Gene transfer into airway epithelial stem cell by using lentiviral vector pseudotyped with rna virus or dna virus spike protein

The present invention relates to lentiviral vectors pseudotyped with RNA or DNA virus spike proteins for introducing genes into airway epithelial stem cells.

Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder among Caucasians. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Such mutations prevent ion transfer across the airway epithelium, which leads to mucosal thickening and attachment of bacteria, ultimately causing the disorder. Therefore, airway epithelial cells are important targets for gene therapy.

For long-term expression of transgenes, it is important to introduce genes into airway epithelial progenitor cells including stem cells. “Stem cells” refers to undifferentiated cells which are multipotent and capable of self-renewal. In the case of the airway, stem cells are present near the ductal epithelia of the sub-mucosal glands and the basal cells of the basement membrane, and the ductal epithelium and the basal cells are protected from external toxins and injuries (see Non-patent Documents 1 and 2). Furthermore, in the mouse trachea, they are also reported to be scattered in the cartilage tissue (see Non-patent Document 1). In general, since epithelial stem cells are very few in number and exist in isolated locations in the sub-mucosal glands, it is difficult to adopt the gene transfer approach and such. Furthermore, it is known that airway epithelial cells including stem cells are tissues where gene transfer is very difficult due to the presence of the mucin layer and mucus.

Moreover, airway epithelial cells have polarity, and the side in contact with outside air is referred to as apical side, and the celomic side is referred to as basolateral side. Since there are no virus receptors on the apical surface of airway epithelial cells, there are hardly any vectors that can be efficiently transduced into intact airway epithelium. Receptors for most viruses including adenovirus are present on the basolateral side; therefore, preconditioning using EGTA, surfactants and such is necessary immediately before gene transfer. In the case of lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G), for example, the epithelial surface must be preconditioned with a detergent to perform efficient transfection. However, clinically it may not be possible to use treatment with these chemical substances.

Vectors for gene therapy and methods for their administration are being developed for genetic diseases of impaired respiratory system, such as cystic fibrosis. For gene transfer into airway epithelial cells, it is necessary to introduce the gene from the apical side of the epithelial cells by passing through the mucin layer and mucus. Invasion of virus vectors that infect from the basolateral side is blocked by tight junctions present between epithelial cells. Accordingly, administration methods that disrupt these tight junctions by the combined use of a calcium chelating agent, EGTA, and various surfactants are employed. However, such treatments (preconditioning) are not desirable for clinical application to humans.

Since VSV-G receptors that are generally used for pseudotyping are present on the basolateral side of airway epithelial cells, treatment with lysophosphatidylcholine (LPC; a type of surfactant) is necessary for gene transfer. When a mouse nasal cavity was infected with a VSV-G-pseudotyped HIV vector (carried gene; lacZ) after LPC treatment, the transgene expression was maintained for at least 92 days (see Non-patent Document 3). The result suggests the gene has been introduced into stem cells because epithelial cells lived three months. As an example of lentiviral vector pseudotyping without preconditioning, an envelope protein of the Ebola virus Zaire strain has been reported (see Non-patent Document 4). The carried gene LacZ was found to be maintained for 63 days in the mouse airway tissues. Although the duration of this experiment was shorter than the survival time of epithelial cells, the gene was confirmed to be transferred into the sub-mucosal gland where the stem cells are said to exist.

The following documents are also known.

[Non-patent Document 1] Borthwick et al., Am J. Respir. Cell Mol. Biol., Vol. 24, pp. 662-670, 2001
[Non-patent Document 2] Engelhardt, Am J. Respir. Cell Mol. Biol., Vol. 24, pp. 649-652, 2001

An objective of the present invention is to provide lentiviral vectors pseudotyped with RNA or DNA virus spike proteins for introducing genes into airway epithelial stem cells.

The present inventors carried out dedicated research to solve the above-mentioned problems. Specifically, to develop vectors that can introduce genes into airway epithelial stem cells, vectors of a simian immunodeficiency virus (SIV), a type of lentivirus, were pseudotyped with envelope glycoproteins F and HN, which are spike proteins of Sendai virus, a type of RNA virus.

The airway epithelium is covered with mucus, and it has been difficult to transfer genes into airway epithelial cells and such with conventional techniques. For introducing genes into these cells, a method that physically removes the extracellular matrix such as mucus by washing had been tried. However, this method was cumbersome and posed the danger of tissue damage.