Nanomaterials-Mediated Co-Stimulation of Toll-Like Receptors and CD40 for Antitumor Immunity.

Toll-like receptors (TLRs) and CD40-related signaling pathways represent critical bridges between innate and adaptive immune responses. Here, an immunotherapy regimen that enables co-stimulation of TLR7/8- and CD40-mediated pathways is developed. TLR7/8 agonist resiquimod (R848) derived amino lipids, RAL1 and RAL2, are synthesized and formulated into RAL-derived lipid nanoparticles (RAL-LNPs). The RAL2-LNPs show efficient CD40 mRNA delivery to DCs both in vitro (90.8 ± 2.7%) and in vivo (61.3 ± 16.4%). When combined with agonistic anti-CD40 antibody, this approach can produce effective antitumor activities in mouse melanoma tumor models, thereby suppressing tumor growth, prolonging mouse survival, and establishing antitumor memory immunity. Overall, RAL2-LNPs provide a novel platform toward cancer immunotherapy by integrating innate and adaptive immunity.

IL-27 Induces CCL5 Production by T Lymphocytes, Which Contributes to Antitumor Activity.

IL-27 is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells including T lymphocytes. In this study, we demonstrate that IL-27 directly induces CCL5 production by T lymphocytes, particularly CD8+ T cells in vitro and in vivo. IL-27-induced CCL5 production is IL-27R-dependent. In CD4+ T cells, IL-27-induced CCL5 production was primarily dependent on Stat1 activation, whereas in CD8+ T cells, Stat1 deficiency does not abrogate CCL5 induction. A chromatin immunoprecipitation assay revealed that in the CCL5 promoter region, both putative Stat3 binding sites exhibit significant binding to Stat3, whereas only one out of four Stat1 binding sites displays moderate binding to Stat1. In tumor-bearing mice, IL-27 induced dramatic production of CCL5 in tumor-infiltrating T cells. IL-27-induced CCL5 appears to contribute to an IL-27-mediated antitumor effect. This is signified by diminished tumor inhibition in anti-CCL5- and IL-27-treated mice. Additionally, intratumor delivery of CCL5 mRNA using lipid nanoparticles significantly inhibited tumor growth. Thus, IL-27 induces robust CCL5 production by T cells, which contributes to antitumor activity.

Intratumoral delivery of IL-12 and IL-27 mRNA using lipid nanoparticles for cancer immunotherapy.

Cytokines are important immunotherapeutics with approved drugs for the treatment of human cancers. However, systemic administration of cytokines often fails to achieve adequate concentrations to immune cells in tumors due to dose-limiting toxicity. Thus, developing localized therapy that directly delivers immune-stimulatory cytokines to tumors may improve the therapeutic efficacy. In this study, we generated novel lipid nanoparticles (LNPs) encapsulated with mRNAs encoding cytokines including IL-12, IL-27 and GM-CSF, and tested their anti-tumor activity. We first synthesized ionizable lipid materials containing di-amino groups with various head groups (DALs). The novel DAL4-LNP effectively delivered different mRNAs in vitro to tumor cells and in vivo to tumors. Intratumoral injection of DAL4-LNP loaded with IL-12 mRNA was most potent in inhibiting B16F10 melanoma tumor growth compared to IL-27 or GM-CSF mRNAs in monotherapy. Furthermore, intratumoral injection of dual DAL4-LNP-IL-12 mRNA and IL-27 mRNA showed a synergistic effect in suppressing tumor growth without causing systematic toxicity. Most importantly, intratumoral delivery of IL-12 and IL-27 mRNAs induced robust infiltration of immune effector cells, including IFN-γ and TNF-α producing NK and CD8+ T cells into tumors. Thus, intratumoral administration of DAL-LNP loaded with IL-12 and IL-27 mRNA provides a new treatment strategy for cancer.

Formulation and Delivery Technologies for mRNA Vaccines.

mRNA vaccines have become a versatile technology for the prevention of infectious diseases and the treatment of cancers. In the vaccination process, mRNA formulation and delivery strategies facilitate effective expression and presentation of antigens, and immune stimulation. mRNA vaccines have been delivered in various formats: encapsulation by delivery carriers, such as lipid nanoparticles, polymers, peptides, free mRNA in solution, and ex vivo through dendritic cells. Appropriate delivery materials and formulation methods often boost the vaccine efficacy which is also influenced by the selection of a proper administration route. Co-delivery of multiple mRNAs enables synergistic effects and further enhances immunity in some cases. In this chapter, we overview the recent progress and existing challenges in the formulation and delivery technologies of mRNA vaccines with perspectives for future development.

Biomimetic nanoparticles deliver mRNAs encoding costimulatory receptors and enhance T cell mediated cancer immunotherapy.

Antibodies targeting costimulatory receptors of T cells have been developed for the activation of T cell immunity in cancer immunotherapy. However, costimulatory molecule expression is often lacking in tumor-infiltrating immune cells, which can impede antibody-mediated immunotherapy. Here, we hypothesize that delivery of costimulatory receptor mRNA to tumor-infiltrating T cells will enhance the antitumor effects of antibodies. We first design a library of biomimetic nanoparticles and find that phospholipid nanoparticles (PL1) effectively deliver costimulatory receptor mRNA (CD137 or OX40) to T cells. Then, we demonstrate that the combination of PL1-OX40 mRNA and anti-OX40 antibody exhibits significantly improved antitumor activity compared to anti-OX40 antibody alone in multiple tumor models. This treatment regimen results in a 60% complete response rate in the A20 tumor model, with these mice being resistant to rechallenge by A20 tumor cells. Additionally, the combination of PL1-OX40 mRNA and anti-OX40 antibody significantly boosts the antitumor immune response to anti-PD-1 + anti-CTLA-4 antibodies in the B16F10 tumor model. This study supports the concept of delivering mRNA encoding costimulatory receptors in combination with the corresponding agonistic antibody as a strategy to enhance cancer immunotherapy.

Advances of nanomaterials-based strategies for fighting against COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 100 million people globally due to its high infectivity. After decades of efforts on the studies of nanomaterials, researchers have applied nanomaterials-based strategies to combat the pandemic of the coronavirus disease 2019 (COVID-19). First, nanomaterials facilitate the development of easy, fast, and low-cost diagnostic assays to detect SARS-CoV-2 and related biomarkers. Second, nanomaterials enable the efficient delivery of viral antigens to antigen-presenting cells or serve as adjuvants in the host, leading to vaccine development at an unprecedented pace. Lastly, nanomaterials-based treatments may inhibit SARS-CoV-2 replication and reduce inflammation. Overall, nanomaterials have played important roles in controlling this COVID-19 pandemic. Here, we provide a brief overview of the representative examples of nanomaterials-based diagnostics, vaccines, and therapeutics in the fight against COVID-19.

Construction of Messenger RNA (mRNA) Probes Delivered By Lipid Nanoparticles to Visualize Intracellular Protein Expression and Localization at Organelles.

Organelles are specialized compartments, where various proteins reside and play crucial roles to maintain essential cellular structures and functions in mammalian cells. A comprehensive understanding of protein expressions and subsequent localizations at each organelle is of great benefit to the development of organelle-based therapies. Herein, a set of single or dual organelle labeling messenger RNAs (SOLAR or DOLAR) is designed as novel imaging probes, which encode fluorescent proteins with various organelle localization signals. These mRNA probes enable to visualize the protein localizations at different organelles and investigate their trafficking from ribosomal machinery to specific organelles. According to the in vitro results, SOLAR probes show organelle targeting capabilities consistent with the design. Moreover, DOLAR probes with different linkers display distinct targeting properties depending on different organelle localization signals. Additionally, these mRNA probes also exhibit organelle labeling ability in vivo when delivered by lipid nanoparticles (LNPs). Therefore, these mRNA-based probes provide a unique tool to study cell organelles and may facilitate the design of organelle-based therapies.

RNA drug discovery: Conformational restriction enhances specific modulation of the T-box riboswitch function.

Antibacterial drug resistance is a global health concern that requires multiple solution approaches including development of new antibacterial compounds acting at novel targets. Targeting regulatory RNA is an emerging area of drug discovery. The T-box riboswitch is a regulatory RNA mechanism that controls gene expression in Gram-positive bacteria and is an exceptional, novel target for antibacterial drug design. We report the design, synthesis and activity of a series of conformationally restricted oxazolidinone-triazole compounds targeting the highly conserved antiterminator RNA element of the T-box riboswitch. Computational binding energies correlated with experimentally-derived Kd values indicating the predictive capabilities for docking studies within this series of compounds. The conformationally restricted compounds specifically inhibited T-box riboswitch function and not overall transcription. Complex disruption, computational docking and RNA binding specificity data indicate that inhibition may result from ligand binding to an allosteric site. These results highlight the importance of both ligand affinity and RNA conformational outcome for targeted RNA drug design.

Functionalized lipid-like nanoparticles for in vivo mRNA delivery and base editing.

Messenger RNA (mRNA) therapeutics have been explored to treat various genetic disorders. Lipid-derived nanomaterials are currently one of the most promising biomaterials that mediate effective mRNA delivery. However, efficiency and safety of this nanomaterial-based mRNA delivery remains a challenge for clinical applications. Here, we constructed a series of lipid-like nanomaterials (LLNs), named functionalized TT derivatives (FTT), for mRNA-based therapeutic applications in vivo. After screenings on the materials, we identified FTT5 as a lead material for efficient delivery of long mRNAs, such as human factor VIII (hFVIII) mRNA (~4.5 kb) for expression of hFVIII protein in hemophilia A mice. Moreover, FTT5 LLNs demonstrated high percentage of base editing on PCSK9 in vivo at a low dose of base editor mRNA (~5.5 kb) and single guide RNA. Consequently, FTT nanomaterials merit further development for mRNA-based therapy.

Leveraging mRNA Sequences and Nanoparticles to Deliver SARS-CoV-2 Antigens In Vivo.

SARS-CoV-2 has become a pandemic worldwide; therefore, an effective vaccine is urgently needed. Recently, messenger RNAs (mRNAs) have emerged as a promising platform for vaccination. In this work, the untranslated regions (UTRs) of mRNAs are systematically engineered in order to enhance protein production. Through a comprehensive analysis of endogenous gene expression and de novo design of UTRs, the optimal combination of 5' and 3' UTR are identified and termed NASAR, which are 5- to 10-fold more efficient than the tested endogenous UTRs. More importantly, NASAR mRNAs delivered by lipid-derived TT3 nanoparticles trigger a dramatic expression of potential SARS-CoV-2 antigens. The antigen-specific antibodies induced by TT3-nanoparticles and NASAR mRNAs are over two orders of magnitude more than that induced by the FDA-approved lipid nanoparticle material MC3 in vaccinated mice. These NASAR mRNAs merit further development as alternative SARS-CoV-2 vaccines.

Leveraging mRNAs sequences to express SARS-CoV-2 antigens in vivo.

SARS-CoV-2 has rapidly become a pandemic worldwide; therefore, an effective vaccine is urgently needed. Recently, messenger RNAs (mRNAs) have emerged as a promising platform for vaccination. Here, we systematically investigated the untranslated regions (UTRs) of mRNAs in order to enhance protein production. Through a comprehensive analysis of endogenous gene expression and de novo design of UTRs, we identified the optimal combination of 5' and 3' UTR, termed as NASAR, which was five to ten-fold more efficient than the tested endogenous UTRs. More importantly, NASAR mRNAs delivered by lipid-derived nanoparticles showed dramatic expression of potential SARS-CoV-2 antigens both in vitro and in vivo. These NASAR mRNAs merit further development as alternative SARS-CoV-2 vaccines.

Author Correction: Vitamin lipid nanoparticles enable adoptive macrophage transfer for the treatment of multidrug-resistant bacterial sepsis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Rational Design of Small Molecules to Enhance Genome Editing Efficiency by Selectively Targeting Distinct Functional States of CRISPR-Cas12a.

CRISPR-Cas12a, a type-V CRISPR-Cas endonuclease, is an effective genome editing platform. To improve the gene editing efficiency of Cas12a, we rationally designed small molecule enhancers through a combined computational approach. First, we used extensive molecular dynamics (MD) simulations to explore the conformational landscape of Cas12a from Acidaminococcus (AsCas12a), revealing distinct conformational states that could be targeted by small molecules to modulate its genome editing function. We then identified 57 compounds that showed different binding behavior and stabilizing effects on these distinct conformational states using molecular docking. After experimental testing 6 of these 57 compounds, compound 1, quinazoline-2,4(1H,3H)-dione, was found particularly promising in enhancing the AsCas12a-mediated genome editing efficiency in human cells. Compound 1 was shown to act like a molecular "glue" at the interface between AsCas12a and crRNA near the 5'-handle region, thus specifically stabilizing the enzyme-crRNA complex. These results provide a new paradigm for future design of small molecules to modulate the genome editing of the CRISPR-Cas systems.

CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides.

CRISPR-Cas12a (CRISPR-Cpf1) was reported to have multiple types of cleavage activities. Without the assistance of CRISPR RNA (crRNA), we investigated DNase activity and substrate specificity of Cas12a orthologs in the presence of diverse divalent metal ions. Cas12a from different species are capable of degrading single-stranded DNA (ssDNA) and/or double-stranded DNA (dsDNA), depending on the metal ions used. In spite of sharing high sequence similarity and functional domains among diverse Cas12a orthologs, only Acidaminococcus sp. Cas12a (AsCas12a) showed a predominant preference for cleaving ssDNA, but no detectable activity toward dsDNA substrate in the presence of magnesium (II) ions. In addition, we found that both AsCas12a and Francisella novicida Cas12a (FnCas12a) caused substantial dsDNA cleavage in the presence of manganese (II) ion. More importantly, the DNase activities can be inhibited by synthetic DNA oligonucleotides with phosphorothioate linkage modifications. Overall, ssDNase activity of the Cas12a orthologs uncovered a distinct approach for DNA cleavage compared with crRNA-guided dsDNA breaks, and provided insights into potential biological and therapeutic applications.

Vitamin lipid nanoparticles enable adoptive macrophage transfer for the treatment of multidrug-resistant bacterial sepsis.

Sepsis, a condition caused by severe infections, affects more than 30 million people worldwide every year and remains the leading cause of death in hospitals1,2. Moreover, antimicrobial resistance has become an additional challenge in the treatment of sepsis3, and thus, alternative therapeutic approaches are urgently needed2,3. Here, we show that adoptive transfer of macrophages containing antimicrobial peptides linked to cathepsin B in the lysosomes (MACs) can be applied for the treatment of multidrug-resistant bacteria-induced sepsis in mice with immunosuppression. The MACs are constructed by transfection of vitamin C lipid nanoparticles that deliver antimicrobial peptide and cathepsin B (AMP-CatB) mRNA. The vitamin C lipid nanoparticles allow the specific accumulation of AMP-CatB in macrophage lysosomes, which is the key location for bactericidal activities. Our results demonstrate that adoptive MAC transfer leads to the elimination of multidrug-resistant bacteria, including Staphylococcus aureus and Escherichia coli, leading to the complete recovery of immunocompromised septic mice. Our work provides an alternative strategy for overcoming multidrug-resistant bacteria-induced sepsis and opens up possibilities for the development of nanoparticle-enabled cell therapy for infectious diseases.

Regulation of OmpA Translation and Shigella dysenteriae Virulence by an RNA Thermometer.

RNA thermometers are cis-acting riboregulators that mediate the posttranscriptional regulation of gene expression in response to environmental temperature. Such regulation is conferred by temperature-responsive structural changes within the RNA thermometer that directly result in differential ribosomal binding to the regulated transcript. The significance of RNA thermometers in controlling bacterial physiology and pathogenesis is becoming increasingly clear. This study combines in silico, molecular genetics, and biochemical analyses to characterize both the structure and function of a newly identified RNA thermometer within the ompA transcript of Shigella dysenteriae First identified by in silico structural predictions, genetic analyses have demonstrated that the ompA RNA thermometer is a functional riboregulator sufficient to confer posttranscriptional temperature-dependent regulation, with optimal expression observed at the host-associated temperature of 37°C. Structural studies and ribosomal binding analyses have revealed both increased exposure of the ribosomal binding site and increased ribosomal binding to the ompA transcript at permissive temperatures. The introduction of site-specific mutations predicted to alter the temperature responsiveness of the ompA RNA thermometer has predictable consequences for both the structure and function of the regulatory element. Finally, in vitro tissue culture-based analyses implicate the ompA RNA thermometer as a bona fide S. dysenteriae virulence factor in this bacterial pathogen. Given that ompA is highly conserved among Gram-negative pathogens, these studies not only provide insight into the significance of riboregulation in controlling Shigella virulence, but they also have the potential to facilitate further understanding of the physiology and/or pathogenesis of a wide range of bacterial species.

Chemotherapy drugs derived nanoparticles encapsulating mRNA encoding tumor suppressor proteins to treat triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is one type of the most aggressive breast cancers with poor prognosis. It is of great urgency to develop new therapeutics for treating TNBC. Based on current treatment guideline and genetic information of TNBC, a combinational therapy platform integrating chemotherapy drugs and mRNA encoding tumor suppressor proteins may become an efficacious strategy. In this study, we developed paclitaxel amino lipid (PAL) derived nanoparticles (NPs) to incorporate both chemotherapy drugs and P53 mRNA. The PAL P53 mRNA NPs showed superior properties compared to Abraxane® and Lipusu® used in the clinic including high paclitaxel loading capacity (24 wt.%, calculated by paclitaxel in PAL), PAL encapsulation efficiency (94.7% ± 6.8%) and mRNA encapsulation efficiency (88.7% ± 0.7%). Meanwhile, these NPs displayed synergetic cytotoxicity of paclitaxel and P53 mRNA in cultured TNBC cells. More importantly, we demonstrated in vivo anti-tumor efficacy of PAL P53 mRNA NPs in an orthotopic TNBC mouse model. Overall, these chemotherapy drugs derived mRNA NPs provide a new platform to integrate chemotherapy and personalized medicine using tumor genetic information, and therefore represent a promising approach for TNBC treatment.

Targeted delivery of atorvastatin via asialoglycoprotein receptor (ASGPR).

Targeted drug delivery platforms can increase the concentration of drugs in specific cell populations, reduce adverse effects, and hence improve the therapeutic effect of drugs. Herein, we designed two conjugates by installing the targeting ligand GalNAc (N-acetylgalactosamine) onto atorvastatin (AT). Compared to the parent drug, these two conjugates, termed G2-AT and G2-K-AT, showed increased hepatic cellular uptake. Moreover, both conjugates were able to release atorvastatin, and consequently showed dramatic inhibition of ß-hydroxy-ß-methylglutaryl-CoA (HMG-CoA) reductase and increased LDL receptors on cell surface.

Lipid Polymer Hybrid Nanomaterials for mRNA Delivery.

INTRODUCTION: In the past decade, messenger RNA (mRNA) has been extensively explored in a wide variety of biomedical applications. However, efficient delivery of mRNA is still one of the key challenges for its broad applications in the clinic. Recently, lipid polymer hybrid nanoparticles (LPNs) are evolving as a promising class of biomaterials for RNA delivery, which integrate the physicochemical properties of both lipids and polymers. We previously developed an N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT) derived lipid-like nanomaterial (TT3-LLN) which was capable of effectively delivering multiple types of mRNA. In order to further improve the delivery efficiency of TT3-LLN, in this study, we focused on studying the effects of incorporating different polymers on establishing LPNs and aimed to develop an optimized lipid polymer hybrid nanomaterial for efficient mRNA delivery. METHODS: We incorporated a series of biodegradable and biocompatible polymer materials into the formulation of TT3-LLNs to develop LPNs. mRNA delivery efficiency of different LPNs were evaluated and a systematic orthogonal optimization was further carried out. RESULTS: Our data indicate that PLGA4 (MW 24,000-38,000 g/mol) dramatically increased delivery efficiency of TT3-LLNs in comparison to other polymers. Further optimization identified PLGA4-7 LPNs (PLGA:mRNA=9:1, mass ratio; TT3:DOPE:Cholesterol:DMG-PEG2000=25:25:45:0.75, molar ratio) as a lead formulation, which displayed significantly enhanced delivery of two types of mRNA in three different human cell lines as compared with TT3-LLNs. CONCLUSIONS: Results from this study potentially provide new insights into developing LPNs for mRNA based therapeutics.

Synthetic Oligonucleotides Inhibit CRISPR-Cpf1-Mediated Genome Editing.

Previously, researchers discovered a series of anti-CRISPR proteins that inhibit CRISPR-Cas activity, such as Cas9 and Cpf1 (Cas12a). Herein, we constructed crRNA variants consisting of chemically modified DNA-crRNA and RNA-crRNA duplexes and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1. More important, without prehybridization, these PS-modified DNA oligonucleotides showed the ability to suppress DNA double-strand breaks induced by two Cpf1 orthologs, AsCpf1 and LbCpf1. Time-dependent inhibitory effects were validated in multiple loci of different human cells. Further studies demonstrated that PS-modified DNA oligonucleotides were able to serve as Cpf1 inhibitors in a sequence-independent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder target DNA binding and recognition by Cpf1. Consequently, these synthetic DNA molecules expand the sources of CRISPR inhibitors, providing a platform to inactivate Cpf1-mediated genome editing.