Efficient Gene Delivery Vectors Research Articles

Mechanism of Cationic Lipid Induced DNA Condensation: Lipid-DNA Coordination and Divalent Cation Charge Fluctuations.

The condensation of nucleic acids by lipids is a widespread phenomenon in biology with crucial implications for drug delivery. However, the mechanisms of DNA assembly in lipid bilayers remain insufficiently understood due to challenges in measuring and assessing each component's contribution in the lipid-DNA-cation system. This study uses all-atom molecular dynamics simulations to investigate DNA condensation in cationic lipid bilayers. Our exhaustive exploration of the thermodynamic factors reveals unique roles for phospholipid head groups and cations. We observed that bridging cations between lipid and DNA drastically reduce charges, while mobile magnesium cations "ping-ponging" between double strands create charge fluctuations. While the first factor stabilizes the DNA-lipid complex, the latter creates attractive forces to induce the spontaneous condensation of DNAs. This novel mechanism not only sheds light on the current data regarding cationic lipid-induced DNA condensation but also provides potential design strategies for creating efficient gene delivery vectors for drug delivery.

GFP chromophore derived amphiphiles containing [12]aneN3 and biotin moieties as bio-imaging and targeting nonviral gene vectors

Gene therapy is a promising treatment, that brings the necessary opportunity to cure human genetic and major diseases, but it is hampered by the lack of safe and efficient gene delivery vectors. In this study, four amphiphiles were derived from GFP chromophore benzylimidazolidinone (G) through modifications with oxypropyl (1) or methylene (2) linked di(triazole-[12]aneN3) (M) in the presence or absence of tumor-targeting biotin (B) moiety, GMB-1/2 and GM-1/2. The four compounds showed excellent aggregation-induced emission (AIE) characteristics, strong DNA condensation abilities, and good biocompatibility. Among them, GMB-1, which containing targeting biotin moiety and a flexible propoxy linker, showed the highest transfection efficiency in three cell lines. The luciferase transfection efficiency of GMB-1 was as high as 15.2 times that of Lipofectamine 2000 (Lipo2000) without auxiliary lipid DOPE, and its gene therapy effect of delivering the p53 gene was also significantly better than that of Lipo2000 in HeLa cells due to its obvious targeting property. Fluorescence tracing experiments indicated that GMB-1 was able to quickly carry DNA into cells, to successfully escape from lysosomes, then to release DNA, and at last to smoothly deliver DNA into the nucleus. Moreover, its EGFP transfection efficiency in zebrafish reached 1.7 times that of Lipo2000. The excellent performance of GMB-1 demonstrated that the appreciative incorporation of GFP chromophore, di(triazole-[12]aneN3), and biotin moieties into the nonviral gene vectors provided a promising strategy for their clinical applications.

Engineered Exosomes with ATF5-Modified mRNA Loaded in Injectable Thermogels Alleviate Osteoarthritis by Targeting the Mitochondrial Unfolded Protein Response.

Osteoarthritis (OA) progression is highly associated with chondrocyte mitochondrial dysfunction and disorders of catabolism and anabolism of the extracellular matrix (ECM) in the articular cartilage. The mitochondrial unfolded protein response (UPRmt), which is an integral component of the mitochondrial quality control (MQC) system, is essential for maintaining chondrocyte homeostasis. We successfully validated the pivotal role of activating transcription factor 5 (ATF5) in upregulating the UPRmt, mitigating IL-1β-induced inflammation and mitochondrial dysfunction, and promoting balanced metabolism in articular cartilage ECM, proving its potential as a promising therapeutic target for OA. Modified mRNAs (modRNAs) have emerged as novel and efficient gene delivery vectors for nucleic acid therapeutic approaches. In this study, we combined Atf5-modRNA (modAtf5) with engineered exosomes derived from bone mesenchymal stem cells (ExmodAtf5) to exert cytoprotective effects on chondrocytes in articular cartilage via Atf5. However, the rapid localized metabolization of ExmodAtf5 limits its application. PLGA-PEG-PLGA (Gel), an injectable thermosensitive hydrogel, was used as a carrier of ExmodAtf5 (Gel@ExmodAtf5) to achieve a sustained release of ExmodAtf5. In vitro and in vivo, the use of Gel@ExmodAtf5 was shown to be a highly effective strategy for OA treatment. The in vivo therapeutic effect of Gel@ExmodAtf5 was evidenced by the preservation of the intact cartilage surface, low OARSI scores, fewer osteophytes, and mild subchondral bone sclerosis and cystic degeneration. Consequently, the combination of ExmodAtf5 and PLGA-PEG-PLGA could significantly enhance the therapeutic efficacy and prolong the exosome release. In addition, the mitochondrial protease ClpP enhanced chondrocyte autophagy by modulating the mTOR/Ulk1 pathway. As a result of our research, Gel@ExmodAtf5 can be considered to be effective at alleviating the progression of OA.

Abstract 15318: A Dene- and Drug-Eluting Peripheral Stent Using a Baculovirus-Eluting PVA Hydrogel Coating

Introduction: Over 20 % of people over 60 have peripheral artery disease (PAD), the narrowing of peripheral arteries due to atherosclerosis. More recent data suggests a surge in PAD in younger people (affecting up to 4 % of people). Atherosclerosis is complex, initially involving fat deposition and endothelial cell dysfunction, leading to inflammation, hyperproliferation of cells, plaque development, and calcification. This can lead to significant pain, discomfort, limb ischemia, stroke, and increases the risk of major cardiovascular events such as myocardial infarction and coronary artery disease. Often a bare-metal or drug-eluting stent will force the artery open. These stents are highly effective but come with complications such as restenosis, thrombosis, and enhanced endothelial dysfunction leading to patency as low as 60 % one-year post-implantation. Hypothesis: A gene- and drug-eluting hydrogel stent coating can promote endothelial recovery while the long-term drug elution prevents restenosis and inflammation, improving the clinical outcome of PAD (Figure 1). Methods & results: The gene encoding nitric oxide had been cloned into a baculovirus, a highly efficient and safe gene delivery vector to arterial cells. Nitric oxide inhibits smooth muscle cell proliferation, promotes endothelial cell proliferation, promotes angiogenesis, and prevents reactive oxygen species production within the diseased artery. The 10-day gene elution has the potential to leave behind a healthy endothelial lining, and the 100-day Everolimus drug elution can prevent restenosis and inflammation. Moreover, the gene- and drug-eluting stent is stable during crimping and expansion, has a high tensile strength, is non-cytotoxic, and is anti-thrombotic by preventing platelet adhesion. Conclusion: Overall, the novel stent exhibits beneficial mechanical and cellular properties with the potential to mitigate current stenting complications using dual gene and drug therapy.

Peptide-Based Vectors for Gene Delivery

Gene therapy is the ultimate therapeutic technology for diseases related to gene abnormality. However, the use of DNA alone has serious problems, such as poor stability and difficulty in entering target cells. The development of a safe and efficient gene delivery system is the cornerstone of gene therapy. Of particular interest, multifunctional peptides are rationally designed as non-viral vectors for efficient gene delivery. As components of gene delivery vectors, these peptides play critically important roles in skeleton construction, the implementation of targeting strategies, cell membrane penetration, endosome rupture, and nuclear transport. In recent years, the research of functional peptide-based gene delivery vectors has made important progress in improving transfection efficiency. The latest research progress and future development direction of peptide-based gene delivery vectors are reviewed in this paper.

Peptide-DNA Co-Assembled Nanoparticles as a Nonviral Vector for Gene Delivery with High Transgene Expression

Nonviral vectors for gene therapy are a safer alternative to viral vectors that elicit a harmful immune response. Artificial viruses combine the strengths of viral and nonviral vectors and have emerged to be safe and efficient gene delivery vectors. Herein, we report the development of peptide–DNA coassembled nanoparticles as a new efficient vector for gene delivery with high transgene efficiency. The key to our success is to introduce several rationally designed functional short peptide modules: (1) a pH-sensitive segment, (2) an endosomal interruption segment, (3) a cell penetration peptide with high positive charges, (4) a nuclear localization signal peptide with positive charges, (5) aromatic groups to tune the hydrophobic/hydrophilic ratio, and (6) redox-sensitive residues. These peptide–DNA coassembled nanoparticles achieved efficient DNA delivery with transfection efficiency over 90% in a serum-containing medium. This work demonstrates the effectiveness of the module-based design approach and lays the groundwork for potential biomedical applications of peptide-based biomaterials.

Hyperbranched Poly(β-amino ester)s (HPAEs) Structure Optimisation for Enhanced Gene Delivery: Non-Ideal Termination Elimination

Many polymeric gene delivery nano-vectors with hyperbranched structures have been demonstrated to be superior to their linear counterparts. The higher delivery efficacy is commonly attributed to the abundant terminal groups of branched polymers, which play critical roles in cargo entrapment, material-cell interaction, and endosome escape. Hyperbranched poly(β-amino ester)s (HPAEs) have developed as a class of safe and efficient gene delivery vectors. Although numerous research has been conducted to optimise the HPAE structure for gene delivery, the effect of the secondary amine residue on its backbone monomer, which is considered the non-ideal termination, has never been optimised. In this work, the effect of the non-ideal termination was carefully evaluated. Moreover, a series of HPAEs with only ideal terminations were synthesised by adjusting the backbone synthesis strategy to further explore the merits of hyperbranched structures. The HPAE obtained from modified synthesis methods exhibited more than twice the amounts of the ideal terminal groups compared to the conventional ones, determined by NMR. Their transfection performance enhanced significantly, where the optimal HPAE candidates developed in this study outperformed leading commercial benchmarks for DNA delivery, including Lipofectamine 3000, jetPEI, and jetOPTIMUS.

An adeno-associated virus vector penetrates the blood-brain barrier in non-human primates.

Developing gene therapy for use in the central nervous system has been hampered by the lack of an efficient vector for gene delivery. We report an adeno-associated virus vector with an enhanced ability to cross the blood-brain barrier in both rodents and non-human primates, and use it to develop systemic anti-tumour gene therapies for glioblastoma.

Bioreducible polyethylenimine core–shell nanostructures as efficient and non-toxic gene and drug delivery vectors

Low molecular weight branched polyethylenimine (LMW bPEIs 1.8 kDa) have received considerable attention for the fabrication of nucleic acid carriers due to their biocompatible and non-toxic nature. However, due to the inadequate nucleic acid complexation ability and transportation across the cell membrane, these show poor transfection efficacy, limiting their clinical applications. Therefore, to overcome these challenges, in this study, we have grafted bPEI 1.8 kDa with a disulfide bond containing hydrophobic moiety, 3-(2-pyridyldithio) propionic acid (PDPA), via amide linkages through EDC/NHS-mediated coupling to obtain N-[3-(2-pyridyldithio)] propionoyl polyethylenimine (PDPP) conjugates. The best formulation for nucleic acid transfection was evaluated after preparing a series of PDPP conjugates by varying the amount of PDPA. In an aqueous environment, these PDPP conjugates self-assembled to form spherical shaped core–shell PDPP nanostructures with size ranging from ∼188–307 nm and zeta-potential from ∼ +3 to +19 mV. The positively charged surface of the core–shell nanocomposites helps in the binding of plasmid DNA (pDNA), its transportation inside the cell, and protection against enzymes. Evaluation of PDPP/pDNA complexes on mammalian cells revealed that all these complexes showed significantly improved transfection efficacy without hampering cytocompatibility. Amongst all, the pDNA complex of PDPP-2 exhibited the best transfection efficiency (i.e. >6-fold) in comparison to pDNA complex of the native bPEI. The nanocomposites exhibited the redox responsive behavior advantageous for therapeutic delivery to the tumor cells. The core of the nanostructures facilitate the encapsulation of a hydrophobic model drug, ornidazole. In vitro drug release analysis showed a faster release rate in response to a reductant mimicking the cellular environment. Altogether, these nanostructures have great potential to co-deliver both drug and gene simultaneously in response to tumor cell reductive microenvironment in vitro and could be used as the next-generation delivery system.

Metallohelix vectors for efficient gene delivery via cationic DNA nanoparticles.

The design of efficient and safe gene delivery vehicles remains a major challenge for the application of gene therapy. Of the many reported gene delivery systems, metal complexes with high affinity for nucleic acids are emerging as an attractive option. We have discovered that certain metallohelices—optically pure, self-assembling triple-stranded arrays of fully encapsulated Fe—act as nonviral DNA delivery vectors capable of mediating efficient gene transfection. They induce formation of globular DNA particles which protect the DNA from degradation by various restriction endonucleases, are of suitable size and electrostatic potential for efficient membrane transport and are successfully processed by cells. The activity is highly structure-dependent—compact and shorter metallohelix enantiomers are far less efficient than less compact and longer enantiomers.

Nicotinic acid-based cationic vectors for efficient gene delivery to glioblastoma cells

A tocopherol-conjugated nicotinic acid-based lipid (NGT) was used for liposomal formation with the co-lipid DOPE and exhibited enhanced transfection of glioblastoma cells for eGFP and β-galactosidase protein expression.

In vitro and in vivo optimization of liposomal nanoparticles based brain targeted vgf gene therapy

Vgf (non-acronymic), a neurotrophin stimulated protein which plays a crucial role in learning, synaptic activity, and neurogenesis, is markedly downregulated in the brain of Alzheimer’s disease (AD) patients. However, since vgf is a large polar protein, a safe and efficient gene delivery vector is critical for its delivery across the blood brain barrier (BBB). This research work demonstrates brain-targeted liposomal nanoparticles optimized for delivering plasmid encoding vgf across BBB and transfecting brain cells. Brain targeting was achieved by surface functionalization using glucose transporter-1 targeting ligand (mannose) and brain targeted cell-penetrating peptides (chimeric rabies virus glycoprotein fragment, rabies virus derived peptide, penetratin peptide, or CGNHPHLAKYNGT peptide). The ligands were conjugated to lipid via nucleophilic substitution reaction resulting in >75% binding efficiency. The liposomes were formed by film hydration technique demonstrating size <200 nm, positive zeta potential (15–20 mV), and polydispersity index <0.3. The bifunctionalized liposomes demonstrated ∼3 pg/µg protein vgf transfection across in vitro BBB, and ∼80 pg/mg protein in mice brain which was 1.5–2 fold (p < 0.05) higher compared to untreated control. The nanoparticles were also biocompatible in vitro and in vivo, suggesting a safe and efficient gene delivery system to treat AD.

Non-invasive gene delivery across the blood-brain barrier: present and future perspectives.

Non-invasive gene delivery across the blood-brain barrier: present and future perspectives.

Evaluation and Optimization of Poly-d-Lysine as a Non-Natural Cationic Polypeptide for Gene Transfer in Neuroblastoma Cells

Cationic polypeptides and cationic polymers have cell-penetrating capacities and have been used in gene transfer studies. In this study, we investigate the capability of a polymer of d-lysine (PDL), a chiral form of α–Poly-lysine, as a possible nonviral vector for releasing genetic materials to neuroblastoma cells and evaluate its stability against proteases. We tested and compared its transfection effectiveness in vitro as a vehicle for the EGFP plasmid DNA (pDNA) reporter in the SH-SY5Y human neuroblastoma, HeLa, and 3T3 cell lines. Using fluorescent microscopy and flow cytometry, we demonstrated high transfection efficiencies based on EGFP fluorescence in SH-SY5Y cells, compared with HeLa and 3T3. Our results reveal PDL as an efficient vector for gene delivery specifically in the SH-SY5Y cell line and suggest that PDL can be used as a synthetic cell-penetrating polypeptide for gene therapy in neuroblastoma cells.

Dual-Functionalized Covalent Triazine Framework Nanosheets as Hierarchical Nonviral Vectors for Intracellular Gene Delivery

Gene therapy is a revolutionary biomedical technique for the treatment of many diseases, and the development of safe and efficient intracellular gene delivery vectors is crucial for successful gene...

Disaccharide-polyethylenimine organic nanoparticles as non-toxic in vitro gene transporters and their anticancer potential

Polyethylenimines (PEIs) have been shown as efficient gene delivery vectors due to their unique properties, however, toxicity as well as non-specific interactions with the tissues/cells because of high charge density have hampered their use in clinical applications. To counter these concerns, here, we have prepared disachharide-PEI organic nanoparticles by mixing PEIs with non-reducing disaccharides, i.e. trehalose (TPONs) and sucrose (SPONs), under mild conditions. The fabricated nanoparticles were complexed with pDNA and size of these complexes was found in the range of ~130–162 nm with zeta potential ~ +8–25 mV. Further evaluation of these nanoparticles revealed that substitution of disaccharides on PEIs successfully augmented cell viability. Transfection efficiency exhibited by these complexes was significantly higher than the unmodified polymer and the standard, Lipofectamine, complexes. Fabrication of organic nanoparticles did not alter the buffering capacity considerably which was found to be instrumental during endosomal escape of the complexes. Among both the series of nanoparticles, trehalose-PEI organic nanoparticles (TPONs) exhibited greater pDNA transportation potential than sucrose-PEI organic nanoparticles (SPONs) which was also established by flow cytometric data, wherein percent cells expressing GFP was higher in case of TP/pDNA complexes as compared to SP/pDNA complexes. Interestingly, TPONs also showed promising anticancer activity on cancer cell lines i.e. Mg63, MCF-7 and HepG2. Overall, the results advocate promising potential of disaccharide-PEI organic nanoparticles as efficient gene delivery agents which can be used effectively in future gene therapy applications along with anti-cancer competence of TPONs.

Development of a nontoxic and efficient gene delivery vector based on histidine grafted chitosan

To achieve biocompatibility and high gene transfection efficiency, developing new cationic polymers with natural functional moieties has been regarded as a promising way. In this work, histidine was grafted on chitosan (CS) to produce CS derivatives (HGCS) for siRNA delivery and the HGCS/siRNA complexes were formed by the self-assembly method. The structures of histidine grafted CS polymers were characterized by FT-IR and XRD. The Acid-base titration assay was used to measure their proton buffering capacities and results indicated HGCS polymers have stronger buffering capacity than unmodified CS. The particle size, zeta potential and the stability of the polymer/siRNA complexes were measured by laser particle size analyzer and gel retardation assay. The cytotoxicity of polymers was determined by MTT assays in RSC96 cells. Moreover, cellular uptake investigation into polymer/siRNA complexes demonstrated that HGCS polymers have efficient siRNA transfection ability in B16F1 cells and HeLa cells. These data suggest HGCS polymers as a hopeful non-viral gene delivery vectors.

Photoluminescent carbon quantum dot/poly-l-Lysine core-shell nanoparticles: A novel candidate for gene delivery

Cationic polymers such as poly-l-lysine (PLL) are able to interact electrostatically with DNA to produce polymeric systems with nanometric diameters due to the neutralization and accumulation of DNA. This study integrates the outstanding properties of carbon quantum dots (CQDs) with PLL to develop a novel gene delivery vehicle with a core-shell hybrid nanostructure. The CQD/PLL core-shell nanoparticles (NPs) were, therefore, synthesized in such a way that they had narrow size distribution and an average diameter under 10 nm, both of which were confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Fourier transform infrared (FTIR) spectroscopy exhibited that the PLL passivation agents were formed on the CQDs through releasing amine groups on their surface. The positive charge of the CQD/PLL core-shell NPs reduced from +15 to nearly zero mV after being loaded with DNA at the weight ratio of 2:1. These traceable, water-soluble, biocompatible, and tunable photoluminescent NPs demonstrated a quantum yield of around 12% and a cellular uptake of nearly 70%. The NPs also showed no considerable toxicity to the human embryonic kidney (HEK)-293T cells. Hence, these novel CQD/PLL core-shell NPs hold great promise as a non-toxic and efficient gene delivery vector.

Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.

Recombinant adeno-associated viruses (rAAVs) are efficient, non-invasive gene delivery vectors via intravenous delivery, however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our previous Cre recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to quickly and accurately identify variants of interest in a given selection landscape through multiple positive and negative selection criteria by incorporating next-generation sequencing, synthetic library generation, and a novel analysis pipeline. In vivo selections for brain endothelial cell-, astrocyte-, and neuron-transducing capsids have identified variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity, or cross the blood-brain barrier across diverse murine strains. Collectively, M-CREATE methodology accelerates the discovery of novel capsids for use in neuroscience and gene therapy applications.

Amino Acid-Linked Low Molecular Weight Polyethylenimine for Improved Gene Delivery and Biocompatibility.

The construction of efficient and low toxic non-viral gene delivery vectors is of great significance for gene therapy. Herein, two novel polycations were constructed via Michael addition from low molecular weight polyethylenimine (PEI) 600 Da and amino acid-containing linkages. Lysine and histidine were introduced for the purpose of improved DNA binding and pH buffering capacity, respectively. The ester bonds afforded the polymer biodegradability, which was confirmed by the gel permeation chromatography (GPC) measurement. The polymers could well condense DNA into nanoparticles and protect DNA from degradation by nuclease. Compared with PEI 25 kDa, these polymers showed higher transfection efficiency, lower toxicity, and better serum tolerance. Study of this mechanism revealed that the polyplexes enter the cells mainly through caveolae-mediated endocytosis pathway; this, together with their biodegradability, facilitates the internalization of polyplexes and the release of DNA. The results reveal that the amino acid-linked low molecular weight PEI polymers could serve as promising candidates for non-viral gene delivery.