While over the past couple of decades, mRNA technology has advanced dramatically and provided one way out of the COVID-19 pandemic, at the same time public perception and favor has polarized, and strong and vocal holdouts have unequivocally affected general interest and uptake.

Successful sa-mRNA encapsulation into lipid nano particles (LNP) typically involves initial development of a small-scale process followed by scale-up. We have scaled up our process from the bench scale to the clinical trial manufacturing (CTM) scale. During scale up, we encountered and solved challenges such as increased processing time, impact of process container material, and foaming during Tangential Flow Filtration (TFF). Furthermore, the impact of peristaltic pumping action on LNP product was investigated to ensure consistent product quality across scales. Our scale-up was successful as measured by process reproducibility and achieved quality attributes of bulk drug product.

Self-replicating RNA (srRNA) technology has been enabled for vaccines, and requires lower doses than conventional mRNA, due to its ability to amplify in situ. Most srRNA approaches are derived from the same alphavirus backbone. Since viruses can differentially impact host cell mechanisms, we have explored alternate alphaviral vectors to assess their biological utility. We show that new synthetic srRNA vectors can enable us to build better next-generation therapies.

The SARS-CoV-2 virus continues to evolve which presents the need to adapt and broaden protection provided by mRNA vaccines. This talk will highlight the importance of robust analytical methods to enable rapid mRNA vaccine development. It will also feature the application of Pfizer’s established mRNA analytical platform in combination with new developments to support next-generation products aimed at addressing the continuously changing viral landscape.

I will discuss a miRNA that has long been known to have anti-oncogenic properties and is derived from a long primary transcript, which we recently discovered has a separate tumor suppressor function. The role and anti-oncogenic mechanism by which this long non-coding RNA functions will be discussed. In addition, the therapeutic implications for therapeutic intervention will also be examined.

Akagera is developing ligand-targeted lipid nanoparticles encapsulating mRNA as vaccines against some of the world's most intractable infectious diseases. Here we describe our efforts to develop novel ionizable lipids to improve endosomal escape and pair them with small molecule lipid ligands to increase their uptake and subsequent expression in dendritic cells. This increased efficiency has significant potential impact on manufacturing capacity, cost of goods, tolerability, and flexibility to incorporate multiple mRNAs.

The value of the RNA modality comes from its nature as a precise, versatile, and real-time instruction coding. Its initial use cases utilize a single action coding – for instance, for the production of a viral antigen component. However, more advanced applications are rapidly developing around coding entire instruction sets for combinational logic and benefit. The promise of such approaches will be discussed along with novel strategies for implementing them.

• The flexibility of mRNA is a challenge for chromatography separation 
•  Structural interconversion upon adsorption is more pronounced than observed with proteins 
•  How pure is pure for mRNA therapeutics and vaccines
  

Following the rapid and successful deployment of mRNA vaccines during the COVID-19 pandemic, over 150 mRNA vaccines are in development, with further therapeutic applications in oncology, cardiovascular and genetic diseases. This presentation will provide an overview of USP updated guidelines based on stakeholder feedback and further assessment and qualification of analytical methods presented therein to address common quality attributes for mRNA products.

• How do we measure dsRNA? – Current analytical methods.
• What are the biological implications for dsRNA in vaccines? 
• What might the non-emergency dsRNA regulatory landscape look like?​

Ribonucleic acids (RNAs) have recently shown success in the vaccine space and promise as candidates for new therapeutics. For these large RNA molecules, RNA integrity is a key critical quality attribute (CQA) related to the translation of the intended RNA sequence. This quality attribute is tested and monitored for drug substance and drug product, both at manufacturing release and on stability. RNA integrity been assessed as a key quality attribute for optimization of the drug substance and drug product processes. This presentation discusses a capillary electrophoresis-based methodology to separate and resolve RNA of varied size and length.

Significant advances have been made over the last three decades in the development, delivery, safety and manufacturing of mRNA constructs for vaccines and therapeutics. This is evident from the tremendous contributions that mRNA vaccines have made in containing the COVID-19 pandemic. The goal of this presentation is to review and address fundamentals and unit operations for mRNA functionality, stability, delivery, safety and manufacturing. Within the context of this review, we will provide insights and novel technologies that may be helpful for furthering the development, delivery, safety and manufacturing of mRNA vaccines and therapeutics for infectious diseases, genetic disorders and cancers.

RNA activation in acquired and genetic diseases Small activating RNAs (saRNA) are double- stranded 21 nucleotide RNA that can target promoters or enhancers leading to mRNA upregulation. MTL-CEBPA is an investigative drug that resulted from the conjugation of saRNA CEBPA with NOV 340 lipsomes that targets tumour associated macrophages in order to alter favourably the tumour microenvironment. MTL-CEBPA has been administered safely in over 140 patients with advanced cancer and improved clinical outcome in a sub-set of patients when co-administered with TKI or checkpoint inhibitor. 

Liposome and LNP formulations have evolved as highly potent drug delivery systems. Recent literature gives valuable insights in developing and manufacturing these formulations, with focus mainly set on delivering different liposome and LNP compositions/sizes. This presentation will give some insight into thermodynamic equilibrium considerations, such as long-term (size) stability of liposomes and LNPs at various temperature, as well as equilibrium radius considerations in manufacturing liposomes and LNPs.

Lipid nanoparticle (LNP) technology, as currently one of the most promising and emerging technologies, shows the ability to deliver nucleic acid therapeutics for non-viral gene therapy (NVGT). Compared to intramuscular vaccine delivery, intravenous (i.v.) delivery of a LNP formulation for gene therapy shows unique challenges. An LNP formulation for gene therapy via i.v. may require different lipid and formulation design. Here, we will discuss the considerations and challenges in early LNP development and impact on tissue targeting for NVGT.

In the development of LNP-based drug product, the choice of manufacturing technology is one of the key factors for success. The choice of technology can have a significant impact on the biophysical properties, structural characteristics, colloidal stability, and efficacy of the LNP. This study describes the impact of different manufacturing parameters and the scale-up consideration to enable successful LNP drug product manufacturing.

In this talk, we discuss de-risking mRNA adduct formation in lipid nanoparticle formulation intended for glycogen storage disease type-1a using analytical tools.