Nonviral gene transfer into the central nervous system (CNS) offers the prospect of providing safe therapies for neurological disorders and manipulating gene expression for studying neuronal function. However, results reported so far have been disappointing. We show that the cationic polymer polyethylenimine (PEI) provides unprecedentedly high levels of transgene expression in the mature mouse brain. Three different preparations of PEI (25-, 50-, and 800-kD) were compared for their transfection efficiencies in the brains of adult mice. The highest levels of transfection were obtained with the 25-kD polymer. With this preparation, DNA/PEI complexes bearing mean ionic charge ratios closest to neutrality gave the best results. Under such conditions, and using a cytomegalovirus (CMV)-luciferase construction, we obtained up to 0.4 10(6) RLU/microgram DNA (equivalent to 0.4 ng of luciferase), which is close to the values obtained using PEI to transfect neuronal cultures and the more easily transfected newborn mouse brain (10(6) RLU/microgram DNA). Widespread expression (over 6 mm3) of marker (luciferase) or functional genes (bcl2) was obtained in neurons and glia after injection into the cerebral cortex, hippocampus, and hypothalamus. Transgene expression was found more than 3 months post-injection in cortical neurons. No morbidity was observed with any of the preparations used. Thus, PEI, a low-toxicity vector, appears to have potential for fundamental research and genetic therapy of the brain.
Generally, cationic vector-based intravenous delivery of at a ratio of 4 nitrogen equivalents per DNA phosphate. DNA is hindered by interactions of positively charged comLower levels of transfection were found in the heart, plexes with serum proteins. However, if optimally formuspleen, liver and kidney. Expression was dose-and timelated, cationic vectors can provide reasonable levels of dependent in all tissues examined. In the lung, -galactotransfection in the lung either by intravenous or intrapulsidase staining showed transgene expression in clusters monary routes. We investigated the in vivo transfection of 10 or more pulmonary cells including the alveolar endocapacity of a cationic polymer: linear, 22 kDa polyethylenithelium, squamous and great alveolar epithelial cells (type mine. PEI/DNA complexes were formulated in 5% glucose I and II pneumocytes) and septal cells. These findings indiand delivered into adult mice through the tail vein. Two cate that the complexes pass the capillary barrier in the marker genes were used, -galactosidase and luciferase.lung. Although the delivery mechanism requires eluciHigh levels of luciferase expression (10 7 RLU/mg protein) dation, linear PEI has promise as a vector for intravenous were found in the lung when DNA was complexed with PEI transfer of therapeutic genes.Keywords: cationic polymers; pneumocytes; plasmid DNA; nonviral; gene therapy A number of generations of cationic vectors have been synthetised and tested in a variety of in vivo models and some have been taken to clinical trials. Most of these vectors are either monocationic or polycationic lipids, but there has been more recent interest in cationic polymers. Indeed, we showed that the branched cationic polymer polyethylenimine (PEI) can provide high levels of transfection in vivo. [1][2][3] In particular the lowest molecular weight preparation then commercially available, the 25 kDa preparation from Aldrich, was shown to be a versatile and efficient vector in the mammalian brain. 2 In a more recent study, 3 we chose to examine the effect of formulation procedures (glucose or saline solutions) on the size and in vivo transfection activity of a mixture of linear polymers with a mean MW of 22 kDa (Exgene 500; Euromedex, Souffleweyersheim, France). We found that the complexes formed in glucose were an order of magnitude smaller than those formed in saline and these complexes provided high levels of gene transfer following dilution into a physiological medium, the cerebrospinal fluid. In the light of these findings we chose to examine the effects of injecting complexes of plasmid DNA formulated with 22 kDa PEI in 5% glucose directly into the blood system and to examine transgene expression in a variety of organs.PEI-DNA complexes with different ratios of PEI nitro- gen to DNA phosphate (N/P ratio) were prepared in 5% glucose using a CMV-Luc plasmid 4 and the 22 kDa linear PEI. This PEI is synthesised to a degree of polymerisation of 510 units. Earlier experiments carried out with the branched 25 kDa PEI (Aldri...
Currently in vivo gene delivery by synthetic vectors is m). Intraventricular injection of complexes formulated in hindered by the limited diffusibility of complexes in extraglu-cose showed the complexes to be highly diffusible in cellular fluids and matrices. Here we show that certain the cerebrospinal fluid of newborn and adult mice, diffusing formulations of plasmid DNA with linear polyethylenimine from a single site of injection throughout the entire brain (22 kDa PEI, ExGene 500) can produce complexes that ventricular spaces. Transfection efficiency was followed by are sufficiently small and stable in physiological fluids so histochemistry of -galactosidase activity and double as to provide high diffusibility. When plasmid DNA was forimmunocytochemistry was used to identify the cells transmulated with 22 kDa PEI in 5% glucose, it produced a fected. Transgene expression was found in both neurons homogeneous population of complexes with mean diamand glia adjacent to ventricular spaces. Thus, this method eters ranging from 30 to 100 nm according to the amount of formulation is promising for in vivo work and may well of PEI used. In contrast, formulation in physiological saline be adaptable to other vectors and physiological models. produced complexes an order of magnitude greater (у1 Keywords: cationic polymers; polyethylenimine; brain; non-viral; gene transfer Gene transfer into the central nervous system (CNS) could provide a valuable technique for exploring the function of neural systems and a potential basis for therapy of neural disease. Until recently, the CNS appeared to present insurmountable barriers to the transfer of genes. First, the blood-brain barrier tends to preclude vascular delivery.1 Second, the principally postmitotic neuronal population prevents the use of retroviruses. Thus, to circumvent these problems and to obtain substantial levels of foreign gene expression in the CNS, progress must be made with vectors suitable for intrathecal delivery.Ideally vectors for gene transfer in the brain should have the following characteristics: they should not incur any safety risks (neither infectious, nor toxic), they should be easy to produce and verify, they should not limit the size of genes to be vectorised and they should be able to transduce post-mitotic neurons. Non-viral vectors can potentially fulfil all of these criteria. [2][3][4][5][6][7] In particular the branched cationic polymer polyethylenimine (PEI) has already been shown to be a versatile and efficient vector in the mammalian brain. In this study, we chose to examine the effect of formulation procedures (glucose or saline solutions) on the size and in vivo transfection activity of a mixture of linear polymers (repeated units of CH 2 -CH 2 -NH) with a mean MW of 22 kDa (Exgene 500; Euromedex, Souffleweyersheim, France). As the complexes formed in glucose were shown to be an order of magnitude smaller than those formed in saline, we examined the diffusibility and gene transfer efficiency of these complexes following injection into the...
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