Gene transfer methods

Abstract

Improved methods for transferring a gene into target cells by using a retrovirus, wherein the gene transfer efficiency is improved and the target cells are efficiently transformed by binding the retrovirus to a functional substance which is immobilized on as carrier and having an activity of binding to retroviruses followed by washing; using an antibody capable of specifically recognizing cells, laminin or mannose-rich type sugar chain as a substance having an activity of binding to the target cells; pre-treating the target cells so as to inactivate transferrin receptor, or introducing a new functional group into the functional substance.

Description

[0001] This is a division of copending parent application Ser. No. 09 / 743,354, which is the national stage under 35 USC 371 of the international application PCT / JP99 / 03403, filed Jun. 25, 1999.[0002] 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.[0003] 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 a...

AI Technical Summary

Benefits of technology

0101] Effect of Enhancing Gene Transfer Efficiency of Laminin

0102] Mouse laminin (Gibco) or human laminin (Takara Shuzo) was used in combination with a functional substance having an activity of binding to a virus to carry out gene transfer experiment. A 24-well non-treated microplate for cell culture (Falcon) used in the experiment was coated with these functional substances according to the following two methods.

0103] The cocktail method: A mixture of two functional substances is added to the plate. The plate is allowed to stand at 4 C. overnight. The plate is blocked with 2% BSA at 37 C. for 20 minutes and then washed with PBS.

0104] The pre-coating method: A solution of a functional substance having an activity of binding to a virus is added to the plate. The plate is allowed to stand at 4 C. overnight. The solution is removed. A laminin solution is added to the plate. The plate is incubated at 37 C. for 2 hours, blocked with 2% BSA, and then washed with PBS.

0105] 0.5 ml of the solution of the functional substance was used to coat each well.

0106] 10.sup.5 L1210 cells and 0.5 ml of Eco-EGFP virus supernatant (10.sup.5 cfu / ml) were added to the well. The plate was incubated for 24 hours. After incubation, the cells were collected by using a cell detachment buffer (Bio Whittaker) and washed. EGFP-expressing cells were analyzed by flow cytometry using FACSVantage (Becton Dickinson) at an excitation wavelength of 488 nm and an emission wavelength of 515-545 nm. The gene transfer efficiency (the ratio of EGFP-expressing cells to total cells) was calculated. The experimental results are shown in Tables 2 to 5.

Problems solved by technology

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.

However, the gene transfer efficiency using a retrovirus is still unsatisfactory for practical clinical application.

However, construction and establishment of virus-producer cells that can produce high titer viruses usually requires much labor.

However, since such concentration can be used only for this vector, it can not be widely used.

However, no convenient and efficient method is known in the current state of the art.

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