Effective polyethylenimine-mediated gene transfer into human endothelial cells

Background The major advantage in choosing non‐viral vectors such as cationic polymers for in vitro and in vivo transfection is their higher biosafety than viral ones. Among the cationic polymers, polyethylenimines (PEIs) are promising molecules for gene delivery to a variety of cells. Efficient transfection of primary endothelial cells using PEIs could be regarded as an interesting strategy of treatment in some ischemic cardiovascular diseases. Methods Efficacies of a 22‐kDa linear PEI (L‐PEI) and its glucose‐grafted derivative (L‐PEI‐Glc4) were compared for gene transfer into human umbilical vein endothelial cells (HUVEC) using the reporter gene luciferase. Cells were incubated for 2, 4 and 24 h with PEI/DNA complexes made in 150 mM sodium chloride (NaCl) or in 5% glucose solution. Luciferase activity was measured 24 h after the onset of transfection. The effects of low (2%) and high (30%) concentrations of serum on transfection efficacy were assessed as well. We then studied the intracellular fate of the PEI/DNA complexes labelled with the DNA intercalator YOYO‐1 using flow cytometry analysis (FACS) and confocal microscopy. Results PEI/DNA complexes formed in NaCl led to a higher transfection efficacy than those made in glucose. The optimal formulation, depending on the incubation time and the presence of serum in the medium, was obtained using DNA complexed to L‐PEI‐Glc4 and incubated for 4 h with the cells. This condition led to 50% fluorescent cells after GFP transfection. A high serum concentration diminished the L‐PEI associated toxicity but decreased L‐PEI‐Glc4 transfection efficiency. FACS analysis using both vectors showed that almost 90% of the cells had internalized the DNA complexes. Confocal microscopic observations showed a fast attachment of the complexes to the cell surface followed by inclusion into vesicles that migrated to the perinuclear region. Conclusions In this work, we defined the optimal conditions for gene delivery in HUVEC. These conditions were obtained when using derivatives L‐PEI and L‐PEI‐Glc4 complexed with DNA in 150 mM NaCl and added to cells for 2 and 4 h, respectively. Cellular trafficking of the complexes suggested that cell entry was not a limiting factor for gene delivery using PEI. This study underlined the interest in PEIs as efficient vectors for gene transfer into human endothelial cells

Transfection endovasculaire du gène du Vascular Endothelial Growth Factor (VEGF) vectorisé par le polyéthylènimine (PEI) dans un modèle d'ischémie périphérique chez le lapin (Optimisation des formulations PEI / ADN dans une stratégie thérapeutique angiogénique)

Le développement d'une circulation collatérale par des néovaisseaux bourgeonnant à partir de capillaires préexistants : angiogenèse, se développe dans les situations d'ischémie mais peut rester insuffisant face à la demande en oxygène et nutriements. Dans l'angiogenèse thérapeutique on administre un facteur angiogénique pour stimuler la vascularisation des tissus ischémiques. Le but de cette étude était de valider in vitro et dans un modèle animal d'ischémie périphérique, une thérapeutique angiogénique par transfection du gène codant pour le vascular endothelial growth factor 165 humain (VEGF165h) vectorisé par le polyéthylènimine (PEI), un polymère cationique. L'efficacité de transfection des dérivés, PEI linéaire (L-PEI) et PEI glycosylé (L-PEI-Glc4) a été comparée dans les HUVEC. Après 4 h d'incubation des complexes L-PEI-Glc4/ADN préparés dans le NaCl, le pourcentage de transfection à 24h atteint 50%, sans toxicité cellulaire majeure. En microscopie confocale, les complexes PEI/ADN s'internalisent par endocytose 2h après le début de la transfection. Ils migrent vers l'aire périnucléaire dans les 4h Lors de la transfection intra-artérielle utilisant un cathéter à ballonnet poreux chez le lapin, la formulation optimale de L-PEI-Glc4/ADN (1 mg/ml), est mise en contact 20 min avec la paroi vasculaire. Environ 10% des cellules endothéliales sont transfectées. Il n'y a pas de diffusion systémique du transgène dans les principaux organes.Chez le lapin après induction d'une ischémie périphérique, les complexes L-PEI-Glc4/VEGF sont transfectés au site de l'occlusion au septième jour. Ex vivo, la sécrétion de VEGF est 5 fois plus importante dans l'artère traitée que dans le contrôle. Trois semaines après la tansfection, le débit artériel, mesuré dans le membre ischemié est significativement plus élevé dans le groupe VEGF165 par rapport au groupe contrôle. L'angiogenèse thérapeutique par transfection génique du VEGF vectorisé par le PEI chez l'animal est efficace et sûre.Transfection endovasculaire du gène du Vascular Endothelial Growth Factor (VEGF) vectorisé par le polyéthylènimine (PEI) dans un modèle d'ischémie périphérique chez le lapin. Optimisation des formulations PEI/ADN dans une stratégie thérapeutique angiogénique.The spontaneous development of new collateral vessels by sprouting of preexisting capillaries: angiogenesis, occurred in ischemic tissues but may be inefficient to allow oxygen and nutriments supplies to tissues suffering ischemia. In therapeutic angiogenesis, angiogenic factors are delivered to tissues in order to stimulate the arterial growth in response to hypoxia. The aim of this work was to evaluate, in vitro and in a model of peripheral ischemia, the efficacy of an angiogenic therapeutic strategy using the gene transfer of the human vascular endothelial growth factor (VEGF165h) vectorised by, polyethylenimine (PEI), a cationic polymerThe transfection efficacies of two PEI derivatives: a linear PEI (L-PEI) and a glycosylated derivative (L-PEI-Glc4) were compared into HUVEC. After 4h of incubation, L-PEI-Glc4/DNA complexed in NaCl induced up to 50% of transfection quantified 24h later, without a major toxicity. Using confocal microscopy, the complexes internalised by endocytosis 2h after the onset of transfection. They were trafficking to the nuclear area 4h later.In rabbit, following arterial transfection with a channel catheter, the optimal L-PEI-Glc4/DNA (1 mg/ml) was infused for 20 min. Ten percent of the endothelial cells was transfected, without any systemic dissemination of the transgene in main organs. In the rabbit ischemic hindlimb model, L-PEI-Glc4/VEGF complexes were transfected at the occlusion site at day 7. Ex vivo secretion of VEGF was 5 times higher in the treated arteries compared to the control. The arterial flow measured 3 weeks after transfection at the occlusion site was significantly higher in the VEGF treated arteries than in control.Therapeutic angiogenesis by gene transfer using VEGF vectorised by PEI is efficient and safe in an animal model of peripheral ischemia.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Subpopulations of somatostatin-immunoreactive non-pyramidal neurons in the amygdala and adjacent external capsule project to the basal forebrain: evidence for the existence of GABAergic projection neurons in the cortical nuclei and basolateral nuclear complex

The hippocampus and amygdala are key structures of the limbic system whose connections include reciprocal interactions with the basal forebrain (BF). The hippocampus receives both cholinergic and GABAergic afferents from the medial septal area of the BF. Hippocampal projections back to the medial septal area arise from nonpyramidal GABAergic neurons that express somatostatin (SOM), calbindin (CB), and neuropeptide Y (NPY). Recent experiments in our lab have demonstrated that the basolateral amygdala, like the hippocampus, receives both cholinergic and GABAergic afferents from the BF. These projections arise from neurons in the substantia innominata and ventral pallidum. It remained to be determined, however, whether the amygdala has projections back to the BF that arise from GABAergic nonpyramidal neurons. This question was investigated in the present study by combining Fluorogold (FG) retrograde tract tracing with immunohistochemistry for GABAergic nonpyramidal cell markers, including SOM, CB, NPY, parvalbumin, calretinin, and glutamic acid decarboxylase (GAD). FG injections into the basal forebrain produced a diffuse array of retrogradely labeled neurons in many nuclei of the amygdala. The great majority of amygdalar FG+ neurons did not express nonpyramidal cell markers. However, a subpopulation of nonpyramidal SOM+ neurons, termed long range nonpyramidal neurons (LRNP neurons), in the external capsule, basolateral amygdala, and cortical and medial amygdalar nuclei were FG+. About one-third of the SOM+ LRNP neurons were CB+ or NPY+, and one-half were GAD+. It remains to be determined if these inhibitory amygdalar projections to the BF, like those from the hippocampus, are important for regulating synchronous oscillations in the amygdalar-BF network

Improved MECP2 gene therapy extends the survival of MeCP2-null mice without apparent toxicity after intracisternal delivery

Intravenous administration of AAV9/hMECP2 has been shown to extend the lifespan of Mecp2-/y mice, but this delivery route induces liver toxicity in wild-type (WT) mice. To reduce peripheral transgene expression, we explored the safety and efficacy of AAV9/hMECP2 injected into the cisterna magna (ICM). AAV9/hMECP2 (1 x 1012 vg; ICM) extended Mecp2-/y survival, but aggravated hindlimb clasping and abnormal gait phenotypes. In WT mice, 1 x 1012 vg of AAV9/hMECP2 induced clasping and abnormal gait. A lower dose mitigated these adverse phenotypes but failed to extend survival of Mecp2-/y mice. Thus, ICM delivery of this vector is impractical as a treatment for Rett syndrome (RTT). To improve the safety of MeCP2 gene therapy, the gene expression cassette was modified to include more endogenous regulatory elements believed to modulate MeCP2 expression in vivo. In Mecp2-/y mice, ICM injection of the modified vector extended lifespan and was well tolerated by the liver, but did not rescue RTT behavioral phenotypes. In WT mice, these same doses of the modified vector had no adverse effects on survival or neurological phenotypes. In summary, we identified limitations of the original vector and demonstrated that an improved vector design extends Mecp2-/y survival without apparent toxicity