GL67A pGM169 - Our first clinical trial product

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A vial of lipid GL67A prior to rehydration (left) and a vial of the plasmid pGM169 (right).

Introduction

GL67A/pGM169 is the combination of cationic liposome (GL67A) and plasmid DNA expressing CFTR (pGM169). 

Aerosolisation of a plasmid DNA vector to the lungs of patients represents an enormous challenge and plasmid DNA cannot be efficiently delivered to the airways on its own. The Consortium has recently completed a major clinical trial programme with this product (Alton EW et al., 2013 Thorax).

Due to these delivery problems, we combine the negatively charged plasmid with positively charged fatty liposomes (GL67A) and these form very small, tighly bound particles which are stable enough to withstand aerosolisation and can be taken up by the cell membrane of cells in the respiratory tract.

Following uptake by the cells plasmid DNA is transported to the nucleus where it starts to express its copy of the CFTR gene.

 

 

pGM169- codon optimised CFTR, CpG-free and improved duration of expression

A map of our clinical trial plasmid pGM169The plasmid vector pGM169 was created in Oxford in mid 2006 during our pre-clinical development work. In a number of studies this plasmid easily established itself as our most promising candidate for clinical testing.

The CFTR cDNA was codon optimised to maximise the efficiency of CFTR protein production (the natural human CFTR sequence is actually really inefficient). This cDNA sequence was coupled with our groundbreaking hCEFI promoter which provides the longest duration of CFTR activity that we have ever observed in our pre-clinical models (Hyde SC et al., 2008 Nature Biotechnology).

In addition, by using technology developed initially by the biotech company Invivogen, we were able to make the plasmid completely devoid of any CG dinucleotides which have been associated with an inflammatory response in-vivo.

 

 

What is Lipid GL67A?

Extensive chemical optimisation of the promising DC-Chol structure by Genzyme Inc. (Cambridge, MA, USA) generated the cationic lipid GL67 with improved gene transfer potency (Lee et al. 1996), well-characterised safety parameters and desirable stability during aerosolisation (Eastman et al. 1998).

GL67 was co-formulated (GL67A) with the neutral lipid dioleoylphosphatidylethanolamine (DOPE), thought to facilitate pDNA endosomal escape, along with small amounts of a polyethylene glycol-containing lipid (DMPE-PEG5000) to stabilise formulations at concentrations sufficient for aerosol delivery to the human lung (Eastman et al. 1997).

A single nasal administration of the GL67A/pDNA formulation was safe, directed vector-derived mRNA expression, and produced an overall ~20% correction of the CFTR Cl- channel defect in the nose (Zabner et al. 1997). Of all the cationic lipids available for non-viral gene transfer, Genzyme Lipid GL67A is still the only non-viral GTA that has been successfully aerosolized to the lungs of patients in phase I clinical trials (Alton et al. 1999) & (Ruiz et al. 2001).

After delivery to the lungs of CF patients via jet nebuliser, vector-derived transgene mRNA was detected in four out of nine bronchoscopic lung samples (Ruiz et al. 2001). Using the same formulation, Alton reported partial correction of the CF Cl- transport defect in the nose, trachea and lower airways of treated CF subjects (Alton et al. 1999), providing proof-of-principle for correction of CFTR-mediated Cl- secretion following gene transfer to the lung.

The Consortium clinical trials that are have just been completed represent the third time that GL67A has been used in a gene therapy clinical trial for CF. The decision to proceed with this plasmid was only reached after an extensive programme of testing where GL67A out-performed many new and established GTAs in mouse and sheep models. Based on these studies, we believe GL67A still represents the 'Gold Standard' for aerosol delivery of a non-viral vector.

 

Making a Clinical Product

A lot has changed since the last clinical trials were undertaken for GL67/pDNA complexes. The Consortium has put a lot of effort into making the lipid DNA combination suitable for a 21st Century clinical trial.

The scale of the planned clinical trials was such that production of GL67A and pGM169 had to be contracted out to biotech firms. The plasmid was manufactured by VGXi in Houston, Texas. In order to conduct our planned multi-dose clinical trials, so much pGM169 is required that it will become the most abundant plasmid molecule ever produced for human clinical use.

Aerosol technology has also changed much in recent years so even the process of selecting an appropriate nebuliser for the clinical trial has become a vital research project requiring several months of testing.

Finally a new method of mixing the lipid and DNA was developed to allow for complete control and reproducibility of every dose (Davies LA et al., 2010 BioTechniques) to the airways.


 

Human airway liquid interface cultures transduced with a lentivirus expressing Luciferase.

 

Proposed 3D model of the CFTR protein.

 

Light microscope image of a human airway liquid interface cultures. Dark patches are mucous.

 

Mouse lung large airway (cell nuclei blue) transduced with an adenoviral vector (green).

 

A cake that only some of us got to enjoy!

 

DNA fragments being cut from an agarose gel exposed to UV.

 

Pellets of DNA following precipitation.

 

Purifying mRNA from tissue samples.