells or direct injection on mRNA is described.

OAS: 2’-5’-oligoadenylate synthetase.

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carcinoma patients are treated with intranodal injections of mRNA

encoding tumor antigens and TriMix adjuvant (EUDRACT

2012-005572-34).

Infectious diseases vaccines

Curevac has expanded their 2-component mRNA vaccine, an

RNA adjuvant and a naked mRNA encoding antigen, to viral

influenza. It was demonstrated to induce strain-specific protective

immune responses in aged and newborn mice, along with long-

lasting protection in pigs and ferrets [128]. Alpha Viruses replicate

their genome by self-amplifying the RNA. An encoded RNA-

dependent RNA polymerase makes reverse (antisense) copies of

the entire genome and then full-length genomic RNA plus sub-

genomic structural proteins. These structural proteins can be

replaced with antigen to form a self-replicating RNA vaccine.

This approach has been used for flaviviruses [129], parainfluenza,

respiratory syncytial virus and influenza viruses [130–132]. Novartis

recently demonstrated that intramuscular injection of an RNA

self-replicating vaccine complexed in LNPs at very low doses

induced protective immune responses against influenza virus in

mice and non-human primates [133,134].

Two Phase I/II clinical trials using mRNA coding for HIV

antigens have been performed. These studies delivered DCs

ex vivo transfected with antigen-encoding mRNA to patients

on antiretroviral therapy. Immunization was deemed safe and

CD4

and CD8

T cell responses against the antigens delivered

were induced, but no antiviral effects were observed [135,136].

Vaccines for IgE hypersensitivity

Environmental allergic diseases are a hypersensitivity disorder of

the immune system mediated by IgE antibodies. Current treat-

ments involve immunization with graded dosing of the allergen to

modulate the type of T cell response and induce IgG antibodies

that compete and inhibit IgE binding to allergens. New and effec-

tive, but very costly, alternatives involve the injection of anti-IgE

monoclonal antibody [137–140]. The molecular identification of the

most common antigens leading to hypersensitivity has allowed

the development of recombinant vaccine methods. mRNA encod-

ing allergen vaccination prompted long-lasting allergen specific

Th1 immune responses that protected mice from allergen

exposure-mediated inflammation of the lung [141–145].

Cancer immunotherapy

Chimeric antigen receptor therapy has shown very good results

in several diseases (reviewed in [146]). Cells transfected with and

expressing such receptors are able to kill tumor cells that

express the targeted antigen. Multiple methods can be used to

load expanded T or NK cells, including mRNA. The advantage

of mRNA delivery is its transient nature, which can reduce the

risk of toxicity by uncontrolled expansion of the adoptively

transferred cells. A variety of antigen-specific receptors have

been delivered using mRNA and tested for antitumor immu-

nity in animal models [147–150]. Expanded T cells loaded with

mRNA encoding chimeric antigen receptors is being used in a

Phase I clinical trial for pancreatic cancer [151].

mRNA encoding engineered nucleases for gene editing

Early forms of gene therapy for the repair of inborn errors of a

particular gene sought to replace the defective gene by delivering

a functional copy containing its own promoter and regulatory

regions and inserting it into the chromosomes using a viral vec-

tor. Genome editing has emerged as a potential alternative for

gene therapy. Zinc finger nucleases and transcription activator-

like effector nucleases use meganucleases linked to protein

sequences that bind to specific DNA sequences that allows site-

specific cutting of DNA in chromatin [152,153]. The CRISPR/

cas9 system is derived from the acquired immune system of cer-

tain bacteria that uses RNA tags linked to a protein with nucle-

ase activity called cas9. The RNA tags identify the site for

cutting [154,155]. The major adverse event encountered in all

forms of gene editing is the risk of nonspecific editing. The

amount of off site effects associates with increasing duration of

functional enzyme as mediated by plasmid or viral delivery sys-

tems [156]. All three of the gene editing technologies only require

the nucleases to be present for a short duration, thus, their tran-

sient expression from encoding mRNA would meet this criteria

and likely minimize the potential for nonspecific effects.

mRNAs encoding Cas9, transcription activator-like effector

nucleases and zinc finger nucleases and ZFNa have been success-

fully used to edit genomes ex vivo in embryonic cells from dif-

ferent species and in vivo in rodents and zebrafish [157–164].

In summary, for all three gene editing approaches, the use of

mRNA either by direct injection in vivo or ex vivo treatment

would allow the fine tuning of dosing that cannot be achieved

with plasmid and viral delivery. The additional advantages of

high transfection efficiency without cell toxicity would be bene-

ficial for the generation of transgenic animals and the treatment

of human genetic diseases with potential application to other

types of diseases, including cancer.

Cell fate reprogramming with mRNA

In 2010, mRNAs coding for the Yamanaka stem cell factors

(Oct3/4, Sox2, Klf4, c-Myc) [165] containing pseudouridine and

5-methylcytydine were used to efficiently reprogram cells to

pluripotency (iPS cells) without any integration events [166].

A number of variants using the nucleoside modified mRNA

approach have been described that claim a more effective

induction of pluripotent stem cells or cell fate conversion

(reviewed in [3]). Prior to the use of nucleoside modified

mRNA with its lack of innate immune signaling, mRNA was

already being used to induce iPS cells [167], because of its high

in vitro transfection efficiency and transient expression with

lack of genomic integration. The transient expression of iPS

factors makes the use of nucleoside-modified mRNA for creat-

ing iPS cells attractive for different fields, including disease

modeling [168] and therapy for a variety of diseases [169] with

potential application to treatment.

Protein replacement therapies

mRNA based therapeutics deliver a natural product, that is, all

of its components are found in an organism and depending on

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how it is made, is indistinguishable from other RNA in the

cell. It allows robust and tunable delivery of the encoded pro-

tein. It avoids the expensive manufacturing of therapeutic pro-

teins in fermentation tanks and protein-specific purification

procedures. All of these attributes allow mRNA the potential to

reduce cost and time for entry into clinical testing, which is

particularly helpful for epidemic vaccines and therapies.

mRNA therapeutics can be viewed as a form of transient

gene therapy without the potential complications of long-term

gene therapy, including insertional mutagenesis, vector immu-

nity and effects of viral replication on cell function. The deliv-

ery of therapeutic proteins by mRNA is an obvious therapeutic

objective. The delivery of such encoded proteins can be initially

divided between extracellular acting and systemic proteins ver-

sus intracellular acting proteins and then further divided based

on; short-term delivery to treat deficient or non-functional pro-

teins; long-term replacement of deficient or non-functional pro-

teins; delivery of exogenous therapeutic proteins (monoclonal

antibodies), and acute site-specific or systemic delivery of a pro-

tein during a medical emergency or therapeutic procedure.

As described earlier, while the first delivery of an mRNA

in vivo used reporter proteins [1], it was quickly followed by

the delivery of vasopressin to the hypothalamus to reverse dia-

betes insipidus [2]. Therapeutic mRNA research outside of vac-

cines became dormant after this description until the

identification that the inclusion of naturally occurring modified

nucleosides in mRNA along with purification procedures

resulted in an mRNA that did not activate any RNA sensors

and was translated in primary cells and in vivo at much greater

levels [15,22,41,170]. These findings led to a reemergence into the

study of mRNA based protein therapy for the treatment of a

wide range of diseases.

The most advanced studies delivered EPO to mice and mac-

aques and used pseudouridine-modified, HPLC-purified

mRNA. As little as 10 ng of mRNA resulted in a physiologic

response, increase in reticulocytes, in mice [41].In vitro tran-

scribed mRNA coding for surfactant protein B and containing

25% of the modified nucleosides 2-thiouridine and m5C were

analyzed in a mouse model of congenital surfactant protein B

deficiency. Surfactant protein B mRNA 20 mg was delivered

two-times a week and was found to protect mice from respira-

tory failure and prolong their lifespan [16]. The intratracheal

delivery of a nucleoside-modified mRNA containing 10% of

the modified nucleosides 2-thiouridine and m5C and coding

for the regulatory T cell transcription factor Foxp3 (20 mg

before of during challenge) protected the mice from allergen-

induced tissue inflammation and airway hyperresponsive-

ness [171]. In a new therapeutic approach that will require much

additional development before it is ready for clinical use, direct

intramyocardial injection of mRNA containing pseudouridine-

and m5C, encoding VEGF-A improved heart function and

enhanced long-term survival in a model of mouse myocardial

infarction [172]. In another report, mesenchymal stem cells were

engineered ex vivo to express functional rolling machinery

(P-selectin glycoprotein ligand-1 and Sialyl-Lewisx) and the

immunosuppressive cytokine IL-10 using pseudouridine-

modified mRNA. When delivered in vivo, the engineered cells

rapidly localized to an inflammatory site in the ear and deliv-

ered IL-10 that resulted in a reduction in inflammation [173].

Extensive studies have so far identified similar levels of transla-

tion after HPLC purification [22,174] of mRNA containing pseu-

douridine and pseudouridine plus m5C, whereas mRNA

containing 1-methyl-pseudouridine consistently gave increased

levels of translation [DREW WEISSMAN,UNPUBLISHED DATA], suggesting the

superiority of this nucleoside modification.

There are multiple factors that must be considered beyond

simply injecting mRNA and hoping for a physiological effect.

Protein signal sequences are optimized for both the protein it

is attached to and the cell type that secretes the protein [175,176].

Relative secretory signal strength differs and optimal signal pep-

tides can be substituted to increase the amount of protein

released per mRNA molecule [177]. Cell type specific differences

in posttranslational modification need to be considered, espe-

cially when the delivered mRNA will not be translated by the

cells that naturally produce the protein. Certain cell types do

not have the capability to properly glycosylate every protein,

especially when highly complex glycosylation is needed [178].

Other types of posttranslational modification include proteo-

lytic processing and cofactor-dependent folding and clearance

of misfolded versions. Certain proteins require chaperones to

be properly folded or cleared, if misfolded, and complete post-

translational processing (reviewed in [179]). Endoprotease proc-

essing is an important part of the maturation of many

polypeptides, including cytokines, growth factors, neuropepti-

des, receptors, hormones, enzymes and plasma proteins [180].

A second form of proteolytic processing uses convertases that

function in the Golgi apparatus and secretory granules [181].

Cells that take up mRNA encoding a protein of interest need

to have any required endoprotease or convertase in order to

process and successfully produce functional proteins.

Clinical development of therapeutic mRNA

The only clinical applications beyond Phase I trials where

mRNA has been injected into patients or patient cells were

transfected ex vivo followed by readministration are vaccine tri-

als. Other uses of therapeutic mRNA, including gene editing,

protein replacement, cell fate reprogramming and cancer

immunotherapy will require the determination of the pharma-

cokinetic characteristics of the drug and the performance of

dose finding, which are only indirectly determined for mRNA

vaccine studies. The principal difficulty in determining mRNA-

encoded protein pharmacokinetics is that what is delivered

(mRNA) is not the final active agent (encoded protein). This

compounds the difficulty in determining potential variability in

the amount of protein produced, which is further compounded

by the bioavailability, defined as the percent of delivered

mRNA that is translated in a cell. Such potential variability

would appear to make the delivery of a protein therapeutic that

needs consistent and exact levels of active protein, such as insu-

lin, more difficult for mRNA. The variation of the protein

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product effect after administration of the same dose to the

same patient and then for different individuals will need to be

investigated. The analysis of interaction with other drugs will

also need to consider the use of drugs that alter mRNA uptake,

such as chloroquine-like drugs, or alter translation or RNA

metabolism, such as cancer and autoimmunity therapeutics.

The FDA and the European equivalent (EMA) acted on

multiple Investigational New Drug applications for the use of

mRNA as a vaccine, but they have not reviewed any protocols

where therapeutic mRNA to replace a protein or edit the

genome will be delivered in vivo. This leaves open many possi-

bilities for what they will require in preclinical testing. One can

assume that since mRNA, unlike DNA and viral vectors, does

not contain promoters or antibiotic resistance genes, and can-

not integrate into the genome and disruption of genes does not

occur, at least for protein replacement, that testing for genome

integration, germline transmission, carcinogenicity and genotox-

icity should not be required.

Potential immune recognition of encoded proteins could

lead to adverse events, including anaphylaxis and other immu-

nologic reactions, as well as local and systemic infusion-related

reactions. Immune responses can not only neutralize the bio-

logical activity of the delivered therapeutic protein, it could

also bind and impair or neutralize the activity of the endoge-

nous protein counterpart [182,183]. The classic examples of this

occurred with EPO. Adeno-associated virus (AAV) delivery of

EPO to cynomolgus macaques led to severe anemia due to

neutralization of encoded and endogenous protein [184].

A change in the formulation of therapeutic EPO protein led to

pure red cell aplasia in treated humans by neutralization of

endogenous EPO [185–187]. The observation that both gene ther-

apy and protein delivery of EPO led to autoimmunity raises

the potential risk for any mRNA-delivered protein. Recombi-

nant proteins require production in cell lines, yeast, plants or

bacteria that can result in altered posttranslational modification

leading to immune recognition; thus, the advantage of delivery

of mRNA encoding the gene is that the protein is made and

processed by the host. The mechanism behind AAV EPO-

induced antibodies remains unknown but serves as a warning

to any genetic delivery of a protein. Current data does not

demonstrate any potential for the induction of immunogenicity

against the mRNA-encoded protein.

A theoretical potential for the induction of antibodies against

the in vitro-transcribed mRNA or the worsening of diseases

where anti-RNA antibodies already exist, such as systemic lupus

erythematosus, needs to be considered. Evidence suggests that

anti-RNA autoantibodies may play a role in both the induction

and progression of autoimmunity in systemic lupus erythema-

tosus patients [188]. This raises the potential that mRNA ther-

apy using in vivo-administered mRNA could initiate or worsen

autoimmune diseases, including systemic lupus erythematosus.

Epitopes bound by autoantibodies have been identified and

potentially such epitopes could be engineered out of therapeutic

mRNA [189], but additional study into the ability of in vitro-

transcribed mRNA to induce anti-RNA autoantibodies and

worsen diseases where anti-RNA antibodies are present and

potentially pathogenic are needed.

The potential for immunogenicity of the mRNA-encoded

protein when delivered as a replacement for a defective or defi-

cient endogenous version needs to be considered. This has been

similarly addressed for other forms of gene therapy where the

delivered protein could potentially be seen as foreign, due to

the lack of the protein in the host.

The manufacturing of mRNA under current day techni-

ques is relatively simple. Therapeutic mRNA can be made in

a single reaction using template DNA and phage polymerase

with NTPs and buffer. There are no animal or cellular com-

ponents; thus, the risks are considerably lower compared to

recombinant proteins. An additional advantage of mRNA

over recombinant proteins for protein replacement therapies

is that the same procedure for making, purifying, and formu-

lating mRNA can be used for just about any mRNA encod-

ing a protein, while different purifications and formulations

are required for recombinant proteins. The manufacturing

process for producing mRNA has low variation and is easy to

control.

Expert commentary

Recently, with the identification of methods to reduce the immu-

nogenicity and increase the translational potential of mRNA,

there has been an expansion in research and development of

protein-encoding mRNA as a new therapeutic drug class. This

has fallen on the heels of a similar expansion of RNA therapeutics

involving siRNA, which has brought many new researchers and

hypotheses into the field. mRNA therapeutics is at the beginning

of its development, which informs us of the possibility that great

success and unexpected findings are likely to appear. The fields

of therapy where mRNA shows promise include vaccines, protein

replacement, gene editing and cell therapy, but new fields of use

are likely to be identified.

There has been a significant increase in interest in mRNA

therapeutics in the biotechnology arena and a number of uni-

versity derived and repurposed companies have developed,

including CureVac [190], BioNTech [191], Argos Therapeu-

tics [192], eTheRNA [193], Factor Bioscience [194], Trilink [195],

Ethris [196], Cellscript [197], and Moderna [198]. They have gar-

nered significant amounts of venture funding. A number of

pharmaceutical companies have also entered into the develop-

ment of mRNA-based therapeutics, including Shire, Novartis,

Astra Zeneca, Takeda, and Sanofi-Pasteur and a number of

others have expressed a peripheral interest and appear to be

waiting for potential products with greater development.

There are currently 33 clinical trials registered that are using

mRNA as a therapeutic (FIGURE 3). Most involve vaccines for a

variety of cancers. The most advanced are at Phase IIIB/IV,

suggesting the possibility of acceptance for clinical use in the

near future. It is expected with the expansion in biotech and

pharmaceutical companies’interest in mRNA therapeutics that

there will be a significant expansion in clinical trails in all

aspects of mRNA therapy.

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Enrolling by invitation

NCT00204516 Vaccination with tumor mRNA in metastatic melanoma ... Completed

NCT00204607 Intradermal vaccination with stabilized tumor mRNA-a

clinical phase l/lI trial in melanoma patients Completed

NCT00228189 Carcinoembryonic antigen-loaded dendritic cells in advanced

colorectal cancer patients

Completed

NCT00243529 Peptide-pulsed vs. RNA-transfected dendritic cell vaccines in

melanoma patients

Completed

NCT00510133 A study of active immunotherapy with GRNVAC1 in patients

with acute myelogenous leukemia (AML) Active, not recruiting

NCT00514189 Feasibility study of acute myelogenous leukemia mRNA plus

lysate loaded dendritic cell vaccines

Terminated

NCT00626483 Basiliximab in treating patients with newly diagnosed

glioblastoma multiforme ...

Recruiting

NCT00639639 Vaccine therapy in treating patients with newly diagnosed

glioblastoma multiforme

Active, not recruiting

NCT00833781 A pilot study of a dendritic cell vaccine in HIV-1 Infected ... Completed

NCT00834002 Dendritic cell vaccination for patients with acute myeloid

leukemia in remission

Completed

NCT00846456 Safe study of dendritic ceil (DC) based therapy targeting

tumor stem cells in glioblastoma Completed

NCT00890032 Vaccine therapy in treating patients undergoing surgery for

recurrent glioblastoma multiforme

Active, not recruiting

NCT00923312 Trial of an RNActive®-derived cancer vaccine in stage IIIB/IV

non small cell lung cancer (NSCLC)

Active, not recruiting

NCT00929019 Messenger ribonucleic acid (mRNA) transfected dendritic

ceil vaccination in high risk uveal melanoma patients

Recruiting

NCT00940004 Toll-like receptor (TLR) ligand matured dendritic cell

vaccination in melanoma patients

Active, not recruiting

NCT00961844 Trial for vaccine therapy with dendritic cells in patients with

metastatic malignant melanoma

Terminated

NCT00965224 Efficacy of dendritic cell therapy for myeloid leukemia and

myeloma

NCT00978913 Transfected dendritic cell based therapy for patients with

breast cancer or malignant melanoma

Recruiting

NCT01066390 A study on the safety and immunogenicity of... administration

of an autologous mRNA electroporated dendritic cell vaccine

in ... unresectable stage III or IV melanoma

Completed

NCT01197625 Vaccine therapy in curative resected prostate cancer

patients

Recruiting

NCT01278914 Trial of vaccine therapy with mRNA-transfected dendritic

cells in patients with androgen resistant metastatic prostate

cancer

Completed

NCT01278940 Trial of vaccine therapy with mRNA-transfected dendritic

cells in patients with advanced malignant melanoma

Completed

NCT01291420 Dendritic cell vaccination for patients with solid tumors Enrolling by invitation

NCT01334047 Trial of vaccine therapy in recurrent platinum sensitive

ovarian cancer patients

Recruiting

NCT01446731 Dendritic cell vaccination and docetaxei for patients with

prostate cancer

Recruiting

NCT01456065 Safety of active immunotherapy in subjects with ovarian

cancer

Active, not recruiting

NCT01456104 Immune responses to autologous langerhans-type dendritic

cells electroporated with mRNA encoding a tumor-associated

antigen in patients with malignancy:... melanoma

Recruiting

NCT01530698 Single-step antigen loading and TLR activation of dendritic

cells in melanoma patients

Completed

NCT01686334 Efficacy study of dendritic ceil vaccination in patients with

acute myeloid leukemia in remission

Recruiting

NCT01973322 Vaccination with autologous dendritic cells loaded with

autologous tumor lysate ... preleukapheresis IFN-alfa in ...

metastatic melano ma...

Recruiting

NCT01995708 CT7, MAGEA3, and WT1 mRNA electroporated autologous

langerhans-type dendritic cells ... multiple myeloma patients

undergoing autologous stem ceil transplantation

Recruiting

NCT02140138 An open label randomised trial of RNActive Cancer

vaccine in high risk and intermediate risk patients with

prostate cancer

Recruiting

Trial no. Title Status

EUDRACT:

2012-005572-34

A phase I study on the feasibility and safety of mRNA

immunotherapy in combination with RFA in patients with

hepatocellular carcinoma

Unknown

Figure 3. Clinical trials registered at clinicaltrials.gov using therapeutic mRNA. The clinicaltrials.gov and European Clinical Trials

Database (EudraCT) websites were queried for mRNA and results were analyzed for the use of mRNA as a therapeutic as of 8 January

2014.

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The choice of a target for mRNA drugs requires a thorough

understanding of the basic science, the clinical applicability and

the market. When an mRNA drug candidate is identified, the

process optimization and scale up to clinical grade GMP pro-

duction can be performed rapidly, because of the simplicity of

mRNA production. The expected costs for producing GMP

grade mRNA for clinical studies are likely 5–10-fold lower

compared to recombinant protein therapeutics that requires

complicated and specialized purification procedures that are dif-

ferent for every protein. The major difficulty for all mRNA

therapeutics that involve in vivo conveyance of the mRNA is

complexing and delivery. Most direct-injected mRNA vaccines

use uncomplexed mRNA for the delivery of mRNA that is to

be translated, which greatly simplifies these protocols. Naked

mRNA for protein replacement therapies or gene editing will

likely fail due to the inefficiency of uptake and the lack of pro-

tection from ubiquitous RNases. This leaves the field of

mRNA complexing open to new innovations. Beyond complex-

ing, the delivery of the mRNA to a particular cell type or tissue

is even more complex given all of the different cell types and

tissues and the complexity of targeting particular cells. The cur-

rent leader for in vivo delivery was borrowed from siRNA ther-

apeutics, LNPs [42–44], that very efficiently target the liver. New

complexing agents that can be directed to other cell types and

organs are needed. At present, directing therapeutic develop-

ment to tissues and cells that can be more easily targeted (liver,

skin and respiratory tact) makes the most sense.

mRNA therapeutics is at an early stage of development, but

with the amount of interest from biotech and pharmaceutical

companies, it is expected to rapidly develop to clinical trials

and new drugs. Many obstacles are present in the path to

development, but the extremely broad distribution of potential

therapeutic uses, vaccines, protein replacement, gene editing,

and cell reprogramming and the wide distribution of potential

drugs within each of these categories, for example, protein

replacement can include both intracellular and extracellular

proteins, replacement versus new therapeutic, transient or

chronic treatment and systemic administration versus organ or

tissue-specific therapy has brought many new resources and a

large amount of capital into the field. I believe that mRNA

therapeutics will have an extremely broad base that will aid in

the development of new therapeutic avenues in the future.

Five-year view

mRNA vaccine therapy has been in clinical trials and has

shown initial promise for a number of different types of

cancer. Phase IIIb/VI clinical trials will start soon and will

be instrumental in propelling the field, if positive. mRNA

therapeutics is at the beginning of its development with no

clinical trials currently being performed. An enormous

amount of research and resources by biotech and pharma-

ceutical companies is involved in the development of

nucleoside-modified mRNA as a therapeutic. It is expected

that many different types of therapeutics will be developed,

including ones for acute treatment, inflammation, tissue

regeneration, gene editing, and chronic therapies, including

hormone, cytokine and monoclonal antibody treatment.

Studies in both fields of use of mRNA will benefit develop-

ment of each other, likely leading to rapidly advancing

expansion of effective therapeutics. It is likely that

nucleoside-modified mRNA encoding a therapeutic protein

will enter clinical trials within 2 years and begin a new

approach to treatment with therapeutic proteins with poten-

tial rapid expansion to other diseases.

Financial & competing interests disclosure

D Weissman is named in patents describing nucleoside modified mRNA as

a therapeutic. The author has no other relevant affiliations or financial

involvement with any organization or entity with a financial interest in

or financial conflict with the subject matter or materials discussed in the

manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Key issues

• mRNA delivers therapeutic proteins to cells in vitro and in vivo, but its intrinsic immunogenicity has limited clinical development to

mainly vaccine studies.

• Nucleoside modification reduces the ability of in vitro transcribed RNA to activate RNA innate immune sensors.

• The further removal of phage polymerase produced contaminants, namely double-stranded RNA, can completely ablate the

immunogenicity of RNA containing certain nucleoside modifications.

• mRNA encoding a variety of antigens is an effective vaccine approach that can be delivered by direct injection of mRNA or ex vivo

treatment of cells.

• A major hurdle to mRNA therapeutics is the development of safe and effective in vivo delivery technologies, which is further

complicated when the targeting of particular cells or organs is required.

• mRNA therapy has therapeutic advantages, no risk of chromosomal integration, high transfection efficiency in both dividing and non-

dividing cells, superior modulation of the amount and duration of protein production, in many different fields, including vaccines, pro-

tein replacement, genome editing, cell fate reprogramming and cancer immunotherapy.

• Nucleoside-modified mRNA has the potential to revolutionize protein therapeutics, but is at an early stage of development and requires

optimization and understanding of all elements of production and use.

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Novel Efficient Lipid-Based Delivery Systems Enable a Delayed Uptake and Sustained Expression of mRNA in Human Cells and Mouse Tissues

Article

Full-text available

May 2024

Over the past decade, mRNA-based therapy has displayed significant promise in a wide range of clinical applications. The most striking example of the leap in the development of mRNA technologies was the mass vaccination against COVID-19 during the pandemic. The emergence of large-scale technology and positive experience of mRNA immunization sparked the development of antiviral and anti-cancer mRNA vaccines as well as therapeutic mRNA agents for genetic and other diseases. To facilitate mRNA delivery, lipid nanoparticles (LNPs) have been successfully employed. However, the diverse use of mRNA therapeutic approaches requires the development of adaptable LNP delivery systems that can control the kinetics of mRNA uptake and expression in target cells. Here, we report effective mRNA delivery into cultured mammalian cells (HEK293T, HeLa, DC2.4) and living mouse muscle tissues by liposomes containing either 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) or the newly applied 1,30-bis(cholest-5-en-3β-yloxycarbonylamino)-9,13,18,22-tetraaza-3,6,25,28-tetraoxatriacontane tetrahydrochloride (2X7) cationic lipids. Using end-point and real-time monitoring of Fluc mRNA expression, we showed that these LNPs exhibited an unusually delayed (of over 10 h in the case of the 2X7-based system) but had highly efficient and prolonged reporter activity in cells. Accordingly, both LNP formulations decorated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) provided efficient luciferase production in mice, peaking on day 3 after intramuscular injection. Notably, the bioluminescence was observed only at the site of injection in caudal thigh muscles, thereby demonstrating local expression of the model gene of interest. The developed mRNA delivery systems hold promise for prophylactic applications, where sustained synthesis of defensive proteins is required, and open doors to new possibilities in mRNA-based therapies.

Recent advances in mRNA-based vaccine for cancer therapy; bench to bedside

Article

Feb 2024
CELL BIOCHEM FUNCT

The messenger RNA (mRNA) vaccines have progressed from a theoretical concept to a clinical reality over the last few decades. Compared to conventional vaccination methods, these vaccines have a number of benefits, such as substantial potency, rapid growth, inexpensive production, and safe administration. Nevertheless, their usefulness was restricted up to now due to worries about the erratic and ineffective circulation of mRNA in vivo. Thankfully, these worries have largely been allayed by recent technological developments, which have led to the creation of multiple mRNA vaccination platforms for cancer and viral infections. The mRNA vaccines have been demonstrated as a powerful alternative to traditional conventional vaccines because of their high potency, safety and efficacy, capacity for rapid clinical development, and potential for rapid, low-cost manufacturing. The paper will examine the present status of mRNA vaccine technology and suggest future paths for the advancement and application of this exciting vaccine platform as a common therapeutic choice.

Strategies to Reduce the Risks of mRNA Drug and Vaccine Toxicity

Article

Jan 2024
NAT REV DRUG DISCOV

mRNA formulated with lipid nanoparticles is a transformative technology that has enabled the rapid development and administration of billions of coronavirus disease 2019 (COVID-19) vaccine doses worldwide. However, avoiding unacceptable toxicity with mRNA drugs and vaccines presents challenges. Lipid nanoparticle structural components, production methods, route of administration and proteins produced from complexed mRNAs all present toxicity concerns. Here, we discuss these concerns, specifically how cell tropism and tissue distribution of mRNA and lipid nanoparticles can lead to toxicity, and their possible reactogenicity. We focus on adverse events from mRNA applications for protein replacement and gene editing therapies as well as vaccines, tracing common biochemical and cellular pathways. The potential and limitations of existing models and tools used to screen for on-target efficacy and de-risk off-target toxicity, including in vivo and next-generation in vitro models, are also discussed.

Transient growth factor expression via mRNA in lipid nanoparticles promotes hepatocyte cell therapy in mice

Article

Full-text available

Jun 2024

Primary human hepatocyte (PHH) transplantation is a promising alternative to liver transplantation, whereby liver function could be restored by partial repopulation of the diseased organ with healthy cells. However, currently PHH engraftment efficiency is low and benefits are not maintained long-term. Here we refine two male mouse models of human chronic and acute liver diseases to recapitulate compromised hepatocyte proliferation observed in nearly all human liver diseases by overexpression of p21 in hepatocytes. In these clinically relevant contexts, we demonstrate that transient, yet robust expression of human hepatocyte growth factor and epidermal growth factor in the liver via nucleoside-modified mRNA in lipid nanoparticles, whose safety was validated with mRNA-based COVID-19 vaccines, drastically improves PHH engraftment, reduces disease burden, and improves overall liver function. This strategy may overcome the critical barriers to clinical translation of cell therapies with primary or stem cell-derived hepatocytes for the treatment of liver diseases.

Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription

Article

Full-text available

May 2024
MOLECULES

: mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5′-cap and a 3′-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.

Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors

Article

Full-text available

Mar 2024

Hypoxia-Inducible Factor-1α (HIF-1α) has presented a new direction for ischemic preconditioning of surgical flaps to promote their survival. In a previous study, we demonstrated the effectiveness of HIF-1a DNA plasmids in this application. In this study, to avoid complications associated with plasmid use, we sought to express HIF-1α through mRNA transfection and determine its biological activity by measuring the upregulation of downstream angiogenic genes. We transfected six different HIF-1a mRNAs–one predominant, three variant, and two novel mutant isoforms–into primary human dermal fibroblasts using Lipofectamine, and assessed mRNA levels using RT-qPCR. At all time points examined after transfection (3, 6, and 10 h), the levels of HIF-1α transcript were significantly higher in all HIF-1α transfected cells relative to the control (all p < 0.05, unpaired Student’s T-test). Importantly, the expression of HIF-1α transcription response genes (VEGF, ANG-1, PGF, FLT1, and EDN1) was significantly higher in the cells transfected with all isoforms than with the control at six and/or ten hours post-transfection. All isoforms were transfected successfully into human fibroblast cells, resulting in the rapid upregulation of all five downstream angiogenic targets tested. These findings support the potential use of HIF-1α mRNA for protecting ischemic dermal flaps.

Unlocking the potential of nanocarrier-mediated mRNA delivery across diverse biomedical frontiers: A comprehensive review

Article

Apr 2024
INT J BIOL MACROMOL

Potential therapeutic strategy for cancer: Multi-dimensional cross-talk between circRNAs and parental genes

Article

Mar 2024

The clinical impact of mRNA therapeutics in the treatment of cancers, infections, genetic disorders, and autoimmune diseases

Article

Feb 2024

mRNA delivery systems for cancer immunotherapy: Lipid nanoparticles and beyond

Article

Jan 2024

Efficient stimulation of HIV-1-specific T cells using dendritic cells electroporated with mRNA encoding autologous HIV-1 Gag and Env proteins

Article

Full-text available

Jan 2006

Infection with human immunodeficiency virus type 1 (HIV-1) is characterized by dysfunction of HIV-1-specific T cells. To control the virus, antigen-loaded den-dritic cells (DCs) might be useful to boost and broaden HIV-specific T-cell responses. In the present study, monocyte-derived DCs from nontreated HIV-1-seropositive patients were electroporated with codon-optimized ("humanized") mRNA encoding consensus HxB-2 (hHXB-2) Gag protein. These DCs elicited a strong HIV-1 Gag-specific interferon-(IFN-) response by an HLA-A2-restricted CD8 T-cell line. Moreover, hHXB-2 gag mRNA-electropo-rated DCs also triggered IFN-secretion by autologous peripheral blood mononuclear cells (PBMCs), CD4 T cells, and CD8 T cells from all patients tested. Next, a novel strategy was developed using autologous virus sequences. Significant specific IFN-T-cell responses were induced in all patients tested by DCs electroporated with patients' autologous polymerase chain reaction (PCR)-amplified and in vitro-transcribed proviral and plasma viral mRNA encoding either Gag or Env. The stimulatory effect was seen on PBMCs, CD8 T cells, and CD4 T cells, demonstrating both major histocompatibility complex (MHC) class I and MHC class II antigen presentation. Moreover, a significant interleukin-2 (IL-2) T-cell response was induced by DCs elec-troporated with hHxB-2 or proviral gag mRNA. These findings open a major perspective for the development of patient-specific immunotherapy for HIV-1 dis

Genetic engineering of human ES and iPS cells using TALE nucleases

Article

Full-text available

Oct 2012

Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator–like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).

Self-coded 3`-Extension of Run-off Transcripts Produces Aberrant Products during in Vitro Transcription with T7 RNA Polymerase

Article

Full-text available

Mar 1995

Marylena Dabrowski

More than 70% of the RNA synthesized by T7 RNA polymerase during run-off transcription in vitro can be incorrect products, up to twice as long as the expected transcripts. Transcriptions with model templates indicate that false transcription is mainly observed when the correct product cannot form stable secondary structures at the 3′-end. Therefore, the following hypothesis is tested: after leaving the DNA template, the polymerase can bind a transcript to the template site and the 3′-end of the transcript to the product site and extend it, if the 3′-end is not part of a stable secondary structure. Indeed, incubation of purified transcripts with the polymerase in transcription conditions triggers a 3′-end prolongation of the RNA. When two RNAs of different lengths are added to the transcription mix, both generate distinct and specific patterns of prolonged RNA products without any interference, demonstrating the self-coding nature of the prolongation process. Furthermore, sequencing of the high molecular weight transcripts demonstrates that their 5′-ends are precisely defined in sequence, whereas the 3′-ends contain size-variable extensions which show complementarity to the correct transcript. Surprisingly, a reduction of the UTP concentration to 0.2-1.0 mM in the presence of 3.5-4.0 mM of the other NTPs leads to faithful transcription and good yields, irrespective of the nucleotide composition of the template.

A Cationic Nanoemulsion for the Delivery of Next-generation RNA Vaccines

Article

Full-text available

Jul 2014

Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here we describe a cationic nanoemulsion delivery system developed to deliver a self-amplifying mRNA vaccine. This non-viral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults and the elderly. We show that non-viral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and non-human primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the cationic nanoemulsion-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted sub-unit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a cationic nanoemulsion is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.Molecular Therapy (2014); doi:10.1038/mt.2014.133.

Processing of Double-Stranded RNA in Mammalian Cells: A Direct Antiviral Role?

Article

Full-text available

Jun 2014
J INTERF CYTOK RES

Michael Gantier

Processing of viral double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) contributes directly to an antiviral effect in plants and invertebrates, which is amplified through the recruitment of RNA interference (RNAi). In mammals, viral dsRNAs are the substrate of the innate immune response and limit viral spread by impacting on cellular translation and cytokine production, as well as promoting cell death. Recent studies suggest that viral siRNAs also exert a direct antiviral activity in mammalian cells. Here, I review the current knowledge of dsRNA processing in mammalian cells and discuss the recent findings in light of the complex interplay between RNAi and dsRNA-driven innate immune responses toward the common goal of virus restriction.

Extracellular dsRNA: Its Function and Mechanism of Cellular Uptake

Article

Full-text available

Jun 2014
J INTERF CYTOK RES

Double-stranded RNA (dsRNA) is arguably the most potent viral trigger of innate immune signaling. Its activity has been recognized for over 5 decades, first as a toxin, then as a central component of the interferon system, as an efficient activator of antiviral responses and an immunomodulator for therapeutic applications. Nucleic acid sensing is the main basis for antiviral defense systems throughout the diverse forms of life from bacteria to plants and animals. Pattern recognition receptors of the host defense system not only sense viral dsRNA as a pathogen-associated molecular pattern in infected cells, but also recognize circulating endogenous dsRNA, a nonmicrobial signal, as a danger-associated molecular pattern, often leading to autoimmunity. Despite the effects of extracellular viral and host dsRNA associated with infection and autoimmunity, respectively, the understanding of cellular mechanisms for its recognition and uptake has only been appreciated in recent years. This review presents an overview of this unique form of nucleic acid, addressing its roles in infection, autoimmunity, and host sensing mechanisms. The goal of this review is to highlight the novel findings with a focus on extracellular recognition and uptake by the cell.

Half-lives of beta and gamma globin messenger RNAs and of protein synthetic capacity in cultured human reticulocytes

Article

Nov 1985

The turnover rates of beta and gamma globin messenger RNAs and of beta and gamma globin protein synthesis in human reticulocytes have been measured. Our goal was to determine whether beta globin mRNA is significantly more stable than gamma globin mRNA during the final stages of erythroid cell maturation. Such a result could explain the reported increase in the beta-gamma protein synthetic ratio during erythroid maturation. As determined by molecular hybridization and cell- free translation assays, the half-lives of both mRNAs are 20 to 29 hours in adult and neonatal reticulocytes. Protein synthetic capacity in intact cells decays with a half-life of six to eight hours, but beta protein synthesis declines at the same rate as gamma. Therefore, the changing ratio of fetal to adult hemoglobin synthesis during late erythroid maturation does not result from differences in mRNA turnover rates or changes in translation efficiencies. These data, coupled with those obtained with immature erythroid cells (Farquhar et al, Dev Biol 85: 403, 1981), suggest that, during erythroid maturation, the gamma- beta globin protein synthesis ratio declines because gamma gene transcription ceases earlier than beta gene transcription. Our results also indicate that the protein synthetic machinery, not the quantity of mRNA, becomes rate-limiting for globin production in cultured reticulocytes.

Humoral Immune Response

Chapter

Jan 2012

Translational control of localized mRNAs: restricting protein synthesis in space and time

Article

Dec 2008
NAT REV MOL CELL BIO

LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1

Article

Oct 2003

Salima Hacein-Bey-Abina

We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as γ chain (γc) deficiency] in 9 out of 10 patients by retrovirus-mediated γc gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with γδ+ or αβ+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.

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