Lysine-Derived Charge-Altering Releasable Transporters: Targeted Delivery of mRNA and siRNA to the Lungs.

Targeted delivery of nucleic acid therapeutics to the lungs could transform treatment options for pulmonary disease. We have previously developed oligomeric charge-altering releasable transporters (CARTs) for in vivo mRNA transfection and demonstrated their efficacy for use in mRNA-based cancer vaccination and local immunomodulatory therapies against murine tumors. While our previously reported glycine-based CART-mRNA complexes (G-CARTs/mRNA) show selective protein expression in the spleen (mouse, >99%), here, we report a new lysine-derived CART-mRNA complex (K-CART/mRNA) that, without additives or targeting ligands, shows selective protein expression in the lungs (mouse, >90%) following systemic IV administration. We further show that by delivering siRNA using the K-CART, we can significantly decrease expression of a lung-localized reporter protein. Blood chemistry and organ pathology studies demonstrate that K-CARTs are safe and well-tolerated. We report on the new step economical, organocatalytic synthesis (two steps) of functionalized polyesters and oligo-carbonate-co-α-aminoester K-CARTs from simple amino acid and lipid-based monomers. The ability to direct protein expression selectively in the spleen or lungs by simple, modular changes to the CART structure opens fundamentally new opportunities in research and gene therapy.

Neoadjuvant Intratumoral Immunotherapy with TLR9 Activation and Anti-OX40 Antibody Eradicates Metastatic Cancer.

The combination of the synthetic TLR9 ligand CpG and agnostic OX40 antibody can trigger systemic antitumor immune responses upon co-injection into the tumor microenvironment, eradicating simultaneous untreated sites of metastatic disease. Here we explore the application of this in situ immunotherapy to the neoadjuvant setting. Current neoadjuvant checkpoint blockade therapy is delivered systemically, resulting in off-target adverse effects. In contrast, intratumoral immunotherapy minimizes the potential for toxicities and allows for greater development of combination therapies. In two metastatic solid tumor models, neoadjuvant intratumoral immunotherapy generated a local T-cell antitumor response that then acted systemically to attack cancer throughout the body. In addition, the importance of timing between neoadjuvant immunotherapy and surgical resection was established, as well as the increased therapeutic power of adding systemic anti-PD1 antibody. The combination of local and systemic immunotherapy generated an additional survival benefit due to synergistic inhibitory effect on tumor-associated macrophages. These results provide a strong rationale for translating this neoadjuvant intratumoral immunotherapy to the clinical setting, especially in conjunction with established checkpoint inhibitors. SIGNIFICANCE: This work demonstrates the ability of neoadjuvant intratumoral immunotherapy to target local and distant metastatic disease and consequently improve survival.

An mRNA SARS-CoV-2 Vaccine Employing Charge-Altering Releasable Transporters with a TLR-9 Agonist Induces Neutralizing Antibodies and T Cell Memory.

The SARS-CoV-2 pandemic has necessitated the rapid development of prophylactic vaccines. Two mRNA vaccines have been approved for emergency use by the FDA and have demonstrated extraordinary effectiveness. The success of these mRNA vaccines establishes the speed of development and therapeutic potential of mRNA. These authorized vaccines encode full-length versions of the SARS-CoV-2 spike protein. They are formulated with lipid nanoparticle (LNP) delivery vehicles that have inherent immunostimulatory properties. Different vaccination strategies and alternative mRNA delivery vehicles would be desirable to ensure flexibility of future generations of SARS-CoV-2 vaccines and the development of mRNA vaccines in general. Here, we report on the development of an alternative mRNA vaccine approach using a delivery vehicle called charge-altering releasable transporters (CARTs). Using these inherently nonimmunogenic vehicles, we can tailor the vaccine immunogenicity by inclusion of coformulated adjuvants such as oligodeoxynucleotides with CpG motifs (CpG-ODN). Mice vaccinated with the mRNA-CART vaccine developed therapeutically relevant levels of receptor binding domain (RBD)-specific neutralizing antibodies in both the circulation and in the lung bronchial fluids. In addition, vaccination elicited strong and long-lasting RBD-specific TH1 T cell responses including CD4+ and CD8+ T cell memory.

An mRNA SARS-CoV-2 vaccine employing Charge-Altering Releasable Transporters with a TLR-9 agonist induces neutralizing antibodies and T cell memory.

The SARS-CoV-2 pandemic has necessitated the rapid development of prophylactic vaccines. Two mRNA vaccines have been approved for emergency use by the FDA and have demonstrated extraordinary effectiveness. The success of these mRNA vaccines establishes the speed of development and therapeutic potential of mRNA. These authorized vaccines encode full-length versions of the SARS-CoV-2 spike protein. They are formulated with Lipid Nanoparticle (LNP) delivery vehicles that have inherent immunostimulatory properties. Different vaccination strategies and alternative mRNA delivery vehicles would be desirable to ensure flexibility of future generations of SARS-CoV-2 vaccines and the development of mRNA vaccines in general. Here, we report on the development of an alternative mRNA vaccine approach using a delivery vehicle called Charge-Altering Releasable Transporters (CARTs). Using these inherently nonimmunogenic vehicles we can tailor the vaccine immunogenicity by inclusion of co-formulated adjuvants such as oligodeoxynucleotides with CpG motifs (CpG-ODN). Mice vaccinated with the mRNA-CART vaccine developed therapeutically relevant levels of RBD-specific neutralizing antibodies in both the circulation and in the lung bronchial fluids. In addition, vaccination elicited strong and long lasting RBD-specific T H 1 T cell responses including CD4 + and CD8 + T cell memory.

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.

Enhancing immunotherapy of STING agonist for lymphoma in preclinical models.

Direct activation of tumor infiltrating antigen-presenting cells (APCs) by intratumoral injection of STING agonists (STINGa) leads to regression of the treated lymphoma tumor. Because STING activation induces apoptosis in lymphoma cells in vitro, we distinguished between the direct therapeutic vs the indirect immunotherapeutic properties of STINGa in vivo. Employing wild-type or STING knockout hosts bearing either wild-type or STING knockout tumor cells, we demonstrated that local tumor regression is totally dependent on STING expression by the host and is therefore immune mediated. However, distant untreated tumors are weakly affected after injection of STINGa to a single tumor site. Therefore, using the STINGa currently being tested in clinical trials, we screened for immunomodulatory agents that could synergize with the STING pathway to induce a systemic antitumor immune response and regression of distant tumors. We combined the STINGa with agents that improve APC or T-cell function. We found that modulation of both APCs and T cells can enhance control of distant lymphoma tumors by STINGa. In particular, adding an anti-GITR antibody induced lymphocyte expansion in the lymph node draining the treated site followed by increased T-cell infiltration in the distant tumor. Furthermore, more of these CD8 T cells at the distant site expressed PD-1. Therefore, blockade of PD-1 further enhanced tumor control at the distant site, leading to cure in 50% of the mice. These preclinical data provide the rationale for testing local injection of STINGa followed by agonistic anti-GITR and anti-PD-1 antibodies as immunotherapy for human lymphoma.

Transient production of artemisinin in Nicotiana benthamiana is boosted by a specific lipid transfer protein from A. annua.

Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although all genes of the artemisinin/arteannuin B (AN/AB) biosynthesis pathway (AN-PW) from Artemisia annua have been identified, ectopic expression of these genes in Nicotiana benthamiana yielded mostly glycosylated pathway intermediates and only very little free (dihydro)artemisinic acid [(DH)AA]. Here we demonstrate that Lipid Transfer Protein 3 (AaLTP3) and the transporter Pleiotropic Drug Resistance 2 (AaPDR2) from A. annua enhance accumulation of (DH)AA in the apoplast of N. benthamiana leaves. Analysis of apoplast and cell content and apoplast exclusion assays show that AaLTP3 and AaPDR2 prevent reflux of (DH)AA from the apoplast back into the cells and enhances overall flux through the pathway. Moreover, AaLTP3 is stabilized in the presence of AN-PW activity and co-expression of AN-PW+AaLTP3+AaPDR2 genes yielded AN and AB in necrotic N. benthamiana leaves at 13 days post-agroinfiltration. This newly discovered function of LTPs opens up new possibilities for the engineering of biosynthesis pathways of high value terpenes in heterologous expression systems.

Comparative proteomics of short and tall glandular trichomes of Nicotiana tabacum reveals differential metabolic activities.

Leaf glandular trichomes (epidermal hairs) actively synthesize secondary metabolites, many of which are the frontline of plant defense. In Nicotiana tabacum, tall and short glandular trichomes have been identified. While the former have been extensively studied and match the classic picture of trichome function, the short trichomes have remained relatively uncharacterized. We have set up a procedure based on centrifugation on Percoll density gradients to obtain separate tall and short trichome fractions purified to >85%. We then investigated the proteome of both trichome types combining 2D-LC fractionation of tryptic peptides and quantification of a set of 461 protein groups using isobaric tags for relative and absolute quantitation. Almost the entire pathway leading to the synthesis of diterpenes was identified in the tall trichomes. Indications for their key roles in the synthesis of cuticular compounds were also found. Concerning the short glandular trichomes, ribosomal proteins and enzymes such phosphoenolpyruvate carboxykinase and polyphenol oxidase were more abundant than in the tall glandular trichomes. These results are discussed in the frame of several hypotheses regarding the respective roles of short and long glandular trichomes.

Isolation of heat shock-induced Nicotiana tabacum transcription promoters and their potential as a tool for plant research and biotechnology.

Transcription promoters of heat shock protein (HSP) genes have been used to control the expression of heterologous proteins in plants and plant cells. To obtain a strong HSP promoter that is functionally active in Nicotiana tabacum BY-2 cells, we set out to identify a promoter of an endogenous gene showing high activation of expression by heat. An N. tabacum BY-2 cell culture was treated for 8 h at 37°C and the cell protein extract analyzed by two-dimensional electrophoresis. A major spot was identified by mass spectrometry as belonging to the small HSP family. The promoter regions and the 5' and 3' untranslated regions of two genes, NtHSP3A and NtHSP3B, with sequences fitting the protein identified were cloned and fused to a hybrid reporter gene coding for ß-glucuronidase (GUS) and a yellow fluorescent protein. These constructs were introduced into N. tabacum BY2 cells by Agrobacterium tumefaciens-mediated transformation. Both promoters conferred similar heat-induced GUS expression. In the best heat shock condition, GUS activity was increased 200 fold and reached 285 pmol min(-1) µg protein(-1). Up-scaling in a 4-l bioreactor resulted in similar heat-induced expression. The NtHSP3A promoter was then used to drive the expression of NtPDR1, a plasma membrane transporter belonging to the pleiotropic drug resistance family. No expression was observed at 25°C, while, at 37°C, expression was similar to that obtained using a strong constitutive promoter. These data show that the HSP promoters isolated are useful for high heat-induced expression in N. tabacum BY-2 cells.