A Novel Mixing Device for the Reproducible Manufacture of Non-Viral Gene Therapy Formulations.

Davies LA, Nunez-Alonso GA, Hebel HL, Scheule RK, Cheng SH, Gill DR, Hyde SC

Molecular Therapy, 17 S1 S188

Download

The American Society of Gene Therapy Annual Conference, San Diego, 2009

The generation of non-viral gene therapy formulations requires the complexation of negatively charged plasmid DNA (pDNA) with cationic gene transfer agents (GTAs) such as lipids, polymers and peptides. Within the laboratory, small volumes of reagent are often prepared by stepwise addition of one reagent to the other. However, this technique is inappropriate for the production of larger amounts of material required for clinical applications because incomplete or variable mixing associated with larger volumes can significantly affect both the physical characteristics and the in vivo performance of the complexes.

We have developed a pneumatic mixing device that allows the reliable and reproducible mixing of large volumes of GTAs and have investigated its suitability for the production of two non-viral gene therapy formulations of interest for treatment of cystic fibrosis lung disease. The LMD2 pneumatic mixer consists of a compressed gas driven system, designed for the controlled mixing of reagents, packaged side-by-side in a dual-lumen syringe attached to an 8-element HDPE static mixer.

The rates of mixing and extrusion are fully adjustable allowing practical liquid mixing rates in the range 0.2 - 20 ml/s. Video analysis of the device in action demonstrated the linearity and reproducibility of extrusion rate over the full range of mixing rates even when using formulations with viscosities in excess of 20cP. The LMD2 was utilised to form complexes between the 5.6 kb luciferase expression plasmid pCIKLux and the cationic lipid GL67A (Genzyme) (0.8 mM pDNA: 0.6 mM lipid), or 25kDa polyethylenimine (0.6mM pDNA, N:P 10:1). A total of 10 ml of each formulation was prepared at mixing rates from 1 - 20 ml/s and the physical characteristics of the resultant complexes compared with those prepared by standard small volume mixing (<500µl). Irrespective of the mixing technique, the measurements of particle size and zeta potential were similar for complexes of pDNA/GL67A (Range 314.83 - 278.63nm; 3.37 - 4.10mV), or pDNA/PEI (Range 79.95 - 112.17 nm; 24.33 - 27.70mV) at the mixing rates tested.

Importantly, agarose gel analysis of dissociated complexes revealed no shear degradation of pDNA. To confirm the biological efficacy of complexes prepared using the LMD2, 10ml of pCIKLux/GL67A (8 mM: 6 mM) or pCIKLux/PEI (0.6mM pDNA, N:P 10:1) were aersolised to the lungs of BALB/c mice using a whole body exposure chamber. Luciferase expression was analysed 24 hr later and equivalent gene expression was observed in mice exposed to aerosols prepared using the LMD2 and those prepared using small volume mixing, for both pCIKLux/GL67A and pCIKLux/PEI.

These data demonstrate that this novel mixing device is suitable for large-scale production of functional gene therapy reagents in a standardised and reproducible manner essential for reproducible clinical administration.