Cargo-free LNP Manufacturing Could Spark a Nucleic Acid Therapy Renaissance

The Elegance of Nucleic Acid Therapy is Compromised by Logistic Complexity

Conventional biologics are static; their structure and mechanism are fixed once they are developed. In contrast, nucleic acid therapies are inherently programmable—putting treatments once thought to be impossible within reach.

The attractiveness of using RNA or DNA as a therapeutic comes from the nature of a nucleic acid itself, which can encode a plethora of biological outcomes based on the type of nucleic acid and the nucleotide sequence.¹ To elicit a targeted immune response, mRNA encoding an antigen can function as a vaccine, while mRNA encoding antibodies can mimic an adaptive immune response. To intervene at the level of disease-causing molecular mechanisms, siRNA can silence disease-causing genes; mRNA can replace missing proteins; and targeted gene editing can be achieved when a guide RNA is paired with mRNA encoding a gene editor.¹

Small adjustments to a nucleic acid design enable further tailoring of a nucleic acid therapy’s mechanism of action, and can lead to improvements in stability, potency, and safety. This enables amendments at the drug substrate level without changing the overall drug product design.¹ For instance, modifying the coding sequence of an antigenic protein can allow an mRNA vaccine platform to be adapted to the biology of a specific recipient or pathogenic target, without changing the overarching approach or mechanism.

This programmability makes nucleic acid therapies uniquely well-suited to rapidly respond to quickly evolving infectious diseases and to the development of personalized medicines. Yet, current manufacturing and delivery models for nucleic acid therapies are based on processes that assume large-batch production of a fixed product, and this limits the inherent flexibility of nucleic acid therapeutics, thus reducing their therapeutic application.² An alternative process that combines cargo-agnostic pre-formed vesicles (PFVs) with the customizable therapeutic agent at the point-of-care could help nucleic acid therapies enable personalized medicines.³

How Are Nucleic Acid Therapies Made Today?

This type of therapy includes a highly fragile and biologically reactive nucleic acid cargo, which acts as the therapeutic agent, and a delivery vehicle, which encapsulates the cargo to protect it and facilitate entry into target cells. A commonly used and clinically validated delivery vehicle for nucleic acid therapies, lipid nanoparticles (LNPs), are constructed from a precise mix of ionizable lipids, structural lipids, and steric lipids—each playing a critical role. These include interacting with, encapsulating, and protecting the nucleic acid payload. Together, the formulation of an LNP determines how well it maintains stability and purity during manufacturing and administration as well as the success of cellular delivery and uptake.⁴ For special applications, LNPs can be decorated with ligands which enable cell-specific delivery.

Current manufacturing and shipping processes for nucleic acid therapies involve loading the delivery vehicle with the nucleic acid cargo upfront. This requires relatively large batch sizes and complex procedures to maintain the integrity and stability of the product over time.² In this loading process, the lipid components of the LNPs are combined and dissolved in an organic solvent (typically ethanol), and rapidly mixed with the nucleic acid cargo suspended in an acidic buffer, assembling themselves into LNPs that completely encapsulate the nucleic acid therapeutic. The ethanol must then be removed and replaced with an isotonic neutral pH buffer to stabilize the loaded LNPs, before the products are prepared for cryopreservation, vialed, frozen, and shipped via cold-chain (typically -20°C to -80°C).²

While working on the COMIRNATY™ vaccine process with Pfizer and BioNTech, we found that this manufacturing model is sensible when large batches of a nucleic acid therapy are needed to address a pandemic-scale response.⁴ However, the resulting products required cold-chain infrastructure and needed to be received and administered by hospital personnel trained in handling cryopreserved products, limiting vaccine access in low-resource settings. The manufacturing model was also wasteful; as the COVID-19 virus mutated, the efficacy of the original vaccine design waned, necessitating a yearly update to the formulation. Re-manufacturing the vaccine with an updated mRNA sequence led to the waste of the previous year’s unused vaccines.

In most contexts where a nucleic acid therapy’s modality is most promising, upfront batch-based production is counterproductive, necessitating over-production that increases the cost of a nucleic acid therapy on a per-dose basis. The excessive minimum-batch sizes of current production methods limit the economic feasibility of single, small-batch therapies and of therapeutic iteration. Meanwhile, pre-loading of the delivery vehicle with nucleic acid cargo ties therapeutic modification-to-administration turnaround times to the combined manufacturing, release, and shipping process which can take upwards of six months.²

Processes that enable smaller-scale production and shorter turnaround times between therapeutic modification and administration are needed to propel rapidly programmable medicines from possibility to clinical reality.

Pre-formed Vesicles Could Resolve LNP Manufacturing Bottlenecks

Loading PFVs with their nucleic acid cargo at the point-of-care has the potential to resolve many of these manufacturing bottlenecks for highly personalized and time-sensitive nucleic acid therapies by enabling much smaller-scale production of nucleic acid cargo and shortening timelines for platform modification.

By manufacturing PFVs separately from their cargo, large batches of PFVs can be manufactured ahead of time, in a process analogous to normal LNP production. PFVs can then be combined at the point-of-care with nucleic acid cargo, which can be developed on much shorter timelines and in appropriate quantities for small-scale administration.³

Moreover, manufacturing and shipping PFVs separately from nucleic acid cargo allows for tailored preservation and storage techniques that are less dependent on cold-chain shipping and storage for the delivery vehicle (The nucleic acid cargo may still require cold-chain shipping and storage). Compared to pre-assembled therapies, PFVs can be stored at higher temperatures and shipped in a liquid format while maintaining stability.

Preclinical Evidence Sets the Stage for Clinical Development

Preclinical testing demonstrates that PFVs can be assembled with nucleic acid cargo within 10–20 minutes at the point-of-care without heat or solvents. With optimized buffer, researchers observed over 90 percent encapsulation efficiency across a variety of payloads, including mRNA, siRNA, self-amplifying RNA (saRNA), and mRNA/gRNA mixes at a 1:1 ratio. Due to the reduced interaction time between LNP and mRNA, the PFV process has shown decreased formation of unwanted RNA-lipid adducts compared to conventional mixing methods.³

Physical and chemical analysis of formulations produced by the conventional and PFV methods show that the particle properties are indistinguishable between both processes. These measurements included particle size and polydispersity index, encapsulation efficiency, RNA content, lipid content, and RNA integrity. Moreover, morphological analysis by cryo-EM imaging showed equivalent particles, and PFV loaded with mRNA demonstrated stability for over 40 weeks in liquid-format storage conditions (2–8^oC).³ The most compelling stability readout showed unloaded PFV’s unique ability to retain encapsulation efficacy across all time points over a 12 month period when stored in non-frozen, liquid form (4^oC) or cryopreserved (-80^oC), while retaining colloidal stability (unchanging size and a polydispersity index under 0.1) across storage conditions.³ The capacity for long-term storage suggests the delivery vehicle of a nucleic acid therapy can be stored in bulk at the point-of-care so that the timing of therapeutic administration is primarily contingent upon the availability of nucleic acid cargo.

In a proof-of-concept where the PFV was produced immediately before administration, researchers demonstrated equivalent performance between the two delivery methods across different applications: prophylactic vaccines and systemically administered therapeutics. For an mRNA vaccine encoding an influenza hemagglutinin (HA) antigen, functional antibody response was statistically equivalent between conventional LNP and PFV. For systemically administered therapeutics, comparable serum antibody expression was also achieved following delivery of mRNA encoding a monoclonal antibody.³

While PFVs have yet to be applied in clinical trials, preclinical evidence to date supports the long-term potential to improve the feasibility of programmable medicine. Drug developers working on vaccine strategies for rapidly evolving pathogens and those working on highly personalized and programmable nucleic acid medicines may benefit from an investment in this novel manufacturing strategy.

A Convergence of Nucleic Acid Therapeutic Innovation

The PFV manufacturing method converges in time with many other advancements that are broadening the accessibility and applications of nucleic acid medicines. Improved understanding of nucleic acid and disease biology is expanding the forms and applications of nucleic acid cargo.^1,4 Change is incremental but hastened by the collaboration of teams who bring together diverse solutions and approaches to better address enduring threats to human health and well-being.