mRNA research has been expanding in many areas of biotechnology and healthcare, from vaccines that helped combat the COVID-19 pandemic to therapies for rare genetic diseases. Evaluating and improving the efficacy and safety of mRNA-based modalities is critical to the continued innovation of mRNA technology. Robust mRNA delivery tools are particularly crucial to address concerns around therapeutic efficiency and adverse effects. The challenge remains to deliver the mRNA construct to the targeted sequence without premature degradation, unwanted edits, and genomic integration.

Types of mRNA Delivery Methods

Physical Methods:

Naked mRNA is repelled by the electrostatic forces present on the cell membrane due to its negative charge, making it difficult for cells to take it up. Physical methods have been developed to manipulate the cell membrane and enable the direct entry of mRNA into cells. Electroporation and microinjection are the most recognized techniques.

In electroporation, electrical pulses are applied to create temporary pores along the cell membrane to facilitate mRNA uptake. While this method can be powerful for cell types that cannot be transfected through other delivery methods, the electrical shock and cell membrane disruption can reduce cell viability. Therefore, electroporation is more suitable for in vitro or ex vivo applications, such as CAR-T engineering, than in vivo applications.

mRNA can also be delivered mechanically through microinjection under a microscope, allowing precise control over dosage when studying gene expression. However, this method is labor-intensive, slow, and requires technical skills, limiting its applicability for in vivo and clinical applications.

Delivery efficiency of these methods may suffer from the instability of mRNA, risk of degradation by RNases, and severe immunogenicity.

Viral Vectors:

Viral vectors are viruses engineered to deliver genetic material into the cells. They harbor the natural viral mechanisms of cell entry without the disease-causing effects. To achieve this, researchers remove the disease-causing genes and insert the custom DNA sequence (e.g., a sequence that would encode a therapeutic protein). These vectors enter the cell either by binding receptors on target cells or through endocytosis. Once in the cytoplasm, they can release the gene cargo, which gets transcribed into mRNA for ribosomal translation.

While adeno-associated (AAV) and lentiviral vectors induce long-term expression beneficial for cancer treatment and gene therapies, adenoviral vectors exhibit transient expression and are preferred for vaccine development.¹

The host-targeting and transduction capacity of adenoviruses makes adenoviral vectors robust gene delivery systems. They can efficiently target several tissues and cell types with substantial cellular uptake and release inside cells. They have rapid protein expression profiles and can induce strong immunogenic responses, which makes them ideal for vaccine development. However, their effectiveness in gene therapies is reduced as the host may develop immunity against adenoviruses.² More importantly, the transient gene expression profile of adenoviral vectors may not be sufficient for gene-replacement therapies. While more stable viral vectors can be used to modify and correct gene defects on a DNA level, they create room for insertional mutagenesis, which may disrupt genome stability with detrimental effects.³

Lipid Nanoparticles (LNPs):

Lipid nanoparticles (LNPs) are widely used to deliver mRNA to the targeted cells by enveloping them in a protective shell. LNPs comprise ionizable cationic lipids that electrostatically bind and self-assemble around mRNA particles to form a lipid membrane. The membrane structure with hydrophilic heads facing outwards and hydrophobic tails facing inward ensures mRNA integrity while promoting cellular uptake through endocytosis. LNP-based mRNA delivery has several advantages, such as protection from RNases, controlled delivery, scalability, biocompatibility, and low immunogenicity compared to viral vectors. Furthermore, tissue-specific delivery can be achieved by tuning the particle size.

On the other hand, they are prone to impurities due to complex manufacturing and storage demands. A key concern is the risk of reactivity between mRNA nucleobases and the lipids. For example, when developing vaccines for COVID-19, Moderna identified lipid-mRNA adducts caused by such reactions, which severely diminished protein expression and product shelf-life.⁴ In addition, due to structure and chemical composition, LNPs tend to accumulate in the liver, making their delivery to other areas of the body challenging.⁵ Finally, repeated or high-dose administration was associated with lipid-induced toxicity.⁶ Therefore, strict care must be taken during LNP formulation and mRNA encapsulation to improve transfection efficiency, stability, and biocompatibility.

Polymeric Nanoparticles

Polymer-based mRNA delivery technologies are like LNPs, as polymers encapsulate, protect, and deliver mRNA into cells. Although there is a great variety of polymers with different physical and chemical properties, these can be divided into three main categories.

Cationic polymers, such as polyethyleneimine (PEI) and poly(amidoamine) (PAMAM), can electrostatically bind negatively charged mRNA in a similar mechanism to LNPs. This interaction condenses mRNA into a nano-complex that can rapidly be taken into cells by endosomes. Once inside the cells, the endosome-polymer interaction facilitates the release of mRNA into the cytoplasm. While cationic polymers often exhibit high transfection efficiency, their cytotoxicity and lack of biocompatibility remain to be solved.⁷

Non-cationic polymers, such as poly (ethylene glycol) (PEG) and polyesters, have reduced cytotoxicity. Their formulation requires covalent attachment to the mRNA. These polymers shield mRNA from degradation by generating steric hindrance against RNases. However, their formulation may compromise the mRNA load capacities and endosomal release efficiency, ultimately impacting their efficacy.⁷

The issues surrounding cytotoxicity and delivery efficiency are addressed through biocompatible and smart polymers. While hybrid nanopolymers are designed to find the sweet spot of safety and efficacy, more biocompatible polymeric alternatives, such as poly(aspartic acid) and poly(amine-co-ester), are also explored for their mRNA delivery efficiencies.^8,9 Furthermore, stimuli-responsive polymers can be exploited to actively deliver mRNA into cells instead of relying on the natural cellular transport mechanisms.¹⁰ These nanopolymers can be designed to respond to endogenous stimuli, such as changes in biological conditions around the targeted cells. Other nanoparticles can also be manipulated exogenously through a magnetic field or ultrasound. Overall, the adverse effects of mRNA delivery can be mitigated with improvements in the selectivity and biocompatibility of polymer nanoparticles.

mRNA Purification and its Significance:

While considerations related to the robustness of mRNA delivery systems are essential, the importance of mRNA purification in successful delivery should not be ignored. Regulatory agencies like the FDA require consistent and reproducible outputs, which is only possible when highly purified mRNA is used.

Purification is necessary for removing various mRNA impurities, including double-stranded RNA and uncapped or truncated mRNA fragments, which may trigger unwanted immune responses and reduce translation efficiency. For instance, dsRNA may activate toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I)-like receptors that can lead to adverse effects, such as inflammation.

Overall, mRNA purification is obligatory for achieving high efficacy and safety from mRNA delivery methods.

Successful Applications of mRNA Delivery

As delivery strategies continue to champion mRNA research, we see the integration of mRNA-based vaccines and therapeutic agents in biomedical applications.

Thanks to lipid nanoparticle-based mRNA delivery, COVID-19 vaccines BNT162b2 (Pfizer-Biontech) and mRNA-1273 (Moderna) were authorized worldwide to immunize large populations by encoding the SARS-CoV-2 spike protein to trigger the immune system.^11,12 Their success was followed by vaccines against influenza and respiratory syncytial virus.¹³

The utility of mRNA vaccines extends beyond infectious diseases, as several mRNA vaccines for cancer immunotherapy are in phase I-III clinical trials. A striking example is the mRNA-4157/V940, which received an FDA breakthrough status in 2023. The vaccine with the lipid-nanoparticle-mRNA delivery system is developed to encode 34 tumor-associated antigens. It can be customized per patient by sequencing tumor samples and identifying mutations. Thus, the vaccine holds immense promise for personalized medicine applications.¹⁴

CAR-T cell engineering is another example of mRNA delivery contributing to cancer treatment. By delivering mRNA to patient-derived T-cells, researchers can prompt T-cells to express chimeric antigen receptors (CARs) that improve their targeting efficiency against tumor cells. Although traditional delivery is conducted ex vivo, in vivo mRNA delivery via LNPs demonstrated successful CAR translation efficiency.¹⁵

Finally, mRNA-based gene therapy methods can be exploited to deliver functional mRNA to patients with genetic disorders. The delivered mRNA can encode the lacking protein to restore healthy function without permanently altering the patient's genome. A group of clinical trials are exploring protein replacement therapies, including a potential treatment for cystic fibrosis involving a functional CFTR mRNA encapsulated in LNPs and delivered in aerosol form.¹⁶

References

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