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Abstract The identification of the cystic fibrosis (CF) gene opened the way for gene therapy. In the ten years since then, proof of principle in vitro and then in animal models in vivo has been followed by numerous clinical studies using both viral and non-viral vectors to transfer normal copies of the gene to the lungs and noses of CF patients. A wealth of data have emerged from these studies, reflecting enormous progress and also helping to focus and define key difficulties that remain unresolved. Gene therapy for CF remains the most promising possibility for curative rather than symptomatic therapy.

Aims Therapeutic angiogenesis is a potential new treatment for patients unsuitable for conventional revascularization strategies. We investigated angiogenesis via a ‘master switch gene’ hypoxia inducible factor (HIF-1α). [ABSTRACT FROM PUBLISHER]

Clinical trials of gene therapy for cystic fibrosis suggest that current levels of gene transfer efficiency are probably too low to result in clinical benefit, largely as a result of the barriers faced by gene transfer vectors within the airways. The respiratory epithelium has evolved a complex series of extracellular barriers (mucus, lack of receptors, immune surveillance, etc.) aimed at preventing penetration of lumenally delivered materials, including gene therapy vectors. In addition, once in the cell, further hurdles have to be overcome, including DNA degradation, nuclear import and the ability to maintain long-term transgene expression. Strategies to overcome these barriers will be addressed in this review and include the use of: (i) clinically relevant adjuncts to overcome the extra- and intracellular barriers; (ii) less-conventional delivery routes, such as intravenous or in utero administration; (iii) more efficient non-viral vectors and ‘stealth’ viruses which can be re-administered; and (iv) new approaches to prolong transgene expression by means of alternative promoters or integrating vectors. These advances have the potential to improve the efficiency of gene delivery to the airway epithelium, thus making gene therapy a more realistic option for cystic fibrosis. [Copyright &y& Elsevier]

Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer. [ABSTRACT FROM AUTHOR]

The identification of the cystic fibrosis (CF) gene opened the way for gene therapy. In the ten years since then, proof of principle in vitro and then in animal models in vivo has been followed by numerous clinical studies using both viral and non-viral vectors to transfer normal copies of the gene to the lungs and noses of CF patients. A wealth of data have emerged from these studies, reflecting enormous progress and also helping to focus and define key difficulties that remain unresolved. Gene therapy for CF remains the most promising possibility for curative rather than symptomatic therapy. [ABSTRACT FROM AUTHOR]

Oral administration of an adeno-associated virus vector that encodes the enzyme ß-galactosidase effectively reverses lactose intolerance in rats (pages 1131?1135). [ABSTRACT FROM AUTHOR]

Comments on gene therapy for asthma. Obstacles to establishing gene therapy as a treatment option in the near future; Gene transfer efficiency; Asthma pathogenesis.

A letter to the editor is presented in response to the article "A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis," by M. R. Knowles,, K. W. Hohneker and Z. Zhou, et al. in the September 28, 1995 issue.

Bactofection has emerged as an approach for gene therapy. Here, we assessed bactofection into airway epithelial cells in vitro and in vivo using E. coli genetically engineered to enter non-phagocytosing cells (invasive strain).CFTE and 293T cells were infected with E. coli - carrying a luciferase (lux) plasmid under control of the CMV promoter/enhancer (pCIKLux) - at MOIs of 50 to 500 for 2 hrs. 48 hrs post infection (p.i.) both cell lines showed dose-related expression of lux even at the lowest MOI used (CFTE-MOI 50: 119.7 ± 24.0, uninfected: 1.3 ± 1.2 RLU/mg protein, n=5, p<0.001) (293T-MOI 50: 628,040 ± 56,474, uninfected: 476 ± 192 RLU/mg protein, n=5, p<0.001).For in vivo experiments the lungs of mice were first inoculated with bacteria expressing GFP under the control of a prokaryotic promoter (5×107–5×109 bacteria/mouse, n=4) to visualise bacterial localisation. 1 hr p.i. the vast majority of GFP-positive bacteria were concentrated around the alveoli suggesting that bacteria are associated with the pneumocytes. However, bacteria were rarely associated with the conducting airway epithelium (the target for cystic fibrosis gene therapy).To assess, if bactofection leads to gene expression in vivo, mice were infected with invasive E. coli carrying pCIKLux (5×107 – 5×109 bacteria/mouse) and culled 48 hrs p.i.. Mice receiving the highest dose died within 2 hrs. Lux expression was detectable in mice receiving 2.5×109 E. coli (E. coli: 25.8 ± 2.7, uninfected: 0.15 ± 0.06 RLU/mg protein; p<0.01; n=4).It has previously been shown that the CMV promoter/enhancer is able to generate low level protein synthesis in gram-negative bacteria. Therefore, two terminations sequences were inserted into the plasmid (pCIKLux-mod) in an attempt to abolish prokaryotic lux expression. To assess, if the observed lux expression in vivo was due to plasmid transfer from bacteria into eukaryotic cells, mice were infected with invasive and non-invasive E. coli carrying the modified pCIKLux-mod as well as invasive and non-invasive E.coli carrying the unmodified pCIKLux (2.5×109 bacteria/mouse, n=8–9/group). 48 hrs p.i. lux expression was determined in lung homogenates. Lux expression was significantly reduced in mice infected with the modified invasive pCIKLux (E. coli/pCIKLux: 30.0 ± 14.4, E. coli/pCIKLux-mod: 2.11 ± 0.45 RLU/mg protein; p<0.01; n=8–9). Thus, the introduction of termination sequences significantly reduced prokaryotic expression from the CMV promoter. Further, lux expression in mice treated with non-invasive E. coli expressed similar levels of lux to their invasive counterparts. Several groups have reported successful bactofection in vitro, however, bacteria-mediated gene transfer does currently not lead to easily detectable gene expression in conducting airways, therefore, further illustrating that efficient transfection of airway epithelial cells is difficult.Molecular Therapy (2006) 13, S270–S271; doi: 10.1016/j.ymthe.2006.08.777 [ABSTRACT FROM AUTHOR]