492. Manipulation of Ultrasound Conditions for Effective Gene Delivery in a Porcine Model
Authors: James Harrang, Shuxian Song, Bryn M. Smith, Kyle Morrison, Carol H. Miao
Journal: Molecular Therapy (2016)

Our group has previously demonstrated that increasing pulse duration lowers the accompanying peak negative pressure (PNP) required for effective gene delivery (UMGD) in mouse and cell models. This effect allows selection of conditions with minimal associated tissue damage and enables tuning to maximize the capabilities of piezo-materials. To eventually extend this finding clinically, we investigated its scalability in a pig model. In parallel, we examined several spatial effects which are important scaling considerations. According to our established UMGD protocol, the liver of each pig was exposed via a midline incision. Next, using contrast diagnostic ultrasound to confirm placement and perfusion, we catheterized a consistent branch of the portal vein. Just prior to therapeutic US exposure, the inferior vena cava was temporarily occluded. US exposure and infusion of a solution containing pGL4 plasmid and phospholipid MBs were initiated simultaneously. Therapeutic US was delivered via either H105, an unfocused 52 mm disc transducer, or H185D, a 49 mm disc transducer with three cylindrical focuses. Our US pulse durations spanned 19 µs-22 ms, with PNPs spanning 0.6-6.9 MPa. 24 hours after surgery, pigs were sacrificed to harvest treated and control liver lobes. After sectioning, spatially-mapped samples were analyzed for luciferase expression. Our ongoing experiments have added further support for a species-generalized model that increasing pulse duration enables the use of lower PNP for effective UMGD. Notably, within a paired study, increasing pulse duration from 19 µs to 200 µs at a constant 6.9 MPa PNP yielded an up to 17-fold increase in sampled luciferase gene expression. Furthermore, in the same paired study, a 200 µs pulse duration at a lower 4.5 MPa PNP still maintained up to a 9-fold increase in sampled expression versus the 19 µs, 6.9 MPa group. Despite these increases, ALT and AST values remained consistent or lowered for both groups when moving from 19 to 200 µs. When comparing spatial effects, H105 yielded significantly increased expression relative to H185D at equivalent pulse duration and focal PNP. This result suggests that focal treatment volume is also an important consideration for UMGD in larger animal models. However, comparing expression relative to energy flux shows no such discrepancy in our data, suggesting that pressure-dependent cavitation dynamics still play a role in UMGD efficacy. By manipulating US pulse durations, our group has successfully achieved increased expression, circumventing the peak power density limitations imposed by piezo-materials used in US transducers. Such tuning has also allowed us to achieve comparable expression at decreased PNPs. Since skin attenuation imposes a barrier to high PNPs in transcutaneous UMGD, this result has promising implications for advancing that modality. Our results demonstrate the advancement of UMGD technology for achieving efficient gene transfer in large animal models.