The ability to transfer nucleic acids into cells has vast experimental and therapeutic implications. Many different chemical, electrochemical and biological approaches have been used for this purpose. In vitro chemical methods include osmotic shock transformation of prokaryotic cells and calcium phosphate transfection and liposome-mediated transfer for eukaryotic cells. Nucleic acids, namely DNA, have also been delivered to cells by electroporation. While this latter approach is amenable to nucleic acid transfer in vitro, it is inherently unsuitable for in vivo use. Biological approaches have focused on viral strategies which include retroviral and most recently adenoviral mediated gene transfer into cells in culture and, in some instances, cells in vivo. A common disadvantage of the above-mentioned strategies is their inability to specifically target cells for nucleic acid delivery. Targeting of cell subsets usually requires the selective harvesting of cells followed by in vitro delivery and re-introduction in vivo.
Viral mediated gene transfer requires the in vitro production of defective viral particles which encapsulate a nucleic acid of a finite size. The encapsulated nucleic acid, usually referred to as a viral vector, is a recombinant nucleic acid which contains a gene(s) of interest cloned between 5′ and 3′ flanking viral cis elements. The cis elements are required for integration into the host genome yet they are also capable of transcriptional regulation. As a result, these elements have the potential to interfere with the transcriptional activity of the cloned gene(s). Another limitation of viral mediated gene transfer is the need for and the difficulty in achieving high titre viral stocks. In vivo infection with viruses, when applicable, is generally not effective given the in vivo dilution of viral particles. Additionally, although both retroviral and adenoviral methods employ replication-defective viral particles, the possibility of producing replication-competent viruses and thereby causing active infection in vivo is an inherent danger of both systems.
For retroviral mediated gene transfer to occur, target cells whether in vitro or in vivo must be in a cycling status. Since retroviruses package nucleic acid in the form of RNA, reverse transcription of the RNA to DNA is required for integration into the host genome from where the gene exerts its effects. Cells which divide infrequently or never at all, such as some classes of stem cells or terminally differentiated end cells, are usually less amenable to gene transfer via retroviral infection as compared to rapidly dividing cells. Thus diseases for which a long-term cure is dependent upon stem cell or end cell manipulation are poor candidates for gene therapy treatment using retroviral transfection. Retroviral use is also limited to the restricted range of host infectivity specific to each strain of virus. In contrast adenoviruses which contain double stranded DNA do not require target cells to be cycling for infection, integration and propagation.
DNA has also been delivered to cells using receptor-mediated endocytosis. In this approach, DNA is initially complexed with polycations such as polylysine for condensation and charge neutralization purposes. Ligands for cell surface receptors, such as transferrin, are then coupled either biochemically or enzymatically to the polylysine moieties. In a further modification, the transferrin molecules are coupled to the outer surface of inactivated adenoviral particles. The adenoviral particles can effect the release of the DNA/polylysine/transferrin complex from endosomes prior to lysosome mediated degradation. The transfer of up to 48 kilobases (kb) of DNA has been reported using this approach. Cotten et al., PNAS v. 89, p.6094-6098 (1992).
In contrast to the use of polycations for complexing DNA, other approaches have incorporated specific DNA binding domains which recognize and bind distinct nucleic acid consensus sequences. An example of this is the use of the GAL4 DNA binding domain of yeast which selectively binds to a 17 bp sequence. Thus a nucleic acid to be delivered must usually be modified to incorporate artificial GAL4 binding sites. Likewise, other approaches which rely on a consensus sequence dependent DNA binding domain will similarly require modification of the transferred nucleic acid.