Biomedical Applications of mRNA Using Nanomedicine: Comparison
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The development and commercialization of mRNA COVID-19 vaccines have pivoted the way towards future applications of mRNA medicines by finally finding a solution to the problem of delivery. Despite concerns from detractors about a lack of applications for nanotechnology, nanomedicine has now demonstrated that it is a translational, relevant and much-needed approach to engineer new genetic drugs. Thus, it is now clear that the full potential of mRNA therapeutics, along with its advantages could not be envisioned without nanomedicine.

  • mRNA
  • LNPs
  • vaccine
  • COVID-19
  • nanomedicine
  • nanotechnology
  • gene therapy
  • CRISPR
  • cancer
  • protein replacement

1. Prospects and Limitations of mRNA

The first isolation of messenger RNA (mRNA) was published in Nature in 1961 [1[1][2],2], starting the path towards a full understanding of this molecule [3]. The potential to synthetically produce mRNA from DNA in the 1980s, i.e., in vitro transcribed mRNA (IVT mRNA), opened a broad spectrum of preclinical applications [4]. Since then, multiple efforts have been performed to understand its mechanism of action and the path towards developing mRNA-based drugs. Therapeutic mRNA’s potential lies in its capacity to induce the expression of proteins [4,5][4][5] for preventing or altering a particular disease state. mRNA therapeutics hold many opportunities, which can depend on the targeted cells, organ selective accumulation and encoded protein of interest. Nowadays, however, two main approaches are considered when using mRNA, both of which will be reviewed and explained in depth. The first is dendritic cell (DC) targeting, so as to achieve immune activation. The second is to exploit the natural capacity of nanoparticles to accumulate in the liver and use it to endogenously produce therapeutic proteins.
Importantly, mRNA has several advantages over other gene therapies such as DNA or pDNA which make this molecule more translational in terms of pharmaceutical properties: (i) mRNA does not need to reach the nucleus of the cell as opposed to DNA, resulting in more efficient delivery; (ii) mRNA does not integrate into the genome of the host cell, a fact associated with risk of mutagenesis; (iii) mRNA can be synthesized in the lab with fairly easy protocols, following scalable processes in agreement with GMP regulations; and (iv) mRNA sequences can be easily modified and updated, which is an important fact to consider in vaccinology (i.e., when mutations of the target protein occur). These advantages are also important when comparing mRNA technology to protein delivery technology, which normally comes with short half-lives and expensive and tedious industrial processes [4,5,6][4][5][6].

2. Biomedical Applications of mRNA Using Nanomedicine

The approval of mRNA vaccines using LNPs can indeed be considered the biggest milestone to foster the clinical translation of many other mRNA nanoformulations that can address current clinical needs. Indeed, the technology can be applied to a broad spectrum of applications and therapeutic areas. Vaccines for infectious diseases might be the first application that has reached the market, but nanoformulated mRNA has the full potential to answer clinical demands related to the needs of therapeutic proteins. In the next sections, we aim to provide a full overview of the most promising developments to date. Biomedical applications for mRNA therapeutics are pictured in Figure 41.
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Figure 41. Biomedical applications for mRNA therapeutics. Immuno-oncology treatments can be envisioned using the liver as a factory of therapeutic proteins such as neoantigens or antibodies, as well as by using the antigen-presenting cells’ physiological function to induce immune activation towards cancerous cells. The latter can also be applied for the development of safe and efficacious mRNA and saRNA vaccines against infectious diseases. Gene delivery for therapeutic protein synthesis or restoration of a particular enzymatic function as well as gene editing using CRISPR cas9 technology can be achieved by targeting the liver. Cardiovascular regeneration can be achieved by intracardial delivery of mRNA encoding for growth factors.

2.1. Infectious Diseases

Despite the fact that COVID-19 vaccines have been the first marketed mRNA formulations, this technology has been researched for decades using different delivery approaches, from LPX to cationic nanoemulsions to polymeric nanoparticles [9][7]. Since mRNA loaded into liposomes demonstrated for the first time that it could trigger a specific T-cell response against influenza virus in mice [141][8], the field has enormously advanced. In fact, only in 2019, 15 mRNA candidates entered clinical trials [10][9]. Because this area has very recently been covered in an extensive review [142][10], wthe researchers will only showcase some of the most promising approaches, redirecting the reader to this article for further information. Nonetheless, all the clinical trials for infectious diseases other than COVID-19 are shown in Table 1. In this regard, clinically, respiratory syncytial virus (RSV) and influenza virus are amongst the most advanced clinical candidates.
Table 1. mRNA Nanomedicines for Infectious Diseases in Clinical Trials. A list of clinical trials for infectious diseases other than COVID-19 is provided in this table.
Infectious Diseases Other than COVID-19
TherapeuticClinical ApplicationFormulationPhaseNCT NumberSponsor
][30].
Today, both strategies described, (1) and (2), are under clinical evaluation in phase 1 or 2. Melanoma, prostate cancer, triple-negative breast cancer and head and neck cancer are some of the biomedical applications in which this technology is being clinically investigated (Table 2). An extensive review on this matter has been recently published [161][31].
Table 2. mRNA Nanomedicines for Oncology in Clinical Trials. A list of clinical trials for oncology applications, including the subsections described above, is provided in this table.
Oncology
Cancer Vaccines
Shared Tumor Antigens
BNT111 Advanced Melanoma Modified mRNA-LPX Phase II NCT04526899 BioNTech
BNT112 Prostate Cancer Modified mRNA-LPX Phase I/II NCT04382898 BioNTech
BNT113 HPV16+ head and neck cancer Modified mRNA-LPX Phase II NCT04534205 BioNTech
BNT114 Triple-Negative Breast Cancer Modified mRNA-LPX Phase I NCT03815058 BioNTech
BNT115 Ovarian Cancer Modified mRNA-LPX Phase I NCT04163094 BioNTech
mRNA-5671 Carcinoma, NSCLC, Pancreatic and colorectal neoplasms Non-disclosed Phase I NCT03948763 Merck/Moderna
Personalized cancer vaccines
mRNA-4157 Melanoma Modified mRNA-LNPs Phase II NCT03897881 Moderna
BNT122 Advanced Melanoma Modified mRNA-LPX Phase I NCT03815058 BioNTech
mRNA-4157 Solid Tumors Modified mRNA-LNPs Phase I NCT03313778 Moderna
mRNA-2752 Solid Tumors Modified mRNA-LNPs Phase I NCT03739931 Moderna
CV8102 Solid Tumors Not disclosed Phase I NCT03291002 CureVac
V941 Solid Tumors Not disclosed Phase I NCT03948763 Merck Sharp & Dohme Corp.
Medi1191 Solid Tumors Modified mRNA-LPNs Phase I NCT03946800 MedImmuneLC
mRNA-2416 Solid Tumors and Lymphoma Modified mRNA-LNPs Phase I/II NCT03323398 Moderna
mRNA-2752 Solid Tumors and Lymphoma Modified mRNA-LNPs Phase I NCT03739931 Moderna
mRNA-2752 Carcinoma Modified mRNA-LNPs Phase I NCT02872025 Moderna
BNT122 Colorectal Cancer Stage II/III Modified mRNA-LPX Phase II NCT04486378 BioNTech
MVT-5873 Pancreatic Cancer Not disclosed Phase I NCT02672917 BioNTech
BNT131 Metastatic Neoplasm Not disclosed Phase I NCT03871348 BioNTech
The Liver as a Bioreactor
Encoding Cytokines
BNT152, BNT153 Solid Tumor Not disclosed Phase I NCT04710043 BioNTech
BNT151 Solid Tumor Not disclosed Phase I/II NCT04455620 BioNTech
mRNA-6231 Autoimmune Disorders Modified mRNA-LNPs Phase I NCT04916431 Moderna
Encoding Antibodies
BNT142 Solid Tumor Encoding Ab Phase I/II NCT04503278 BioNTech
mRNA-1944 Chikungunya Modified mRNA-LNPs Phase I NCT03829384 Moderna
mRNA-1653hMPV/PIV3Modified mRNA-LNPsPhase INCT04144348

NCT03392389		Moderna
mRNA-1345RSVModified mRNA-LNPsPhase INCT04528719Moderna
mRNA-11777RSVModified mRNA-LNPsPhase IUnregisteredModerna, Merck
mRNA-1172RSVModified mRNA-LNPsPhase IUnregisteredModerna, Merck
mRNA-1893ZikaModified mRNA-LNPsPhase INCT04064905Moderna
mRNA-1325ZikaModified mRNA-LNPsPhase INCT03014089Moderna
mRNA-1647CMVModified mRNA-LNPsPhase IINCT04232280Moderna
mRNA-1647CMVModified mRNA-LNPsPhase IINCT03382405Moderna
mRNA-1443CMVModified mRNA-LNPsPhase INCT03382405Moderna
MRT5400Influenza A (H3N2)Not disclosedPhase IUnregisteredTranslateBio, Sanofi
MRT5401Influenza A (H3N2)Not disclosedPhase IUnregisteredTranslateBio, Sanofi
mRNA-1010Influenza A, Influenza BNot disclosedPhase II/IIINCT04956575Moderna
mRNA-1851Influenza A (H7N9)Modified mRNA-LNPsPhase INCT03345043Moderna
mRNA-1440Influenza A (H7N8)Modified mRNA-LNPsPhase INCT03076385Moderna
mRNA-1388ChikungunyaModified mRNA-LNPsPhase INCT03829384Moderna
CV7201RabiesNon-modified mRNA- in RNActivePhase INCT02241135CureVac
CV7202RabiesUnmodified mRNA-LNPsPhase INCT03713086CureVac
GSK3903133ARabiessaRNA–cationic nanoemulsionPhase INCT04062669GSK
Abbreviations: hMPV/PIV3—human metapneumovirus/parainfluenzavirus type 3; RSV—respiratory syncytial virus; CMV—cytomegalovirus; HPV—human papillomavirus.
Furthermore, preclinically, one of the most important applications is the HIV vaccine. HIV currently affects 38 million people; these numbers are expected to increase by 2030 [143][11]. This virus has been extensively researched for decades; however, its retroviral nature, along with the fact that it possesses a very high antigen diversity in its envelope, make it very difficult to achieve good outcomes [144][12]. In recent years, several studies have tried to deliver mRNA-vaccine encoding for HIV proteins; however, their success has not been as expected [127,129,145][13][14][15]. Nevertheless, a recently published study has shown broad neutralization and reduction in the risk of infection in rhesus macaques upon the administration of mRNA encoding for both HIV-1 envelope protein and simian immunodeficiency virus Gag proteins, which would be responsible for forming virus-like particles (VLP) [146][16]. A different approach based on targeting hepatocytes, which will be further discussed for immuno-oncology applications in Section 3.2.2below, has achieved good results. This sentudry involved the delivery of mRNA encoding the light and heavy chains of VRC01, a neutralizing antibody against HIV-1. Using LNPs, they were able to achieve robust protein expression in the liver. Furthermore, prophylactic immunization was also achieved at a dose of 0.7 mg/kg as opposed to the 10–20 mg/kg needed if the recombinant protein is administered [147][17].
Another important preclinical advancement was achieved with the Ebola vaccine. Ebola virus (EbolV) caused the deaths of more than 11,000 lives across West Africa from 2014 to 2016 [148][18]. This lethal virus causes a hemorrhagic fever that is fatal in up to 50% of the patients [148][18]. In 2019, one viral vector-based Ebola vaccine was approved by the FDA; however, clinical trials reported some safety concerns. As explained in the introduction, from this point of view, mRNA vaccination presents an advantage. In a study on guinea pigs, 20 μg of glycoprotein-encoding mRNA was administered using LNPs as a delivery vehicle, resulting in good antibody titers and protection from death [149][19].
Lastly, it is important to note the advancements achieved in other infectious diseases other than viruses. In this regard, Plasmodium, the causal agent of Malaria, is highlighted next. Very recently, the first malaria vaccine was approved by WHO [150][20]. The RTS, S malaria vaccine is a recombinant protein vaccine which has shown to prevent lethal disease, and is especially efficacious when given together with pharmacological treatment [150][20]. Several studies have also been conducted towards the development of an mRNA vaccine against malaria. The first is an saRNA-based [151][21] encoding for Plasmodium-secreted cytokine macrophage migrating inhibitory factor, which had previously shown efficacy [152][22]. In this study, anti-Plasmodium antibodies and protective T-cell memory were achieved [151][21]. In another study, Plasmodium falciparum acid-rich protein (PfGARP) mRNA-encoded LNPs were dosed to infected aotus monkeys, showing reduced levels of the parasite after three doses [153][23].
Altogether, these advancements provide strong evidence of the possibilities ahead regarding mRNA vaccinology, which seems to provide necessary advantages as the next vaccine technology, especially when considering future pandemics.

2.2. Immuno-Oncology

RNAs are critical for gene expression in their multiple forms, whether in non-coding forms such as miRNA, siRNA or tRNAs that regulate transcription and translation, or in coding forms such as mRNA [154][24]. Cancer immunotherapy has been a game changer since the approval of the immune checkpoint inhibitor ipilimumab in 2011 [155][25]. The number of immuno-oncology (IO) drugs in the development pipeline in 2020 grew by 22% compared to 2019, accentuating the high interest in these therapies. Immunomodulators, cell therapies and cancer vaccines represent the most important trends [156][26]. In fact, these therapies are often given simultaneously, with one example of this being TriMiX, whose composition is CD70, CD40 ligand and TLR4 [157][27]. However, scientists have met a few challenges when it comes to the new development of these immunotherapeutic agents, mostly related to off-target toxicity [158][28]. Thus, mRNA technology can offer advantages in terms of scalability, cost and production issues, widening immunotherapy use worldwide. Altogether, immunotherapy combined with nanomedicine could bring a potential solution: to engineer the delivery of immunomodulators, focusing their action on target tissues such as tumors or tumor-draining lymph nodes, or specific cell types, so as to control the timing and location of immunomodulation [158][28]. To achieve this, two main strategies can be performed: the first, by targeting DCs and taking advantage of their natural immune role; and the second, by making use of liver capacity to synthesize proteins. In the following sections we will describe both mechanisms in depth.

2.2.1. Cancer Vaccines: Targeting Dendritic Cells

The most well-known strategy for treating cancer with mRNA is cancer vaccines, in which mRNA is transcribed into a protein which in turn trains the immune system to fight against the tumor. Thus, rather than being prophylactic, cancer vaccines seek to stimulate cell-mediated responses. Therapeutically, this technology can be applied (1) by using shared antigens for the same tumor to stimulate immune activation, or (2) by designing a personalized cancer vaccine which possesses the individual’s neoantigens, i.e., proteins preferentially expressed in cancerous cells but not in the patients’ healthy tissue, such as growth-associated factors or antigens [9][7].
The way the latter strategy (2) is approached involves the most advanced tissue gene sequencing: a patients’ tumor and healthy tissue samples are taken and analyzed looking for tumor-associated antigens, as pictured in Figure 52.
/media/item_content/202203/6225dc28d870cpharmaceutics-14-00460-g005.png
Figure 52. Personalized mRNA cancer vaccines. Healthy and tumor tissue are isolated from the patient sample, analyzed and compared in order to find mutations present in the tumor tissue. Bioinformatic tools are used to predict the MCH Class I and II binding. A tandem gene encoding for several neoantigens is then designed and retrotranscribed to mRNA. Finally, by making use of nanotechnology, the mRNA is formulated and delivered. Adapted with permission from [159][29], published by Nature Reviews Drug Discovery, 2018.
The technology relies on a special algorithm prediction of binding strength to self-major histocompatibility complex (MHC) receptors. After mutation identification, neoantigens are ranked based on how well they would bind to this receptor, which will then drive the activation of T cells, followed by the immunological response against that specific neoantigen. Upon administration of the mRNA into a suitable, stable and efficient formulation, DCs are transfected, and due to their antigen-presenting cells’ nature, they localize the neoantigen onto their MHC receptor located in their surface, which will be then presented to the T cells, starting the immune cascade [159,160][29
Abbreviations: Ab—antibodies; NSCLC—non-small-cell lung cancer.

2.2.2. The Liver as a Factory of Immunomodulatory Proteins

As commented in the previous sectionly, targeting DCs is the main strategy towards the development of cancer vaccines. Nonetheless, this is not the only approach in which mRNA can be used for immunotherapy. This approach is based on the on the fact that nanoparticles have the intrinsic capacity to accumulate in the liver [162][32], which has been for many years the core center of the research. Thus, targeting the liver can be achieved by exploiting its capacity for protein production in order to build up an endogenous factory of therapeutic proteins, which will then be released into systemic circulation. In fact, the feasibility of targeting the liver for mRNA delivery for the endogenous production of immunomodulators can be envisioned by considering advances in other research areas, such as protein replacement therapies with mRNA, which will also be covered in Section 3.3below. Furthermore, this strategy also overcomes the main disadvantages of many immunomodulator administrations, such as their short half-life and subsequent long perfusions and recurrent visits to the medical doctor’s office, as well as their high production cost [163][33].
This mRNA application has been studied for two types of recombinant proteins: immunomodulators and antibodies. The production of immunomodulators, such as cytokines, has already been proven to be an achievable strategy. For instance, intratumoral administration of LNPs with mRNA-encoding cytokines IL-23 and IL-36 and the T-cell costimulatory OX40L was proven to lead to tumor regression in colon cancer and melanoma models and is now under clinical trials (NCT03739931) [164][34]. In another study using IL-12, a well-known cytokine [165][35], Lai et al. reported mRNA-encoding IL-12 LNPs to be a good candidate for slowing down the progression of MYC oncogene-driven human hepatocellular carcinoma, with good biodistribution within the tumor and no toxicity. Treatment reduced liver-tumor burden and increased survival in MYC-induced HCC transgenic mice [166][36].
Interestingly, co-delivery of cytokines and antibodies has proven to have potential for treating different types of cancer [166,167,168][36][37][38]. In fact, the use of mRNA to endogenously produce antibodies has also been achieved. Apart from the already-commented HIV study inbefore Section 3.1 [147][17], another study with trastuzumab aimed to apply this strategy to tackle oncology. Rybakova et al. succeeded in efficiently delivering mRNA encoding for trastuzumab, a monoclonal antibody widely used for breast cancer. They found a dose-dependent increase in trastuzumab protein levels in mouse serum after 24 h administration. When the administration of the monoclonal antibody Herceptin® was compared, results revealed similar half-lives for both antibodies (i.e., endogenously produced and Herceptin); however, the mRNA-based delivery resulted in a 64% higher antibody serum concentration over the course of 30 days. Overall, a single dose of LNPs with the mRNA at 2 mg/kg showed a favorable pharmacokinetic profile when compared to a single injection of Herceptin at 8 mg/kg [169][39]. This technology has also been proven to be efficient for the delivery of bispecific antibodies [170][40]. Stadler et al. succeeded in achieving a promising therapeutic outcome by generating his-tagged bispecific antibodies directed against the T-cell receptor-associated molecule CD3, a tumor-associated antigen [170][40].

2.2.3. CAR-T Cells

On a last note on immunotherapy, the CAR-T-cell field can also take advantage of mRNA technology. Chimeric antigen receptor (CAR) T-cell therapy is considered by many the next promise of cancer immunotherapy. In August 2017, the FDA approved the first CAR-T treatment, Kymriah® (tisagenlecleucel), for the treatment of certain patients, both pediatric and young adults with B-cell acute lymphoblastic leukemia (B-ALL). Several months after that, in October 2017, the FDA approved Yescarta® (axicabtagene ciloleucel) for adult patients with certain types of refractory B-cell non-Hodgkin lymphoma (B-NHL) [171][41] This approach consists of engineering lymphocyte T surfaces with tumor specific antigens that would bind to tumor-specific receptors. Right now, CD19-targeting CAR-T-cell therapy is approved for both B-cell acute lymphoblastic leukemia (B-ALL) as well as non-Hodgkin lymphoma (NHL), where it has demonstrated up to 90% complete remission when rayed in chemo-refractory B-cell malignancies [171][41]. Despite the promising positive outcomes on liquid tumors, CAR-T therapy has challenges in its application to solid tumors, including limited cancer-specific targets and non-persistence of the transferred CAR-T cells, allowing for low side effects but efficient tumor eradication [172][42], and research needs to be carried out to tackle solid tumors such as breast, melanoma or ovarian. With regard to solid tumors, in a recent paper published, T-cells were bioengineered with nanoparticle-encapsulating mRNA encoding for a single-chain variable fragment (scFv) that can specifically bind to CLDN6, an oncofetal cell-surface antigen for CAR-T-cell targeting. Results demonstrated that RNA nanoparticles can efficiently stimulate CAR-T cells [173][43].

2.3. RNA-Based Protein Replacement Therapies

As mentioned before, attempts to deliver therapeutic antibodies as mRNA molecules have been carried out with success with the aim of overcoming the drawbacks of administering the therapeutic protein. So far, protein replacement therapies are mainly focused on secreted proteins or proteins of bacterial origin. It is important to understand the processes following the translation of mRNA into proteins, which include folding, post-translational modification, aggregation into secretory granules and transport outside the cell. One of the most important things to consider are the signal peptides, as they take a great role in directing the protein secretion. Unless the signal sequence is optimized, extracellular mRNA should be transfected ideally into cells that the encoded protein is naturally secreted in [174][44]. A table with current clinical trials on going is provided in this section (Table 3).
Table 3. mRNA Nanomedicines for Protein Replacement Therapies in Clinical Trials. A list of clinical trials for protein replacement applications is provided in this table.
mRNA Delivery for Protein Replacement Therapies
mRNA-3927 Propionic Acidemia Modified mRNA-LNPs Phase 1/2 NCT04159103 Moderna
MRT5005 Cystic Fibrosis Modified mRNA-LNPs Phase 1/2 NCT03375047 Translate Bio

2.3.1. Cystic Fibrosis

Cystic fibrosis (CF) is a genetic disease which affects over 90,000 individuals all over the world. The F508del is the most prevalent CF-causing mutation, and affects approximately 82% of the CF population. This mutation leads to CFTR-protein misfolding, which is then arrested by the cell machinery and targeted to the proteasome, leading to its premature degradation [175][45]. Thus, mRNA-mediated replacement therapy can be applied to produce copies of the cystic fibrosis conductance receptor (CFTR), which is defective in CF patients. However, when it comes to delivery of mRNA nanoparticles to the lung, these nanoformulations not only need to be endocytosed, escape the endosome and release the cargo inside the cytoplasm, but they also need to migrate across viscous mucus, avoid clearance and uptake by the macrophages within this mucus and the airways and finally reach the pulmonary epithelial cells [175,176][45][46]. These additional biological barriers make the delivery of mRNA-loaded nanoparticles to the lung especially challenging. To date, the most advanced mRNA therapy for cystic fibrosis comes from Translate Bio and it is currently in Phase 1/2 (NCT03375047) [177][47].

2.3.2. Rare Metabolic Diseases

An important area which would be enormously impacted by this technology are rare metabolic diseases. These disorders lack financial support, and thus, effective treatments. From this point of view, mRNA technology can entail an enormous advantage due to the cost-efficient matters already commented. Furthermore, the previous idea and background given on endogenously produced proteins can also be applied in this section.

Acute Intermittent Porphyria

Acute intermittent porphyria is a chronic disease caused by the hepatic deficiency of porphobilinogen deaminase (PBGD), the enzyme which catalyzes the transformation of porphobilinogen into protoporphyrinogen during the heme synthesis pathway [178][48]. This causes the accumulation of the porphyrin precursors γ-aminolevulinic acid (ALA) and porphobilinogen (PBG), which entails acute neurovisceral attacks associated with a high production of potentially neurotoxic porphyrin precursors [178][48]. Treatments so far are composed of hemin replacement therapy, which restores the regulatory heme pool in the liver and suppresses the accumulation of precursors; however, these reductions occur days after the injection and the levels of enzymes should be monitored closely. Although this is an efficacious treatment, 5% of patients suffer recurrent attacks over the years. Prophylactic hemin treatment is being more commonly used for chronic symptoms; however, repeated administrations have been shown to have important side effects [179][49] RNA therapies seem to give an answer to the treatment of this disease. The siRNA drug Givosiran® aims to silence the ALAS1 enzyme, stopping the production of ALA and PBG. mRNA therapies are still in preclinical development. In this regard, Jiang et al. evaluated, along with the Moderna, the potential treatment of this disease with mRNA-encoding PBGD enzyme into LNPs in two models of acute hepatic porphyria. Furthermore, they also performed a multiple-dose study on non-human primates to further prove its translatability to humans. The results showed that administration of hPBGD mRNA led to expression of the therapeutic protein after 2 h post-administration, and most importantly, maintained its activity throughout the entire duration of the attack [180][50]. Overall, this sentudry reported sustained efficacy and tolerability of the hPBGD mRNA after single and repeated administrations in mice, rabbit and non-human primates [180][50], which further evidenced the potential of mRNA for the treatment of this disease.

Propionic Acidaemia (PA)

Propionic Acidaemia (PA) is a rare pediatric disorder caused by propionyl-CoA carboxylase (PCC) deficiency, which results in impaired propionate metabolism and accumulation of toxic metabolites, leading to a life-threatening condition. There are two disease subtypes, PCCA deficiency and PCCB deficiency, each of them referring to the different subunits forming PCC, respectively A (of 72 kDa) or B (of 54 kDa). Current treatments for this disease include fluid therapies or actions to remove the excess of toxic metabolites, but liver transplant is also a potential outcome for these patients. In this term, mRNA-therapeutic encoding for PCC enzymes can provide a solution. In this study, Jiang L. et al. report the use of two mRNAs encoding both human PCCA and PCCB. Results showed the restoration of ammonia levels and the function of the PCC enzyme in the liver; reducing toxic metabolites associated with this condition up to 6 months upon repeated administrations in mice [181][51]. Currently, mRNA-3927 from Moderna Therapeutics is under clinical trials (NCT04159103).

Fabry Disease

Fabry Disease is an X-linked lysosomal storage disorder which results from the mutation of the gene GLA encoding for the lysosomal enzyme α-galactosidase A, responsible for glycolipids’ metabolism. Its loss of activity leads to fat accumulation within the lysosomes of multiple tissues, which in turn leads to clinical disease with fatal renal and cardiac function alteration [182][52]. Current treatments for this metabolic disease include enzyme replacement therapies named Replagal® and Fabrazyme® [183][53]; and migalastat [184][54], a small molecule chaperone. While these treatments have shown to slow the progression of the disease, the variability of response, immunogenicity and difficulties with the manufacturing of these therapies are needs which have not yet been met. In this context, the main studies with mRNA have been published so far, showing some advancements. Using mRNA encoding for α-galactosidase, DeRosa et al. demonstrated it is possible to achieve sustained delivery in a Fabry model mouse and in a non-human primate with a resulting reduction of relevant biomarkers [185][55]. Upon delivery, serum protein levels reached as high as 1330-fold over normal physiological values. Another study conducted by Moderna also showed promising results upon intravenous administration of LNPs, with a dose-dependent protein activity and substrate reduction maintained for up to 6 weeks in mice. Upon repeated administration, the pharmacodynamic response was sustained and the administration to non-human primates confirmed safety and translatability [186][56].

Methylmalonic Acidemia (MMA)

Methylmalonic Acidemia (MMA) is a rare metabolic disease which is characterized by mutations in the gene coding of the vitamin B12-dependent enzyme methyl-malonyl-CoA (MUT), causing partial or complete loss of activity. This enzyme catalyzes important metabolic reactions linked to the Krebs cycle, playing a central role in intermediary metabolism. Its partial or total lack of activity causes the accumulation of toxic metabolites and the alteration of mitochondrial oxidative phosphorylation. Clinically, patients affected with this disorder experience multisystemic complications which are more common in organs with high metabolic demand, such as the brain or heart [187][57].
There is currently no effective treatment for these patients, and this disorder is often treated by carnitine supplementation and protein-limited diets; however, mortality is still around 20%, and survivors can develop systemic complications. mRNA therapy encoding for human MUT using LNPs as delivery vehicles was studied by An et al. in two clinically relevant models. In both, a decrease in the enzyme substrate methylmalonic acid blood levels were achieved, with good pharmacokinetic properties. Importantly, none of the clinical models developed any hMUT antibodies upon repeated administration or hepatic inflammation [188][58].

Ornithine Transcarbamylase Deficiency (OTD)

Ornithine transcarbamylase deficiency (OTD) affects 6 in 100,000 people, and is the most frequent inborn error of the urea cycle. As other urea-cycle disorders, it results in ammonia accumulation which clinically translates to fatal neurological disorders. OTD is more specifically caused by a deficiency in ornithine transcarbamylase (OTC). Therapeutic options for these patients include a protein-restricted diet for life, with arginine and citrulline supplementation, but liver transplantation is the only therapeutic option in cases of severe OTC deficiency [189][59]. In this study, the mRNA administration resulted in increased OTC enzymatic activity up to 10 days after dosing, along with increased mice survival [190][60].

2.4. Gene Editing

Clustered regularly interspaced short palindromic repeat (CRISPR)-related systems have become the main tool when considering therapeutic approaches for the cure of genetic diseases. CRISPR-Cas9 complex is composed of two main elements: Cas9 protein, an endonuclease that is capable of DNA cleaving; and a single-guide RNA (sgRNA), which guides the Cas9 protein to the site of interest. This gene-editing tool is, therefore, based on the recognition of PAM (protospacer-adjacent motif) sequence and a complementary 20-nucleotide genomic sequence that induces DNA brakes, which are repaired by error-prone nonhomologous end-joining or precise homology-directed repair [191][61]. To date, CRISPR/Cas9-based systems are versatile and have extensively been used for biotechnological purposes such as knockout/knock-in animal models or cells. From a drug-delivery point of view, it is important to understand that intracellular delivery of both components is needed. Therefore, various in vivo studies addressing differing delivery approaches such as viral vectors, mRNA or recombinant protein have been performed. In this section we will focus solely on mRNA delivery will be the focus, as it is the purpose of this reviewentry [191,192,193,194,195][61][62][63][64][65].
One of the approaches that have been most extensively researched are hepatic diseases. In 2016, a study on human hereditary tyrosinemia (HHT) showed slightly promising results on correcting the fumarylacetoacetate hydrolase (FAH) mutation. This was performed by the systemic delivery of Cas9 mRNA encapsulated in LNPs and sgRNA encapsulated in AAV particles. The final results demonstrated an overall gene correction of 6%, suggesting that this could be an effective approach for the treatment of a range of metabolic liver diseases. However, authors pointed out that one potential reason for the mild gene correction could be that the nanoformulations were not co-delivered, hypothesizing that the sgRNA suffered degradation during mRNA translation, and thus decreasing efficacy [195][65]. Following up on this, another study conducted by the same team aimed to deliver both Cas9 mRNA and sgRNA using LNPs for both components. In order to make it possible for the sgRNA to be efficiently delivered, they conducted several chemical modifications on the sequence, i.e., including the addition of 2’ hydroxyl (OH) groups on nucleotides, which resulted in highly improved results with an efficacy of 80% [196][66] This restudyearch is especially important as it highlights the importance of chemical modifications as well as the possibility of using synthetic nanoparticles, broadening up the industrial applications of gene editing. Similarly, Miller et al. reported successful results with a 95% protein knockdown in vitro, by making use of a zwitterionic lipid as a delivery vector by also co-delivering the different components [197][67] Adding to this latter study, Finn et al. reported a 97% reduction in serum protein levels upon the administration of Cas9 mRNA co-formulated with modified sgRNA into LNPs for gene editing of the mouse transthyretin gene in the liver [198][68]. To sum up, probably the most important advancement with regards to gene editing is the NTLA-2001 clinical study (NCT04601051), the first clinical trial so far with CRISPR-Cas9, conducted by Intellia Therapeutics for patients with hereditary ATTR amyloidosis suffering from polyneuropathy. This gene-editing medicine is based on the CRISPR Cas9 system and it uses mRNA-encoding Cas9 protein and the guide RNA encapsulated in a LNPs. Results showed mild adverse effects, and the administration led to a decrease in TTR after a single dose [199][69]. In October 2021, Intellia Therapeutics announced the beginning of a new CRISPR clinical trial, NTLA-2002, for the treatment of hereditary angioedema (HE), a rare autosomal dominant disorder that causes unexpected inflammatory attacks [200][70]. Altogether, these results present an undeniable precedent for gene editing. A current landscape on CRISPR-Cas9 clinical trials is provided next (Table 4).
Table 4. mRNA Nanomedicines for Gene editing in Clinical Trials. A list of clinical trials for gene editing applications is provided in this table. Abbreviations: sg, single-guide.
Gene Editing
NTLA-2001 Hereditary Transthyretin Amyloidosis Cas9 mRNA and sg mRNA-LNPs Phase 1 NCT04601051 Intellia Therapeutics
NTLA-2002 Hereditary Angioedema Cas9 mRNA and sg mRNA LNPs Phase 1/2 Not registered yet Intellia Therapeutics

2.5. Autoimmune Diseases

Autoimmune diseases are chronic and debilitating conditions which are caused by immune response against self-antigens. Disorders such as multiple sclerosis or diabetes type I affect millions of people worldwide. Current treatments available do not target the origin of the disease and are not curative. However, immune tolerance—defined as the state of unresponsiveness of the immune system (IS) to any substance that has the potential to trigger a response—has been described before as a potential therapeutic tool [201,202][71][72]. In this term, mRNA technology could potentially be used to induce self-tolerance, by administering a self-antigen encoded in mRNA. In a recent study, Krienke et al. [203][73] showed that it is possible to deliver myelin oligodendrocyte glycoprotein (MOG)-encoded mRNA by making use of the lipoplex-type nanocarrier to multiple-sclerosis mice models. Upon intravenous administration, modified mRNA was capable of triggering the proliferation of T-reg cells without inducing immune activation, preventing myelin damage and paralysis [203][73]. This is, to the best of our knowledge, the first proof of concept using mRNA technology for immune tolerance.

2.6. Cardiovascular Diseases

Another potential for the development of mRNA therapeutics is treating myocardial infarction (MI) and heart failure (HF), the leading causes of death in many countries. MI leads to a massive loss of cardiomyocytes (CM), which are replaced by highly proliferating fibroblasts, resulting in loss of healthy tissue. These changes are irreversible, and along with oxidative stress and inflammation, the ultimate result is heart failure. Gene therapy with DNA-based or viral vectors has been used to try to induce cardiac regeneration, but has not had great success due to poor and uncontrolled gene expression. However, mRNA technology could give a plausible response for the treatment of ischemic injury, mainly by three different mechanisms: (1) inducing CM proliferation; (2) inhibiting heart cell death and attenuating inflammation; and (3) supporting cardiovascular regeneration. It is important to note that although mRNA applications in cardiology are progressing quickly, right now research is focused on intracardiac administration, as it is the most effective delivery method so far; however, it causes stress and local injury to the tissue. Therefore, it is crucial to develop a delivery vehicle that can target the heart and that can be then administered s.c. or i.v. [204,205][74][75]. mRNA encoding for vascular endothelial growth factor A (VEGF-A) is currently under clinical phase 2 (Table 5) in a collaboration between AstraZeneca and Moderna Therapeutics after showing the induction of cardiovascular regeneration in several studies. In addition, Magadum et al. were able to induce CM proliferation by using mRNA encoding for FSTL1 and Pkm2 [204][74].
Table 5. mRNA Nanomedicines for cardiovascular diseases in Clinical Trials. A list of clinical trials for cardiovascular diseases is provided in this table.
Cardiovascular Diseases
AZD8601 Heart Failure Modified mRNA-LNPs Phase II NCT03370887 AstraZeneca

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