Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer. Hypoxia-activated prodrugs (HAPs) have shown promise as potential therapeutic agents for TNBC. While increasing hypoxia levels may promote the HAP activation, it raises concerns regarding HIF1α-dependent drug resistance. It is desirable to develop a targeted approach that enhances tumor hypoxia for HAP activation without promoting HIF1α-dependent drug resistance in TNBC treatment. Herein, we proposed a multi-responsive carrier-free self-assembled nanomedicine named AQ4N@CA4T1ASO. This nanomedicine first targeted tumors by the TNBC-targeting aptamers (T1), and then disassembled in the reductive and acidic conditions within tumors. The released Combretastatin 4 (CA4) could exacerbate hypoxia, thereby promoting the conversion of inactive Banoxantrone (AQ4N) to its active form, AQ4. Simultaneously, the released antisense oligonucleotide (ASO) could attenuate hypoxia-induced HIF1α mRNA expression, thereby sensitizing the tumor to chemotherapy. Overall, this smart nanomedicine represents a profound targeted therapy strategy, combining "hypoxia-potentiating, hypoxia-activated, chemo-sensitization" approaches for TNBC treatment. In vivo study demonstrated significant suppression of tumor growth, highlighting the promising potential of this nanomedicine for future clinical translation.

Sclerostin emerges as a novel target for bone anabolic therapy in bone diseases. Osteogenesis imperfecta (OI) and X-linked hypophosphatemia (XLH) are rare bone diseases in which therapeutic potential of sclerostin inhibition cannot be ignored. In OI, genetic/pharmacologic sclerostin inhibition promoted bone formation of mice, but responses varied by genotype and age. Serum sclerostin levels were higher in young OI-I patients, while lower in adult OI-I/III/IV. It's worth investigating whether therapeutic response of OI to sclerostin inhibition could be clinically predicted by genotype and age. In XLH, preclinical/clinical data suggested factors other than identified FGF23 contributing to XLH. Higher levels of circulating sclerostin were detected in XLH. Sclerostin inhibition promoted bone formation in Hyp mice, while restored phosphate homeostasis in age-/gender-dependent manner. The role of sclerostin in regulating phosphate metabolism deserves investigation. Sclerostin/FGF23 levels of XLH patients with/without response to FGF23-antibody warrants study to develop precise sclerostin/FGF23 inhibition strategy or synergistic/additive strategy. Notably, OI patients were associated with cardiovascular abnormalities, so were XLH patients receiving conventional therapy. Targeting sclerostin loop3 promoted bone formation without cardiovascular risks. Further, blockade of sclerostin loop3-LRP4 interaction while preserving sclerostin loop2-ApoER2 interaction could be a potential precise sclerostin inhibition strategy for OI and XLH with cardiovascular safety. The Translational Potential of this Article. Preclinical data on the molecular understanding of sclerostin inhibition in OI and therapeutic efficacy in mouse models of different genotypes, as well as clinical data on serum sclerostin levels in patients with different phenotypes of OI, were reviewed and discussed. Translationally, it would facilitate to develop clinical prediction strategies (e.g. based on genotype and age, not just phenotype) for OI patients responsive to sclerostin inhibition. Both preclinical and clinical data suggested sclerostin as another factor contributing to XLH, in addition to the identified FGF23. The molecular understanding and therapeutic effects of sclerostin inhibition on both promoting bone anabolism and improving phosphate homostasis in Hyp mice were reviewed and discussed. Translationaly, it would facilitate the development of precise sclerostin/FGF23 inhibition strategy or synergistic/additive strategy for the treatment of XLH. Cardiovascular risk could not be ruled out during sclerostin inhibition treatment, especially for OI and XLH patients with cardiovascular diseases history and cardiovascular abnormalities. Studies on the role of sclerostin in inhiting bone formation and protecting cardiovascular system were reviewed and discussed. Translationaly, blockade of sclerostin loop3-LRP4 interaction while preserving sclerostin loop2-ApoER2 interaction could be a potential precise sclerostin inhibition strategy for OI and XLH with cardiovascular safety.

Sclerostin, encoded by the SOST gene, is a novel bone anabolic target for bone diseases. Humanized anti-sclerostin antibody, romosozumab, was approved for treatment of postmenopausal osteoporosis by the US Food and Drug Administration (FDA), but with a black-box warning on cardiovascular risk. The clinical data regarding cardiovascular events from various pre-marketing and post-marketing studies of romosozumab were inconsistent. Overall, the cardiovascular risk of sclerostin inhibition could not be excluded. The restriction of romosozumab in patients with cardiovascular disease history would be necessary. Moreover, genome-wide association study (GWAS) analyses of SOST variants revealed inconsistent results of the association between SOST variations and cardiovascular diseases. Further research incorporating larger sample sizes and functional analyses are necessary. In analyses of serum/tissue sclerostin levels in patients with cardiovascular diseases, the results were controversial but indicated an association between sclerostin and the presence/severity/outcomes of cardiovascular diseases. Nonclinical studies in rodents indicated the inhibitory effect of sclerostin on inflammation, aortic aneurysm, atherosclerosis, and vascular calcification. Sclerostin loop3 participated in the inhibitory effect of sclerostin on bone formation, while the cardiovascular protective effect of sclerostin was independent of sclerostin loop3. Macrophagic sclerostin loop2–apolipoprotein E receptor 2 (ApoER2) interaction participated in the inhibitory effect of sclerostin on inflammation in vitro. Sclerostin in human aortic smooth muscle cells participated in the reduction in calcium deposition. The role of sclerostin in cardiovascular system deserves further investigation.

Duchenne muscular dystrophy (DMD), a fatal X-linked disorder, features progressive muscle fibrosis as a key driver of mortality. While CTGF represents a therapeutic target for DMD, its VWC-domain-targeting antibody (FG-3019) failed in clinical trials. Through experimental validation, we identified CT-domain as a superior target domain, as it contributed more fibrosis activity of CTGF than VWC-domain without elevating compensatory TGF-β1 level. Aptamers are synthetic oligonucleotides identified through SELEX, which can specifically bind to flexible protein domains through their unique 3D conformations. Their small molecular size enables effective tissue penetration while maintaining high target specificity, making them ideal CT-domain inhibitors. Nevertheless, conventional SELEX involves time-consuming and inefficient multiple screening rounds. Here, we employed our generative AI model, AptGEN, to rapidly discover a potent CT-domain specific aptamer within 42 days. This chemically modified aptamer (Apc003OA) distributed and remained in muscle tissues for an extended period, whereas FG-3019 could not. Importantly, it demonstrated better fibrosis inhibitory activity in vitro and in mdx mice when compared to FG-3019. Furthermore, Apc003OA demonstrated a favorable safety profile in mdx mice. Within 10 months, we progressed from target domain discovery, aptamer drug discovery, and then obtained both Orphan Drug Designation and Pediatric Rare Disease Designation by US Food and Drug Administration.

Mechanical unloading leads to bone loss and cardiovascular deconditioning, accompanied by elevated sclerostin expression. Genetic Sost knockout or pharmacologic sclerostin antibody treatment was reported to counteract bone loss during mechanical unloading in mice. However, severe cardiovascular events were reported in postmenopausal osteoporotic patients treated with commercially available sclerostin antibody targeting loop2. It is desirable to develop a precise sclerostin inhibition strategy to counteract unloading-induced bone loss, without increasing cardiovascular risk. In a previously published rodent studies under normal loading condition, it was found that sclerostin loop3 participated in the inhibitory effect of sclerostin on bone formation, while the preventive action of sclerostin against cardiovascular events was independent of sclerostin loop3. Nevertheless, whether and how sclerostin loop3 contributes to bone formation reduction and bone loss under mechanical unloading condition remains unclear. In this study under mechanical unloading condition, either sclerostin loop3-specific deficiency in Sost^loop3−/− mice or sclerostin loop3-specific inhibition by our tailor-made aptamer Apc001 counteracted unloading-induced bone loss without increasing arterial stiffness, whereas either Sost knockout or romosozumab treatment significantly increased unloading-induced arterial stiffness in mice. These findings indicated sclerostin loop3 as a therapeutic target with cardiovascular safety against unloading-induced bone loss. Mechanistically, we identified that sclerostin loop3 bound to LRP4 in osteoblasts under mechanical unloading condition. Osteoblast-specific Lrp4 knockout counteracted unloading-induced bone formation reduction and bone loss in OB. Lrp4^−/− mice. Further, blocking the interaction of sclerostin loop3 with LRP4 via mutation of the interaction residues (Lrp4m) or pharmacologic inhibition with LRP4 peptide tool (LRP4-Pep) dramatically attenuated binding of sclerostin to LRP6, counteracted decrease of Wnt/β-catenin signaling activity and osteogenic potential in osteoblasts under mechanical unloading condition in vitro. Consistently, Lrp4m counteracted unloading-induced bone formation reduction and bone loss in mice in vivo. In Lrp4m/OB-Lrp4 mice, osteoblast-conditional correction of Lrp4m to wild-type Lrp4 attenuated the counteractive effect of Lrp4m on unloading-induced bone loss. Pharmacologically, osteoblasts-targeted LRP4-Pep counteracted bone formation reduction and bone loss during mechanical unloading in wild-type mice. Sclerostin loop3-mediated anchoring of sclerostin to LRP4 facilitated its binding to LRP6 in osteoblasts, contributing to bone formation reduction and bone loss under mechanical unloading condition. Specifically blocking the interaction of sclerostin loop3 with LRP4 in osteoblasts would offer a precise strategy with cardiovascular safety for treatment of unloading-induced bone loss.

Aptamers, synthetic oligonucleotide ligands, have shown significant promise for therapeutic and diagnostic applications owing to their high specificity and affinity for target molecules. However, the conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) for aptamer selection is time-consuming and often yields limited candidates. To address these limitations, we introduce AptaGPT, a novel computational strategy that leverages a Generative Pre-trained Transformer (GPT) model to design and optimize aptamers. By training on SELEX data from early rounds, AptaGPT generated a diverse array of aptamer sequences, which were then computationally screened for binding using molecular docking. The results of this study demonstrated that AptaGPT is an effective tool for generating potential high-affinity aptamer sequences, significantly accelerating the discovery process and expanding the potential for aptamer research. This study showcases the application of generative language models in bioengineering and provides a new avenue for rapid aptamer development.