Unlocking the therapeutic applicability of LNP-mRNA: chemistry, formulation, and clinical strategies

Background

mRNA therapy has recently emerged as a prominent field in the treatment landscape for various clinical diseases. As far as in 1978, mRNA has been used as a genetic material deliver to cell to induce protein expression via liposome ^[1]. While this approach has become the spotlight since the remarkable success of Pfizer-BioNTech and Moderna, two lipid-nanoparticle-mRNA vaccines against covid-19 pandemic. Due to the instability nature of mRNA, the indispensable role of lipid nanoparticle in enabling the effect of mRNA has drawn scientist attention. To date, lipid nanoparticle (LNP), which composed of cholesterol, phosphate lipid, and PEGylated lipid, are the most clinical advanced non-vial platforms for mRNA delivery. LNPs have garnered considerable attention for their ability to encapsulate and protect mRNA molecules from degradation while facilitating their delivery into target cells. However, despite their promise, LNP mRNA therapy still faces several challenges that need to be addressed for its successful clinical translation such as, 1 specific targeting non-muscle and non-hepatocyte; 2. quality control when scale up LNP 3. Safety concern

Research progress

LNP as the novel drug delivery system enhancing mRNA therapeutic efficacy has been proved for its delivery ability. This review provides valuable insights into the current state-of-the-art of LNP mRNA-based therapeutics and highlights the remaining unmet needs in translating mRNA into clinics. These include optimizing the dosage of mRNA therapeutics, addressing Chemistry, Manufacturing, and Controls (CMC) requirements, ensuring safety, and enhancing targeting efficiency to improve therapeutic outcomes (Fig. 1).

First, the author illustrated and categorized ionizable lipid, cholesterol, phospholipid and PEGylated lipid in the decade that has been reported in the publications. The principle of ionizable lipid design and synthesis follows the combination of a positive charged head usually contains an amine, guanidine, and heterocyclic group, a linker, and a hydrophobic lipid tail (Fig.2)

High throughput synthesis of ionizable lipid is a chemistry method to achieve novel highly efficient ionizable lipid design and selection. For example, Dr Anderson and Langer’s group use has found significant higher cytokine section from two ionizable lipid, A2-Iso5-2DC18 and A12-Iso5-2DC18. Through the selection from 1080 ionizable lipid, the top two ionizable lipid showed their potential ability to active STING immune-mediated pathway in the use of cancer vaccine (Fig. 3).

Cholesterol and phospholipid play a significant role in the formation of LNP and its targeting efficiency. Recently, Dr. Whitehead’s group showed a organ specific delivery by increasing the amount of phospholipid in the LNP. They increased the amount of phospholipid lipids ranging from 10% to 40% to achieve a higher spleen targeting. on the other hand, they also found that negatively charged phospholipid have trend to target spleen while positively charged helper lipid trend to target lung (Fig. 4).

Besides, scientists have found the organ selective targeting by changing the net charge of the LNP. For instance, the addition of fifth compound, has been investigated by the Dr. Siegwart group to achieve liver, spleen, and lung selective targeting via formulating LNP with 18PA and DOTAP (Fig 5.)

Apart from elucidation the complexity of LNP development and formation in modifying chemistry, the review also highlights the alternative strategies to enhance LNP-mRNA delivery by surface modification. Surface-modified LNPs, such as those incorporating PEGylated lipids or ligand-mediated targeting moieties, have shown promise in improving circulation time, reducing nonspecific interactions, and enhancing cellular uptake (Fig. 6).

The clearance and circulation time of LNP in vivo is usually controlled by the content and length of PEGylated lipid. The strategy of PEGlation the surface of LNP has been widely used to stabilize nanoparticles and decrease the nonspecific interaction with serum protein. However, increase the length of PEG will also decrease the fusion between LNP and endosome membrane, which is known as “PEG dilemma”. PEG Shielding is another factor that influence the biodistribution efficiency of LNP. Modification of PEG length and content will impact biomolecular corona adsorption, in which influence the LNP therapeutic efficiency.

Besides PEGlation, the conjugation of ligands or antibody on the surface of LNP is another strategy to increase organ specific targeting. As shown in the Fig. 7, 4% T cell can be transfected in vivo via LNP with 16% CD3 antibody conjugated on the surface. The therapeutic effect of antibody conjugated LNP has been proved by Rurik et al. they achieved 81.1% of splenic CD4+ T cell and 75.6% of splenic CD8+T cells reporter protein expression in in Cre-loxP reporter mice via CD5 antibody conjugated LNP. And extended their research to deliver FAPCAR expressing mRNA into T cell in the treatment of cardiac injury and fibrosis (Fig. 8).

These advancements hold significant potential for overcoming existing challenges in LNP-mediated mRNA therapy and advancing the clinical translation of these innovative treatments.

Another aspect of LNP-mRNA in vivo performance remains unclear. Upon injection, nanoparticles interact with proteins, forming a corona effect will influence organ targeting. Different LNPs develop unique protein coronas, targeting diverse tissues. For example, ApoE aids liver targeting, vitronectin enhances tumor cell-mediated delivery, and fibrinogen boosts lung targeting. Corona adsorption is closely tied to nanocarrier coating and dynamics. In LNPs, PEG length and alkyl chain affect surface chemistry and coronation, influencing tissue targeting (Fig. 9)。

The current obstacle of scaling up LNP to meet commercial manufacturing standard of pharmaceutical industry has been discussed in the review.

Storage: commercialized LNP-mRNA vaccines require low-temperature storage and cold chain transportation (-80 °C) limiting their widespread use to some extent. Thus incorporating suitable cryoprotectants and achieving stable lyophilization or frozen storage is essential for the development of mRNA therapeutics.

Characterization: conventional dynamic light scattering (DLS) instruments in typical laboratories cannot meet the stringent standards required for drug production regarding LNP size, zeta potential, polydispersity index (PDI), and encapsulation efficiency. There is also no precise method for assessing the proportion of empty LNPs. Research has reported a method based on multi-laser cylindrical illumination confocal spectroscopy (CICS) for detecting mRNA and lipid content in LNP formulations at the single nanoparticle level which could provide a paradigm for commercial production (Fig. 10).

Safety: currently, the absorption, distribution, metabolism, and excretion (ADME) properties of LNPs in vivo lack of deep research. In the clinical translation of mRNA therapy, larger doses of LNP-mRNA drugs are needed, posing higher requirements for biosafety.

Despite the progress made in preclinical studies, the transition of LNP mRNA therapy into clinical practice has been met with certain obstacles. While mRNA vaccines have demonstrated success in infectious diseases like COVID-19, their application in other therapeutic areas, such as oncology, has faced setbacks in clinical trials. For instance, Moderna's mRNA2752, intended for treating solid malignant tumors/lymphoma, terminated at phase I trials (NCT03739931), highlighting the need for further research to address the remaining barriers to clinical translation.

Future prospect

Looking ahead, this review emphasizes the importance of gaining a deeper understanding of the biological pathways and metabolism underlying mRNA modalities and LNP biodistribution systems. This knowledge will be instrumental in guiding the rational design of ionizable lipids, optimizing delivery precision, and ensuring acceptable safety profiles, thereby meeting the unmet clinical needs, and realizing the full potential of LNP-mediated mRNA therapy.