Nucleic acid transfer phage

Details Nucleic acid transfer phage Nucleic acid transfer phage

2001-09-04

2004-05-25

Mosher, Mary E. (Department: 1648)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

Introduction of a polynucleotide molecule into or...

C530S350000, C435S320100, C435S252300, C435S252330, C435S235100, C435S069700, C435S975000, C536S023400

Reexamination Certificate

active

06740524

ABSTRACT:

TECHNICAL FIELD
The present invention belongs to the gene engineering, and relates to a technique for transporting a foreign substance such as nucleic acid by using virus particles, in particular.
BACKGROUND ART
The technology by which a foreign gene is delivered into animal cells is important for analysis of various biological phenomena, and for practical applications including gene therapy and creation of valuable animals. There are two major categories among the techniques used for DNA transfer: one is a biological approach relying on recombinant animal viruses carrying a foreign gene, and the other is a non-biological approach relying on physical force for delivering DNA into cells.
The former based on a principle by which recombinant viruses deliver their genome into the cells and integrate the viral genome into the host genome. This strategy is currently employed as a principal technique for gene therapy of Lesch-Nyhan syndrome and of adenosine deaminase (ADA) deficiency.
The strategies dependent on recombinant viruses, however, have been criticized for many disadvantages including pathogenicity of its own because it utilizes the biological features of intact viruses. In case of recombinant retrovirus vectors, the sequences involved in pathogenicity or replication are deleted from their genome for vector production. Nevertheless, gene-engineered vectors have still potentials of recovering infectivity through homologous recombination with endogenous retrovirus sequence in host cell genome. In addition, retrovirus vectors can only be applied to dividing cells due to the characteristics of the life cycle of the virus. Adenovirus vector, another virus vector, is proved to be effective in delivering genes into non-dividing cells like neurons. However, this vector has other problems in cytotoxicity and ntigenicity. The need of cultured mammalian cells for viral vector production is thought to be another disadvantage of viral vectors in terms of manufacturing.
Accordingly, non-viral strategies for DNA delivery have been used in parallel with those relying on recombinant viruses. Various methods have been established, in which purified DNA was delivered into the cells with the help of cationic compounds such as calcium phosphate, DEAE-dextran, cationic polymer and cationic liposome. However, due to a number of reasons, the transfection efficiency of these procedures is generally very low, comparing to the strategies using recombinant viruses. For example, the efficiency of nuclear delivery of DNA is quite insufficient in non-viral strategies. Thus, these non-viral strategies also have many disadvantages, which have to be overcome for the medical application such as gene therapy.
Gene delivery into mammalian cells consists of the following three major steps. Firstly, genetic materials should adsorb to and traverse across the cell membrane. Secondly, genetic materials should be transported from the cytoplasm into the nucleus. Thirdly, genetic materials should be properly transcribed to messenger RNA in the nucleus. Furthermore, if the genetic material is a piece of nucleic acid used for controlling the expression of endogenous genes, it should be localized to the site of transcription properly. All of these steps are processed relatively efficiently in the retrovirus-mediated gene transfer systems but inefficiently in non-viral gene delivery systems.
In order to improve the efficiency of gene transfer by non-viral delivery systems, scientists have tried to obtain the clues to facilitate each of these steps. For facilitating the nuclear delivery of a gene, application of nuclear localization signal (NLS) has been investigated. NLS is a stretch of amino acid residues that functions as a transport signal to the nucleus (Garcia-Bustos G. et al. Biochem. Biophys. Acta 1071:83-101 (1991)). It has been reported that NLS could promote the nuclear transport of various cytoplasmic proteins when attached to these proteins covalently or with recombinant DNA technology (Lanford R. E. et al. Cell 46:575-582 (1986); Yoneda Y. et al. Exp. Cell Res. 170:439-452 (1987); Chelsky D. et al. Mol. Cell. Biol. 9:2487-2492 (1989)). In most of the approaches to facilitate the nuclear delivery of DNA, NLS was attached to the DNA packed into a complex as small as the inner diameter of the nuclear pore. Poly-L-lysine (Perales J. C. et al. Eur. J. Biochem. 266:255-266 (1994)) and cationic lipids (Zabner J. et al. J. Biol. Chem. 270:18997-19007 (1995)) has been employed to pack the DNA, as well as proteins such as HMG-1 and histons.
These approaches using synthetic complexes, however, have several disadvantages as practical gene delivery tools. For example, the complex might be insoluble and heterogeneous in size, and the degree of the compaction is generally affected by salt concentration. Technical restriction to make a small complex may also limit the size of a gene to be delivered. In addition, current non-viral vector cannot be supplied as a product ready for use, because the complex containing DNA is unstable and should be prepared just before use. This is a big obstacle in consideration of commercial supply of the vector. Moreover, harsh procedures required for purifying bacterial plasmid DNA from impurities, such as alkaline-lysis method, hot phenol method, ethidium bromide/cesium chloride ultracentrifugation and chromatography, may damage the recovered DNA.
Recently, it was reported that the intrinsic membrane-penetrating activity of HIV TAT protein could be applied to the intracellular delivery of various substances (WO94/04686). It is established that a specific region of TAT protein consisting the amino acid residues, LGISYGRKKRRQRRRPPQ (SEQ ID NO: 1), is essential for transport into cells (Vives E. et al. J. Biol. Chem. 272:16010-16017 (1997)). In this prior art, this segment was linked to various proteins and double-stranded nucleic acids to assist their delivery across the cell membrane. However, this technology can be applied only to the delivery of small piece of nucleic acid that can regulate the expression of endogenous genes through competitive inhibition, and the enhancement of the delivery of DNA fragments large enough to contain intact exogenous genes has never been established in this system.
Capsid proteins of animal viruses, such as adenovirus and SV40, contain intrinsic NLS, which is implicated in the active nuclear transport of viral genome in the early phase of infection (Greberand U. F. and Kasamatsu I. Trends Cell Biol. 16:189-195 (1996)). In addition, SV40 particle with 50 nm in diameters was suggested to be transported into the nucleus as an intact particle (HumMeler K. et al. J. Virol. 6:87-93 (1970)). Another transport system in which an exogenous gene is packaged in the capsid MS-2 phage was also reported (Published Japanese Translation of International Patent Application No. Hei 7-508168). However, no reproducible system has been established in which the intracellular delivery of a large fragment of DNA is facilitated by NLS.
To overcome these problems, the present inventors previously established a novel transport system utilizing phage particles which displays NLS peptide on their head (WO98/06828). In this system, DNA fragments were encapsulated in the phage head and were transported into the nucleus efficiently. As a consequence, expression of encapsulated genes was stimulated significantly. However, the phage particles have to be delivered into the cytoplasm by microinjection for efficient nuclear transport, because NLS could not assist the penetration of nucleic acids across the cell membrane by itself.
Incorporation of a ligand to cell surface receptors into the gene transfer vehicles is another approach for improving the efficiency. The adsorption and uptake of the phage particles by cells can be improved when a binding ligand was displayed on the tail of lambda phage (WO96/21007). However, this approach could not improve the efficiency to transport DNA into the nucleus. In case of the recombinant lambda phage displaying the cell surface ligand (RGD peptide) on the

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