Synthetic mRNAs are artificially engineered molecules that can deliver genetic information to cells and direct the synthesis of proteins. Unlike conventional DNA-based therapies, which rely on the integration of foreign genes into the host genome, synthetic mRNAs do not alter the genetic makeup of the cells and thus avoid the risk of insertional mutagenesis and immune rejection. Synthetic mRNAs also offer several advantages over other RNA-based therapies, such as antisense oligonucleotides and small interfering RNAs, which mainly function as gene silencers. Synthetic mRNAs can be used to express any desired protein, either to supplement a missing or defective protein or to induce a specific cellular response, such as differentiation, proliferation, or apoptosis.
The concept of synthetic mRNAs dates back to the 1960s, when scientists first demonstrated that synthetic polynucleotides could be translated into proteins in vitro. However, it was not until the 1990s that synthetic mRNAs were successfully delivered and expressed in living cells. Since then, synthetic mRNAs have emerged as a powerful tool for various applications in biotechnology and biomedicine, such as vaccine development, gene editing, and protein replacement therapy. For example, synthetic mRNAs encoding antigens from pathogens or tumors can elicit potent and protective immune responses and thus serve as effective vaccines for infectious diseases and cancer. Synthetic mRNAs encoding nucleases, such as CRISPR-Cas9, can be used to introduce precise and targeted modifications in the genome and thus correct genetic defects or create novel traits. Synthetic mRNAs encoding therapeutic proteins, such as erythropoietin, factor VIII, or insulin, can be used to treat diseases caused by protein deficiency or dysfunction, such as anemia, hemophilia, or diabetes.
The design and synthesis of synthetic mRNAs are crucial steps for determining their functionality and efficacy. Synthetic mRNAs are prepared by in vitro transcription (IVT) using a DNA template and a bacteriophage RNA polymerase, such as T7, SP6, or T3. The DNA template contains a promoter sequence recognized by the RNA polymerase, followed by the coding sequence of the desired protein, and optionally a poly(A) tail sequence. The IVT reaction also requires nucleoside triphosphates (NTPs), magnesium ions, and buffer solution. The resulting synthetic mRNAs are then purified by various methods, such as polyacrylamide gel electrophoresis (PAGE), high-performance liquid chromatography (HPLC), or affinity chromatography.
Fig.1 Preparation process of IVT mRNA.1
One of the most important features of synthetic mRNAs is their 5' cap structure, which is essential for their stability, translation efficiency, and immune response. The 5' cap is a modified guanosine nucleotide that is linked to the first nucleotide of the mRNA by a 5'-5' triphosphate bridge. The 5' cap protects the mRNA from degradation by exonucleases, facilitates its transport from the nucleus to the cytoplasm, and enhances its binding to the translation initiation complex. However, the IVT reaction does not produce capped mRNAs, and therefore, additional steps are required to introduce the cap structure. There are two main methods for capping synthetic mRNAs: co-transcriptional capping and post-transcriptional capping.
Co-transcriptional capping involves adding a cap analog, such as m7G(5')ppp(5')G or ARCA (anti-reverse cap analog), to the IVT reaction. The cap analog competes with the GTP for the initiation of the transcription, and thus, a fraction of the synthetic mRNAs will have the cap analog at their 5' end. The advantage of this method is that it is simple and efficient, but the disadvantage is that it produces a mixture of capped and uncapped mRNAs, as well as mRNAs with inverted cap orientation, which can reduce the translation efficiency and increase the immunogenicity.
Post-transcriptional capping involves adding a capping enzyme, such as vaccinia virus capping enzyme (VCE) or Arabidopsis thaliana capping enzyme (AtCE), to the purified synthetic mRNAs. The capping enzyme catalyzes the removal of the 5' phosphate group and the addition of the GMP and the methyl group to form the cap structure. The advantage of this method is that it produces fully capped mRNAs with the correct cap orientation, but the disadvantage is that it requires additional purification steps and may cause degradation or damage to the mRNAs.
Besides the natural cap structure (cap0), synthetic mRNAs can also have modified cap structures, such as cap1 (methylated at the 2'-O position of the first nucleotide), cap2 (methylated at the 2'-O position of the second nucleotide), or pseudouridine cap (containing pseudouridine instead of uridine). These modified cap structures can improve the stability, translation efficiency, and immune response of synthetic mRNAs by increasing their resistance to decapping enzymes, enhancing their affinity for the translation initiation factor elF4E, and reducing their recognition by innate immune sensors.
The design and synthesis of synthetic mRNAs also involve other aspects, such as sequence optimization, chemical modification, and quality control. Sequence optimization aims to increase the expression and functionality of the encoded protein by adjusting the codon usage, GC content, secondary structure, and regulatory elements of the mRNA. Chemical modification aims to increase the stability and reduce the immunogenicity of the mRNA by replacing some of the natural nucleotides with modified ones, such as 1-methylpseudouridine, 5-methylcytidine, or 2-thiouridine. Quality control aims to ensure the purity, integrity, and identity of the synthetic mRNAs by using various analytical methods, such as gel electrophoresis, mass spectrometry, or sequencing.
The design and synthesis of synthetic mRNAs are challenging and require careful optimization and validation. The choice of the cap structure, the method of capping, and the other parameters of the mRNA synthesis depend on the specific application and the desired outcome of the synthetic mRNAs. The ultimate goal is to produce synthetic mRNAs that can efficiently and safely deliver the genetic information and the protein of interest to the target cells.
The delivery and expression of synthetic mRNAs are critical steps for determining their functionality and efficacy in vivo. Synthetic mRNAs need to overcome several barriers, such as extracellular degradation, cellular uptake, endosomal escape, cytoplasmic transport, and translation initiation, to reach the ribosomes and produce the desired proteins. Therefore, synthetic mRNAs require suitable delivery vehicles that can protect them from nuclease degradation, enhance their cellular uptake and endosomal escape, and facilitate their cytoplasmic transport and translation initiation.
Various strategies and vehicles have been developed for delivering synthetic mRNAs into target cells, such as liposomes, nanoparticles, and viral vectors. Among them, lipid nanoparticles (LNPs) are the most widely used and clinically successful delivery systems for synthetic mRNAs, especially for vaccines and therapeutics against infectious diseases and cancer. LNPs are composed of a lipid bilayer that encapsulates the synthetic mRNAs and can be modified with targeting ligands, such as antibodies, peptides, or aptamers, to increase their specificity and affinity for the target cells. LNPs can protect the synthetic mRNAs from extracellular degradation and enhance their cellular uptake by endocytosis. LNPs can also facilitate the endosomal escape of the synthetic mRNAs by using ionizable lipids, such as DOTAP or MC3, that can disrupt the endosomal membrane at an acidic pH. LNPs can also improve the cytoplasmic transport and translation initiation of the synthetic mRNAs by using helper lipids, such as cholesterol or DOPE, that can increase membrane fluidity and fusion.
The expression and regulation of synthetic mRNAs are influenced by several factors and mechanisms, such as mRNA stability, translation efficiency, and protein degradation. The stability of synthetic mRNAs depends on their cap structure, poly(A) tail length, and nucleotide modifications, which can affect their susceptibility to exonucleases, such as Xrn1 and Dcp2. The translation efficiency of synthetic mRNAs depends on their 5' untranslated region (UTR), coding region, and 3' UTR, which can affect their binding to the translation initiation complex, their codon usage and secondary structure, and their interaction with microRNAs and RNA-binding proteins, respectively. The protein degradation of synthetic mRNAs depends on their amino acid sequence, post-translational modifications, and subcellular localization, which can affect their recognition and ubiquitination by the proteasome, their glycosylation and folding by the endoplasmic reticulum, and their targeting and import by the mitochondria, respectively.
The delivery and expression of synthetic mRNAs are challenging and require careful optimization and validation. The choice of the delivery vehicle, the mRNA design, and the expression regulation depend on the specific application and the desired outcome of the synthetic mRNAs. The ultimate goal is to deliver and express synthetic mRNAs that can efficiently and safely produce the protein of interest in the target cells.
Synthetic mRNAs have a wide range of applications in various fields of biomedicine, such as infectious diseases, cancer, genetic disorders, and regenerative medicine. Synthetic mRNAs can be used to encode antigens, antibodies, cytokines, enzymes, hormones, or transcription factors and thus modulate the immune system, correct genetic defects, or restore physiological functions.
One of the most successful and prominent applications of synthetic mRNAs is the development of vaccines against infectious diseases, such as COVID-19, influenza, Zika, rabies, and HIV. Synthetic mRNAs encoding antigens from pathogens can induce potent and protective immune responses, both humoral and cellular, and thus confer immunity and prevent infection. Synthetic mRNAs have several advantages over traditional vaccines, such as inactivated, attenuated, or subunit vaccines, in terms of safety, efficacy, and scalability. Synthetic mRNAs do not contain any infectious material and thus eliminate the risk of reversion, contamination, or transmission. Synthetic mRNAs can elicit both neutralizing antibodies and cytotoxic T cells and thus provide broad and durable protection. Synthetic mRNAs can be rapidly and easily produced and adapted to emerging variants or strains, enabling a flexible and timely response to pandemics.
Another promising and emerging application of synthetic mRNAs is the treatment of cancer, either as monotherapy or in combination with other modalities, such as surgery, chemotherapy, radiotherapy, or immunotherapy. Synthetic mRNAs can be used to encode tumor antigens, monoclonal antibodies, checkpoint inhibitors, or cytokines, and thus activate or enhance the anti-tumor immune response or suppress or overcome the tumor immune evasion. Synthetic mRNAs can also be used to encode nucleases, such as CRISPR-Cas9, or transcription factors, such as p53, and thus introduce targeted and specific modifications in the tumor genome or restore tumor suppressor function. Synthetic mRNAs can also be used to encode therapeutic proteins, such as pro-apoptotic factors, anti-angiogenic factors, or anti-metastatic factors, and thus induce tumor cell death, inhibit tumor growth, or prevent tumor invasion.
Synthetic mRNAs also have potential applications in the treatment of genetic disorders, such as cystic fibrosis, hemophilia, or muscular dystrophy. Synthetic mRNAs can be used to encode functional proteins that are missing or defective due to mutations in the corresponding genes and thus supplement or replace the deficient proteins or correct the physiological consequences. Synthetic mRNAs can also be used to encode nucleases, such as CRISPR-Cas9, or base editors, such as ABE or CBE, and thus repair or correct the mutations in the target genes or introduce beneficial mutations in the compensatory genes. Synthetic mRNAs can also be used to encode transcription factors, such as OCT4, SOX2, KLF4, or MYC, and thus reprogram the somatic cells into induced pluripotent stem cells (iPSCs), which can then be differentiated into various cell types for cell replacement therapy.
Synthetic mRNAs also have potential applications in the field of regenerative medicine, such as wound healing, tissue engineering, or organ transplantation. Synthetic mRNAs can be used to encode growth factors, such as VEGF, FGF, or PDGF, or extracellular matrix proteins, such as collagen, elastin, or fibronectin, and thus promote angiogenesis, cell proliferation, or matrix deposition, and facilitate wound healing or tissue regeneration. Synthetic mRNAs can also be used to encode transcription factors, such as OCT4, SOX2, KLF4, or MYC, and thus reprogram the somatic cells into iPSCs, which can then be differentiated into various cell types for tissue engineering or organ transplantation. Synthetic mRNAs can also be used to encode immunomodulatory factors, such as IL-10, TGF-β, or IDO, and thus suppress or modulate the immune response and prevent or reduce the rejection or inflammation of the transplanted tissues or organs.
In conclusion, synthetic mRNAs have emerged as a novel and versatile biotechnology platform for disease prevention and treatment, with many advantages over other modalities such as DNA, protein, or viral vectors. However, synthetic mRNAs also face some challenges and limitations, such as stability, delivery, expression, regulation, safety, and efficacy, which require further optimization and validation. Moreover, synthetic mRNAs also encounter some ethical, regulatory, and social issues, such as patent, approval, access, and acceptance, which require further discussion and resolution. Nevertheless, synthetic mRNAs have shown great promise and potential in various preclinical and clinical studies and have demonstrated remarkable results and benefits in the fight against COVID-19. Synthetic mRNAs are expected to revolutionize the field of biomedicine and to provide new and better solutions for many unmet medical needs.
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