Artificial Intelligence (AI)-Aided Structure Optimization for Enhanced Gene Delivery: The Effect of the Polymer Component Distribution (PCD).

Gene therapy has emerged as a significant advancement in medicine in recent years. However, the development of effective gene delivery vectors, particularly polymer vectors, remains a significant challenge. Limited understanding of the internal structure of polymer vectors has hindered efforts to enhance their efficiency. This work focuses on investigating the impact of polymer structure on gene delivery, using the well-known polymeric vector poly(ß-amino ester) (PAE) as a case study. For the first time, we revealed the distinct characteristics of individual polymer components and their synergistic effects-the appropriate combination of different components within a polymer (high MW and low MW components) on gene delivery. Additionally, artificial intelligence (AI) analysis was employed to decipher the relationship between the polymer component distribution (PCD) and gene transfection performance. Guided by this analysis, a series of highly efficient polymer vectors that outperform current commercial reagents such as jetPEI and Lipo3000 were developed, among which the transfection efficiency of the PAE-B1-based polyplex was approximately 1.5 times that of Lipo3000 and 2 times that of jetPEI in U251 cells.

Exploring the Bottom-Up Growth of Anisotropic Gold Nanoparticles from Substrate-Bound Seeds in Microfluidic Reactors.

We developed an unconventional seed-mediated in situ synthetic method, whereby gold nanostars are formed directly on the internal walls of microfluidic reactors. The dense plasmonic substrate coatings were grown in microfluidic channels with different geometries to elucidate the impacts of flow rate and profile on reagent consumption, product morphology, and density. Nanostar growth was found to occur in the flow-limited regime and our results highlight the possibility of creating shape gradients or incorporating multiple morphologies in the same microreactor, which is challenging to achieve with traditional self-assembly. The plasmonic-microfluidic platforms developed herein have implications for a broad range of applications, including cell culture/sorting, catalysis, sensing, and drug/gene delivery.

Branch Unit Distribution Matters for Gene Delivery.

As a key nonviral gene therapy vector, poly(ß-amino ester) (PAE) has demonstrated great potential for clinical application after two decades of development. However, even after extensive efforts in structural optimizations, including screening chemical composition, molecular weight (MW), terminal groups, and topology, their DNA delivery efficiency still lags behind that of viral vectors. To break through this bottleneck, in this work, a thorough investigation of highly branched PAEs (HPAEs) was conducted to correlate their fundamental internal structure with their gene transfection performance. We show that an essential structural factor, branch unit distribution (BUD), plays an important role for HPAE transfection capability and that HPAEs with a more uniform distribution of branch units display better transfection efficacy. By optimizing BUD, a high-efficiency HPAE that surpasses well-known commercial reagents (e.g., Lipofectamine 3000 (Lipo3000), jetPEI, and Xfect) can be generated. This work opens an avenue for the structural control and molecular design of high-performance PAE gene delivery vectors.

Delivering the CRISPR/Cas9 system for engineering gene therapies: Recent cargo and delivery approaches for clinical translation.

Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9) has transformed our ability to edit the human genome selectively. This technology has quickly become the most standardized and reproducible gene editing tool available. Catalyzing rapid advances in biomedical research and genetic engineering, the CRISPR/Cas9 system offers great potential to provide diagnostic and therapeutic options for the prevention and treatment of currently incurable single-gene and more complex human diseases. However, significant barriers to the clinical application of CRISPR/Cas9 remain. While in vitro, ex vivo, and in vivo gene editing has been demonstrated extensively in a laboratory setting, the translation to clinical studies is currently limited by shortfalls in the precision, scalability, and efficiency of delivering CRISPR/Cas9-associated reagents to their intended therapeutic targets. To overcome these challenges, recent advancements manipulate both the delivery cargo and vehicles used to transport CRISPR/Cas9 reagents. With the choice of cargo informing the delivery vehicle, both must be optimized for precision and efficiency. This review aims to summarize current bioengineering approaches to applying CRISPR/Cas9 gene editing tools towards the development of emerging cellular therapeutics, focusing on its two main engineerable components: the delivery vehicle and the gene editing cargo it carries. The contemporary barriers to biomedical applications are discussed within the context of key considerations to be made in the optimization of CRISPR/Cas9 for widespread clinical translation.

Non-viral delivery of CRISPR-Cas9 complexes for targeted gene editing via a polymer delivery system.

Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(ß-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15-20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR-Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

Identification of Key Genes Driving Tumor Associated Macrophage Migration and Polarization Based on Immune Fingerprints of Lung Adenocarcinoma.

The identification of reliable indicators in the tumor microenvironment (TME) is critical for tumor prognosis. Tumor associated macrophages (TAMs) are the major component of non-tumor stromal cells in TME and have increasingly been recognized as a predictive biomarker for lung adenocarcinoma (LUAD) prognosis. Here, we report the development of a prognosis model for LUAD using three immune-related genes (IRGs) detected in The Cancer Genome Atlas (TCGA) which potentially regulate TAMs in TME. In 497 LUAD patients, higher immune scores conferred better overall survival (OS). We identified 93 hub IRGs out of 234 for further prognostic significance. Among them, three IRGs (BTK, Cd1c, and S100P) were proved to be closely correlated to the prognosis of patients with LUAD. Moreover, the immune risk score (IRS) based on the gene expression level of the three IRGs was an independent prognostic factor for OS. Higher IRS predicted lower OS, higher mortality and worse tumor stage. With a good predictive ability [area under the ROC curve (AUC) in TCGA = 0.701, AUC in GEO = 0.722], the IRS contributed to a good risk stratification ability of the nomogram. Immunologically, the three IRGs were related to M1 macrophages and NK cell subsets in TME. Interestingly, by characterizing these immune components in situ we found that S100P is a driver for tumor cells to induce TAM migration and M2 polarization in the immunosuppressive tumor niche. We identified the key genes driving TAM migration and transformation and elucidated the immune landscape of LUAD. The data suggest that IRGs from TME have the potential to become indicators for estimating cancer prognosis and guiding individualized treatment.

Reactive oxygen species (ROS): utilizing injectable antioxidative hydrogels and ROS-producing therapies to manage the double-edged sword.

Reactive oxygen species (ROS) are generated in cellular metabolism and are essential for cellular signalling networks and physiological functions. However, the functions of ROS are 'double-edged swords' to living systems that have a fragile redox balance between ROS generation and elimination. A modest increase of ROS leads to enhanced cell proliferation, survival and benign immune responses, whereas ROS stress that overwhelms the cellular antioxidant capacity can damage nucleic acids, proteins and lipids, resulting in oncogenic mutations and cell death. ROS are therefore involved in many pathological conditions. On the other hand, ROS present selective toxicity and have been utilised against cancer and pathogens, thus also acting as a double-edged sword in the healthcare field. Injectable antioxidative hydrogels are gel precursors that form hydrogel constructs in situ upon delivery in vivo to maintain an antioxidative capacity. These hydrogels have been developed to counter ROS-induced pathological conditions, with significant advantages of biocompatibility, excellent moldability, and minimally invasive delivery. The intrinsic, readily controllable ROS-scavenging ability of the functionalised hydrogels overcomes many drawbacks of small molecule antioxidants. This review summarises the roles of ROS under pathological conditions and describes the state-of-the-art of injectable antioxidative hydrogels. A particular emphasis is also given to current ROS-producing therapeutic interventions, enabling potential application of injectable antioxidant hydrogels to prevent the adverse effects of many cancer and infection treatments.

Demodex and rosacea revisited.

Demodex mites are part of the vast microbiome living on and within human skin. The interaction of the various microorganisms with the skin plays a key role in the maintenance of homeostasis. The precise role and function of Demodex mites within normal and diseased human skin remains elusive. The emergence of ivermectin as a key therapy for rosacea has refocused interest in the role of Demodex mites in the pathogenesis of this skin disease and the ability of Demodex to modulate the host immune system.

Clinical potential of gene-directed enzyme prodrug therapy to improve radiation therapy in prostate cancer patients.

Despite the advances in prostate cancer diagnosis and treatment, current therapies are not curative in a significant proportion of patients. Gene-directed enzyme prodrug therapy (GDEPT), when combined with radiation therapy, could improve the outcome of treatment for prostate cancer, the second leading cause of cancer death in the western world. GDEPT involves the introduction of a therapeutic transgene, which can be targeted to the tumour cells. A prodrug is administered systemically and is converted to its toxic form only in those cells containing the transgene, resulting in cell kill. This review will discuss the clinical trials which have investigated the potential of GDEPT at various stages of prostate cancer progression. The advantages of using GDEPT in combination with radiotherapy will be examined, as well as some of the recent advances which enhance the potential utility of GDEPT.

Androgen hypersensitivity in prostate cancer: molecular perspectives on androgen deprivation therapy strategies.

Androgen deprivation therapy is initially successful in treating advanced prostate cancer. However, after a period of time tumors inevitably recur. Improved understanding of the various biochemical causes of resistance to hormonal therapy is of crucial importance for developing more effective therapeutic strategies in this cohort of patients. This review discusses the preclinical evidence for androgen hypersensitivity (AH), as a mechanism by which tumors become hormone-refractory (HR). We propose that the growth of some such tumors may be not only stimulated by, but also dependent on low hormone levels, and furthermore, that normal hormone concentrations can have an inhibitory effect on growth. The incidence and importance of AH merits further investigation both in preclinical studies and during clinical trials of intermittent androgen withdrawal or testosterone replacement. We suggest that a subset of HR prostate cancer patients who have androgen-hypersensitive tumors could be particularly amenable to these treatments. Finally, potential approaches for developing biomarkers to identify such patients are explored.

The HIF-1alpha C1772T polymorphism may be associated with susceptibility to clinically localised prostate cancer but not with elevated expression of hypoxic biomarkers.

We investigated the role of the C1772T polymorphisms in exon 12 of the Hypoxia-inducible factor-1 alpha (HIF-1alpha) gene C1772T genotype in prostate cancer (PCa) and amplification of the hypoxic response. We identified the heterozygous germline CT genotype as an increased risk factor for clinically localised prostate cancer (Odds ratio = 6.2; p < 0.0001). While immunostaining intensity for HIF-1alpha and VEGF was significantly enhanced in 75% of PCa specimens when compared to matched benign specimens (p < 0.0001), the CT genotype did not modulate the kinetics of HIF-1alpha protein expression in hypoxia in vitro, and was not associated with enhanced expression of hypoxic biomarkers. This study provides the first evidence of an increased risk for clinically localised prostate cancer in men carrying the C1772T HIF-1alpha gene polymorphism. Although our results did not suggest an association between expression of hypoxic biomarkers and genotype status, the correlation may merit further investigation.

Hypoxia response element-driven cytosine deaminase/5-fluorocytosine gene therapy system: a highly effective approach to overcome the dynamics of tumour hypoxia and enhance the radiosensitivity of prostate cancer cells in vitro.

BACKGROUND: We proposed to exploit hypoxia-inducible factor (HIF)-1alpha overexpression in prostate tumours and use this transcriptional machinery to control the expression of the suicide gene cytosine deaminase (CD) through binding of HIF-1alpha to arrangements of hypoxia response elements. CD is a prodrug activation enzyme, which converts inactive 5-fluorocytosine to active 5-fluorouracil (5-FU), allowing selective killing of vector containing cells. METHODS: We developed a pair of vectors, containing either five or eight copies of the hypoxia response element (HRE) isolated from the vascular endothelial growth factor (pH5VCD) or glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (pH8GCD) gene, respectively. The kinetics of the hypoxic induction of the vectors and sensitization effects were evaluated in 22Rv1 and DU145 cells in vitro. RESULTS: The CD protein as selectively detected in lysates of transiently transfected 22Rv1 and DU145 cells following hypoxic exposure. This is the first evidence of GAPDH HREs being used to control a suicide gene therapy strategy. Detectable CD levels were sustained upon reoxygenation and prolonged hypoxic exposures. Hypoxia-induced chemoresistance to 5-FU was overcome in both cell lines treated with this suicide gene therapy approach. Hypoxic transfectants were sensitized to prodrug concentrations that were ten-fold lower than those that are clinically relevant. Moreover, the surviving fraction of reoxygenated transfectants could be further reduced with the concomitant delivery of clinically relevant single radiation doses. CONCLUSIONS: This strategy thus has the potential to sensitize the hypoxic compartment of prostate tumours and improve the outcome of current therapies.

The emerging roles of DNA methylation in the clinical management of prostate cancer.

Aberrant DNA methylation is one of the hallmarks of carcinogenesis and has been recognized in cancer cells for more than 20 years. The role of DNA methylation in malignant transformation of the prostate has been intensely studied, from its contribution to the early stages of tumour development to the advanced stages of androgen independence. The most significant advances have involved the discovery of numerous targets such as GSTP1, Ras-association domain family 1A (RASSF1A) and retinoic acid receptor beta2 (RARbeta2) that become inactivated through promoter hypermethylation during the course of disease initiation and progression. This has provided the basis for translational research into methylation biomarkers for early detection and prognosis of prostate cancer. Investigations into the causes of these methylation events have yielded little definitive data. Aberrant hypomethylation and how it impacts upon prostate cancer has been less well studied. Herein we discuss the major developments in the fields of prostate cancer and DNA methylation, and how this epigenetic modification can be harnessed to address some of the key issues impeding the successful clinical management of prostate cancer.

Molecular pathology of prostate cancer: the key to identifying new biomarkers of disease.

Microarray technology has recently accelerated the study of the molecular events involved in prostate cancer, offering the prospect of more precise prognosis and new therapeutic strategies. This review summarises current knowledge of the molecular pathology of prostate cancer. The expression and function of numerous genes have been shown to be altered in prostate cancer. Many of these genes are involved in cell cycle regulation, steroid hormone metabolism or regulation of gene expression. The mechanisms by which androgen independence arises are discussed, including cross-activation, gene amplification and point mutations of the androgen receptor. Analysis of changes in the levels of expression of large numbers of genes during prostate cancer progression have provided a better understanding of the basis of the disease, yielding new molecular markers, such as hepsin, with potential use in diagnosis and prognosis.

Gene-based therapy in prostate cancer.

Prostate cancer is one of the commonest causes of illness and death from cancer. Radical prostatectomy, radiotherapy, and hormonal therapy are the main conventional treatments. However, gene therapy is emerging as a promising adjuvant to conventional strategies, and several clinical trials are in progress. Here, we outline several approaches to gene therapy for prostate cancer that have been investigated. Methods of gene delivery are described, particularly those that have commonly been used in research on prostate cancer. We discuss efforts to achieve tissue-specific gene delivery, focusing on the use of tissue-specific gene promoters. Finally, the present use of gene therapy for prostate cancer is evaluated. The ability to deliver gene-therapy vectors directly to prostate tissue, and to regulate gene expression in a tissue-specific manner, offers promise for the use of gene therapy in prostate cancer.