PLATFORM TECHNOLOGY – The PTXΔLNP® Platform: On the Promise of Developing New LNPs for Tomorrow’s mRNA Therapies
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.
- L. Brannon-Peppas and J. O. Blanchette, ‘Nanoparticle and targeted systems for cancer therapy’, Advanced Drug Delivery Reviews, vol. 56, no. 11. Elsevier, pp. 1649-1659, Sep. 22, 2004.doi:10.1016/j.addr.2004.02.014.
- E. Rohner, R. Yang, K. S. Foo, A. Goedel, and K. R. Chien, ‘Unlocking the promise of mRNA therapeutics’, Nature Biotechnology, vol. 40, no. 11. Nature Research, pp. 1586–1600, Nov. 01, 2022. doi: 10.1038/s41587-022-01491-z.
- H. Zu and D. Gao, ‘Non-viral Vectors in Gene Therapy: Recent Development, Challenges, and Prospects’, AAPS Journal, vol. 23, no. 78. Springer Science and Business Media Deutschland GmbH, pp. 1-12, Jul. 01, 2021. doi: 10.1208/s12248-021-00608-7.
- S. Qin et al., ‘mRNA-based therapeutics: powerful and versatile tools to combat diseases’, Signal Transduction and Targeted Therapy, vol. 7, no. 1. Springer Nature, Dec. 01, 2022. doi: 10.1038/s41392-022-01007-w.
- H. Daraee, A. Etemadi, M. Kouhi, S. Alimirzalu, and A. Akbarzadeh, ‘Application of liposomes in medicine and drug delivery’, Artificial Cells, Nanomedicine and Biotechnology, vol. 44, no. 1. Taylor and Francis Ltd., pp. 381-391, Jan. 01, 2016. doi: 10.3109/21691401.2014.953633.
- H. Xing, K. Hwang, and Y. Lu, ‘Recent developments of liposomes as nanocarriers for theranostic applications’, Theranostics, vol. 6, no. 9. Ivyspring International Publisher, pp. 1336-1352, 2016. doi: 10.7150/thno.15464.
- Akinc, A., Maier, M. A., Manoharan, M., Fitzgerald, K., Jayaraman, M., Barros, S., Cullis, P. R. (2019). The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nature Nanotechnology, 14(12), 1084-1087. doi:10.1038/s41565-019-0591-y.
- M. Mendonça, A. Kont, P S. Kowalski, and C. O’Driscoll, ‘Design of lipid-based nanoparticles fordelivery of therapeutic nucleic acids’, Drug Discov Today, vol. 28, no. 3, pp. 1-17, 2023.
- L. M Lewis, A. V Badkar et al., ‘The Race to Develop the Pfizer-BioNTech COVID-19 Vaccine: From the Pharmaceutical Scientists’ Perspective‘, Journal of Pharmaceutical Sciences 112 (2023) 640−647.
- M. A. Matthay et al., ‘Acute respiratory distress syndrome’, Nat Rev Dis Primers, vol. 5, no. 1, 2018, doi: 10.1038/s41572-019-0069-0.
- K. Radloff, B. Gutbier, C. Dunne, et al., ‘Spatial expression of an mRNA encoding Tie2-agonist in the capillary endothelium of the lung prevents pulmonary vascular leakage’, bioRxiv 2022.10.12.511878, 2022, doi: 10.1101/2022.10.12.511878.
- Evaxion and Pantherna announce promising preclinical mRNA vaccine data. EVAXION. Retrieved May 12, 2023, from https://www.evaxion-biotech.com/press-releases/ 080223-evaxion-and-pantherna-announce-promising-preclinical-mrna-vaccine-data.
- W. T. Ma, F. Gao, K. Gu, and D. K. Chen, ‘The role of monocytes and macrophages in autoimmune diseases: A comprehensive review’, Frontiers in Immunology, vol. 10, no. 1140. Frontiers Media S.A., pp. 1-24, 2019. doi: 10.3389/fimmu.2019.01140.
- Chaudhary, N., Weissman, D. & Whitehead, K.A. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat Rev Drug Discov 20, 817–838 (2021). https://doi.org/10.1038/s41573-021-00283-5.
- Köhl, U., Arsenieva, S., Holzinger, A. & Abken, H. ’CAR T cells in trials: recent achievements and challenges that remain in the production of modified T cells for clinical applications’, Hum. Gene Ther, 29, 559-568 (2018).
- Depil S, Duchateau P, Grupp SA, Mufti G, Poirot L. “Off-the-shelf” allogeneic CAR T cells: development and challenges’, Nat Rev Drug Discov, 2020 Mar;19(3):185-199. doi: 10.1038/s41573-019-0051-2. Epub 2020 Jan 3. PMID: 31900462.
- Nawaz, W., Huang, B., Xu, S. et al. AAV-mediated in vivo CAR gene therapy for targeting human T-cell leukemia. Blood Cancer J. 11, 119 (2021). https://doi.org/10.1038/ s41408-021-00508-1.
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.
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