Cationic lipid formulations alter the in vivo tropism of AAV2/9 vector in lung

Abstract

Physicochemical properties of gene transfer vectors play an important role in both transduction efficiency and biodistribution following airway delivery. Adeno-associated virus (AAV) vectors are currently used in many gene transfer applications; however, the respiratory epithelium remains a challenging target. We synthesized two cationic sterol-based lipids, dexamethasone-spermine (DS) and disubstituted spermine (D(2)S) for pulmonary gene targeting. Scanning and transmission electron micrographs (TEM) confirmed that AAV/lipid formulations produced submicron-sized clusters. When AAV2/9 or AAV2/6.2 were formulated with these cationic lipids, the complexes had positive zeta potential (zeta) and the transduction efficiency in cultured A549 cells increased by sevenfold and sixfold, respectively. Transduction of cultured human airway epithelium with AAV2/6.2-lipid formulations also showed approximately twofold increase in green fluorescence protein (GFP) positive cells as quantified by flow cytometry. Intranasal administration of 10(11) genome copies (GC) of AAV2/9 and AAV2/6.2 coformulated with lipid formulations resulted in an average fourfold increase in transgene expression for both vectors. Formulation of AAV2/9 with DS changed the tropism of this vector for the alveolar epithelium, resulting in successful transduction of conducting airway epithelium. Our results suggest that formulating AAV2/9 and AAV2/6.2 with DS and D(2)S can lead to improved physicochemical characteristics for in vivo gene delivery to lung.

Figure 1

Coformulation of AAV with cationic lipids results in formation of virion clusters. (a) Scanning electron micrographs of AAV2/9, (b) AAV2/9 coformulated with DS, and (c) AAV2/9 coformulated with D₂S. (d) Transmission electron micrographs of AAV2/6.2, and (e) AAV2/6.2 coformulated with 60/40 mol% DS/D₂S with DOPE. All samples with cationic lipids contain ~7 × 10⁻¹⁴ µmol/GC and were incubated with AAV particles for 15 minutes followed by the preparation procedure for microscopy described in the materials and methods. (f) Surface areas of clusters were calculated from multiple micrographs for AAV2/9. The bar represents the mean value from at least 28 measurements for each condition. All coformulations resulted in statistical increases in effective particle size with respect to AAV2/9 control calculated using the Mann–Whitney U-test (*P < 0.01). AAV, adeno-associated virus; DS, dexamethasone-spermine; D₂S, disubstituted spermine; GC, genome copy.

Figure 2

Transduction efficiency in A549 cells with DS and D₂S liposomes (1:1 molar ratio with DOPE) at three charge ratios with ffluc2 transgene at 48 hours after exposure to AAV. (a) Coformulations with AAV2/6.2, (b) Coformulations with AAV2/9. Results presented as the mean and error bars represent SD from 48 replicates for control and 8 replicates of each experimental condition. All coformulations resulted in statistical increases in luminescence with respect to the positive control (AAV alone) as calculated using the Mann–Whitney U-test (*P < 0.01). AAV, adeno-associated virus; DS, dexamethasone-spermine; D₂S, disubstituted spermine; ffluc2, firefly luciferase; RLU, relative light unit.

Figure 3

Transduction efficiency of AAV2/6.2 expressing GFP transgene coformulated with DS and D₂S in cultures of human airway measured 72-hours after exposure. (a) All coformulations were prepared at 1.86 × 10⁻¹³ µmol/GC. Representative fluorescent images of each condition are shown. (b) Flow cytometric analysis for each condition with positive gate shown as vertical line shows an increase in positive population with coformulation. AAV, adeno-associated virus; DS, dexamethasone-spermine; D₂S, disubstituted spermine.

Figure 4

AAV2/6.2 and AAV2/9 gene expression with lipid coformulations in mouse lung. Vectors were delivered to mice by intranasal instillation. (a) Lungs were analyzed for β-gal expression after 35–41 days. Representative histological cross-sections from each group (n = 5) stained for β-gal expression (blue) and counterstained with nuclear fast red are shown. (b) Lung homogenates from all animals were analyzed for total expression of β-gal normalized to total protein (solid circles) with groups means shown as horizontal solid bars. Increases in total expression for both viral vectors were observed in some lipid-formulated groups with increased expression of transgene in the conducting airway compared to control. Scales for each subset of conditions were set independently to show all data points clearly due to the variability in maximum transgene expression. AAV, adeno-associated virus; β-gal, β-galactosidase; DOPE, 1,2 dioleoylphosphatidylethanolamine; DS, dexamethasone-spermine; D₂S, disubstituted spermine; GC, genome copy.

Figure 5

Bioluminescent imaging showing AAV2/6.2 and AAV2/9 gene expression with lipid coformulations in mouse lung. Vectors were delivered to mice by intranasal instillation. (a) AAV2/6.2 imaged in vivo after 29 days. (b) AAV2/9 imaged in vivo after 14 days. Scales for each vector were set independently to show full range of expression. Representative images for each group (n = 5) are shown. Increases in total expression for both viral vectors were observed in all lipid-formulated groups compared to control. Higher systemic transduction was noted for AAV2/9 coformulated with both lipids. AAV, adeno-associated virus; DOPE, 1,2 dioleoylphosphatidylethanolamine; DS, dexamethasone-spermine; D₂S, disubstituted spermine.

Figure 6

Gene transfer with AAV2/9 after formulation with lipid and MC gels in mouse lung. Mice were treated by intranasal instillation and analyzed for β-gal expression after 30 days. (a) Representative histological cross-sections from each group (n = 5) stained for β-gal expression (blue) and counterstained with nuclear fast red are shown. (b) Lung homogenates from all animals were analyzed for total expression of β-gal normalized to total protein. Transgene expression in the conducting airway was observed for all groups indicating a change in tropism for this serotype; however, coformulation with DS/DOPE liposomes resulted in greater total β-gal expression in lung homogenate. Results presented as the mean and error bars represent standard deviations from two replicates for each condition (n = 5). AAV, adeno-associated virus; β-gal, β-galactosidase; DOPE, 1,2 dioleoylphosphatidylethanolamine; DS, dexamethasone-spermine; D₂S, disubstituted spermine; MC, methylcellulose.