Aerosol-Mediated Non-Viral Lung Gene Therapy: The Potential of Aminoglycoside-Based Cationic Liposomes.

Aerosol lung gene therapy using non-viral delivery systems represents a credible therapeutic strategy for chronic respiratory diseases, such as cystic fibrosis (CF). Progress in CF clinical setting using the lipidic formulation GL67A has demonstrated the relevance of such a strategy while emphasizing the need for more potent gene transfer agents. In recent years, many novel non-viral gene delivery vehicles were proposed as potential alternatives to GL67 cationic lipid. However, they were usually evaluated using procedures difficult or even impossible to implement in clinical practice. In this study, a clinically-relevant administration protocol via aerosol in murine lungs was used to conduct a comparative study with GL67A. Diverse lipidic compounds were used to prepare a series of formulations inspired by the composition of GL67A. While some of these formulations were ineffective at transfecting murine lungs, others demonstrated modest-to-very-efficient activities and a series of structure-activity relationships were unveiled. Lipidic aminoglycoside derivative-based formulations were found to be at least as efficient as GL67A following aerosol delivery of a luciferase-encoding plasmid DNA. A single aerosol treatment with one such formulation was found to mediate long-term lung transgene expression, exceeding half the animal's lifetime. This study clearly supports the potential of aminoglycoside-based cationic lipids as potent GL67-alternative scaffolds for further enhanced aerosol non-viral lung gene therapy for diseases such as CF.

Limitations of the murine nose in the development of nonviral airway gene transfer.

A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials.

Coating of adeno-associated virus with reactive polymers can ablate virus tropism, enable retargeting and provide resistance to neutralising antisera.

BACKGROUND: Copolymers based on poly-[N-(2-hydroxypropyl) methacrylamide] (HPMA) have been used previously to enable targeted delivery of adenovirus. Here we demonstrate polymer-coating techniques can also be used to modify and retarget adeno-associated virus (AAV) types 5 and 8. METHODS: Three strategies for modifying transductional targeting of AAV were employed. The first involved direct reaction of AAV5 or AAV8 with amino-reactive HPMA copolymer. The second approach used carbodiimide (EDC) chemistry to increase the number of surface amino groups on the AAV5 capsid, thereby improving coating efficiency. In the third approach, the AAV5 genome was isolated from capsid proteins and delivered in a synthetic polyplex consisting of polyethylenimine (PEI) and HPMA. RESULTS: Efficient covalent attachment of HPMA copolymer to AAV5 could only be achieved following modification of the virus with EDC. Coating inhibited sialic acid dependent infection and provided a platform for retargeting via new ligands, including basic fibroblast growth factor. Retargeted infection was shown to be partially resistant to neutralising antisera. Delivery of AAV5 genomes using PEI and HPMA was efficient and provided absolute control of tropism and protection from antisera. In contrast AAV8 could be reacted directly with HPMA copolymer and allowed specific retargeting via the epidermal growth factor receptor, but gave no protection against neutralising antisera. CONCLUSIONS: Reactive HPMA polymers can be used to ablate the natural tropism of both AAV8 and EDC-modified AAV5 and enable receptor-specific infection by incorporation of targeting ligands. These data show transductional targeting strategies can be used to improve the versatility of AAV vectors.