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

a technology of lentiviral vectors and airway epithelium, which is applied in the direction of peptide/protein ingredients, drug compositions, and genetically modified cells, can solve the problems of difficult gene transfer, hardly any vectors that can be efficiently transduced into intact airway epithelium, and difficult to adopt gene transfer approaches. achieve the effect of stable maintenance of gene expression and efficient introduction

Inactive Publication Date: 2016-09-29
ID PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about developing vectors that can deliver genes into airway epithelial stem cells without needing to first condition them. These vectors use proteins from airway-infecting viruses to create an efficient way to introduce genes into these cells. This technique allows for stable expression of genes beyond the life of the epithelial cells.

Problems solved by technology

Such mutations prevent ion transfer across the airway epithelium, which leads to mucosal thickening and attachment of bacteria, ultimately causing the disorder.
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.
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.
However, clinically it may not be possible to use treatment with these chemical substances.
However, such treatments (preconditioning) are not desirable for clinical application to humans.

Method used

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  • Gene transfer into airway epithelial stem cell by using lentiviral vector pseudotyped with RNA virus or DNA virus spike protein
  • Gene transfer into airway epithelial stem cell by using lentiviral vector pseudotyped with RNA virus or DNA virus spike protein
  • Gene transfer into airway epithelial stem cell by using lentiviral vector pseudotyped with RNA virus or DNA virus spike protein

Examples

Experimental program
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Effect test

example 1

Construction of Sendai Virus Envelope Protein Expression Plasmids

(1) Construction of Cytoplasmic Domain-Substituted HN Expression Plasmid

[0145]An HN expression plasmid was constructed, where the cytoplasmic domain of the HN protein was substituted with the cytoplasmic domain of the SIV envelope protein (FIG. 1). After annealing three sets of synthetic oligonucleotides (Xho+Xma / Xma−Xho, Xma+131 / 135−Xma, 132+Bam / Bam−136), they were incorporated in turn into the XhoI-BamHI site of pBluescript KS+. A purified synthetic oligonucleotide-linked fragment obtained by digesting the aforementioned recombinant plasmid with XhoI and DraIII and a purified fragment comprising the 3′ side of the HN protein obtained by digesting the HN protein expression plasmid pCAGGS-HN with DraIII and Bsu36I were incorporated into the XhoI-Bsu36I site of pCAGGS (Gene, vol. 108, pp. 193-200, 1991). The plasmid obtained by the above-mentioned method was used as the SIV cytoplasmic domain-substituted HN expression p...

example 2

Gene Transfer into Mouse Nasal Cavity Epithelial Cells Using the F / HN Pseudotyped SIV Vector

[0151]An F / HN pseudotyped SIV vector carrying eGFP was administered using a thin catheter to the mouse nasal cavity, the left nostril, without preconditioning (n=3, 4×10e8 TU per animal; 100 μl. The duration of expression of the transgenes was day 3 to day 360, and anatomical analysis of the mouse nasal cavity sections was performed according to the method of Mery et al. (Mery et al. Toxicol. Pathol. Vol. 22, p.353-3′72, 1994). GFP expression was evaluated in the nasal cavity sections located 1 to 4 mm (sections corresponding to distances of 1, 2, 3, and 4 mm) from the tip of the mouse nasal bone.

[0152]As a result, without preconditioning in airway epithelial cells, efficient transfection and continuous GFP expression was observed for at least 160 days. Expression of eGFP was found to be maintained even on day 220 and day 360. Since the survival time of mouse nasal cavity epithelial cells is ...

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Abstract

The present inventors successfully introduced genes into stem cells of airway epithelial tissues using simian immunodeficiency virus vectors pseudotyped with F and HN, which are envelope glycoproteins of Sendai virus. Gene transfer into airway epithelial tissue stem cells using a vector of the present invention is useful for gene therapy of genetic respiratory diseases such as cystic fibrosis. Furthermore, it is possible to select respiratory organs such as the lungs as production tissues for providing proteins that are deficient due to genetic diseases.

Description

STATEMENT REGARDING SEQUENCE LISTING[0001]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 270077_401C2_SEQUENCE LISTINGtxt. The text file is 6 KB, was created on Feb. 4, 2015, and is being submitted electronically via EFS-Web.TECHNICAL FIELD[0002]The present invention relates to lentiviral vectors pseudotyped with RNA or DNA virus spike proteins for introducing genes into airway epithelial stem cells.BACKGROUND ART[0003]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 t...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/86A61K38/17A61K35/76A61K48/00A61P11/00C12N5/074C12N5/10C12N7/00C12N7/01C12N15/09
CPCC12N15/86C12N2740/15043A61K48/00A61K38/177A61K38/1709C07K14/4712C12N5/0688C12N2510/00C12N2740/15045C12N2810/6072C12N2810/609A61K38/162A61P11/00C12N15/867C12N5/10A61K35/76A61K9/0043C12N7/00C12N2740/15033
Inventor MITOMO, KATSUYUKIINOUE, MAKOTOIWASAKI, HITOSHIHASEGAWA, MAMORUALTON, ERIC W.GRIESENBACH, UTA
Owner ID PHARMA
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