Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-01-24
2001-09-11
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S235100, C435S320100, C435S325000, C424S093200
Reexamination Certificate
active
06287864
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a gene transfer method with the use of a serum-free medium, more specifically, to a method that elevates the gene transfer efficiency into target cells and enables efficient transformation of the target cells, and to a series techniques concerned therewith in the field of medicine, cell technology, gene technology, 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.
Most of the gene transfer trials in humans approved by Food and Drug Administration (FDA) to date are ones in which a gene is transferred into cells with the use of a recombinant retrovirus vector. Since the retrovirus vector efficiently transfers the foreign gene of interest into cells and stably integrates the gene into their chromosomal DNAs, it is a preferable means of gene transfer particularly for the gene therapy in which a long-term gene expression is desired. This vector has been subjected to various modifications so as not to have a bad influence on the organism with the transferred gene.
For example, the replication function of the vector is deleted 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 replicate by itself, a retrovirus in which the vector is encapsidated in a virus particle is generally prepared by using retrovirus producer cells (packaging cells).
Bone marrow cells are the preferable target cells for the somatic gene therapy since they can be handled in vitro and contain hematopoietic stem cells that are capable of self-replicating. It has been demonstrated that umbilical cord blood also contains a number of progenitor cells including hematopoietic stem cells. By performing the gene therapy in which a gene is transferred into such target cells, which are then transplanted into an organism, the transferred gene can be expressed in blood cells for a long period, and a disease can be healed for life.
However, the hematopoietic stem cell is one of the cells into which a gene is not readily transferred with high efficiency, despite of the studies by many groups. To date, the most efficient protocol for gene transfer with respect to hematopoietic stem cells from mice or other animals have been the method in which the hematopoietic stem cells are co-cultured with retrovirus producer cells. However, under the apprehension about safety, gene transfer in cell-free system with a lower risk of contamination of the retrovirus producer cells has been desired for clinical gene therapy methods for humans. Unfortunately, it is not easy to efficiently transfer a gene into hematopoietic stem cells without co-culturing with retrovirus producer cells.
Recently, it was reported that fibronectin, which is a component of the extracellular matrix, or a fragment thereof alone elevates the gene transfer efficiency into cells by 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).
Furthermore, it is disclosed in WO 97/18318 that a functional substance other than fibronectin (such as fibroblast growth factor, collagen etc.) elevates the gene transfer efficiency and that similar increase in the gene transfer efficiency is observed when a mixture of a functional substance having a retrovirus-binding activity and another functional substance having target cell-binding activity is used.
It is believed that the increase in gene transfer efficiency caused by these functional substances is due to the increase in chance of interaction between the retrovirus and the target cells which are co-localized with the aid of the substances.
OBJECTS OF THE INVENTION
In the gene transfer methods using the retrovirus as described above, infection with the retrovirus (i.e., gene transfer) occurs when the target cells are cultured in a medium containing the retrovirus. A medium containing a serum from an animal (in many cases, fetal calf serum (FCS)) is used in this step. Since the serum contains constituents that can serve as nutrients for cells and various growth factors, it is believed that the serum is highly effective for maintaining cells in vitro.
It is believed that the component or the contents of constituents in the serum derived from an animal vary depending on the condition of the health or the like of the animal individual from which the serum was collected. Therefore, reproducible results are not always obtained if the cultivation of the cells and/or the gene transfer is carried out using sera of different lots. Furthermore, since a serum from a heterologous organism other than, for example, a human contains substances antigenic against a human, suitable washing steps are required to decrease the contents of the antigenic substances when the cells maintained in the presence of such a serum are transplanted into a human. In addition, the quality of the serum must be strictly controlled such that the serum does not contain viruses, mycoplasmas or the like.
As described above, the gene transfer methods with the use of serum-containing media have problems, of which the solution is desired.
SUMMARY OF THE INVENTION
The present inventors have studied intensively and surprisingly found that the use of serum-free medium elevates the gene transfer efficiency as compared with a conventional serum-containing medium and that a particularly high gene transfer efficiency is accomplished using a medium to which low-density lipoprotein is added. Thus, the present invention has been completed.
The first invention of the present invention relates to a method for transferring a gene into target cells by a retrovirus, comprising infecting target cells with a retrovirus in serum-free culture medium in the presence of a functional substance in an amount effective in elevating the gene transfer efficiency of the retrovirus into the target cells by co-localizing the retrovirus and the target cells.
There is no limitation regarding the functional substance to be used in the present invention. For example, a substance that has a retrovirus-binding site and a target cell-binding site in a single molecule or a mixture of a molecule that has a retrovirus-binding site and another molecule that has a target cell-binding site can be used. For example, a functional substance such as fibronectin, fibroblast growth factor, collagen and polylysin, or a substance having a retrovirus-binding activity equivalent thereto (e.g., a heparin-binding substance other than those described above) can be used as the functional substance that has the retrovirus-binding site. As the functional substance that has the target cell-binding site, for example, a substance having a ligand that binds to the target cell can be used.
The functional substance preferable to the present invention includes, for example, a fibronectin fragment having a retrovirus-binding site such as a heparin-II-binding region and a cell-binding site such as a binding region to VLA-5 and/or VLA-4. For example, the polypeptide of which the amino acid sequence is shown in the SEQ ID NO:1 of the Sequence Listing (CH-296) is a fibronectin fragment that has a heparin-II-binding region and binding regions to VLA-5 and VLA-4.
There is no limitation regarding a culture medium to be used in the method of the present invention as long as it does not contain a serum. A medium prepared by mixi
Bagnis Claude
Imbert Anne-Marie
Mannoni Patrice
Browdy and Neimark
Guzo David
Leffers, Jr. Gerald G.
Takara Shuzo Co. Ltd.
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