Evgeniy Leyvi

Enhanced gene expression of systemically administered plasmid DNA in the liver with therapeutic ultrasound and microbubbles

Ultrasound-mediated delivery (USMD) of novel therapeutic agents in the presence of microbubbles is a potentially safe and effective method for gene therapy offering many desired characteristics, such as low toxicity, potential for repeated treatment, and organ specificity. In this study, we tested the capability of USMD to improve gene expression in mice livers using glycogen storage disease Type Ia as a model disease under systemic administration of naked plasmid DNA. Image-guided therapeutic ultrasound was used in two studies to provide therapeutic ultrasound to mice livers. In the first study, involving wild-type mice, control animals received naked plasmid DNA (pG6Pase 150 μg) via the tail vein, followed by an infusion of microbubbles; the treated animals additionally received therapeutic ultrasound (1 MHz). Following the procedure, the animals were left to recover and were subsequently euthanized after 2 d and liver samples were extracted. Reverse transcription polymerase chain reaction (RT-PCR) assays were performed on the samples to quantify mRNA expression. In addition, Western blot assays of FLAG-tagged glucose-6-phosphatase (G6Pase) were performed to evaluate protein expression. Ultrasound-exposed animals showed a 4-fold increase in G6Pase RNA in the liver, in comparison with control animals. Furthermore, results from Western blot analysis demonstrated a 2-fold increased protein expression in ultrasound-exposed animals after two days ( p <; 0.05). A second pilot study was performed with G6Pase knockout mice, and the animals were monitored for correction of hypoglycemia over a period of 3 weeks before tissue analysis. The RT-PCR assays of samples from these animals demonstrated increased G6Pase RNA in the liver following ultrasound treatment. These results demonstrate that USMD can increase gene expression of systemically injected naked pDNA in the liver and also provide insight into the development of realistic approaches that can be translated into clinical practice.

Enhanced gene transfection of plasmid DNA in the liver with ultrasound and microbubbles

Ultrasound mediated delivery (USMD) in the presence of microbubbles is a potentially safe and effective method for gene therapy, offering many desired characteristics such as low toxicity, potential for repeated treatment, as well as organ specificity. In this study we tested the capability of USMD to improve gene transfection to mice livers for treating a rare genetic disorder, Glycogen Storage Disease Type 1a. An image guided therapeutic ultrasound system (TIPS, Philips) was used to provide therapeutic ultrasound to mice liver under IACUC approved protocols. Anesthetized healthy mice were placed supine on a heated pad and coupled to the TIPS transducer. Therapeutic plasmid DNA (FLAG-pG6Pase) was injected and microbubbles infused via the tail vein. Treated animals (n=4) received therapeutic ultrasound pulses (1 MHz, 2.5 MPa) over the entire liver. Control animals (n=5) received pDNA but not ultrasound treatment. Post treatment the animals were left to recover and subsequently sacrificed after 5 days. Tissues from the left, middle, caudal, right anterior, and right posterior liver lobes were harvested and stored. Quantitative PCR assays were then performed on the samples to quantify gene transfection. Ultrasound treated animals showed significantly higher levels of G6Pase transfection compared to control animals (p<0.05) in all five lobes of the liver. On average, the treated animals showed 5.4 times more pDNA accumulation in the liver compared to controls. Immunohistochemistry staining for FLAG tag showed increased transgene expression especially around the blood vessels in treated animals. No evidence of toxicity was found up to 5 days post treatment.

Acoustic Accessibility Investigation for Ultrasound Mediated Treatment of Glycogen Storage Disease Type Ia Patients

Glycogen storage disease type Ia (GSDIa) is caused by an inherited defect in the glucose-6-phosphatase gene. The recent advent of targeted ultrasound-mediated delivery (USMD) of plasmid DNA (pDNA) to the liver in conjunction with microbubbles may provide an alternative treatment option. This study focuses on determining the acoustically accessible liver volume in GSDIa patients using transducer models of various geometries with an image-based geometry-driven approach. Results show that transducers with longer focal lengths and smaller apertures (up to an f/number of 2) are able to access larger liver volumes in GSDIa patients while still being capable of delivering the required ultrasound dose in situ (2.5 MPa peak negative pressure at the focus). With sufficiently large acoustic windows and the ability to use glucose to easily assess efficacy, GSD appears to be a good model for testing USMD as proof of principle as a potential therapy for liver applications in general.

Ultrasound and Microbubble Mediated Doxil Delivery in a Murine Breast Cancer Model: Therapeutic Efficacy Dependence on Tumor Growth Rate

The effect of tumor growth rate and treatment repeats are examined as parameters in pressure-mediated ultrasound treatments with microbubbles and Doxil in a murine breast cancer model. For this purpose, mice with a tumor doubling time of 8 and 13 days respectively received either a single or two ultrasound treatments (at 1 MHz/1 MPa) in conjunction with Definity microbubbles (1:1 dilution) and Doxil (3 mg/Kg dose) after the tumor size reached 150 mm3. The tumor model was generated using MDA-MB-231-luc cells implanted into the lower mammary fat pad of SCID beige mice. At 15 days post-treatment, tumor size was reduced by 3±18%, 8±14%, and 20±10% as compared to control for the Doxil only, ultrasound + microbubbles + Doxil (single treatment), and ultrasound + microbubbles + Doxil (2 treatments) groups, respectively, in the mice with the slower growing tumors. The mice with the faster growing tumor yielded tumor size reductions of 46±27%, 71±10%, and 61±26%, respectively, for the same groups. We hypothesize that treatment efficacy is dependent on the dynamics of the tumor itself, even within the same cell line.