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Adeno-associated virus (AAV) is a promising viral vector and meets most requirements of being a safe biological agent. However, the commercialization of AAV has been hampered due to the limitation of large-scale production, and only a small number of clinical trials have been launched. In recent years, progresses in scalable manufacturing of AAV have dramatically improved AAV-based clinical researches, and have assisted the development of investigational drug products. An AAV1-based investigational product, Glybera, has been formally approved by European Commission for the treatment of lipoprotein lipase deficiency (LPLD). Glybera was the first gene therapy product in the western world, and the production process involves a scalable baculovirus-insect cell system. However, many problems still need to be solved to improve the productivity and quality of AAV. The present review gives critical insights into current state-of-the-art scalable producing methodologies of AAV, such as baculovirus-insect cell system, HSV complementation system, and Ad complementation system, along with a discussion on the problems, solutions, and developmental trends. Novel AAV-producing platforms in Saccharomyces cerevisiae and vaccinia virus complementation system will also be discussed. [ABSTRACT FROM AUTHOR]

Lung infections with Pseudomonas aeruginosa and other pathogens in cystic fibrosis (CF) cause progressive airway obstruction and tissue damage, the predominant cause of morbidity and mortality in CF. We investigated whether a recombinant adeno-associated virus type 5 (AAV5) vector expressing murine interleukin (IL)-10 (AAV5.Cβ-mIL-10), a regulatory/anti-inflammatory cytokine, could decrease airway inflammation in IL-10 knockout mice chronically infected with mucoid P. aeruginosa. Mice that received AAV5.Cβ-mIL10 through intratracheal inoculation produced IL-10 at an average of 25 000 pg/ml in the epithelial lining fluid (ELF) and 12 000 pg/g-lung tissue 6 weeks post-vector delivery, significantly higher levels than in placebo-treated mice. At 3 days post-infection, proinflammatory cytokines (IL-1β, tumor necrosis factor (TNF)-α, macrophage inhibitory protein (MIP)-1α and (KC) in the ELF and lung homogenate were decreased (1–9 folds) in the AAV5.Cβ-mIL10-treated mice accompanied by less pronounced and more localized neutrophil infiltration in lung sections, when compared with placebo-treated mice. These results suggest that AAV5.Cβ-mIL10 induces IL-10 levels in the lungs mediating a significant anti-inflammatory response and making AAV-IL-10 gene transfer a potentially useful therapy in the treatment of CF lung disease. [ABSTRACT FROM AUTHOR]

Fragile X syndrome (FXS) is caused by a mutation that silences the fragile X mental retardation gene (FMR1), which encodes the fragile X mental retardation protein (FMRP). To determine whether FMRP replacement can rescue phenotypic deficits in a fmr1-knockout (KO) mouse model of FXS, we constructed an adeno-associated virus-based viral vector that expresses the major central nervous system (CNS) isoform of FMRP. Using this vector, we tested whether FMRP replacement could rescue the fmr1-KO phenotype of enhanced long-term depression (LTD), a form of synaptic plasticity that may be linked to cognitive impairments associated with FXS. Extracellular excitatory postsynaptic field potentials were recorded from CA3–CA1 synaptic contacts in hippocampal slices from wild-type (WT) and fmr1-KO mice in the presence of AP-5 and anisomycin. Paired-pulse low-frequency stimulation (PP-LFS)-induced LTD is enhanced in slices obtained from fmr1 KO compared with WT mice. Analyses of hippocampal synaptic function in fmr1-KO mice that received hippocampal injections of vector showed that the PP-LFS-induced LTD was restored to WT levels. These results indicate that expression of the major CNS isoform of FMRP alone is sufficient to rescue this phenotype and suggest that post-developmental protein replacement may have the potential to improve cognitive function in FXS. [ABSTRACT FROM AUTHOR]

A number of preclinical studies have shown the adeno-associated virus (AAV) to be an efficient vehicle for gene therapy. Clinical studies successfully demonstrated its potential for in vivo gene transfer. The complexity of host–vector interactions when progressing from small to large animal models, and eventually to humans, has impeded translation of AAV technology to the clinic. One approach to address this complexity has been to explore the biological characteristics of variations in AAV capsid structure. Initial strategies characterized the naturally occurring capsid variants from mammalian species. The structural and functional knowledge gathered on these natural AAV variants as vectors has led to the first series of second-generation vectors that aim at specifically improving certain properties by rational design of the capsid. A third exciting approach uses directed evolution to isolate vectors that are able to overcome selective pressures applied in the laboratory and thereby steer the capsid to evolve toward improved functionality.Gene Therapy (2009) 16, 311–319; doi:10.1038/gt.2008.170; published online 4 December 2008 [ABSTRACT FROM AUTHOR]

Inborn errors of metabolism are collectively common, frequently severe and in many instances difficult or impossible to treat. Accordingly, there is a compelling need to explore novel therapeutic modalities, including gene therapy, and examine multiple phenotypes where the risks of experimental therapy are outweighed by potential benefits to trial participants. Among available gene delivery systems recombinant AAV shows special promise for the treatment of metabolic disease given the unprecedented efficiencies achieved in transducing key target tissues, such as liver and muscle, in small animal models. To date over 30 metabolic disease phenotypes have been investigated in small animal studies with complete phenotype correction being achieved in a substantial proportion. Achieving adequately widespread transduction within the central nervous system, however, remains a major challenge, and will be critical to realization of the therapeutic potential of gene therapy for many of the most clinically troubling metabolic disease phenotypes. Despite the relatively low immunogenicity of AAV vectors, immune responses are also emerging as a factor requiring special attention as efforts accelerate toward human clinical translation. Four metabolic disease phenotypes have reached phase I or I/II trials with one, targeting lipoprotein lipase deficiency, showing exciting early evidence of efficacy.Gene Therapy (2008) 15, 831–839; doi:10.1038/gt.2008.64; published online 10 April 2008 [ABSTRACT FROM AUTHOR]

Autonomous parvoviruses possess an intrinsic oncotropism based on viral genetic elements controlling gene expression and genome replication. We constructed a hybrid vector consisting of the H1 parvovirus-derived expression cassette comprising the p4 promoter, the ns1 gene and the p38 promoter flanked by the adeno-associated viruses 2 (AAV2) inverted terminal repeats and packaged into AAV2 capsids. Gene transduction using this vector could be stimulated by coinfection with adenovirus, by irradiation or treatment with genotoxic agents, similar to standard AAV2 vectors. However, the latter were in most cases less efficient in gene transduction than the hybrid vector. With the new vector, tumor cell-selective increase in transgene expression was observed in pairs of transformed and non-transformed cells, leading to selective killing of the transformed cells after expression of a prodrug-converting enzyme. Preferential gene expression in tumor versus normal liver tissue was also observed in vivo in a syngeneic rat model. Comparative transduction of a panel of different tumor cell lines with the H1 and the H1/AAV hybrid vector showed a preference of each vector for distinct cell types, probably reflecting the dependence of the viral tropism on capsid determinants.Cancer Gene Therapy (2008) 15, 252–267; doi:10.1038/sj.cgt.7701113; published online 18 January 2008 [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis (MPS) IIIB is an inherited lysosomal storage disease, caused by the deficiency of α-N-acetylglucosaminidase (NaGlu), resulting in severe global neurological involvement with high mortality. One major hurdle in therapeutic development for MPS IIIB is the presence of the blood–brain barrier, which impedes the global central nervous system (CNS) delivery of therapeutic materials. In this study, we used a minimal invasive strategy, combining an intravenous (i.v.) and an intracisternal (i.c.) injection, following an i.v. infusion of mannitol, to complement the CNS delivery of adeno-associated viral (AAV) vector for treating MPS IIIB in young adult mice. This treatment resulted in a significantly prolonged lifespan of MPS IIIB mice (11.1–19.5 months), compared with that without treatment (7.9–11.3), and correlated with significantly improved behavioral performances, the restoration of functional NaGlu, and variable correction of lysosomal storage pathology in the CNS, as well as in different somatic tissues. This study demonstrated the great potential of combining i.v. and i.c. administration for improving rAAV CNS gene delivery and developing rAAV gene therapy for treating MPS IIIB in patients.Gene Therapy (2007) 14, 1065–1077; doi:10.1038/sj.gt.3302961; published online 26 April 2007 [ABSTRACT FROM AUTHOR]

Sjögren–Larsson syndrome (SLS) is an autosomal recessive neurocutaneous disorder caused by mutation in the ALDH3A2 gene that codes for human fatty aldehyde dehydrogenase (FALDH). Sjögren–Larsson syndrome patients lack FALDH, which catalyzes the oxidation of long-chain aliphatic aldehydes to fatty acids. The impaired FALDH activity leads to congenital ichthyosis, mental retardation and spasticity. The current lack of treatment is an impetus to develop gene therapy strategies by introducing functional FALDH into defective cells. We delivered human FALDH into keratinocytes of SLS patients using recombinant adeno-associated virus-2 vectors. Transduction of SLS keratinocytes resulted in an augmentation of FALDH activity comparable to phenotypically normal heterozygous carriers. Toxicity of long-chain aldehydes for FALDH-deficient cells decreased almost to the level of unaffected keratinocytes. Three-dimensional culture of corrected SLS keratinocytes revealed an ameliorated FALDH expression. These studies demonstrate the restoration of FALDH in human SLS cells supporting the concept of gene therapy as a potential future treatment option for SLS.Gene Therapy (2006) 13, 1021–1026. doi:10.1038/sj.gt.3302743; published online 9 March 2006 [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Age-related obesity is associated with impaired hypothalamic pro-opiomelanocortin (Pomc) gene expression. We assessed whether overproduction of POMC in the hypothalamus ameliorates age-related obesity in rats. Methods: Recombinant adeno-associated virus (rAAV) encoding Pomc (rAAV-Pomc) or control vector was delivered bilaterally into the basomedial hypothalamus of aged obese rats with coordinates targeting the arcuate nucleus. Energy balance, glucose metabolism, brown adipose tissue thermogenesis and mRNA levels of hypothalamic neuropeptides and melanocortin receptors were assessed. Results: Forty-two days after Pomc gene delivery, hypothalamic Pomc expression increased 12-fold while agouti-related protein and neuropeptide Y mRNA levels remained unchanged. Using a punch technique, we detected the highest Pomc RNA level in the arcuate nucleus. Pomc overexpression reduced food consumption from day 10 after vector injection, but this anorexic effect abated by day 30. In contrast, there was a steady decrease in body weight without apparent attenuation. Pomc gene delivery decreased visceral adiposity and induced uncoupling protein 1 in brown adipose tissue in aged rats. Serum NEFA and triglyceride levels were also diminished by rAAV-Pomc treatment. Improved glucose metabolism and insulin sensitivity were observed on day 36 but not day 20 after Pomc gene delivery. The expression of hypothalamic melanocortin 3 and 4 receptor decreased by 17% and 25%, respectively in rAAV-Pomc rats. Conclusions/interpretation: This study demonstrates that targeted Pomc gene therapy in the hypothalamus reduces body weight and visceral adiposity, and improves glucose and fat metabolism in aged obese rats. Thus long-term activation of the central melanocortin system may be a viable strategy to combat age-related obesity and diabetes. [ABSTRACT FROM AUTHOR]

Sjögren-Larsson Syndrome (SLS) is caused by an autosomal recessive defect in the gene coding for fatty aldehyde dehydrogenase (FALDH), an enzyme necessary for the oxidation of long-chain aliphatic aldehydes to fatty acid as one enzyme of the fatty alcohol:nicotinamide-adenine dinucleotide (NAD+)-oxidoreductase complex (FAO). The impaired activity of FALDH leads to the clinical symptom triad of generalized ichthyosis, mental retardation, and spastic diplegia or tetraplegia. Treatment options are primarily symptomatic. Gene therapy by means of genetic reintroduction of the functional FALDH gene into defective cells has so far not been considered as a therapeutic modality. In order to pursue such an approach for SLS, we constructed a recombinant adeno-associated virus-2 vector containing the human cDNA of functional FALDH and evaluated its capability to restore the enzyme-deficiency in a FALDH-deficient cell line resembling the gene defect of SLS. rAAV-2 transduction of FALDH-deficient cells, usually exhibiting less than 10% of normal FALDH activity, resulted in an increase of FALDH activity within the range of unaffected cells. Moreover, FALDH-transduced cells regained resistance over exposure to long chain aldehydes, which are otherwise toxic to FALDH-deficient cells. These results indicated that rAAV-2 vectors are able to restore FALDH-deficiency in a cell system resembling SLS. The findings give the first support to the concept that gene therapy might be a future option for the treatment of SLS. [ABSTRACT FROM AUTHOR]