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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.

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.

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.

International audience; Background: Gene delivery within the central nervous system at postnatal age is one of the most challenging tasks in neuroscience and currently only a few effective methods are available. Comparison with existing methods: For postnatal central nervous system cells, viral approaches are commonly used for genetic engineering but they face several biosafety requirements for production and use making them less accessible to the community. Conversely, lipid-based methods are widely used in cell culture but face limitation in vivo mainly due to the inflammatory responses they induce. To this aspect, the use of a transgenic mouse line can represent a credible answer to the community working on rat models still requires an effective and successful solution to circumvent these difficulties. New method: We describe a new polymer-based gene delivery system allowing persistent and robust in vivo transfection with low DNA amount, reduced inflammation and high diffusion. The expression profile along the brain, the stability, the diffusion of the DNA together with the quantity of cells transfected were evaluated through in vivo approaches. Results: With a single low-volume injection, we targeted different cell types within the rat brain. We measured the diffusion rate ranging from 1 to 5 mm based on the injected volume, in the three-dimensions axis. Finally, we modified brain susceptibility to epileptic seizures using a specific knock-down of the neuronal specific potassium chloride transporter 2. Conclusions: This safe and easy system opens perspectives for non viral gene delivery in the rat brain with perspectives to study brain function in vivo.

Summary Objective SCN1A encodes the alpha subunit of the voltage-gated sodium channel and plays a crucial role in several epilepsy syndromes. The common SCN1A splice-site polymorphism rs3812718 (IVS5N+5 G>A) might contribute to the pathophysiology underlying genetic generalized epilepsies and is associated with electrophysiologic properties of the channel and the effect of sodium-channel blocking antiepileptic drugs. We assessed the effects of the rs3812718 genotype on cortical excitability at baseline and after administration of carbamazepine in order to investigate the mechanism of this association. Methods Paired-pulse transcranial magnetic stimulation (TMS) was applied in 92 healthy volunteers with the homozygous genotypes AA or GG of rs3812718 at baseline and after application of 400 mg of carbamazepine or placebo in a double-blind, randomized, crossover design. Resting motor threshold (RMT), short interval intracortical inhibition (SICI), intracortical facilitation (ICF), and cortical silent period (CSP) were determined. Results At baseline there was no significant difference in any TMS parameter. Genotype GG was associated with a higher carbamazepine-induced increase in CSP duration as compared to AA (multivariate analysis of covariance [MANCOVA], p = 0.013). An expected significant increase in RMT was genotype independent. Significance We found that the rs3812718 genotype modifies the effect of carbamazepine on CSP duration (mainly reflecting modulation of γ-aminobutyric acid (GABA)ergic inhibition), but not on RMT (mainly reflecting modulation of voltage-gated sodium channels). This provides evidence that rs3812718 affects the pharmacoresponse to carbamazepine via an effect on GABAergic cortical interneurons. Our results also confirm that TMS is useful to investigate the effect of genetic variants on cortical excitability and pharmacoresponse.

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.

Cationic liposomes are a useful in vitro but as yet unproven in vivo delivery system for oligonucleotides. An understanding of the mechanism of delivery mediated by cationic lipid/oligonucleotide complexes has been lacking. In this review, we describe recent results concerning several steps of the delivery process, including the formation of complexes, the intracellular distribution of the oligonucleotide and the lipid, as well as the uptake pathway and site of intracellular release. Cationic liposomes form a polyelectrolyte complex with the oligonucleotides, protect them from nuclease degradation, enhance their cellular uptake and improve the oligonucleotide potency. In the majority of cell types studied, cationic lipids deliver oligonucleotides into the cell predominately via an endocytotic pathway rather then by fusion with the plasma membrane. We propose that the oligonucleotide is released from the complex when anionic lipids from the cytoplasmic facing lipid monolayer of the cell flip into c...

Encapsulation of oligonucleotides in antibody-targeted liposomes (immunoliposomes) which bind to target cells permits intracellular delivery of the oligonucleotides. This approach circumvents problems of extracellular degradation by nucleases and poor membrane permeability which free phosphodiester oligonucleotides are subject to, but leaves unresolved the inefficiency of encapsulation of oligonucleotides in liposomes. We have coupled oligonucleotides to cholesterol via a reversible disulfide bond. This modification of oligonucleotides improved their association with immunoliposomes by a factor of about 10 in comparison to unmodified oligonucleotides. The presence of cholesteryl-modified oligonucleotides incorporated in the bilayer of liposomes did not interfere with the coupling of the targeting protein to the liposome surface. Free or cholesterol coupled oligonucleotides associated with liposomes and directed against the tat gene of HIV-1 were tested for inhibition of HIV-1 proliferation in acutely infected cells. We demonstrate that the cholesteryl-modified as well as unmodified oligonucleotides acquire the target specificity of the antibody on the liposome. Their antiviral activity when delivered into cells is sequence-specific. The activity of these modified or unmodified oligonucleotides to inhibit the replication of HIV was the same on an equimolar basis (EC50 around 0.1 microM). Cholesterol coupled oligonucleotides thus offer increased liposome association without loss of antiviral activity.

Experiments performed in our laboratory or in collaboration with other groups demonstrating the delivery to cells of mono-, oligo- and polynucleotides from antibody-targeted small liposomes are reviewed. Biologically-active molecules delivered into cells via these liposomes include: phosphorylated derivatives of dideoxyuridine, which have activity against the human immunodeficiency virus; the oligonucleotide (2'-5') (A)n and various analogues, which have anti-viral and antiproliferative activity; and antisense phosphodiester and phosphorothioate oligodeoxynucleotides, which may have both gene-specific and nonspecific effects. Polynucleotides include: the RNA duplex poly (rI:rC), and related molecules, which are inducers of interferon and other cytokines; long RNA antisense molecules and plasmids. Advantages for delivery by liposomes, as compared to use of the same molecules free in solution include: protection against degradation, reduction of toxicity, improved pharmacokinetics and the possibilit...

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. 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. 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. 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.

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.

We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.

Among the 2′,3′-dideoxynucleoside 5′-triphosphates containing a physiological base, 2′,3′-dideoxyuridine 5′-triphosphate (ddUTP) has been reported to be among the most powerful inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT) in cell-free systems. However, in contrast to other dideoxynucleosides, 2′,3′-dideoxyuridine (ddU) is inactive in treatment of HIV-infected cells in culture, since it is a poor substrate for cellular nucleoside kinases. This problem cannot be overcome by the use of phosphorylated ddU because such compounds are unable to cross cell membranes. To promote entry and thus bypass the limiting steps of intracellular phosphorylation, we have encapsulated mono- and tri-phosphorylated ddU in liposomes coupled to monoclonal antibodies (immunoliposomes). We investigated antiviral effects in two human T cell lines (MT-4, CEM). We observed that ddU nucleotides remain phosphorylated for several weeks after encapsulation in immunoliposomes, and potent antiviral activity is obtained when these drugs are delivered into infected cells by cell-specific antibodies (ED50 ⩽1 μM on CEM). In contrast, no inhibition was observed with non-targeted liposomes containing phosphorylated ddU, or with empty liposomes, whether targeted or not.

We have examined two T lymphocyte cell surface molecules, CD4 and CD7, as targets for specific delivery of drugs from antibody-directed liposomes. The efficiency of uptake by peripheral lymphocytes, thymocytes, and two CEM sublines (CEM.MRS and CEM-T4) of anti-CD4 and anti-CD7 liposomes containing methotrexate was evaluated by the methotrexate-mediated inhibition of the incorporation of d -[ 3 H]Urd into DNA. This was compared with similar liposomes targeted to MHC-encoded HLA class I molecules, which are known to be efficiently taken up by T cells. Despite the lower expression of CD7 molecules relative to HLA class I on most cell lines, CD7 was shown to be a good target for drug delivery. The results of an internalization study using radiolabeled Protein A showed that a higher proportion of CD7 molecules was internalized than HLA class I molecules. CD4-targeted liposomes, in contrast, were relatively ineffective for drug delivery for lymphoid cells, and only partially inhibited CEM-T4 cells. The lack of toxicity correlated with poor internalization of the target molecule on most cell lines. The drug effect of anti-CD4 liposomes was more pronounced on HeLa-T4, which is an epithelial cell line transfected with the CD4 gene. In contrast to lymphoid cells, these cells efficiently internalized CD4 molecules. PMA is known to down-regulate surface expression of CD4 molecules on various T cells. Internalization of CD4 was induced by PMA, but PMA failed to induce cytotoxicity of CD4-targeted liposomes for CEM.MRS. The internalized drug was probably degraded rapidly because internalized anti-CD4 antibody-bound Protein A was degraded very rapidly.