Noninvasive Imaging Reveals Stable Transgene Expression in Mouse Airways After Delivery of a Nonintegrating Recombinant Adeno-Associated Viral Vector

Abstract

Gene therapy holds promise to cure a wide range of genetic and acquired diseases. Recent successes in recombinant adeno-associated viral vector (rAAV)-based gene therapy in the clinic for hereditary disorders such as Leber's congenital amaurosis and hemophilia B encouraged us to reexplore an rAAV approach for pulmonary gene transfer. Only limited clinical successes have been achieved for airway gene transfer so far, underscoring the need for further preclinical development of rAAV-based gene therapy for pulmonary disorders. We sought to determine the preclinical potential of an airway-tropic serotype, rAAV2/5, encoding reporter genes when delivered to mouse airways. Although several groups have assessed the stability of gene transfer using a nonintegrating rAAV in mouse airways, long-term stability for more than a year has not been reported. Additionally, an extensive quantitative analysis of the specific cell types targeted by rAAV2/5 using cell-specific markers is lacking. We obtained sustained gene expression in upper and lower airways up to 15 months after vector administration, a substantial proportion of the lifespan of a laboratory mouse. In addition, we demonstrated that readministration of rAAV2/5 to the airways is feasible and increases gene expression 14 months after primary vector administration, despite the presence of circulating neutralizing antibodies. Finally, identification of transduced cell types revealed different subpopulations being targeted by rAAV2/5, with 64% of β-galactosidase-positive cells being ciliated cells, 34% club cells in the conducting airways, and 75% alveolar type II cells in the alveoli at 1 month postinjection. This underscores the therapeutic potential of a nonintegrating rAAV vector to develop a gene therapeutic drug for a variety of pulmonary disorders, such as cystic fibrosis, primary ciliary dyskinesia, and surfactant deficiencies.

Roadmap for an early gene therapy for cystic fibrosis airway disease

“…Furthermore, mouse models have been instrumental in addressing generic outcomes of gene therapy, ie, transduction efficiency, cell‐specific tropism, immune responses to vector and transgene, and longevity of gene expression (reviewed in Mingozzi and High). In non‐CF mouse airways, we have shown sustained reporter expression till 15 months after rAAV2/5 delivery, a substantial proportion of the lifespan of a laboratory mouse . Nevertheless, this question remains to be re‐investigated in an animal model spontaneously developing lung disease, such as the β‐ENaC overexpressing mouse or the CF rat, ferret, and pig .…”

Cystic Fibrosis Gene Therapy – Not Low-hanging Fruit

“…1 The research community has not yet reached a consensus for adeno-associated vectors (AAV), which is the current vector of choice for a range of eye disorders and haemophilia, for example. [5][6] However, in the context of lung gene transfer, we have not seen any convincing evidence that AAV can be re-administered without loss of efficacy and, therefore, currently question the further development of this vector for CF gene therapy. These data led the UK CF Gene Therapy Consortium (GTC; http://cfgenetherapy.org.uk/), which is currently the largest group pursuing translational CF gene therapy, to focus on non-viral gene transfer.…”

Barriers to inhaled gene therapy of obstructive lung diseases: A review

“…Recently, a pseudotyped (hybrid) AAV gene vector, bearing AAV2 rep and AAV5 cap expression cassette (AAV2/5), achieved persistent gene transfer lasting up to 15 months in the airways and alveoli of mice following intratracheal administration (Figs. 1A, B) [105]. They also found that re-administration of AAV2/5 14 months after the initial administration did not significantly reduce the gene transfer efficacy, presumably due to the long dosing interval [105].…”

“…They also found that re-administration of AAV2/5 14 months after the initial administration did not significantly reduce the gene transfer efficacy, presumably due to the long dosing interval [105]. As a result of this dosing interval, neutralizing antibody levels were reduced by more than 50% compared to peak levels after the first administration [105]. The process and rationale of pseudotyping viral gene vectors is discussed in detail in section 5.1.…”

“…(B) Bioluminescence imaging of firefly luciferase expression in the lung and nose of mice at 1, 3, 6, 12, and 15 months post-administration. Reprinted from [105] with permission of Mary Ann Liebert, Inc. (C, D) A simian immuno-deficiency virus pseudotyped with the respiratory pathogen Sendai virus (F/HN-SIV) demonstrates sustained transgene expression in the nose and lungs of mice after intranasal administration, lasting 22 months postadministration. (C) Fluorescent microscopy images of GFP expression mediated by F/HN-SIV in mouse lungs.…”