Lysosomal cystine governs ferroptosis sensitivity in cancer via cysteine stress response.

The amino acid cysteine and its oxidized dimeric form cystine are commonly believed to be synonymous in metabolic functions. Cyst(e)ine depletion not only induces amino acid response but also triggers ferroptosis, a non-apoptotic cell death. Here, we report that unlike general amino acid starvation, cyst(e)ine deprivation triggers ATF4 induction at the transcriptional level. Unexpectedly, it is the shortage of lysosomal cystine, but not the cytosolic cysteine, that elicits the adaptative ATF4 response. The lysosome-nucleus signaling pathway involves the aryl hydrocarbon receptor (AhR) that senses lysosomal cystine via the kynurenine pathway. A blockade of lysosomal cystine efflux attenuates ATF4 induction and sensitizes ferroptosis. To potentiate ferroptosis in cancer, we develop a synthetic mRNA reagent, CysRx, that converts cytosolic cysteine to lysosomal cystine. CysRx maximizes cancer cell ferroptosis and effectively suppresses tumor growth in vivo. Thus, intracellular nutrient reprogramming has the potential to induce selective ferroptosis in cancer without systematic starvation.

Accelerating diabetic wound healing by ROS-scavenging lipid nanoparticle-mRNA formulation.

Current treatment options for diabetic wounds face challenges due to low efficacy, as well as potential side effects and the necessity for repetitive treatments. To address these issues, we report a formulation utilizing trisulfide-derived lipid nanoparticle (TS LNP)-mRNA therapy to accelerate diabetic wound healing by repairing and reprogramming the microenvironment of the wounds. A library of reactive oxygen species (ROS)-responsive TS LNPs was designed and developed to encapsulate interleukin-4 (IL4) mRNA. TS2-IL4 LNP-mRNA effectively scavenges excess ROS at the wound site and induces the expression of IL4 in macrophages, promoting the polarization from the proinflammatory M1 to the anti-inflammatory M2 phenotype at the wound site. In a diabetic wound model of db/db mice, treatment with this formulation significantly accelerates wound healing by enhancing the formation of an intact epidermis, angiogenesis, and myofibroblasts. Overall, this TS LNP-mRNA platform not only provides a safe, effective, and convenient therapeutic strategy for diabetic wound healing but also holds great potential for clinical translation in both acute and chronic wound care.

Cas13d knockdown of lung protease Ctsl prevents and treats SARS-CoV-2 infection.

SARS-CoV-2 entry into cells requires specific host proteases; however, no successful in vivo applications of host protease inhibitors have yet been reported for treatment of SARS-CoV-2 pathogenesis. Here we describe a chemically engineered nanosystem encapsulating CRISPR-Cas13d, developed to specifically target lung protease cathepsin L (Ctsl) messenger RNA to block SARS-CoV-2 infection in mice. We show that this nanosystem decreases lung Ctsl expression in normal mice efficiently, specifically and safely. We further show that this approach extends survival of mice lethally infected with SARS-CoV-2, correlating with decreased lung virus burden, reduced expression of proinflammatory cytokines/chemokines and diminished severity of pulmonary interstitial inflammation. Postinfection treatment by this nanosystem dramatically lowers the lung virus burden and alleviates virus-induced pathological changes. Our results indicate that targeting lung protease mRNA by Cas13d nanosystem represents a unique strategy for controlling SARS-CoV-2 infection and demonstrate that CRISPR can be used as a potential treatment for SARS-CoV-2 infection.

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.

STING Agonist-Derived LNP-mRNA Vaccine Enhances Protective Immunity Against SARS-CoV-2.

Lipid nanoparticle (LNP)-mediated delivery of messenger RNA (mRNA) COVID-19 vaccines has provided large-scale immune protection to the public. To elicit a robust immune response against SARS-CoV-2 infections, antigens produced by mRNAs encoding SARS-CoV-2 Spike glycoprotein need to be efficiently delivered and presented to antigen-presenting cells such as dendritic cells (DCs). As concurrent innate immune stimulation can facilitate the antigen presentation process, a library of non-nucleotide STING agonist-derived amino lipids (SALs) was synthesized and formulated into LNPs for mRNA delivery. SAL12 lipid nanoparticles (SAL12-LNPs) were identified as most potent in delivering mRNAs encoding the Spike glycoprotein (S) of SARS-CoV-2 while activating the STING pathway in DCs. Two doses of SAL12 S-LNPs by intramuscular immunization elicited potent neutralizing antibodies against SARS-CoV-2 in mice.

Canopy Homolog 2 contributes to liver oncogenesis by promoting unfolded protein response-dependent destabilization of tumor protein P53.

BACKGROUD AND AIMS: Abnormalities in the tumor protein P53 (p53) gene and overexpression of mouse double minute 2 homolog (MDM2), a negative regulator of p53, are commonly observed in cancers. p53 destabilization is regulated by endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in cancer. However, the mechanisms remain enigmatic. Canopy homolog 2 (CNPY2) is a key UPR initiator that primarily involved in ER stress and is highly expressed in the liver, but its functional role in regulating liver carcinogenesis is poorly understood. Therefore, we aimed to investigate the role of CNPY2 in hepartocarcinogenesis through URP-dependent p53 destabilization. APPROACH AND RESULTS: Here, we showed that CNPY2 expression is up-regulated in HCC and negatively correlated with survival rate in liver cancer patients. Deletion of Cnpy2 obliterates diethylnitrosamine (DEN)-induced HCC in mice. Mechanistic studies demonstrated that CNPY2 binds and prevents ribosome proteins from inhibiting MDM2 and enhances the UPR activity of protein kinase RNA-like endoplasmic reticulum kinase and inositol-requiring transmembrane kinase endoribonuclease-1α, leading to p53 destabilization and cell-cycle progression. In addition, transcriptome analyses uncovered that CNPY2 is also required for DEN-induced expression of oncogenes, including c-Jun and fibroblast growth factor 21. Intratumoral injection of nanoparticle-based CRISPR single-guide RNA/CRISPR-associated protein 9 mRNA against Cnpy2 has antitumor effects in HCC. CONCLUSIONS: These findings demonstrate that CNPY2 is crucial for liver oncogenesis through UPR-dependent repression of p53 and activation of oncogenes, providing insights into the design of a therapeutic target for HCC.

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.

Lipids and Lipid Derivatives for RNA Delivery.

RNA-based therapeutics have shown great promise in treating a broad spectrum of diseases through various mechanisms including knockdown of pathological genes, expression of therapeutic proteins, and programmed gene editing. Due to the inherent instability and negative-charges of RNA molecules, RNA-based therapeutics can make the most use of delivery systems to overcome biological barriers and to release the RNA payload into the cytosol. Among different types of delivery systems, lipid-based RNA delivery systems, particularly lipid nanoparticles (LNPs), have been extensively studied due to their unique properties, such as simple chemical synthesis of lipid components, scalable manufacturing processes of LNPs, and wide packaging capability. LNPs represent the most widely used delivery systems for RNA-based therapeutics, as evidenced by the clinical approvals of three LNP-RNA formulations, patisiran, BNT162b2, and mRNA-1273. This review covers recent advances of lipids, lipid derivatives, and lipid-derived macromolecules used in RNA delivery over the past several decades. We focus mainly on their chemical structures, synthetic routes, characterization, formulation methods, and structure-activity relationships. We also briefly describe the current status of representative preclinical studies and clinical trials and highlight future opportunities and challenges.

Lipid Nanoparticle-mRNA Formulations for Therapeutic Applications.

After decades of extensive fundamental studies and clinical trials, lipid nanoparticles (LNPs) have demonstrated effective mRNA delivery such as the Moderna and Pfizer-BioNTech vaccines fighting against COVID-19. Moreover, researchers and clinicians have been investigating mRNA therapeutics for a variety of therapeutic indications including protein replacement therapy, genome editing, and cancer immunotherapy. To realize these therapeutics in the clinic, there are many formidable challenges. First, novel delivery systems such as LNPs with high delivery efficiency and low toxicity need to be developed for different cell types. Second, mRNA molecules need to be engineered for improved pharmaceutical properties. Lastly, the LNP-mRNA nanoparticle formulations need to match their therapeutic applications.In this Account, we summarize our recent advances in the design and development of various classes of lipids and lipid derivatives, which can be formulated with multiple types of mRNA molecules to treat diverse diseases. For example, we conceived a series of ionizable lipid-like molecules based on the structures of a benzene core, an amide linker, and hydrophobic tails. We identified N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3) as a lead compound for mRNA delivery both in vitro and in vivo. Moreover, we tuned the biodegradability of these lipid-like molecules by introducing branched ester or linear ester chains. Meanwhile, inspired by biomimetic compounds, we synthesized vitamin-derived lipids, chemotherapeutic conjugated lipids, phospholipids, and glycolipids. These scaffolds greatly broaden the chemical space of ionizable lipids for mRNA delivery. In another section, we highlight our efforts on the research direction of mRNA engineering. We previously optimized mRNA chemistry using chemically-modified nucleotides to increase the protein expression, such as pseudouridine (ψ), 5-methoxyuridine (5moU), and N1-methylpseudouridine (me1ψ). Also, we engineered the sequences of mRNA 5' untranslated regions (5'-UTRs) and 3' untranslated regions (3'-UTRs), which dramatically enhanced protein expression. With the progress of LNP development and mRNA engineering, we consolidate these technologies and apply them to treat diseases such as genetic disorders, infectious diseases, and cancers. For instance, TT3 and its analog-derived lipid-like nanoparticles can effectively deliver factor IX or VIII mRNA and recover the clotting activity in hemophilia mouse models. Engineered mRNAs encoding SARS-CoV-2 antigens serve well as vaccine candidates against COVID-19. Vitamin-derived lipid nanoparticles loaded with antimicrobial peptide-cathepsin B mRNA enable adoptive macrophage transfer to treat multidrug resistant bacterial sepsis. Biomimetic lipids such as phospholipids formulated with mRNAs encoding costimulatory receptors lead to enhanced cancer immunotherapy.Overall, lipid-mRNA nanoparticle formulations have considerably benefited public health in the COVID-19 pandemic. To expand their applications in clinical use, research work from many disciplines such as chemistry, engineering, materials, pharmaceutical sciences, and medicine need to be integrated. With these collaborative efforts, we believe that more and more lipid-mRNA nanoparticle formulations will enter the clinic in the near future and benefit human health.

Recent advances of biomaterials in stem cell therapies.

Stem cells have been utilized as 'living drugs' in clinics for decades. Their self-renewal, differentiation, and immunomodulating properties provide potential solutions for a variety of malignant diseases and disorders. However, the pathological environment may diminish the therapeutic functions and survival of the transplanted stem cells, causing failure in clinical translation. To overcome these challenges, researchers have developed biomaterial-based strategies that facilitatein vivotracking, functional engineering, and protective delivery of stem cells, paving the way for next-generation stem cell therapies. In this perspective, we briefly overview different types of stem cells and the major clinical challenges and summarize recent progress of biomaterials applied to boost stem cell therapies.

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.

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.

Design, synthesis and evaluation of anti-CD38 antibody drug conjugate based on Daratumumab and maytansinoid.

Daratumumab, an FDA approved antibody drug, displays specific targeting ability to abnormal white blood cells overexpressing CD38 and provides efficacious therapy for multiple myeloma. Here, in order to achieve enhanced remission of multiple myeloma, we designed Dara-DM4, antibody drug conjugates (ADCs) by conjugating Daratumumab and DM4 via a disulfide linker. Dara-DM4 showed significantly higher cellular uptake and inhibitory efficacy on MM1S cells that overexpressing CD38 with an IC50 of 0.88 µg/mL post 72 hr treatment. These results support a promising ADCs strategy for multiple myeloma treatment.

GlcNAc Conjugated Atorvastatin with Enhanced Water Solubility and Cellular Internalization.

Targeting ligands facilitate cell specific drug delivery and improve pharmaceutical properties. Herein, we designed two ligand drug conjugates by conjugating GlcNAc (N-acetylglucosamine) with atorvastatin. These two conjugates, termed G-AT and G-K-AT, exhibited enhanced water solubility and cellular uptake. Moreover, both G-AT and G-K-AT were able to release atorvastatin and consequently achieve significant inhibition against 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase.

Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates.

siRNA therapeutics have promise for the treatment of a wide range of genetic disorders. Motivated by lipoproteins, we report lipopeptide nanoparticles as potent and selective siRNA carriers with a wide therapeutic index. Lead material cKK-E12 showed potent silencing effects in mice (ED50 ∼ 0.002 mg/kg), rats (ED50 < 0.01 mg/kg), and nonhuman primates (over 95% silencing at 0.3 mg/kg). Apolipoprotein E plays a significant role in the potency of cKK-E12 both in vitro and in vivo. cKK-E12 was highly selective toward liver parenchymal cell in vivo, with orders of magnitude lower doses needed to silence in hepatocytes compared with endothelial cells and immune cells in different organs. Toxicity studies showed that cKK-E12 was well tolerated in rats at a dose of 1 mg/kg (over 100-fold higher than the ED50). To our knowledge, this is the most efficacious and selective nonviral siRNA delivery system for gene silencing in hepatocytes reported to date.

Poly(glycoamidoamine) Brushes Formulated Nanomaterials for Systemic siRNA and mRNA Delivery in Vivo.

Safe and effective delivery is required for siRNA and mRNA-based therapeutics to reach their potential. Here, we report on the development of poly(glycoamidoamine) brush nanoparticles as delivery vehicles for siRNA and mRNA. These polymers were capable of significant delivery of siRNA against FVII and mRNA-encoding erythropoietin (EPO) in mice. Importantly, these nanoparticles were well-tolerated at their effective dose based on analysis of tissue histology, systemic cytokine levels, and liver enzyme chemistry. The polymer brush nanoparticles reported here are promising for therapeutic applications.

Effects of Chemically Modified Messenger RNA on Protein Expression.

Chemically modified nucleotides play significant roles in the effectiveness of mRNA translation. Here, we describe the synthesis of two sets of chemically modified mRNAs [encoding firefly Luciferase (FLuc) and enhanced green fluorescent protein (eGFP), respectively], evaluation of protein expression, and correlation analysis of expression level under various conditions. The results indicate that chemical modifications of mRNAs are able to significantly improve protein expression, which is dependent on cell types and coding sequences. Moreover, eGFP mRNAs with N1-methylpseudouridine (me(1)ψ), 5-methoxyuridine (5moU), and pseudouridine (ψ) modifications ranked top three in cell lines tested. Interestingly, 5moU-modified eGFP mRNA was more stable than other eGFP mRNAs. Consequently, me(1)ψ, 5moU, and ψ are promising nucleotides for chemical modification of mRNAs.

Injectable and Glucose-Responsive Hydrogels Based on Boronic Acid-Glucose Complexation.

Injectable hydrogels have been widely used for a number of biomedical applications. Here, we report a new strategy to form an injectable and glucose-responsive hydrogel using the boronic acid-glucose complexation. The ratio of boronic acid and glucose functional groups is critical for hydrogel formation. In our system, polymers with 10-60% boronic acid, with the balance being glucose-modified, are favorable to form hydrogels. These hydrogels are shear-thinning and self-healing, recovering from shear-induced flow to a gel state within seconds. More importantly, these polymers displayed glucose-responsive release of an encapsulated model drug. The hydrogel reported here is an injectable and glucose-responsive hydrogel constructed from the complexation of boronic acid and glucose within a single component polymeric material.

Design, synthesis and evaluation of anti-CD123 antibody drug conjugates.

Leukemia stem cells (LSCs) account for the development of drug resistance and increased recurrence rate in acute myeloid leukemia (AML) patients. Targeted drug delivery to leukemia stem cells remains a major challenge in AML chemotherapy. Overexpressed interleukin-3 receptor alpha chain, CD123, on the surface of leukemia stem cells was reported to be a potential target in AML treatment. Here, we designed and developed an antibody drug conjugate (CD123-CPT) by integrating anti-CD123 antibody with a chemotherapeutic agent, Camptothecin (CPT), via a disulfide linker. The linker is biodegradable in the presence of Glutathione (GSH, an endogenous component in cells), which leads to release of CPT. Anti-CD123 antibody conjugates showed significant higher cellular uptake in CD123-overexpressed tumor cells. More importantly, CD123-CPT demonstrated potent inhibitory effects on CD123-overexpressed tumor cells. Consequently, these results provide a promising targeted chemotherapeutical strategy for AML treatment.