Gene Delivery Platform Research Articles

Reactive Oxygen Species (ROS)-Degradable Polymeric Nanoplatform for Hypoxia-Targeted Gene Delivery: Unpacking DNA and Reducing Toxicity.

Smart polymers as ideal gene carriers have drawn increasing attentions due to the effective DNA release once triggered by intrinsic stimuli, as well as reduced cytotoxicity. Herein, a stimulus-responsive, positively charged and water-soluble polymer (OEI-TK x) was facilely engineered by cross-linking low molecular weight oligoethylenimine (OEI) via thioketal (TK) linkages that would cleave selectively in reactive oxygen species (ROS)-rich environments induced by hypoxia. Agarose gel electrophoresis assay demonstrated that the threshold N/P ratios for complete retardation of negatively charged DNA migration were above 5 for OEI-TK x. The reduction in DNA-condensing capability and the changes in particle size, size distribution and particle morphology all illustrated that OEI-TK x possessed excellent ROS responsiveness. OEI-TK x/DNA polyplexes showed lower toxicity and higher gene transfection efficiency compared with PEI/DNA polyplexes. Theoptimum formulation, OEI-TK x/DNA polyplexes (N/P = 40), showed a little better performance than PEI/DNA polyplexes in cellular uptake profile. Furthermore, OEI-TK x/DNA polyplexes could escape from endosomes to the cytosol as efficiently as PEI/DNA polyplexes. Confocal images confirmed that OEI-TK x/DNA polyplexes could more effectively release DNA than PEI/DNA polyplexes, mainly owing to the valid cleavage of thioketal linkages induced by characteristic rich-ROS in Hela cells. These results suggested that OEI-TK x could represent an on-demand stimulus-responsive gene delivery platform.

Novel facile thermosensitive hydrogel as sustained and controllable gene release vehicle for breast cancer treatment

The current research process in gene therapy for cancer treatment has brought much attention due to its great potential for both inherited and acquired diseases. Precise accumulation in target site and on-demand release of drug is critical factors for the efficient gene therapy. Since the delivery of suitable gene largely depends on the delivery carrier, the design of suitable gene delivery vehicle for the sustained gene release in target site are attracting increasingly interest among the researchers. In this report, an effective and relatively convenient gene delivery platform is developed by the electrostatic interaction between negative charged survivin antisense oligonucleotide (Sur-ASON) and positive charged PHB-b-PDMAEMA (PHB-P) co-polymer and then the induction of thermosensitive PF127 hydrogel. The prepared hydrogel could achieve a sustained gene release property in the tumor region after injection, thus to enhance the effect of Survivin antisense oligonucleotide and inhibit P-gp impaired drug uptake simultaneously. In vivo anti-tumor efficacy and H&E staining indicated that Sur-ASON/PHB-P/PF127 hydrogel was greatly effective in enhancing the treatment effects of Sur-ASON while reducing the degradation and the possible adverse side effects, and this novel hydrogel could achieve the controlled gene release up to maximum 16 days. The aforementioned properties indicated that the novel hydrogel could be applied as a promising and convenient anti-cancer agent for anticancer therapy with minimum injection frequency to possibly increase patient compliance.

Delivery of gene therapy to resting immune cells for an HIV cure.

The utilization of genetically modified T cells to therapeutically target to various previously incurable diseases such, as cancer, has expanded exponentially in recent years. This success now provides the motivating force in applying the same technology for incurable infectious diseases including HIV. The common bottleneck in gene therapy continues to be at the level of gene delivery. Although present approaches adapt the cell to the delivery technology, emerging techniques now focus on leaving cells in their phenotypically resting state. In doing so, engraftment and proliferation potential are retained and in turn increase the efficacy of this approach at a lowered cost. This review will outline the main efforts of gene delivery using viral vectors or nonviral vectors and challenges moving forward not only in resting T cells, but also in other resting immune cells including hematopoietic stem cells. In focusing on HIV cure efforts using gene therapy, progress on solving the challenges of gene delivery will be described for both viral and nonviral vectors. Advances in the basic virology of lentiviruses have led to the proposal of many next generation lentiviral vector platforms for resting immune cells. Moreover, we will also highlight the progress made in nonviral approaches using nanotechnology as alternatives and/or synergistic technologies to be used alongside lentiviral platforms. The innovative approaches described in these recent studies, particularly those using the natural mechanisms employed by HIV to enhance for example virus entry or virus latency, will enable future optimization of gene delivery platforms and therapeutics, which will importantly, provide a pathway toward translation into clinical practice.

Adeno-associated virus vector as a platform for gene therapy delivery.

Adeno-associated virus (AAV) vectors are the leading platform for gene delivery for the treatment of a variety of human diseases. Recent advances in developing clinically desirable AAV capsids, optimizing genome designs and harnessing revolutionary biotechnologies have contributed substantially to the growth of the gene therapy field. Preclinical and clinical successes in AAV-mediated gene replacement, gene silencing and gene editing have helped AAV gain popularity as the ideal therapeutic vector, with two AAV-based therapeutics gaining regulatory approval in Europe or the United States. Continued study of AAV biology and increased understanding of the associated therapeutic challenges and limitations will build the foundation for future clinical success.

A TRAIL-Delivered Lipoprotein-Bioinspired Nanovector Engineering Stem Cell-Based Platform for Inhibition of Lung Metastasis of Melanoma.

Genetically engineered mesenchymal stem cells (MSCs), as non-viral gene delivery platforms, are rapidly evolving in tumor therapy due to their low immunogenicity and natural tumor-homing capacity.Methods: In this paper, we selected reconstituted high-density lipoprotein (rHDL), a lipoprotein-bioinspired nanovector with specific binding ability to scavenger receptor B type I (SR-BI) expressed on MSCs, as a transfection agent to genetically modify MSCs. pDNA encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was used as a functional gene to be transfected into the nucleus of MSCs for TRAIL expression. Lauric acid-coupled polyethyleneimine (PEI-LA) as an amphiphilic cationic polymer was synthesized to electrostatically bind to pDNA, and then incorporated into rHDL to form rHDL/PEI-LA/pDNA nanoparticles.Results: The nanoparticles exhibited homogenous particle size and excellent serum stability in vitro. Meanwhile, this SR-BI-targeted rHDL performed efficient intracellular gene delivery, specific lysosome-independent mechanism of cellular uptake and high transfection of pDNA towards MSCs. Moreover, high TRAIL expression in MSCs was detected after rHDL-mediated transfection. In vitro and in vivo results indicated that genetically engineered MSCs could accurately target to B16F10 cells, thereby producing significant apoptosis-inducing effect on aggressive melanoma.Conclusion: TRAIL-expressing MSCs engineered by rHDL nanovector was an efficient and hypotoxic method for stem cells-based pulmonary melanoma metastasis-targeting therapy.

Continuous Vector-free Gene Transfer with a Novel Microfluidic Chip and Nanoneedle Array.

Delivery of foreign cargoes into cells is of great value for bioengineering research and therapeutic applications. In this study, we proposed and established a carrier-free gene delivery platform utilizing staggered herringbone channel and silicon nanoneedle array, to achieve high-throughput in vitro gene transfection. With this microchip, fluidic micro vortices could be induced by the staggered-herringboneshaped grooves within the channel, which increased the contact frequency of the cells with the channel substrate. Transient disruptions on the cell membrane were well established by the nanoneedle array on the substrate. Compared to the conventional nanoneedle-based delivery system, proposed microfluidic chip achieved flow-through treatment with high gene transfection efficiency (higher than 20%) and ideal cell viability (higher than 95%). It provides a continuous processing environment that can satisfy the transfection requirement of large amounts of biological molecules, showing high potential and promising prospect for both basic research and clinical application.

Characterization of abscopal effects of intratumoral electroporation-mediated IL-12 gene therapy

Intratumoral electroporation-mediated IL-12 gene therapy (IT-pIL12/EP) has been shown to be safe and effective in clinical trials, demonstrating systemic antitumor effects with local delivery of this potent cytokine. We recently optimized our IL-12 gene delivery platform to increase transgene expression and efficacy in preclinical models. Here we analyze the immunological changes induced with the new IT-pIL12/EP platform in both electroporated and distant, non-electroporated lesions. IT-pIL12/EP-treated tumors demonstrated rapid induction of IL-12-regulated pathways, as well as other cytokines and chemokines pathways, and upregulation of antigen presentation machinery. The distant tumors showed an increase in infiltrating lymphocytes and gene expression changes indicative of a de novo immune response in these untreated lesions. Flow cytometric analyses revealed a KLRG1hi CD8+ effector T-cell population uniquely present in mice treated with IT-pIL12/EP. Despite being highly activated, this population expressed diminished levels of PD-1 when re-exposed to antigen in the PD-L1-rich tumor. Other T-cell exhaustion markers appeared to be downregulated in concert, suggesting an orchestrated “armoring” of these effector T cells against T-cell checkpoints when primed in the presence of IL-12 in situ. These cells may represent an important mechanism by which local IL-12 gene therapy can induce a systemic antitumor immune response without the associated toxicity of systemic IL-12 exposure.

Facile Gene Delivery Derived from Branched Low Molecular Weight Polyethylenimine by High Efficient Chemistry.

Convenient methods for the preparation of gene delivery platforms based on branched low molecular weight polyethylenimine (PEI) were described. Firstly, PEI lipids, with a low molecular weight PEI headgroup and hexadecyl chain tail group, were prepared through a highly efficient ring-opening reaction of glycidyl hexadecyl ether (EpoxyC16) by amine from PEI. Then, the PEI lipids were used as a component of cationic liposomes and as a surfactant for the preparation of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticle (NP) via solvent extraction/evaporation method. As potential effective gene delivery platforms, their preparation, size, size distribution, toxicities, plasmid DNA loading, in vitro transfection and intracellular trafficking were studied. Both facile platforms showed less toxicity and higher transfection efficacy when compared to high molecular weight PEI in vitro, and may have further versatile applications in the gene delivery field.

Targeting Root Cause by Systemic scAAV9-hIDS Gene Delivery: Functional Correction and Reversal of Severe MPS II in Mice

No treatment is available to address the neurological need and reversibility of MPS II. We developed a scAAV9-hIDS vector to deliver the human iduronate-2-sulfatase gene and test it in mouse model. We treated MPS II mice at different disease stages with an intravenous injection of scAAV9-mCMV-hIDS at different doses. The treatments led to rapid and persistent restoration of IDS activity and the reduction of glycosaminoglycans (GAG) throughout the CNS and somatic tissues in all cohorts. Importantly, the vector treatment at up to age 6 months improved behavior performance in the Morris water maze and normalized the survival. Notably, vector treatment at age 9 months also resulted in persistent rIDS expression and GAG clearance in MPS II mice, and the majority of these animals survived within the normal range of lifespan. Notably, the vector delivery did not result in any observable adverse events or detectable systemic toxicity in any treated animal groups. We believe that we have developed a safe and effective gene therapy for treating MPS II, which led to recent IND approval for a phase 1/2 clinical trial in MPS II patients, further supporting the extended potential of the demonstrated systemic rAAV9 gene delivery platform for broad disease targets.

Histidine and arginine conjugated starch-PEI and its corresponding gold nanoparticles for gene delivery

The high surface energy of gold nanoparticles (GNPs) along with its unique physical and chemical properties delivers it as an effective nonviral gene delivery platform for anticancer treatments. GNPs were synthesized by using starch-PEI in which PEI act as reducing agent and starch as a stabilizer. Cytocompatibility studies carried out in C6 cells revealed that gold modification significantly improved the percentage cell viability even at higher polymer concentration. Irrespective of excellent cellular internalization, the transfection efficiency studied with p53 plasmid was found to be compromised with gold modification. For better transfection efficiency, we further modified starch-PEI with amino acids (l-Arginine, l-Histidine) and synthesized the corresponding GNPs. Though starch-PEI gold nanoparticles exhibited low transfection, its amino acid modified counterparts were found to have good transfection efficiency along with low cytotoxicity. It was found that the GNPs containing l-Arginine showed improved transfection efficiency. Hence, it can be inferred that selective amino acid modification is beneficial for improving the transfection efficiency.

IL-10 Gene Transfection in Primary Endothelial Cells via Linear and Branched Poly(β-amino ester) Nanoparticles Attenuates Inflammation in Stimulated Macrophages

Poly(β-amino esters) or PBAEs are highly efficient synthetic polymers optimized for gene delivery, a complicated process dependent on polymer properties such as hydrophobicity, charge, and degradability. The modular design of PBAEs has allowed for the identification of which polymer and nanoparticle properties significantly affect gene delivery efficiency in various cell types. However, these investigations need to be extended to more difficult-to-transfect cells such as primary endothelial cells, which hold enormous potential for atherosclerosis. Here a small library of 6 different PBAEs were screened for efficacy and safety in two types of primary endothelial cells (ECs). Nearly all polymers were more efficient than commercial transfection reagents (p < 0.05), reaching 60% and 15% transfection efficiency in human and mouse primary ECs, respectively. The top performing PBAE was used to deliver a plasmid encoding the anti-inflammatory cytokine interleukin-10 (IL-10), which has the potential to reduce inflammation in atherosclerosis. Significant increases in IL-10 mRNA and protein were detectable in ECs 72 h after transfection with PBAE:IL-10 nanoparticles. Macrophages cultured in conditioned medium from IL10-transfected ECs showed activation of anti-inflammatory signaling pathways. In addition, these macrophages secreted significantly less (25%) tumor necrosis factor α (TNFα) when challenged with lipopolysaccharide (LPS). These results underline the capabilities of PBAEs to be expanded as a fine-tunable platform for anti-inflammatory gene delivery within the context of atherosclerosis.

Enhancement of adenovirus infection and adenoviral vector-mediated gene delivery by bromodomain inhibitor JQ1

Adenovirus-based vectors are among the most commonly used platforms for gene delivery and gene therapy studies. One of the obstacles for potential application is dose-related toxicity. We show here that adenovirus infection and Ad-mediated gene delivery can be enhanced by inhibitors of bromodomain and extra-terminal (BET) family proteins. We showed that JQ1, but not its inactive enantiomer (−)-JQ1, dose-dependently promoted Ad infection and Ad-mediated gene delivery in both epithelial and lymphocyte cells. Given orally, JQ1 also enhanced transgene expression in a murine tumor model. Inhibitors of histone deacetylases (HDACi) are among the commonly reported small molecule compounds which enhance Ad-mediated gene delivery. We found that JQ1 treatment did not cause histone acetylation nor expression of Ad attachment receptor CAR. Instead, JQ1 treatment induced an increase in BRD4 association with CDK9, a subunit of P-TEFb of transcription elongation. Concurrently, we showed that CDK9 inhibition blocked Ad infection and JQ1 enhancement on the infection. The study exemplifies the potentials of BET inhibitors like JQ1 in oncolytic virotherapy.

Dual functionalized liposome-mediated gene delivery across triple co-culture blood brain barrier model and specific in vivo neuronal transfection

Gene therapy has become a promising approach for neurodegenerative disease treatment, however there is an urgent need to develop an efficient gene carrier to transport gene across the blood brain barrier (BBB). In this study, we strategically designed dual functionalized liposomes for efficient neuronal transfection by combining transferrin (Tf) receptor targeting and enhanced cell penetration utilizing penetratin (Pen). A triple cell co-culture model of BBB confirmed the ability of the liposomes to cross the barrier layer and transfect primary neuronal cells. In vivo quantification of PenTf-liposomes demonstrated expressive accumulation in the brain (12%), without any detectable cellular damage or morphological change. The efficacy of these nanoparticles containing plasmid β-galactosidase in modulating transfection was assessed by β-galactosidase expression in vivo. As a consequence of accumulation in the brain, PenTf-liposomes significantly induced gene expression in mice. Immunofluorescence studies of brain sections of mice after tail vein injection of liposomes encapsulating pDNA encoding GFP (pGFP) illustrate the superior ability of dual-functionalized liposomes to accumulate in the brain and transfect neurons. Taken together, the multifunctional liposomes provide an excellent gene delivery platform for neurodegenerative diseases.

Zinc Coordination Substitute Amine: A Noncationic Platform for Efficient and Safe Gene Delivery.

Amines have been extensively involved in vector design thus far, however, their clinical translation has been impeded by several obstacles: cytotoxicity, polyplex serum instability and low efficacy in vivo. In pursuit of functional groups to substitute amines in vector design to address these disadvantages is of great significance. Herein, we report well-tailored noncationic copolymers that contain hydrophilic, hydrophobic, and zinc coordinative moieties through reversible addition-fragmentation chain transfer (RAFT) polymerization for efficient and safe gene delivery. These polymers are capable of condensing DNA, enabling the formation of uncharged polyplexes. Especially, the zinc coordinative ligand can simultaneously benefit strong DNA binding, robust cellular uptake, efficacious endosomal destabilization, low cytotoxicity, and avoidance of serum protein adsorption. The coordinative module holds great promise to substitute amines and inspires the development of next-generation gene vectors. More importantly, the coordinative copolymers illuminate the possibility and potential of noncationic gene delivery systems for clinical applications.

Incorporation of Polycaprolactone to Cyclodextrin‐Based Nanocarrier for Potent Gene Delivery

AbstractStability of polyplex and safety are key factors to achieve stable gene transfection and high transfection efficiency. In this report, a star‐like amphiphilic biocompatible cyclodextrin‐poly(ε‐caprolactone)‐poly(2‐(dimethylamino) ethyl methacrylate), β‐CD‐g‐(PCL‐b‐PDMAEMA) x copolymer, consisting of biocompatible cyclodextrin core, biodegradable and stable poly(ε‐caprolactone) PCL segments, cationic and hydrophilic PDMAEMA blocks, is synthesized to achieve high efficiency of gene transfection with enhanced stability, due to the micelle formation by hydrophobic PCL segments. In comparison with polyethylenimine (PEI‐25k), a golden standard for nonviral vector gene delivery, this copolymer shows higher encapsulated plasmid desoxyribose nucleic acid (pDNA) ability and the persistence of transgene expression. More interestingly, this gene delivery platform by β‐CD‐g‐(PCL‐b‐PDMAEMA) x shows lower toxicity but better gene transfection efficiency at low N/P ratios, indicating high potential in gene therapy applications.

MicroRNA-Regulated Gene Delivery Systems for Research and Therapeutic Purposes.

Targeted gene delivery relies on the ability to limit the expression of a transgene within a defined cell/tissue population. MicroRNAs represent a class of highly powerful and effective regulators of gene expression that act by binding to a specific sequence present in the corresponding messenger RNA. Involved in almost every aspect of cellular function, many miRNAs have been discovered with expression patterns specific to developmental stage, lineage, cell-type, or disease stage. Exploiting the binding sites of these miRNAs allows for construction of targeted gene delivery platforms with a diverse range of applications. Here, we summarize studies that have utilized miRNA-regulated systems to achieve targeted gene delivery for both research and therapeutic purposes. Additionally, we identify criteria that are important for the effectiveness of a particular miRNA for such applications and we also discuss factors that have to be taken into consideration when designing miRNA-regulated expression cassettes.

Biodegradable Polymers Grafted onto Multifunctional Mesoporous Silica Nanoparticles for Gene Delivery

Biodegradable polymer possesses significant potential for applications in different fields, since flexibility gives rise to materials with great physical and mechanical property diversity. The poly-caprolactone (PCL) and chitosan derivatives (CS) have the ability to form scaffolds, which adhere to the surface of mesoporous silica nanoparticles (MSNs) and its porous networks. The novel characteristics of the developed PCL/MSNs and CS/MSNs, such as very low in vivo degradation rate, ordered pore network, uniform and tunable size and shape of the particles, high pore volume and surface area, non-toxicity, and biocompatibility, among others, are responsible for its favorable gene delivery device and makes this conjugation a very good biomaterial for this application. In the present study, we investigated the synthesis of silica nanoparticles MCM-41 covalently grafted with PCL and CS and their use as a potential small interfering RNA (siRNA) carrier. The physical–chemical and morphological characterizations, as well as the applicability of functionalized MSNs as platforms for gene delivery, were assessed. Our results confirmed that MSNs that were successfully functionalized with PCL and CS kept their typical morphology and pore arrangement. Furthermore, their surface modification was successfully held. In vitro biocompatibility and cytotoxicity assays suggest the ability of MSNs to support passive uptake and indicated the potential of this material as a gene delivery system for cervical cancer cells (HeLa).

An ultrasonic nanobubble-mediated PNP/fludarabine suicide gene system: A new approach for the treatment of hepatocellular carcinoma.

ObjectiveThe purpose of this study is to generate an ultrasonic nanobubble (NB)-mediated purine nucleoside phosphorylase (PNP)/fludarabine suicide gene system for the treatment of human hepatocellular carcinoma (HCC).MethodsNBs were prepared from a mixture the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), perfluoropropane gas and other materials using the high shear dispersion method. NBs treated with ultrasound irradiation functioned as a gene-transfer system, and a self-constructed suicide gene expression plasmid, pcDNA3.1(+)/PNP, treated with fludarabine functioned as a therapeutic gene. This system was used to determine the cytotoxic effects of PNP/fludarabine on HepG2 cells and SMMC7721 cells.Results1. NBs with a small diameter (208–416 nm) and at a high concentration and fine homogeneity were prepared under the optimal method. 2. The pcDNA3.1(+)/PNP plasmid was efficiently transfected into HCC cells using ultrasonic NBs. 3. At 0.75μg/ml fludarabine, PNP/fludarabine showed marked cytotoxic effects toward HepG2 and SMMC7721 cells. PNP/fludarabine achieved the same effect against both SMMC7721 and HepG2 cells but at a lower concentration of fludarabine for the latter. 4. Bystander effects: a 10–20% decrease in the cell survival rate was observed when only 5–10% of transfected cells were PNP positive.ConclusionsNBs constitute a non-toxic, stable and effective gene-delivery platform. The PNP/fludarabine suicide gene system inhibited the growth of HCC cells, induced HCC cell apoptosis, and caused a notable bystander effect at a low fludarabine concentration. This study establishes an important new method for miniaturizing microbubbles and improving a new NB-mediated approach for gene therapy of HCC.

Slow Infusion of Recombinant Adeno-Associated Viruses into the Mouse Cerebrospinal Fluid Space.

Recombinant adeno-associated viruses (rAAVs) are the leading in vivo gene delivery platform, and have been extensively studied in gene therapy targeting various tissues, including the central nervous system (CNS). A single-bolus rAAV injection to the cerebrospinal fluid (CSF) space has been widely used to target the CNS, but it suffers from several drawbacks, such as leakage to peripheral tissues. Here, a protocol is described using an osmotic pump to infuse rAAV slowly into the mouse CSF space. Compared to the single-bolus injection technique, pump infusion can lead to higher CNS transduction and lower transduction in the peripheral tissues.

Bioinspired Star-Shaped Poly(l-lysine) Polypeptides: Efficient Polymeric Nanocarriers for the Delivery of DNA to Mesenchymal Stem Cells.

The field of tissue engineering is increasingly recognizing that gene therapy can be employed for modulating in vivo cellular response thereby guiding tissue regeneration. However, the field lacks a versatile and biocompatible gene delivery platform capable of efficiently delivering transgenes to mesenchymal stem cells (MSCs), a cell type often refractory to transfection. Herein, we describe the extensive and systematic exploration of three architectural variations of star-shaped poly(l-lysine) polypeptide (star-PLL) with varying number and length of poly(l-lysine) arms as potential nonviral gene delivery vectors for MSCs. We demonstrate that star-PLL vectors are capable of self-assembling with pDNA to form stable, cationic nanomedicines. Utilizing high content screening, live cell imaging, and mechanistic uptake studies we confirm the intracellular delivery of pDNA by star-PLLs to MSCs is a rapid process, which likely proceeds via a clathrin-independent mechanism. We identify a star-PLL composition with 64 poly(l-lysine) arms and five l-lysine subunits per arm as a particularly efficient vector that is capable of delivering both reporter genes and the therapeutic transgenes bone morphogenetic protein-2 and vascular endothelial growth factor to MSCs. This composition facilitated a 1000-fold increase in transgene expression in MSCs compared to its linear analogue, linear poly(l-lysine). Furthermore, it demonstrated comparable transgene expression to the widely used vector polyethylenimine using a lower pDNA dose with significantly less cytotoxicity. Overall, this study illustrates the ability of the star-PLL vectors to facilitate efficient, nontoxic nucleic acid delivery to MSCs thereby functioning as an innovative nanomedicine platform for tissue engineering applications.