Non-viral Gene Therapy Research Articles

Dipeptide-functionalized polyamidoamine dendrimer-mediated apoptin gene delivery facilitates apoptosis of human primary glioma cells

Glioblastoma multiform (GBM) is the most frequent and aggressive form of brain tumors in adults. However, the development of more efficient and safe nonviral vector gene therapy represents a promising therapeutic approach, using a tumor-specific killer gene, named apoptin. In this study, we describe the efficacy of non-viral gene delivery vectors, the amino acid-conjugated PAMAM derivatives (PAMAM-H-R and PAMAM-H-K) in delivering a therapeutic gene, displaying affinity toward human primary glioma cells (GBL-14 cells) and dermal fibroblasts. We analyzed transfection efficiency, using luciferase (Luci) and a pDNA encoding for enhanced fluorescent protein (EGFP), and cytotoxicity in both cells. The results show that transfection efficiency of PAMAM-H-R improved compared to native PAMAM dendrimer, but cytotoxicity of PAMAM-H-R and PAMAM-H-K were very low. We treated both cells with a polyplex formation of PAMAM-H-R or PAMAM-H-K/apoptin, and analyzed their cellular uptake and localization by flow cytometry and confocal microscopy. Furthermore, we analyzed the endosomal escape effect using TEM images, and found that PAMAM-H-R showed very fast escape from endosome to the cytosol. Caspase 3 activity assay, cell cycle distribution, and JC-1 analysis showed apoptosis induced by apoptin in GBL-14 cells. This indicates that PAMAM-H-R can be a potential nonviral vector gene delivery carrier for brain tumor therapy. The present study demonstrates that PAMAM-H-R/apoptin gene polyplex can be used as an effective therapeutic candidate for GBM due to its selective induction of apoptosis in primary glioma cells as a potential nonviral gene delivery carrier for brain tumor therapy.

Different strategies of gene delivery for treatment of cancer and other disorders

Gene therapy is the gene transfer into host cells for treatment of acquired and genetic disorders. For this purpose, there are awide variety of gene delivery methods with special properties including viral and non-viral vectors. The non-viral methods usephysical forces or chemical compounds (natural or synthetic) to transfer DNA into a cell. The efficiency of the non-viral genetherapy depends on conquering four different intra- and extra-cellular barriers such as cellular uptake, endosomal escape, nuclearentry, and gene expression. Among various gene carriers, some viral vectors such as Adenovirus, Lentivirus, Vaccinia as well asgene gun and lipofection achieved to clinical trials. In this mini-review, we briefly describe different approaches for gene deliveryand their applications in various phases of clinical trials.

Neuroprotective Effect of Non-viral Gene Therapy Treatment Based on Tetanus Toxin C-fragment in a Severe Mouse Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a hereditary childhood disease that causes paralysis and progressive degeneration of skeletal muscles and spinal motor neurons. SMA is associated with reduced levels of full-length Survival of Motor Neuron (SMN) protein, due to mutations in the Survival of Motor Neuron 1 gene. Nowadays there are no effective therapies available to treat patients with SMA, so our aim was to test whether the non-toxic carboxy-terminal fragment of tetanus toxin heavy chain (TTC), which exhibits neurotrophic properties, might have a therapeutic role or benefit in SMA. In this manuscript, we have demonstrated that TTC enhance the SMN expression in motor neurons “in vitro” and evaluated the effect of intramuscular injection of TTC-encoding plasmid in the spinal cord and the skeletal muscle of SMNdelta7 mice. For this purpose, we studied the weight and the survival time, as well as, the survival and cell death pathways and muscular atrophy. Our results showed that TTC treatment reduced the expression of autophagy markers (Becn1, Atg5, Lc3, and p62) and pro-apoptotic genes such as Bax and Casp3 in spinal cord. In skeletal muscle, TTC was able to downregulate the expression of the main marker of autophagy, Lc3, to wild-type levels and the expression of the apoptosis effector protein, Casp3. Regarding the genes related to muscular atrophy (Ankrd1, Calm1, Col19a1, Fbox32, Mt2, Myod1, NogoA, Pax7, Rrad, and Sln), TTC suggest a compensatory effect for muscle damage response, diminished oxidative stress and modulated calcium homeostasis. These preliminary findings suggest the need for further experiments to depth study the effect of TTC in SMA disease.

Optimizing a Method for the Quantification by Quantitative Real-Time Polymerase Chain Reaction of Host Cell DNA in Plasmid Vector Batches Used in Human Gene Therapy.

Gene therapy products are very complex advanced therapy medicinal products produced using different processes that require many chemical and biological reagents and production intermediates, such as producing cells. The quantification of residual impurities in gene therapy vectors is a major quality control step when these vectors are used for therapeutic purposes, whether or not they are derived from viruses. Indeed, in nonviral gene therapy products, particularly plasmid vectors used to transfer genetic material, the presence of host-cell DNA (HCDNA) from the bacterial cells used for the vector production is an important concern because of the risk of immunogenicity and insertional mutagenesis. Several methods have been developed to quantify residual HCDNA, but real-time quantitative polymerase chain reaction (qPCR) seems to be most suitable because it allows detecting traces of "contaminating" DNA. The French National Agency for Medicines and Health Products Safety (ANSM) ensures the quality and safety of gene transfer medicinal products and must be able to quantify, in its own laboratories, the amount of HCDNA present in plasmid vector batches. Therefore, we developed and validated a qPCR method to quantify at the femtogram level the presence of Escherichia coli residual DNA in plasmid vectors. This approach uses the capillary-based LightCycler 1.5 System (Roche) with SYBR Green I, a primer pair against the E. coli 23S ribosomal RNA gene and different concentrations of a linearized plasmid that contains the 23S target sequence, as standard. This qPCR method is linear on an 8-decade logarithmic scale, accurate, reproducible, and sensitive (quantification of up to 10 copies of 23S target sequence per reaction, or 1.4 E. coli genome, or 7 fg of bacterial DNA). This technique allows ensuring that batches of plasmid vectors to be used in clinical trials comply with the specifications on HCDNA content.

Highly branched poly(β-amino ester)s for skin gene therapy

Poly(β-amino ester)s (PAEs) have emerged as a promising class of gene delivery vectors with performances that can even be compared to viruses. However, all of the transfection studies (over 2350 PAEs) have been limited to linear poly(β-amino ester)s (LPAEs) despite increasing evidence that polymer structure significantly affects performance. Herein, we describe the development of highly branched poly(β-amino ester)s (HPAEs) via a new “A2+B3+C2” Michael addition approach demonstrating 2 to 126-fold higher in vitro transfection efficiencies of different cell types in comparison to their linear LPAE counterparts as well as greatly out-performing the leading transfection reagents SuperFect and the “gold-standard” polyethyleneimine (PEI) - especially on skin epidermal cells. More importantly, the ability to correct a skin genetic defect is demonstrated in vivo utilizing a recessive dystrophic epidermolysis bullosa (RDEB) knockout mouse model. Our results provide evidence that the “A2+B3+C2” approach can be controlled and offers sufficient flexibility for the synthesis of HPAEs. The branched structures can significantly improve the transfection efficiency and safety of PAEs highlighting the great promise for the successful application of non-viral gene therapy in skin disease.

Behavioral assessment of neuropathic pain, fatigue, and anxiety in experimental autoimmune encephalomyelitis (EAE) and attenuation by interleukin-10 gene therapy

Relapsing-remitting multiple sclerosis is commonly associated with motor impairments, neuropathic pain, fatigue, mood disorders, and decreased life expectancy. However, preclinical pharmacological studies predominantly rely on clinical scoring of motor deficit as the sole behavioral endpoint. Thus, the translational potential of these studies is limited. Here, we have assessed the therapeutic potential of a novel anti-inflammatory interleukin-10 (IL-10) non-viral gene therapy formulation (XT-101-R) in a rat relapsing remitting experimental autoimmune encephalomyelitis (EAE) model. EAE induced motor deficits and neuropathic pain as reflected by induction of low-threshold mechanical allodynia, suppressed voluntary wheel running, decreased social exploration, and was associated with markedly enhanced mortality. We also noted that voluntary wheel running was depressed prior to the onset of motor deficit, and may therefore serve as a predictor of clinical symptoms onset. XT-101-R was intrathecally dosed only once at the onset of motor deficits, and attenuated each of the EAE-induced symptoms and improved survival, relative to vehicle control. This is the first pharmacological assessment of such a broad range of EAE symptoms, and provides support for IL-10 gene therapy as a clinical strategy for the treatment of multiple sclerosis.

Synthesis of intracellular reduction-sensitive amphiphilic polyethyleneimine and poly(ε-caprolactone) graft copolymer for on-demand release of doxorubicin and p53 plasmid DNA

This study aims to present a new intelligent polymeric nano-system used for combining chemotherapy with non-viral gene therapy against human cancers. An amphiphilic copolymer synthesized through the conjugation of low molecular weight polyethyleneimine (LMw-PEI) and poly(ε-caprolactone) (PCL) via a bio-cleavable disulfide linkage was successfully employed for the simultaneous delivery of drug and gene molecules into target cells. Compared to the conventional PCL copolymerization pathway, this paper represents a straightforward and efficient reaction pathway including the activation of PCL-diol hydroxyl end groups, cystamine attachment and LMw-PEI conjugation which are successfully performed at mild conditions as confirmed by FTIR and 1H NMR. Thermal, morphological characteristics as well as biocompatibility of the copolymer were investigated. The copolymer showed great tendency to form positively charged nanoparticles (∼163.1nm, +35.3mV) with hydrophobic core and hydrophilic shell compartments implicating its potential for encapsulation of anti-cancer drug and plasmid DNA, respectively. The gel retardation assay confirmed that the nanoparticles could successfully inhibit the migration of pDNA at ∼5 nanoparticle/pDNAw/w. The in vitro cytotoxicity tests and LDH assay revealed that the cationic amphiphilic copolymer was essentially non-toxic in different carcinoma cell lines in contrast to branched PEI 25K. Moreover, the presence of redox sensitive disulfide linkages provided smart nanoparticles with on-demand release behavior in response to reducing agents such as cytoplasmic glutathione (GSH). Importantly, confocal microscopy images revealed that in contrast to free Dox, the nanoparticles were capable of faster internalizing into the cells and accumulating in the perinuclear region or even in the nucleus. Finally, the co-delivery of Dox and p53-pDNA using the copolymer displayed greater cytotoxic effect compared with the Dox-loaded nanoparticle counterpart as revealed by cell viability and Caspase 3 expression assay. These results suggest the copolymer as a promising candidate for the development of smart delivery systems. Statement of SignificanceWe employed cystamine dihydrochloride as a disulfide linkage for the conjugation of PCL diol and low molecular weight PEI segments through a straightforward and efficient reaction pathway at mild conditions. The new copolymer was essentially non-toxic in different carcinoma cell lines and showed great tendency to form positively charged nanoparticles. Therefore, it can be utilized as a promising platform for simultaneous drug and gene delivery to aggressive cancers. The results of drug and gene co-delivery experiments confirmed the pivotal role of disulfide linkage on the controlled release of both drug and gene molecules in response to glutathione concentration gradient between extracellular and intracellular microenvironments. In addition, the co-delivery of doxorubicin and p53 plasmid DNA using the new copolymer displayed greater cytotoxic effect compared with single agent (i.e. Dox) loaded counterpart, which indicated the significance of rapid dissociation of therapeutic agents from the carrier for synergistic cytotoxic effects on cancer cells.

534. Preparation for a First-in-Man Lentivirus Trial in Cystic Fibrosis Patients

Background: We have recently shown that non-viral gene therapy can stabilise the decline of lung function in cystic fibrosis (CF) patients. However, the effect was modest, and it is important to develop more potent gene transfer agents in parallel. F/HN-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in pre-clinical models. In preparation for a first-in-man CF trial using the lentiviral vector we have undertaken key translational pre-clinical studies. Methods: Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air liquid interface cultures to select the lead candidate; CFTR expression and function were assessed in CF models (knockout mice and human intestinal organoids) using this lead candidate vector. Toxicity was assessed and “benchmarked” against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. Results: A hybrid promoter consisting of the elongation factor 1α promoter and the CMV enhancer was most efficacious in both murine lungs and human air liquid interface cultures. The efficacy, toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector is stable in clinically relevant delivery devices. Conclusions: The data support progression of the F/HN pseudotyped lentiviral vector into a first-in-man CF trial due to start in Q2 2017. Regulatory-compliant toxicology studies are currently being performed.

Bioreducible-Cationic Poly(amido amine)s for Enhanced Gene Delivery and Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

The development of efficient and safe gene delivery carriers has been a major challenge in the clinical application of non-viral gene therapy. Herein, we report novel bioreducible poly(amido amine)s for the efficient delivery of genetic material such as plasmid DNA. A library of 34 different bioreducible polymer compounds was synthesized and screened to find lead materials for in vitro gene transfection. Our lead material (CBA-106) allows effortless polyplex formation with genetic materials by electrostatic interactions at the weight ratio of 1:5 (DNA/polymer). Polyplexes were further characterized by DLS and AFM analysis. Enhanced serum stability and bioreducibility under physiological conditions were confirmed, in addition to low cellular cytotoxicity. When compared with a commercially available gene delivery carrier (Lipofectamine 2000), CBA-1 06 shows comparable or even surpassing gene transfection efficiency. Furthermore, BMP-2 plasmids were efficiently delivered to tonsil-derived mesenchymal stem cells (TMSCs) for osteogenic commitment in vitro and in vivo. Taken together, our results clearly demonstrate the potential of novel bioreducible polymeric systems for gene delivery applications. We suggest that our system can provide a valuable platform for the broad application of gene regulation in cell therapy and regenerative medicine.

439. Prolonged Expression of Secreted Enzymes in Dogs After Liver Delivery of Sleeping Beauty Transposons: Implications for Non-Viral Gene Therapy of Mucopolysaccharidoses Types I and VII

The non-viral integrating vector the Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic diseases in mice including mucopolysaccharidosis (MPS) types I and VII caused by α-iduronidase (IDUA) and β-glucuronidase (GUSB) deficiency, respectively. More recently we have used modified approaches of the hydrodynamic procedure to deliver therapeutic transposons to dog liver. Reproducible delivery and transposition in dogs are about 1% the levels in mice. Using a transgenic canine reporter secreted alkaline phosphatase (cSEAP), in the absence of immune suppression we can detect transgenic protein for up to six weeks post infusion using catheter-mediated hydrodynamic delivery. A proof-of-principle immunomodulation using GdCl3 to block macrophages in liver and spleen prolonged the presence of the cSEAP protein in circulation from 6 weeks to up to at least 5 months after a single vector infusion. We achieved stabilized activity in one dog at about 2-fold of baseline values. Durability of cSEAP in serum was inversely correlated with transient increase of liver enzymes ALT and AST in response to the vector delivery procedure, pointing to the deleterious effect of hepatocellular toxicity on transgene maintenance. However, GdCl3 immunomodulation was ineffective for repeat vector infusions, suggesting a possibility of an alternative, more potent immunosuppression regimen. Evidence of transposition was obtained with the most efficient transposase SB100X but not with SB11. For transgenic IDUA and GUSB, therapeutic activity in serum peaked at 50-350% of wild-type at 2-4 days post-treatment, but lasted only a few days. The differences in levels and duration of detection of cSEAP in the blood compared to those of IDUA and GUSB may be in part due to the facilitated uptake of lysosomal enzymes into cells compared to cSEAP. Longer endurance of transgenic proteins at therapeutic levels may be possible in SB-treated dogs using alternative immunosuppressive regimens.

628. Transposons Expressing Full-Length Human Dystrophin Enable Genetic Correction of Dystrophic Mesoangioblasts and iPS-Derived Mesoangioblast-Like Cells

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. Alternatively, dystrophic mesoangioblast-like cells were generated from iPS of DMD patients. These iPS-derived mesoangioblasts, constitute an essentially unlimited supply of stem/progenitor cells that could be genetically corrected using PB transposons expressing dystrophin. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.

Comparison of two silica based nonviral gene therapy vectors for breast carcinoma: evaluation of the p53 delivery system in Balb/c mice

Silica nanoparticles as a nonviral vector for in vivo gene therapy neither surface functionalized SiNp1 is neither “a cationic ion” nor a surface (encapsulation) nor SiNp2 (adsorption). p53 gene expression in the breast upon (i.v) administration. SiNp1 showed a 50- and 100-fold transfection activity, tumor growth inhibition, animal survival (80%), and high levels of p53 and Bax were detected in the sera of treated animals compared to SiNp2 or naked pCMV/p53, respectively. These results demonstrate for improvements in the both systems. This study suggests that nonviral vector systems will have important roles in achieving the impermanent gene transfer in vivo.

Gene therapy as a potential tool for treating neuroblastoma-a focused review.

Neuroblastoma, a solid tumor caused by rapid division of undifferentiated neuroblasts, is the most common childhood malignancy affecting children aged <5 years. Several approaches and strategies developed and tested to cure neuroblastoma have met with limited success due to different reasons. Many oncogenes are deregulated during the onset and development of neuroblastoma and thus offer an opportunity to circumvent this disease if the expression of these genes is restored to normalcy. Gene therapy is a powerful tool with the potential to inhibit the deleterious effects of oncogenes by inserting corrected/normal genes into the genome. Both viral and non-viral vector-based gene therapies have been developed and adopted to deliver the target genes into neuroblastoma cells. These attempts have given hope to bringing in a new regime of treatment against neuroblastoma. A few gene-therapy-based treatment strategies have been tested in limited clinical trials yielding some positive results. This mini review is an attempt to provide an overview of the available options of gene therapy to treat neuroblastoma.

Systematically probing the bottom-up synthesis of AuPAMAM conjugates for enhanced transfection efficiency

BackgroundGold nanoparticles (AuNPs) have shown great promise as scaffolds for gene therapy vectors due to their attractive physiochemical properties which include biocompatibility, ease of functionalization via the nearly covalent gold-sulfur dative bond, and surface plasmon optical properties. Previously, we synthesized stable AuNP-polyamidoamine (AuPAMAM) conjugates and showed their success in vitro as non-viral gene delivery vectors.ResultsIn this study, we systematically perturbed each component of the AuPAMAM conjugates and analyzed the resulting effect on transfection efficiency. Due to the modular, bottom-up nature of the AuPAMAM synthesis, we were able to probe each step of the fabrication process. The relationship between each conjugation parameter and the function of the final vector were investigated. More than fourfold enhanced transfection efficiency was achieved by modifying the PAMAM concentration, PAMAM core chemistry, PAMAM terminus chemistry, and self-assembled monolayer composition of the AuPAMAM conjugates.ConclusionsThis work suggest that AuPAMAM synthesis platform is a promising non-viral gene therapy approach and highlights the importance of inspecting the role of each individual constituent in all nanotechnology hybrid materials.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0178-9) contains supplementary material, which is available to authorized users.

Transgene sequences free of CG dinucleotides lead to high level, long-term expression in the lung independent of plasmid backbone design

Non-viral aerosol gene therapy offers great potential for treating chronic lung diseases of the airways such as cystic fibrosis (CF). Early clinical trials showed that transgene expression in the airways was transient whereas maximal duration of transgene expression is essential in order to minimise the frequency of aerosol treatments. Improved vector design, such as careful selection of the promoter/enhancer, can lead to more persistent levels of transgene expression, but multiple factors affect expression in vivo. Following aerosol delivery to the lungs of mice, we measured reporter gene expression from a CpG-free luciferase transgene cassette in the context of both a plasmid and minicircle vector configuration and showed that the vector backbone had no effect on expression. Transgene activity was affected by the vector backbone however, when a similar, but sub-optimal CpG-containing transgene was used, suggesting that aspects of the plasmid backbone had a negative impact on transgene expression. Similar studies were performed in Toll-like receptor-9 (TLR9) knockout mice to investigate a potential role for the TLR9 signalling pathway in detecting CpGs in the vector sequence. Even in the absence of TLR9, persistent expression could only be achieved with a CpG-free transgene. Together, these data indicate that in order to achieve high levels of persistent expression in vivo, a CpG-free transgene cassette is required.

Improved osteogenic vector for non-viral gene therapy.

Therapeutic compensation of deficient bone regeneration is a challenging task and a topic of on-going search for novel treatment strategies. One promising approach for improvement involves non-viral gene delivery using the bone morphogenetic protein-2 (BMP-2) gene to provide transient, local and sustained expression of the growth factor. However, since efficiency of non-viral gene delivery is low, this study focused on the improvement of a BMP-2 gene expression system, aiming for compensation of poor transfection efficiency. First, the native BMP-2 gene sequence was modified by codon optimisation and altered by inserting a highly truncated artificial intron (96 bp). Transfection of multiple cell lines and rat adipose-derived mesenchymal stem cells with plasmids harbouring the improved BMP-2 sequence led to a several fold increased expression rate and subsequent osteogenic differentiation. Additionally, comparing expression kinetics of elongation factor 1 alpha (EF1α) promoter with a state of the art CMV promoter revealed significantly higher BMP-2 expression when under the influence of the EF1α promoter. Results obtained by quantification of bone markers as well as osteogenic assays showed reduced sensitivity to promoter silencing effects of the EF1α promoter in rat adipose-derived mesenchymal stem cells. Finally, screening of several protein secretion signals using either luciferase or BMP-2 as reporter protein revealed no superior candidates for potential replacement of the native BMP-2 secretion signal. Taken together, by enhancing the exogenous BMP-2 expression system, low transfection efficiencies in therapeutic applications can be compensated, making safe non-viral systems even more suitable for tissue regeneration approaches.

The effect of lance geometry and carbon coating of silicon lances on propidium iodide uptake in lance array nanoinjection of HeLa 229 cells

Connecting technology to biologic discovery is a core focus of non-viral gene therapy biotechnologies. One approach that leverages both the physical and electrical function of microelectromechanical systems (MEMS) in cellular engineering is a technology previously described as lance array nanoinjection (LAN). In brief, LAN consists of a silicon chip measuring 2 cm by 2 cm that has been etched to contain an array of 10 μm tall, solid lances that are spaced every 10 μm in a grid pattern. This array of lances is used to physically penetrate hundreds of thousands of cells simultaneously and to then electrically deliver molecular loads into cells. In this present work, two variables related to the microfabrication of the silicon lances, namely lance geometry and coating, are investigated. The purpose of both experimental variables is to assess these parameters’ effect on propidium iodide (PI), a cell membrane impermeable dye, uptake to injected HeLa 229 cells. For the lance geometry experimentation, three different microfabricated lance geometries were used which include a flat/narrow (FN, 1 μm diameter), flat/wide (FW, 2–2.5 μm diameter), and pointed (P, 1 μm diameter) lance geometries. From these tests, it was shown that the FN lances had a slightly better cell viability rate of 91.73% and that the P lances had the best PI uptake rate of 75.08%. For the lance coating experimentation, two different lances were fabricated, both silicon etched lances with some being carbon coated (CC) in a <100 nm layer of carbon and the other lances being non-coated (Si). Results from this experiment showed no significant difference between lance types at three different nanoinjection protocols (0V, +1.5V DC, and +5V Pulsed) for both cell viability and PI uptake rates. One exception to this is the comparison of CC/5V Pul and Si/5V Pul samples, where the CC/5V Pul samples had a cell viability rate 5% higher. Both outcomes were unexpected and reveal how to better optimize LAN parameters for future transfection experimentation.

Interactions between DNA and Gemini surfactant: impact on gene therapy: part I.

Nonviral gene therapy using gemini surfactants is a unique approach to medicine that can be adapted toward the treatment of various diseases. Recently, gemini surfactants have been utilized as candidates for the formation of nonviral vectors. The chemical structure of the surfactant (variations in the alkyl tail length and spacer/head group) and the resulting physicochemical properties of the lipoplexes are critical parameters for efficient gene transfection. Moreover, studying the interaction of the surfactant with DNA can help in designing an efficient vector and understanding how transfection complexes overcome various cellular barriers. Part I of this review provides an overview of various types of gemini surfactants designed for gene therapy and their transfection efficiency; and Part II will focus on different novel methods utilized to understand the interactions between the gemini and DNA in a lipoplex.

Cystic Fibrosis Gene Therapy – Not Low-hanging Fruit

The last 25 years have shown that it has been comparatively slow and difficult to develop cystic fibrosis (CF) gene therapy; the lung is a complex target organ. However, research has steadily progressed and recently it was shown that non-viral gene therapy can stabilise CF lung disease. These data, in addition to the development of potent lentiviral vectors, have renewed interest in CF gene therapy within academia and industry.

Hopes for Curing Cystic Fibrosis Lung Disease by Non-Viral Gene Therapy

Hopes for Curing Cystic Fibrosis Lung Disease by Non-Viral Gene Therapy