Nonviral ultrasound-mediated gene delivery in small and large animal models

Abstract

Ultrasound-mediated gene delivery (sonoporation) is a minimally invasive, nonviral and clinically translatable method of gene therapy. This method offers a favorable safety profile over that of viral vectors and is less invasive as compared with other physical gene delivery approaches (e.g., electroporation). We have previously used sonoporation to overexpress transgenes in different skeletal tissues in order to induce tissue regeneration. Here, we provide a protocol that could easily be adapted to address various other targets of tissue regeneration or additional applications, such as cancer and neurodegenerative diseases. This protocol describes how to prepare, conduct and optimize ultrasound-mediated gene delivery in both a murine and a porcine animal model. The protocol includes the preparation of a microbubble-DNA mix and in vivo sonoporation under ultrasound imaging. Ultrasound-mediated gene delivery can be accomplished within 10 min. After DNA delivery, animals can be followed to monitor gene expression, protein secretion and other transgene-specific outcomes, including tissue regeneration. This procedure can be accomplished by a competent graduate student or technician with prior experience in ultrasound imaging or in performing in vivo procedures.

Sonodelivery in Skeletal Muscle: Current Approaches and Future Potential

“…Injections are therefore difficult to standardize, as optimal techniques require training and extensive practice. One method that has been used to increase injection accuracy when delivering a variety of payloads is the utilization of imaging ultrasound to guide the injection [ 10 , 11 , 12 , 13 ], although this too requires expertise and extensive practice. Another complication associated with increased test animal size is that of decreased gene uptake/expression.…”

“…Although it is a significant challenge to produce good manufacturing practice (GMP) levels of virus at high enough titers for clinical trials, localized and systemic gene transfer methods utilizing AAVs have been developed, each with its own advantages and disadvantages [ 19 , 24 ]. Systemic viral delivery (i.e., intravenous injection) poses great risk to patients due to potential immune response and off-target delivery [ 13 ]. Localized delivery (i.e., direct intramuscular injection) tends to be much safer than systemic delivery because it induces a lower level of immune response and has decreased off-target delivery, but it is often less efficient for transducing large muscle masses due to the inability of the viral vector to disperse throughout the muscle tissue.…”

“…This method can be applied to any portion of the body and is a relatively rapid process. One of the main challenges involved with electroporation is the invasiveness of the technique in vivo; electrodes are typically inserted into the subject [ 13 , 31 ]. Another major challenge with electroporation is the resultant low cell viability in vivo [ 32 ].…”

“…Although some of the general mechanisms will be discussed herein, a description of the basic methodology used for sonoporation will not be provided. We would encourage anyone interested in a thorough description of in vitro and in vivo sonoporation methodologies to refer to the previously published works available, including [ 13 , 34 , 35 ], among others.…”

“…Patient safety and therapeutic expense are two major factors to consider in any treatment type. Consequently, ultrasound-based strategies hold great promise for gene delivery or other therapeutics, due to their low cost and clinically established safety [ 13 ]. Ultrasound has been used on patients since the late 1940s and has been considered safe for pregnant women since the 1950s [ 36 ].…”