FROM CONCEPT TO REALITY: THE RISE OF MESSENGER RIBONUCLEIC ACID IN MEDICAL MARVELS

FROM CONCEPT TO REALITY: THE RISE OF MESSENGER RIBONUCLEIC ACID IN MEDICAL MARVELS

Sakshi Dharane, Pallavi Hangargekar *, Dhanashri Bhujange, Dhanashri Yawalkar, Sujata Bhosale and Amol Joshi

Department of Quality Assurance, K. T. Patil College of Pharmacy, Dharashiv, Maharashtra, India.

ABSTRACT: Recent years have witnessed remarkable advancements in biomedical sciences, particularly in the realm of mRNA (messenger ribonucleic acid) technology. This manuscript offers a detailed overview of the burgeoning field of mRNA and its profound implications in science and medicine. Beginning with an introduction that elucidates the fundamental concepts and historical evolution of mRNA technology, the discussion progresses to an in-depth exploration of its structure, function, and pivotal role in modern medicine. The differences between mRNA and DNA are clarified to underscore the unique attributes of mRNA. A significant focus is given to mRNA vaccines, hailed as breakthroughs in preventive medicine. The exploration extends to mRNA therapeutics, highlighting their potential in targeted drug delivery and their ability to overcome traditional pharmaceutical challenges. Issues surrounding the efficacy and safety of mRNA applications are addressed, emphasizing importance of safety and ethical considerations. Present difficulties and potential paths in mRNA technology are discussed, underscoring ongoing research and innovative prospects. This review elucidates the transformative potential of mRNA technology, offering insights into its current applications, challenges, and promising future perspectives. As mRNA continues to revolutionize medicine, understanding its intricacies is vital for navigating the forefront of biomedical innovation.

Keywords: mRNA technology, mRNA vaccines, Genetic disorders, Targeted drug delivery

INTRODUCTION:

Current Trends in mRNA Research: The "blueprint" of human cells is found in messenger RNA, a naturally occurring molecule (mRNA). It can create immunogens that target proteins for therapeutic purposes and trigger immune responses in-vivo to fight a range of pathogens ¹.

The development of vaccines intended to treat as well as prevent disease has recently placed a significant emphasis on based on RNA technologies. As a result of this, mRNA vaccines have developed greatly in recent years and are now an invaluable tool for treating and preventing infections, especially in the case of SARS-CoV-2 infection.

By using targets present in viral genome, mRNA vaccines can be developed and produced more quickly than other common vaccines. Dendritic cells have the ability to deliver mRNA vaccines ex-vivo. Polymers, peptides, lipid nanoparticles, free mRNA in solution, and delivery carriers can also be used to encapsulate them. Because mRNA technology can prevent cancer and infectious diseases, it has shown a great deal of promise in clinical applications ². There has been a lot of interest in mRNA technology due to its amazing therapeutic potential. The wide application of mRNA molecules depends on the development of stabilization methods. Although mRNA delivery has been reviewed extensively, relatively little of it has addressed the root causes of mRNA instability and strategies for mitigating its problems. Every living cell must contain mRNA, which acts as a mediator in the genetic information transfer process. Research interest in mRNA therapeutics has increased significantly due to mRNA's inherent versatility ³.

Factors like toxicity, vector size, and unwanted immune responses limit its ability to spread. An important advancement in mRNA delivery has been the rising interest in different non-viral nanovehicles, including lipid-based nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, and more ¹. It is essential to remember that the transient effects of mRNA are short-lived and easily controlled, which lowers the likelihood of unforeseen consequences and long-term toxicity ⁴. Because of its effectiveness, low cost, quick development, and safety, scientists believe mRNA vaccine technology will soon become the norm in the field ⁵. Since, lipid nanoparticle-based messenger RNA (mRNA) vaccines showed remarkable clinical results in the COVID-19 pandemic, mRNA has gained recognition as a potent therapeutic agent for a variety of human diseases, especially cancerous tumors. mRNAs are used in immunomodulatory proteins, cancer vaccines, therapeutic antibodies, and adoptive T-cell therapies, among other applications, in the fight against cancer ⁶. The three areas of mRNA vaccine technology that have seen the most significant advancements recently are: 1) mRNA sequence engineering; 2) the creation of techniques that facilitate the easy, quick, and large-scale cGMP production of mRNA; and 3) the creation of extremely effective and secure mRNA vaccine delivery materials ⁷.

Key Milestones: First identified in the early 1960s, messenger RNA (mRNA) was introduced enter cells to express proteins in 1970s ⁸. They found that RNA taken from tumour cells carrying the Mengo encephalitis virus could be used to create infectious viral particles. Malone and associates proposed drug application of mRNA in 1989. Then, Zhou and associates demonstrated in 1994 that mRNA could be utilized to create vaccines. Ying and colleagues proposed the use of mRNA as a cancer-fighting tool in 1999. An mRNA vaccine may stimulate the body's defenses against prostate cancer, according to a 2003 trial. The first mRNA-based cancer vaccine, AVX701, started clinical trials in 2007. New strategies for enhancing mRNA vaccines were created in 2019. RNA vaccines were also delivered via lipid nanoparticles.

A study on a COVID-19 mRNA vaccine in 2021 produced encouraging findings. Research on mRNA vaccines for cancer and the flu is currently underway, demonstrating their potential as an effective preventive and therapeutic measure ⁹. The concept of using mRNA to encode proteins for immunisation or protein replacement was first validated in-vivo in 1990 by Wolff et al., who demonstrated that mice could produce a target protein after intramuscular injection. But decades passed before the promise of this technology could be clinically validated. Technical problems with mRNA stability and delivery as well as a brief shift in industry focus, funding, and research priorities towards DNA vaccines in the 2000s contributed to this delay4. Meanwhile, some dedicated researchers continued to work on mRNA, a single-stranded nucleic acid, because of its potential advantages as a vaccine component ¹⁰.

FIG. 1: KEY MILESTONES

Basics of Vaccination: Vaccination is the most effective means of controlling and preventing illness; it has saved many lives from cancer and infectious diseases ².

Each year, vaccinations save countless lives and prevent millions of illnesses. Because of the widespread use of vaccines, the smallpox virus has been eradicated and the prevalence of measles, polio, and other childhood diseases has drastically decreased globally. Conventional vaccination strategies offer long-lasting protection against a range of serious illnesses. These strategies include live, attenuated, and inactivated pathogens as well as subunit vaccines ¹¹.

One of the best public health tactics we have in the fight against infectious diseases is vaccination. Since then, vaccines that prevent 30 deadly illnesses have replaced the live cowpox virus that vaccination pioneer Edward Jenner used in 1798 to prevent smallpox. These vaccines save 6 million lives annually, extend life expectancy, and improve health for people of all ages by providing immune protection against multiple pathogen types.

Additionally, utilization of immunization in prevention and treatment of cancer is growing. Additionally, it can lessen the need for antibiotics to treat bacterial infections, thereby reducing the likelihood that antibiotic resistance will arise. However, licensed vaccinations against several serious, chronic, and life-threatening infectious diseases are still not available ⁶. Nucleic acid therapeutics appear to be promising alternatives to conventional vaccination strategies ¹¹.

The review explores the groundbreaking advancements in mRNA (messenger ribonucleic acid) technology and its profound impact on modern medicine. The purpose of the review is to provide insight into the revolutionary potential of mRNA in transforming disease prevention and treatment. As a recent and rapidly evolving topic, mRNA technology shows its promising applications in combating infectious diseases, genetic disorders, and cancer.

Understanding MRNA: A Simple Overview: The poly (A) tail, a stop signal-containing coding sequence, 3' untranslated region (3' UTR), 5' untranslated region (5' UTR), also referred to as leader RNA, and a 5' cap make up mRNA. Each mRNA template yields multiple copies of the corresponding protein when translated by ribosomes and tRNA in the cytoplasm, providing a quantitative advantage over individual protein production ¹².

The 5' UTR is where protein translation starts, and ribosome binding depends on the preinitiation complex's formation. As per the RNA translation scanning model, the 5' UTR's secondary structures and sequence have an impact on both translation efficiency and mRNA stability. Additionally, some viruses, such as the encephalomyocarditis virus, can induce cap-independent expression of proteins because a 5' UTR internal ribosome entry site (IRES). IRES primarily responsible for translating the 5' UTR's downstream open reading frame (ORF) sequence.

For better translation efficiency, the Kozak sequence is typically placed next to the 5' UTR. The elements of the 3' UTR affect the stability of mRNA. Eukaryotic mRNAs typically have mRNA degradation signals in their 3' UTR, which have an impact on the mRNAs' stability. Iron-responsive elements (IREs), a separate but equally important segment of the 3' UTR, regulate the translation and stability of mRNA ¹³.

mRNA vs. DNA: Basic Differences: More benefits than DNA therapy are associated with mRNA therapy. While mRNA delivery can achieve strong transfection efficiency using non-viral vectors including lipids and polymers, viral vectors are necessary to produce high transfection effectiveness in DNA therapies. Additionally, using in-vitro transcription in cell-free environment guarantees production of mRNA vaccines. On the contrary, viruses or particles that resemble viruses, mRNA does not elicit immune responses specific to vectors or carriers (VLPs). Furthermore, since mRNA functions directly in the cytoplasm and transcription requires DNA entry into the nucleus to occur before protein synthesis, antigens in mRNA vaccines are generated more quickly. In cells, mRNA experiences molecular alterations like deadenylation and decappingbefore being hydrolyzed by RNase. These changes ensure the transient expression of exogenous mRNA treatments, enhancing safety ^{12, 13}.

TABLE 1: mRNA VS. DNA

 Top of Form

mRNA Vaccines: A Breakthrough in Preventive Medicine:

How do mRNA Vaccines Work: For an mRNA vaccine to work, the target cells must be successfully injected with it. Once it enters cytoplasm, translated into the appropriate proteins. mRNA cannot enter the cytoplasm without first passing through negatively charged phospholipid bilayer of cell membrane. Molecules that can enter cells through passive diffusion are usually limited to molecular weights of less than 1000 Da. Thus, a carrier is needed to aid mRNA passage into cytoplasm of the cell. Presently, LNPs administer mRNA vaccines the most frequently; these vaccinations have four difficult components and inflammatory side effects.

It makes sense to conduct a thorough investigation into novel delivery strategies. Between translating DNA-encoded proteins and having cytoplasmic ribosomes synthesize proteins, mRNA is in an intermediate stage of the protein synthesis process. The two main forms of RNA that are being studied as potential vaccines are self-amplifying virally generated RNA and non-replicating mRNA. Self-amplifying RNAs enable high levels of protein synthesis and RNA amplification inside cells because they carry the genetic information necessary for both antigen and the viral replication machinery. In addition to target antigen, traditional mRNA-based vaccinations also have 5' and 3' untranslated regions (UTRs) ⁹. The anatomical and physiological traits of immunization sites, like muscles, lymphatic organs, and epidermis, affect vaccine effectiveness, making the delivery route of mRNA vaccines essential. For optimal effects, vaccinations can be administered either locally or systemically ¹. A virus-specific T-cell response was successfully elicited in mice injected with liposome-coated mRNA encoding an influenza nucleoprotein in the 1990s, highlighting potential of mRNA therapeutics as novel vaccination approach ¹⁴.

Innovations in Delivery Systems: Viral and non-viral vector delivery methods are the two primary categories for mRNA vaccines. Enhancing delivery techniques to target particular tissues or cells increases efficacy and safety. Key non-viral vectors are cationic liposomes (LNPs), which were identified in the 1960s. FDA-approved lipid and LNP medications, like ion is able cationic lipids (iLNPs), use proteases, polymers, LNPs, and cell-based administration to efficiently deliver RNA vaccines into the cytosol. Both viral and non-viral tactics increase efficacy and shield mRNA from deterioration. On the other hand, immune responses and insertional mutagenesis are safety concerns associated with viral vectors. Major challenges are the instability and vulnerability of RNA.

Direct injection of naked mRNA was one of the early techniques; although simple to produce, it is less immunogenic and stable. Because of their stability, protection, and biocompatibility, LNPs have emerged as the go-to carriers. Electrostatic interactions between polymers and mRNA form polyplexes, which enhance mRNA's immunogenicity and cellular absorption while also protecting it. Target specificity, stability, safety, and transfection efficiency are all improved by the use of different encapsulation techniques in polymeric nanoparticles, which are derived from polymers like polylactic-co-glycolic acid and PEG. The choice of delivery mechanism depends on the mRNA vaccine type, desired attributes, and target audience ¹⁵.

Routes of Administration: The amount, speed, and efficacy of immune response are directly impacted by the distribution and formulation of mRNA vaccines. The ways in which LNP-mRNA is administered affect its distribution, rate of expression, and therapeutic outcomes. Intramuscular (IM), subcutaneous (SC), intradermal (ID), and intravenous (IV) are common routes of administration. IV injections enable high protein production, while IM is the predominant method due to efficient absorption by myocytes and larger injection volumes, reducing site reactions but increasing systemic absorption. SC injections also allow larger volumes, reducing pressure and discomfort. Each route has unique benefits and considerations for vaccine delivery and patient experience ¹³.

Success Stories: mRNA Vaccines in Action: Attempts to develop vaccines against parasites like Plasmodium and Schistosoma, and pathogens like HIV and Mycobacterium tuberculosis, have not yet produced effective immunity. Conventional vaccine platforms often fail to respond quickly, safely, effectively, and economically to epidemic outbreaks. The need for vaccines utilising in-host mRNA translation and in vitro mRNA transcription was brought to light by the COVID-19 pandemic. BioNTech and Pfizer's BNT162b2 trial represented a major advancement in public health. mRNA vaccines are safe, avoid integration hazards, and are highly effective in inducing strong humoral and T-cell responses. They share a quick, simple, and cell-free manufacturing process with DNA vaccines, making mRNA an ideal candidate for rapid therapeutic or preventive vaccines in new pandemics ¹⁶.

MRNA Vaccine Applications: Treatment of a number of refractory conditions, such as infectious diseases, cancer, metabolic genetic disorders, and heart and brain disorders, may be possible with the utilization of mRNA-based therapeutics. In-depth research on mRNA vaccines has been conducted over the last 20 years, looking at both their potential for treating and preventing cancer as well as their capacity to combat infectious diseases ¹³.

A stable and adaptable platform for cancer vaccines is offered by mRNA. A perfect mRNA vaccine would include several neoantigens, such as driver gene mutations and both predicted and confirmed mutations and be prepared in suitable nanoparticles and administered with suitable adjuvants. The most potent antigen-presenting cell, DCs, should be the target of a perfect mRNA vaccine. When it is successfully applied to clinical settings, our ability to fight cancer will be greatly enhanced ⁹. The rapid development and utilizations of mRNA vaccines in the fight against the COVID-19 pandemic is one recent illustration of the significant impact that RNA therapeutics have had on medicine ¹⁴. Pre-clinical and clinical studies are looking into using IVT mRNAs to replace missing or damaged proteins caused by genetic illnesses or in circumstances where protein delivery may be therapeutically beneficial. The potential of IVT mRNA to treat hepatic disorders, generate human stem cells, and regenerate cardiac tissues has been investigated ¹³.

Pharmacological Advancement of mRNA Vaccines: Messenger RNA vaccines offer many benefits over DNA plasmids, viral vectors, and traditional live-attenuated viruses. Unlike DNA, mRNA does not risk chromosomal integration, enhancing safety. Its short half-life and rapid degradation in host cells further improve safety and effectiveness. RNA vaccines can rapidly introduce mutations for tailored therapies or pandemic responses, showing high adaptability. Unlike live-attenuated and vaccinations using viral vectors, mRNA vaccines are not contagious and do not incorporate into host genome. Additionally, producing mRNA in-vitro is quick and cost-efficient, underscoring the practical benefits of mRNA-based vaccinations ¹⁷.

mRNA in Therapeutics: mRNA therapeutics control pathogenic processes and provide therapeutic effects by using RNA molecules to translate particular proteins inside target cells. Protein replacement therapy holds promise for treating a range of illnesses, including cancer, heart disease, neurological disorders, and metabolic diseases. It replaces faulty proteins in diseases resulting in protein deficiencies. mRNA therapies that increase the production of Cas protein for gene editing are advantageous for gene editing techniques like the CRISPR/Cas system. Because mRNA therapies allow for the temporary production of transcription factors without the risk of mutagenesis, they are also used in stem cell engineering and the treatment of CAR T cells. Clinical trials are presently being conducted on several mRNA therapies ¹⁸.

Current Applications:

Beyond Vaccines: To fight infectious diseases, mRNA vaccines could be developed with potential uses in both therapy and prevention. mRNA vaccines containing the antigen of a pathogenic microorganism elicit robust and notable humoral and T-cell immune responses ¹³. mRNA vaccines have surfaced as a novel approach to address this problem and have the potential to effectively combat infectious diseases ¹.

SARS-CoV-2: The COVID-19 pandemic has caused severe harm to the world, with over 772 million cases of infection and over 6.9 million deaths by November 22, 2023. The FDA approved mRNA-1273 from Moderna and BNT162b2 from Pfizer-BioNTech in 2020 as vaccine candidates that have shown promise in combating the virus. BNT162b21 is composed of the ionizable lipid ALC-0315 and nucleoside-modified mRNA. An infectious virus known as single-stranded RNA (ssRNA) is the cause of SARS-CoV-2. Since the disease was first discovered in Wuhan, China in December 2019, it has shown signs of spreading throughout the world. The pharmaceutical sector has created several vaccines against the virus using various vector technologies ¹⁹. Numerous illnesses, fatalities, and significant societal disruptions were brought on by COVID-19 pandemic. Main goals for public health were to develop "safe and effective" vaccines ¹⁶. Rapid progress in mRNA vaccine development has been essential in controlling the coronavirus disease 2019 pandemic (COVID-19), indicating that such technology may be applied to other infectious disease outbreaks in the future. mRNA vaccines are particularly well-suited for vaccine development because of their adaptability, which allows antigens to be easily modified by changing the sequence within the mRNA coding region. This is especially true during crises of rapidly spreading infectious diseases ¹⁸. Several vaccine technologies, including messenger RNA (mRNA) vaccine technology, were quickly developed in response to the 2019 COVID-19 outbreak to combat the pandemic and stop the disease's spread ⁵.

As it has prevented countless infections, hospitalizations, and deaths, the COVID-19 vaccination is most effective intervention in the fight against the global pandemic caused by SARS-CoV-2 infection. The application of current mRNA vaccine technology has enabled the development of vaccines that are highly immunogenic and efficient. Acute Respiratory Syndrome Severe The third new coronavirus that has caused serious illness in several nations over the past 20 years is SARS-CoV-2. COVID-19 has overtaken global healthcare systems, with over 250 million infections.

The most effective method of pandemic control to date has been the quick development and testing of highly effective vaccines made possible by the use of proven vaccine technology. In a broad vaccination campaign, authorized COVID-19 vaccines make use of a variety of platforms, including in-vitro transcribed messenger ribonucleic acid (mRNA) for the first time ²⁰.

For those without underlying medical conditions, the majority of SARS-CoV2 infections do not represent a significant risk to life. In cases of severe infections, immune system may overreact in the lungs, resulting in the death of epithelial cells and alveoli, pulmonary edema, dangerously high vascular permeability, and even death. The spike protein present on the surface of SARS-CoV-2 binds to ACE2 receptors on host cells, enabling the virus to enter the cell ¹⁵.

Influenza Viruses: Influenza virus, a common respiratory pathogen that affects people worldwide, is a major threat to public health and world economy. mRNA-based vaccinations may prove to be an effective means of enhancing influenza immunity. mRNA-based influenza vaccinations have been demonstrated in numerous studies to elicit a strong and long-lasting immune response against the virus ¹. Because it is easy to test the effectiveness of mRNA vaccines against influenza virus in small animal models, possesses devices for evaluating T and B cell reactions., and has potential benefits, mRNA vaccines against influenza virus are among most studied. It may take at least six months to produce traditional, FDA-approved vaccinations against novel influenza pandemic viruses, during which time the public would not be protected ²¹.

Rabies Virus: Rabies, a fatal neurological illness affecting humans and other warm-blooded animals, remains a significant concern. CureVac has created CV7201, an mRNA vaccine for rabies, using cationic polypeptide protamine as delivery method. Preclinical studies involving mice and pigs have demonstrated that CV7201 effectively induces humoral immune responses and T-cell responses ¹. Recently, results from a study using a protamine-formulated, sequence-optimized conventional mRNA vaccine that encodes the glycoprotein of the rabies virus were published for the first time in humans (RABV-G). Strong neutralising antibody responses in pigs and protective immunity against a fatal intracerebral virus challenge in mice had previously been shown to result from this vaccination ^{22, 23}.

mRNA Vaccines Targeting Zika Virus Infection: Pardi et al. independently reported the effectiveness of their nucleoside-modified conventional mRNA, formulated with LNP, against Zika infection. After two 10-mg i.m. or one 30-mg i.d. vaccination, respectively, both groups showed remarkable levels of neutralizing titers and protection against lethal challenge in mice, and after a single 50-mg i.d. vaccination in NHPs. To potentially increase the vaccine's safety, Richner et al. also tested an mRNA vaccine encoding a modified Zika prM-E antigen that contained mutations that destroyed the conserved fusion-loop epitope in domain II of the E protein ^{24, 25, 26}.

HIV Virus: HIV poses a global health challenge, compromising immunity by targeting immune cells and increasing susceptibility to diseases and infections. According to estimates from Global Statistics, approximately 39.0 million individuals worldwide will be living with HIV by the end of 2022. Despite extensive research efforts, effective treatment for HIV remains elusive, highlighting the urgent need for prevention measures. Moderna has created an experimental mRNA HIV vaccine using the same platform technology as their effective COVID-19 mRNA vaccine. The vaccine has shown positive results in mice and non-human primates, proving its safety and effectiveness in producing a strong immune response against HIV. Moreover, the vaccination has been demonstrated to stimulate the generation of neutralizing antibodies ¹.

mRNA Immunisations Against Parasitic and Bacterial Diseases: Recently, the efficacy of self-amplifying mRNA against parasitic diseases has been evaluated with malaria as the target disease. plasmodium macrophage migration inhibitory factor (PMIF), which suppresses memory T cells and allows the parasites to evade the immune system, is encoded by a self-amplifying mRNA vaccine. Self-amplifying mRNA vaccination stimulated humoral and cellular immune responses against PMIF, and anti-PMIF immunoglobulin G (IgG) blocked PMIF's pro-inflammatory effects. Tfh cell and GC response were enhanced, blood-stage latency after sporozoite infection was delayed, and memory CD4+ T cells with antigen experience and liver-resident CD8+ T cells were better differentiated. In addition, mice that had recovered from their infection but had been exposed to sporozoites once more were completely immune to contracting the infection again. Adoptive transfer of CD8+ or CD4+ T cells recapitulated protection against reinfection. This study showed how effective self-amplifying mRNA vaccination is at preventing the parasite's immune-evasion strategy^{27, 28}.

mRNA in Cancer Treatment: The emergence of mRNA-based cancer immunotherapy as a promising cancer treatment approach is due to the combination of mRNA and nanoparticle technology. Clinical trials have shown that mRNA-based cancer immunotherapy is safe and effective, leading to significant tumor remission in certain patients. Because of its adaptability, mRNA can be successfully employed in a variety of cancer immunotherapy methods ⁴. Because they have therapeutic as well as preventive benefits, cancer vaccinations can be a good substitute for cancer immunotherapy.

Tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), which can be used to produce long-lasting therapeutic effects by attacking and killing cancer cells that over-express these antigens, are the specific targets of vaccines that stimulate immune memory. As a result, it is expected that mRNA vaccines will be an effective cancer treatment modality ¹. Immune checkpoint inhibitors are expected to be the future of mRNA cancer immunotherapy in conjunction with mRNA-based personalized therapeutic modalities such as tailored vaccines ⁴.

mRNA in Genetic disorders: Hereditary diseases like hemophilia, cystic fibrosis, and muscular dystrophy can benefit from the replacement or enhancement of deficient proteins or genes through messenger RNA therapy. Gene abnormalities affecting clotting factors are the cause of hemophilia, a bleeding disorder. Mutations in any gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis, also referred to as lung disease. Mutations in the gene that produces the protein dystrophin result in muscular dystrophy. By introducing mRNA that encodes specific proteins or genes to those targeted regions, mRNA treatment can improve the function of diseased cells or tissues and reduce the symptoms of the underlying illness ²⁰.

Genome Editing: Through genome editing, genetic diseases may be effectively treated by precisely adding, removing, or replacing DNA sequences at a specific locus in genomic DNA. Since, the groundbreaking discovery of the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9), gene editing has gained enormous popularity. Co-delivery of Cas9 mRNA and sgRNA can overcome the limitations of conventional CRISPR/Cas9 delivery by plasmid, minimise off-target effects, and achieve transient Cas9 protein expression ²⁹.

Quality, safety, and efficacy of mRNA-Based Vaccines:

Safety of mRNA-Based Vaccines: A phase 1 dose-escalation, open-label trial with participants aged 18 to 70 or older examined the safety of mRNA vaccines using mRNA-1273. Two groups underwent the investigation: one comprised of individuals aged 18–55 who received a dose of 250 µg, and another group comprised older subjects who received either 25 µg or 100 µg. After the vaccination, a number of negative effects were noted, including the emergence of new chronic illnesses, unforeseen side effects, systemic and local side effects, and major side effects. Headaches, exhaustion, chills, muscle stiffness, and injection site soreness were a few of the frequent side effects. Antibody levels that neutralise viruses are linked to animal and human defence against SARS-CoV-2 and other viruses ³⁰.

One benefit of mRNA manufacturing over most biologicals is that it doesn't require the use of cell cultures during production. Compared to more complex vaccine production methods, this trait's quick response time reduces the chance of contamination. RNA-based vaccinations against infectious diseases are evaluated for quality, safety, and efficacy by current regulations. Right now, the emphasis is on creating manufacturing procedures that can reliably yield products of superior quality. As a result, requirements need to be set for various crucial process steps, drug substances (DS), drug products (DP), intermediates, and acceptance criteria. Considerations include things like product yields and analytical technologies that enable accurate product characterization and quantification. These include things like product identity, purity, and quality. The quality of mRNA is assessed using gel electrophoresis and HPLC (high-performance liquid chromatography). Moreover, reverse transcription polymerase chain reaction (RT-PCR) or next-generation sequencing can be used to confirm the identity of the sample ²⁹.

Efficacy of mRNA Vaccines: After the second vaccination dose, the efficacy of mRNA-1273 was evaluated primarily for its ability to prevent symptomatic COVID-19 infection, with the secondary goal being prevention of severe COVID-19 infection.

The vaccination showed 94.1% efficacy against symptomatic COVID-19, and no severe cases were reported among those who received the shot. The vaccination showed high efficacy against various COVID-19 variants in Qatar, including 88.1% against the alpha variant after the first dose and 100% after the second dose. It also showed 95.7% efficacy against severe COVID-19. Efficacy rates against the beta strain were 61.3% and 96.4% following first and second doses, respectively, demonstrating the vaccine's ability to successfully prevent infections, hospitalizations, and fatalities ³⁰.

When compared to the majority of biological processes, mRNA manufacturing has the advantage of not requiring the use of cell cultures. The risk of contamination is lower than with other complex vaccine manufacturing processes because of its quick reaction time. Furthermore, the mRNA safety profile is favored by the transient expression and non-integrative nature of the cellular expression ^{31, 32}. Guidelines for regulations governing the assessment of RNA-based prophylactic vaccines for infectious diseases in terms of their efficacy, safety, and quality are currently under consideration. These days, focus is on developing manufacturing procedures that can produce goods of a high caliber and consistency ³³.

Current Challenges and Future Perspectives:

Challenges in mRNA Technology: mRNA vaccines offer a promising substitute for conventional vaccines because of their quick growth, great potency, and inexpensive manufacturing. However, its physiochemical properties might affect organ distribution, cellular transport, and mRNA stability. High-quality mRNA is less likely to be degraded by RNAse during synthesis. The current generation of mRNA-based vaccines against SARS-CoV-2, when administered in combination, inhibit Th2-biased immune responses, a crucial step in preventing vaccine-associated enhanced respiratory disease. Viral infections are frequently associated with acute myocarditis; cases have been documented in otherwise healthy people following vaccination against smallpox or the flu. Despite the fact that lipid-based nanomaterials are used in several vaccines to transport mRNA, each vaccine requires a different storage temperature. The BioNTech/Pfizer BNT162b2 vaccine needs to be stored at –80^oC, while the Moderna mRNA-1273 vaccine has a six-month shelf life and needs to be kept at –20^oC. There are still many obstacles to overcome, even though mRNA vaccines are safer and can be made fast using easily obtained materials. Immunogenicity, instability, low transfection efficiency, inefficient targeted delivery, and bio-incompatibility are still problems. The physical characteristics of messenger RNA (mRNA) present various obstacles to its conversion into specific antigens. These features include its high molecular weight, negative charge, susceptibility to RNases, and presence of both intracellular and extracellular barriers. Moreover, a large amount of mRNA is ambushed in endosomes at the time of entry, which prevents it from entering the cytoplasm and carrying out its intended functions. IVT mRNA transcripts have a brief half-life of five minutes in sera, indicating that they are unstable and readily broken down by primers ¹³.

Future Perspectives: The recent success in developing mRNA-based COVID-19 vaccinations has led to an explosion in preclinical and human studies exploring the potential of mRNA vaccines. Current preclinical and clinical research has demonstrated the effectiveness of mRNA technology in treating range of viral infections, like Ebola, Zika, Streptococcus, and Toxoplasma gondii. The development of mRNA vaccines against numerous cancer types has advanced. Recent developments in innate immune monitoring and in-vivo delivery technologies have sped up the development of mRNA vaccines, as demonstrated by the rapid development of mRNA-based COVID-19 vaccines. There are currently only two vaccines against cancer that are approved by the FDA, and they are for hepatitis B and human papilloma viruses ¹⁷.

CONCLUSION: Starting from its early discovery in the 1950s and 1960s and continuing for more than 50 years, the development of mRNA technology is a remarkable example of scientific innovation in action. During this journey, scientists have discovered the profound potential of messenger ribonucleic acid (mRNA), revealing avenues for ground-breaking discoveries that serve as cornerstones of contemporary medical innovation. The development of mRNA vaccines, which provide faster development and scalability than those of traditional vaccine approaches, marks a turning point in preventive medicine. The efficacy and potential of mRNA vaccines to transform disease prevention globally are demonstrated by their success stories, especially in the fight against the COVID-19 pandemic. mRNA therapies have great potential to treat a wide range of illnesses, including cancer and genetic disorders, in addition to vaccinations. But as is the case with any new technology, safety and morality must always come first. Fostering public trust and confidence in these ground-breaking therapies requires ensuring the safety of mRNA applications through stringent testing and adherence to regulatory guidelines. It seems that mRNA technology has a bright future ahead of it, with continued research efforts poised to overcome present obstacles and unlock even greater therapeutic potential. We can use mRNA to address some of the most urgent medical issues facing humanity and usher in a new era of universal health and well-being by continuing to innovate and work across disciplines.

ACKNOWLEDGMENT: The authors would like to thank Dr. Akhilesh Patel, Department of Pharmacy, NIMS institute of pharmacy, Jaipur, Rajasthan, for their kind support.

Funding: This work did not require any external funding.

Ethics Approval and Consent to Participate: As this is a review article, no ethics approval or consent to participate was required.

CONFLICT OF INTEREST: The authors declare no conflict of interest

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    How to cite this article:

    Dharane S, Hangargekar P, Bhujange D, Yawalkar D, Bhosale S and Joshi A: From concept to reality: the rise of messenger ribonucleic acid in medical marvels. Int J Pharm Sci & Res 2025; 16(3): 644-54. doi: 10.13040/IJPSR.0975-8232.16(3).644-54.

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