Non-viral Plasmid Research Articles

Plasmid DNA Delivery into the Skin via Electroporation with a Depot-Type Electrode.

Objectives: Non-viral mediated plasmid DNA transfection by electroporation (EP) is an established method for gene transfection. In this study, the usefulness of direct EP at an intradermal (i.d.) site (DEP) with implanted electrodes to achieve a high protein expression level was investigated. In addition, DEP application with various intervals with a low application voltage was also evaluated to confirm its effect on protein expression. Methods: Green fluorescent protein (GFP)- and luciferase-encoding DNA were administrated, and GFP and luciferase were evaluated. Results: A higher protein expression level was observed after green fluorescent protein (GFP)- and luciferase-encoding DNA were delivered by i.d. injection followed by DEP application. When luciferase expression was observed with an in vivo imaging system, continuous expression was confirmed over 21 days after i.d. injection followed by DEP at 100 V. This approach provided increased gene expression levels compared with conventional EP methods via the stratum corneum layer. In addition, the effect of application voltage on luciferase expression was investigated; two-time applications (repeated DEP) at 20 V with 5 min intervals showed similar luciferase expression level to single DEP application with 100 V. Histological observations showed the skin became thicker after a single DEP at 100 V, whereas no apparent thickness changes were confirmed after repeated DEP at 20 V with 5 min intervals. Conclusions: This study revealed that direct i.d. voltage application achieved high protein expression levels even at low voltages. Skin is a promising administration site for DNA vaccines, so this approach may be effective for DNA vaccine delivery into skin tissue.

A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors.

Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for a sufficient time. iPSCs are typically generated using viruses or virus-based methods, which have drawbacks such as vector persistence, risk of insertional mutagenesis, and oncogenesis. The application of less harmful nonviral vectors is limited as conventional plasmids cannot deliver the levels or duration of the factors necessary from a single transfection. Hence, plasmids that are most often used for reprogramming employ the potentially oncogenic Epstein-Barr nuclear antigen 1 (EBNA-1) system to ensure adequate levels and persistence of expression. In this study, we explored the use of nonviral SMAR DNA vectors to reprogram human fibroblasts into iPSCs. We show for the first time that iPSCs can be generated using nonviral plasmids without the use of EBNA-1 and that these DNA vectors can provide sufficient expression to induce pluripotency. We describe an optimised reprogramming protocol using these vectors that can produce high-quality iPSCs with comparable pluripotency and cellular function to those generated with viruses or EBNA-1 vectors.

Enhancing In Vivo Electroporation Efficiency through Hyaluronidase: Insights into Plasmid Distribution and Optimization Strategies.

Electroporation (EP) stands out as a promising non-viral plasmid delivery strategy, although achieving optimal transfection efficiency in vivo remains a challenge. A noteworthy advancement in the field of in vivo EP is the application of hyaluronidase, an enzyme with the capacity to degrade hyaluronic acid in the extracellular matrix, which thereby enhances DNA transfer efficiency by 2- to 3-fold. This paper focuses on elucidating the mechanism of hyaluronidase's impact on transfection efficiency. We demonstrate that hyaluronidase promotes a more uniform distribution of plasmid DNA (pDNA) within skeletal muscle. Additionally, our study investigates the effect of the timing of hyaluronidase pretreatment on EP efficiency by including time intervals of 0, 5, and 30 min between hyaluronidase treatment and the application of pulses. Serum levels of the pDNA-encoded transgene reveal a minimal influence of the hyaluronidase pretreatment time on the final serum protein levels following delivery in both mice and rabbit models. Leveraging bioimpedance measurements, we capture morphological changes in muscle induced by hyaluronidase treatment, which result in a varied pDNA distribution. Subsequently, these findings are employed to optimize EP electrical parameters following hyaluronidase treatment in animal models. This paper offers novel insights into the potential of hyaluronidase in enhancing the effectiveness of in vivo EP, as well as guides optimized electroporation strategies following hyaluronidase use.

PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry.

The term 'gene doping' is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: EPO, FST, GH1, IGF1, and ILRN1. The test is based on real-time polymerase chain reaction (PCR) and includes separate screening and confirmation assays that detect different unique targets in each transgene. For doping material, we used nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA for the targeted genes; the vectors were accurately quantified by digital PCR. To reduce non-specific amplification from genomic DNA observed in some assays, a restriction digest step was introduced in the PCR protocol prior to cycling to cut the amplifiable targets within the endogenous genes. We made the screening stage of the test simpler and faster by multiplexing PCR assays for four transgenes (EPO, FST, IGF1, and ILRN1), while the GH1 assay is performed in simplex. Both stages of the test reliably detect at least 20 copies of each transgene in a background of genomic DNA equivalent to what is extracted from two milliliters of equine blood. The test protocol was documented and tested with equine blood samples provided by an official doping control authority. The developed tests will form the basis for screening official horseracing samples in Australia.

Antibiotic-Free Gene Vectors: A 25-Year Journey to Clinical Trials.

Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors' production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical delivery techniques and to the refinement of their structure, non-viral plasmid DNA are now under intensive consideration as pharmaceutical drugs. Plasmids traditionally carry an antibiotic resistance gene for providing the selection pressure necessary for maintenance in a bacterial host. Nearly a dozen different antibiotic-free gene vectors have now been developed and are currently assessed in preclinical assays and phase I/II clinical trials. Their reduced size leads to increased transfection efficiency and prolonged transgene expression. In addition, associating non-viral gene vectors and DNA transposons, which mediate transgene integration into the host genome, circumvents plasmid dilution in dividing eukaryotic cells which generate a loss of the therapeutic gene. Combining these novel molecular tools allowed a significantly higher yield of genetically engineered T and Natural Killer cells for adoptive immunotherapies due to a reduced cytotoxicity and increased transposition rate. This review describes the main progresses accomplished for safer, more efficient and cost-effective gene and cell therapies using non-viral approaches and antibiotic-free gene vectors.

Non-invasive, targeted, and non-viral ultrasound-mediated brain-derived neurotrophic factor plasmid delivery for treatment of autism in a rat model.

Autism has clinical manifestations such as social interaction disorder, speech and intellectual development disorder, narrow interest range, and stereotyped and repetitive behavior, all of which bring considerable economic and mental burden to society and families, and represent a public health problem requiring urgent attention. Brain-derived neurotrophic factor (BDNF) plays an important role in supporting survival, differentiation, growth, and synapse formation of neurons and participates in the plasticity of nerves. However, it is difficult for BDNF to penetrate the blood-brain barrier (BBB) due to its large molecular weight. Low-frequency focused ultrasound (FUS) combined with microbubbles (MBs) has been demonstrated to be a promising method for opening the BBB non-invasively, transiently, and locally. Here, we studied the therapeutic effect of FUS combined with BDNF plasmid-loaded cationic microbubbles (BDNFp-CMBs) in a rat model of autism. BDNF-CMBs were prepared and the transfection efficiency of FUS combined with BDNF-CMBs was tested in vitro. A rat model of autism was established from the juvenile male offspring of Sprague-Dawley (SD) pregnant rats treated with sodium valproate (VPA) solution through intraperitoneal injection. The autism rats were randomized into three groups: the VPA group, which received no treatment, the BDNFp group, which was treated by injection of BDNFp, and the FUS + BDNFp-CMBs group, which was administered FUS combined with BDNFp-CMBs. Age-matched normal rats served as the control group (Con). Following treatment, stereotyped, exploratory, and social–behavioral tests were performed on the animals in each group. The rat brains were then collected for subsequent histological examination, and the changes in synaptic structures in the prefrontal cortex (PFC) were detected under transmission electron microscopy. The results showed that the constructed BDNFp could be loaded onto CMBs with high loading efficiency. The BDNFp-CMBs prepared in this study showed good stability in vivo. FUS combined BDNFp-CMBs could effectively and non-invasively open the BBB of rats. The stereotyped, exploratory, and social behaviors of the FUS + BDNFp-CMBs group were significantly improved. Compared to the VPA group, the abnormality of neuronal morphology and number in the PFC of the FUS + BDNFp-CMBs was alleviated to a certain extent and was accompanied by restoration of the damaged synapses in the encephalic region. Our work demonstrates the positive therapeutic effect of BDNF delivered by FUS non-invasively across the BBB into the PFC in a rat model of autism, offering a potential strategy for treating autism.

Deagglomeration of DNA nanomedicine carriers using controlled ultrasonication

Control over the agglomeration state of manufactured particle systems for drug and oligonucleotide intracellular delivery is paramount to ensure reproducible and scalable therapeutic efficacy. Ultrasonication is a well-established mechanism for the deagglomeration of bulk powders in dispersion. Its use in manufacturing requires strict control of the uniformity and reproducibility of the cavitation field within the sample volume to minimise within-batch and batch-to-batch variability. In this work, we demonstrate the use of a reference cavitating vessel which provides stable and reproducible cavitation fields over litre-scale volumes to assist the controlled deagglomeration of a novel non-viral particle-based plasmid delivery system. The system is the Nuvec delivery platform, comprising polyethylenimine-coated spiky silica particles with diameters of ∼ 200 nm. We evaluated the use of controlled cavitation at different input powers and stages of preparation, for example before and after plasmid loading. Plasmid loading was confirmed by X-ray photoelectron spectroscopy and gel electrophoresis. The latter was also used to assess plasmid integrity and the ability of the particles to protect plasmid from potential degradation caused by the deagglomeration process. We show the utility of laser diffraction and differential centrifugal sedimentation in quantifying the efficacy of product de-agglomeration in the microscale and nanoscale size range respectively. Transmission electron microscopy was used to assess potential damages to the silica particle structure due to the sonication process.

Recent Progress in the Discovery and Development of Monoclonal Antibodies against Viral Infections.

Monoclonal antibodies (mAbs), the new revolutionary class of medications, are fast becoming tools against various diseases thanks to a unique structure and function that allow them to bind highly specific targets or receptors. These specialized proteins can be produced in large quantities via the hybridoma technique introduced in 1975 or by means of modern technologies. Additional methods have been developed to generate mAbs with new biological properties such as humanized, chimeric, or murine. The inclusion of mAbs in therapeutic regimens is a major medical advance and will hopefully lead to significant improvements in infectious disease management. Since the first therapeutic mAb, muromonab-CD3, was approved by the U.S. Food and Drug Administration (FDA) in 1986, the list of approved mAbs and their clinical indications and applications have been proliferating. New technologies have been developed to modify the structure of mAbs, thereby increasing efficacy and improving delivery routes. Gene delivery technologies, such as non-viral synthetic plasmid DNA and messenger RNA vectors (DMabs or mRNA-encoded mAbs), built to express tailored mAb genes, might help overcome some of the challenges of mAb therapy, including production restrictions, cold-chain storage, transportation requirements, and expensive manufacturing and distribution processes. This paper reviews some of the recent developments in mAb discovery against viral infections and illustrates how mAbs can help to combat viral diseases and outbreaks.

Dual CAR-T cells to treat cancers co-expressing NKG2D and PD1 ligands in xenograft models of peritoneal metastasis.

While the expression of either NKG2D ligands or PD-1 ligands has been reported in various types of cancers, the co-expression of the two sets of ligands in the same tumour tissues is still un-investigated. After examining 68 primary ovarian cancer samples, we observed around 80% of the co-expression in low grade serous and endometrioid ovarian cancer samples. We then constructed a dual CAR system that splits the conventional single-input of a 2nd generation CAR into two independent chimeric receptors, one composed of the NKG2D extracellular domain linked with DAP12 for T cell activation and another using the PD-1 extracellular domain linked with 4-1BB for costimulatory signal 2 input. Given the limitation of the low-affinity PD-1 receptor in recognizing cancer cells with low levels of PD-1 ligands, we also used a high-affinity scFv specific to PD-L1 in our combinatorial approach to expand the range of target cancer cells with different expression levels of PD-L1. The two types of dual CAR-T cells were generated through electroporation of non-viral piggyBac transposon plasmids and were effective in eliminating the target cancer cells. Especially, the dual CAR-T cells with anti-PD-L1 scFv were capable of eradicating established tumors in mouse models of peritoneal metastasis of colorectal cancer and ovarian cancer. Since both NKG2D ligands and PD-1 ligands have been marked as favourable cancer therapeutic targets, the new dual CAR-T cells developed in this study hold attractive application potential in treating metastatic peritoneal carcinoma.

High-throughput 5\u2032 UTR engineering for enhanced protein production in non-viral gene therapies

Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5′ UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5′ UTRs with high translation efficiencies and use this information with in silico genetic algorithms to generate synthetic 5′ UTRs. A total of ~12,000 5′ UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identify three synthetic 5′ UTRs that outperform commonly used non-viral gene therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5′ UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.

Gene Therapy for Overactive Bladder: A Review of BK-Channel α-Subunit Gene Transfer.

A need exists for local (ie, bladder-specific) interventions to treat overactive bladder (OAB) with low risk of unwanted postprocedural outcomes. Gene therapy targeted to leverage endogenous physiology in bladder cells may assist in restoring normal cell and organ function. Herein, we review the potential promise of gene therapy for treating OAB, focusing on gene transfer of URO-902, a non-viral naked plasmid DNA expressing the big potassium (BK) channel. We searched PubMed for articles concerning functional aspects of the BK channel and its potential use for gene transfer as local OAB treatment. Results from preclinical, phase 1, and phase 2 studies of URO-902 for erectile dysfunction and phase 1 studies of URO-902 for OAB are included. The BK channel has been extensively studied; however, URO-902 is the first gene therapy used in clinical trials directed toward treating OAB via the BK channel. In both URO-902 studies, there were no serious adverse events considered treatment related and no adverse events leading to early withdrawal. Both studies included secondary efficacy endpoints with promising results suggesting improvement in OAB symptoms, and quality of life, with use of URO-902 versus placebo. Gene therapy involving the BK channel, such as gene transfer with URO-902, has demonstrated promising safety and efficacy results in women with OAB. Findings warrant further investigation of the use of URO-902 for OAB treatment.

Evaluation of BMP2/miRNA co-expression systems for potent therapeutic efficacy in bone-tissue regeneration.

Reconstruction of bone defects and compensation of deficient repair mechanisms represent important goals within the field of regenerative medicine and require novel safe strategies for translation into the clinic. A non-viral osteogenic gene therapeutic vector system ('hybrid vectors') was generated, combining an improved bone morphogenetic protein 2 (BMP2) gene cassette and single pro-osteogenic microRNAs (miR-148b-3p, miR-20-5p, miR-590b-5p), driven by the U6 promoter. The vectors were tested in vitro for their osteogenic differentiation potential in C2C12 and C3H/10T1/2 cell lines, using BMP2 alone as control. After confirming BMP2 expression and miRNA transcription, increased osteogenic differentiation was observed by all hybrid vectors, but most consistently by BMP2/miR-590-5p, using alkaline phosphatase enzyme activity assays and osteogenic marker mRNA quantitation, including runt-related transcription factor 2 (Runx2), collagen type 1 (Col1a1) and osteocalcin. To visualise target mRNAs of the respective miRNAs, next generation sequencing was performed, confirming down-regulation of mRNA targets of the hybrid vectors. Since the hybrid vector consisting of BMP2 and miR-590-5p showed the largest increase in osteogenic differentiation in vitro, this was tested in a mouse ectopic-bone model. Mineralisation was more than with BMP2 alone. The present study showed hybrid vectors as a novel non-viral gene therapeutic plasmid system for combining therapeutic effects of recombinant protein expression and miRNA transcription that did not add to the burden of the translation machinery, while improving the therapeutic efficacies. In vivo proof-of-principle in the context of bone regeneration suggested that such hybrid vectors will be applicable in a wide array of gene therapeutic strategies.

An Epidermal Growth Factor Motif of Developmental Endothelial Locus 1 Protein Inhibits Efficient Angiogenesis in Explanted Squamous Cell Carcinoma In Vivo.

Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have longterm effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model. In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors. Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis. All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins. These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis.

Combination Cancer Therapy of a Del1 Fragment and Cisplatin Enhanced Therapeutic Efficiency In Vivo.

Combination cancer therapy is currently under investigation. This study examined the effect of cancer combination therapy using the E3 and C1 (E3C1) domains of developmental endothelial locus-1 (Del1) and cisplatin (CDDP) in murine transplanted tumors. Mice with transplanted tumors (A431, SCCKN or SCC-4 cells) were injected intraperitoneally with CDDP and injected locally with nonviral plasmid vectors encoding E3C1. Histochemical analysis of the transplanted tumors was then performed to assess the effects on prognosis. The CDDP+E3C1 injected group had reduced tumor growth and longer survival compared to the CDDP injected group. In addition, cell death was observed in the tumor of the CDDP+E3C1 group.. Furthermore, angiogenesis and increased blood vessels were observed together with stromal development. The CDDP+E3C1 treatment resulted in improved survival and poor tumor stromal development in mice with transplanted tumors.

Stromal Cell–Derived Factor-1 Plasmid Treatment for Patients With Peripheral Artery Disease (STOP-PAD) Trial: Six-Month Results

Purpose: To present the 6-month results of the Stromal Cell–Derived Factor-1 Plasmid Treatment for Patients with Peripheral Artery Disease (STOP-PAD) trial. The trial was an attempt to alter the course of chronic limb-threatening ischemia (CLTI) with a biological agent vs placebo after successful arterial revascularization at or below the knee. Materials and Methods: The multicenter, randomized, double-blinded, placebo-controlled, phase 2B STOP-PAD trial (ClinicalTrials.gov identifier NCT02544204) randomized 109 patients (mean age 71 years; 68 men) with Rutherford category 5 or 6 CLTI and evidence of persistent impaired forefoot perfusion following recent successful revascularization to 8- (n=34) or 16-mg (n=36) intramuscular injections of a non-viral DNA plasmid–based treatment vs placebo (n=34). The primary efficacy outcome was the 6-month wound healing score evaluated by an independent wound core laboratory; the primary safety endpoint was major adverse limb events (MALE), a composite of major amputation plus clinically-driven target lesion revascularization at 6 months. Results: Only one-third of the patients had complete wound healing at 6 months in the placebo (31%), 8-mg injection (33%), and 16-mg injection (33%) groups. In addition, the observed increase in the toe-brachial index from baseline to 6 months was statistically significant in each group; however, this did not result in lower rates of MALE at 6 months (24% in the placebo, 29% in the 8-mg injection, and 11% in the 16-mg injection groups). During the 6-month period, 6 patients (6%) died, and 24 patients (23%) had an amputation [only 4 (4%) major]. Conclusion: Combining revascularization and biological therapy failed to improve outcomes in CLTI at 6 months. STOP-PAD has provided insights for future trials to evaluate biological therapy.

Effect of cryopreservation of mesenchymal stromal cells after non-viral transfection on cell viability and transgene expression

Background & Aim Mesenchymal stromal cells (MSCs) have been shown to exert important immunomodulatory effects in both acute and chronic diseases. In acute inflammatory conditions, an “off-the-shelf” cryopreserved, allogeneic cell product might be better suited as it can be ready for administration to patients without delay. Genetically modified cells have recently gained tremendous interest given the successes in chimeric antigen receptors (CAR T) cell therapy, in which T cells from patients can be genetically engineered to produce an artificial T-cell receptor for treating cancer. However, whether a genetically modified, allogeneic MSC can have sustained transgene expression post cryopreservation has not yet been well studied. Herein we assess the effect of non-viral transfection on cell viability and protein expression in cryopreserved-and-then-thaw MSC. Methods, Results & Conclusion MSCs were derived from bone marrow of healthy donors and characterized using the ISCT criteria (surface marker expression and tri-lineage differentiation potentials). MSCs were transfected with non-viral plasmid using two protocols: 1) transfection 24 hours after cell seeding, or 2) transfection after 6 days of culture. Transfected MSCs were cryopreserved at 24 hours after transfection. Viability (Trypan blue), cell number recovery and transgene expression (Western blot) were examined. Prior to cryopreservation, the cell viability was 87% ± 3% from protocol 1 (n=2) and 93% ± 1.2% from protocol 2 (n=3). After one week of storage in liquid nitrogen, cell viability was 71% ± 4% and 82% ± 5% for Protocol 1 and 2 respectively, while the percentage of cell recovery post thaw was 72% ± 9% and 95% ± 2%. Thawed cells were sub-cultured to confirm no abnormality in cell growth and morphology. To determine levels of transgene protein expression, cell pellets were collected pre- and post-cryopreservation. Western blot analysis showed that `transgene protein could be detected 2 days after cells were thawed, and the protein expression was modestly reduced to 78% ± 6.7% to that of pre-cryopreservation in protocol 1 and 93%± 1.7% in protocol 2. Our results address the concerns that transgene expression might be lost during the process of cryopreservation by showing a sustained but slightly reduced level of proteins overexpression to that of pre-cryopreservation. Further optimization will be conducted to minimize the loss of protein expression and to improve viability after thaw.

In Vivo Delivery of Nucleic Acid-Encoded Monoclonal Antibodies.

Antibody immunotherapy is revolutionizing modern medicine. The field has advanced dramatically over the past 40 years, driven in part by major advances in isolation and manufacturing technologies that have brought these important biologics to the forefront of modern medicine. However, the global uptake of monoclonal antibody (mAb) biologics is impeded by biophysical and biochemical liabilities, production limitations, the need for cold-chain storage and transport, as well as high costs of manufacturing and distribution. Some of these hurdles may be overcome through transient in vivo gene delivery platforms, such as non-viral synthetic plasmid DNA and messenger RNA vectors that are engineered to encode optimized mAb genes. These approaches turn the body into a biological factory for antibody production, eliminating many of the steps involved in bioprocesses and providing several other significant advantages, and differ from traditional gene therapy (permanent delivery) approaches. In this review, we focus on nucleic acid delivery of antibody employing synthetic plasmid DNA vector platforms, and RNA delivery, these being important approaches that are advancing simple, rapid, in vivo expression and having an impact in animal models of infectious diseases and cancer, among others.

A Non-Viral Plasmid DNA Delivery System Consisting on a Lysine-Derived Cationic Lipid Mixed with a Fusogenic Lipid.

The insertion of biocompatible amino acid moieties in non-viral gene nanocarriers is an attractive approach that has been recently gaining interest. In this work, a cationic lipid, consisting of a lysine-derived moiety linked to a C12 chain (LYCl) was combined with a common fusogenic helper lipid (DOPE) and evaluated as a potential vehicle to transfect two plasmid DNAs (encoding green fluorescent protein GFP and luciferase) into COS-7 cells. A multidisciplinary approach has been followed: (i) biophysical characterization based on zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and cryo-transmission electronic microscopy (cryo-TEM); (ii) biological studies by fluorescence assisted cell sorting (FACS), luminometry, and cytotoxicity experiments; and (iii) a computational study of the formation of lipid bilayers and their subsequent stabilization with DNA. The results indicate that LYCl/DOPE nanocarriers are capable of compacting the pDNAs and protecting them efficiently against DNase I degradation, by forming Lα lyotropic liquid crystal phases, with an average size of ~200 nm and low polydispersity that facilitate the cellular uptake process. The computational results confirmed that the LYCl/DOPE lipid bilayers are stable and also capable of stabilizing DNA fragments via lipoplex formation, with dimensions consistent with experimental values. The optimum formulations (found at 20% of LYCl content) were able to complete the transfection process efficiently and with high cell viabilities, even improving the outcomes of the positive control Lipo2000*.

Electroactive composite scaffold with locally expressed osteoinductive factor for synergistic bone repair upon electrical stimulation

Tissue engineering is a promising strategy for the repair of large-scale bone defects, in which scaffolds and growth factors are two critical issues influencing the efficacy of bone regeneration. Unfortunately, the broad application of growth factors is limited by their poor stability in the scaffolds. In the present study, the strictly controlled expression of human bone morphogenetic protein-4 (hBMP-4) in the presence of doxycycline is achieved by adding an hBMP-4 gene fragment into a non-viral artificial restructuring plasmid vector (pSTAR) to form the pSTAR-hBMP-4 plasmid (phBMP-4). Furthermore, the controlled release of phBMP-4 is obtained with an electroactive tissue engineering scaffold, generated by combining a triblock copolymer of poly(l-lactic acid)-block-aniline pentamer-block-poly(l-lactic acid) (PLA-AP) with poly(lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA). This PLGA/HA/PLA-AP/phBMP-4 composite scaffold, with controlled gene release and Dox-regulated gene expression upon electrical stimulation, operating synergistically, exhibits an improved cell proliferation ability, enhanced osteogenesis differentiation in vitro, and effective bone healing in vivo in a rabbit radial defect model. Taking these results together, the proposed smart PLGA/HA/PLA-AP/phBMP-4 scaffold lays a solid theoretical and experimental basis for future applications of such multi-functional materials in bone tissue engineering to help patients in need.

Secreting oviduct epithelial cells of Coturnix coturnix japonica (QOEC) and changes to their proteome after nonviral transfection.

The quail oviduct (Coturnix c. japonica) is a natural candidate avian bioreactor, while the secretive quail oviduct epithelial cells (QOECs) are potential in vitro producers of recombinant proteins and vaccines. In view of the need for highly performing and transformable cell lines, QOEC may potentially act as an alternative bioreactor platform to the existing ones, for example, to the Chinese hamster ovary. The aim of this work was to characterize QOECs and their response to nucleofection with a nonviral plasmid DNA carrying the human interferon-α 2a gene (hIFNλ2a), in vitro. Primary QOEC cultures from laying quails (10-15 weeks old) were characterized by their proliferation rate, doubling time, and multilineage differentiation. Electroporation to cell nuclei (nucleofection) was used to deliver nonviral plasmid DNA containing a reporter GFP and hIFN under the ovalbumin promoter. The posttransfection analysis included polymerase chain reaction, Western blot analysis, and liquid chromatography coupled to tandem mass spectrometry. QOEC showed a typical epithelial characteristic in a primary 2D monolayer culture system and retained secretive potential up to the first passage. QOEC showed differentiation into osteoblastic lineage after stimulation. The nucleofection mean efficiency was low (2.3%). Differences of up to 10% in the proteomic profiles between nontransfected and transfected QOEC were found, the most important of these were related to the absence of keratins and cell-adhesion proteins in the transfected QOEC. Concluding, with the practical information provided here, QOEC have the potential to serve as an avian secreting cellular platform. QOEC may be further transformed to cell lineage to meet the requirement for a stable, electrocompetent, and transfectable model. The first proteomic comparison of QOEC delivered in this study showed, in the majority, a stable proteome of the nontransfected vs transfected QOEC.