Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Broad Aspects of Chemotherapeutic mRNA

Amrita Mohanty1, Gowhar Iqbal 1*, Lukram Sushil Singh, Viral Kumar Ganpatbhai,
1
ICAR-Central Institute of Fisheries Education, Panch Marg, Yari Road, Mumbai-400061

Corresponding author: Gowhar Iqbal1*,

Abstract:- Naturally, mRNA is a single-stranded  Progress of key problems of mRNA therapeutics -

structure consisting of an (m7g) 7-methyl-guanosine  increasing stability
position at 5’end and 3’end consisting of a poly (A) tail.  improving translation efficiency
mRNA has emerged as a novel potential drug candidate  avoiding immunogenicity of mRNA
and has wide applications in cancer immunotherapy,  enhancing mRNA delivery
gene therapy, infectious disease vaccines, and cellular  One of the major challenges of naked mRNA-based
genetic engineering. Extension of the half-life of mRNA therapy is its short half-life which is caused by the rapid
can be achieved by introducing some stabilizing elements degradation by abundant extracellular RNAse, and this
into UTR regions. Employing mRNA for its half-life of in-vitro transcribed mRNA and also its
therapeutical prospects holds various characteristics protein products is a crucial factor affecting the
such as simple modifications, faster and transitory pharmacokinetics and pharmacodynamics properties of
presentation, and compatibility lacking mutagenesis, mRNA.
which are important for treating various diseases.
mRNA has emerged as a novel potential drug candidate  Structural elements of mRNA –
and has wide applications in cancer immunotherapy, Naturally, mRNA is a single-stranded structure
gene therapy, infectious disease vaccines, and cellular consisting of a (m7g) 7-methyl-guanosine position at 5’end
genetic engineering. In recent years, mRNA has proved and 3’end consisting of a poly (A) tail. It consists of an ORF
itself a novel therapeutic drug. (open reading frame) that is highlighted by a start codon and
a stop codon. The structure also consists of (UTR’s)
Keywords:- mRNA, Therapeutics, Immunotherapy, mRNA untranslated regions positioned in the middle of the cap/tail
Delivery, and ORF. Naturally, mRNA is a single-stranded structure
which consists of a (m7G) 7-methyl-guanosine positioned at
I. INTRODUCTION 5’ end and at 3’ end consists of a poly (A) tail. It consists of
an ORF (open reading frame) highlighted by the start and
mRNA has emerged as a wonderful field of applied stop codon. The structure also consists of UTRs
research since it was discovered in the 1960s. It acts as (untranslated regions) positioned in the middle of the
connecting link between DNA and protein and evolves as a cap/tail and ORF.
molecule which can regulate the function of genes in all
living organisms [1]. Ex vivo mRNA-transfected dendritic The circular DNA template and double-stranded
cells entered clinical trials for the first time. It has also faced oligonucleotide are generally preferred as templates for
many hindrances, such as instability and immunogenicity invitro mRNA synthesis. The bacteriophage RNA
and has created a stake in its development, making it less polymerase (T7, T3, SP6) along with rNTP (ribonucleoside
pursued in comparison to DNA in gene therapy. These triphosphate) are essential for producing complementary
raising issues have been resolved by introducing modified RNA strands. So, the 5’cap has a vital role in the translation
nucleosides into mRNA sequences, developing various process, maturation of mRNA, splicing process, and
RNA packaging delivery systems, improving translation nonsense-mediated decay, and the 3’ end consists of a poly
efficiency, and avoiding immunogenicity. (A) tail that plays a crucial role in the translation process
and stability of mRNA.
It includes various benefits, such as; mRNA does not
need to enter the nucleus to be functional. It does not insert  5’ cap modification -
into the genome. Therefore, it places a better, safer choice During transcription 7-methyl-guanosine (m7G) is
for researchers with a low risk of insertional mutagenesis. present in the mRNA cap linked to the first transcribed RNA
Recent therapeutics have enabled the production of mRNA nucleotide by a 5’, 5’-Triphosphate bridge (PPP) (m7GpppN
in cost-effective ways, can be produced quickly and have structure). It binds with the translation initiation factor
more flexibility because the invitro transcription process (EIF4E) and enhances RNA translational; hence binds with
produces them. Henceforth, mRNA has emerged as a novel DCP1/DCP2 complex and mediates mRNA decay [3].
potential drug candidate and has wide applications in cancer
immunotherapy, gene therapy, infectious disease vaccines, They recently reported that a cap analogue includes the
and cellular genetic engineering [3,4]. anti-reverse cap analogues (ARCAs) that are modified
within the ribose moiety of m7G. The ARCA-capped

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Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
mRNA inhibits incorrect cap positioning during mRNA  mRNA delivery -
synthesis and also shows superior translation efficiency. Some desired properties of mRNA delivery
systems;
 Poly (A) tail –  Protecting mRNA from digestion by extracellular
Generally, the 3’end of mature mRNA in eukaryotes nuclease
consists of a poly (A) tail. It has been marked that the poly  Should have less toxicity
(A) tail plays a crucial role in regulating the stability and  Efficient cell-specific uptake
enhancing the translational efficiency of mRNA. Recent  Delivery vehicle components should be efficiently
research demonstrated that the dendritic cells showed cleared
positive results of protein translation along with a poly (A)  Delivery vehicles should help in the easy endosomal
tail of 100 nucleotides and a 5’ARCA cap analogue. It has escape of mRNA.
been reported that the mRNA with longer tails showed
enhanced protein expression in cells [3]. A critical challenge in the development of mRNA
based therapeutics is the safe and efficient delivery of
 3’UTR of mRNA mRNA. The N-acetylgalactosamine (GalNAC)- oligo
Extension of the half-life of mRNA can be achieved by conjugated shows excellent safety hepatic targeted delivery
introducing some stabilizing elements into UTR regions. invivo and is ineffective for mRNA delivery. Naked mRNA
The β and α globin of 3’UTRs of mRNAs are principal cannot easily move through the cell membrane because of
factors for the improvement of mRNA half-life to more than its charge, size and degradability and it also leaks into the
1 day. To increase the stability and translational efficiency, cytoplasm. It is also reported that many cells have low
several IVT mRNA were conjugated with the 3’UTR, while uptake efficiency of mRNA. There is an exception with
two β globins were incorporated altogether in a head-to-tail immature dendritic cells, which can easily uptake mRNA
position and enhanced the stabilizing effect of mRNA [3]. through the macropinocytosis pathway and efficiently
accumulate mRNA [5].
 5’UTR of mRNA -
Several UTR, like as the 5’ UTR of human heat shock II. APPLICATIONS
protein 70, internal ribosomal entry sites (IRES), and 3’
UTR of eukaryotic elongation factor (EEDF1A1), have been  mRNA vaccines for treating cancer immunotherapy-
visualized for therapeutics mRNA applications. Trimix generally combines three mRNA molecules
coding for CD401, CD70 immunomodulators and truncated
 Avoiding immunogenicity of mRNA - TLR4. When the DCs (dendritic cells) were transfected by
One major stake with IVT mRNA is its immunogenic in-situ methods or mRNA encoding antigens when injected
property because extracellular RNA will be identified as an into lymph nodes or combined along with Trimix gave
epitome of viral infection. Hence, IVT mRNA activates the better results [7].
immune cells and initiates inflammation in the presence of
toll-like receptors. The RNA sequences are rich in uridine  mRNA vaccines provide protection in case of infectious
residues and potentially activators of toll-like receptors. So, disease -
this immunogenicity issues are resolved by decreasing the Various mRNA-based vaccines for healing several
uridine content of mRNA. To date, various strategies for infectious diseases such as HIV, zika virus infection, and
nucleotide chemical modification have been developed for rabies are still under examination. Modern Inc. has
reducing the immunogenic property without impeding the developed an influenza vaccine, H10N8, an altered mRNA
translation efficiency of mRNA, e.g. replacing natural vaccine conjugated with LNP that codes for viral antigenic
adenosine with N’-methyl adenosine (m1A) or N6-methyl hemagglutinin (HA) protein.
adenosine (m6A)’, replacing natural uridine with 5-methyl
uridine, 2-thiouridine (s2U), 5-methyloxyuridine (5moU),  mRNA-based protein replacement therapy -
N1-methyl pseudouridine (m1𝜑), pseudouridine (𝜑). Out of In this mRNA-based therapy, they tried transcriptional
these, m5C and 𝜑 gave the most promising results as they replacement therapy and generated an additional copy
reduced the immunogenic properties of mRNA and number of CFTR (cystic fibrosis transmembrane
enhanced the translational efficiency in both in vitro and in conductance regulator) protein which is absent in patients
vivo conditions [5]. suffering from Cystic fibrosis. In 2016, phase AstraZeneca
and Moderna started a clinical trial for AZD8601; here, they
It is also demonstrated that if the length of the poly(A) examined the therapy that codes for vascular endothelial
tail is increased, and will recreate mRNA with low mRNA factor-A (VEGF-A).
immunogenicity properties because the uridine content is
reduced or embedded in the sequence. It is studied that after  Gene editing using mRNA-based therapy-
purification processes like HPLC, the mRNA with 𝜑 Genome editing technology using mRNA therapeutical
modification is visualized with non-immunogenic properties techniques has also gained success in T cells, progenitor
and the protein translational efficiency is also enhanced. cells (HSPC), and hematopoietic stem cells. They
introduced adeno-associated virus (AAV) serotype-6 vectors
and zinc finger nucleases (ZFN) mRNA by electroporation
into the cells. But it raises an issue associated with the

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Volume 8, Issue 7, July – 2023 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
efficacy and safety of this editing technology. In a study,  In the case of mRNA, the translation is completed in the
they reported that when hematopoietic stem cells and cytoplasm, but in a plasmid, DNA translation occurs in
progenitor cell genome were edited based upon CRISPR/ the nucleus.
Cas 9 AAV6, they noticed that Cas9 mRNA cited changes  The probability of insertional mutagenesis can be
related to transcription, evoked viral property and altogether overcome by the application of mRNA-based gene
presented transcription down-regulation [4]. therapy
 Transfection of mRNA into the cells of the host is quite
 mRNA-based cell programming and their fates- easier as they have simpler construction than that of
mRNA can be used for programming cells and DNA.
reorienting the fates. iPSC’s (induced pluripotent stem cells)  Translation process also occurs suddenly just after the
have become a middle tool by giving a layout of cell’s transfection of mRNA
potentiality to construct disease models and for tissue
bioengineering. For a generation of IPS cells, the somatic IV. MANUFACTURE OF INVITRO SYNTHETIC
cells are generally transfected by mRNA and DNA-based mRNA –
methodologies. While the application value of IVT mRNA
has proven better results in gene expression and producing Synthetic mRNA structure is similar to the natural mRNA
IPSCs. A scientist also reported that Venezuelan equine construction. Due to their therapeutic values, the eukaryotic
encephalitis reprogramming factors (VEE-RF) RNA mRNA containing cap in 5’end is recently being studied.
replicon had expressed 4 reprogramming factors. When The presence of polymerases, NTPs and regular cap
(VEE-RF) RNA was transfected into fibroblasts of human (m7GpppGNpN) direct reverse orientation capping [6].
beings, it produced iPS cells. Applying IVT mRNA for
guiding the iPSC’s differentiation into differentiated cells. But this reverse capping exhibits decreased rate of
translation in mRNA. Whereas, 3’H, 2’-O methyl, 3’-O
Recently, from various trials of the mRNA-1273 methyl anti-reverse cap (ARCA) present in m7Guanine
vaccine developed by an American biotech company, exhibit 100% present in the proper orientation and hence,
Moderna Inc. showed good promising results against the also enhances the efficiency of translation of synthetically
Covid-19 pandemic and has entered phase 3 clinical trial. designed mRNA. It also prevents the enzymatic
Despite these, biotech and pharma companies have made degeneration of mRNA. In recent years, ARCA capping has
advances in technology, but quiet has not wholly resolved been the most commonly employed method of invitro
the major problems of mRNA, such as; immunogenicity, mRNA synthesis. In recent years, ARCA capping has been
delivery and off-target effects. In cancer immunotherapy, a the most commonly employed method of invitro mRNA
major concern may be the appropriate selection of mRNA synthesis. 3’UTR plays a vital role in controlling the
antigens. Additional challenges include the quick escaping pharmacokinetic property of mRNA. 3’UTRs of human
of mRNA from endosomes and the efficient and safe origin belonging to the globin family are most widely
delivery of mRNA into most cells. Another issue lies with employed in the field of synthesis of mRNA based upon the
protein replacement therapy; the dosage should be planned characteristic property of erythrocytes.
approximately as the quantity of protein liberated by a
similar mRNA dose differs greatly in discrete populations. Various ways of delivery also have consequences, like
And finally, a major long-lasting challenge would be the the different mean half-life of production of protein from
mRNA’s tissue selection. mRNA transfection varies between 7-30 hr invivo and 50hr
in case of in-vitro. Basically, the decaying of mRNA starts
Since the last few generations, mRNA acted as very when the poly (A) tail deadenylation approaches equal to
few explored drug invention fields. While compared with nearly 10 nucleotides.
conventional protein pharmaceuticals, mRNA depicts
smaller productivity, the least cost and pollution check.  Synthetic mRNA delivery –
Several problems correlated to DNA vaccines can also be Mostly the intracellular delivery of synthetic mRNA is
circumvented by implementing RNA vaccines. However, a major stake as the cell membrane is composed of the lipid
some of the major troublesome matters of mRNA, like bilayer. Due to its bigger size (300-500) KDa and
stability and immunogenicity, are checked to a certain extent polyanionic feature, nanoparticle encapsulation is required
by chemical modification of choosen nucleotides. for mRNA delivery [8].

III. ADVANTAGES OF MRNA-BASED GENE There are various mRNA delivery tools-
TREATMENT –  Naked mRNA delivery
 mRNA-based polyplex and lipoplex transfection
Synthetic mRNA has arisen as a beneficial transfection
 Virus-assisted delivery
instrument with various therapeutic values in this developed
 Polypeptide intervened deliver
era. Retaintivity of mRNA inside the cell is a major
requirement of synthetic mRNA for successful application
 Naked mRNA delivery
of gene therapy—comparison between mRNA based
Generally, intake of naked mRNA by cells is not
delivery and delivery of plasmid DNA.
encouraged due to charge, size etc. Cells that can uptake
naked mRNA using the electroporation method of delivery

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are also affected by many factors such as permeability of the have tumour-residing characteristics and rapidly enlarge
membrane, electric field, type of cell, and medium’s ionic by exvivo methods.
strength [9].  Molecular level: at certain instances, these potent
molecules counteract the tumor-producing cells like
Maintaining the ratio between healthy cells and TRAIL (tumor necrosis factor-related apoptosis inducing
synthetic mRNA is essential as they play key points in ligand). These molecules influence programmed cell
electroporation methods. Previously, mRNA transfections death basically in tumour-producing cells by binding
by electroporation method were performed on immature receptors that are dead.
dendritic cells and haematopoietic cells. Basically, to  Exosome level: the exosomes carry the anticancerous
successfully conduct this electroporation transfection, mRNAs and can identify the tumour-producing cells
highly-priced equipment and more cells are required. But, governed by membranes with targeting molecules.
by employing the electroporation method, the mortality rate  Cell-based modality treatment: [12]. The target
of cells is at a high level. So, there is a requirement for mRNA was prefixed by employing resected tumour
refinement to get better efficiency. tissue or detection by the real-time method. Next, they
prepared the vehicle cells (mesenchymal stem cells, bone
 mRNA-based polyplex and lipoplex transfection – marrow-derived cells) from the patient’s body. Further,
Components of mRNA, along with cationic lipids or by transfection method, synthetic mRNA was delivered
polymers, form polyplex and lipoplexes. Carriers used for into vehicle cells. Associated exosomes and selected
delivering synthetic mRNA can be employed for a particular mRNA-carrying cells were collected for the purpose of
cell or tissue to transform their composition. Polyplex and administration. Chemotherapeutics such as
lipoplex-controlled transfection of synthetic mRNA is Temozolomide (TMZ) were introduced directly into
mostly applicable to diverse preclinical researchers. Cationic selected exosomes. They visualized the target modes of
liposomes are generally more cytotoxic and create mRNA on tumour sites and the translated products in
obstruction in the cellular metabolism in various cells. So, vehicle cells as well as in recipient cell-to-cell
they possess hindrance in invitro studies [11]. connection. The techniques developed were beneficial
Recently, complicated vectors carrying synthetic mRNA for cellular-based cancer therapy.
have been designed like nanoparticle polymers that exhibit
pH reactions and effectively transported invivo. V. CONCLUSION

 Polypeptide-directed delivery Employing mRNA for its therapeutical prospects holds

Generally, polypeptides are designed to transfer various supreme characteristics such as simple
synthetic mRNA to the cytoplasm of cells. Polypeptides that modifications, faster and transitory presentation, and
are amphiphilic and cationic in nature play a key role in compatibility lacking mutagenesis, and these features are
delivering mRNA. The peptide such as GALA functionaries important for treating glioblastoma’s heterogeneity and
the polyplexes mRNA (PPx-GALA) in cells of dendrites, complications. Synthetically designed mRNA’s that are
and hence, the intake of target mRNA (PPx-GALA) by the transferred by many vectors are powerful weapons for
cells is increased by 18 times in comparison to attacking tumours over various biological barriers (blood-
lipofectamine and exhibits no cytotoxicity. Such designed brain and brain tumour barriers). Choosing vectors to
target mRNA mediated by polypeptides enhances the transport mRNA and methods of administration are
expression of mRNA along with whether they are tissue- completely patient-customized options. In recent years,
specific or cells specific [10]. mRNA has proved itself a novel therapeutics drug and has
been tried in various clinical and preclinical trials, including
 mRNA delivery by virus particles – control against infectious disease and immunotherapy
Target cells are transfected with synthetic mRNA with related to cancer. But among these, it gave potential results
the help of virus-like particles. Generally, in such delivery for oncology fields. Henceforth, mRNA therapeutics
systems via viral infections, cloning of specific genes in a effectively treat patients battling glioblastoma disease.
particular viral system is essential, packing particular cells
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