Lipid Nanoparticle Formulations Research Articles

Accelerating ionizable lipid discovery for mRNA delivery using machine learning and combinatorial chemistry.

To unlock the full promise of messenger (mRNA) therapies, expanding the toolkit of lipid nanoparticles is paramount. However, a pivotal component of lipid nanoparticle development that remains a bottleneck is identifying new ionizable lipids. Here we describe an accelerated approach to discovering effective ionizable lipids for mRNA delivery that combines machine learning with advanced combinatorial chemistry tools. Starting from a simple four-component reaction platform, we create a chemically diverse library of 584 ionizable lipids. We screen the mRNA transfection potencies of lipid nanoparticles containing those lipids and use the data as a foundational dataset for training various machine learning models. We choose the best-performing model to probe an expansive virtual library of 40,000 lipids, synthesizing and experimentally evaluating the top 16 lipids flagged. We identify lipid 119-23, which outperforms established benchmark lipids in transfecting muscle and immune cells in several tissues. This approach facilitates the creation and evaluation of versatile ionizable lipid libraries, advancing the formulation of lipid nanoparticles for precise mRNA delivery.

Ionizable lipid nanoparticles for RAS protease delivery to inhibit cancer cell proliferation

Mutations in RAS, a family of proteins found in all human cells, drive a third of cancers, including many pancreatic, colorectal, and lung cancers. However, we lack clinical therapies that can effectively prevent RAS from causing tumor growth. Recently, a protease was engineered that specifically degrades active RAS, offering a promising new tool for treating these cancers. However, like many other intracellularly acting protein-based therapies, this protease requires a delivery vector to be delivered to its site of action within the cell. In this study, we explored the incorporation of cationic lipids into ionizable lipid nanoparticles (LNPs) to develop a RAS protease delivery platform capable of inhibiting cancer cell proliferation in vitro and in vivo. A library of 13 LNPs was designed, and each formulation was evaluated for in vitro delivery efficiency and toxicity. A subset of 4 top performing LNP formulations was identified and further explored for their impact on cancer cell proliferation in human colorectal cancer cells with mutated KRAS in vitro and in vivo, as well as their in vivo biodistribution and toxicity. In vivo, both the concentration of cationic lipid and type of cargo influenced LNP and cargo distribution. All top LNPs showed RAS protease functionality in vitro, and the top formulation achieves effective intracellular delivery in vivo, decreasing cancer cell proliferation in an in vivo xenograft model, significantly reducing tumor growth and size. Ultimately, this LNP platform demonstrates potential to advance cancer therapeutics, proving the efficacy of the RAS protease when delivered in this fashion.

Dendritic-cell-targeting virus-like particles as potent mRNA vaccine carriers.

Messenger RNA vaccines lack specificity for dendritic cells (DCs)-the most effective cells at antigen presentation. Here we report the design and performance of a DC-targeting virus-like particle pseudotyped with an engineered Sindbis-virus glycoprotein that recognizes a surface protein on DCs, and packaging mRNA encoding for the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or for the glycoproteins B and D of herpes simplex virus 1. Injection of the DC-targeting SARS-CoV-2 mRNA vaccine in the footpad of mice led to substantially higher and durable antigen-specific immunoglobulin-G titres and cellular immune responses than untargeted virus-like particles and lipid-nanoparticle formulations. The vaccines also protected the mice from infection with SARS-CoV-2 or with herpes simplex virus 1. Virus-like particles with preferential uptake by DCs may facilitate the development of potent prophylactic and therapeutic vaccines.

Localization of Intramuscular mRNA Delivery Using Deep Eutectic-Lipid Nanocomposites.

Messenger ribonucleic acid (mRNA) has long been touted as a next-generation therapeutic modality for infectious disease, cancer, and genetic disorders. Lipid nanoparticles (LNPs) provide an elegant delivery strategy for mRNA cargo to help realize this potential for vaccination. However, systemic exposure seen with traditional LNP formulations can have significant implications on efficacy and safety. Efforts to mitigate this have largely been focused on laborious lipid or LNP redesign. Here, the use of a deep eutectic-lipid nanocomposite delivery system for the tuning of mRNA expression for intramuscular injections in vivo is reported. One deep eutectic, cholinium malonate, allows for the linear control of percent expression at the muscular injection site based solely on its concentration in the formulation. The same deep eutectic solvent (DES) can increase local muscle expression by 68% and significantly decrease off-target liver expression by 72%. Physico-chemical studies suggest that the DES incorporates into or onto the pre-formed LNPs thus impacting endosomal escape and in situ interactions. These nanocomposites provide new possibilities for previously approved LNP formulations and without the need for lipid redesign to induce localized expression.

Poly(2-methyl-2-oxazoline) as a polyethylene glycol alternative for lipid nanoparticle formulation

Lipid nanoparticles (LNPs) have emerged as the platform of choice for mRNA delivery. Polyethylene glycol (PEG) is considered a key component of currently approved LNP-based delivery systems as it ensures particle stability and shapes various facets of LNP behavior in biological systems. Whilst PEG has numerous characteristics that are favorable for delivery systems, there is a growing body of evidence that suggests that it is immunogenic. Thus, next-generation mRNA therapeutics are likely to benefit from the identification of PEG alternatives. Towards this end, we have assessed the suitability of poly(2-methyl-2-oxazoline) (PMOZ) for LNP-based mRNA delivery. We compared the properties and bioactivities of PMOZ-containing LNPs to that of a standard composition that includes PEG. Decreasing the percentage of PMOZ in formulations improved transfection efficiency and enhanced the immunostimulatory potential. Reducing the PMOZ density was shown to enhanced antigen-specific T-cell responses in vivo. Interestingly, we found that this was not the case for antibody responses. A direct comparison between LNPs that contain the same amount of PEG or PMOZ strongly suggests that the former induces stronger CD8+ T-cell responses while the latter induces superior neutralizing titers. These findings augur well for the further development of PMOZ as a PEG replacement for LNP-based mRNA delivery approaches.

Facile generation of heterotelechelic poly(2-oxazoline)s towards accelerated exploration of poly(2-oxazoline)-based nanomedicine.

Controlling the end-groups of biocompatible polymers is crucial for enabling polymer-based therapeutics and nanomedicine. Typically, end-group diversification is a challenging and time-consuming endeavor, especially for polymers pre-pared via ionic polymerization mechanisms with limited functional group tolerance. In this study, we present a facile end-group diversification approach for poly(2-oxazoline)s (POx), enabling quick and reliable production of hetero-telechelic polymers to facilitate POxylation. The approach relies on the careful tuning of reaction parameters to establish differential reactivity of a pentafluorobenzyl initiator fragment and the living oxazolinium chain-end, allowing the selective introduction of N-, S-, O-nucleophiles via the termination of the polymerization, and a consecutive nucleophilic para-fluoro substitution. The value of this approach for the accelerated development of nanomedicine is demonstrated through the synthesis of well-defined lipid-polymer conjugates and POx-polypeptide block-copolymers, which are well-suited for drug and gene delivery. Furthermore, we investigated the application of a lipid-POx conjugate for the formulation and delivery of mRNA-loaded lipid nanoparticles for immunization against the SARS-COV-2 virus, underscoring the value of POx as a biocompatible polymer platform.

Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in quadrivalent influenza vaccine vaccinated mice.

There are considerable avenues through which currently licensed influenza vaccines could be optimized. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus-derived defective interfering (SDI) RNA, a RIG-I agonist; and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), a TLR7/8 agonist. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating direct delivery of SDI RNA to the cytosol, where RIG-I sensing induces inflammatory and type I interferon responses. We previously tested SDI RNA and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here, we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The LNPs were incorporated with SDI RNA to determine its potential as a combination adjuvant with IMDQ-PEG-Chol by evaluating the host immune response to vaccination and infection in immunized BALB/c mice. Adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated with quadrivalent inactivated influenza vaccine (QIV), showing robust induction of antibody titers and T-cell responses. Depending on the adjuvant combination and LNP formulation, humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with the protective responses to viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was tested by challenging mice with a vaccine-matched strain of influenza A virus A/Singapore/gp1908/2015 IVR-180 (H1N1). Groups that received either LNP formulated with SDI or IMDQ-PEG-Chol, or both, showed very low levels of viral replication in their lungs at 5 days post-infection (DPI). These studies provide evidence that the combination of vaccines with LNPs and/or adjuvants promote antigen-specific cellular responses that can contribute to protection upon infection. Interestingly, we observed differences in humoral and cellular responses to vaccination between different groups receiving K-Ac7-Dsa or S-Ac7-Dog lipids in LNP formulations. The differences were also reflected in inflammatory responses in lungs of vaccinated animals to infection, depending on LNP formulations. Therefore, this study suggests that the composition of the LNPs, particularly the ionizable lipid, plays an important role in inducing inflammatory responses in vivo, which is important for vaccine safety and to prevent adverse effects upon viral exposure.

Development of Lipid Nanoparticle Formulation for the Repeated Administration of mRNA Therapeutics

Development of Lipid Nanoparticle Formulation for the Repeated Administration of mRNA Therapeutics

Dopamine- and Grape-Seed-Extract-Loaded Solid Lipid Nanoparticles: Interaction Studies between Particles and Differentiated SH-SY5Y Neuronal Cell Model of Parkinson's Disease.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, primarily associated with dopaminergic neuron depletion in the Substantia Nigra. Current treatment focuses on compensating for dopamine (DA) deficiency, but the blood-brain barrier (BBB) poses challenges for effective drug delivery. Using differentiated SH-SY5Y cells, we investigated the co-administration of DA and the antioxidant Grape Seed Extract (GSE) to study the cytobiocompability, the cytoprotection against the neurotoxin Rotenone, and their antioxidant effects. For this purpose, two solid lipid nanoparticle (SLN) formulations, DA-co-GSE-SLNs and GSE-ads-DA-SLNs, were synthesized. Such SLNs showed mean particle sizes in the range of 187-297 nm, zeta potential values in the range of -4.1--9.7 mV, and DA association efficiencies ranging from 35 to 82%, according to the formulation examined. The results showed that DA/GSE-SLNs did not alter cell viability and had a cytoprotective effect against Rotenone-induced toxicity and oxidative stress. In addition, this study also focused on the evaluation of Alpha-synuclein (aS) levels; SLNs showed the potential to modulate the Rotenone-mediated increase in aS levels. In conclusion, our study investigated the potential of SLNs as a delivery system for addressing PD, also representing a promising approach for enhanced delivery of pharmaceutical and antioxidant molecules across the BBB.

Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing

Nonviral delivery of the CRISPR/Cas9 system provides great benefits for in vivo gene therapy due to the low risk of side effects. However, in vivo gene editing by delivering the Cas9 ribonucleoprotein (RNP) is challenging due to the poor delivery into target tissues and cells. Here, we introduce an effective delivery method for the CRISPR/Cas9 RNPs by finely tuning the formulation of ionizable lipid nanoparticles. The LNPs delivering CRISPR/Cas9 RNPs (CrLNPs) are demonstrated to induce gene editing with high efficiencies in various cancer cell lines in vitro. Furthermore, we show that CrLNPs can be delivered into tumor tissues with high efficiency, as well as induce significant gene editing in vivo. The current study presents an effective platform for nonviral delivery of the CRISPR/Cas9 system that can be applied as an in vivo gene editing therapeutic for treating various diseases such as cancer and genetic disorders.

Abstract LB019: Comprehensive therapeutic impact: Follistatin mRNA-loaded lipid nanoparticles halt lung metastases and prolong survival in murine head & neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) poses a significant public health challenge due to its aggressive nature, late-stage diagnosis, and association with cancer-associated cachexia. Activin A (ActA), a member of the TGF-β superfamily, has been implicated in HNSCC pathogenesis and cachexia development. Our study proposes a novel therapeutic approach utilizing follistatin (FST) mRNA-loaded lipid nanoparticles (LNPs) to inhibit activin A and address HNSCC progression and cancer cachexia. We engineered LNPs with pseudouridine-substituted FST mRNA for enhanced stability and translational efficiency. In vitro experiments on murine cell lines demonstrated successful FST protein upregulation and subsequent reduction in ActA expression, particularly in HNSCC cells (7.75 ng/mL ActA levels decreased to 6.12 ng/mL by adding 500ng/mL FST mRNA LNP, p = 0.0184). Scratch assays further indicated decreased cellular mobility (26% increase in open “wound area,” p=0.0029), suggesting potential implications for decreased metastatic behavior. For in vivo validation, C57BL/6 mice bearing MLM3 HNSCC tumors were chosen as the experimental model. Our FST mRNA LNP therapy exhibited tolerability with minimal impact on food intake, body weight, and reactogenic cytokine levels. Comprehensive blood analyses revealed no significant alterations in red blood cell parameters, while liver and spleen samples confirmed successful mRNA delivery and elevated FST protein levels (180-times increase, p = 0.0243) with reduced ActA levels (3-times decrease, p= 0.0467) in the serum. Subcutaneous administration of FST mRNA LNP formulation ipsilateral to MLM3 HNSCC tumors resulted in a significant reduction in tumor size (1.7 times reduction, p=0.0418). Intraperitoneal injections yielded a similar outcome with a significant decrease in tumor burden. Our findings revealed a remarkable and unprecedented outcome: the FST mRNA LNP formulation completely inhibited the development of lung metastases originating from primary MLM3 tumors. Survival analysis demonstrated a significant extension of the lifespan of treated mice compared to control groups, emphasizing the therapeutic efficacy of the FST mRNA LNP formulation in preventing lung metastatic spread. Notably, the administration of FST mRNA LNP demonstrated a significant muscle-sparing effect in the context of cancer-associated cachexia. In conclusion, our study introduces a novel and safe therapeutic strategy for HNSCC, leveraging LNPs to deliver FST mRNA for ActA inhibition. This approach holds promise for future clinical applications, addressing the dual challenges of HNSCC progression and cancer-associated cachexia. Citation Format: Oleh Taratula, Tetiana Korzun, Antony Jozic, Vladislav Grigoriev, Gaurav Sahay, Daniel L. Marks. Comprehensive therapeutic impact: Follistatin mRNA-loaded lipid nanoparticles halt lung metastases and prolong survival in murine head & neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB019.

Genetic and functional correction of argininosuccinate lyase deficiency using CRISPR adenine base editors

Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production.

In vitro uptake of an MC3 lipid nanoparticle formulation by mice, non-human primate, and human hepatocytes and Kupffer cells

In vitro uptake of an MC3 lipid nanoparticle formulation by mice, non-human primate, and human hepatocytes and Kupffer cells

Analytical Characterization of Heterogeneities in mRNA-Lipid Nanoparticles Using Sucrose Density Gradient Ultracentrifugation.

Rational design and robust formulation processes are critical for optimal delivery of mRNA by lipid nanoparticles (LNPs). Varying degrees of heterogeneity in mRNA-LNPs can affect their biophysical and functional properties. Given the profound complexity of mRNA-LNPs, it is critical to develop comprehensive and orthogonal analytical techniques for a better understanding of these formulations. To this end, we developed a robust ultracentrifugation method for density-based separation of subpopulations of mRNA-LNPs. Four LNP formulations encapsulating human erythropoietin (hEPO) with varying functionalities were synthesized using two ionizable lipids, A and B, and two helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), along with cholesterol and DMG-PEG-2K. Upon ultracentrifugation on a sucrose gradient, a distinct pattern of "fractions" was observed across the gradient, from the less dense topmost fraction to the increasingly denser bottom fractions, which were harvested for comprehensive analyses. Parent LNPs, A-DOPE and B-DOPE, were resolved into three density-based fractions, each differing significantly in the hEPO expression following intravenous and intramuscular routes of administration. Parent B-DEPE LNPs resolved into two density-based fractions, with most of the payload and lipid content being attributed to the topmost fraction compared to the lower one, indicating some degree of heterogeneity, while parent A-DEPE LNPs showed remarkable homogeneity, as indicated by comparable in vivo potency, lipid numbers, and particle count among the three density-based fractions. This study is the first to demonstrate the application of density gradient-based ultracentrifugation (DGC) for a head-to-head comparison of heterogeneity as a function of biological performance and biophysical characteristics of parent mRNA-LNPs and their subpopulations.

Abstract 6740: Next-generation LNPs induce effective anti-tumor T cell responses

Abstract mRNA immunotherapies have gained interest for the treatment of tumors from various origins, however, their ability to provide long -term durable tumor control has been underwhelming. Here we report that novel mRNA lipid nanoparticle (LNP) vaccinations induce robust expansion of antigen-specific T cells, although this does not always correlate with their ability to infiltrate and kill solid tumors. Using a portfolio of novel ionizable lipids and a robust in vivo model that reliably identifies LNPs with prominent anti-tissue T cell activity we have identified a putative LNP candidate that provides long term tumor control in a murine syngeneic tumor model. We utilized the model antigen glycoprotein (Gp), from the lymphocytic choriomeningitis virus (LCMV), and produced mRNA encoding the immunodominant epitopes gp3333-41, gp6161-80 and gp276276-286 (3GP-mRNA). This mRNA was encapsulated in various novel LNP formulations and administered i.m. into naïve C57Bl/6 mice to track the induction of a robust CD8+ T cell response identified using tetramers to gp33 and gp276. Successful candidate LNPs were then tested in the RIPgp model of autoimmune diabetes. RIPgp transgenic mice express LCMV-Gp in the pancreatic β cells and offer a robust assay for the ability to induce antigen-specific T cells capable of breaking self-tolerance and infiltrating and killing target tissue. Utilizing the RIPgp model to screen LNP candidates we identified a lead candidate, C2-5A, that both induced a robust expansion of antigen-specific T cells and tissue destruction. When utilized in a colon adenocarcinoma model expressing the LCMV-Gp antigen (MC38-Gp), therapeutic dosing of LNP C2-5A as a monotherapy lead to long term tumor clearance in 40% of the treated animals. We believe this screening platform allows the selection of LNP candidates capable of breaking self-tolerance, and one such candidate C2-5A provides a potent vehicle and adjuvant for mRNA based cancer vaccines. Citation Format: Douglas G. Millar, Matthew J. Gold, Kirsten Olsen, Yu Wu, Yury Karpov, Robert Nechanitzky, Haritha Menon, Rajesh Krishnan, Robert Georgantas, Pamela Ohashi, Natalia Martin-Orozco. Next-generation LNPs induce effective anti-tumor T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6740.

Abstract 5924: RNAi mediated inhibition of beta-catenin demonstrates anti-tumor efficacy and immune microenvironment modulation in preclinical hepatocellular carcinoma models

Abstract Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide, and the third most common cause of cancer-related deaths. For the 40% to 50% of HCC patients who are candidates for systemic therapies, the landscape has evolved significantly in the last 10 years, with most recently, immunotherapies such as combination of atezolizumab and bevacizumab emerging as the current first-line systemic therapy for patients with intermediate or advanced disease. However, there are no predictive biomarkers for patients with HCC to enable selection of those most likely to benefit from these therapies and this remains a disease with high unmet medical need. Activation of the WNT pathway via mutational events or indirect pathway activation is implicated in many cancers including up to 50% of HCC patient tumors. High expression of beta-catenin, encoded by the CTNNB1 gene, has been shown to correlate with decreased survival and more rapid disease progression in HCC patients. In addition, WNT/beta-catenin pathway activation has been shown to result in immune exclusion and resistance to immunotherapy in HCC. ALN-BCAT comprises a chemically modified siRNA encapsulated in a lipid nanoparticle formulation which generates robust and highly specific reductions of CTNNB1 mRNA, downstream biomarkers and tumor cell proliferation in in vitro model systems. Anti-tumor activity has been demonstrated in multiple in vivo orthotopic HCC mouse models, in both early and late treatment settings as measured by liver to body weight ratio. Reductions in tumor burden were orthogonally confirmed by histology analysis and shown to be associated with changes in CTNNB1-related biomarkers. Finally, ALN-BCAT treatment results in changes in the tumor microenvironment in a mouse syngeneic HCC model, with increases in CD4+ and CD8+ T cells and reductions in M2 macrophages, consistent with a shift to a more immune stimulatory anti-tumor microenvironment. Here, we demonstrate that ALN-BCAT provides a novel approach to the inhibition of the WNT pathway as a therapeutic approach for treatment of HCC patients and WNT pathway activated tumors. Citation Format: Tulin Dadali, Spencer Miller, Gaurav Saawant, Stephen Abbott, Mikyung Yu, Svetlana Morskaya, Brandon Lehrich, Satdarshan Monga, Gloria Lau, Martin Maier, Wendy Broom. RNAi mediated inhibition of beta-catenin demonstrates anti-tumor efficacy and immune microenvironment modulation in preclinical hepatocellular carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5924.

Abstract 6752: Targeting of neoantigen cancer vaccines to antigen presenting cells results in faster and broader T cell responses whether encoded in DNA or mRNA

Abstract Individualized cancer vaccines based on patient-specific neoantigens represent a groundbreaking approach in oncology, offering a highly individualized treatment option for cancer patients. Unlike traditional vaccines, which are designed to prevent diseases by stimulating an immune response against a specific pathogen, personalized cancer vaccines are therapeutic, aiming to treat cancer by training the immune system to recognize and attack the patient’s own cancer cells. The key is to induce a fast, strong, broad, and persistent CD8+ T cell response. Nykode Therapeutics has developed a platform that targets antigens directly to antigen presenting cells (APCs) using a modular dimeric protein format known as a VaccibodyTM. Individualized neoantigen vaccines delivered in a DNA-encoded Vaccibody format have been shown to induce strong and broad T cell responses in pre-clinical and clinical settings. Here, we wanted to determine if the APC-targeted approach leads to superior T cell responses if Vaccibody vaccines are encoded in mRNA and administered in a lipid nanoparticle (LNP) formulation. For this purpose, we designed and evaluated a model cancer vaccine composed of 20 unstructured 27 amino acid long neoantigens derived from the murine MC38 tumor model. A DNA-encoded Vaccibody vaccine elicited faster T cell responses compared to an untargeted mRNA-LNP-encoded vaccine. DNA-encoded Vaccibody responses were also broader, targeting a substantially greater number of neoantigens. Next, we compared the APC-targeting Vaccibody vaccine to an untargeted vaccine when both were formulated in an mRNA-LNP format. The APC-targeting Vaccibody vaccine elicited a greater breath and magnitude of T cell responses compared to the untargeted vaccine. Further studies to evaluate the functional differences associated with the various formats, in terms of T cell phenotype and response to tumor challenge, are ongoing. In summary, the data demonstrate that an APC-targeted Vaccibody vaccine containing MC38 neoantigens can be encoded in both DNA and mRNA-LNP to induce faster, broader, and stronger T cell responses compared to a non-targeted neoantigen vaccine. Citation Format: Brian T. Weinert, Lise Skullerud, Eirik Solbakken, Ana Textor, Mirjam Dürkoop, Inga Patrycja Winge, Gunnstein Norheim, Elisabeth Stubsrud, Stine Granum, Mikkel W. Pedersen, Håkan Norell. Targeting of neoantigen cancer vaccines to antigen presenting cells results in faster and broader T cell responses whether encoded in DNA or mRNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6752.

Simple Scattering: Lipid nanoparticle structural data repository.

Lipid nanoparticles (LNPs) are being intensively researched and developed to leverage their ability to safely and effectively deliver therapeutics. To achieve optimal therapeutic delivery, a comprehensive understanding of the relationship between formulation, structure, and efficacy is critical. However, the vast chemical space involved in the production of LNPs and the resulting structural complexity make the structure to function relationship challenging to assess and predict. New components and formulation procedures, which provide new opportunities for the use of LNPs, would be best identified and optimized using high-throughput characterization methods. Recently, a high-throughput workflow, consisting of automated mixing, small-angle X-ray scattering (SAXS), and cellular assays, demonstrated a link between formulation, internal structure, and efficacy for a library of LNPs. As SAXS data can be rapidly collected, the stage is set for the collection of thousands of SAXS profiles from a myriad of LNP formulations. In addition, correlated LNP small-angle neutron scattering (SANS) datasets, where components are systematically deuterated for additional contrast inside, provide complementary structural information. The centralization of SAXS and SANS datasets from LNPs, with appropriate, standardized metadata describing formulation parameters, into a data repository will provide valuable guidance for the formulation of LNPs with desired properties. To this end, we introduce Simple Scattering, an easy-to-use, open data repository for storing and sharing groups of correlated scattering profiles obtained from LNP screening experiments. Here, we discuss the current state of the repository, including limitations and upcoming changes, and our vision towards future usage in developing our collective knowledge base of LNPs.

Abstract 3252: Anti-tumor effect of the combination of a lipid nanoparticle suspension of an AKT-1 antisense oligonucleotide (WGI-0301) with tyrosine kinase inhibitors in an orthotopic murine tumor model

Abstract Background: Archexin is a fully phosphorothioated 20-mer antisense oligonucleotide, complementary to AKT-1 mRNA, which leads to the inhibition of translation and the downstream pathway activities of AKT-1 mRNA. WGI-0301 is a proprietary lipid nanoparticle formulation (QTsomeTM) of Archexin designed to enhance delivery. A phase I clinical study of WGI-0301 as monotherapy in patients with advanced solid tumors is currently ongoing in the U.S. Given the limited efficacy of TKIs compared to immunotherapy and growing incidence resistance, WGI-0301 combined with TKIs may augment the therapeutic response to TKIs by enhancing angiogenic blockade, inhibiting AKT-1, and overcome resistance. Methods: The anti-tumor effect of combining WGI-0301 with Lenvatinib/Sorafenib/Cabozantinib was assessed using a Hep3B-Luciferase orthotopic mouse tumor model. We divided sixty-four female Balb/c nude mice into groups (8 mice per group), including vehicle control, WGI-0301 (8 mg/kg), Lenvatinib (10 mg/kg), Sorafenib (20 mg/kg), Cabozantinib (20 mg/kg), WGI-0301+Lenvatinib (8+10 mg/kg), WGI-0301+Sorafenib (8+20 mg/kg), and WGI-0301+Cabozantinib (8+20 mg/kg). We administered treatments via tail vein injection (vehicle/WGI-0301, QW4 weeks) or oral administration (Lenvatinib/Sorafenib/Cabozantinib, QD28 days). We monitored body weight twice weekly and measured tumor burden using bioluminescence with an IVIS (Lumina III) imaging system twice weekly as well. Treatment was suspended between Day 29 and Day 70 for survival observation. Results: The tumor growth inhibition (TGI) rates of the combination of WGI-0301 with Lenvatinib/Sorafenib/Cabozantinib were 66.05%, 86.81%, and 75.89%, respectively. These values were higher than those of the Lenvatinib/Sorafenib/Cabozantinib single drug group with TGI rate of 50.84%, 55.68%, and 47.84%. The tumor bioluminescence signal intensities of the two drugs combination (56.74*108, 22.28*108, and 40.40*108 photon/s) were slightly lower than that of the Lenvatinib/Sorafenib/Cabozantinib single drug group (81.97*108, 73.95*108, and 86.96*108 photon/s). The median survival time was 35.0, 46.0, 55.5, and 51.5 days in the vehicle, WGI-0301+Lenvatinib, WGI-0301+Sorafenib and WGI-0301+Cabozantinib, respectively. The median survival time of Sorafenib, the two-drug combinations were 55.5, 55.5, 58.0, and 53.5 days, which was significantly prolonged compared to the vehicle in the mice bearing the Hep3B-luc orthotopic tumor model. No severe adverse effects were observed in the drug combination groups. Conclusion: The combination of WGI-0301 with TKIs demonstrated superior anti-tumor efficacy over TKI monotherapy in the absence of substantial increase in toxicity. This finding warrants further robust clinical investigation in patients with advanced HCC. Citation Format: Fengyang Xie, Tao Lin, Chao Wang, Jia Wei, Robert J. Lee, Ben Zhao, Yuxin Angela Men. Anti-tumor effect of the combination of a lipid nanoparticle suspension of an AKT-1 antisense oligonucleotide (WGI-0301) with tyrosine kinase inhibitors in an orthotopic murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3252.

Abstract 7249: Enhancing mRNA- lipid nanoparticles formulations to improve transfection efficiency in macrophages and dendritic cells in cancer immunotherapy

Abstract Cancer poses a global health challenge, driving the quest for innovative therapies. Messenger ribonucleic acid (mRNA) lipid nanoparticles (LNPs) are a promising strategy in cancer immunotherapy. However, a major obstacle lies in efficiently transfecting immune cells like macrophages and dendritic cells (DCs) with LNPs. This study seeks to improve cancer immunotherapy by optimizing mRNA-LNP formulations. Therefore, we systematically evaluated three components of LNPs, namely ionizable lipids, phospholipids, and sterols, and fine-tuned the components and their molar ratios. A comprehensive screening process was conducted to determine the efficiency of LNPs, involving in vitro experiments on macrophages and DCs, as well as in vivo assessments. Among six ionizable lipids assessed in different cell lines (RAW264.7 and DC2.4), SM-102 significantly enhanced the transfection efficiency in macrophages and DCs. Subsequently, we evaluated these LNPs in an in vivo setting. We quantified the bioluminescence signal after administering LNPs loaded with luciferase mRNA. The results demonstrated a significant increase in luciferase expression when using SM-102 and ALC-0315 lipids. To validate this finding, we conducted a T cell response study, which revealed an increased percentage of CD8+ T cells with SM-102-based LNPs compared to other formulations. Having identified SM-102 as a potent ionizable lipid, we proceeded to evaluate three phospholipids in vitro. The results indicated a significant enhancement in transfection efficiency with DOPE lipids compared to DSPC. Consequently, we selected DOPE as the phospholipid for subsequent screening. Lastly, we explored different types of sterols with varying molar ratios in macrophages and DC cells. Our findings unveiled a notable improvement in mRNA delivery, particularly in DCs, when the molar ratio of β-sitosterol was increased to 19.5%, while concurrently decreasing the molar ratio of cholesterol to 19% within the same formulation. It is noteworthy that the addition of β-sitosterol alone led to reduced transfection efficiency in DCs but resulted in a significant enhancement in macrophages. Lastly, we evaluated the top four formulations in vivo, and the results demonstrated a significant increase when β-sitosterol was added to the formulation with cholesterol, at molar ratios of 19.5% and 19%, respectively. Overall, altering lipid composition and their molar ratios profoundly impact mRNA-LNP transfection. The optimized formulations, comprising SM-102, DOPE, and cholesterol with or without β-sitosterol, outperformed the alternatives. These refined formulations hold promise for clinical applications, potentially enhancing passive therapeutic agent delivery into macrophages and DCs, offering promising prospects for advancing cancer immunotherapy. Citation Format: Yasir Alshehry, Matthew Fernandez, Raneem Aldaqqa, Asma Al-Terawi, Douglas Sweet, Sandro da Rocha. Enhancing mRNA- lipid nanoparticles formulations to improve transfection efficiency in macrophages and dendritic cells in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7249.