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Objective(s): Zirconium-based metal-organic frameworks (MOFs) nanostructures, due to their capability of easy surface modification, are considered interesting structures for delivery. In the present study, the surfaces of UIO-66 and NH2-UIO-66 MOFs were modified by polyethyleneimine (PEI) 10000 Da, and their efficiency for plasmid delivery was evaluated. Materials and Methods: Two different approaches, were employed to prepare surface-modified nanoparticles. The physicochemical characteristics of the resulting nanoparticles, as well as their transfection efficiency and cytotoxicity, were investigated on the A549 cell line. Results: The sizes of DNA/nanocarriers for PEI-modified UIO-66 (PEI-UIO-66) were between 212-291 nm and 267-321 nm for PEI 6-bromohexanoic acid linked UIO-66 (PEI-HEX-UIO-66). The zeta potential of all was positive with the ranges of +16 to +20 mV and +23 to +26 mV for PEI-UIO-66 and PEI-HEX-UIO-66, respectively. Cellular assay results showed that the PEI linking method had a higher rate of gene transfection efficiency with minimal cytotoxicity than the wet impregnation method. The difference between transfection of modified nanoparticles compared to the PEI 10 kDa was not significant but the PEI-HEX-UIO-66 showed less cytotoxicity. Conclusion: The present study suggested that the post-synthetic modification of MOFs with PEI 10000 Da through EDC/NHS+6-bromohexanoic acid reaction can be considered as an effective approach for modifying MOFs' structure in order to obtain nanoparticles with better biological function in the gene delivery process. [ABSTRACT FROM AUTHOR]

Currently, many types of non-linear topological structure polymers, such as brush-shaped, star, branched and dendritic structures, have captured much attention in the field of gene delivery and nanomedicine. Compared with linear polymers, non-linear topological structural polymers offer many advantages, including multiple terminal groups, broad and complicated spatial architecture and multi-functionality sites to enhance gene delivery efficiency and targeting capabilities. Nevertheless, the complexity of their synthesis process severely hampers the development and applications of nonlinear topological polymers. This review aims to highlight various synthetic approaches of non-linear topological architecture polymers, including reversible-deactivation radical polymerization (RDRP) including atom-transfer radical polymerization (ATRP), nitroxide-mediated polymerization (NMP), reversible addition-fragmentation chain transfer (RAFT) polymerization, click chemistry reactions and Michael addition, and thoroughly discuss their advantages and disadvantages, as well as analyze their further application potential. Finally, we comprehensively discuss and summarize different non-linear topological structure polymers for genetic materials delivering performance both in vitro and in vivo, which indicated that topological effects and nonlinear topologies play a crucial role in enhancing the transfection performance of polymeric vectors. This review offered a promising guideline for the design and development of novel nonlinear polymers and facilitated the development of a new generation of polymer-based gene vectors. [ABSTRACT FROM AUTHOR]

Metal–organic frameworks (MOFs) are porous materials with unique characteristics that make them well-suited for drug delivery and gene therapy applications. Among the MOFs, zeolitic imidazolate framework-8 (ZIF-8) has emerged as a promising candidate for delivering exogenous DNA into cells. However, the potential of ZIF-8 as a vector for sperm-mediated gene transfer (SMGT) has not yet been thoroughly explored. This investigation aimed to explore the potential of ZIF-8 as a vector for enhancing genetic transfer and transgenesis rates by delivering exogenous DNA into sperm cells. To test this hypothesis, we employed ZIF-8 to deliver a plasmid expressing green fluorescent protein (GFP) into mouse sperm cells and evaluated the efficiency of DNA uptake. Our findings demonstrate that ZIF-8 can efficiently load and deliver exogenous DNA into mouse sperm cells, increasing GFP expression in vitro. These results suggest that ZIF-8 is a valuable tool for enhancing genetic transfer in SMGT, with important implications for developing genetically modified animals for research and commercial purposes. Additionally, our study highlights the potential of ZIF-8 as a novel class of vectors for gene delivery in reproductive biology. Overall, our study provides a foundation for further research into using ZIF-8 and other MOFs as gene delivery systems in reproductive biology and underscores the potential of these materials as promising vectors for gene therapy and drug delivery. [ABSTRACT FROM AUTHOR]

[Display omitted] The aim of the study was to evaluate the effect of thiolation of lipid-based nanoparticles (LNPs) on cellular uptake of nucleic acids. A thiolated surfactant was synthesized by binding palmitic acid covalently to cysteine. Green fluorescent protein (GFP) encoding plasmid DNA (pDNA) was used as model nucleic acid and incorporated via hydrophobic ion-pairing with a cationic cholesterol derivate (DcCholesterol) in LNPs that were prepared by solvent injection method using the thiolated surfactant for surface decoration. LNPs were characterized regarding size, polydispersity index, zeta potential and stability in biorelevant media. The endosomal escape properties of LNPs were evaluated by erythrocytes interaction studies. Cell viability and transfection efficiency on HEK293 cells were investigated. The structure of the thiolated surfactant was confirmed by 1H NMR and FT-IR. LNPs containing the nucleic acid-DcCholesterol complex with and without the thiolated surfactant were developed and displayed sizes in the range from 173 nm to 233 nm with a narrow size distribution (PDI < 0.3) and a negative zeta potential. LNPs showed no significant increase in size after 4 h of incubation in artificial body fluids. Erythrocytes interaction studies revealed enhanced endosomal escape properties for thiolated LNPs compared to non-thiolated LNPs. LNPs showed in concentrations lower than 0.74 mg/mL cell viability ≥ 80 %. Transfection studies on HEK cells with thiolated LNPs compared to non-thiolated LNP showed a 4.6-fold higher expression of GFP. Surface thiolation of LNPs represents a promising tool for enhancing intracellular nucleic acid delivery of LNPs. [ABSTRACT FROM AUTHOR]

Genetic disorders of the central nervous system (CNS) comprise a significant portion of disability in both children and adults. Several preclinical animal models have shown effective adeno-associated virus (AAV) mediated gene transfer for either treatment or prevention of autosomal recessive genetic disorders. Owing to the intricacy of the human CNS and the blood–brain barrier, it is difficult to deliver genes, particularly since the expression of any given gene may be required in a particular CNS structure or cell type at a specific time during development. In this review, we analyzed delivery methods for AAV-mediated gene therapy in past and current clinical trials. The delivery routes analyzed were direct intraparenchymal (IP), intracerebroventricular (ICV), intra-cisterna magna (CM), lumbar intrathecal (IT), and intravenous (IV). The results demonstrated that the dose used in these routes varies dramatically. The average total doses used were calculated and were 1.03 × 1013 for IP, 5.00 × 1013 for ICV, 1.26 × 1014 for CM, and 3.14 × 1014 for IT delivery. The dose for IV delivery varies by patient weight and is 1.13 × 1015 IV for a 10 kg infant. Ultimately, the choice of intervention must weigh the risk of an invasive surgical procedure to the toxicity and immune response associated with a high dose vector. [ABSTRACT FROM AUTHOR]

Gene delivery to, and expression in, the mouse brain is important for understanding gene functions in brain development and disease, or testing gene therapies. Here, we describe an approach to express a transgene in the mouse brain in a cell-type-specific manner. We use stereotaxic injection of a transgene-expressing adeno-associated virus into the mouse brain via the intracerebroventricular route. We demonstrate stable and sustained expression of the transgene in neurons of adult mouse brain, using a reporter gene driven by a neuron-specific promoter. This approach represents a rapid, simple, and cost-effective method for global gene expression in the mouse brain, in a cell-type-specific manner, without major surgical interventions. The described method represents a helpful resource for genetically engineering mice to express a therapeutic gene, for gene therapy studies, or to deliver genetic material for genome editing and developing knockout animal models. [ABSTRACT FROM AUTHOR]

The proto‐oncogene c‐Src is a protein‐tyrosine kinase that is associated with increased risk of acute and chronic kidney diseases, cardiovascular diseases, neurological diseases, and cancers. The purpose of this study was to develop a gene delivery carrier responding to activated c‐Src and to evaluate its efficacy in vitro and in vivo. The polymeric gene carrier consists of a neutral main chain bearing a peptide substrate of c‐Src or negative control peptide. Complexes of the polymer containing c‐Src‐targeting peptides and DNA were phosphorylated in the presence of c‐Src, resulting in release of the DNA. Green fluorescent protein (GFP) expression was observed after microinjection of complexes of polymer containing c‐Src‐targeting peptides and GFP‐encoding DNA into A431 cells at the nitrogen/phosphate (N/P) ratio of 1.0. GFP was not expressed in cells microinjected with negative control polymer/DNA complex. Direct injection of complexes of polymer containing c‐Src‐targeting peptides and luciferase‐encoding DNA at N/P ratios of 1.0 and 2.0 into tumor‐bearing mice resulted in luciferase expression in A431 tumors but not in normal skin tissues. No luciferase expression was observed in A431 tumors or skin tissues after injection of control polymer/DNA complex. These results indicate that our gene delivery carrier enables activated c‐Src‐specific gene expression. [ABSTRACT FROM AUTHOR]

There has been considerable interest in gene vectors and their role in regulating cellular activities and treating diseases since the advent of nucleic acid drugs. MicroRNA (miR) therapeutic strategies are research hotspots as they regulate gene expression post‐transcriptionally and treat a range of diseases. An original tetrahedral framework nucleic acid (tFNA) analog, a bioswitchable miR inhibitor delivery system (BiRDS) carrying miR inhibitors, is previously established; however, it remains unknown whether BiRDS can be equipped with miR mimics. Taking advantage of the transport capacity of tetrahedral framework nucleic acid (tFNA) and upgrading it further, the treatment outcomes of a traditional tFNA and BiRDS at different concentrations on TGF‐β‐ and bleomycin‐induced fibrosis simultaneously in vitro and in vivo are compared. An upgraded traditional tFNA is designed by successfully synthesizing a novel BiRDS, carrying a miR mimic, miR‐27a, for treating skin fibrosis and inhibiting the pyroptosis pathway, which exhibits stability and biocompatibility. BiRDS has three times higher efficiency in delivering miRNAs than the conventional tFNA with sticky ends. Moreover, BiRDS is more potent against fibrosis and pyroptosis‐related diseases than tFNAs. These findings indicate that the BiRDS can be applied as a drug delivery system for disease treatment. [ABSTRACT FROM AUTHOR]

Nanotechnology helps design innovative and effective medicine delivery methods. Different particle characteristics are used in targeted delivery to elicit therapeutic responses. Recently, gene therapy nanoparticles have interested researchers. Regardless of their different atomic structures, they can penetrate cell walls effectively. Encapsulating various genetic materials for treatment has resulted in effective cellular uptakes. Nanoparticles have some unique properties in biological and non-biological systems, and the alteration of some physical and chemical properties of materials, such as their toxicity, has put nanomaterials in the focus of researchers. In addition to its main properties, shredded nanoparticles and their effects on surface properties can be formed into materials with important and sometimes different biological activities. These particles also have properties of the surface causing those biological responses such as inflammatory effects, DNA damage, cytotoxicity, hormonal changes, and so on. Changes in the biological effects of particles are due to: (1) nanoparticles are closely linked to the biological system and thus can easily enter the biological system of the cells; (2) their structural properties, which suddenly change when nanoparticles lose their capacity. Because of this, these particles have biological impacts on biological systems and are employed in cancer therapy, plant hormone operation, gene transfer, cell labeling, organic absorption, imaging, and more. To innovate, nanoscience research must be Manus. In an attempt to show the amazing advances in gene therapy, this review will define gene therapy and the vital role of nanoparticles in packaging and transferring genes for cancers. [ABSTRACT FROM AUTHOR]

Gene therapy displays great promise in the treatment of cervical cancer. The occurrence of cervical cancer is highly related to persistent human papilloma virus (HPV) infection. The HPV oncogene can be cleaved via gene editing technology to eliminate carcinogenic elements. However, the successful application of the gene therapy method depends on effective gene delivery into the vagina. To improve mucosal penetration and adhesion ability, quaternized chitosan was introduced into the poly(β-amino ester) (PBAE) gene-delivery system in the form of quaternized chitosan-g-PBAE (QCP). At a mass ratio of PBAE:QCP of 2:1, the polymers exhibited the highest green fluorescent protein (GFP) transfection efficiency in HEK293T and ME180 cells, which was 1.1 and 5.4 times higher than that of PEI 25 kD. At this mass ratio, PBAE–QCP effectively compressed the GFP into spherical polyplex nanoparticles (PQ–GFP NPs) with a diameter of 255.5 nm. In vivo results indicated that owing to the mucopenetration and adhesion capability of quaternized CS, the GFP transfection efficiency of the PBAE–QCP hybrid system was considerably higher than those of PBAE and PEI 25 kD in the vaginal epithelial cells of Sprague–Dawley rats. Furthermore, the new system demonstrated low toxicity and good safety, laying an effective foundation for its further application in gene therapy. [ABSTRACT FROM AUTHOR]

Over the last decade, the clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most promising gene editing tool and is broadly utilized to manipulate the gene for disease treatment, especially for cancer, which involves multiple genetic alterations. Typically, CRISPR/Cas9 machinery is delivered in one of three forms: DNA, mRNA, or ribonucleoprotein. However, the lack of efficient delivery systems for these macromolecules confined the clinical breakthrough of this technique. Therefore, a variety of nanomaterials have been fabricated to improve the stability and delivery efficiency of the CRISPR/Cas9 system. In this context, the natural biopolymer-based carrier is a particularly promising platform for CRISPR/Cas9 delivery due to its great stability, low toxicity, excellent biocompatibility, and biodegradability. Here, we focus on the advances of natural biopolymer-based materials for CRISPR/Cas9 delivery in the cancer field and discuss the challenges for their clinical translation. [ABSTRACT FROM AUTHOR]

Effective process development towards intensified processing for gene delivery applications using Hepatitis B core Antigen (HBcAg) virus-like particles (VLPs) relies on analytical methods for the absolute quantification of HBcAg VLP proteins and bound nucleic acids. We investigated a silica spin column (SC)-based extraction procedure, including proteinase K lysis and silica chromatography, for the absolute quantification of different species of nucleic acids bound to HBcAg VLPs analyzed by dye-based fluorescence assays. This revealed load-dependent nucleic acid recoveries of the silica-SC-based extraction. We also developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method to separate and quantify the HBcAg proteins and the bound nucleic acids simultaneously without prior sample treatment by dissociation reagents. The method demonstrated sufficient linearity, accuracy, and precision coefficients and is suited for determining absolute protein and nucleic acid concentrations and HBcAg protein purities at various purification stages. Both the silica-SC-based extraction and the RP-based extraction presented overcome the limitations of analytical techniques, which are restricted to relative or qualitative analyses for HBcAg VLPs with bound nucleic acids. In combination with existing analytics, the methods for an absolute quantification of HBcAg VLPs and bound nucleic acids presented here are required to evaluate downstream purification steps, such as the removal of host cell-derived nucleic acids, concurrent protein loss, and efficient loading with therapeutic nucleic acids. Hence, the methods are key for effective process development when using HBcAg VLP as potential gene delivery vehicles. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeat activation (CRISPRa) technology has emerged as a precise genome editing tool for activating endogenous transgene expression. While it holds promise for precise cell modification, its translation into tissue engineering has been hampered by biosafety concerns and suboptimal delivery methods. To address these challenges, we have developed a CRISPRa non-viral gene delivery platform by immobilizing non-viral CRISPRa complexes into a biocompatible hydrogel/nanofiber (Gel/NF) composite scaffold. The Gel/NF scaffold facilitates the controlled and sustained release of CRISPRa complexes and also promotes cell recruitment to the scaffold for efficient and localized transfection. As a proof of concept, we employed this CRISPRa delivery platform to activate the vascular endothelial growth factor (VEGF) gene in a rat model with full-thickness skin defects. Our results demonstrate sustained upregulation of VEGF expression even at 21 days post-implantation, resulting in enhanced angiogenesis and improved skin regeneration. These findings underscore the potential of the Gel/NF scaffold-based CRISPRa delivery platform as an efficient and durable strategy for gene activation, offering promising prospects for tissue regeneration. Translation of clustered regularly interspaced short palindromic repeat activation (CRISPRa) therapy to tissue engineering is limited by biosafety concerns and unsatisfactory delivery strategy. To solve this issue, we have developed a CRISPRa non-viral gene delivery platform by immobilizing non-viral CRISPRa complexes into a biocompatible hydrogel/nanofiber (Gel/NF) composite scaffold. This scaffold enables controlled and sustained release of CRISPRa and can induce cell recruitment for localized transfection. As a proof of concept, we activated vascular endothelial growth factor (VEGF) in a rat model with full-thickness skin defects, leading to sustained upregulation of VEGF expression, enhanced angiogenesis and improved skin regeneration in vivo. These findings demonstrate the potential of this platform for gene activation, thereby offering promising prospects for tissue regeneration. [Display omitted] [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is an incurable X‐linked recessive genetic disease caused by mutations in the dystrophin gene. Many researchers aim to restore truncated dystrophin via viral vectors. However, the low packaging capacity and immunogenicity of vectors have hampered their clinical application. Herein, we constructed four lentiviral vectors with truncated and sequence‐optimized dystrophin genes driven by muscle‐specific promoters. The four lentiviral vectors stably expressed mini‐dystrophin in C2C12 muscle cells in vitro. To estimate the treatment effect in vivo, we transferred the lentiviral vectors into neonatal C57BL/10ScSn‐Dmdmdx mice through local injection. The levels of modified dystrophin expression increased, and their distribution was also restored in treated mice. At the same time, they exhibited the restoration of pull force and a decrease in the number of mononuclear cells. The remissions lasted 3–6 months in vivo. Moreover, no integration sites of vectors were distributed into the oncogenes. In summary, this study preliminarily demonstrated the feasibility and safety of lentiviral vectors with mini‐dystrophin for DMD gene therapy and provided a new strategy to restore truncated dystrophin. [ABSTRACT FROM AUTHOR]

Non-viral gene delivery systems are typically designed vector systems with contradictory properties, namely sufficient stability before cellular uptake and instability to ensure the release of nucleic acid cargoes in the transcription process after being taken up into cells. We reported previously that poly-(L-lysine) terminally bearing a multi-arm PEG (maPEG-PLL) formed nanofiber-polyplexes that suppressed excessive DNA condensation via steric repulsion among maPEGs and exhibited effective transcriptional capability in PCR amplification experiments and a cell-free gene expression system. In this study, the reversible stabilization of a nanofiber-polyplex without impairing the effective transcriptional capability was investigated by introducing cross-links between the PLL side chains within the polyplex using a cross-linking reagent with disulfide (SS) bonds that can be disrupted under reducing conditions. In the presence of dextran sulfate and/or dithiothreitol, the stability of the polyplex and the reactivity of the pDNA were evaluated using agarose gel electrophoresis and real-time PCR. We succeeded in reversibly stabilizing nanofiber-polyplexes using dithiobis (succinimidyl propionate) (DSP) as the cross-linking reagent. The effect of the reversible stabilization was confirmed in experiments using cultured cells, and the DSP-crosslinked polyplexes exhibited gene expression superior to that of polyethyleneimine polyplexes, which are typical polyplexes. [ABSTRACT FROM AUTHOR]

BACKGROUND: Traditional bone tissue engineering techniques for treating critical bone defects suffer from low osteogenic efficiency and poor safety. Geneenhanced bone tissue engineering grafts constructed with non-viral nanoparticles have attracted widespread attention from scholars both domestically and internationally due to their higher osteogenic rates and safety, leading to extensive research in this field. OBJECTIVE: To review new technologies, methods, and challenges in the research of nanoparticles in gene therapy for bone tissue engineering, aiming to provide a reference for research on gene therapy mediated by nanoparticles in bone tissue engineering. METHODS: The first author searched PubMed, Web of Science, and CNKI. The Chinese and English search terms were “bone defect repair, bone tissue engineering, gene delivery, nanoparticles, non-viral gene vector, sustained release technology, sequential release, targeted delivery.” Finally, 84 articles were included for summary. RESULTS AND CONCLUSION: (1) Targeted gene delivery at various physiological stages of bone defect healing can significantly enhance bone repair efficacy. In the early inflammatory stage, delivering anti-inflammatory genes via nanoparticles to regulate the inflammatory response lays the foundation for subsequent bone healing. During the angiogenesis phase, local delivery of vascularization target genes aids in forming a highly organized vascular system, significantly accelerating bone healing. As vascularization progresses, neural re-innervation of the bone begins; at this stage, delivering functional genes promoting nerve regeneration facilitates neuro-osteogenic regeneration. During the osteogenic phase, constructing nanoparticle-bone gene complexes directly enhances the efficiency of bone formation on scaffold and in vivo. (2) Non-viral nanocarriers such as various organic and inorganic nanoparticles, metal-organic frameworks, and exosomes show immense potential in gene therapy for bone tissue engineering. Each of these carriers has its unique advantages and limitations. Therefore, in practical applications, selection of the appropriate type primarily depends on factors such as gene transfection efficiency, biocompatibility, and osteogenic properties. (3) To comprehensively improve the efficiency of gene delivery, the gene transfection efficiency of nanocarriers is mainly enhanced through various functional designs, including enhancing the temporal regulation ability such as slow release and multi-gene delivery sequence, enhancing the spatial targeting ability of bone tissue and osteoblast-related cells, enhancing the transmembrane transport efficiency and nuclear targeting ability. (4) Numerous challenges need to be overcome in order to further promote the clinical application of nanoparticle-mediated gene therapy for bone tissue engineering, including improving gene transfection efficiency of organic carriers, reducing biosafety risks of inorganic carriers, optimizing the production process of new types of nanocarriers, and promoting interactions between other physiological processes and osteogenesis. These are also research hotspots and trends of gene therapy for bone tissue engineering in the future. [ABSTRACT FROM AUTHOR]

The clinical utility of emulsions as delivery vehicles is hindered by a dependence on passive release. Stimuli-responsive emulsions overcome this limitation but rely on external triggers or are composed of nanoparticle-stabilized droplets that preclude sizes necessary for biomedical applications. Here, we employ cleavable poly(2-oxazoline) diblock copolymer surfactants to form perfluorocarbon (PFC) nanoemulsions that release cargo upon exposure to glutathione. These surfactants allow for the first example of redox-responsive nanoemulsions in cellulo. A noncovalent fluorous tagging strategy is leveraged to solubilize a GFP plasmid inside the PFC nanoemulsions, whereupon protein expression is achieved selectively when employing a stimuli-responsive surfactant. This work contributes a methodology for non-viral gene delivery and represents a general approach to nanoemulsions that respond to endogenous stimuli.

Nanodiamonds are innovative nanocrystalline carbon particles able to deliver chemically conjugated miRNAs. In oncology, the use of miRNA-based therapies may represent an advantage, based on their ability to simultaneously target multiple intracellular oncogenic targets. Here, nanodiamonds were tested and optimized to deliver miR-34a, a miRNA playing a key role in inhibiting tumor development and progression in many cancers. The physical-chemical properties of nanodiamonds were investigated suggesting electrical stability and uniformity of structure and size. Moreover, we evaluated nanodiamond cytotoxicity on two breast cancer cell models and confirmed their excellent biocompatibility. Subsequently, nanodiamonds were conjugated with miR-34a, using the chemical crosslinker polyethyleneimine; real-time PCR analysis revealed a higher level of miR-34a in cancer cells treated with the different formulations of nanodiamonds than with commercial transfectant. A significant and early nanodiamond-miR-34a uptake was recorded by FACS and fluorescence microscopy analysis in MCF7 and MDA-MB-231 cells. Moreover, nanodiamond-miR-34a significantly inhibited both cell proliferation and migration. Finally, a remarkable anti-tumor effect of miR-34a-conjugated nanodiamonds was observed in both heterotopic and orthotopic murine xenograft models. In conclusion, this study provides a rationale for the development of new therapeutic strategies based on use of miR-34a delivered by nanodiamonds to improve the clinical treatment of neoplasms.

Abstract Despite the recent advances in the development of bone graft substitutes, treatment of critical size bone defects continues to be a significant challenge, especially in the elderly population. A current approach to overcome this challenge involves the creation of bone-mimicking scaffolds that can simultaneously promote osteogenesis and angiogenesis. In this context, incorporating multiple bioactive agents like growth factors, genes, and small molecules into these scaffolds has emerged as a promising strategy. To incorporate such agents, researchers have developed scaffolds incorporating nanoparticles, including nanoparticulate carriers, inorganic nanoparticles, and exosomes. Current paper provides a summary of the latest advancements in using various bioactive agents, drugs, and cells to synergistically promote osteogenesis and angiogenesis in bone-mimetic scaffolds. It also discusses scaffold design properties aimed at maximizing the synergistic effects of osteogenesis and angiogenesis, various innovative fabrication strategies, and ongoing clinical studies. Graphical Abstract

Gene therapy holds great promise for curing cancer by editing the deleterious genes of tumor cells, but the lack of vector systems for efficient delivery of genetic material into specific tumor sites in vivo has limited its full therapeutic potential in cancer gene therapy. Over the past two decades, increasing studies have shown that lentiviral vectors (LVs) modified with different glycoproteins from a donating virus, a process referred to as pseudotyping, have altered tropism and display cell-type specificity in transduction, leading to selective tumor cell killing. This feature of LVs together with their ability to enable high efficient gene delivery in dividing and non-dividing mammalian cells in vivo make them to be attractive tools in future cancer gene therapy. This review is intended to summarize the status quo of some typical pseudotypings of LVs and their applications in basic anti-cancer studies across many malignancies. The opportunities of translating pseudotyped LVs into clinic use in cancer therapy have also been discussed.

Baculoviral vectors (BVs) derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) are an attractive tool for multigene delivery in mammalian cells, which is particularly relevant for CRISPR technologies. Most applications in mammalian cells rely on BVs that are pseudotyped with vesicular stomatitis virus G-protein (VSV-G) to promote efficient endosomal release. VSV-G expression typically occurs under the control of the hyperactive polH promoter. In this study, we demonstrate that polH-driven VSV-G expression results in BVs characterised by reduced stability, impaired morphology, and VSV-G induced toxicity at high multiplicities of transduction (MOTs) in target mammalian cells. To overcome these drawbacks, we explored five alternative viral promoters with the aim of optimising VSV-G levels displayed on the pseudotyped BVs. We report that Orf-13 and Orf-81 promoters reduce VSV-G expression to less than 5% of polH, rescuing BV morphology and stability. In a panel of human cell lines, we elucidate that BVs with reduced VSV-G support efficient gene delivery and CRISPR-mediated gene editing, at levels comparable to those obtained previously with polH VSV-G-pseudotyped BVs (polH VSV-G BV). These results demonstrate that VSV-G hyperexpression is not required for efficient transduction of mammalian cells. By contrast, reduced VSV-G expression confers similar transduction dynamics while substantially improving BV integrity, structure, and stability.

Immunotherapy combined with chemicals and genetic engineering tools is emerging as a promising strategy to treat triple-negative breast cancer (TNBC), which is more aggressive with poorer progress than other breast cancer subtypes. In this study, lipid-based nanoparticles (LNPs) possessed an NK cell-like function that could deliver tumor-specific therapeutics and inhibit tumor growth. LNPs fused with an NK cell membrane protein system (NK-LNP) have three main features: (i) hydrophilic plasmid DNA can inhibit TNBC metastasis when encapsulated within LNPs and delivered to cells; (ii) the lipid composition of LNPs, including C18 ceramide, exhibits anticancer effects; (iii) NK cell membrane proteins are immobilized on the LNP surface, enabling targeted delivery to TNBC cells. These particles facilitate the targeted delivery of HIC1 plasmid DNA and the modulation of immune cell functions. Delivered therapeutic genes can inhibit metastasis of TNBC and then induce apoptotic cell death while targeting macrophages to promote cytokine release. The anticancer effect is expected to be applied in treating various difficult-to-treat cancers with LNP fused with NK cell plasma membrane proteins, which can simultaneously deliver therapeutic chemicals and genes.

Gene delivery therapy has emerged as a popular approach for the treatment of various diseases. However, it still poses the challenges of accumulation in target sites and reducing off-target effects. Aerosol gene delivery for the treatment of pulmonary diseases has the advantages of high lung accumulation, specific targeting and fewer systemic side effects. However, the key challenge is selecting the appropriate formulation for aerosol gene delivery that can overcome physiological barriers. There are numerous existing gene carriers under study, including viral vectors and non-viral vectors. With the development of biomaterials, more biocompatible substances have applied gene delivery via inhalation. Furthermore, many types of genes can be delivered through aerosol inhalation, such as DNA, mRNA, siRNA and CRISPR/Cas9. Aerosol delivery of different types of genes has proven to be efficient in the treatment of many diseases such as SARS-CoV-2, cystic fibrosis and lung cancer. In this paper, we provide a comprehensive review of the ongoing research on aerosol gene delivery therapy, including the basic respiratory system, different types of gene carriers, different types of carried genes and clinical applications.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly intratumorally heterogeneous disease that includes several subtypes and is highly plastic. Effective gene delivery to all PDAC cells is essential for modulating gene expression and identifying potential gene-based therapeutic targets in PDAC. Most current gene delivery systems for pancreatic cells are optimized for islet or acinar cells. Lentiviral vectors are the current main gene delivery vectors for PDAC, but their transduction efficiencies vary depending on pancreatic cell type, and are especially poor for the classical subtype of PDAC cells from both primary tumors and cell lines. Methods: We systemically compare transduction efficiencies of glycoprotein G of vesicular stomatitis virus (VSV-G)-pseudotyped lentiviral and Sendai viral vectors in human normal pancreatic ductal and PDAC cells. Results: We find that the Sendai viral vector gives the most robust gene delivery efficiency regardless of PDAC cell type. Therefore, we propose using Sendai viral vectors to transduce ectopic genes into PDAC cells.

Abstract Rapid growth in nanoparticles (NPs) as delivery systems holds vast promise to promote therapeutic approaches for cancer treatment. Presently, a diverse array of NPs with unique properties have been developed to overcome different challenges and to achieve sophisticated delivery routes for enhancement of a series of therapies. Inspiring advances have been achieved in the field of cancer therapy using NPs. In this review, we aim to summarize the up‐to‐date progression of NPs for addressing various challenges, and expect to elicit novel and potential opportunities alternatively. We first introduce different sorts of NP technologies, illustrate their mechanisms, and present their applications. Then, the achievements made by NPs to break obstacles in delivering cargoes to specific sites through particular routes are highlighted, including long‐circulation, tumor targeting, responsive release, and subcellular localization. We subsequently retrospect recent research of NPs in different cancer treatments from single therapy, like chemotherapy, to combination therapy, like chemoradiotherapy, and to integrative therapy. Finally, the challenges and perspectives of NPs in cancer therapy, and their potential impact on the field of oncology are discussed. We believe this review can offer a deeper understanding of the challenges and opportunities of NPs for cancer therapy.

Self-assembly of various amphipathic copolymers is a simple method that allows the preparation of complex nanoparticles with several useful properties. Therefore, the aim of this research was to develop nanoparticles with better biocompatibility, biodegradability, and prolonged circulation time in the bloodstream to deliver drugs and genes into breast cancer tissues in a controlled and targeted manner. In this study, the copolymers PLA-chitosan- PEG -folic acid (COPA), PLA-chitosan- PEG -glucose (COPB), COPA & COPB (COPAB) and chitosan- PLA-PEG FA /Glu/VEGF/siRNA/PTX (NPsAB/siRNA/Paclitaxel) were synthesized, to control the release of paclitaxel (PTX) and siRNA and the circulation time of nanoparticles in blood. This was confirmed by 1H NMR and FTIR spectroscopy. The particle size, zeta potential and morphology of NPsAB /siRNA/PTX were studied by DLS and TEM, respectively. The results showed that the NPsAB/siRNA/PTX had spherical morphology with particle size and zeta potential about 200 nm and −7.8 mV, respectively. In vitro cytotoxicity assay results showed that the nanoparticles had good biocompatibility and low toxicity. Also demonstrated that NPsAB in the serum medium improved the efficiency of drug and siRNA delivery more than two-fold compared to COPA, COPB, and COPAB nanoparticles. Due to results the release pattern of siRNA and PTX from NPsAB nanoparticles under an acidic environment was significantly higher than that of their release rate in a neutral medium. Therefore, due to the acidity of tumor tissue, this property of NPsAB nanoparticles seems to be useful in the treatment of cancer.

Abstract Duchenne muscular dystrophy (DMD) is an incurable X‐linked recessive genetic disease caused by mutations in the dystrophin gene. Many researchers aim to restore truncated dystrophin via viral vectors. However, the low packaging capacity and immunogenicity of vectors have hampered their clinical application. Herein, we constructed four lentiviral vectors with truncated and sequence‐optimized dystrophin genes driven by muscle‐specific promoters. The four lentiviral vectors stably expressed mini‐dystrophin in C2C12 muscle cells in vitro. To estimate the treatment effect in vivo, we transferred the lentiviral vectors into neonatal C57BL/10ScSn‐Dmdmdx mice through local injection. The levels of modified dystrophin expression increased, and their distribution was also restored in treated mice. At the same time, they exhibited the restoration of pull force and a decrease in the number of mononuclear cells. The remissions lasted 3–6 months in vivo. Moreover, no integration sites of vectors were distributed into the oncogenes. In summary, this study preliminarily demonstrated the feasibility and safety of lentiviral vectors with mini‐dystrophin for DMD gene therapy and provided a new strategy to restore truncated dystrophin.

Chimeric antigen receptor T‐cell (CAR‐T) therapy has emerged as a highly efficacious treatment modality for refractory and relapsed hematopoietic malignancies in recent years. Furthermore, CAR technologies for cancer immunotherapy have expanded from CAR‐T to CAR‐natural killer cell (CAR‐NK), CAR‐cytokine‐induced killer cell (CAR‐CIK), and CAR‐macrophage (CAR‐MΦ) therapy. Nevertheless, the high cost and complex manufacturing processes of ex vivo generation of autologous CAR products have hampered broader application. There is an urgent need to develop an efficient and economical paradigm shift for exploring new sourcing strategies and engineering approaches toward generating CAR‐engineered immune cells to benefit cancer patients. Currently, researchers are actively investigating various strategies to optimize the preparation and sourcing of these potent immunotherapeutic agents. In this work, the latest research progress is summarized. Perspectives on the future of CAR‐engineered immune cell manufacturing are provided, and the engineering approaches, and diverse sources used for their development are focused upon. [ABSTRACT FROM AUTHOR]

Hydrogels have unique properties such as water retainer, softness, flexibility, and biocompatibility that cause their application in biology. Among the various hydrogel structures can be referred to intelligent hydrogels. These structures can respond to environmental stimuli. Environmental stimuli can be physical, including temperature or chemicals, such as pH. In this study, the application of smart hydrogels in the fields of medicine, protein, and gene delivery, as well as tissue engineering has been divided and the results of this study show that different types of smart hydrogels with different capabilities have been identified. The mechanical strength of hydrogels is suitable for physiological function and various purposes such as biomedical applications, tendon and ligament repair, cartilage replacement, and tissue engineering. Currently, hydrogels are used to make contact lenses, health products, tissue engineering scaffolds, drug delivery systems, and wound dressing. Most studies in the field of smart drug delivery have been reported, especially in anticancer drugs, and also tissue engineering that can achieve more desirable results in the near future. [ABSTRACT FROM AUTHOR]

This study aims to explore the stability of lipo-polymeric niosomes/niosome-based pCMS-EGFP complexes under different storage temperatures (25 °C, 4 °C, and −20 °C). To date, the question of nucleic acid-complex stability is one of the most vital issues in gene delivery applications. The need for stable vaccines during the COVID-19 pandemic has merely highlighted it. In the case of niosomes as gene carriers, the scientific literature still lacks comprehensive stability studies. In this study, the physicochemical features of niosomes/nioplexes in terms of size, surface charge, and polydispersity index (PDI), along with transfection efficiency, and cytotoxicity in NT2 cells were evaluated for 8 weeks. Compared to day 0, the physicochemical features of the niosomes stored at 25 °C and −20 °C changed dramatically in terms of size, zeta potential, and PDI, while remaining in reasonable values when stored at 4 °C. However, niosomes and nioplexes stored at 4 °C and −20 °C showed nearly stable transfection efficiency values, yet an obvious decrease at 25 °C. This article provides a proof of concept into the stability of polymeric cationic niosomes and their nioplexes as promising gene delivery vehicles. Moreover, it highlights the practical possibility of storing nioplexes at 4 °C for up to 2 months, as an alternative to niosomes, for gene delivery purposes. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) system is a powerful genomic DNA editing tool. The increased applications of gene editing tools, including the CRISPR-Cas system, have contributed to recent advances in biological fields, such as genetic disease therapy, disease-associated gene screening and detection, and cancer therapy. However, the major limiting factor for the wide application of gene editing tools is gene editing efficiency. This review summarizes the recent advances in factors affecting the gene editing efficiency of the CRISPR-Cas9 system and the CRISPR-Cas9 system optimization strategies. The homology-directed repair efficiency-related signal pathways and the form and delivery method of the CRISPR-Cas9 system are the major factors that influence the repair efficiency of gene editing tools. Based on these influencing factors, several strategies have been developed to improve the repair efficiency of gene editing tools. This review provides novel insights for improving the repair efficiency of the CRISPR-Cas9 gene editing system, which may enable the development and improvement of gene editing tools. [ABSTRACT FROM AUTHOR]

Deeper knowledge about the role of the tumor microenvironment (TME) in cancer development and progression has resulted in new strategies such as gene-based cancer immunotherapy. Whereas some approaches focus on the expression of tumoricidal genes within the TME, DNA-based vaccines are intended to be expressed in antigen-presenting cells (e.g., dendritic cells, DCs) in secondary lymphoid organs, which in turn induce anti-tumor T cell responses. Besides effective delivery systems and the requirement of appropriate adjuvants, DNA vaccines themselves need to be optimized regarding efficacy and selectivity. In this work, the concept of DC-focused transcriptional targeting was tested by applying a plasmid encoding for the luciferase reporter gene under the control of a derivative of the human fascin1 gene promoter (pFscnLuc), comprising the proximal core promoter fused to the normally more distantly located DC enhancer region. DC-focused activity of this reporter construct was confirmed in cell culture in comparison to a standard reporter vector encoding for luciferase under the control of the strong ubiquitously active cytomegalovirus promoter and enhancer (pCMVLuc). Both plasmids were also compared upon intravenous administration in mice. The organ- and cell type-specific expression profile of pFscnLuc versus pCMVLuc demonstrated favorable activity especially in the spleen as a central immune organ and within the spleen in DCs. [ABSTRACT FROM AUTHOR]

In this study, N'-(3-aminopropyl)-N-(30-(carbamoyl cholesteryl) propyl)-glycine amide (A) and 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE, D) (AD) liposomes were synthesised at molar ratios of 50:25 (AD5025), 50:50 (AD5050) and 50:75 (AD5075) and complexed with plasmid, pTRAIL-EGFP. AD liposome/pTRAIL-EGFP were evaluated for their complex ability, particle size, polydispersity index, zeta potential, expression of pTRAIL-EGFP, cytotoxicity, cell growth inhibition and apoptosis induction in KB cells. AD liposomes complexed completely with pTRAIL-EGFP at AD liposome/DNA ratios of above 4.5/1. The particle size of AD liposome/pTRAIL-EGFP ranged from 180±8 to 1,072±657nm depending on the proportion of lipid composition and liposome/DNA ratio. The extent of gene expression of pTRAIL-EGFP via AD liposome/pTRAIL-EGFP was significantly higher than that of the cells treated with pTRAIL-EGFP and depended on the AD liposome/DNA ratio. Cytotoxicity of AD liposomes was dependent on A and D molar ratio. Cell growth inhibition of AD liposome/pTRAIL-EGFP was significantly higher than that of the cells treated with pTRAIL-EGFP. The amount of late apoptotic and dead cells of AD liposome/pTRAIL-EGFP was significantly higher than that of cells treated with pTRAIL-EGFP. From this study that one can conclude that AD liposomes can carry and deliver pTRAIL-EGFP into KB cells resulting in cell growth inhibition and cell death. [ABSTRACT FROM AUTHOR]

Carbon nanotubes (CNTs) are nanostructures, allotropes of carbon which are made up of graphene sheets wrapped around it forming cylindrical structures. CNTs have been regarded to have interesting and attractive physical and chemical properties and have been tremendously used in genetic engineering. Understanding the role of CNTs in development of transgenic plants, review of research papers in the field was done. CNTs are classified into two categories: the single‐walled and multiwalled (MWCNTs) structures. They are valuable vectors in various biomedicine fields such as Gene delivery, Drug delivery, Immunotherapy, Tissue engineering, and Biomedical imaging and also, they deliver the DNA without damaging the cells. Based on recent studies, the functionalization of CNTs when combined with some other suitable molecules can drastically subside their toxic effects. Having unique properties such as small size, larger surface area is useful in delivering DNA into mammalian cells as well. Modifications in CNTs can make nucleic acids adhere to them even more efficiently. Also, MWCNTs are crucial in delivery DNA into the cytoplasm. Based on other methods, the CNTs‐DNA are a preferred choice and the inclination toward double‐stranded DNA is used over single‐stranded DNA in gene delivery shows effective results. The only downside of CNTs is that they are hydrophobic and are difficult to form an aqueous solution, thus limiting their applicability. This review will aid you in comprehending useful knowledge related to a general overview of topics related to CNTs. [ABSTRACT FROM AUTHOR]

Oncolytic viruses have emerged as new powerful therapeutic agents for cancer therapy by specifically lysing cancer cells while activating innate immune responses at the same time. However, due to the thorny issues of safety concerns and host immune reaction, the clinical application of oncolytic viruses is still limited. Herein, we report a rationally designed oncolytic virus-like nanoparticles (OV-NPs) composed of stimulator of interferon genes (STING)-stimulating polymer loaded with therapeutic genes for cancer immunotherapy. After injection into tumor, the OV-NPs carrying OX40L plasmid could reprogram tumor cells to express OX40L immune checkpoint molecules and activate the STING pathway for cooperatively enhancing antitumor immunity, with a tumor suppression rate of 92.3% in B16F10 tumor model and 78.7% in MC38 tumor model without causing any toxicity. The OV-NPs could be further applied in carrying other plasmids (IL-12) and utilization in gene combination therapy. This study should inspire designing synthetic OV-NPs as alternative strategies for extending oncolytic virus application in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) delivery by extracellular vesicles (EVs) has recently inspired tremendous developments in cancer treatments. However, hybridization between miRNA and its target mRNA is still difficult to be imaged in vivo to assess the therapeutic effects in time. Herein we design a nano-scale fluorescent “off–on” complex encapsulated by small extracellular vesicles (sEVs) for real-time visualization and evaluation of gene therapy efficiency in human gastric cancer cells and murine xenograft tumor models. The complex is formed by π–π stacking between graphene quantum dots (GQDs) and tumor suppressor miR-193a-3p conjugated fluorescent tag whose signals remain off when binding to GQDs. Loaded into sEVs using tunable sonication techniques, the GQDs/Cy5-miR particles enter the tumor cells and promote miR-193a-3p escape from endosomes. The miR-193a-3p in GQDs/Cy5-miR is unleashed to pair the specific target oncogene cyclin D1 (CCND1), therefore turning on the fluorescence of miRNA tags. We find out that GQDs/Cy5-miR@sEVs can activate the “turn-on” fluorescent signal and exhibit the longest retention time in vivo, which suggests a minimized degradation of miR-193a-3p in dynamic processes of miRNA-mRNA binding. More importantly, GQDs/Cy5-miR@sEVs significantly promote cancer apoptosis in vitro and in vivo via the enhanced cellular uptake. Our study demonstrates that GQDs/Cy5-miR@sEVs represent an efficient and refined theranostic platform for gene therapy in cancers. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking immunotherapy for cancer. However, the intricate and costly manufacturing process remains a hurdle. Improving the transduction rate is a potential avenue to cut down costs and boost therapeutic efficiency. Peptide nanofibrils (PNFs) serve as one such class of transduction enhancers. PNFs bind to negatively charged virions, facilitating their active engagement by cellular protrusions, which enhances virion attachment to cells, leading to increased cellular entry and gene transfer rates. While first-generation PNFs had issues with aggregate formation and potential immunogenicity, our study utilized in silico screening to identify short, endogenous, and non-immunogenic peptides capable of enhancing transduction. This led to the discovery of an 8-mer peptide, RM-8, which forms PNFs that effectively boost T cell transduction rates by various retroviral vectors. A subsequent structure-activity relationship (SAR) analysis refined RM-8, resulting in the D4 derivative. D4 peptide is stable and assembles into smaller PNFs, avoiding large aggregate formation, and demonstrates superior transduction rates in primary T and NK cells. In essence, D4 PNFs present an economical and straightforward nanotechnological tool, ideal for refining ex vivo gene transfer in CAR-T cell production and potentially other advanced therapeutic applications. [ABSTRACT FROM AUTHOR]

Hemophilic arthropathy (HA) due to repeated bleeding into the joint cavity is a major cause of morbidity in patients with hemophilia. The molecular mechanisms contributing to this condition are not well characterized. MicroRNAs (miRs) are known to modulate the phenotype of multiple joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Since miR125a is known to modulate disease progression in OA and RA, we performed a targeted screen of miR125a-5p and its target genes in a murine model of chronic HA. A digital PCR analysis demonstrated significant downregulation of miR125a-5p (2-fold vs control joint). Further molecular evaluation revealed elevated expression of the immunological markers STAT1 (7.6-fold vs control joint) and TRAF6 (10.6 fold vs control joint), which are direct targets of miR125a-5p. We then studied the impact of targeted overexpression of miR125a-5p using an Adeno-associated virus (AAV) vector in modulating the molecular mediators of HA. AAV5-miR125a vectors were administered intra-articularly either alone or in combination with a low dose of AAV8-based human factor 8 (F8) gene in a murine model of HA. We observed significantly increased expression of miR125a-5p in AAV5-miR125a administered mice (~12 fold vs injured joint) or in combination with AAV8-F8 vectors (~44 fold vs injured joint). The activity assay revealed ~17 %–20 % FVIII levels in mice that received low dose liver-directed F8 gene therapy. Further immunohistochemical analysis, demonstrated a decrease in inflammatory markers (STAT1 and TRAF6) and cartilage-degrading matrix metalloproteinases (MMPs) 3, 9, 13 in the joints of treated animals. These data highlight the crucial role of miR125a-5p in the development of HA. Targeted profiling and validation by AAV based miR125a delivery identifies the role of miR125a-5p-TRAF6- STAT1 axis, in the sustenance of inflammatory pathophysiology in a chronic hemophilic arthropathy model. Created using Biorender.com. [Display omitted] • miR125a-5p is a potential molecular mediator of hemophilic arthropathy • The molecular mediators, STAT1 and TRAF6, the direct targets of miR125a-5p, regulates MMP activity in the arthritic joints. • AAV based miR125a-5p and F8 vectors alleviates the pathogenesis of HA by downregulation of MMPs 3, 9 and 13. [ABSTRACT FROM AUTHOR]