Contrast ultrasound imaging and therapy is a compelling technology, as it is inexpensive, widely used, portable, and permits real-time anatomical and molecular imaging. Targeted agents are now in human molecular imaging trials in the US and Europe and therapeutic uses of microbubbles are also expanding into clinical trials. MB oscillation can enhance vascular and cell membrane permeability and can accomplish local therapeutic transfection. Here, we will apply US-guided transfection to enhance suicide gene therapy. In suicide gene therapy, cell suicide-inducing transgenes (e.g. cytosine deaminase/5-fluorocytosine and the herpes simplex virus/ganciclovir) are introduced into cancer cells. The therapeutic transgenes locally convert a non-toxic small molecule pro-drug into a cytotoxic drug or create a toxic gene expression product. Recent game-changers that make our work feasible include: programmable US systems combining imaging and therapy, commercially-available (human quality) ionizable lipids, immunotherapy reagents, and new adeno- associated viruses (AAV) with organ-specific serotypes, and all are used here. Both viral and non-viral approaches are feasible and will be compared. We have recently explored the use of image-guided microbubble oscillation to transfect cells in vivo and achieve a therapeutic response. We find that ultrasound- mediated transfection can be highly effective and endeavor to extend this technique to delivery and transfection in cancer. Translational delivery protocols for human medicine involve transducers with center frequencies between 1 MHz and 220 kHz. The use of such low frequencies is highly desirable to facilitate 3D beam steering both for applications in the brain and for image-guided transfection. Practical therapeutic ultrasound systems and transducers use 1000 or less transducer elements and random element geometries result in grating lobes at higher frequencies. The highly nonlinear effect of frequency on microbubble oscillation has not yet been carefully evaluated in this frequency range. Here, we will compare in vivo delivery, transfection and suicide gene therapy achieved with MHz and hundreds of kHz transducers. Our overall goal in the renewal is to safely enhance delivery and transfection in vitro and in vivo in a clinically relevant manner. We will specifically assess suicide gene therapy with viral and non-viral vectors and will combine the local ultrasound therapy with immunotherapy in breast cancer.
Our specific aims are to: develop and validate models for contrast agent oscillation and received echoes with driving frequencies in the hundreds of kHz; maximize the delivery of unique AAVs and non-viral vectors in liver and breast cancer by insonation with a frequency selected within the hundreds of kHz to MHz, and assess safety and efficacy of a) suicide gene therapy in mice with and without immunotherapy and b) transfection in canines.
Gene therapy is rapidly advancing with three relevant treatments recently approved for use in cancer. We advance suicide gene therapy for liver and breast cancer by developing ultrasound- based methods for transfection. Here, we will explore ultrasound transmission frequencies in the hundreds of kilohertz and transfection with newly developed viral and non-viral vehicles to enhance treatment, and will combine this treatment with immunotherapy.
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