Production and purification of recombinant adeno-associated vectors.

The use of recombinant adeno-associated virus (rAAV) vectors in gene therapy for preclinical studies in animal models and human clinical trials is increasing, as these vectors have been shown to be safe and to mediate persistent transgene expression in vivo. Constant improvement in rAAV manufacturing processes (upstream production and downstream purification) has paralleled this evolution to meet the needs for larger vector batches, higher vector titer, and improved vector quality and safety. This chapter provides an overview of existing production and purification systems used for adeno-associated virus (AAV) vectors, and the advantages and disadvantages of each system are outlined. Regulatory guidelines that apply to the use of these systems for clinical trials are also presented. The methods described are examples of protocols that have been utilized for establishing rAAV packaging cell lines, production of rAAV vectors using recombinant HSV infection, and for chromatographic purification of various AAV vector serotypes. A protocol for the production of clinical-grade rAAV type 2 vectors using transient transfection and centrifugation-based purification is also described.

RNAi inhibition of mineralocorticoid receptors prevents the development of cold-induced hypertension.

The objective was to determine whether the mineralocorticoid receptor (MR) plays a role in the initiation and development of cold-induced hypertension (CIH) by testing the hypothesis that the RNA interference (RNAi) inhibition of the MR attenuates CIH. The recombinant adeno-associated virus (AAV) carrying a short-hairpin small-interference RNA for MR (MRshRNA) or a scrambled sequence (ControlshRNA) was constructed. Six groups of albino mice were used (6 mice/group). Three groups were exposed to cold (6.7 degrees C), whereas the remaining three groups were kept at room temperature (RT; warm) as controls. In each temperature condition, three groups received an intravenous injection of MRshRNA, ControlshRNA, or virus-free PBS, respectively, before exposure to cold. The viral complexes (0.35 x 10(11) particles/mouse, 0.5 ml) or PBS (0.5 ml) was delivered into the circulation via the tail vein. The blood pressure (BP) of the mice treated with ControlshRNA or PBS increased significantly during exposure to cold, whereas the BP of the cold-exposed MRshRNA-treated mice did not increase and remained at the level of the control group kept at RT. Thus AAV delivery of MRshRNA prevented the initiation of CIH. MRshRNA significantly attenuated cardiac and renal hypertrophy. MRshRNA decreased the cold-induced increase in MR protein expression to the control level in the hypothalamus, kidneys, and heart, indicating an effective prevention of the cold-induced upregulation of MR. RNAi inhibition of MR resulted in significant decreases in the plasma level of norepinephrine, plasma renin activity, and plasma level of aldosterone in cold-exposed mice. MR played a critical role in the initiation and development of CIH. AAV delivery of MRshRNA may serve as a new approach for the prevention of cold-induced hypertension.

Relationships between the physicochemical properties of an amphiphilic triblock copolymers/DNA complexes and their intramuscular transfection efficiency.

Poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer (PEO-PPO-PEO) based plasmid delivery systems are increasingly drawing attention in the field of nonviral gene transfer because of their proven in vivo transfection capability. They result from the simple association of DNA molecules with uncharged polymers. We examined the physicochemical properties of PEO-PPO-PEO/DNA mixtures, in which the PEO-PPO-PEO is Lutrol (PEO75-PPO30-PEO75), formulated under various conditions. We found that interactions between PEO-PPO-PEO and DNA are mediated by the central hydrophobic block within the block copolymer. Dynamic light scattering and cryo-electron microscopy showed that the mean diameter of transfecting particles as well as their stability depended on the PEO-PPO-PEO/DNA ratio and on the ionic composition of the formulating medium. The most active formulation promoting a good tissue-distribution and an optimal transfection was characterized by a reduced electrophoretic mobility, a mean hydrodynamic diameter of approximately 250-300 nm and by a conserved B-DNA form as shown by circular dichroism studies. Our study also revealed that the stability of these formulations strongly depended on a concentration balance between the DNA and the PEO-PPO-PEO, over which the DNA conformation was modified, micron-sized particles were generated, and the transgene expression was declined. We showed that the physicochemical properties of PEO-PPO-PEO/DNA formulations directly impact the level of gene expression in transfected muscles.

Amphiphilic block copolymers promote gene delivery in vivo to pathological skeletal muscles.

We reported that amphiphilic block copolymers hold promise as nonviral vectors for the delivery of plasmid DNA, ranging from 4.7 to 6.2 kb, to healthy muscle for the production of local or secreted proteins. To evaluate the efficiency of these vectors to deliver large plasmid DNA molecules to pathological muscles, plasmid DNAs of various lengths were complexed with Lutrol or poloxamine 304 and injected intramuscularly into dystrophic muscles. Lutrol-DNA and poloxamine 304-DNA complexes promoted gene transfer into muscles of the naturally occurring mouse model for DMD (mdx) in a dose- and plasmid DNA size-dependent manner. For small plasmid DNAs encoding reporter genes, this improvement over naked DNA was smaller in mdx than in the wild-type control strain. By contrast, Lutrol enabled us to deliver the large plasmid (16.1 kb) encoding the rod-deleted dystrophin in mdx mouse muscle, whereas the same amount of naked DNA did not lead to dystrophin expression, under the same experimental conditions. Lutrol-treated mdx mice showed the production of dystrophin in large numbers of muscle fibers. More importantly, we also found that expressing dystrophin with Lutrol led to restoration of the dystrophin-associated protein complex. Thus, we conclude that block copolymers constitute a novel class of vectors for the delivery of large plasmid DNA not only to healthy muscles but also to pathological muscle tissues.

Nonionic amphiphilic block copolymers promote gene transfer to the lung.

Various pulmonary disorders, including cystic fibrosis, are potentially amenable to a treatment modality in which a therapeutic gene is directly delivered to the lung. Current gene delivery systems, either viral or nonviral, need further improvement in terms of efficiency and safety. We reported that nonionic amphiphilic block copolymers hold promise as nonviral gene delivery systems for transfection of muscular tissues. To evaluate the efficiency of these vectors in the lung, intratracheal instillation or aerosolization of reporter genes complexed with Lutrol or PE6400 was performed. Lutrol-DNA and, to a lesser extent, PE6400-DNA complexes promoted efficient gene transfection into mouse airways in a dose-dependent manner. This improvement over naked DNA was observed irrespective of the reporter gene. Lutrol enabled us to deliver significantly higher DNA amounts than current nonviral vectors, with even greater increases in gene expression and without the formation of colloidally unstable complexes. Time course studies showed that Lutrol-DNA complexes permitted prolonged gene expression for up to 5 days whereas with poly(ethylenimine) (PEI)-DNA polyplexes, expression peaked on days 1-2 postinstillation, was strongly reduced by day 5, and reached background levels on day 7. Aerosolized delivery of Lutrol-DNA complexes, a less invasive approach to deliver genes to the lung, gave 5- to 15-fold higher reporter gene expression compared with PEI-DNA polyplexes administered via the same delivery route. After intratracheal instillation of Lutrol-DNA complexes, histochemical staining for beta-galactosidase expression showed the presence of large blue areas. Histopathological analysis showed that Lutrol alone did not elicit inflammation, and that the inflammatory response after intratracheal instillation of Lutrol-DNA complexes was reversible and was observed only with the highest amounts of DNA. We also found that Lutrol can efficiently deliver genes to the airways of cystic fibrosis mice. Thus, we conclude that Lutrol is a highly promising vector for gene delivery to the lung.

Inducible production of erythropoietin using intramuscular injection of block copolymer/DNA formulation.

BACKGROUND: We have previously shown that intramuscular injection of plasmid DNA formulated with a non-ionic amphiphile synthetic vector [poly(ethylene oxide)(13)-poly(propylene oxide)(30)-poly(ethylene oxide)(13) block copolymer; PE6400] increases reporter gene expression compared with naked DNA. We have now investigated this simple non-viral formulation for production of secreted proteins from the mouse skeletal muscle. METHODS: Plasmids encoding either constitutive human secreted alkaline phosphatase or murine erythropoietin inducible via a Tet-on system were formulated with PE6400 and intramuscularly injected into the mouse tibial anterior muscle. RESULTS: PE6400/DNA formulation led to an increased amount of recombinant alkaline phosphatase secreted from skeletal muscle as compared with naked DNA. In the presence of doxycycline, a single injection of 10 microg plasmid encoding inducible murine erythropoietin formulated with PE6400 significantly increased the hematocrit, whereas the same amount of DNA in the absence of PE6400 had no effect. The increase in the hematocrit was stable for 42 days. The tetracycline-inducible promoter permitted pharmacological control of hematocrit level after DNA intramuscular injection. However, 4 months post-injection the hematocrit returned to its pre-injection value, even in the presence of doxycycline. This phenomenon was likely caused by an immune response against the tetracycline-activated transcription factor. CONCLUSIONS: Intramuscular injection of plasmid DNA formulated with PE6400 provides an efficient and simple method for secretion and production of non-muscle proteins.

Negatively charged self-assembling DNA/poloxamine nanospheres for in vivo gene transfer.

Over the past decade, numerous nonviral cationic vectors have been synthesized. They share a high density of positive charges and efficiency for gene transfer in vitro. However, their positively charged surface causes instability in body fluids and cytotoxicity, thereby limiting their efficacy in vivo. Therefore, there is a need for developing alternative molecular structures. We have examined tetrabranched amphiphilic block copolymers consisting of four polyethyleneoxide/polypropyleneoxide blocks centered on an ethylenediamine moiety. Cryo-electron microscopy, ethidium bromide fluorescence and light and X-ray scattering experiments performed on vector-DNA complexes showed that the dense core of the nanosphere consisted of condensed DNA interacting with poloxamine molecules through electrostatic, hydrogen bonding and hydrophobic interactions, with DNA molecules also being exposed at the surface. The supramolecular organization of block copolymer/DNA nanospheres induced the formation of negatively charged particles. These particles were stable in a solution that had a physiological ionic composition and were resistant to decomplexation by heparin. The new nanostructured material, the structure of which clearly contrasted with that of lipoplexes and polyplexes, efficiently transferred reporter and therapeutic genes in skeletal and heart muscle in vivo. Negatively charged supramolecular assemblies hold promise as therapeutic gene carriers for skeletal and heart muscle-related diseases and expression of therapeutic proteins for local or systemic uses.

Ligand-mediated transcription elongation control using triplex-based padlock oligonucleotides.

Triplex-forming oligonucleotides (TFOs) provide useful tools for the artificial regulation of gene expression at the transcriptional level. They can become topologically linked to their DNA target upon circularization, thereby forming very stable triple helical structures. These "padlock oligonucleotides" are able to interfere with transcription elongation when their target site is located in the transcribed region of a gene. In vitro transcription experiments showed that a bacterial RNA polymerase was stopped at the site of triple-helix formation, whereas expression of a reporter gene was inhibited in live cells. In both cases, the padlock oligonucleotide was more efficient at inhibiting transcription elongation than a linear TFO, and the inhibition was observed only in the presence of a triplex stabilizing agent. These results provide new insights into the ligand-modulated locking of padlock oligonucleotides around their DNA target.