PLATFORM TECHNOLOGY - The PTXΔLNP® Platform: On the Promise of Developing New LNPs for Tomorrow’s mRNA Therapies

INTRODUCTION

The implementation of nanomedicine to the medical field has led to significant breakthroughs in the targeted and effective delivery of small molecules and oligonucleotide therapeutics to affected diseased areas.¹ The COVID-19 pandemic has given rise to a new era of promising modalities in the biopharmaceutical sector, with messenger-RNA (mRNA) technologies prominently taking center stage as the next generation of therapeutics and vaccines against severe pathologies.²

Throughout the past few decades, non-viral engineered vec­tors – including lipid or polymer-based ones – have been exten­sively developed to effectively encapsulate and protect synthetic RNA/mRNA species during systemic administration for gene de­livery purposes.³ The rationale behind the design of such nanos­tructured delivery systems is to achieve advanced cellular uptake at the affected site with subsequent functional endosomal escape of the nucleic acid payload into the cell cytoplasm. Additionally, these systems should exhibit low immunogenic and toxicological profiles, prolonged circulation properties, serum stability, chem­ical versatility, organ specificity, and facile scale-up manufacturing among the others.⁴ The first well-studied and FDA-approved de­livery system was liposome-based, whereas the first siRNA-LNP approved formulation, Onpattro paved the way for the newest generation of nanovectors composed of lipids encapsulating mRNAs called lipid-based nanoparticles (LNPs).^5-7 Principally, these are referred to as 3D nano-constructs comprising three, four, or even more different lipid moieties, such as ionizable and/or cationic lipids, phospholipids, cholesterol, and polyethyl­ene glycol (PEG)-lipids (Figure 1).⁸

Pantherna Therapeutics engages in the development of two distinct scientific pillars, including both mRNA and LNP technol­ogy platforms as a basis for novel top-notch mRNA therapeutics against a wide spectrum of possible target diseases. The com­pany´s PTXmRNA® technology has demonstrated superior results in terms of the expression of the desired target protein compared to commercially available mRNA. Best performance of PTXm­RNA® was achieved through modifications in the codon sequence, incorporating state-of-the-art capping structures, and by employing spe­cific untranslated sequence regions flank­ing the coding region. These modifications have demonstrated superior expression most likely through optimized ribosome loading and scanning for multiple mRNA protein targets. Additionally, the PTXΔLNP® platform offers a synergistic sister technol­ogy to the mRNA platform to obtain potent mRNA-LNPs for therapeutic applications.

The LNP formulation and manufactur­ing process is of essential importance for the precise control and prediction of the particle’s intrinsic characteristics, especially when aiming for clinical translation. Con­ventional preparative methods, such as solvent-injection, and automated microflu­idic systems are widely implemented for cost-effective and facile scaled-up produc­tion of LNPs nowadays. Progress in the de­velopment of flow chemistry mixing has proven to adequately meet the require­ments for ease and feasibility over the in­dustrial scalability of LNPs. Various peers, such as BioNTech/Acuitas and Moderna, invested into the research development of mRNA LNP formulas for over a decade, which during the COVID-19 pandemic, resulted in the well-known formulated COVID-19 vaccine suspension. The un­derlying mRNA-LNP as in the case of BNT162b2 (Comirnaty®) consists of four structural lipids, namely the ionizable lipid ALC-0315, the two helpers Cholesterol and Distearoylphosphatidylcholine (DSPC), and the PEGylated lipid ALC-0159, and the formula is approved for intramuscular injection.⁹

Pantherna’s proprietary formulations entail chemically versatile lipidic systems that bear ultimately three different physic­ochemical attributes, namely their overall surface charge: neutral (nLNPs), cationic (cLNPs), and anionic (aLNPs) (Figure 2). nLNPs and aLNPs are basically catego­rized in the standardized four-lipid com­positional scheme, whereas the cLNP corresponding derivatives comprise mainly of three lipid moieties, which practically fa­vors more cost-effective manufacturing. Through continuous fine-tuning over the established manufacturing processes, the overall objective of Pantherna is to ensure high levels of scalability, reproducibility, and cost-effectiveness for their LNP pipeline platform. The design simplicity of their LNP systems ensures robust physico­chemical stability of the lipid suspensions produced (monodisperse sizes, versatile surface charges, batch-to-batch consis­tency, high oligonucleotide encapsulation rates), and primarily a safe toxicological profile paving the way toward promising clinical translation.

Pantherna’s delivery platform assets are organ selectivity and cell type speci­ficity of the LNPs. Figure 3 illustrates the in vivo biodistribution profile of selected LNP formulations derived from the platform, upon intravenous administration in mice models. Pantherna’s formulations can con­fer more direct organ-specificity with their cLNPs (orange bars) demonstrating high expression in lung tissue and with one aLNP formula (blue bars) showing selec­tive expression in the liver tissue but with­out any prominent activity in the remaining organs. The nLNPs (grey bars) demon­strate prominent liver-directed expression rates, with one nLNP candidate formula­tion also effectively targeting the pancre­atic tissue.

The dynamic of Pantherna’s technol­ogy platforms is exemplified by PAN004, which is the lead candidate formulation for systemic administration. PAN004 is a novel synthetic, nucleoside-modified mRNA encoding COMP-Ang1 (mRNA-76b) formulated with PTX cationic lipid nanoparticle (PTXcLNP002) making use of a proprietary cationic lipid within a three-lipid moiety formulation. The highly selec­tive delivery to the lung was demonstrated in suitable in vivo experiments, sparing other vascular beds, and bypassing the liver. Single cell RNA-sequencing con­firmed lung endothelial delivery specificity of the therapeutically active component COMP-Ang1 PTXmRNA® after PAN004 was administered intravenously. PAN004 is intended to enable high spatial expres­sion and thereby positioning of a hyperac­tive Tie2- agonist to counteract the progression of acute respiratory distress syndrome (ARDS), which is composed of leaky lung endothelium and is caused by various events, such as sepsis, pneumonia, or COVID-19. In the acute phase of ARDS, pulmonary edema is a hallmark patho­physiological event that is accompanied by neutrophil influx and inflammatory cytokine production that leads later to fi­brosis, epithelial damage, and life-threat­ening respiratory dysfunction. There is an unmet medical need as lethality of ARDS is still high at 30%-40%.¹⁰ PAN004 acts in the acute phase by stabilizing the endothe­lial barrier and therefore counteracts edema and neutrophil influx exemplifying the therapeutic potential of mRNA-thera­pies beyond vaccination.¹¹

In addition to Pantherna’s lead devel­opment program, further innovative mRNA and LNP combinations from the platform exhibited promising prospects across a range of pathologies. Alongside lung, liver, and pancreas, the PTX portfolio possesses LNP formulations also suitable for local delivery, such as intramuscular administration or even for ex vivo applica­tion. In addition to applications for my­ocyte-directed target gene expression, eg, in regenerative medicine, the PTXΔLNP® platform orients particularly toward cancer vaccination for efficient immunization. The functionality of PTXΔLNPs for immuniza­tion was recently disclosed through a col­laboration with Evaxion Biotech.¹² The combination of PTXΔLNPs and Evaxion’s AI-identified cancer vaccine antigens en­coded by a PTXmRNA® induced a robust T cell response against the tumor antigens in vivo while simultaneously eliminating the tumor growth of the syngeneic tumors in mice. The results of this study establish the potential of PTXΔLNPs as very efficient mRNA-LNP cancer vaccine tools.

Immune cell targeting is important in cancer vaccines, infectious diseases, and in autoimmune disorders. Pantherna holds promising data that demonstrate selective immune cell uptake of our developed LNPs with positive uptake in monocytes and macrophages. Specific targeting of these immune cells can be envisioned for many different purposes and provides prospects for their use in immunization against infectious diseases and in autoimmune disorders, such as systemic sclerosis, rheumatoid arthritis, primary biliary cholangitis, Sjogren’s syndrome, and in­flammatory bowel disease.^13,14 Currently, immune cell utilization in therapies in­volves time and expensive processes with the isolation of T cells or monocytes di­rectly from each individual patient, genetic editing/ re-programming or re-activation of the cells, and re-introduction back to the patient.¹⁵ This method comes with chal­lenges, including difficulties in viral trans­duction and transfection of cells and keeping them free from contamination by treating them only in a closed-circuit envi­ronment. Efforts are being made in the de­velopment of allogeneic “off-the-shelf” cell therapies, using cryopreserved cells mod­ified from donors, although, these thera­pies would reduce costs and offer faster treatments to patients; however, the risk of graft-rejection from allogenic cells may be life-threatening.¹⁶ The use of Pantherna’s LNP-platforms for non-viral ex vivo appli­cation could increase the number of trans­fected cells and the subsequent production of the target mRNA, enhancing the effi­ciency and reducing toxicity during the production of autologous and allogenic cell therapies. Circumvention of ex vivo transfection with systemic administration of adenosine-associated virus (AAV) has demonstrated in vivo generated CAR-T cells leading to positive in vivo tumor re­gression.¹⁷ However, the known downside of viral therapies is that the effects are per­manent, whereas LNPs can offer an alter­native transient therapeutic approach. In the long run, optimized PTXΔLNPs prospectively bypass ex vivo cellular han­dling altogether by intravenous injection to specifically target and transfect the desired immune cell population, potentially cir­cumventing the costly and risky processes of adoptive immune cell therapies.

In summary, the PTXmRNA® and PTXΔLNP® platforms offer a readily usable technology platform for selective delivery to different organs and immune cells for any given administration route providing the basis for promising future novel mRNA therapies.

REFERENCES

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Dr. Charlotte Dunne a scientist in the R&D department at Pantherna Therapeutics. She recently joined the company in 2023. Prior to that, she worked as a Postdoc at Helmholtz-Zentrum Hereon and Berlin Center for Regenerative Therapies at Charité, Germany. She earned her PhD in Pharmacology from the University of Auckland at the Centre for brain research, New Zealand in 2022. She has experience in working with vascular cells and degenerative diseases.

Dr. Katrin Radloff is a scientist in the R&D Department at Pantherna Therapeutics. She joined the company in 2020 working on the development of PAN004. Prior to that, she has worked as a Postdoc on NMR-based metabolomics in the Department of Physiology and Biochemistry of Nutrition and the Max-Rubner-Institut in Karlsruhe, Germany. During her PhD, she investigated inflammation resolution pathways in gastrointestinal cancer at the institute of biomedical science at the University of São Paulo, Brazil.

Dr. Leonidas Gkionis is a scientist in the R&D Department at Pantherna Therapeutics. He joined the company in 2022 working on the design and production of cutting-edge formulations. Prior to that, he worked as a Galenical Formulation Scientist at different pharmaceutical industries. He earned his PhD in Nanomedicine from the University of Manchester, UK, as part of the Graphene NOWNano CDT sponsored by EPSRC. He holds hands-on professional experience in the pharmaceutical manufacturing of small drug molecules and recently of oligonucleotides.