In situ production and secretion of proteins endow therapeutic benefit against psoriasiform dermatitis and melanoma.

Genetic medicines have the potential to treat various diseases; however, certain ailments including inflammatory diseases and cancer would benefit from control over extracellular localization of therapeutic proteins. A critical gap therefore remains the need to develop and incorporate methodologies that allow for posttranslational control over expression dynamics, localization, and stability of nucleic acid-generated protein therapeutics. To address this, we explored how the body's endogenous machinery controls protein localization through signal peptides (SPs), including how these motifs could be incorporated modularly into therapeutics. SPs serve as a virtual zip code for mRNA transcripts that direct the cell where to send completed proteins within the cell and the body. Utilizing this signaling biology, we incorporated secretory SP sequences upstream of mRNA transcripts coding for reporter, natural, and therapeutic proteins to induce secretion of the proteins into systemic circulation. SP sequences generated secretion of various engineered proteins into the bloodstream following intravenous, intramuscular, and subcutaneous SP mRNA delivery by lipid, polymer, and ionizable phospholipid delivery carriers. SP-engineered etanercept/TNF-α inhibitor proteins demonstrated therapeutic efficacy in an imiquimod-induced psoriasis model by reducing hyperkeratosis and inflammation. An SP-engineered anti-PD-L1 construct mediated mRNA encoded proteins with longer serum half-lives that reduced tumor burden and extended survival in MC38 and B16F10 cancer models. The modular nature of SP platform should enable intracellular and extracellular localization control of various functional proteins for diverse therapeutic applications.

Spleen SORT LNP Generated in situ CAR T Cells Extend Survival in a Mouse Model of Lymphoreplete B Cell Lymphoma.

Chimeric Antigen Receptor (CAR) T cell immunotherapy is revolutionizing treatment for patients suffering from B-cell lymphoma (BL). However, the current method of CAR T cell production is complicated and expensive, requiring collection of patient blood to enrich the T cell population, ex vivo engineering/activation, and quality assessment before the patient can receive the treatment. Herein we leverage Spleen Selective ORgan Targeted (SORT) Lipid Nanoparticles (LNPs) to produce CAR T cells in situ and bypass the extensive and laborious process currently used. Optimized Spleen SORT LNPs containing 10 % 18 : 1 PA transfected CD3+, CD8+, and CD4+ T cells in wild-type mice. Spleen SORT LNPs delivered Cre recombinase mRNA and CAR encoding mRNA to T cells in reporter mice and in a lymphoreplete B cell lymphoma model (respectively) after intravenous injection without the need for active targeting ligands. Moreover, in situ CAR T cells increased the overall survival of mice with a less aggressive form of B cell lymphoma. In addition, in situ transfected CAR T cells reduced tumor metastasis to the liver by increasing tumor infiltrating lymphocytes. Overall, these results offer a promising alternative method for CAR T cell production with pre-clinical potential to treat hematological malignancies.

Optimization of phospholipid chemistry for improved lipid nanoparticle (LNP) delivery of messenger RNA (mRNA).

Lipid nanoparticles (LNPs) have been established as an essential platform for nucleic acid delivery. Efforts have led to the development of vaccines that protect against SARS-CoV-2 infection using LNPs to deliver messenger RNA (mRNA) coding for the viral spike protein. Out of the four essential components that comprise LNPs, phospholipids represent an underappreciated opportunity for fundamental and translational study. We investigated this avenue by systematically modulating the identity of the phospholipid in LNPs with the goal of identifying specific moieties that directly enhance or hinder delivery efficacy. Results indicate that phospholipid chemistry can enhance mRNA delivery by increasing membrane fusion and enhancing endosomal escape. Phospholipids containing phosphoethanolamine (PE) head groups likely increase endosomal escape due to their fusogenic properties. Additionally, it was found that zwitterionic phospholipids mainly aided liver delivery, whereas negatively charged phospholipids changed the tropism of the LNPs from liver to spleen. These results demonstrate that the choice of phospholipid plays a role intracellularly by enhancing endosomal escape, while also driving organ tropism in vivo. These findings were then applied to Selective Organ Targeting (SORT) LNPs to manipulate and control spleen-specific delivery. Overall, selection of the phospholipid in LNPs provides an important handle to design and optimize LNPs for improved mRNA delivery and more effective therapeutics.

All-In-One Dendrimer-Based Lipid Nanoparticles Enable Precise HDR-Mediated Gene Editing In Vivo.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein gene editing is poised to transform the treatment of genetic diseases. However, limited progress has been made toward precise editing of DNA via homology-directed repair (HDR) that requires careful orchestration of complex steps. Herein, dendrimer-based lipid nanoparticles (dLNPs) are engineered to co-encapsulate and deliver multiple components for in vivo HDR correction. BFP/GFP switchable HEK293 cells with a single Y66H amino acid mutation are employed to assess HDR-mediated gene editing following simultaneous, one-pot delivery of Cas9 mRNA, single-guide RNA, and donor DNA. Molar ratios of individual LNP components and weight ratios of the three nucleic acids are systematically optimized to increase HDR efficiency. Using flow cytometry, fluorescence imaging, and DNA sequencing to quantify editing, optimized 4A3-SC8 dLNPs edit >91% of all cells with 56% HDR efficiency in vitro and >20% HDR efficiency in xenograft tumors in vivo. Due to the all-in-one simplicity and high efficacy, the developed dLNPs offer a promising route toward the gene correction of disease-causing mutations.

Quantitative characterization of all single amino acid variants of a viral capsid-based drug delivery vehicle.

Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes-a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.

First Total Synthesis of ω-Phenyl Δ6 Fatty Acids and their Leishmanicidal and Anticancer Properties.

INTRODUCTION: The first total synthesis of ω-phenyl Δ6 fatty acids (FA) and their cytotoxicity (A549) and leishmanicidal (L. infantum) activities are described. The novel 16-phenyl-6-hexadecynoic acid (1) and the known 16-phenylhexadecanoic acid (2) were synthesized in 7-8 steps with overall yields of 46 % and 41 %, respectively. The syntheses of the unprecedented 10-phenyl-6-decynoic acid (3), 10-cyclohexyl-6-decynoic acid (4) and 10-(4-methoxyphenyl)-6-decynoic acid (5) was also performed in 3 steps with 73-76 % overall yields. The use of lithium acetylide coupling enabled the 4-step synthesis of 10-phenyl-6Z-decenoic acid (6) with a 100 % cis-stereochemistry. The cytotoxicity of these novel FA was determined against A549 cells and L. infantum promastigotes and amastigotes. Among the ω-phenylated FA, the best cytotoxicity towards A549 was displayed by 1, with an IC50 of 18 ± 1 µM. On the other hand, among the C10 acids, the ω-cyclohexyl acid 4 presented the best cytotoxicity (IC50 = 40 ± 2 µM) towards A549. RESULTS: Based on caspase-3/7 studies neither of the FA induced apoptosis in A549, thus implying other mechanisms of cell death. CONCLUSION: The antileishmanial studies were performed with the top Leishmania donovani topoisomerase IB (LdTopIB) inhibitors, namely 1 and 2 (EC50 between 14 and 36 µM, respectively), acids that did not stabilize the cleavage complexes between LdTopIB and DNA. Acids 1 and 2 displayed cytotoxicity towards L. infantum amastigotes (IC50 = 3-6 µM) and L. infantum promastigotes (IC50 = 60- 70 µM), but low toxicity towards murine splenocytes. Our results identified 1 as the optimum ω- phenylated acid of the series.