Improving the efficiency of cystic fibrosis airway gene therapy
Dr Martin Donnelley
Women’s and Children’s Health Network
Treatments for cystic fibrosis (CF) have improved in recent years, enhancing survival and quality of life, but these interventions are not curative. Gene therapy is likely the only mutation-class-independent method of overcoming CF lung disease, however achieving therapeutic levels of gene transduction is a challenge. This study will assess whether altering our current gene vector pseudotype – the coating that controls the types of cells transduced – can result in higher levels of airway gene expression. This will allow us to assess which pseudotype to progress towards clinical trials.
Martin Donnelley, Nathan Rout-Pitt, David Parsons, Alexandra McCarron, Chantelle McIntyre
The addition of a properly functioning CF gene into affected CF airway cells is recognised as the only biologically rational way to prevent or treat CF airway disease, for all classes of CF mutation. This ultimately means that in the future, if a baby born with CF is treated with gene therapy at birth, they will never develop CF lung disease; and for a person currently living with CF, that the progression of their lung disease would be halted – improving both quality and length of life.
This comparison between the VSV-G and HA pseudotype has shown that VSV-G produces significantly higher levels of LacZ transduction in the trachea than HA, regardless of wither LPC conditioning is used. Although both pseudotypes were able to produce long-term-sustained Luc gene expression over 18 months, the ability to produce high titres and large volumes of vector required for human clinical studies, is more feasible with the VSV-G pseudotype compared to HA, thus reducing time and cost of vector production for pre-clinical and clinical studies. Together, these results indicate that LPC may not be required for its tight-junction properties, but instead for its mucolytic and cilia-reduction properties, which increases access to cellular receptors and vector residence time, both of which are important for efficient gene expression levels.
Cystic fibrosis (CF) is a debilitating disease that results in poor quality of life and premature death, primarily from lung disease. Lentiviral (LV) vectors are a promising option for treating CF airway disease by adding functional copies of the CF gene into the airway epithelial and airway stem cells. The outer coating of LV vectors can easily be changed by a process called pseudotyping, to alter the type of cells targeted by the vector. The VSV-G envelope protein is the most widely used pseudotype in airway gene therapy due to its effectiveness in achieving short and long-term gene expression in respiratory cells. VSV-G receptors are located on the basolateral surface (inward-facing side) of the airway cells, and access to these is prevented by epithelial tight-junctions. Our team previously showed that conditioning the nasal airways of mice with the compound LPC (lysophosphatidlcholine) prior to VSV-G LV delivery improves gene transfer effectiveness. However, a study in 2013 demonstrated the effectiveness of a HA pseudotyped LV vector – which targets receptors on the apical (outward-facing) cell surface – in the nasal and lung airways of mice without the need of an airway conditioning compound such as LPC. The ability to achieve high levels of gene expression without conditioning is advantageous, however these studies only assessed the short-term effectiveness of HA in mice.
These two pseudotypes enter airway epithelial cells differently; therefore, this study was designed to examine and compare the short- and long-term gene transfer levels produced by both pseudotypes in the trachea and upper conducting airways of normal mice using our HIV-1 LV vector system with and without our two-step LPC and LV vector dosing method.
Ciliated and airway stem cells are a primary target for CF gene therapy. The VSV-G pseudotype has shown to be extremely effective in targeting both ciliated and airway stem cells in the nasal and lung airways of mice following LPC conditioning. Although the HA pseudotype has been shown to be efficient at transducing ciliated airway cells, the effectiveness of HA in the basolateral region has yet been determined. Access to this region is important since it is the location of the airway stem cells.
To determine the location and type of cells transduced by both pseudotypes, and the effectiveness of HA in the basolateral region, 46 mice were separated into four treatment groups, and received either PBS (control) or LPC as the airway-conditioning agent, followed one hour later by either a HA or VSV-G pseudotyped LV vector containing the LacZ gene. One-week later, their lungs were harvested and X-gal processed for en face and histological assessment.
En face examination revealed that LacZ transduction was typically more pronounced in the trachea and bronchioles for both pseudotypes, while H & E stained trachea and lung sections indicated that both pseudotypes transduced ciliated airway cells. Quantification of the number of transduced cells indicates that regardless of airway conditioning, VSV-G produces significantly higher levels of LacZ transduction in the trachea than HA. Although LPC conditioning is required for VSV-G transduction in the nasal airways, these results indicate that VSV-G is capable of transducing lung airway cells in the absence of LPC conditioning, suggesting that in the lungs its use may not be essential for tight-junction opening. LPC may also be useful for its mucolytic and/or cilia-reduction properties, both of which might increase vector access to receptors and vector residence time, but this should be assessed in future studies. Further assessment of LacZ transduced lung samples is ongoing.
We have previously demonstrated that long-term gene expression can be achieved in the nasal airways of CF mice with the VSV-G pseudotype following LPC airway conditioning. Unexpectedly, we also detected long-term reporter gene expression in the upper conducting airways (from fluid moving down the trachea from the nose) and found that expression was present irrespective of whether LPC conditioning was used.
This part of study aimed to determine whether the HA pseudotype could produce sustained long-term gene expression in the lungs, and whether the addition of LPC enhances gene expression levels for either pseudotype over-time. To achieve this, 36 mice were dosed with either PBS (control) or LPC, followed one hour later by either a HA or VSV-G pseudotyped LV vector carrying the luciferase gene. The mice underwent bioluminescent imaging (BLI) at various time-points for 18-months to assess luciferase expression.
Long-term BLI revealed that at one week, the VSV-G pseudotype produced significantly higher levels of Luc gene expression than HA, regardless of whether LPC was used. The difference in initial expression levels produced by VSV-G might have been the result of a higher titre than HA (1.56 x 108 TU/ml and 1.56 x 108 TU/ml respectively). Gene expression levels for all groups decreased at 1 month as expected, and interestingly, despite the lower titre of HA, there was no statistical differences between the pseudotypes or the conditioning for up to 12 months. Further expert biostatistical analysis is currently underway for the remainder of the imaging time-points. The level of luminescence produced by VSV-G (which was consistent with previous findings) and HA, remained steady for approximately 18 months, indicating that the Luc reporter gene was stably integrated into the genome of the host airway cells and is stably expressed, a primary goal for LV-mediated gene transfer for the treatment of CF.
Conference Poster Abstracts:
(ASGCT 2018) C. Carpentieri, N. Farrow, P. Cmielewski, C. McIntyre, N. Rout-Pitt, A. McCarron, D. Parsons & M. Donnelley, “Airway gene-addition therapy for cystic fibrosis: Comparative efficiency of HA and VSV-G pseudotyped lentiviral vectors”, Molecular Therapy, vol. 26, 5s1, pp. 126, 2018.
(TSANZ, 2018) C. Carpentieri, N. Farrow, P. Cmielewski, C. McIntyre, A. McCarron, N. Rout-Pitt, D. Parsons & M. Donnelley, “Comparative efficiency of HA and VSV-G pseudotyped lentiviral vectors for cystic fibrosis airway gene therapy”, Respirology, vol. 23, s1, pp. 139, 2018.
(NACFC, 2017) C. Carpentieri, N. Farrow, P. Cmielewski, C. McIntyre, A. McCarron, N. Rout-Pitt, D. Parsons & M. Donnelley, “Comparative efficiency of HA and VSV-G pseudotyped lentiviral vectors for cystic fibrosis airway gene therapy”, Pediatric Pulmonology, vol. 52, s47, pp. s323, 2017.
(ACFC/ASMR, 2017) C. Carpentieri, N. Farrow, P. Cmielewski, C. McIntyre, A. McCarron, N. Rout-Pitt, D. Parsons & M. Donnelley, “Testing the efficiency of a HA pseudotyped lentiviral vector for treating cystic fibrosis lung disease”, presented at the Australian Cystic Fibrosis Conference and Australian Society for Medical Research conferences, 2017
Current Research Paper Underway:
C. Carpentieri, N. Farrow, P. Cmielewski, C. McIntyre, N. Rout-Pitt, D. Parsons & M. Donnelley, “Choosing the right lentiviral pseudotype for cystic fibrosis airway gene therapy: Balancing production and transduction efficiencies”