Biocompatible polymer enhances the in vitro and in vivo transfection efficiency of HVJ envelope vector

BACKGROUND: Vector development is critical for the advancement of human gene therapy. However, the use of viral vectors raises many safety concerns and most non-viral methods are less efficient for gene transfer. One of the breakthroughs in vector technology is the combination of the vector with various polymers. METHODS: HVJ (hemagglutinating virus of Japan) envelope vector (HVJ-E) has been developed as a versatile gene transfer vector. In this study, we combined HVJ-E with cationized gelatin to make it a more powerful tool and assessed its transfection efficiency in vitro and in vivo. In addition, we investigated the mechanism of the gene transfer by means of the inhibition of fusion or endocytosis. RESULTS: The combination of both protamine sulfate and cationized gelatin with HVJ-E, referred to as PS-CG-HVJ-E, further enhanced the in vitro transfection efficiency. In CT26 cells, the luciferase gene expression of PS-CG-HVJ-E was approximately 10 times higher than that of the combination of protamine sulfate with HVJ-E or the combination of cationized gelatin with HVJ-E, referred to as PS-HVJ-E or CG-HVJ-E, respectively. Furthermore, the luciferase gene expression in liver mediated by intravenous administration of CG-HVJ-E was much higher than the luciferase gene expression mediated by PS-HVJ-E or PS-CG-HVJ-E and approximately 100 times higher than that mediated by HVJ-E alone. CONCLUSIONS: Cationized gelatin-conjugated HVJ-E enhanced gene transfection efficiency both in vitro and in vivo. These results suggest that low molecular weight cationized gelatin may be appropriate for complex formation with various envelope viruses, such as retrovirus, herpes virus and HIV.     

Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action

BACKGROUND: Rational design of gene vectors for therapeutic applications requires understanding of transfection mechanisms. In this study, multiple transfection assays revealed complementary mechanisms between two commonly used transfection agents. This finding was then exploited to produce improved transfection outcomes. METHODS AND RESULTS: Rat C6 glial cells, adult rat hippocampal progenitor cells and primary astrocytes were transfected using Lipofectamine (LA) or polyethylenimine (PEI), in vitro. Although LA- and PEI-transfected populations expressed the same total level of transgene product, LA transfected considerably more cells than PEI (approximately 20 vs. 14%). A fluorescently labelled plasmid and time-course analysis, involving both flow cytometry and confocal microscopy, were used to explain this apparent discrepancy. Results showed that LA delivered more plasmid DNA to the cytoplasm and achieved transgene expression in more cells than PEI. In contrast, PEI transfected fewer cells but, on average, produced more transgene product per transfected cell. CONCLUSIONS: A comparative transfection model was developed to explain these different characteristics. According to this model, transfection is a multistage process with different transfection agents exerting their primary effect at different stages in this process. This model forecast that it should be possible to prepare a chimeric complex with a transfection efficiency that exceeded that achievable with Lipofectamine or polyethylenimine alone. This prediction was tested and shown to hold for glioma cells, primary astrocytes, and adult neural stems cells.     

Non‐viral nucleic acid delivery to the central nervous system and brain tumors

Gene therapy is a rapidly emerging remedial route for many serious incurable diseases, such as central nervous system (CNS) diseases. Currently, nucleic acid medicines, including DNAs encoding therapeutic or destructive proteins, small interfering RNAs or microRNAs, have been successfully delivered to the CNS with gene delivery vectors using various routes of administration and have subsequently exhibited remarkable therapeutic efficiency. Among these vectors, non‐viral vectors are favorable for delivering genes into the CNS as a result of their many special characteristics, such as low toxicity and pre‐existing immunogenicity, high gene loading efficiency and easy surface modification. In this review, we highlight the main types of therapeutic genes that have been applied in the therapy of CNS diseases and then outline non‐viral gene delivery vectors.     

The use of absorption enhancers to enhance the dispersibility of spray-dried powders for pulmonary gene therapy

BACKGROUND: Pulmonary gene therapy requires aerosolisation of the gene vectors to the target region of the lower respiratory tract. Pulmonary absorption enhancers have been shown to improve the penetration of pharmaceutically active ingredients in the airway. In this study, we investigate whether certain absorption enhancers may also enhance the aerosolisation properties of spray-dried powders containing non-viral gene vectors. METHODS: Spray-drying was used to prepare potentially respirable trehalose-based dry powders containing lipid-polycation-pDNA (LPD) vectors and absorption enhancers. Powder morphology and particle size were characterised using scanning electron microscopy and laser diffraction, respectively, with gel electrophoresis used to assess the structural integrity of the pDNA. The biological functionality of the powders was quantified using in vitro cell (A549) transfection. Aerosolisation from a Spinhaler dry powder inhaler into a multistage liquid impinger (MSLI) was used to assess the in vitro dispersibility and deposition of the powders. RESULTS: Spray-dried powder containing dimethyl-beta-cyclodextrin (DMC) demonstrated substantially altered particle morphology and an optimal particle size distribution for pulmonary delivery. The inclusion of DMC did not adversely affect the structural integrity of the LPD complex and the powder displayed significantly greater transfection efficiency as compared to unmodified powder. All absorption enhancers proffered enhanced powder deposition characteristics, with the DMC-modified powder facilitating high deposition in the lower stages of the MSLI. CONCLUSIONS: Incorporation of absorption enhancers into non-viral gene therapy formulations prior to spray-drying can significantly enhance the aerosolisation properties of the resultant powder and increase biological functionality at the site of deposition in an in vitro model.     

The use of amino acids to enhance the aerosolisation of spray-dried powders for pulmonary gene therapy

BACKGROUND: Pulmonary delivery of gene therapy offers the potential for the treatment of a range of lung conditions, including cystic fibrosis, asthma and lung cancer. Spray-drying may be used to prepare dry powders for inhalation; however, aerosolisation of such powders is limited, resulting in poor lung deposition and biological functionality. In this study, we examine the use of amino acids (arginine, aspartic acid, threonine, phenylalanine) to enhance the aerosolisation of spray-dried powders containing model non-viral gene vectors. METHODS: Lipid/polycation/pDNA (LPD) vectors, in the presence or absence of amino acids, were dispersed in lactose solutions, and spray-dried to produce appropriately sized dry powders. Scanning electron microscopy and laser diffraction were used to determine particle morphology and diameter, respectively. Gel electrophoresis was used to examine the influence of amino acids on the structural integrity of the LPD complex. In vitro cell (A549) transfection was used to determine the biological functionality of the dry powders, and the in vitro aerosolisation performance was assessed using a multistage liquid impinger (MSLI). RESULTS: Both gel electrophoresis and in vitro cell transfection indicated that certain amino acids (aspartic acid, threonine) can adversely affect the integrity and biological functionality of the LPD complex. All amino acids significantly increased the aerosolisation of the powder, with the arginine and phenylalanine powders showing optimal deposition in the lower stages of the MSLI. CONCLUSIONS: Amino acids can be used to enhance the aerosolisation of spray-dried powders for respiratory gene delivery, allowing the development of stable and viable formulations for pulmonary gene therapy.     

Interaction of polyamine gene vectors with RNA leads to the dissociation of plasmid DNA-carrier complexes

BACKGROUND: Plasmid DNA (pDNA) dissociation from polyamine gene vectors after cellular uptake has not been well characterized. A more detailed understanding of this process could lead to more efficient gene transfer agents. Since RNA is present in the cytoplasm at high concentrations and due to its structural similarity to DNA, we were interested in its conceivable interaction with polyamine gene vectors. METHODS: In a first set of experiments gene vectors were incubated in cell lysate and pDNA release was investigated by Southern blot analysis with or without RNase A pretreatment and by confocal laser scanning microscopy. Further, interaction of polyamine gene vectors with RNA was investigated by fluorescence quenching assay. These methods were complemented by a functionality assay using isolated nuclei. RESULTS: The incubation of gene vectors with cell lysate resulted in the dissociation of pDNA from the complexes. This effect was abolished when the cell lysate was pretreated with RNase A. The addition of RNA in the absence of cell lysate led also to a dissociation of pDNA. This process commenced instantaneously after the addition of RNA as analyzed by fluorescence quenching. When gene vectors were incubated in cell lysate containing isolated nuclei, the dissociation of pDNA from the polyamine gene vectors occurred preferentially extranuclearally as confirmed by confocal laser scanning microscopy. These results were further corroborated in a functional assay. CONCLUSIONS: These data suggest that RNA induces pDNA dissociation from the polyamine gene vectors. Furthermore, this process apparently occurs in the cytoplasm before the gene vectors enter the nucleus.     

High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system

Background

Gene therapy strategies for the treatment of vascular disease such as the prevention of post‐angioplasty restenosis require efficient, non‐toxic transfection of vascular cells. In vitro studies in these cells contribute to vector development for in vivo use and for the evaluation of genes with therapeutic potential. The aim of this project was to evaluate a novel synthetic vector consisting of a liposome (L), an integrin targeting peptide (I), and plasmid DNA (D), which combine to form the LID vector complex.

Methods

Cultures of porcine smooth muscle cells and endothelial cells were established and then transfected with the LID vector, using the reporter genes luciferase and green fluorescent protein and the metalloprotease inhibitor TIMP‐1.

Results

The LID vector system transfected primary porcine vascular smooth muscle cells and porcine aortic endothelial cells with efficiency levels of 40% and 35%, respectively. By increasing the relative DNA concentration four‐fold, incubation periods as short as 30 min achieved the same levels of luciferase transgene expression as 4 h incubations at lower DNA concentrations. The transfection did not affect cell viability as measured by their proliferative potential. Serum levels of up to 20% in the transfection medium had no adverse affect on the efficiency of transfer and gene expression in either cell type. Transfections with the cDNA for TIMP‐1 produced protein levels that peaked at 130 ng/ml per 24 h and persisted for 14 days at 10 ng/ml per 24 h.

Conclusion

This novel vector system has potential for studies involving gene transfer to cardiovascular cells in vitro and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Analysis of the intracellular barriers encountered by nonviral gene carriers in a model of spatially controlled delivery to neurons

BACKGROUND: Neuron-specific, nonviral gene delivery vehicles are useful tools for the potential treatment of neurological disease and spinal cord injury. For minimally invasive, peripheral administration, gene carriers must efficiently mediate uptake at axon terminals, retrograde axonal transport, vesicular escape, and nuclear entry. The design of improved vehicles will benefit from an understanding of the barriers that limit nonviral delivery to neurons. Here, we demonstrate a detailed analysis of intracellular trafficking of both a lipid-based and a polymer-based delivery vehicle following site-specific exposure to neuron-like cells. METHODS: Site-specific exposure of gene carriers to soma or neurites of neuron-like PC-12 cells was accomplished using a microfluidic, compartmented culture chamber. Binding and internalization of vehicles at neurites and soma were quantified using an environmentally sensitive fluorescent marker. The intracellular transport of gene carriers was analyzed by time-lapse particle tracking in live cells, and transfection efficiencies were measured using green fluorescent protein (GFP) as a reporter gene. RESULTS: While the lipid-based carrier mediated measurable transfection when delivered to neuronal soma, neuritic delivery of this formulation failed to produce reporter gene expression due to limited internalization and transport. In contrast, the polymeric nanoparticles displayed active retrograde transport toward neuronal soma, but failed to produce measurable reporter gene expression. CONCLUSIONS: These results highlight distinct intracellular barriers preventing efficient neuronal transfection by the nonviral carriers examined, and provide a basis for the rational improvement of existing nonviral systems.     

In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage

BACKGROUND: Understanding the mechanisms underlying gene electrotransfer muscle damage can help to design more effective gene electrotransfer strategies for physiological and therapeutical applications. The present study investigates the factors involved in gene electrotransfer associated muscle damage. METHODS: Histochemical analyses were used to determine the extent of transfection efficiency and muscle damage in the Tibialis anterior muscles of Sprague-Dawley male rats after gene electrotransfer. RESULTS: Five days after gene electrotransfer, features of muscle degeneration and regeneration were consistently observed, thus limiting the extent of transfection efficiency. Signs of muscle degeneration/regeneration were no longer evident 21 days after gene electrotransfer except for the presence of central myonuclei. Neither the application of electrical pulses per se nor the extracellular presence of plasmid DNA per se contributed significantly to muscle damage (2.9 +/- 1.0 and 2.1 +/- 0.7% of the whole muscle cross-sectional area, respectively). Gene electrotransfer of a plasmid DNA, which does not support gene expression, increased significantly muscle damage (8.7 +/- 1.2%). When plasmid DNA expression was permitted (gene electrotransfer of pCMV-beta-galactosidase), muscle damage was further increased to 19.7 +/- 4.5%. Optimization of cumulated pulse duration and current intensity dramatically reduced gene electrotransfer associated muscle damage. Finally, mathematical modeling of gene electrotransfer associated muscle damage as a function of the number of electrons delivered to the tissue indicated that pulse length critically determined the extent of muscle damage. CONCLUSION: Our data suggest that neither the extracellular presence of plasmid DNA per se nor the application of electric pulses per se contributes significantly to muscle damage. Gene electrotransfer associated muscle damage mainly arises from the intracellular presence and expression of plasmid DNA.     

Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection

BACKGROUND: The mechanism of gene transfer into hepatocytes by the hydrodynamics-based transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the transfection. METHODS: We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with (35)S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA. RESULTS: When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of (35)S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time. CONCLUSIONS: Our results show that the efficiency of hydrodynamics-based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure.     

Spatial and acoustic pressure dependence of microbubble-mediated gene delivery targeted using focused ultrasound

BACKGROUND: Ultrasound/microbubble-mediated gene delivery has the potential to be targeted to tissue deep in the body by directing the ultrasound beam following vector administration. Application of this technology would be minimally invasive and benefit from the widespread clinical experience of using ultrasound and microbubble contrast agents. In this study we evaluate the targeting ability and spatial distribution of gene delivery using focused ultrasound. METHODS: Using a custom-built exposure tank, Chinese hamster ovary cells in the presence of SonoVue microbubbles and plasmid encoding beta-galactosidase were exposed to ultrasound in the focal plane of a 1 MHz transducer. Gene delivery and cell viability were subsequently assessed. Characterisation of the acoustic field and high-resolution spatial analysis of transfection were used to examine the relationship between gene delivery efficiency and acoustic pressure. RESULTS: In contrast to that seen in the homogeneous field close to the transducer face, gene delivery in the focal plane was concentrated on the ultrasound beam axis. Above a minimum peak-to-peak value of 0.1 MPa, transfection efficiency increased as acoustic pressure increased towards the focus, reaching a maximum above 1 MPa. Delivery was microbubble-dependent and cell viability was maintained. CONCLUSIONS: Gene delivery can be targeted using focused ultrasound and microbubbles. Since delivery is dependent on acoustic pressure, the degree of targeting can be determined by appropriate transducer design to modify the ultrasound field. In contrast to other physical gene delivery approaches, the non-invasive targeting ability of ultrasound makes this technology an attractive option for clinical gene therapy.