testing-vitro-vivo-efficiency-mrna-lipid-nanoparticles-formulated

Testing the <em>In Vitro</em> and <em>In Vivo</em> Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and ...

University of Pennsylvania

The development of therapeutic cancer vaccines as a means to generate immune reactivity against tumors has been explored since the early discovery of tumor-specific antigens by Georg Klein in the 1960s. However, challenges including weak immunogenicity, systemic toxicity, and off-target effects of cancer vaccines remain as barriers to their broad clinical translation. Advances in the design and implementation of biomaterials are now enabling enhanced efficacy and reduced toxicity of cancer vaccines by controlling the presentation and release of vaccine components to immune cells and their microenvironment. Here, we discuss the rational design and clinical status of several classes of cancer vaccines (including DNA, mRNA, peptide/protein, and cell-based vaccines) along with novel biomaterial-based delivery technologies that improve their safety and efficacy. Further, strategies for designing new platforms for personalized cancer vaccines are also considered.

Biomaterials for vaccine-based cancer immunotherapy.

Messenger RNA (mRNA) has recently emerged as a promising class of nucleic acid therapy, with the potential to induce protein production to treat and prevent a range of diseases. However, the widespread use of mRNA as a therapeutic requires safe and effective in vivo delivery technologies. Libraries of ionizable lipid nanoparticles (LNPs) have been designed to encapsulate mRNA, prevent its degradation, and mediate intracellular delivery. However, these LNPs are typically characterized and screened in an in vitro setting, which may not fully replicate the biological barriers that they encounter in vivo. Here, we designed and evaluated a library of engineered LNPs containing barcoded mRNA (b-mRNA) to accelerate the screening of mRNA delivery platforms in vivo. These b-mRNA are similar in structure and function to regular mRNA, and contain barcodes that enable their delivery to be quantified via deep sequencing. Using a mini-library of b-mRNA LNPs formulated via microfluidic mixing, we show that these different formulations can be pooled together, administered intravenously into mice as a single pool, and their delivery to multiple organs (liver, spleen, brain, lung, heart, kidney, pancreas, and muscle) can be quantified simultaneously using deep sequencing. In the context of liver and spleen delivery, LNPs that exhibited high b-mRNA delivery also yielded high luciferase expression, indicating that this platform can identify lead LNP candidates as well as optimal formulation parameters for in vivo mRNA delivery. Interestingly, LNPs with identical formulation parameters that encapsulated different types of nucleic acid barcodes (b-mRNA versus a DNA barcode) altered in vivo delivery, suggesting that the structure of the barcoded nucleic acid affects LNP in vivo delivery. This platform, which enables direct barcoding and subsequent quantification of a functional mRNA, can accelerate the in vivo screening and design of LNPs for mRNA therapeutic applications such as CRISPR-Cas9 gene editing, mRNA vaccination, and other mRNA-based regenerative medicine and protein replacement therapies.

Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening.

Chimeric antigen receptor (CAR) T cell therapy relies on the  manipulation of patient T cells to create potent, cancer-targeting therapies, shown to be capable of inducing remission in patients with acute lymphoblastic leukemia and large B cell lymphoma. However, current CAR T cell engineering methods use viral delivery vectors, which induce permanent CAR expression and could lead to severe adverse effects. Messenger RNA (mRNA) has been explored as a promising strategy for inducing transient CAR expression in T cells to mitigate the adverse effects associated with viral vectors, but it most commonly requires electroporation for T cell mRNA delivery, which can be cytotoxic. Here, ionizable lipid nanoparticles (LNPs) were designed for  mRNA delivery to human T cells. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for luciferase mRNA delivery to Jurkat cells, revealing seven formulations capable of enhanced mRNA delivery over lipofectamine. The top-performing LNP formulation, C14-4, was selected for CAR mRNA delivery to primary human T cells. This platform induced CAR expression at levels equivalent to electroporation, with substantially reduced cytotoxicity. CAR T cells engineered via C14-4 LNP treatment were then compared to electroporated CAR T cells in a coculture assay with Nalm-6 acute lymphoblastic leukemia cells, and both CAR T cell engineering methods elicited potent cancer-killing activity. These results demonstrate the ability of LNPs to deliver mRNA to primary human T cells to induce functional protein expression, and indicate the potential of LNPs to enhance mRNA-based CAR T cell engineering methods.

Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering.

In recent years, the development of nanoparticles has expanded into a broad range of clinical applications. Nanoparticles have been developed to overcome the limitations of free therapeutics and navigate biological barriers - systemic, microenvironmental and cellular - that are heterogeneous across patient populations and diseases. Overcoming this patient heterogeneity has also been accomplished through precision therapeutics, in which personalized interventions have enhanced therapeutic efficacy. However, nanoparticle development continues to focus on optimizing delivery platforms with a one-size-fits-all solution. As lipid-based, polymeric and inorganic nanoparticles are engineered in increasingly specified ways, they can begin to be optimized for drug delivery in a more personalized manner, entering the era of precision medicine. In this Review, we discuss advanced nanoparticle designs utilized in both non-personalized and precision applications that could be applied to improve precision therapies. We focus on advances in nanoparticle design that overcome heterogeneous barriers to delivery, arguing that intelligent nanoparticle design can improve efficacy in general delivery applications while enabling tailored designs for precision applications, thereby ultimately improving patient outcome overall.

Engineering precision nanoparticles for drug delivery.

Nucleic acids, such as messenger RNAs, antisense oligonucleotides, and short interfering RNAs, hold great promise for treating previously 'undruggable' diseases. However, there are numerous biological barriers that hinder nucleic acid delivery to target cells and tissues. While lipid nanoparticles (LNPs) have been developed to protect nucleic acids from degradation and mediate their intracellular delivery, it is challenging to predict how alterations in LNP formulation parameters influence delivery to different organs. In this study, we utilized high-throughput in vivo screening to probe for structure-function relationships of intravenously administered LNPs along with quartz crystal microbalance with dissipation monitoring (QCM-D) to measure the binding affinity of LNPs to apolipoprotein E (ApoE), a protein implicated in the clearance and uptake of lipoproteins by the liver. High-throughput in vivo screening of a library consisting of 96 LNPs identified several formulations containing the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) that preferentially accumulated in the liver, while identical LNPs that substituted DOPE with the helper lipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) preferentially accumulated in the spleen. Using QCM-D, it was found that one DOPE-containing LNP formulation (LNP 42) had stronger interactions with ApoE than an identical LNP formulation that substituted DOPE with DSPC (LNP 90). In order to further validate our findings, we formulated LNP 42 and LNP 90 to encapsulate Cy3-siRNA or mRNA encoding for firefly luciferase. The DSPC-containing LNP (LNP 90) was found to increase delivery to the spleen for both siRNA (two-fold) and mRNA (five-fold). In terms of liver delivery, the DOPE-containing LNP (LNP 42) enhanced mRNA delivery to the liver by two-fold and improved liver transfection by three-fold. Understanding the role of the helper lipid in LNP biodistribution and ApoE adsorption may aid in the future design of LNPs for nucleic acid therapeutics.

Helper lipid structure influences protein adsorption and delivery of lipid nanoparticles to spleen and liver.

T-cell-based immunotherapies hold promise for the treatment of many types of cancer, with three approved products for B-cell malignancies and a large pipeline of treatments in clinical trials. However, there are several challenges to their broad implementation. These include insufficient expansion of adoptively transferred T cells, inefficient trafficking of T cells into solid tumours, decreased T-cell activity due to a hostile tumour microenvironment and the loss of target antigen expression. Together, these factors restrict the number of therapeutically active T cells engaging with tumours. Nanomaterials are uniquely suited to overcome these challenges, as they can be rationally designed to enhance T-cell expansion, navigate complex physical barriers and modulate tumour microenvironments. Here, we present an overview of nanomaterials that have been used to overcome clinical barriers to T-cell-based immunotherapies and provide our outlook of this emerging field at the interface of cancer immunotherapy and nanomaterial design.

Nanomaterials for T-cell cancer immunotherapy.

Clinical advances enable the prenatal diagnosis of genetic diseases that are candidates for gene and enzyme therapies such as messenger RNA (mRNA)-mediated protein replacement. Prenatal mRNA therapies can treat disease before the onset of irreversible pathology with high therapeutic efficacy and safety due to the small fetal size, immature immune system, and abundance of progenitor cells. However, the development of nonviral platforms for prenatal delivery is nascent. We developed a library of ionizable lipid nanoparticles (LNPs) for in utero mRNA delivery to mouse fetuses. We screened LNPs for luciferase mRNA delivery and identified formulations that accumulate within fetal livers, lungs, and intestines with higher efficiency and safety compared to benchmark delivery systems, DLin-MC3-DMA and jetPEI. We demonstrate that LNPs can deliver mRNAs to induce hepatic production of therapeutic secreted proteins. These LNPs may provide a platform for in utero mRNA delivery for protein replacement and gene editing.

Ionizable lipid nanoparticles for in utero mRNA delivery.

While initial approaches to adoptive T cell therapy relied on the identification and expansion of rare tumour-reactive T cells, genetic engineering has transformed cancer immunotherapy by enabling the modification of primary T cells to increase their therapeutic potential. Specifically, gene editing technologies have been utilized to create T cell populations with improved responses to antigens, lower rates of exhaustion, and potential for use in allogeneic applications. In this review, we provide an overview of T cell therapy gene editing strategies and the delivery technologies utilized to genetically engineer T cells. We also discuss recent investigations and clinical trials that have utilized gene editing to enhance the efficacy of T cells and broaden the application of cancer immunotherapies.

Delivery technologies for T cell gene editing: Applications in cancer immunotherapy.

Efficient viral or nonviral delivery of nucleic acids is the key step of genetic nanomedicine. Both viral and synthetic vectors have been successfully employed for genetic delivery with recent examples being DNA, adenoviral, and mRNA-based Covid-19 vaccines. Viral vectors can be target specific and very efficient but can also mediate severe immune response, cell toxicity, and mutations. Four-component lipid nanoparticles (LNPs) containing ionizable lipids, phospholipids, cholesterol for mechanical properties, and PEG-conjugated lipid for stability represent the current leading nonviral vectors for mRNA. However, the segregation of the neutral ionizable lipid as droplets in the core of the LNP, the "PEG dilemma", and the stability at only very low temperatures limit their efficiency. Here, we report the development of a one-component multifunctional ionizable amphiphilic Janus dendrimer (IAJD) delivery system for mRNA that exhibits high activity at a low concentration of ionizable amines organized in a sequence-defined arrangement. Six libraries containing 54 sequence-defined IAJDs were synthesized by an accelerated modular-orthogonal methodology and coassembled with mRNA into dendrimersome nanoparticles (DNPs) by a simple injection method rather than by the complex microfluidic technology often used for LNPs. Forty four (81%) showed activity  and 31 (57%) . Some, exhibiting organ specificity, are stable at 5 °C and demonstrated higher transfection efficiency than positive control experiments  and . Aside from practical applications, this proof of concept will help elucidate the mechanisms of packaging and release of mRNA from DNPs as a function of ionizable amine concentration, their sequence, and constitutional isomerism of IAJDs.

One-Component Multifunctional Sequence-Defined Ionizable Amphiphilic Janus Dendrimer Delivery Systems for mRNA.

Viral engineered chimeric antigen receptor (CAR) T cell therapies are potent, targeted cancer immunotherapies, but their permanent CAR expression can lead to severe adverse effects. Nonviral messenger RNA (mRNA) CAR T cells are being explored to overcome these drawbacks, but electroporation, the most common T cell transfection method, is limited by cytotoxicity. As a potentially safer nonviral delivery strategy, here, sequential libraries of ionizable lipid nanoparticle (LNP) formulations with varied excipient compositions were screened in comparison to a standard formulation for improved mRNA delivery to T cells with low cytotoxicity, revealing B10 as the top formulation with a 3-fold increase in mRNA delivery. When compared to electroporation in primary human T cells, B10 LNPs induced comparable CAR expression with reduced cytotoxicity while demonstrating potent cancer cell killing. These results demonstrate the impact of excipient optimization on LNP performance and support B10 LNPs as a potent mRNA delivery platform for T cell engineering.

Orthogonal Design of Experiments for Optimization of Lipid Nanoparticles for mRNA Engineering of CAR T Cells.

Congenital disorders resulting in pathological protein deficiencies are most often treated postnatally with protein or enzyme replacement therapies. However, treatment of these disorders in utero before irreversible disease onset could significantly minimize disease burden, morbidity, and mortality. One possible strategy for the prenatal treatment of congenital disorders is in utero delivery of messenger RNA (mRNA). mRNA is a nucleic acid therapeutic that has previously been investigated as a platform for protein replacement therapies and gene editing technologies. While viral vectors have been explored to induce intracellular expression of mRNA, they are limited in their clinical application due to risks associated with immunogenicity and genomic integration. As an alternative to viral vectors, safe and efficient in utero mRNA delivery can be achieved using ionizable lipid nanoparticles (LNPs). While LNPs have demonstrated potent in vivo mRNA delivery to the liver following intravenous administration, intra-amniotic delivery has the potential to deliver mRNA to cells and tissues beyond those in the liver, such as in the skin, lung, and digestive tract. However, LNP stability in fetal amniotic fluid and how this stability affects mRNA delivery has not been previously investigated. Here, we engineered a library of LNPs using orthogonal design of experiments (DOE) to evaluate how LNP structure affects their stability in amniotic fluid ex utero and whether a lead candidate identified from these stability measurements enables intra-amniotic mRNA delivery in utero. We used a combination of techniques including dynamic light scattering (DLS), transmission electron microscopy (TEM), and chromatography followed by protein content quantification to screen LNP stability in amniotic fluids. These results identified multiple lead LNP formulations that are highly stable in amniotic fluids ranging from small animals to humans, including mouse, sheep, pig, and human amniotic fluid samples. We then demonstrate that stable LNPs from the ex utero screen in mouse amniotic fluid enabled potent mRNA delivery in primary fetal lung fibroblasts and in utero following intra-amniotic injection in a murine model. This exploration of ex utero stability in amniotic fluids demonstrates a means by which to identify novel LNP formulations for prenatal treatment of congenital disorders via in utero mRNA delivery.

Amniotic fluid stabilized lipid nanoparticles for in utero intra-amniotic mRNA delivery.

Nanoparticles, mRNA, and ultraviolet light combine to reprogram specific immune cells directly in the body.

Lighting the way to personalized mRNA immune cell therapies.

Delivery of nucleic acids, such as mRNA, to immune cells has become a major focus in the past decade with ionizable lipid nanoparticles (LNPs) emerging as a clinically-validated delivery platform. LNPs-typically composed of ionizable lipids, cholesterol, phospholipids, and polyethylene glycol lipids -have been designed and optimized for a variety of applications including cancer therapies, vaccines, and gene editing. However, LNPs have only recently been investigated for delivery to T cells, which has various therapeutic applications including the engineering of T cell immunotherapies. While several LNP formulations have been evaluated for mRNA delivery, recent work has demonstrated that the utilization of cholesterol analogs may enhance mRNA delivery. Other studies have shown that cholesterols modified with hydroxyl groups can alter endocytic recycling mechanisms. Here, we engineered a library of LNPs incorporating hydroxycholesterols to evaluate their impact on mRNA delivery to T cells by leveraging endosomal trafficking mechanisms. Substitution of 25% and 50% 7α-hydroxycholesterol for cholesterol in LNPs enhanced mRNA delivery to primary human T cells ex vivo by 1.8-fold and 2.0-fold, respectively. Investigation of endosomal trafficking revealed that these modifications also increase late endosome production and reduce the presence of recycling endosomes. These results suggest that hydroxyl modification of cholesterol molecules incorporated into LNP formulations provides a mechanism for improving delivery of nucleic acid cargo to T cells for a range of immunotherapy applications.

Hydroxycholesterol substitution in ionizable lipid nanoparticles for mRNA delivery to T cells.

Lipid nanoparticle-mediated RNA delivery holds great potential to treat various liver diseases. However, targeted delivery of RNA therapeutics to activated liver-resident fibroblasts for liver fibrosis treatment remains challenging. Here, we develop a combinatorial library of anisamide ligand-tethered lipidoids (AA-lipidoids) using a one-pot, two-step modular synthetic method and adopt a two-round screening strategy to identify AA-lipidoids with both high potency and selectivity to deliver RNA payloads to activated fibroblasts. The lead AA-lipidoid AA-T3A-C12 mediates greater RNA delivery and transfection of activated fibroblasts than its analog without anisamide and the FDA-approved MC3 ionizable lipid. In a preclinical model of liver fibrosis, AA-T3A-C12 enables ~65% silencing of heat shock protein 47, a therapeutic target primarily expressed by activated fibroblasts, which is 2-fold more potent than MC3, leading to significantly reduced collagen deposition and liver fibrosis. These results demonstrate the potential of AA-lipidoids for targeted RNA delivery to activated fibroblasts. Furthermore, these synthetic methods and screening strategies open a new avenue to develop and discover potent lipidoids with targeting properties, which can potentially enable RNA delivery to a range of cell and tissue types that are challenging to access using traditional lipid nanoparticle formulations.

Margaret M. Billingsley

Department of Bioengineering

University of Pennsylvania

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Margaret M. Billingsley

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University of Pennsylvania

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Department of Bioengineering