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1. Charge-altering releasable transporters enhance mRNA delivery in vitro and exhibit in vivo tropism

   https://doi.org/10.1038/s41467-023-42672-x  

   Li, Zhijian; Amaya, Laura; Pi, Ruoxi; Wang, Sean K; Ranjan, Alok; Waymouth, Robert M; Blish, Catherine A; Chang, Howard Y; Wender, Paul A (December 2023, Nature Communications)

   Abstract The introduction of more effective and selective mRNA delivery systems is required for the advancement of many emerging biomedical technologies including the development of prophylactic and therapeutic vaccines, immunotherapies for cancer and strategies for genome editing. While polymers and oligomers have served as promising mRNA delivery systems, their efficacy in hard-to-transfect cells such as primary T lymphocytes is often limited as is their cell and organ tropism. To address these problems, considerable attention has been placed on structural screening of various lipid and cation components of mRNA delivery systems. Here, we disclose a class of charge-altering releasable transporters (CARTs) that differ from previous CARTs based on their beta-amido carbonate backbone (bAC) and side chain spacing. These bAC-CARTs exhibit enhanced mRNA transfection in primary T lymphocytes in vitro and enhanced protein expression in vivo with highly selective spleen tropism, supporting their broader therapeutic use as effective polyanionic delivery systems. 

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2. mRNA lipid nanoparticle-mediated pyroptosis sensitizes immunologically cold tumors to checkpoint immunotherapy

   https://doi.org/10.1038/s41467-023-39938-9  

   Li, Fengqiao; Zhang, Xue-Qing; Ho, William; Tang, Maoping; Li, Zhongyu; Bu, Lei; Xu, Xiaoyang (July 2023, Nature Communications)

   Abstract Synergistically improving T-cell responsiveness is promising for favorable therapeutic outcomes in immunologically cold tumors, yet current treatments often fail to induce a cascade of cancer-immunity cycle for effective antitumor immunity. Gasdermin-mediated pyroptosis is a newly discovered mechanism in cancer immunotherapy; however, cleavage in the N terminus is required to activate pyroptosis. Here, we report a single-agent mRNA nanomedicine-based strategy that utilizes mRNA lipid nanoparticles (LNPs) encoding only the N-terminus of gasdermin to trigger pyroptosis, eliciting robust antitumor immunity. In multiple female mouse models, we show that pyroptosis-triggering mRNA/LNPs turn cold tumors into hot ones and create a positive feedback loop to promote antitumor immunity. Additionally, mRNA/LNP-induced pyroptosis sensitizes tumors to anti-PD-1 immunotherapy, facilitating tumor growth inhibition. Antitumor activity extends beyond the treated lesions and suppresses the growth of distant tumors. We implement a strategy for inducing potent antitumor immunity, enhancing immunotherapy responses in immunologically cold tumors. 

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3. Quantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs

   https://doi.org/10.1016/j.molcel.2024.11.030  

   Lewis, Cole JT; Xie, Li H; Bhandarkar, Shivani Milind; Jin, Danni; Abdallah, Kyrillos; Draycott, Austin S; Chen, Yixuan; Thoreen, Carson C; Gilbert, Wendy V (December 2024, Molecular Cell)

   mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5′ untranslated regions (UTRs) including alternative isoforms and identify sequences that mediate 200-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3–6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-methylpseudouridine (m1Ψ) selectively enhances translation by specific 5′ UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissecting mechanisms of human translation initiation and engineering more potent therapeutic mRNAs. 

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4. Charge‐Reversed Exosomes for Targeted Gene Delivery to Cartilage for Osteoarthritis Treatment

   https://doi.org/10.1002/smtd.202301443  

   Zhang, Chenzhen; Pathrikar, Tanvi_V; Baby, Helna_M; Li, Jun; Zhang, Hengli; Selvadoss, Andrew; Ovchinnikova, Arina; Ionescu, Andreia; Chubinskaya, Susan; Miller, Rachel_E; et al (April 2024, Small Methods)

   Abstract Gene therapy has the potential to facilitate targeted expression of therapeutic proteins to promote cartilage regeneration in osteoarthritis (OA). The dense, avascular, aggrecan‐glycosaminoglycan (GAG) rich negatively charged cartilage, however, hinders their transport to reach chondrocytes in effective doses. While viral vector mediated gene delivery has shown promise, concerns over immunogenicity and tumorigenic side‐effects persist. To address these issues, this study develops surface‐modified cartilage‐targeting exosomes as non‐viral carriers for gene therapy. Charge‐reversed cationic exosomes are engineered for mRNA delivery by anchoring cartilage targeting optimally charged arginine‐rich cationic motifs into the anionic exosome bilayer by using buffer pH as a charge‐reversal switch. Cationic exosomes penetrated through the full‐thickness of early‐stage arthritic human cartilage owing to weak‐reversible ionic binding with GAGs and efficiently delivered the encapsulated eGFP mRNA to chondrocytes residing in tissue deep layers, while unmodified anionic exosomes do not. When intra‐articularly injected into destabilized medial meniscus mice knees with early‐stage OA, mRNA loaded charge‐reversed exosomes overcame joint clearance and rapidly penetrated into cartilage, creating an intra‐tissue depot and efficiently expressing eGFP; native exosomes remained unsuccessful. Cationic exosomes thus hold strong translational potential as a platform technology for cartilage‐targeted non‐viral delivery of any relevant mRNA targets for OA treatment. 

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5. TGF-βR2 signaling coordinates pulmonary vascular repair after viral injury in mice and human tissue

   https://doi.org/10.1126/scitranslmed.adg6229  

   Zhao, Gan; Xue, Lulu; Weiner, Aaron I; Gong, Ningqiang; Adams-Tzivelekidis, Stephanie; Wong, Joanna; Gentile, Maria E; Nottingham, Ana N; Basil, Maria C; Lin, Susan M; et al (January 2024, Science Translational Medicine)

   Disruption of pulmonary vascular homeostasis is a central feature of viral pneumonia, wherein endothelial cell (EC) death and subsequent angiogenic responses are critical determinants of the outcome of severe lung injury. A more granular understanding of the fundamental mechanisms driving reconstitution of lung endothelium is necessary to facilitate therapeutic vascular repair. Here, we demonstrated that TGF-β signaling through TGF-βR2 (transforming growth factor–β receptor 2) is activated in pulmonary ECs upon influenza infection, and mice deficient in endothelialTgfbr2exhibited prolonged injury and diminished vascular repair. Loss of endothelialTgfbr2prevented autocrineVegfa(vascular endothelial growth factor α) expression, reduced endothelial proliferation, and impaired renewal of aerocytes thought to be critical for alveolar gas exchange. Angiogenic responses through TGF-βR2 were attributable to leucine-rich α-2-glycoprotein 1, a proangiogenic factor that counterbalances canonical angiostatic TGF-β signaling. Further, we developed a lipid nanoparticle that targets the pulmonary endothelium, Lung-LNP (LuLNP). Delivery ofVegfamRNA, a critical TGF-βR2 downstream effector, by LuLNPs improved the impaired regeneration phenotype of ECTgfbr2deficiency during influenza injury. These studies defined a role for TGF-βR2 in lung endothelial repair and demonstrated efficacy of an efficient and safe endothelial-targeted LNP capable of delivering therapeutic mRNA cargo for vascular repair in influenza infection. 

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