Challenges in the Delivery of mRNA-Based Vaccines

Opportunities and Challenges in the Delivery of
mRNA-Based Vaccines
Abishek Wadhwa , Anas Aljabbari , Abhijeet Lokras , Camilla Foged and
Aneesh Thakur *
Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen,
Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark; [email protected] (A.W.);
[email protected] (A.A.); [email protected] (A.L.); [email protected] (C.F.)
* Correspondence: [email protected]; Tel.: + 45-3533-3938; Fax: +45-3533-6001
Received: 28 December 2019; Accepted: 26 January 2020; Published: 28 January 2020

Abstract: In the past few years, there has been increasing focus on the use of messenger RNA
(mRNA) as a new therapeutic modality. Current clinical eorts encompassing mRNA-based drugs are
directed toward infectious disease vaccines, cancer immunotherapies, therapeutic protein replacement
therapies, and treatment of genetic diseases. However, challenges that impede the successful
translation of these molecules into drugs are that (i) mRNA is a very large molecule, (ii) it is
intrinsically unstable and prone to degradation by nucleases, and (iii) it activates the immune system.
Although some of these challenges have been partially solved by means of chemical modification of
the mRNA, intracellular delivery of mRNA still represents a major hurdle. The clinical translation
of mRNA-based therapeutics requires delivery technologies that can ensure stabilization of mRNA
under physiological conditions. Here, we (i) review opportunities and challenges in the delivery of
mRNA-based therapeutics with a focus on non-viral delivery systems, (ii) present the clinical status of
mRNA vaccines, and (iii) highlight perspectives on the future of this promising new type of medicine.
Keywords: mRNA; vaccines; therapeutic; prophylactic; drug delivery systems; lipids; polymers;
nanoparticles; nanomedicine
1. Introduction
Vaccination has had a tremendous impact on global health and the quality of human life by reducing
the mortality and morbidity caused by infectious diseases. The development of vaccines is predicated
on the classical 3I’s paradigm of “isolating, inactivating and injecting” the causative microorganism,
coined by Louis Pasteur [1]. Vaccines can be prophylactic or therapeutic and can broadly be classified
as live attenuated vaccines (weakened microorganisms), inactivated vaccines (killed microorganisms),
subunit vaccines (purified antigens), or toxoid vaccines (inactivated bacterial toxins). As opposed to the
conventional concept of injecting live-attenuated or inactivated pathogens, modern vaccine approaches,
i.e., subunit vaccines, focus on exhibiting ecacy similar to conventional vaccines while obviating
the safety risks associated with whole-cell vaccines. However, subunit antigens often display lower
immunogenicity, which can be rectified by employing delivery systems and/or immunopotentiating
compounds as adjuvants to boost immunogenicity. The modern genome-based rational vaccine design
oers tremendous potential over conventional whole-organism-based vaccine approaches.
Pharmaceutics 2020, 12, 102; doi:10.3390/pharmaceutics12020102
Pharmaceutics 2020, 12, 102 2 of 27
Nucleic acid-based vaccines, i.e., DNA (as plasmids) and RNA (as messenger RNA (mRNA))
vaccines, pave the way for safe and ecacious biologics to mimic inoculation with live organism-based
vaccines, particularly for stimulation of cell-mediated immunity [2]. This technology exhibits promising
potential for the development of novel vaccines against a wide variety of indications and diseases,
extending from prophylactics to therapeutics for infectious diseases, cancer, autoimmune diseases,
and hypersensitivities. While nucleic-acid-based vaccines demonstrate significant advantages over
traditional vaccines [3] in terms of safety, ecacy, induction of both B- and T-cell responses and
specificity, it is noteworthy to mention that mRNA vaccines have advantages when compared to
vaccines based on other types of nucleic acids. A technical challenge associated with DNA vaccines
is to ensure delivery into the cell nucleus, where antigen transcription takes place prior to nuclear
export and translation into protein in the cytoplasm. In addition, DNA vaccines carry a potential risk
of integration into the host genome, which may result in insertional mutagenesis. In contrast, mRNA
vaccines are only targeted for cytoplasmic delivery, circumventing the risk of genomic integration [4].
The relatively short half-life results in transient and more controlled expression of the encoded antigen.
Moreover, mRNA can be produced in a cell-free environment by in vitro transcription (IVT), thereby
eschewing the use of microbes or cultured cells for production, and avoiding the associated quality
and safety issues in the production. This permits simple downstream purification and rapid and
cost-eective manufacturing [5]. However, mRNA is often promulgated on the grounds of the popular
opinion that when using mRNA, unlike DNA, the stringent gene-therapy regulations are bypassed
because mRNA does not integrate into the host genome. However, in reality, this only holds true in the
US since in Europe, any active pharmaceutical ingredient, which contains or consists of a recombinant
nucleic acid, used in or administered to human beings, falls under the scope of the regulation for
advanced therapy medicinal products [6]. Therefore, mRNA-based therapeutics are categorized as
gene therapy. The burgeoning field of mRNA vaccines is very exciting [3,7] and considerable amounts
of relevant preclinical data have been generated, and several clinical trials have been initiated during
the last decade. This gives rise to the vision of translating the mRNA vaccines into human application
for prophylaxis and therapy. In this review, we discuss the current trends in mRNA vaccine approaches
and various strategies and systems for delivering mRNA vaccines.
2. mRNA as Vaccines
The central dogma of molecular biology states that DNA is transcribed into mRNA, which
is subsequently translated into protein [8]. The flow of genetic information in time and space is
orchestrated by complex regulatory mechanisms. Gene therapy represents the introduction of genetic
material into an individual’s cells and biological tissues. Techniques like insertion, alteration, or
removal of genes are employed for correcting defective genes responsible for disease development,
which then cures a disease or ameliorates the clinical status of a patient [9]. Several vectors have been
utilized for gene therapy and they are generally classified as non-viral and viral vectors. Non-viral
vectors possess several advantages compared to the viral vectors, including low host immunogenicity
and potential for scale-up [10]. However, the success of non-viral gene therapy has been very limited in
the past, primarily due to the barriers existing for plasmid DNA (pDNA) delivery, e.g., the necessity to
cross the nuclear membrane before translation, the presence of antibiotic resistance genes in pDNA, and
most importantly, the diculty in controlling and regulating long-term expression. The lack of control
of long-term expression of pDNA poses a huge disadvantage in terms of the duration of treatment and
possible side eects, which is in contrast to conventional drugs, where the treatment can be stopped
instantaneously. These disadvantages of pDNA can possibly be overcome by using mRNA [11]. The
mRNA carries genetic information from the DNA in the nucleus to the cytosol, where it is used by the
ribosomes as a template for protein synthesis. As opposed to pDNA, the mRNA is ecacious in both
mitotic and non-mitotic cells because mRNA exerts its function in the cytoplasm, hence its function
is not dependent on active cell division. Furthermore, unlike pDNA or viral vectors, mRNA does
not contain additional foreign genes, which makes mRNA a safer vector. The challenge of long-term
Pharmaceutics 2020, 12, 102 3 of 27
expression posed by pDNA can also be overcome by using mRNA, since mRNA mediates a rapid,
transient expression of the encoded protein and the duration of the production is well-defined (usually
a few days or weeks, depending on the specific mRNA platform). This makes mRNA expression easier
to control than the gene expression from pDNA and viral vectors [11]. In addition, the manufacturing
of mRNA is cell-free, which strongly reduces the chance of mRNA contamination with bacterial
components. This makes it easier to produce mRNA than pDNA under good manufacturing practice
conditions [12]. Finally, vector-induced immunogenicity can be avoided for mRNA therapeutics, unlike
for viral vectors or virus-like particles, which may elicit a specific immune response against the exposed
viral proteins [13]. Specific therapeutic applications of mRNA, which are currently being explored
include (i) vaccination against cancer and infectious diseases, (ii) protein-replacement therapy, and (iii)
gene editing. Table 1 summarizes examples of ongoing clinical trials of mRNA-based therapeutic and
prophylactic vaccine candidates.
Two classes ofmRNAs, i.e., non-replicating andself-amplifyingmRNA, are commonly usedas vaccine
vectors. Non-replicating mRNA encodes only the protein antigen(s) of interest, while self-amplifying
mRNA also encodes proteins enabling RNA replication [14]. Vaccines based on self-amplifying mRNA
encode the RNA genome of a single-stranded RNA virus, e.g., an alphavirus, a flavivirus [15], or a
picornavirus [7]. They are engineered to increase the duration and level of expression, as well as the
subsequent immune response induced by the encoded antigen(s). They efficiently amplify the production
of sub-genomic mRNA encoding antigen(s) of interest subsequent to a single round of replication. While
both self-amplifying mRNA and non-replicating mRNA find application in prophylactic vaccines for
infectious diseases, non-replicating mRNA is used for cancer vaccines.
3. Fundamental Pharmacology of mRNA Vaccines
In vitro transcribed (IVT) mRNA is employed therapeutically as it mimics fully mature native
mRNApresent in the eukaryotic cytosol [16]. This may be achieved either by ex vivo transfection of cells
with mRNA that are then adoptively transferred or by direct in vivo delivery of the IVT mRNA to the
cytosol [17]. These approaches are explored for genome engineering, genetic reprogramming, adoptive
T cell and dendritic cell (DC) based cancer and infectious disease immunotherapies, tolerization
regimens to treat allergies, and protein replacement therapies. Both ex vivo transfection and direct
in vivo transfection enable the target cells to synthesize the encoded protein(s) in situ, where mRNA is
used as a template and the protein(s) represents the active product. The open reading frame (ORF)
of mature mRNA encoding the protein(s) of interest (the active product) marked by start and stop
codons, respectively, is flanked by untranslated regions (UTRs), and ideally consists of a 5’ cap and a
poly(A) tail [3].
The pharmacodynamic activity of both native and IVT mRNA takes place in the cytosol (Figure 1).
However, in contrast to endogenous mRNA, which is transcribed from DNA in the nucleus and enters
the cytosol through nuclear export, IVT mRNA enters the cytosol from an extracellular source [18].
Once the IVT mRNA is delivered to the cytosol, its pharmacology is governed by the same complex
cellular mechanisms that regulate the stability and translation of endogenous mRNA. The engineered
IVT mRNA resembles the endogenous mRNA so closely that the cellular translation machinery
is seamlessly utilized to synthesize a protein that may undergo post-translational modifications,
eventually resulting in mature protein product(s). In the case of vaccines, this mature protein product(s)
represents the antigen(s), which may elicit potent pathogen-specific humoral and cell-mediated immune
responses. However, the final intracellular destination is determined by the natural or engineered
sequence(s) of the signal peptide or the transmembrane domain [7]. Therefore, mRNA vaccines can be
designed for the delivery of the encoded protein(s) to the desired cellular compartment for proper
presentation and/or function [19].

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