DNA molecules, preparation and use in gene therapy

Details DNA molecules, preparation and use in gene therapy DNA molecules, preparation and use in gene therapy

2001-10-19

2004-11-30

Priebe, Scott D. (Department: 1632)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S091100, C435S091400, C435S455000, C435S320100, C435S325000, C435S252330, C435S254110

Reexamination Certificate

active

06825012

ABSTRACT:

Gene therapy consists in correcting a deficiency or an abnormality by introducing genetic information into the affected cell or organ. This information may be introduced either in vitro into a cell extracted from the organ and then reinjected into the body, or in vivo, directly into the tissue concerned. Being a high molecular weight, negatively charged molecule, DNA has difficulties in passing spontaneously through the phospholipid cell membranes. Different vectors are hence used in order to permit gene transfer: viral vectors on the one hand, natural or synthetic, chemical and/or biochemical vectors on the other hand. Viral vectors (retroviruses, adenoviruses, adeno-associated viruses, etc.) are very effective, in particular, in passing through membranes, but present a number of risks, such as pathogenicity, recombination, replication, immunogenicity, etc.
Chemical and/or biochemical vectors enable these risks to be avoided (for reviews, see Behr et al., Acc.Chem Res., 26, 274-278 (1993), Cotton et al., Curr. Biol., 4:705-710, 1993). These vectors are, for example, cations (calcium phosphate, DEAE-dextran, etc.) which act by forming precipitates with DNA. These precipitates can be “phagocytosed” by the cells. These vectors can also be liposomes in which DNA is incorporated and which fuse with the plasma membrane. Synthetic gene transfer vectors are generally lipids or cationic polymers that complex DNA and form a particle therewith carrying positive surface charges. These particles are capable of interacting with the negative charges of the cell membrane and then of crossing the latter. Dioctadecylamidoglycylspermine (DOGS, Transfectam™) or N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA, Lipofectin™) may be mentioned as examples of such vectors. Chimeric proteins have also been developed: they consist of a polycationic portion which condenses DNA, linked to a ligand which binds to a membrane receptor and carries the complex into the cells by endocytosis. It is thus theoretically possible to “target” a tissue or certain cell populations so as to improve the in vivo bioavailability of the transferred gene.
However, the use of chemical and/or biochemical vectors or of naked DNA implies the possibility of producing large amounts of DNA of pharmacological purity. In effect, in these gene therapy techniques, the medicinal product consists of the DNA itself, and it is essential to be able to manufacture, in appropriate amounts, DNAs having suitable properties for therapeutic use in man.
The plasmids currently used in gene therapy carry (i) an origin of replication, (ii) a selection marker gene such as a gene for resistance to an antibiotic (kanamycin, ampicillin, etc.) and (iii) one or more transgenes with sequences required for their expression (enhancer(s), promoter(s), polyadenylation sequences, etc.). These plasmids currently used in gene therapy (in clinical trials such as the treatment of melanomas, Nabel et al., Human Gene Therapy, 3:399-410, 1992, or in experimental studies) display, however, some drawbacks associated, in particular, with their dissemination in the body. Thus, as a result of this dissemination, a competent bacterium present in the body can, at a low frequency, receive this plasmid. The chance of this occurring is all the greater for the fact that the treatment in question entails in vivo gene therapy in which the DNA may be disseminated in the patient's body and may come into contact with bacteria which infect this patient or alternatively with bacteria of the commensal flora. If the bacterium which is a recipient of the plasmid is an enterobacterium such as
E. coli,
this plasmid may replicate. Such an event then leads to the dissemination of the therapeutic gene. Inasmuch as the therapeutic genes used in gene therapy treatments can code, for example, for a lymphokine, a growth factor, an anti-oncogene, or a protein whose function is lacking in the host and hence enables a genetic defect to be corrected, the dissemination of some of these genes could have unforeseeable and worrying effects (for example, if a pathogenic bacterium were to acquire the gene for a human growth factor).
Furthermore, the plasmids used in non-viral gene therapy also possess a marker for resistance to an antibiotic (ampicillin, kanamycin, etc.). Hence the bacterium acquiring such a plasmid has an undeniable selective advantage, since any therapeutic antibiotic treatment using an antibiotic of the same family as the one selecting the resistance gene of the plasmid will lead to the selection of the plasmid in question. In this connection, ampicillin belongs to the &bgr;-lactams, which is the family of antibiotics most widely used in the world.
It is hence necessary to seek to limit as far as possible the dissemination of the therapeutic genes and the resistance genes. Moreover, the genes carried by the plasmid, corresponding to the vector portion of the plasmid (function(s) required for replication, resistance gene), also run the risk of being expressed in the transfected cells. There is, in effect, a transcription background, which cannot be ruled out, due to the host's expression signals on the plasmid. This expression of exogenous proteins may be thoroughly detrimental in a number of gene therapy treatments, as a result of their potential immunogenicity and hence of the attack of the transfected cells by the immune system. In addition, immunostimulatory DNA sequences present in the plasmid backbone have been shown to trigger immune responses (Sato et al., 1996 Science 273: 352-354).
Hence, it is especially important to be able to have at one's disposal medicinal DNA molecules having a genetic purity suitable for therapeutic use. It also is especially important to have at one's disposal methods enabling these DNA molecules to be prepared in amounts appropriate for pharmaceutical use. The present invention provides a solution to these problems.
The present invention describes, in effect, DNA molecules that can be used in gene therapy, having greatly improved genetic purity and impressive properties of bioavailability. The invention also describes especially effective methods for the preparation of these molecules and for their purification.
The present invention lies, in particular, in the development of DNA molecules which can be used in gene therapy, virtually lacking any non-therapeutic region. The DNA molecules according to the invention, also designated minicircles on account of their circular structure, their small size, and their supercoiled form, display many advantages.
They make it possible, in the first place, to eliminate the risks associated with dissemination of the plasmid, such as (1) replication and dissemination which may lead to an uncontrolled overexpression of the therapeutic gene, (2) the dissemination and expression of resistance genes, and (3) the expression of genes present in the non-therapeutic portion of the plasmid, which are potentially immunogenic and/or inflammatory, and the like and (4) presence of immunostimulatory sequences. The genetic information contained in the DNA molecules according to the invention is limited, in effect, essentially to the therapeutic gene(s) and to the signals for regulation of its/their expression (neither origin of replication nor gene for resistance to an antibiotic or the like). The probability of these molecules (and hence of the genetic information they contain) being transferred to a microorganism and being stably maintained is almost zero.
Furthermore, due to their small size, DNA molecules according to the invention potentially have better bioavailability in vivo. In particular, they display improved capacities for cell penetration and cellular distribution. Thus, it is recognized that the coefficient of diffusion in the tissues is inversely proportional to the molecular weight (Jain, Cancer Res. 47: 3039-3051, 1987). Similarly, at the cellular level, high molecular weight molecules have inferior permeability through the plasma membrane. In addition, for the pla

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