Introduction: A comprehensive analysis of Critical Quality Attributes (CQAs) is crucial for extended culture conditions across sources and time points. Methods: The rAAV9-eGFP vectors were produced. Further, collected lysates and culture supernatants (sources) were purified by affinity chromatography (AC) at two time points (72 and 120 hours). Detailed CQA analysis, including vector genome titer, capsid protein abundance, purity, aggregates, and transduction efficiency, was conducted. Results: The viral titer yield from the 72-h media supernatant was 1.29×1013 ± 7.62×1011 vgc, whereas the lysate yielded 5.28 × 10¹² ± 7.64×1011 vgc. At the 120-h harvest, the supernatant again yielded 7.22 × 10¹² ± 8.97 × 10¹¹ vgc, while the lysate yielded 1.39 × 10¹¹ ± 5.25 × 1010 vgc. ACbased purification showed the absence of degraded capsid proteins, with no detectable host-cell protein contamination across time points. Notably, the SEC revealed pronounced higher-order dimers in 120-h (23% & 55%) lysate-derived vectors purified by AC and CsCl. Transduction efficiency was significantly reduced in the 120-h lysate-purified vectors compared with 72-h lysate (p=0.001) and corresponding 72- and 120-h media supernatants (p=0.004 and p=0.001, respectively). Discussion: Substantial variation was observed in total vector yield, with media supernatants consistently yielding higher titers than lysates at both time points. Future studies should focus on process parameters to suppress dimer formation under extended culture conditions and elucidate the impairment of transduction in late-harvest, lysate-derived AAV particles. Conclusion: The current study established rAAV9 media supernatant harvested at 72-hr and 120-hr as a preferred source selection for improving yield, purity, and potency during scale-up processes.
Despite the remarkable specificity of small interfering RNA (siRNA)-based treatments for gene silencing, biological limitations like poor stability, rapid systemic clearance, immunogenicity, and inefficient intracellular delivery hinder their clinical translation. A promising strategy for overcoming these challenges and enabling precise, site-specific gene silencing is the use of SMART nanocarriers, including stimuli-responsive, adaptable, and multifunctional delivery platforms. Controlled siRNA release, enhanced cellular uptake, and effective endosomal escape are enabled by these novel nanocarriers' ability to respond dynamically to exogenous triggers such as light, ultrasound, and magnetic fields as well as endogenous stimuli such as pH gradients, redox potential, enzymatic activity, and hypoxia. Recent advances have combined rational material design, surface functionalization with targeted ligands, and modular architectures to gain spatiotemporal control over gene silencing while limiting unintended targets and systemic toxicity. This study examines the critical design concepts, fabrication methodologies, and molecular pathways governing SMART nanocarrier-mediated siRNA distribution, with a focus on polymeric, lipid-based, inorganic, and bioinspired platforms. The article highlights key preclinical and emerging clinical breakthroughs, as well as issues with scalability, repeatability, immunocompatibility, and regulatory translation. This review emphasizes the critical significance of stimulus-responsive nanocarriers in developing siRNA treatments towards precision medicine applications in oncology, neurological disorders, and inflammatory illnesses by consolidating recent studies and future prospects.
Abstract: MicroRNAs (miRNAs) have emerged as promising therapeutic agents in cancer because of their ability to simultaneously regulate multiple oncogenic pathways and gene networks. Early preclinical studies demonstrated substantial anti-tumor activity, generating considerable enthusiasm for the rapid clinical translation of miRNA-based therapeutics. However, despite strong mechanistic rationale and encouraging experimental results, the successful clinical application of miRNA therapeutics has remained limited. To date, only a small number of miRNA-based candidates have entered clinical trials, and none have achieved regulatory approval for cancer treatment. This review summarizes advances in miRNA-based cancer therapeutics and discusses the major factors limiting their translation into clinical practice. We discuss current strategies for miRNA restoration and miRNA inhibition, together with advances in delivery technologies, including lipid nanoparticles, polymer-based carriers, viral vectors, exosomes, and ligand-conjugated systems. Furthermore, evidence from preclinical and clinical studies to assess the efficacy, safety, and translational potential of these approaches have been summarized. Special emphasis was placed on the biological and technical barriers which continue to restrict the clinical success, including inefficient and nonspecific delivery, off-target effects, immune-related toxicities, tumor heterogeneity, and the contextdependent nature of miRNA function within complex tumor microenvironments. Overall, the available evidence suggests that miRNA-based therapeutics continues to hold a significant promise in oncology. Although substantial biological and technical challenges still remains to be addressed to achieve their widespread clinical implementation.
Transformative advances in biomarker science are reshaping the landscape of precision medicine, offering powerful tools for early disease detection, accurate prognostication, and individualized therapeutic design. This review synthesizes recent progress in biomarker discovery, classification, and clinical application across multiple medical domains, emphasizing their pivotal role in enhancing diagnostic precision and personalizing treatment strategies. Central to these advances is the integration of next-generation sequencing, advanced mass spectrometry, artificial intelligence, and multi-omics technologies, which collectively strengthen biomarker reliability and reproducibility. The review also explores the contributions of non-coding RNAs, tumor microenvironment dynamics, and immune-inflammatory pathways in constructing disease-specific biomarker profiles. In fields such as oncology, neurology, and infectious diseases, biomarker-driven approaches are transforming drug development and clinical decision-making. However, challenges such as biological variability, lack of standardization, and ethical concerns necessitate coordinated, multidisciplinary solutions. Emerging innovations, including machine learning-driven analytics, single-cell technologies, and wearable biosensors, signal a shift toward more dynamic diagnostics and adaptive interventions. By charting the current and emerging biomarker landscape, this review underscores the shift toward patient-centric care models. It concludes by highlighting the importance of cross-sector collaboration to accelerate the clinical translation of biomarker innovations, ultimately advancing health equity and improving global healthcare outcomes.
Introduction: Therapeutic strategies for advanced Peripheral Artery Disease (PAD), including angiogenic gene therapy trials, have yielded limited clinical benefit, partly because target selection has relied on empirical biological reasoning rather than genetic causal evidence. Since the progression from Intermittent Claudication (IC) to Critical Limb Ischemia (CLI) represents a critical stage of PAD progression, identifying genetically supported causal targets underlying this transition is essential for developing effective molecular interventions. Methods: An integrative analytical framework was applied, combining bulk transcriptomics, Weighted Gene Co-expression Network Analysis (WGCNA), differential expression analysis, Protein- Protein Interaction (PPI) network construction, and machine-learning-based feature selection. Candidate genes were further characterized using single-cell RNA sequencing (scRNA-seq) and validated in an in vivo murine Hindlimb Ischemia model (HLI). Immune infiltration was assessed by CIBERSORT. Genetic causal evidence was obtained through GWAS colocalization and bidirectional Mendelian Randomization (MR). Computational gene perturbation analysis was performed to predict downstream transcriptional consequences. Phenome-Wide Association Study (PheWAS) and drug-gene interaction analyses were conducted to explore pleiotropy and therapeutic relevance. Results: Multi-stage screening consistently identified CDKN1A as a central hub gene associated with ischemic severity. At single-cell resolution, CDKN1A was broadly upregulated across multiple cell types within the ischemic niche and positively correlated with M2 macrophage infiltration. This ischemia-dependent induction was confirmed at the protein level in vivo. Integrative genetic analyses, including GWAS colocalization (PP.H4 = 0.90) and bidirectional MR, provided evidence supporting a causal link between genetically predicted CDKN1A expression and PAD risk, with no evidence of reverse causation. Computational gene perturbation analysis identified downstream transcriptional programs associated with extracellular matrix remodeling, TGF-beta signaling, and angiogenesis. PheWAS showed no genome-wide significant associations across 19 phenotype categories, and drug-gene interaction analysis identified resveratrol as a candidate modulator. Discussion: The current findings indicate that CDKN1A-mediated stress response represents a key mechanism in PAD progression, linking ischemic injury to maladaptive remodeling and supporting its relevance as a genetically supported candidate therapeutic target in PAD. Conclusion: Our findings provide convergent transcriptomic, cellular, and genetic evidence supporting CDKN1A as a genetically supported candidate causal target associated with PAD progression. These results suggest that CDKN1A-mediated cell-cycle arrest and associated stress-response programs are associated with ischemic tissue remodeling, providing a rationale for future genedirected or pharmacological intervention strategies aimed at attenuating the transition to CLI.
