Characterisation of plasmid DNA conjugates as a basis for their processing J.T. Tsai, E. Keshavarz-Moore, J.M. Ward, M. Hoare, P. Ayazi Shamlou, P. Dunnill Abstract Plasmid DNA complexes have been used by several research groups for gene therapy applications and have given promising results during preliminary clinical trials. However, their eventual adoption for the treatment of a wide range of genetic diseases requires considerable progress with regards to carrier formulation and pro- cessing. Amongst the key parameters that are known to be important and need further elucidation are the size and surface charge of the conjugates and the binding ef®ciency of the carrier to the DNA. The present study has focused on two synthetic carriers with different formulation and charge characteristics, both of which have been recommended in the literature as having the potential to deliver genes to target cells. These were a cationic lipid (DC-chol/DOPE) and a polylysine- condensed anionic liposome (OA/DOPE/Chol). Conjugate size, zeta potential, stability and binding ef®ciency were measured for conjugates of DNA plasmids of molecular weights between 6.9 kb and 29 kb. Plasmids were con- densed by polylysine of two different molecular weights with or without cationic lipids. 1 Introduction Novel methods for the processing of new biomaterials are needed if the ever increasing scienti®c discoveries in the area of gene therapy are to be translated into approved biopharmaceutical medicines. Human gene therapy is perceived to have the potential to revolutionise the treat- ment of a wide range of life-threatening genetic disorders and acquired diseases (Marquet et al., 1995). Two ap- proaches have been attempted. With ex vivo treatments, cells are removed from the donor and reintroduced to the patient following insertion of the desired genetic material. The ex vivo method however has limited applications and currently research is focused on in vivo treatments during which gene vectors are delivered to the recipient cells di- rectly, for example by injection into muscle tissues or by aerosolisation into the lungs (Wolff et al., 1990; Caplen et al., 1995). The transport of genetic materials to the cells, their transfer through the cell membrane and their uptake by cell nuclei have posed considerable research challenges. Natural and synthetic systems are currently being assessed as carriers to protect and to deliver DNA for human and animal gene therapy applications (for review see Sokol and Gewirtz, 1996 and Ledley, 1996). Viral-based vectors cap- italise on the natural capacity of viruses to transport rapidly and ef®ciently their heterologous DNA to target cells. Although high levels of expression have been re- ported, progress however has been hampered by safety considerations and post-treatment complications such as immunogenicity (Stewart et al., 1992). Additionally, viral vector carriers currently have limited capacity to accom- modate gene expression systems (Crystal, 1995). These concerns have led to the development of a range of non- viral mediated vesicles (Eastman et al., 1997a; Zhang et al., 1997; Wasan et al., 1996). The present investigation fo- cuses on the process issues related to the physio-chemical properties of synthetic complexes suitable for conjugation to plasmid DNA for gene transfer applications. DNA macromolecules are relatively large; their theo- retical hydrodynamic diameter is between 300 nm for a 7 kb plasmid and close to 700 nm for a 29 kb plasmid (Ledley, 1996). They are also mechanically ``delicate'' and naked DNA is prone to shear damage during stages of processing (Levy et al., 1998) and delivery, for example in hypodermic injection syringes, nebulisers and high ve- locity liquid jets (Ledley, 1996). Synthetic gene vector delivery systems prepared by nano-encapsulation of plasmid DNA within lipid bilayers are inherently safer to administer compared to viral-mediated systems, but suffer from relatively low DNA uptake rate by tissue cells (Crystal, 1995). Several approaches are currently being pursued aimed at improving the rate of non-viral gene transfer to cell nuclei. These include the use of pH sen- sitive lipid bilayers (Zhou et al., 1992), liposome en- trapped, polylysine condensed DNA (Lee and Huang, 1997), and the assembling of DNA macromolecules with cationic polymers and dendrimers (Malik and Duncan, 1998; Wiwattanapatapee et al., 1998; Katayose and Ka- taoka, 1997; Maruyama et al., 1997; Wolfert et al., 1996; Tomalia et al., 1990). Additionally, such complexes and assemblies have been conjugated to monoclonal antibod- Bioprocess Engineering 21 (1999) 279±286 Ó Springer-Verlag 1999 279 Received: 26 October 1998 J.T. Tsai, E. Keshavarz-Moore, J.M. Ward, M. Hoare, P. Ayazi Shamlou (&), P. Dunnill The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE The Advanced Centre for Biochemical Engineering is sponsored by the Biotechnology and Biological Sciences Research Council and the Council's support is gratefully acknowledged.