(629) Assessing the efficacy of airway gene therapy in neonatal rats

Thursday, November 3, 2022

12:45 PM – 1:25 PM ET

Presenting Author(s)

Alexandra McCarron, PhD

Post-doctoral researcher
University of Adelaide, South Australia, Australia

Co-Author(s)

PC

Patricia Cmielewski

Research Fellow
University of Adelaide, South Australia, Australia
DP

David Parsons

Chief Medical Scientist
Women's and Children's Hospital, United States
Martin Donnelley

Senior Research Fellow
University of Adelaide, South Australia, United States

Background: Airway gene-addition therapy is a mutation agonistic approach that has potential to treat lung disease. Gene-addition involves using a vehicle such as a lentiviral vector to deliver copies of the CFTR gene to the airway cells in order to restore normal ion channel activity. Performing airway gene therapy early in life has several potential benefits. The lung environment is virtually disease free, thereby avoiding mucus and bacterial infection, the presence of which are known to reduce gene transfer efficacy. Neonates also have a naive immune system and it is possible that airway stem cells are more accessible during this early period. These factors may improve initial gene transfer levels and expression persistence, respectively. Ultimately, a gene therapy given early in life will provide the best opportunity to halt further progression of lung disease. The aim of this study was to assess the feasibility and efficacy of performing nasal gene transfer in neonatal rats.

Methods: Five day old rat pups (n=6/group) received 4 μL of 0.3% lysophosphatidylcholine to the right nostril followed one hour later by 10 μL of lentiviral vector carrying the LacZ reporter gene (titre: 2 x 109 TU/mL). Neonatal rats were assessed at either 1-week or 8 weeks post gene transfer. A separate group of 8-week old adult rats (n=6) were anaesthetised and gene vector delivery was performed as for the neonates, except that 20 μL of lentiviral vector was used. The adult group was assessed 1-week following gene transfer. For all animals, nasal tissues were harvested and X-gal stained to enable en face assessment of LacZ positive cells. Tissues were decalcified, embedded in wax, sectioned, and stained with hematoxylin and eosin for histological analyses.

Results: Qualitative assessment of en face LacZ staining revealed that rats treated during the neonatal phase had gene expression present in the nose at 1-week and 8 weeks post-delivery (Figure 1). Comparatively, performing the same gene delivery protocol in adult rats resulted in very low levels of LacZ positive cells. Histological assessment of the nasal tissue from the treated neonates indicated that LacZ positive cells were isolated primarily to the regions where respiratory epithelium was present.

Conclusions: Performing nasal gene transfer to rat pups is feasible and results in high levels of gene expression that persists for up to 8 weeks post-delivery. Interestingly, adult rats demonstrated substantially lower gene expression levels when compared to rats dosed as neonates, suggesting that early treatment may be more effective. Subsequent quantitative studies assessing CFTR gene correction in neonatal CF rats will determine whether nasal ion channel function can be restored when performing gene therapy at this early stage in life.

Acknowledgements: This work was supported by the Women's and Children's Hospital Foundation.

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