Genetic Engineering Assignment

mRNA VACCINES
RA2132001010057
 TABLE OF CONTENTS

What? Why?
01 What is an mRNA vaccine 02 Why shift from traditional
and how does it work? vaccines to mRNA
vaccines?

How? Other Applications

03 How are mRNA vaccines 04
manufactured?
 01
What?
What is an mRNA vaccine and how does it
work?
mRNA VACCINES
Messenger ribonucleic acid (mRNA) vaccines
are a novel technology that stimulates the
body’s own immune response.

mRNA transfers the information for the

production of the antigen to our cell
machinery to make viral proteins.

Cells in our body then present the antigen on

their surface and thus trigger the desired
specific immune response.

When the body comes into contact with the

virus, the immune system recognizes the
specific antigen and can fight the viral
infection quickly and in a targeted manner.
Source: https://biontech.de/covid-19-portal/mrna-vaccines
 Types of mRNA Vaccines

Self-amplifying or replicon Non-replicating mRNA vaccines

mRNA vaccines
Dendritic cell Direct injection of
Most currently used self- mRNA vaccines non-replicating
amplifying mRNA (SAM) mRNA vaccines
vaccines are based on an Autologous DCs
alphavirus genome, where electroporated Liposome-
the genes encoding the RNA with mRNA complexed mRNA is
replication machinery are encoding for injected either
intact but the genes encoding antigens and intradermally or
the structural proteins generate cellular intramuscularly to
are replaced with the antigen Immune response elicit an immune
of interest. response
 RESEARCH BREAKTHROUGH

Suppression of RNA
recognition by Toll-
like receptors: the
impact of nucleoside
modification and the
evolutionary origin of
RNA (2005)
Dr. Katalin Karikó Dr. Drew Weissman
University of Pennsylvania and
BioNTech SE
 How does it work?

Selected natural RNA isolated from mammalian and

bacterial cells and RNA transcribed in vitro or
synthesized chemically, activate an immune response by
expressing human TLR3, TLR7, or TLR8.

Such activation was reduced or completely eliminated

with RNA containing naturally occurring modified
nucleosides, such as m5C, m6A, m5U, pseudouridine,
or 2′-O-methyl-U.
 Nucleic Acids Toll-like receptors
(TLRs) Activated

DNA containing
unmethylated- cytosine–
guanine dinucleotide (CpG) TLR9
motifs (bacterial and viral
DNA)

Double-stranded (ds) RNA

TLR3
(viral genome)

Single-stranded (ss) RNA TLR7

RNA oligonucleotides with

phosphorothioate TLR8
internucleotide linkages
 Bacterial mRNA v/s Mammalian
mRNA
Bacterial mRNA contains no nucleoside modifications, whereas mammalian
mRNAs have modified nucleosides such as:

• 5-methylcytidine (m5C)

• N6-methyladenosine (m6A)

• 5-methyluridine (m5U)

• N7-methylguanosine (m7G) (part of the 5′-terminal cap)

• Many 2′-O-methylated nucleosides (2′-O-Me)

• 2-thiouridine or pseudouridine (s2U)

GOALS PROTEIN TRANSLATION
The mRNA insert should have the ability to
translate into the protein of choice inside the
host cell.

IMMUNOGENICITY
The protein hence produced should be able
to elicit an effective immune response.

IMPROVE DELIVERY
The delivery molecules should be such that
the host cell incorporates it without
degrading the mRNA.
 Strategies for optimizing mRNA
pharmacology
• Synthetic cap analogues and capping enzymes stabilize mRNA and
increase protein translation via binding to eukaryotic translation initiation
factors

• Regulatory elements in the 5ʹ‑untranslated region (UTR) and the

3ʹ‑UTR stabilize mRNA and increase protein translation

• Poly(A) tail stabilizes mRNA and increases protein translation

• Modified nucleosides decrease innate immune activation and

increase translation

• Modulation of target cells: co‑delivery of translation initiation factors

and other methods alters translation and immunogenicity
 02
Why?
Why shift from traditional vaccines to mRNA
vaccines?
 Types of Vaccines

Live- Subunit and Toxoid

Inactivated attenuated

Viral vector DNA-based Messenger RNA

(mRNA)
 Traditional Vaccines
1. Time-consuming
The manufacturing of new vaccines is typically a lengthy (6 to 36 months)

2. Expensive for Large scale production

To deliver effective, precise, and consistent vaccines it is imperative to use good
manufacturing practice (GMP) compliant equipment, facilities, and procedures.
which is costly and difficult to implement at a large scale.

3. Less-effective
Vaccines developed on the basis of traditional technology have failed to respond
effectively to several diseases, such as malaria, tuberculosis, AIDS or flu.

Furthermore, SARS and Ebola epidemic outbreaks and, more recently, the CODVID-19
pandemic, show that many of the current platforms are not well suited for a very fast,
efficient, and cost-effective response.
 Advantages of mRNA Vaccines

1. mRNA is precise (unlike attenuated or inactivated vaccines) as it will only express a specific
antigen and induce a directed immune response.

2. Promotes both humoral and cellular immune response and induces the
innate immune system.

3. mRNA is more effective and safe (as compared with DNA-based vaccine) since
expression does not require nuclear entry, and safer, since the probability of random genome
integration is virtually zero.

4. Expression of the coded antigens is transient since mRNA is quickly degraded

by cellular processes, with no traces found after 2–3 days.

5. Minimized Safety Concerns as production is based on an in vitro cell-free transcription

reaction and so cell-derived impurities and viral contaminants are absent.
 03
How?
How are mRNA vaccines manufactured?
 Manufacturing
Pull DNA from Cold Storage

Grow the Cells in Bioreactors

Harvest and Purify the DNA Plasmids

Cut the Plasmids by RE Digestion

 Purify the DNA

Transcribe the DNA into mRNA

Test the mRNA for Accuracy

Prepare the mRNA and lipids

Assemble the mRNA Vaccine

 Major delivery methods for mRNA
vaccines
a) naked mRNA
b) naked mRNA with in vivo electroporation
c) protamine (cationic peptide)-complexed mRNA
d) mRNA associated with a positively charged oil-in-water cationic nanoemulsion
e) mRNA associated with a chemically modified dendrimer and complexed with
polyethylene glycol (PEG)-lipid
f) protamine-complexed mRNA in a PEG-lipid nanoparticle
g) mRNA associated with a cationic polymer such as polyethylenimine (PEI)
h) mRNA associated with a cationic polymer such as PEI and a lipid component
i) mRNA associated with a polysaccharide (for example, chitosan)
j) mRNA in a cationic lipid nanoparticle
k) mRNA complexed with cationic lipids and cholesterol
l) mRNA complexed with cationic lipids, cholesterol and PEG-lipid
 04
Other Applications
 Other Applications of mRNA
1) Replacement Therapy
mRNA is administered to the patient to compensate for a defective
gene/protein, or to supply therapeutic proteins;

2) Cell Therapy
mRNA is transfected into the cells ex vivo to alter cell phenotype or function,
and then these cells are delivered into the patient.

3) Gene Editing
mRNA can be used to introduce non-native proteins into cells, including
tools for gene editing such as the CRISPR-Cas family of nucleases

4) Personalized Cancer Vaccines

mRNA can be used to express tumor-associated epitopes (neoantigens) in
host cells
 Future Aspects
Despite the fact that the immune stimulatory activity of
RNA was discovered decades before such was identified for
DNA and that RNA contains numerous modified
nucleosides, the effect of nucleoside modifications on RNA
immunity has not been explored.

These factors can be used to advance the understanding of

autoimmune diseases where nucleic acids play a
prominent role in the pathogenesis and also determine a
role for nucleoside modifications in viral RNA, and give
future directions into the design of therapeutic RNAs.
 References
1. Karikó K, Buckstein M, Ni H, Weissman D. Suppression of RNA recognition by Toll-like receptors: the
impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 2005
Aug;23(2):165-75. doi: 10.1016/j.immuni.2005.06.008. PMID: 16111635.
2. Rosa, S. S., Prazeres, D., Azevedo, A. M., & Marques, M. (2021). mRNA vaccines manufacturing:
Challenges and bottlenecks. Vaccine, 39(16), 2190–2200.
https://doi.org/10.1016/j.vaccine.2021.03.038
3. Pardi, N., Hogan, M., Porter, F. et al. mRNA vaccines — a new era in vaccinology. Nat Rev Drug
Discov 17, 261–279 (2018). https://doi.org/10.1038/nrd.2017.243
4. https://www.nytimes.com/interactive/2021/health/pfizer-coronavirus-vaccine.html
5. https://www.frontiersin.org/articles/10.3389/fbioe.2021.628137/full
6. https://www.trilinkbiotech.com/applications-for-mrna
THANKS!
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