Chemistry: molecular biology and microbiology – Vector – per se
Patent
1997-05-16
2000-07-04
Campell, Bruce R.
Chemistry: molecular biology and microbiology
Vector, per se
536 231, 530330, 530317, 530378, 530333, 530350, C12N 1563, C12N 1511, C07K 500, C07K 1400
Patent
active
060837411
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to compositions comprising DNA associated with a polycation entity which is itself linked to an integrin specific binding moiety. The compositions of the invention can be used to deliver DNA to cells for internalisation and expression therein. In particular, therefore, the invention relates to methods of obtaining gene expression in cells to overcome genetic deficiencies.
In recent years, with the continuing identification of specific genes responsible for certain disease conditions, the concept of "gene therapy" has attracted a great deal of attention. The potential to deliver a new gene, or even part of a gene sequence, to a defective cell in order to correct such an inherent deficiency is an attractive one. There are, of course, inherent problems in such an approach. For instance, the DNA must be delivered in a form that will be taken up, or internalised, by the target cell. Furthermore, the DNA itself must be expressed effectively in the cell in order to overcome the genetic deficiency. Inherent in these problems is the additional one that the DNA itself, after having entered the cell, must be protected in some way to prevent its damage, or even destruction, by, for example, cellular enzymes.
One potential approach to this problem of internalisation, and protection, of DNA is disclosed in Hart et al (J. Biol. Chem., 269, No 176: 12468-12474 (1994)). This approach exploits the presence of integrin receptors on cell surfaces for achieving internalisation of filamentous phage. Integrins are a super family of heterodimeric cell adhesion molecules that consist of several different .alpha. an .beta. subunits. Their cellular function is to mediate the movement, shape and polarity of cells through binding with proteins of the extracelluar matrix. In addition, integrins are exploited as receptors for cell entry by pathogenic bacteria, such as Yersinia pseudotuberculosis (Isberg, R., Science, 252: 934-938 (1991)) and Bordatella pertussis (Relamna et al, Cell, 61: 1375-1382 (1990)).
Hart et al (supra) found that displaying an integrin-binding peptide sequence on the surface of bacteriophage fD particles enabled the phage particles to be internalised by mammalian cells. However, no effective expression of DNA carried by phage particles was shown.
In addition, there are certain problems associated with the use of such particles to deliver DNA in this fashion. Firstly, there is a packaging size limitation governed by the size of the phage particle itself. Only genetic material up to a particular particle size could be delivered in this fashion. Secondly, the phage itself will only package single stranded genetic material, and this would not be effectively expressed in a mammalian cell system. Finally, the phage itself consists of other proteins and is a somewhat "messy" system for delivering DNA. It is possible that these additional components would have a material effect on whether or not genetic material was expressed.
Other approaches to delivering DNA into mammalian cells are disclosed in WO-A-9418834. Here, DNA was conjugated with a polyelectrolyte to form a complex which was then inserted into an embryonic cell, a germ cell or a germ precursor cell. This method was disclosed primarily for producing transgenic animals. The methods disclosed in this document rely on either microinjection of the complex directly into the germ cell, or by having the polycation/DNA complex present in the culture medium and relying on uptake by the cells.
Cotton et al (PNAS USA, 87: 4033-4037 (1990)) used the natural iron-delivery protein transferrin, coupled to DNA binding polycations such as polylysine or protamine, to deliver DNA into human leukaemic cells. However, they also found that they required the use of other agents to effect the survival of the transfected DNA or to modulate transferrin receptor levels so as to increase the internalisation or uptake of the DNA itself. These steps included increasing the transferring receptor density through treatment of the cells with the cell-permeable ion
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Harbottle Richard P.
Hart Stephen L.
Beckerleg AnneMarie S.
Campell Bruce R.
Imperial College of Science Technology And Medicine
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