Local Delivery of Ox40l, Cd80, and Cd86 mRNA Kindles Global Anticancer Immunity.

Localized expression of effector molecules can initiate antitumor responses through engagement of specific receptors on target cells in the tumor microenvironment. These locally induced responses may also have a systemic effect, clearing additional tumors throughout the body. In this study, to evoke systemic antitumor responses, we utilized charge-altering releasable transporters (CART) for local intratumoral delivery of mRNA coding for costimulatory and immune-modulating factors. Intratumoral injection of the CART-mRNA complexes resulted in mRNA expression at the site of administration, transfecting a substantial proportion of tumor-infiltrating dendritic cells, macrophages, and T cells in addition to the tumor cells, resulting in a local antitumor effect. Using a two-tumor model, we further show that mRNA therapy locally administered to one tumor stimulated a systemic antitumor response, curing both tumors. The combination of Ox40l-, Cd80-, and Cd86-encoding mRNA resulted in the local upregulation of proinflammatory cytokines, robust local T-cell activation, and migration of immune cells to local draining lymph node or to an anatomically distant tumor. This approach delayed tumor growth, facilitated tumor regression, and cured tumors in both A20 and CT26 tumor models. These results highlight mRNA-CART therapy as a viable approach to induce systemic antitumor immunity from a single localized injection. SIGNIFICANCE: The mRNA-CART system is a highly effective delivery platform for delivering immunostimulatory genes into the tumor microenvironment for potential therapeutic development.

Delivery of Inorganic Polyphosphate into Cells Using Amphipathic Oligocarbonate Transporters.

Inorganic polyphosphate (polyP) is an often-overlooked biopolymer of phosphate residues present in living cells. PolyP is associated with many essential biological roles. Despite interest in polyP's function, most studies have been limited to extracellular or isolated protein experiments, as polyanionic polyP does not traverse the nonpolar membrane of cells. To address this problem, we developed a robust, readily employed method for polyP delivery using guanidinium-rich oligocarbonate transporters that electrostatically complex polyPs of multiple lengths, forming discrete nanoparticles that are resistant to phosphatase degradation and that readily enter multiple cell types. Fluorescently labeled polyPs have been monitored over time for subcellular localization and release from the transporter, with control over release rates achieved by modulating the transporter identity and the charge ratio of the electrostatic complexes. This general approach to polyP delivery enables the study of intracellular polyP signaling in a variety of applications.

mRNA vaccination with charge-altering releasable transporters elicits human T cell responses and cures established tumors in mice.

In vivo delivery of antigen-encoding mRNA is a promising approach to personalized cancer treatment. The therapeutic efficacy of mRNA vaccines is contingent on safe and efficient gene delivery, biological stability of the mRNA, and the immunological properties of the vaccine. Here we describe the development and evaluation of a versatile and highly efficient mRNA vaccine-delivery system that employs charge-altering releasable transporters (CARTs) to deliver antigen-coding mRNA to antigen-presenting cells (APCs). We demonstrate in human peripheral blood mononuclear cells that CART vaccines can activate a robust antigen-specific immune response against mRNA-encoded viral epitopes. In an established mouse model, we demonstrate that CARTs preferentially target professional APCs in secondary lymphoid organs upon i.v. injections and target local APCs upon s.c. injection. Finally, we show that CARTs coformulated with mRNA and a Toll-like receptor ligand simultaneously transfect and activate target cells to generate an immune response that can treat and cure mice with large, established tumors.

Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters.

We report a strategy for generating a combinatorial library of oligonucleotide transporters with varied lipid domains and their use in the efficient transfection of lymphocytes with mRNA in vitro and in vivo. This library is based on amphiphilic charge-altering releasable transporters (CARTs) that contain a lipophilic block functionalized with various side-chain lipids and a polycationic α-amino ester mRNA-binding block that undergoes rearrangement to neutral small molecules, resulting in mRNA release. We show that certain binary mixtures of these lipid-varied CARTs provide up to a ninefold enhancement in mRNA translation in lymphocytes in vitro relative to either a single-lipid CART component alone or the commercial reagent Lipofectamine 2000, corresponding to a striking increase in percent transfection from 9-12% to 80%. Informed by the results with binary mixtures, we further show that CARTs consisting of optimized ratios of the two lead lipids incorporated into a single hybrid-lipid transporter molecule maintain the same delivery efficacy as the noncovalent mixture of two CARTs. The lead lipid CART mixtures and hybrid-lipid CARTs show enhanced lymphocyte transfection in primary T cells and in vivo in mice. This combinatorial approach for rapidly screening mRNA delivery vectors has provided lipid-varied CART mixtures and hybrid-lipid CARTs that exhibit significant improvement in mRNA delivery to lymphocytes, a finding of potentially broad value in research and clinical applications.

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals.

Functional delivery of mRNA to tissues in the body is key to implementing fundamentally new and potentially transformative strategies for vaccination, protein replacement therapy, and genome editing, collectively affecting approaches for the prevention, detection, and treatment of disease. Broadly applicable tools for the efficient delivery of mRNA into cultured cells would advance many areas of research, and effective and safe in vivo mRNA delivery could fundamentally transform clinical practice. Here we report the step-economical synthesis and evaluation of a tunable and effective class of synthetic biodegradable materials: charge-altering releasable transporters (CARTs) for mRNA delivery into cells. CARTs are structurally unique and operate through an unprecedented mechanism, serving initially as oligo(α-amino ester) cations that complex, protect, and deliver mRNA and then change physical properties through a degradative, charge-neutralizing intramolecular rearrangement, leading to intracellular release of functional mRNA and highly efficient protein translation. With demonstrated utility in both cultured cells and animals, this mRNA delivery technology should be broadly applicable to numerous research and therapeutic applications.

Cellular delivery and photochemical release of a caged inositol-pyrophosphate induces PH-domain translocation in cellulo.

Inositol pyrophosphates, such as diphospho-myo-inositol pentakisphosphates (InsP7), are an important family of signalling molecules, implicated in many cellular processes and therapeutic indications including insulin secretion, glucose homeostasis and weight gain. To understand their cellular functions, chemical tools such as photocaged analogues for their real-time modulation in cells are required. Here we describe a concise, modular synthesis of InsP7 and caged InsP7. The caged molecule is stable and releases InsP7 only on irradiation. While photocaged InsP7 does not enter cells, its cellular uptake is achieved using nanoparticles formed by association with a guanidinium-rich molecular transporter. This novel synthesis and unprecedented polyphosphate delivery strategy enable the first studies required to understand InsP7 signalling in cells with controlled spatiotemporal resolution. It is shown herein that cytoplasmic photouncaging of InsP7 leads to translocation of the PH-domain of Akt, an important signalling-node kinase involved in glucose homeostasis, from the membrane into the cytoplasm.

Cell-Penetrating, Guanidinium-Rich Oligophosphoesters: Effective and Versatile Molecular Transporters for Drug and Probe Delivery.

The design, synthesis, and biological evaluation of a new family of highly effective cell-penetrating molecular transporters, guanidinium-rich oligophosphoesters, are described. These unique transporters are synthesized in two steps, irrespective of oligomer length, by the organocatalytic ring-opening polymerization (OROP) of 5-membered cyclic phospholane monomers followed by oligomer deprotection. Varying the initiating alcohol results in a wide variety of cargo attachment strategies for releasable or nonreleasable transporter applications. Initiation of oligomerization with a fluorescent probe produces, upon deprotection, a transporter-probe conjugate that is shown to readily enter multiple cell lines in a dose-dependent manner. These new transporters are superior in cell uptake to previously studied guanidinium-rich oligocarbonates and oligoarginines, showing over 2-fold higher uptake than the former and 6-fold higher uptake than the latter. Initiation with a protected thiol gives, upon deprotection, thiol-terminated transporters which can be thiol-click conjugated to a variety of probes, drugs and other cargos as exemplified by the conjugation and delivery of the model probe fluorescein-maleimide and the medicinal agent paclitaxel (PTX) into cells. Of particular significance given that drug resistance is a major cause of chemotherapy failure, the PTX-transporter conjugate, designed to evade Pgp export and release free PTX after cell entry, shows efficacy against PTX-resistant ovarian cancer cells. Collectively this study introduces a new and highly effective class of guanidinium-rich cell-penetrating transporters and methodology for their single-step conjugation to drugs and probes, and demonstrates that the resulting drug/probe-conjugates readily enter cells, outperforming previously reported guanidinium-rich oligocarbonates and peptide transporters.