Search Results (20)

Background/Objectives: While CRISPR/Cas9 technology offers a revolutionary approach for correcting genetic ocular blindness, efficient and safe delivery remains the primary bottleneck. Traditional viral vectors, despite their efficacy, face challenges regarding cargo size limitations and potential genomic integration risks. Non-viral vectors offer distinct comparative advantages, including large cargo capacity for diverse CRISPR tools and transient expression to minimize off-target effects, but must overcome the eye’s formidable static and dynamic barriers, specifically the corneal epithelium, vitreous humor, and the inner limiting membrane. In this review, we present an anatomically guided framework for non-viral CRISPR/Cas9 delivery, mapping engineering strategies to specific ocular tissue targets. We first delineate the mechanisms of key physiological barriers, including the corneal stroma, aqueous humor circulation, and the vitreous–retina interface. Subsequently, we critically evaluate the latest advancements in non-viral platforms, such as pH-responsive lipid nanoparticles and engineered virus-like particles. The core focus of this review is on site-specific breakthrough strategies: from utilizing mucoadhesive polymers to counteract tear clearance in the cornea to exploiting specialized administration routes, such as suprachoroidal space and subretinal injection, to bypass retinal barriers, and deep-penetrating intravitreal carriers for targeting the photoreceptor-RPE complex. By integrating material science with precise administration routes, this review highlights feasible translational pathways for next-generation, carrier-free, or biomimetic ocular gene editing therapies. Full article

Neuroblastoma is a highly aggressive pediatric malignancy originating from neural crest progenitor cells, predominantly in the adrenal medulla. Amplification of the MYCN oncogene occurs in 20–30% of all neuroblastoma cases and approximately 50% of high-risk tumors, strongly correlating with poor prognosis, relapse, and multidrug resistance. MYCN-driven oncogenesis promotes tumor progression by suppressing apoptotic signaling and enhancing survival pathways, including autophagy—a key mechanism underlying resistance to chemotherapy and immunotherapy. This review examines current therapeutic strategies and resistance mechanisms in MYCN-amplified neuroblastoma, while introducing emerging approaches utilizing exosomes as precision drug delivery systems. Exosomes, nanoscale extracellular vesicles secreted by the tumor cells, exhibit natural tropism and can be engineered to selectively target neuroblastoma-specific biomarkers such as glypican-2 (GPC2), which is highly expressed in MYCN-amplified tumors. Leveraging this property, neuroblastoma-derived exosomes can be purified, modified, and loaded with small interfering RNA (siRNA) to silence MYCN expression, combined with chloroquine—an FDA-approved autophagy inhibitor—to simultaneously inhibit autophagy and induce apoptotic signaling. This dual-targeted approach aims to overcome drug resistance, reduce off-target toxicity, and enhance therapeutic efficacy through exosome-mediated specificity. Furthermore, gut dysbiosis has emerged as a critical factor influencing tumor progression and diminishing treatment efficacy in MYCN-amplified neuroblastoma. We propose integrating microbiota-derived exosomes engineered to deliver anti-inflammatory microRNAs (miRNAs) to the gut mucosa, restoring eubiosis and potentiating systemic anti-tumor responses. Collectively, exosome-based strategies represent a paradigm shift in formulating combination therapies, offering a multifaceted approach to target MYCN amplification, inhibit autophagy, induce apoptosis, and modulate the tumor-microbiome axis. These innovations hold significant promise for improving clinical outcomes in high-risk MYCN-amplified neuroblastoma patients. Full article

Chimeric Antigen Receptor (CAR)-T cell therapy has transformed the treatment landscape of cancer, yet major challenges remain in enhancing efficacy, reducing adverse effects, and expanding accessibility. Autologous CAR-T cells, derived from individual patients, have achieved remarkable clinical success in hematologic malignancies; however, their highly personalized nature limits scalability, increases costs, and delays timely treatment. Allogeneic CAR-T cells generated from healthy donors provide an “off-the-shelf” alternative but face two critical immune barriers: graft-versus-host disease (GvHD), caused by donor T-cell receptor (TCR) recognition of host tissues, and host-versus-graft rejection, mediated by recipient immune responses against donor HLA molecules. Recent advances in genome engineering, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, allow precise modification of donor T cells to overcome these limitations. For example, TRAC gene knockout eliminates TCR expression, preventing GvHD, while disruption of HLA molecules reduces immunogenicity without impairing cytotoxicity. Beyond hematologic cancers, CRISPR-edited allogeneic CAR-T cells targeting the NKG2D receptor have shown promise in preclinical studies and early-phase trials. NKG2D CAR-T cells recognize stress ligands (MICA/B, ULBP1–6) expressed on over 80% of diverse solid tumors, including pancreatic and ovarian cancers, thereby broadening therapeutic applicability. Nevertheless, the genomic editing process carries risks of off-target effects, including potential disruption of tumor suppressor genes and oncogenes, underscoring the need for stringent safety and quality control. This review examines the distinguishing features of allogeneic versus autologous CAR-T therapy, with a particular focus on NKG2D-based allogeneic CAR-T approaches for solid tumors. We summarize current strategies to mitigate immune barriers, discuss practical manufacturing challenges, and analyze available clinical data on NKG2D CAR-T trials. Collectively, these insights underscore both the promise and the hurdles of developing safe, universal, and scalable allogeneic CAR-T therapies for solid malignancies. Full article

Efflux is one of the key mechanisms used by Gram-negative bacteria to reduce internal antibiotic concentrations. These active transport systems recognize and expel a wide range of toxic molecules, including antibiotics, thereby contributing to reduced antibiotic susceptibility and allowing the bacteria to acquire additional resistance mechanisms. To date, unlike other resistance mechanisms such as enzymatic modification or target mutations/masking, efflux is challenging to detect and counteract in clinical settings, and no standardized methods are currently available to diagnose or inhibit this mechanism effectively. This review first outlines the structural and functional features of major efflux pumps in Gram-negative bacteria and their role in antibiotic resistance. It then explores various strategies used to curb their activity, with a particular focus on efflux pump inhibitors under development, detailing their structural classes, modes of action, and pharmacological potential. We discuss the main obstacles to their development, including the structural complexity and substrate promiscuity of efflux mechanisms, the limitations of current screening methods, pharmacokinetic and tissue distribution issues, and the risk of off-target toxicity. Overcoming these multifactorial barriers is essential to the rational development of less efflux-prone antibiotics or of efflux pump inhibitors. Full article

Adoptive cell therapies have transformed the treatment landscape for hematologic malignancies. Yet, translation to solid tumors remains constrained by antigen heterogeneity, an immunosuppressive tumor microenvironment (TME), and poor persistence of conventional CAR-T cells. In response, innate immune cell platforms, particularly chimeric antigen receptor–engineered natural killer (CAR-NK) cells and chimeric antigen receptor–macrophages (CAR-MΦ), have emerged as promising alternatives. This review summarizes recent advances in the design and application of CAR-NK and CAR-MΦ therapies for solid tumors. We highlight key innovations, including the use of lineage-specific intracellular signaling domains (e.g., DAP12, 2B4, FcRγ), novel effector constructs (e.g., NKG7-overexpressing CARs, TME-responsive CARs), and scalable induced pluripotent stem cell (iPSC)-derived platforms. Preclinical data support enhanced antitumor activity through mechanisms such as major histocompatibility complex (MHC)-unrestricted cytotoxicity, phagocytosis, trogocytosis, cytokine secretion, and cross-talk with adaptive immunity. Early-phase clinical studies (e.g., CT-0508) demonstrate feasibility and TME remodeling with CAR-MΦ. However, persistent challenges remain, including transient in vivo survival, manufacturing complexity, and risks of off-target inflammation. Emerging combinatorial strategies, such as dual-effector regimens (CAR-NK⁺ CAR-MΦ), cytokine-modulated cross-support, and bispecific or logic-gated CARs, may overcome these barriers and provide more durable, tumor-selective responses. Taken together, CAR-NK and CAR-MΦ platforms are poised to expand the reach of engineered cell therapy into the solid tumor domain. Full article

Lentiviral vectors (LVs) have become a fundamental tool in gene therapy due to their unique ability to transduce both dividing and non-dividing cells, transfer large genes of up to 10 kb, and facilitate stable, long-term expression of therapeutic genes into target cells. A key application of LVs is the ex vivo genetic modification of patient-derived cells, such as the production of CAR-T cells by transducing isolated T cells with LVs to express the CAR gene, enabling them to target and destroy cancer cells once infused back into the patient. However, these ex vivo gene therapy drugs are often dismally unaffordable due to the complex procedures involved, including cell isolation, genetic modification, and expansion, along with the significant risks associated with immune conditioning to ensure successful engraftment. To overcome these barriers, direct in vivo transgene delivery to physiologically relevant cells has been explored, bypassing the need for ex vivo manipulations and reducing costs. Yet, a major challenge in this approach is engineering LV cell tropism to ensure the precise targeting of specific cells while avoiding off-target effects. Recent advances in modifying LV surface proteins have shown promise, including the successful in vivo generation of CAR T cells and ensuing clinical trials. This review is aimed at providing an up-to-date account of the progress in engineering LV tropism, covering the utility of different heterologous viral envelopes and their engineering to achieve cell-type-specific delivery and host immune evasion, and highlighting the potential of in vivo gene therapy to improve the affordability and accessibility of life-saving treatments. Full article

The CRISPR (clustered regularly interspaced short palindromic repeats) system has emerged as a revolutionary gene-editing tool with immense potential in gene therapy, functional genomics, and beyond. However, achieving precise spatiotemporal control of gene editing in specific cells and tissues while effectively mitigating potential risks, such as off-target effects, remains a key challenge for its clinical translation. To overcome these limitations, researchers have developed innovative strategies based on chemical modifications of oligonucleotides to enhance the precision, efficiency, and controllability of CRISPR/Cas9-mediated gene editing. By introducing conditional responsive elements, such as photosensitive groups, small-molecule responsive units, and supramolecular structures, they have successfully achieved precise spatiotemporal and dose-dependent regulation of CRISPR/Cas9 function. This review provides a comprehensive overview of recent advancements in gRNA regulation strategies based on chemical modifications of oligonucleotides, discussing their applications in improving the efficiency, specificity, and controllability of CRISPR/Cas9 editing. We also highlight the challenges associated with the conditional control of gRNA and offer insights into future directions for the chemical regulation of gRNA to further advance CRISPR/Cas9 technology. Full article

Background: Multiple myeloma (MM) is a hematological malignancy originating from the plasma cells present in the bone marrow. Despite significant therapeutic advancements, relapse and drug resistance remain major clinical challenges, highlighting the urgent need for novel therapeutic targets. Methods: To identify potential druggable genes associated with MM, we performed Mendelian randomization (MR) analysis. Causal candidates were further validated using a single-tissue transcriptome-wide association study (TWAS), and colocalization analysis was conducted to assess shared genetic signals between gene expression and disease risk. Potential off-target effects were assessed through an MR phenome-wide association study (MR-PheWAS). Additionally, molecular docking and functional assays were used to evaluate candidate drug efficacy. Results: The MR analysis identified nine druggable genes (FDR < 0.05), among which Orosomucoid 1 (ORM1) and Oviductal Glycoprotein 1 (OVGP1) were supported by both TWAS and colocalization evidence (PPH4 > 0.75). Experimental validation demonstrated the significant downregulation of ORM1 and OVGP1 in MM cells (p < 0.05). Pregnenolone and irinotecan, identified as agonists of ORM1 and OVGP1, respectively, significantly inhibited MM cell viability, while upregulating their expression (p < 0.05). Conclusions: Our study highlights ORM1 and OVGP1 as novel therapeutic targets for MM. The efficacy of pregnenolone and irinotecan in suppressing MM cell growth suggests their potential for clinical application. These findings provide insights into MM pathogenesis and offer a promising strategy for overcoming drug resistance. Full article

The clinical management of malignant tumours is challenging, often leading to severe adverse effects and death. Drug resistance (DR) antagonises the effectiveness of treatments, and increasing drug dosage can worsen the therapeutic index (TI). Current efforts to overcome DR predominantly involve the use of drug combinations, including applying multiple anti-cancerous drugs, employing drug sensitisers, which are chemical agents that enhance pharmacokinetics (PK), including the targeting of cellular pathways and regulating pertinent membrane transporters. While combining multiple compounds may lead to drug–drug interactions (DDI) or polypharmacy effect, the use of drug sensitisers permits rapid attainment of effective treatment dosages at the disease site to prevent early DR and minimise side effects and will reduce the chance of DDI as lower drug doses are required. This review highlights the essential use of TI in evaluating drug dosage for cancer treatment and discusses the lack of a unified standard for TI within the field. Commonly used benefit–risk assessment criteria are summarised, and the critical exploration of the current use of TI in the pharmaceutical industrial sector is included. Specifically, this review leads to the discussion of drug sensitisers to facilitate improved ratios of effective dose to toxic dose directly in humans. The combination of drug and sensitiser molecules might see additional benefits to rekindle those drugs that failed late-stage clinical trials by the removal of detrimental off-target activities through the use of lower drug doses. Drug combinations and employing drug sensitisers are potential means to combat DR. The evolution of drug combinations and polypharmacy on TI are reviewed. Notably, the novel binary weapon approach is introduced as a new opportunity to improve TI. This review emphasises the urgent need for a criterion to systematically evaluate drug safety and efficiency for practical implementation in the field. Full article

Wet age-related macular degeneration (AMD) is an end-stage event in a complex pathogenesis of macular degeneration, involving the abnormal growth of blood vessels at the retinal pigment epithelium driven by vascular endothelial growth factor (VEGF). Current therapies seek to interrupt VEGF signaling to halt the progress of neovascularization, but a significant patient population is not responsive. New treatment modalities such as integrin-binding peptides (risuteganib/Luminate/ALG-1001) are being explored to address this clinical need but these treatments necessitate the use of intravitreal injections (IVT), which carries risks of complications and restricts its availability in less-developed countries. Successful systemic delivery of peptide-based therapeutics must overcome obstacles such as degradation by proteinases in circulation and off-target binding. In this work, we present a novel dendrimer-integrin-binding peptide (D-ALG) synthesized with a noncleavable, “clickable” linker. In vitro, D-ALG protected the peptide payload from enzymatic degradation for up to 1.5 h (~90% of the compound remained intact) in a high concentration of proteinase (2 mg/mL) whereas ~90% of free ALG-1001 was degraded in the same period. Further, dendrimer conjugation preserved the antiangiogenic activity of ALG-1001 in vitro with significant reductions in endothelial vessel network formation compared to untreated controls. In vivo, direct intravitreal injections of ALG-1001 and D-ALG produced reductions in the CNV lesion area but in systemically dosed animals, only D-ALG produced significant reductions of CNV lesion area at 14 days. Imaging data suggested that the difference in efficacy may be due to more D-ALG remaining in the target area than ALG-1001 after administration. The results presented here offer a clinically relevant route for peptide therapeutics by addressing the major obstacles that these therapies face in delivery. Full article

The bacteria-derived CRISPR/Cas (an acronym for regularly interspaced short palindromic repeats/CRISPR-associated protein) system is currently the most widely used, versatile, and convenient tool for genome engineering. CRISPR/Cas-based technologies have been applied to disease modeling, gene therapies, transcriptional modulation, and diagnostics. Nevertheless, some challenges remain, such as the risk of immunological reactions or off-target effects. To overcome these problems, many new methods and CRISPR/Cas-based tools have been developed. In this review, we describe the current classification of CRISPR systems and new precise genome-editing technologies, summarize the latest applications of this technique in several fields of research, and, finally, discuss CRISPR/Cas system limitations, ethical issues, and challenges. Full article

Statins, the intestinal cholesterol transporter inhibitor (ezetimibe), and PCSK9 inhibitors can reduce serum LDL-C levels, leading to a significant reduction in cardiovascular events. However, these events cannot be fully prevented even when maintaining very low LDL-C levels. Hypertriglyceridemia and reduced HDL-C are known as residual risk factors for ASCVD. Hypertriglyceridemia and/or low HDL-C can be treated with fibrates, nicotinic acids, and n-3 polyunsaturated fatty acids. Fibrates were demonstrated to be PPARα agonists and can markedly lower serum TG levels, yet were reported to cause some adverse effects, including an increase in the liver enzyme and creatinine levels. Recent megatrials of fibrates have shown negative findings on the prevention of ASCVD, which were supposed to be due to their low selectivity and potency for binding to PPAR α. To overcome the off-target effects of fibrates, the concept of a selective PPARα modulator (SPPARMα) was proposed. Kowa Company, Ltd. (Tokyo, Japan), has developed pemafibrate (K-877). Compared with fenofibrate, pemafibrate showed more favorable effects on the reduction of TG and an increase in HDL-C. Fibrates worsened liver and kidney function test values, although pemafibrate showed a favorable effect on liver function test values and little effect on serum creatinine levels and eGFR. Minimal drug–drug interactions of pemafibrate with statins were observed. While most of the fibrates are mainly excreted from the kidney, pemafibrate is metabolized in the liver and excreted into the bile. It can be used safely even in patients with CKD, without a significant increase in blood concentration. In the megatrial of pemafibrate, PROMINENT, for dyslipidemic patients with type 2 diabetes, mild-to-moderate hypertriglyceridemia, and low HDL-C and LDL-C levels, the incidence of cardiovascular events did not decrease among those receiving pemafibrate compared to those receiving the placebo; however, the incidence of nonalcoholic fatty liver disease was lower. Pemafibrate may be superior to conventional fibrates and applicable to CKD patients. This current review summarizes the recent findings on pemafibrate. Full article

Major depression disorder (MDD) is a severe mental alteration with a multifactorial origin, and chronic stress is one of the most relevant environmental risk factors associated with MDD. Although there exist some therapeutical options, 30% of patients are still resistant to any type of treatment. GSK3β inhibitors are considered very promising therapeutic tools to counteract stress-related affectations. However, they are often associated with excessive off-target effects and undesired secondary alterations. Meridianins are alkaloids with an indole framework linked to an aminopyrimidine ring from Antarctic marine ascidians. Meridianins could overcome several of the aforementioned limitations since we previously demonstrated that they can inhibit GSK3β activity without the associated neurotoxic or off-target effects in rodents. Here, we show that meridianins delivered into the lateral ventricle inhibited GSK3β in several brain regions involved with stress-related symptoms. We also observed changes in major signaling pathways in the prefrontal cortex (Akt and PKA) and hippocampus (PKC and GluR1). Moreover, meridianins increased synaptic activity, specifically in the CA1 but not in the CA3 or other hippocampal subfields. Finally, we chronically treated the mice subjected to an unpredictable mild chronic stress (CUMS) paradigm with meridianins. Our results showed improvements produced by meridianins in behavioral alterations provoked by CUMS. In conclusion, meridianins could be of therapeutic interest to patients with stress-related disorders such as MDD. Full article

The systemic delivery of doxorubicin (DOX) to treat osteosarcoma requires an adequate drug concentration to be effective, but in doing so, it raises the risk of increasing organ off-target toxicity and developing drug resistance. Herein, this study reveals a multiple therapeutic nanocarrier delivery platform that overcomes off-target toxicity by providing good specificity and imparting enhanced tumor penetration in a three-dimensional (3D) human MG-63 spheroid model. By synthesizing PEG-PLGA nanoparticles by the double emulsion method, encapsulating DOX and Na¹³¹I in the inner core, and conjugating with an epidermal growth factor receptor (EGFR) antibody, it is intended to specifically target human MG-63 cells. The nanocarrier is biocompatible with blood and has good stability characteristics. Na¹³¹I encapsulation efficiency was >96%, and radiochemical purity was >96% over 96 h. A DOX encapsulation efficacy of ~80% was achieved, with a drug loading efficiency of ~3%, and a sustained DOX release over 5 days. The nanocarrier EGFR antibody achieved a ~80-fold greater targeting efficacy to MG-63 cells (EGFR+) than fibroblast cells (EGFR−). The targeted multiple therapeutic DIE-NPs have a higher penetration and uptake of Na¹³¹I to the 3D model and a ~3-fold higher cytotoxicity than the DOX monotherapy (D-NPs). The co-administration of DOX and Na¹³¹I (DIE-NPs) disrupts DNA repair and generates free radicals resulting in DNA damage, triggering the activation of apoptosis pathways. This leads to inhibition of MG-63 cell proliferation and promotes cell cycle arrest in the G0/G1 phase. Furthermore, the PEGylated anti-EGFR functionalized DIE-NPs were found to be biocompatible with red blood cells and to have no adverse effects. This anti-EGFR targeted multifunctional I-131 radio-nanotherapeutic signifies a customizable specific targeted treatment for osteosarcoma. Full article

Glioblastoma Multiforme (GBM) is a devastating disease with a low survival rate and few efficacious treatment options. The fast growth, late diagnostics, and off-target toxicity of currently used drugs represent major barriers that need to be overcome to provide a viable cure. Nanomedicines (NMeds) offer a way to overcome these pitfalls by protecting and loading drugs, increasing blood half-life, and being targetable with specific ligands on their surface. In this study, the FDA-approved polymer poly (lactic-co-glycolic) acid was used to optimise NMeds that were surface modified with a series of potential GBM-specific ligands. The NMeds were fully characterised for their physical and chemical properties, and then in vitro testing was performed to evaluate cell uptake and GBM cell specificity. While all targeted NMeds showed improved uptake, only those decorated with the-cell surface vimentin antibody M08 showed specificity for GBM over healthy cells. Finally, the most promising targeted NMed candidate was loaded with the well-known chemotherapeutic, paclitaxel, to confirm targeting and therapeutic effects in C6 GBM cells. These results demonstrate the importance of using well-optimised NMeds targeted with novel ligands to advance delivery and pharmaceutical effects against diseased cells while minimising the risk for nearby healthy cells. Full article