Question:

Site-specificity and potent gene transfer are the challenging requirements of intravasal gene delivery. We investigated whether coupling of lentiviruses (LVs) and pDNA to magnetic microbubbles (MMB) could establish efficient local gene transfer in vivo after systemic application and subsequent magnetic field (MF) and ultrasound (US) exposure.

Methods:

Coupling of pDNA and LVs to MMB was verified by flow cytometry and fluorescence microscopy of reporter gene expression (DsRed or GFP respectively). Targeted gene transfer in vivo was assessed in the mouse (C57BL/6) dorsal skin fold chamber after intra-arterial injection of MMB coupled to pDNA (4µl/g body weight) or LVs (1.6x10⁶-1.5x10⁷ infectious particles) and MF and US (1MHz, 2W/cm², DC50%, 30sec) exposure. Efficiency of reporter gene expression was assessed by intravital microscopy and real-time PCR 48-96h after treatment. Viral particle amount in blood, urine, stool and saliva 48-96h after treatment was analyzed with p24 ELISA and cell culture.

Results:

pDNA and LVs readily associate with MMB in vitro (n=3). In vivo, MMB specifically delivered the coupled genetic material to dorsal skin arterioles and venules after MF and US application (n=7, p<0.05) compared to MMB injection only. LV-associated MMB gene transfer efficiency was enhanced 120-fold compared to pDNA-associated MMB, as assessed by reporter gene expression (p<0.05, GFP n=4; DsRed n=10). After treatment no residual LVs were detected in the collected biological samples (n=3).

Conclusions:

The MMB technology offers specific tissue targeting with enhanced gene transfer efficiencies using LV-associated MMB compared to pDNA-coupled MMB and may therefore represent a valuable tool for vascular gene therapy.