Gene transfer methods

Details Gene transfer methods Gene transfer methods

2001-01-09

2004-09-07

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

Introduction of a polynucleotide molecule into or...

C435S455000, C435S320100, C435S005000, C435S006120, C435S007100, C435S007200, C435S325000, C435S366000, C435S395000

Reexamination Certificate

active

06787359

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a method that increases the efficiency of gene transfer into target cells and enables efficient transduction of the target cells, as well as a series of related techniques therewith, in the fields of medicine, cell technology, genetic engineering, developmental technology and the like.
BACKGROUND ART
Mechanisms of a number of human diseases have been elucidated. The recombinant DNA techniques and the techniques for transferring a gene into cells have rapidly progressed. Under these circumstances, protocols for somatic gene therapies for treating severe genetic diseases have been recently developed. More recently, attempts have been made to apply the gene therapy not only to treatment of the genetic diseases but also to treatment of viral infections such as AIDS and cancers.
In most of the gene therapies which have been examined for clinical application to humans to date, a gene is transferred into cells by using a recombinant retrovirus vector. The retrovirus vector efficiently transfers the foreign gene of interest into cells and stably integrates the gene into their chromosomal DNA. Therefore, it is a preferable means of gene transfer particularly for the gene therapy in which a long-term gene expression is desired. Such a vector has been subjected to various modifications so as not to have a harmful influence on the organism with the transferred gene. For example, the replication function of the vector is eliminated such that the vector used for the gene transfer does not replicate in the cells while repeating unlimited infection (gene transfer).
Since such a vector (a replication-deficient retrovirus vector) cannot autonomously replicate, a retrovirus vector encapsidated in a virus particle is generally prepared by using retrovirus-producer cells (packaging cells). The simplest method for efficiently transferring a gene into target cells comprises co-culturing the target cells with the retrovirus-producer cells. However, the retrovirus-producer cells may be contaminated in the gene transferred target cells which will be transplanted to a living body in this method.
Recently, it was reported that the presence of fibronectin, a component of the extracellular matrix, or a fragment thereof increases the efficiency of gene transfer into cells using a retrovirus (J. Clin. Invest., 93:1451-1457 (1994); Blood, 88:855-862 (1996)). Also, it has been demonstrated that a fibronectin fragment produced by genetic engineering technique has similar properties and can be utilized to efficiently transfer a foreign gene into hematopoietic stem cells (WO 95/26200). It is suggested that the binding of a heparin-binding region in fibronectin to a retrovirus is involved in the increase in gene transfer efficiency due to fibronectin.
Furthermore, it is disclosed in WO 97/18318 that functional substances other than fibronectin such as fibroblast growth factor increase the gene transfer efficiency. The publication also discloses that similar increase in the gene transfer efficiency is also observed when a mixture of a functional substance having an activity of binding to a retrovirus and another functional substance having an activity of binding to cells is used.
The gene transfer methods using functional substances enable an efficient gene transfer without co-cultivating retrovirus-producer cells and target cells. It is believed that the increase in gene transfer efficiency by the methods is due to the increase in chance of interaction between the retrovirus and the target cells which are closely co-localized with the aid of the functional substances.
In gene transfer using a retrovirus, target cells are infected with the retrovirus, resulting in gene transfer as described above. However, the gene transfer efficiency using a retrovirus is still unsatisfactory for practical clinical application. Thus, it is desired to further increase the infection efficiency.
Increased infection efficiency or gene transfer efficiency may be accomplished by increasing the concentration (titer) of the retrovirus in the virus suspension (supernatant) used. However, construction and establishment of virus-producer cells that can produce high titer viruses usually requires much labor. A pseudo-type virus vector utilizing an envelope protein from vesicular stomatitis virus [Proc. Natl. Acad. Sci. USA, 90:8033-8037 (1993)] can be concentrated by centrifugation. However, since such concentration can be used only for this vector, it can not be widely used.
Additionally, specific infection of target cells with a retrovirus in gene transfer may accomplish high gene transfer efficiency even if the purity of target cells is low. However, no convenient and efficient method is known in the current state of the art.
OBJECTS OF INVENTION
In view of the circumstances as described above, the main object of the present invention is to provide an improved method for transferring a gene into target cells using a retrovirus, in which the gene transfer efficiency is increased and the target cells are efficiently transduced.


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