Method for transferring nucleic acid into multicelled...

Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...

Reexamination Certificate

Rate now

  [ 0.00 ] – not rated yet Voters 0   Comments 0

Details

C435S006120, C435S091100, C435S455000, C536S023100, C536S024500

Reexamination Certificate

active

06528315

ABSTRACT:

The present invention relates to a very remarkable improvement in the in vivo transfer of nucleic acids into the cells of pluricellular eukaryotic organisms or of nucleic acids combined with products which make it possible to increase the yield of such transfers using weak electric fields of between 1 and 600 V/cm, and to the combination of a nucleic acid and the method of transfer according to the invention for their use in gene therapy.
The transfer of genes into a given cell is at the root of gene therapy. However, one of the problems is to succeed in causing a sufficient quantity of nucleic acid to penetrate into cells of the host to be treated; indeed, this nucleic acid, in general a gene of interest, has to be expressed in transfected cells. One of the approaches selected in this regard has been the integration of the nucleic acid into viral vectors, in particular into retroviruses, adenoviruses or adeno-associated viruses. These systems take advantage of the cell penetration mechanisms developed by viruses, as well as their protection against degradation. However, this approach has disadvantages, and in particular a risk of production of infectious viral particles capable of dissemination in the host organism, and, in the case of retroviral vectors, a risk of insertional mutagenesis. Furthermore, the capacity for insertion of a therapeutic or vaccinal gene into a viral genome remains limited.
In any case, the development of viral vectors capable of being used in gene therapy requires the use of complex techniques for defective viruses and for complementation cell lines.
Another approach (Wolf et al. Science 247, 1465-68, 1990; Davis et al. Proc. Natl. Acad. Sci. USA 93, 7213-18, 1996) has therefore consisted in administering into the muscle or into the blood stream a nucleic acid of a plasmid nature, combined or otherwise with compounds intended to promote its transfection, such as proteins, liposomes, charged lipids or cationic polymers such as polyethylenimine, which are good transfection agents in vitro (Behr et al. Proc. Natl. Acad. Sci. USA 86, 6982-6, 1989; Felgner et al. Proc. Natl. Acad. Sci. USA 84, 7413-7, 1987; Boussif et al. Proc. Natl. Acad. Sci. USA 92, 7297-301, 1995).
As regards the muscle, since the initial publication by J. A. Wolff et al. showing the capacity of muscle tissue to incorporate DNA injected in free plasmid form (Wolff et al. Science 247, 1465-1468, 1990), numerous authors have tried to improve this procedure (Manthorpe et al., 1993, Human Gene Ther. 4, 419-431; Wolff et al., 1991, BioTechniques 11, 474-485). A few trends emerge from these tests, such as in particular:
the use of mechanical solutions to force the entry of DNA into cells by adsorbing the DNA onto beads which are then propelled onto the tissues (“gene gun”) (Sanders Williams et al., 1991, Proc. Natl. Acad. Sci. USA 88, 2726-2730; Fynan et al., 1993, BioTechniques 11, 474-485). These methods have proved effective in vaccination strategies but they affect only the top layers of the tissues. In the case of the muscle, their use would require a surgical approach in order to allow access to the muscle because the particles do not cross the skin tissues;
the injection of DNA, no longer in free plasmid form but combined with molecules capable of serving as vehicle facilitating the entry of the complexes into cells. Cationic lipids, which are used in numerous other transfection methods, have proved up until now disappointing, because those which have been tested have been found to inhibit transfection (Schwartz et al., 1996, Gene Ther. 3, 405-411). The same applies to cationic peptides and polymers (Manthorpe et al., 1993, Human Gene Ther. 4, 419-431). The only case of a favourable combination appears to be the mixing of poly(vinyl alcohol) or polyvinylpyrrolidone with DNA. The increase resulting from these combinations only represents a factor of less than 10 compared with DNA injected in naked form (Mumper et al., 1996, Pharmaceutical Research 13, 701-709);
the pretreatment of the tissue to be injected with solutions intended to improve the diffusion and/or the stability of DNA (Davis et al., 1993, Hum. Gene Ther. 4, 151-159), or to promote the entry of nucleic acids, for example the induction of cell multiplication or regeneration phenomena. The treatments have involved in particular the use of local anaesthetics or of cardiotoxin, of vasoconstrictors, of endotoxin or of other molecules (Manthorpe et al., 1993, Human Gene Ther. 4, 419-431; Danko et al., 1994, Gene Ther. 1, 114-121; Vitadello et al., 1994, Hum. Gene Ther. 5, 11-18). These pretreatment protocols are difficult to manage, bupivacaine in particular requiring, in order to be effective, being injected at doses very close to lethal doses. The preinjection of hyperosmotic sucrose, intended to improve diffusion, does not increase the transfection level in the muscle (Davis et al., 1993).
Other tissues have been transfected in vivo either using plasmid DNA alone or in combination with synthetic vectors (reviews by Cotten and Wagner (1994), Current Opinion in Biotechnology 4, 705; Gao and Huang (1995), Gene Therapy, 2, 710; Ledley (1995), Human Gene Therapy 6, 1129). The principal tissues studied were the liver, the respiratory epithelium, the wall of the vessels, the central nervous system and tumours. In all these tissues, the levels of expression of the transgenes have proved to be too low to envisage a therapeutic application (for example in the liver, Chao et al. (1996) Human Gene Therapy 7, 901), although some encouraging results have recently been obtained for the transfer of plasmid DNA into the vascular wall (Iires et al. (1996) Human Gene Therapy 7,959 and 989). In the brain, the transfer efficiency is very low, likewise in tumours (Schwartz et al. 1996, Gene Therapy 3, 405; Lu et al. 1994, Cancer Gene Therapy 1, 245; Son et al. Proc. Natl. Acad. Sci. USA 91, 12669).
Electroporation, or use of electric fields to permeabilize cells, is also used in vitro to promote the transfection of DNA into cells in culture. However, it has up until now been accepted that this phenomenon responded to an effect which is dependent on a threshold and that this electropermeabilization could only be observed for electric fields of relatively high intensity, of the order of 800 to 1200 volts/cm for animal cells. This technique has also been proposed in vivo to improve the efficacy of antitumour agents, such as bleomycin, in solid tumours in man (American Patent No. 5, 468,228, L. M. Mir). With pulses of very short duration (100 microseconds), these electrical conditions (800 to 1200 volts/cm) are very well suited to the intracellular transfer of small molecules. These conditions (pulses of 100 microseconds) have been applied with no improvement for the transfer of nucleic acids in vivo into the liver, where fields of less than 1000 volts/cm have proved completely ineffective, and even inhibitory compared with the injection of DNA in the absence of electrical impulses (Patent WO 97/07826 and Heller et al. FEBS Letters, 389, 225-8, 1996).
There are in fact difficulties with applying this technique in vivo because the administration of fields of such an intensity may cause extensive tissue lesions to a greater or lesser extent which do not represent a problem for the treatment of cancer patients but which may have a major disadvantage for the healthy subject or the sick subject when the nucleic acid is administered into tissues other than tumour tissues.
Whereas all the studies cited mention the need for high electric fields, of the order of 1000 volts/cm, to be effective in vivo, in a truly unexpected and remarkable manner, the applicants have now shown that the transfer of nucleic acids into tissues in vivo could be very substantially increased, without undesirable effects, by subjecting the tissue to electrical pulses of low intensity, for example 100 or 200 volts/cm and of a relatively long duration. Furthermore, the applicants have observed that the high variability in the expression of the transgene observed in the prior art for th

LandOfFree

Say what you really think

Search LandOfFree.com for the USA inventors and patents. Rate them and share your experience with other people.

Rating

Method for transferring nucleic acid into multicelled... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method for transferring nucleic acid into multicelled..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for transferring nucleic acid into multicelled... will most certainly appreciate the feedback.

Rate now

     

Profile ID: LFUS-PAI-O-3020551

  Search
All data on this website is collected from public sources. Our data reflects the most accurate information available at the time of publication.