Search

Aim: To develop a safe and efficient method for targeted, anti-apoptotic gene therapy of corneal endothelial cells (CECs). Materials & methods: Magnetofection (MF), a combination of lipofection with magnetic nanoparticles (MNPs; PEI-Mag2, SO-Mag5, PalD1-Mag1), was tested in human CECs and in explanted human corneas. Effects on cell viability and function were investigated. Immunocompatibility was assessed in human peripheral blood mononuclear cells. Results: Silica iron-oxide MNPs (SO-Mag5) combined with X-tremeGENE-HP achieved high transfection efficiency in human CECs and explanted human corneas, without altering cell viability or function. Magnetofection caused no immunomodulatory effects in human peripheral blood mononuclear cells. Magnetofection with anti-apoptotic P35 gene effectively blocked apoptosis in CECs. Conclusion: Magnetofection is a promising tool for gene therapy of corneal endothelial cells with potential for targeted on-site delivery. [ABSTRACT FROM AUTHOR]

Abstract: Nucleic acids carry the building plans of living systems. As such, they can be exploited to make cells produce a desired protein, or to shut down the expression of endogenous genes or even to repair defective genes. Hence, nucleic acids are unique substances for research and therapy. To exploit their potential, they need to be delivered into cells which can be a challenging task in many respects. During the last decade, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as magnetofection. In this review we summarize the progress and achievements in this field of research. We discuss magnetic formulations of vectors for nucleic acid delivery and their characterization, mechanisms of magnetofection, and the application of magnetofection in viral and nonviral nucleic acid delivery in cell culture and in animal models. We summarize results that have been obtained with using magnetofection in basic research and in preclinical animal models. Finally, we describe some of our recent work and end with some conclusions and perspectives. [Copyright &y& Elsevier]

Adenoviral transduction of the VEGF gene in an oversized skin flap increases flap survival and perfusion. In this study, we investigated the potential of magnetofection of magnetic lipospheres containing VEGF165-cDNA on survival and perfusion of ischemic skin flaps and evaluated the method with respect to the significance of applied magnetic field and ultrasound. We prepared perfluoropropane-filled magnetic lipospheres (‘magnetobubbles’) from Tween60-coated magnetic nanoparticles, Metafectene, soybean-oil and cDNA and studied the effect in an oversized random-pattern-flap model in the rats ( n= 46). VEGF-cDNA-magnetobubbles were administered under a magnetic field with simultaneously applied ultrasound, under magnetic field alone and with applied ultrasound alone. Therapy was conducted 7 days pre-operative. Flap survival and necrosis were measured 7 days post-operatively. Flap perfusion, VEGF-protein concentration in target and surrounding tissue, formation and appearance of new vessels were analysed additionally. Magnetofection with VEGF-cDNA-magnetobubbles presented an increased flap survival of 50% and increased flap perfusion ( P < 0.05). Without ultrasound and without magnetic field, the effect is weakened. VEGF concentration in target tissue was elevated ( P < 0.05), while underlying muscle was not affected. Our results demonstrate the successful VEGF gene therapy by means of magnetobubble magnetofection. Here, the method of magnetofection of magnetic lipospheres is equally efficient as adenoviral transduction, but has a presumable superior safety profile. [ABSTRACT FROM AUTHOR]

Explores the attractive forces of magnetic gradient fields on superparamagnetic nanoparticles to potentiate the efficacies, improve the kinetics and dose response profiles of gene transfer processes and target delivery by application of magnetic fields. Decrease in the incubation time required for transfection/transduction efficiency; Advantage of magnetofection for ex vivo gene therapy approaches.

Recently, chemically modified mRNA (cmRNA) therapeutics have been the subject of extensive application-oriented research in both academia and industry as a safer alternative for gene and recombinant protein therapies. However, the lack of an efficient delivery system hinders widespread application. Here we used ∼100-nm lipoplexes and magnetic lipoplexes that can protect cmRNA from RNases and efficiently deliver it into muscle and fat tissues as well as to the endothelium of the carotid artery. Establishing magnetofection for ex vivo cmRNA delivery for the first time, we suggest this method for potential enhanced and targeted delivery of cmRNA. This study introduces optimal cmRNA complexes with high ex vivo efficiency as good candidates for further in vivo cmRNA delivery. [ABSTRACT FROM AUTHOR]

Bone regeneration using stem cells and growth factors has disadvantages while needing to use supraphysiological growth factors concentrations. Gene therapy has been proposed as alternative, but also has limitation. Messenger RNA (mRNA)-based transcript therapy is a novel approach that may solve plasmid DNA-based gene therapy limitations. Although much more efficient in delivering genes into the cell, mRNA is unfortunately unstable and immunogenic. However, recent reports indicated that chemical modifications of the mRNA molecule can improve stability and toxicity. In this study, we have combined biomaterials and chemically modified mRNA (cmRNA) encoding Metridia luciferase, eGFP, and bone morphogenetic protein (BMP)-2 to develop transcript-activated matrices (TAMs) for gene transfer to stem cells. BMP-2 cmRNA was produced to evaluate its feasibility in stimulating osteogenic differentiation. Fibrin gel and micro-macro biphasic calcium phosphate (MBCP) granules were used as biomaterials. A sustained release of hBMP-2 cmRNA from both biomaterials was observed during 7 days. This occurred significantly faster from the MBCP granules compared to fibrin gels (92% from MBCP and 43% from fibrin after 7 days). Stem cells cultured in hBMP-2 cmRNA/fibrin or on hBMP-2 cmRNA/MBCP were transfected and able to secrete significant amounts of hBMP-2. Furthermore, transfected cells expressed osteogenic markers in vitro. Interestingly, although both TAMs promoted gene expression at the same level, hBMP-2 cmRNA/MBCP granules induced significantly higher collagen I and osteocalcin gene expression. This matrix also induced more mineral deposition. Overall, our results demonstrated the feasibility of developing efficient TAMs for bone regeneration by combining biomaterials and cmRNAs. MBCP synergistically enhances the hBMP-2 cmRNA-induced osteogenic pathway. [ABSTRACT FROM AUTHOR]

Gene therapy is a promising approach for chronic disorders that require continuous treatment such as cardiovascular disease. Overexpression of vasoprotective genes has generated encouraging results in animal models, but not in clinical trials. One major problem in humans is the delivery of sufficient amounts of genetic vectors to the endothelium which is impeded by blood flow, whereas prolonged stop-flow conditions impose the risk of ischemia. In the current study we have therefore developed a strategy for the efficient circumferential lentiviral gene transfer in the native endothelium under constant flow conditions. For that purpose we perfused vessels that were exposed to specially designed magnetic fields with complexes of lentivirus and magnetic nanoparticles thereby enabling overexpression of therapeutic genes such as endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF). This treatment enhanced NO and VEGF production in the transduced endothelium and resulted in a reduction of vascular tone and increased angiogenesis. Thus, the combination of MNPs with magnetic fields is an innovative strategy for site-specific and efficient vascular gene therapy. [ABSTRACT FROM AUTHOR]

Abstract: The use of scaffolds in skin tissue engineering is accompanied with low regeneration rates and high risk of infection. In this study, we activated an FDA-approved collagen scaffold for dermal regeneration by incorporation of copolymer-protected gene vectors (COPROGs) to induce a temporary release of VEGF. In vitro results show that the presence of COPROGs did not affect the distribution, attachment, proliferation and viability of cells in the scaffold. A transient release of VEGF was observed for up to 3 weeks. Moreover a high amount of VEGF was also found in the cells and associated with the scaffold. In a full skin defect model in nude mice, VEGF levels were significantly increased compared to controls in VEGF gene activated scaffolds 14 d after implantation, but not in skin from the wound edge. Results showed an increased amount of non-adherent cells, especially erythrocytes, and von Willebrandt factor (vWF) and a yellow red appearance of gene activated scaffolds in relation to controls. This suggests the presence of leaky vessels. In this work we show that the bioactivation of collagen scaffolds with COPROGs presents a new technology that allows a local release of therapeutic proteins thus enhancing the regenerative potential in vivo. [ABSTRACT FROM AUTHOR]

Abstract: The lung is an important target of gene therapeutic interventions. In contrast to intratracheal instillation, inhalation would be the most practical route of administration in clinical applications. Here we show that aerosolized nanogram quantities of pDNA complexed to PEI (350 ng) yielded transfection levels 15-fold higher than a 140-fold higher dose (50 μg) of the same vector applied directly to the lungs of mice via intratracheal intubation. An important efficacy parameter is the osmolarity of the aerosol and not biophysical properties of the nebulized vector. Vectors formulated and nebulized in hypoosmotic distilled water yielded 57- and 185-fold higher expression levels than those in isotonic 5% glucose or Hepes-buffered saline, respectively. Pretreatment of mice with nebulized indomethacin, which prevents water-induced airway alteration, resulted in lower gene expression, whereas pretreatment with EGTA or polidocanol, which modulate tight-junction activity, had no effect. These results, together with histological analysis of regional lung deposition and gene expression, suggest that a temporary water-induced hypoosmotic shock permeabilizes the epithelium sufficiently to allow vector uptake. The so far observed inefficiency of nonviral gene delivery to the airways may be the result of an inappropriate method of vector administration. [Copyright &y& Elsevier]

Abstract: Magnetofection is nucleic acid delivery to cells supported and site-specifically guided by the attractive forces of magnetic fields acting on nucleic acid shuttles (vectors) which are associated with magnetic nanoparticles. Recent progress with the method confirms its general applicability with small and large nucleic acids and viruses. The method''s therapeutic application as well as mechanistic studies will be discussed. [Copyright &y& Elsevier]

Feline fibrosarcoma is one of the most common feline tumors. It arises spontaneously, is rarely metastatic and relapses within 6 months after standard therapy (surgical resection) in 75 % of the cases. This makes it an ideal model for evaluating immunostimulatory therapeutic strategies. Moreover, besides aggressive surgical excision no effective and versatile treatment is available.Here we report preliminary results from a comparative clinical study where the genes for feline GM-CSF, IFN-g and IL-2 in combination, feline GM-CSF alone or human GM-CSF were administered. The study design is prospective, randomized, placebo- controlled (= standard therapy) and includes five arms: (1) standard therapy, i.e. surgery alone; (2) nonviral Magnetofection of the feline GM-CSF, IFN-g and IL-2 genes into the tumor before surgery or nonviral administration by Magnetofection of feline (3) or human GM-CSF (4) gene alone and (5) nonviral application of the feline GM-CSF, IFN-g and IL-2 genes by a gene-activated-matrix. The procedure included phase I dose finding studies for the gene therapy groups followed by a phase II. Preliminary clinical endpoint is relapse-free survival for one year.Magnetofection, which has been developed in our laboratory, is the association of vectors with magnetic particles (chemicell, Berlin, Germany) and gene delivery under the influence of a magnetic field (Scherer et al. 2002, Gene Ther., Plank et al. 2003, Biol. Chem). It was applied here in order to achieve improved retention of the injected vector dose in the tumor.Copolymer-protected gene vectors (Scherer et al. 2002, J. Gene Med. 4:634-643) were immobilized on a collagen sponge to achieve sustained localized matrix-mediated gene delivery. After tumor excision the gene-activated matrices were implanted in the tumor bed intrasurgically.Pre- and postsurgical diagnosis included complete clinical monitoring of the cats. All gene-therapeutic treatments were well tolerated and led to prolonged relapse-free survival (one year time points: 50 % for intrasurgical implantation of the vector-loaded collagen sponge in the tumor bed, and 52 % for presurgical Magnetofection with human GM-CSF versus 23 % for surgery alone). Additional patients have been admitted to the Magnetofection group such that long-term follow-up will warrant a profound assessment of the benefits of this treatment.Molecular Therapy (2006) 13, S105–S105; doi: 10.1016/j.ymthe.2006.08.327 [ABSTRACT FROM AUTHOR]