Inhaled Gene Therapy

Inhaled gene therapy approaches to treat cystic fibrosis have been largely unsuccessful using approaches such as attenuated adenovirus vectors to deliver the CFTR gene [40]. There is broad consensus that treatment of CF lung disease will require prolonged expression of CFTR for many months. However, only three trials using adenovirus and adeno-associated virus vectors [41-43] have assessed duration in the lung. These vectors were shown to be unsuitable because of adaptive immune responses to these viral vectors [44].

However, newer approaches using lipid-based carriers of CFTR DNA are underway. An inhalation study was carried out in mice to determine the efficacy and safety of administering of cationic lipid formulation GL67A complexed with pGM169, a CpG-free plasmid encoding human CFTR complementary DNA [45]. Twelve biweekly inhalation exposures were carried out over a period of 6 months. Results showed that repeated administration of pGM169/GL67A to murine lungs resulted in a NOAEL at the lowest dose of 1.2 mg pGM169/kg and 6.7 mg GL67A/kg that was estimated to be approximately fivefold the anticipated clinical dose. Reproducible, dose-related, and persistent gene expression (>140 days after each dose) was achieved using an aerosol generated by a clinically relevant nebulizer. This study supported progression into the first nonviral multidose lung trial in CF patients [46].

Approaches have also been used with polyethylenimine (PEI) carriers. The intermittent exposure of Balb/c mice (sex not reported) to PEI (branched)-DNA via an aerosol delivery system resulted in a higher expression of DNA in the lungs of treated animals compared to control aerosol mice (no exposure) or to mice exposed to lipid— DNA vectors [47]. Treatment involved exposure of the mice to 1 min of aerosol followed by a 9 min delay to allow the animals to breathe the aerosol before beginning the cycle again; this cycle was repeated until all of the nebulizer fluid (40 mL in total) was used (approximately 16 h).

While clinical signs of toxicity were not reported in the previous study, a similar study in which female Balb/c mice were exposed to a PEI (branched)-DNA vector via an aerosol delivery system for 30 min did not result in any signs of lung toxicity (including any signs of acute inflammation) [48]. Following the 30 min exposure period, gene expression (as measured by the levels of chloramphenicol acetyl transferase [CAT] in the lung) persisted for 10 days postexposure, although CAT levels decreased to approximately 50% of peak levels at 7 days postexposure [48]. These data suggest that aerosol administration of branched PEI results in lung deposition but does not result in clinical signs of acute toxicity.

A study in which PEI (branched)-DNA or lipid-DNA complexes were delivered to female C57BL/6 mice via an aerosol delivery system (duration of exposure was 30 min) reported a lower level of lung and serum cytokines (specifically, tumor necrosis factor-а [TNF-a] and interleukin-1p [IL-1p] levels) in these mice compared to mice receiving an equivalent amount of PEI (branched)-DNA via the IV route (at a level from 6.45 pg of branched PEI to 5 pg of DNA in a 200 pL solution, based on a PEI-DNA weight ratio of 1.29:1) or compared to mice exposed to the lipid-DNA complex [49]. Additionally, the levels of TNF-a and IL-1p in the lungs peaked at 5-8 h postaerosol exposure and resolved to control levels within 24 h postaerosol exposure, while levels in the bronchoalveolar lavage fluid (BALF) peaked at 24 h and resolved to control levels within 48 h postaerosol exposure. There was no neutrophil infiltration into the BALF at the timepoints evaluated, suggesting that the cytokine levels in the BALF may not be high enough to stimulate an acute inflammatory response. The data suggest that the aerosol delivery of genetic material using branched PEI as a vector decreases the cytokine responses associated with plasmid delivery when compared to cytokine levels via the IV route or via aerosol delivery of genetic material using lipid vectors.

There were no significant differences noted in the levels of CAT in the liver, spleen, kidney, thymus, brain, and blood compared to untreated controls following aerosol delivery of a PEI (branched)-DNA complex to female Balb/c mice for 30 min, suggesting a lack of systemic gene delivery and, by inference, PEI delivery following aerosol exposure to the vector [48]. Delivery of an aerosolized labeled PEI (branched)-DNA complex to female Balb/c mice (exposure duration of 30-120 min) resulted in localization of the label mainly in the epithelial cells lining the airways, suggesting that PEI is concentrated in the lung tissue following aerosol administration [48,50].

Repeated administration of a branched PEI complex also did not result in signs of toxicity [51]. Following the injection of B16-F10 cells (a metastatic melanoma cell line) via the lateral tail vein, male nude mice (strain not reported) received PEI (branched)-p53 via an aerosol delivery system twice a week for 5 weeks, starting at 6 weeks postinoculation of the cancer cells [52]. Control groups included untreated mice, mice treated with PEI (branched; alone) or with PEI (branched)-CAT complexes. There were no signs of acute inflammatory responses, including neutrophil infiltration or tissue damage, in any of the tissues examined from any of the treatment groups (tissues that were examined included lung, liver, kidney, spleen, brain, and heart). In addition, there was no significant loss of body weight in treated animals when compared to the control animals [52].

Intranasal administration of PEI (branched)-DNA at a level from 51.6 pg of PEI (branched) to 40 pg of DNA (based on a PEI-DNA weight ratio of 1.29:1) to female Balb/c mice resulted in a higher expression of DNA in the lungs of treated animals compared to the control (no aerosol exposure) or compared to mice exposed to lipid- DNA vectors [47]. Note that nasal delivery of droplets in mice results in good lung delivery of the solution. Clinical signs of toxicity were not reported in this study; however, it is unclear if the study authors examined the animals for signs of toxicity.

For novel gene therapy vectors, information on biodistribution measurements is needed to determine PK/PD of vector. Usually qPCR is measured in tissues including blood, liver, kidneys, heart, brain, testes, ovaries, and spleen as suggested in FDA guidance [53]. Levels in reproductive organs are important since to assess possible risk of transmission of gene therapy vectors from parent to offspring. These measurements can be conducted along with measurement of the desired transcribed therapeutic protein carried by the vector.

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