Method for siRNA delivery in retina explants.

Several barriers prevent the delivery of nucleic acids to the retina and limit the application of established technologies, such as RNA interference (RNAi), in the study of retinae biology. Organotypic culture of retinal explants is a convenient method to decrease the complexity of the biological environment surrounding the retina while preserving most of its physiological features. Nevertheless, eliciting significant, non-toxic RNAi in retina explants is not straightforward. Retina explants are mainly constituted by neurons organized in discrete circuits embedded within a complex 3D extracellular matrix. About 70% of these neurons are post-mitotic ciliated cells that respond to light. Unfortunately, like the other cells of the retina, photoreceptors are refractory to transfection, and a toxic delivery of nucleic acid often results in permanent cell loss. RNAi has been applied to retina explants using electroporation, viral, and non-viral vectors but with reproducible, poor gene silencing efficiency. In addition, only a few superficial cells can be transduced/transfected in adult retina explants. Therefore, viruses are often injected into the eye of embryos prior to excision of the retina. However, embryonic explants are not the best model to study most retina diseases since even if they are viable for several weeks, the pathological phenotype often appears later in development. We describe a robust and straightforward method to elicit significant RNAi in adult retina explant using Reverse Magnetofection. This transfection method offers a simple tool for non-toxic gene knockdown of specific genes in adult retina explants by using cationic magnetic nanoparticles (MNPs) to complex and deliver short interfering-RNAs (siRNA) in retina cells under the action of a magnetic field.

Magnetically Assisted Drug Delivery of Topical Eye Drops Maintains Retinal Function In Vivo in Mice.

Barded-Biedl syndrome (BBS) is a rare genetic disorder with an unmet medical need for retinal degeneration. Small-molecule drugs were previously identified to slow down the apoptosis of photoreceptors in BBS mouse models. Clinical translation was not practical due to the necessity of repetitive invasive intravitreal injections for pediatric populations. Non-invasive methods of retinal drug targeting are a prerequisite for acceptable adaptation to the targeted pediatric patient population. Here, we present the development and functional testing of a non-invasive, topical, magnetically assisted delivery system, harnessing the ability of magnetic nanoparticles (MNPs) to cargo two drugs (guanabenz and valproic acid) with anti-unfolded protein response (UPR) properties towards the retina. Using magnetic resonance imaging (MRI), we showed the MNPs' presence in the retina of Bbs wild-type mice, and their photoreceptor localization was validated using transmission electron microscopy (TEM). Subsequent electroretinogram recordings (ERGs) demonstrated that we achieved beneficial biological effects with the magnetically assisted treatment translating the maintained light detection in Bbs-/- mice (KO). To our knowledge, this is the first demonstration of efficient magnetic drug targeting in the photoreceptors in vivo after topical administration. This non-invasive, needle-free technology expands the application of SMDs for the treatment of a vast spectrum of retinal degenerations and other ocular diseases.

New Method for Efficient siRNA Delivery in Retina Explants: Reverse Magnetofection.

The prevalence of retinal disorders associated with visual impairment and blindness is increasing worldwide, while most of them remain without effective treatment. Pharmacological and molecular therapy development is hampered by the lack of effective drug delivery into the posterior segment of the eye. Among molecular approaches, RNA-interference (RNAi) features strong advantages, yet delivering it to the inner layer of the retina appears extremely challenging. To address this, we developed an original magnetic nanoparticles (MNPs)-based transfection method that allows the efficient delivery of siRNA in all retinal layers of rat adult retinas through magnetic targeting. To establish delivery of RNAi throughout the retina, we have chosen organotypic retinal explants as an ex vivo model and for future high content screening of molecular drugs. Conversely to classic Magnetofection, and similar to conditions in the posterior chamber of the eye, our methods allows attraction of siRNA complexed to MNPs from the culture media into the explant. Our method termed "Reverse Magnetofection" provides a novel and nontoxic strategy for RNAi-based molecular as well as gene therapy in the retina that can be transferred to a wide variety of organ explants.

Efficient Ocular Delivery of VCP siRNA via Reverse Magnetofection in RHO P23H Rodent Retina Explants.

The use of synthetic RNA for research purposes as well as RNA-based therapy and vaccination has gained increasing importance. Given the anatomical seclusion of the eye, small interfering RNA (siRNA)-induced gene silencing bears great potential for targeted reduction of pathological gene expression that may allow rational treatment of chronic eye diseases in the future. However, there is yet an unmet need for techniques providing safe and efficient siRNA delivery to the retina. We used magnetic nanoparticles (MNPs) and magnetic force (Reverse Magnetofection) to deliver siRNA/MNP complexes into retinal explant tissue, targeting valosin-containing protein (VCP) previously established as a potential therapeutic target for autosomal dominant retinitis pigmentosa (adRP). Safe and efficient delivery of VCP siRNA was achieved into all retinal cell layers of retinal explants from the RHO P23H rat, a rodent model for adRP. No toxicity or microglial activation was observed. VCP silencing led to a significant decrease of retinal degeneration. Reverse Magnetofection thus offers an effective method to deliver siRNA into retinal tissue. Used in combination with retinal organotypic explants, it can provide an efficient and reliable preclinical test platform of RNA-based therapy approaches for ocular diseases.

Self-Amplifying Replicon RNA Delivery to Dendritic Cells by Cationic Lipids.

Advances in RNA technology during the past two decades have led to the construction of replication-competent RNA, termed replicons, RepRNA, or self-amplifying mRNA, with high potential for vaccine applications. Cytosolic delivery is essential for their translation and self-replication, without infectious progeny generation, providing high levels of antigen expression for inducing humoral and cellular immunity. Synthetic nanoparticle-based delivery vehicles can both protect the RNA molecules and facilitate targeting of dendritic cells-critical for immune defense development. Several cationic lipids were assessed, with RepRNA generated from classical swine fever virus encoding nucleoprotein genes of influenza A virus. The non-cytopathogenic nature of the RNA allowed targeting to dendritic cells without destroying the cells-important for prolonged antigen production and presentation. Certain lipids were more effective at delivery and at promoting translation of RepRNA than others. Selection of particular lipids provided delivery to dendritic cells that resulted in translation, demonstrating that delivery efficiency could not guarantee translation. The observed translation in vitro was reproduced in vivo by inducing immune responses against the encoded influenza virus antigens. Cationic lipid-mediated delivery shows potential for promoting RepRNA vaccine delivery to dendritic cells, particularly when combined with additional delivery elements.

Magnetofection is superior to other chemical transfection methods in a microglial cell line.

BACKGROUND: Microglia, the resident phagocytic cells of the brain, have recently been the subject of intense investigation given their role in pathology and normal brain physiology. In general, phagocytic cells are hard to transfect with plasmid DNA. The BV2 cell line is a murine cell line of microglial origin which is often used to study this cell type in vitro. Unfortunately, this microglial cell line is, like other phagocytic cells, resistant to transfection. NEW METHOD: Magnetofection is a well-established transfection method that combines DNA with magnetic particles which, under the influence of a magnetic field, ensures a high concentration of particles in proximity of cultured cells. Only recently, Glial-Mag was specifically developed for efficient transfection of microglia and microglial cell lines. RESULTS: Magnetofection with Glial-Mag yielded a transfection efficiency of 34.95% in BV2 cells, 24h after transfection with an eGFP-expressing plasmid. Efficient gene delivery caused a modest and short-lived cell activation (as measured by IL6 secretion) that ceased by 24h after transfection. COMPARISON WITH EXISTING METHODS: Here we show that Glial-Mag magnetofection of BV2 cells yielded a significantly higher transfection efficiency (34.95%) compared to other chemical transfection methods including calcium-phoshate precipication (0.34%), X-tremeGENE (3.30%) and Lipofectamine 2000 (12.51%). CONCLUSION: Transfection of BV2 cells using Glial-Mag magnetofection is superior compared to other chemical transfection methods and could be considered as the method of choice to chemically transfect microglial cell lines.

3D-fection: cell transfection within 3D scaffolds and hydrogels.

BACKGROUND: 3D matrices are widely used as cell growth supports in basic research, regenerative medicine or cell-based drug assays. In order to genetically manipulate cells cultured within 3D matrices, two novel non-viral transfection reagents allowing preparation of matrices for in situ cell transfection were evaluated. RESULTS: Two lipidic formulations, 3D-Fect™ and 3D-FectIN™, were assessed for their ability to transfect cells cultured within 3D solid scaffolds and 3D hydrogels, respectively. These reagents showed good compatibility with the most widespread types of matrices and enabled transfection of a wide range of mammalian cells of various origins. Classical cell lines, primary cells and stem cells were thus genetically modified while colonizing their growth support. Importantly, this in situ strategy alleviated the need to manipulate cells before seeding them. CONCLUSION: Results presented here demonstrated that 3D-Fect and 3D-FectIN reagents for 3D transfection are totally compatible with cells and do not impair matrix properties. 3D-Fect and 3D-FectIN, therefore, provide valuable tools for achieving localized and sustained transgene expression and should find versatile applications in fundamental research, regenerative medicine and cell-based drug assays.

Magnetic nanoparticles enhance adenovirus transduction in vitro and in vivo.

PURPOSE: Adenoviruses are among the most powerful gene delivery systems. Even if they present low potential for oncogenesis, there is still a need for minimizing widespread delivery to avoid deleterious reactions. In this study, we investigated Magnetofection efficiency to concentrate and guide vectors for an improved targeted delivery. METHOD: Magnetic nanoparticles formulations were complexed to a replication defective Adenovirus and were used to transduce cells both in vitro and in vivo. A new integrated magnetic procedure for cell sorting and genetic modification (i-MICST) was also investigated. RESULTS: Magnetic nanoparticles enhanced viral transduction efficiency and protein expression in a dose-dependent manner. They accelerated the transduction kinetics and allowed non-permissive cells infection. Magnetofection greatly improved adenovirus-mediated DNA delivery in vivo and provided a magnetic targeting. The i-MICST results established the efficiency of magnetic nanoparticles assisted viral transduction within cell sorting columns. CONCLUSION: The results showed that the combination of Magnetofection and Adenoviruses represents a promising strategy for gene therapy. Recently, a new integrated method to combine clinically approved magnetic cell isolation devices and genetic modification was developed. In this study, we validated that magnetic cell separation and adenoviral transduction can be accomplished in one reliable integrated and safe system.

Magnetically enhanced nucleic acid delivery. Ten years of magnetofection-progress and prospects.

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.

High transfection efficiency of neural stem cells with magnetofection.

Primary neural stem cells (NSCs) can be cultivated and differentiated in vitro but are difficult to transfect using conventional methods. We describe a simple and rapid magnetofection-based method suitable for the lab bench as well as for high-throughput projects. Our method yields high transfection efficiency and can be used for deciphering the genetic control of neural cell differentiation.

Nucleic acid delivery using magnetic nanoparticles: the Magnetofection technology.

In recent years, gene therapy has received considerable interest as a potential method for the treatment of numerous inherited and acquired diseases. However, successes have so far been hampered by several limitations, including safety issues of viral-based nucleic acid vectors and poor in vivo efficiency of nonviral vectors. Magnetofection has been introduced as a novel and powerful tool to deliver genetic material into cells. This technology is defined as the delivery of nucleic acids, either 'naked' or packaged (as complexes with lipids or polymers, and viruses) using magnetic nanoparticles under the guidance of an external magnetic field. This article first discusses the principles of the Magnetofection technology and its benefits as compared with standard transfection methods. A number of relevant examples of its use, both in vitro and in vivo, will then be highlighted. Future trends in the development of new magnetic nanoparticle formulations will also be outlined.

Magnetically guided lentiviral-mediated transduction of airway epithelial cells.

BACKGROUND: Lentiviral (LV) vectors are able to only slowly and inefficiently transduce nondividing cells such as those of the airway epithelium. To address this issue, we have exploited the magnetofection technique in in vitro models of airway epithelium. METHODS: Magnetofectins were formed by noncovalent interaction between LV particles and polycation-coated iron oxide nanoparticles. Efficiency of LV-mediated transduction (as evaluated through green fluorescent protein (GFP) expression by cytofluorimetric analysis) was measured in bronchial epithelial cells in the presence or absence of a magnetic field. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) release; cell monolayer integrity by measurement of transepithelial resistance (TER) and evaluation of correct zonula occludens-1 (ZO-1) localization at tight junctions (TJs) by immunofluorescence and confocal microscopy. RESULTS: In nonpolarized cells, magnetofectins enhanced LV-mediated transduction at multiplicity of infection (MOI) of 50 up to 3.9-fold upon a 24-h incubation, to levels that approached those achieved at MOI of 200 for LV alone, in the presence or absence of the magnetic field. Magnetofection significantly increased the percentage of transduced cells up to 186-fold already after 15 min of incubation. In polarized cells, magnetofection increased GFP+ cells up to 24-fold compared to LV alone. Magnetofection did not enhance LDH release and slightly altered TER but not ZO-1 localization at the TJs. CONCLUSIONS: We conclude that magnetofection can facilitate in vitro LV-mediated transduction of airway epithelial cells, in the absence of overt cytotoxicity and maintaining epithelial integrity, by lowering the necessary vector dose and reducing the incubation time required to achieve efficient transduction.

[In vitro and in vivo Magnetofection : a move towards gene therapy]. / Magnétofection in vitro et in vivo : une voie vers la thérapie génique.

Gene therapy offers exciting opportunities for the treatment of innate or acquired genetic diseases. However, there is still a need for a safe and efficient strategy to deliver nucleic acids into cells while overcoming the current limitations faced with standard viral vectors. Intensive researches have been carried out over the past decade, focusing both on viral and non-viral (i.e. physical or chemical) strategies. Of these numerous attempts, magnetofection, defined as the combination of nucleic acid vectors with magnetic nanoparticles, holds the promise to achieve high transfection efficiency with reduced toxicity by magnetically focusing the genetic material to be delivered on its cellular target. In vitro as well as in vivo results already demonstrated that this strategy may become a valuable tool towards practical gene therapy.

Liposomal magnetofection.

In a magnetofection procedure, self-assembling complexes of enhancers like cationic lipids with plasmid DNA or small interfering RNA (siRNA) are associated with magnetic nanoparticles and are then concentrated at the surface of cultured cells by applying a permanent inhomogeneous magnetic field. This process results in a considerable improvement in transfection efficiency compared to transfection carried out with nonmagnetic gene vectors. This article describes how to synthesize magnetic nanoparticles suitable for nucleic acid delivery by liposomal magnetofection and how to test the plasmid DNA and siRNA association with the magnetic components of the transfection complex. Protocols are provided for preparing magnetic lipoplexes, performing magnetofection in adherent and suspension cells, estimating the association/internalization of vectors with cells, performing reporter gene analysis, and assessing cell viability. The methods described here can be used to screen magnetic nanoparticles and formulations for the delivery of nucleic acids by liposomal magnetofection in any cell type.

Recent advances in magnetofection and its potential to deliver siRNAs in vitro.

This chapter describes how to design and conduct experiments to deliver siRNA to adherent mammalian cells in vitro by magnetic force-assisted transfection using self-assembled complexes of small interfering RNA (siRNA) and cationic lipids or polymers that are associated with magnetic nanoparticles. These magnetic complexes are targeted to the cell surface by the application of a magnetic gradient field. In this chapter, first we describe the synthesis of magnetic nanoparticles for magnetofection and the association of siRNA with the magnetic components of the transfection complex. Second, a simple protocol is described in order to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Third, protocols are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA, magnetofection, downregulation of gene expression, and the determination of cell viability. The addition of INF-7 peptide, a fusogenic peptide, to the magnetic transfection triplexes improved gene silencing in HeLa cells. The described protocols are also valuable for screening vector compositions and novel magnetic nanoparticle preparations to optimize siRNA transfection by magnetofection in every cell type.

siRNA delivery by magnetofection.

Magnetofection is defined as the magnetically enhanced delivery of nucleic acids associated with magnetic nanoparticles and has been utilized to deliver synthetic siRNAs to cultured cells. Certain magnetic nanomaterials associate with siRNAs and are suitable for siRNA delivery, either alone or in combination with cationic polymers or cationic lipid enhancers; these complexes are targeted to the cell surface by application of a gradient magnetic field. In this review methods are described to examine siRNA incorporation into magnetic complexes, to evaluate their magnetic responsiveness and to characterize their association with, and uptake into cells. These methods can be utilized to screen magnetic siRNA complexes for their suitability in functional siRNA delivery. Data, obtained since the first description of magnetofection in 2000, and novel results on the characterization of magnetic complexes containing synthetic siRNA are described. In addition, the benefits of siRNA delivery in vitro via magnetofection compared with standard non-magnetic methods of transfection using lipoplexes and polyplexes are highlighted.

Transcriptionally active polymerase chain reaction (TAP): high throughput gene expression using genome sequence data.

An approach is described for making transcriptionally active PCR (TAP) fragments that were used directly in in vitro and in vivo expression experiments. TAP fragments encoding reporter genes were amplified in 1 day using typical PCR methodology and were expressed in cultured cells and in mice at levels comparable with a widely used cytomegalovirus promoter-based plasmid expression vector. Following intramuscular injection, a TAP fragment encoding hepatitis B surface antigen (HBsAg) induced anti-HBsAg antibody titers comparable with those induced by supercoiled plasmid encoding the same antigen. Epitope-tagged TAP fragments were generated and transfected into cells for rapid, high throughput immunocytochemical analysis of the tagged gene products. TAP fragments were also transferred directly into expression vectors by in vivo homologous recombination without conventional cloning, affording a high throughput cloning approach that does not require restriction enzyme digestion, ligations, or thymidine adenine complementation cloning. The methodology has been adapted to a robotic work station enabling the high throughput generation of transcriptionally active genes at the rate of more than 400 different genes per day. This technology offers a practical approach to directly utilize genome sequence data to generate functional proteomes.