rAAV2 traffics through both the late and the recycling endosomes in a dose-dependent fashion.

Inefficient trafficking of recombinant adeno-associated virus type-2 (rAAV2) to the nucleus is a major barrier for transduction. Using imaging and subcellular fractionation techniques, we evaluated the extent of rAAV2 movement through the late (Rab7) and recycling (Rab11) endosomes. Following rAAV2 infection of HeLa cells, immunoisolation of HA-Rab7- or HA-Rab11-tagged endosomes and intracellular colocalization of Cy3-labeled rAAV2 with EGFP-Rab7 or EGFP-Rab11 markers demonstrated dose-dependent trafficking of rAAV2 through the recycling and late endosomal compartments. At low multiplicities of infection (m.o.i. 100 genomes/cell), rAAV2 predominantly trafficked to the Rab7 compartment. In contrast, rAAV2 predominantly trafficked to the recycling endosome at 100-fold higher m.o.i. siRNA studies inhibiting either Rab7 or Rab11 demonstrated that reducing Rab11 protein levels more significantly inhibited rAAV2 transduction on a per genome basis compared to inhibition of Rab7. Dose-response curves, comparing the m.o.i. of AV2Luc infection to relative transduction, also supported the hypothesis that viral movement through the Rab11 compartment at high m.o.i. is more competent for transgene expression ( approximately 100-fold) than virus that moves through the Rab7 compartment at low m.o.i. These findings suggest that strategies to shunt viral movement from the late to the recycling endosome may be effective at increasing viral transduction for gene therapy.

Nox2 and Rac1 regulate H2O2-dependent recruitment of TRAF6 to endosomal interleukin-1 receptor complexes.

Reactive oxygen species (ROS) generated by NADPH oxidases (Nox) have been implicated in the regulation of signal transduction. However, the cellular mechanisms that link Nox activation with plasma membrane receptor signaling remain poorly defined. We have found that Nox2-derived ROS influence the formation of an active interleukin-1 (IL-1) receptor complex in the endosomal compartment by directing the H2O2-dependent binding of TRAF6 to the IL-1R1/MyD88 complex. Clearance of both superoxide and H2O2 from within the endosomal compartment significantly abrogated IL-1beta-dependent IKK and NF-kappaB activation. MyD88-dependent endocytosis of IL-1R1 following IL-1beta binding was required for the redox-dependent formation of an active endosomal receptor complex competent for IKK and NF-kappaB activation. Small interfering RNAs to either MyD88 or Rac1 inhibited IL-1beta induction of endosomal superoxide and NF-kappaB activation. However, MyD88 and Rac1 appear to be recruited independently to IL-1R1 following ligand stimulation. In this context, MyD88 binding was required for inducing endocytosis of IL-1R1 following ligand binding, while Rac1 facilitated the recruitment of Nox2 into the endosomal compartment and subsequent redox-dependent recruitment of TRAF6 to the MyD88/IL-1R1 complex. The identification of Nox-active endosomes helps explain how subcellular compartmentalization of redox signals can be used to direct receptor activation from the plasma membrane.

Spliceosome-mediated RNA trans-splicing with recombinant adeno-associated virus partially restores cystic fibrosis transmembrane conductance regulator function to polarized human cystic fibrosis airway epithelial cells.

We previously reported that spliceosome-mediated RNA trans-splicing (SMaRT), using recombinant adenoviral vectors expressing pre-trans-splicing molecules (PTMs), could partially restore cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity to polarized human DeltaF508 CF airway epithelia. Although these studies proved that SMaRT could correct CFTR mRNA defects, recombinant adenoviral infection from the basolateral surface was required because of inefficient infection from the apical membrane. Hence, applications of SMaRT technology for CF gene therapy require further testing with alternative, more clinically viable, vector systems. Furthermore, because recombinant adeno-associated virus (rAAV) vectors have packing limitations with respect to the size of the CFTR transgene insert, SMaRT correction of CFTR has the added attraction of a smaller transgene cassette. In the present study, we investigated whether rAAV vectors could effectively rescue CFTR chloride conductance in polarized human CF airway epithelial cells, using a SMaRT approach. AAV vectors were generated to carry a PTM engineered to bind intron 9 of CFTR pre-mRNA and then trans-splice the normal sequence for human CFTR exons 10-24 into the endogenous pre-mRNA. Human CF polarized airway epithelia were infected from the apical membrane with rAAV2 or rAAV5 CFTR-PTM vectors in the presence of proteasome-modulating agents (doxorubicin and N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal) to enhance transduction. Epithelia were then evaluated for cAMP-sensitive short-circuit currents 2 weeks postinfection. Levels of CFTR correction seen with rAAV2 (1.07 +/- 0.24 microA) and rAAV5 (0.90 +/- 0.20 microA) CFTR-PTM vectors were similar, representing conductance equivalent to 14.2 and 13.6% of that observed in non-CF human polarized epithelia, respectively. RT-PCR analysis demonstrated the existence of wild-type CFTR transcript in CFTR-PTM-corrected epithelia, whereas only DeltaF508 mRNA was detected in polarized cells infected with control rAAV LacZ-PTM vectors. These results provide evidence that rAAV vectors are capable of using SMaRT to correct CFTR function after apical infection of human CF airway epithelia. The ability of CFTR-PTM-mediated correction to maintain endogenous CFTR regulation of the transgene product may further improve the efficacy of gene therapy for CF.

Dual therapeutic utility of proteasome modulating agents for pharmaco-gene therapy of the cystic fibrosis airway.

Pharmacologic- and gene-based therapies have historically been developed as two independent therapeutic platforms for cystic fibrosis (CF) lung disease. Inhibition of the dysregulated epithelial Na channel (ENaC) is one pharmacologic approach to enhance airway clearance in CF. We investigated pharmacologic approaches to enhance CFTR gene delivery with recombinant adeno-associated virus (rAAV) and identified compounds that significantly improved viral transduction while simultaneously inhibiting ENaC activity through an unrelated mechanism. Treatment of human CF airway epithelia with proteasome modulating agents (LLnL and doxorubicin) at the time of rAAV2 or rAAV2/5 infection dramatically enhanced CFTR gene delivery and correction of CFTR-mediated short-circuit currents. Surprisingly, these agents also facilitated long-term (15-day) functional inhibition of ENaC currents independent of CFTR vector administration. Inhibition of ENaC activity was predominantly attributed to a doxorubicin-dependent decrease in gamma-ENaC subunit mRNA expression and an increase in gamma-ENaC promoter methylation. This is the first report to describe the identification of compounds with dual therapeutic action that are able to enhance the efficacy of CFTR gene therapy to the airway while simultaneously ameliorating primary aspects of CF disease pathophysiology. The identification of such compounds mark a new area for drug development, not only for CF, but also for other gene therapy disease targets.