A recent perspective article published in the journal Nature Reviews Drug Discovery discussed the evolution of lipid nanoparticles (LNPs) for nucleic acid delivery. The authors, Pieter Cullis, Professor of Biochemistry and Molecular Biology at the University of British Columbia, and Philip Felgner, Professor of Physiology and Biophysics at the University of California, Irvine, are co-inventors of lipid nanoparticle technology and pioneers in their use in gene therapy and vaccine delivery.

Perspective: The 60-year evolution of lipid nanoparticles for nucleic acid delivery. Image Credit: Kateryna Kon / Shutterstock

Early studies on in vivo gene delivery showed that functional delivery can be achieved by injecting naked plasmids containing viral genomes cloned into eukaryotic expression vectors. However, the clinical utility of naked plasmid delivery strategies has been limited due to concerns about spontaneous DNA integration into the human genome and inefficient transfection.

Besides, viral vectors for gene therapy were frequently associated with immune responses. Most gene therapies currently in development use viral delivery systems, e.g., adeno-associated virus vectors. Despite considerable progress, concerns regarding manufacturing, genetic capacity, and immunogenicity impede progress for viral vectors.

The authors believe lipid-based delivery systems, such as LNPs, may become dominant due to their enhanced tolerability, safety, genetic capacity, ease of design, and manufacturing. These systems have evolved with research on two related domains: the discovery of lipoplexes’ transfection properties and the advent of LNPs. In the present study, the authors explored the evolution of these two streams of research over the past six decades.

Liposomes and lipoplexes

In 1964, it was discovered that ovolecithin dispersion in aqueous media produces multilamellar systems of concentric lipid bilayers. This led to intensive research to characterize the biophysical and functional properties of lipids. Substantial effort was also invested in developing liposomal systems containing nucleic acid cargos for delivery into cells.

In 1987, it was hypothesized that positively charged liposomes may improve the encapsulation efficiency of negatively charged polymers of nucleic acids in lipid-based systems. However, positively charged bilayer-forming lipids do not exist in nature, and their synthetic counterparts were nonexistent then.

Building upon liposome research, several cationic lipids were synthesized, with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) being the lead compound. Mixing and sonicating DOTMA with equimolar concentrations of helper lipids, such as dioleoyl-phosphatidylethanolamine or dioleoyl-phosphatidylcholine, generates stable, positively charged liposomes.

Lipoplexes form when liposomes are mixed with plasmid DNA (pDNA). Lipoplexes represent a significant milestone and a starting point for generating transfection-competent nanoparticles. Besides, they can efficiently transfect messenger RNA (mRNA) and pDNA into cultured cells without requiring additional functional groups.

LNP delivery systems

The evolution of LNPs containing polar regions (comprising nucleic acid cargo) and a hydrophobic core (of neutral ionizable lipid) was predicated on understanding bilayer liposomal systems and experience from liposomal formulations of anti-cancer drugs. Research on lipid polymorphism and asymmetry provided crucial insights and tools to develop LNP delivery systems.

The advent of scalable processes for formulating and loading liposomal systems and the observation that polyethylene glycol (PEG) coating confers a long circulation half-life prompted intense efforts to produce anti-cancer liposomal formulations for clinical application. In the 1990s, lipid-based formulations of nucleic acid delivery systems were described as exhibiting the long circulation half-lives necessary for accessing disease sites.

Research on liposomes for drug delivery indicated that liposomes with no/little surface charge could have long half-lives. Therefore, efforts ensued to develop lipid-based systems using small amounts of cationic lipids to entrap nucleic acids or develop new entrapment protocols, allowing for a net-neutral delivery system.

The first such system was reported in 1999, wherein pDNA was encapsulated into stabilized plasmid lipid particles (SPLPs) via a detergent dialysis method. Subsequent studies revealed that SPLPs had remarkably longer circulation lifetimes and lower toxicity than complexes. However, the detergent dialysis process was not scalable. Further, encapsulation of antisense oligonucleotides within lipid-based systems was reported in 2001.

These stabilized antisense lipid particles (SALPs) demonstrated long half-lives and lower toxicity than complexes. Further, one study showed that a small interfering RNA (siRNA) designed to silence apolipoprotein B could be encapsulated in stabilized nucleic acid-lipid particles (SNALPs), a version of SALP, with promising results in non-human primates and mice. However, the therapeutic index and potency were inadequate for clinical application.

In 2010, it was revealed that the silencing potency of siRNA LNP formulations could be enhanced, by which time, SALPs, SNALPs, and SPLPs were described as part of LNPs. In 2013, phase 1 clinical trials revealed that LNPs containing siRNA and 4-(N,N-dimethylamino) butyric acid (dilinoleyl) methyl ester could rapidly and robustly downregulate circulating transthyretin.

Phase 3 trials also had excellent results for transthyretin-induced amyloidosis treatment. Throughout development, the ratios of lipids in LNPs have changed, and the best LNP composition is still contentious. Further, it was demonstrated in 2012 that LNPs used for siRNA delivery could be formulated to encapsulate self-amplifying RNA, followed by studies revealing in 2015 that erythropoietin-encoding mRNA could be encapsulated in LNPs to transfect the liver.

Subsequent work showed that LNP mRNA systems could transfect diverse tissues via different routes. In 2017, LNP mRNA encoding a viral protein was demonstrated to be highly protective against the Zika virus. This prompted collaborative efforts to create an influenza vaccine based on the LNP mRNA system. However, in 2020, efforts were diverted to develop a vaccine for coronavirus disease 2019 (COVID-19), resulting in the mRNA vaccine, Comirnaty.

Concluding remarks

The success of LNP systems for nucleic acid-based vaccines and therapeutics ushers in a new era of gene therapies. These systems have overwhelming benefits over viral and other delivery systems regarding cargo capacity, scalability, manufacturing, costs, and personalized therapeutics. The future of LNP-based therapeutics will be contingent on advances in the development of sophisticated LNPs with extrahepatic delivery and progress in molecular biology, which will allow for precise manipulation of the delivered cargo.

With the continual advancement in LNP technology, these systems hold promise for increasingly sophisticated gene therapies, enabling precise and effective treatment options.