Non-Viral Vectors

We are also actively involved in the production and development of delivery systems for non-viral gene therapy as an alternative to the use of viral vectors. Viruses have several problems including insertional mutagenesis, risk of viral infection and replication after recombination events that reconstitute wild type viral functions, induction of host immune responses, and the great cost of production and safety monitoring for clinical applications. The risks of problems associated with viral vectors is eliminated with the use of plasmid DNA.

Non-viral methods make use of cationic lipids, polymers, targeting proteins, and calcium phosphate, as well as mechanical methods such as electroporation to deliver plasmid or “naked DNA” to cells for gene delivery. There is no single best method for all applications. Instead, different delivery vehicles are needed for different applications, target organs, tissues, and cell types. This is exemplified in studies which utilize liposomes to deliver virus in order to avoid rapid immune clearance. Understanding the strengths and weaknesses of the different gene delivery methods is critical in deciding which one to use for a given experiment. To this end the VDL has established a number of methodologies for production of these types of materials.

The VDL offers “Clean DNA” from its plasmid production. One tentative problem plaguing delivery of naked DNA has been the large amounts of contaminants that exist in standard DNA preparations. These contaminants belong to a class of molecules known to inhibit both DNA and RNA polymerase activities. Therefore, gene expression post-transfection can be increased by orders of magnitude if they are removed from the preparations. The presence of these contaminants in DNA also precludes high dose delivery of DNA-liposome complexes intravenously. These contaminants co-purify with DNA by anion exchange chromatography and by cesium chloride density gradient centrifugation and are not endotoxin-free. The VDL has developed methods for their detection in plasmid DNA preparations and can now make plasmid DNA that does not contain these contaminants. Hence this DNA provides greater efficacy and gene expression at lower doses compared to use of other plasmid DNA preparations of the identical plasmid.

Naked DNA has been reported to work exceptionally well in mouse models however when this methodology is translated to the clinic, the efficiency is lost. Recently, we reported the use of highly efficient liposomes that increased gene delivery and expression by two orders of magnitude in all organs (Nature Biotechnology 15:647-652, 1997). These improved liposomes and nucleic acid/liposome complexes have extended half-life in the circulation, are stable in serum, have broad biodistribution, efficiently encapsulate all types of nucleic acids, are targetable to specific organs and cell types, are able to penetrate through tight barriers in several organs, and have been optimized for nucleic acid: lipid ratio and colloidal suspension in vivo. We can efficiently encapsulate nucleic acids of any size including small oligonucleotides or large mammalian artificial chromosomes. We have encapsulated DNA, RNA, ribozymes, DNA-RNA hybrids, proteins with low isoelectric points, and mixtures of nucleic acid and protein. These complexes have been injected into mice, rats, rabbits, pigs, and nonhuman primates. Furthermore, we have demonstrated efficacy in various animal models of disease including cancer, restenosis, and AIDS.

Further improvements in our nonviral delivery system are required for optimal use in humans to treat diseases, including improving the targeting efficiency to specific organs. We now have the technology for adding specific ligands either by ionic interactions or covalent attachments. These ligands include monoclonal antibodies, Fab fragments, peptides, peptide mimetics, small molecule drugs, proteins, and parts of proteins. We can also size fractionate our complexes before adding ligands to produce a totally homogenous population of complexes prior to injection. Furthermore, we can change the charge on the surface of these complexes using polysaccharides in order to avoid nonspecific charge interactions with nontarget cells.

A second goal is to increase the persistence of gene expression for applications that require sustained expression. This can be accomplished by making improvements in the design of plasmid vectors. Sequence elements can be added to nonintegrating vectors for attachment to the nuclear matrix, thereby insuring plasmid persistence in the nucleus. Specific promoters and enhancers can also be used in these vectors to avoid the rapid elimination of gene expression in certain tissues. Plasmids can also be designed to provide specific integration or homologous recombination into the host chromosomes. These sequences can be used to correct host chromosome mutations or to eliminate harmful sequences in the chromosome.

To initiate service for non-viral vector production, fill out the Service Request Form and return it to us by fax (713-798-1230) or by e-mail (vector@bcm.edu).