Ultrasound-mediated gene delivery (or sonodelivery) holds great promise for use in gene therapy because it is inexpensive, widely available, reusable, minimally invasive, and safe. 

1. introduction

Gene therapy shows increasing promise for the treatment of a wide range of diseases, from cancer to musculoskeletal and immunological disorders^[1] [1]. Although the basic premise of gene therapy is relatively simple on the surface—to introduce a gene or genes into a patient’s cells/tissue that will allow the body to produce a therapeutic protein or facilitate a genetic modification—every step of the process holds its own challenges. One of the major challenges associated with making gene therapy widely (and safely) accessible is that of gene delivery in vivo. There are currently many methods for delivering DNA in vitro at high efficiencies, including electroporation, lipid-based transfection, and viral delivery. Each of these methods presents potential challenges for clinical translation, including the cost and time of production, the invasiveness of the technique, the efficiency of delivery, and/or safety concerns related to an immune response or unintended genomic integration of the therapeutic gene. The development of a gene delivery technique that is inexpensive, widely available, reusable, minimally invasive, and safe will catalyze progress in gene therapy. One promising technique that meets all of these criteria has been under investigation for many years but has not yet reached widespread use: ultrasound-mediated gene delivery, more frequently referred to as sonoporation or sonodelivery^[2][3] [2,3]. As its name suggests, this approach involves using ultrasound to deliver non-viral vectors encoding therapeutic proteins into skeletal muscle, allowing the body to then produce its own therapeutic. 

2. Previous Work

Sonodelivery typically requires the addition of microbubbles (MBs) - common echo-contrast agents used in medical procedures in humans that typically consist of an inert gaseous bubble encapsulated by a biocompatible material^{[4][5][6][7][8][9][10][11]}. The ultrasound waves cause the MBs to burst, leading to the formation of transient pores in the cell membrane. It has also been shown that MB-mediated sonoporation leads to disassembly of the cytoskeleton^[12]. Sonoporation has been used to deliver a variety of cargos to a variety of cell types^{[8][13][14][15][16][17][18][19][20][21][22][23][24]}. It has also been used

Sonodelivery typically requires the addition of microbubbles (MBs) - common echo-contrast agents used in medical procedures in humans that typically consist of an inert gaseous bubble encapsulated by a biocompatible material [4–11]. The ultrasound waves cause the MBs to burst, leading to the formation of transient pores in the cell membrane. It has also been shown that MB-mediated sonoporation leads to disassembly of the cytoskeleton [12]. Sonoporation has been used to deliver a variety of cargos to a variety of cell types [8,13–24]. It has also been used

in vivo to deliver cargo in a site-specific manner to multiple targets, including skeletal muscles, the abdomen, and the inner ear^{[4][6][25][26][27][28][29]}. In all of these former studies, there was either no or minimal off-target delivery. Due to variations in sonoporation conditions, ranging from the instrument to the settings to the type of MBs and cargo, the results vary significantly between projects. Because of this, more work is needed before sonodelivery can become a mainstream treatment method.

to deliver cargo in a site-specific manner to multiple targets, including skeletal muscles, the abdomen, and the inner ear [4,6,25–29]. In all of these former studies, there was either no or minimal off-target delivery. Due to variations in sonoporation conditions, ranging from the instrument to the settings to the type of MBs and cargo, the results vary significantly between projects. Because of this, more work is needed before sonodelivery can become a mainstream treatment method.

3. Future Directions

The most pressing future direction for sonodelivery is that of standardization. With so many different sonoporators and sonoporation conditions, even for the same target tissue or cell type, and with such disparity in the results of the gene delivery, it is difficult to determine a standardized treatment method. Developing a standard unit of measure could help to expedite this process. Sonoporation conditions are typically reported by duty cycle, frequency, time, and intensity. Many reports also include probe size and some reports include other conditions, ranging from wave type to power output. Unfortunately, with the differences between instruments, the conditions that are typically reported can lead to a variable power output, meaning that the delivery power is likely not the same, even if the conditions are very similar. Although more optimization is needed to find the average optimal conditions for delivering DNA into muscle using sonoporation, establishing a standard method for reporting sonoporation conditions, such as the inclusion of the power output, which can be calculated for each set of conditions, may help to ameliorate some of the disparities in results.

An interesting future direction for the sonodelivery field would include more work on combining sonoporation with other approaches that might increase the efficacy of gene delivery. Some work has already been done along this vein, including polymer-mediated sonodelivery^[30][31][32]; adjustments to cell membrane permeability by adding enzymes, such as hyaluronidase^[33], and by adding drugs such as lidocaine and adjusting temperature^[14]; and electro-sonoporation^[34][35][36]. Electro-sonoporation is a particularly interesting procedure in which electroporation and sonoporation are applied simultaneously to facilitate cell membrane pore formation. In a proof of concept investigation, Longsine-Parker et al. found that cell viability was 97.3% and membrane pore formation efficiency was 95.6% in mammalian cells after application^[36]. These promising results were further explored with

An interesting future direction for the sonodelivery field would include more work on combining sonoporation with other approaches that might increase the efficacy of gene delivery. Some work has already been done along this vein, including polymer-mediated sonodelivery [30–32]; adjustments to cell membrane permeability by adding enzymes, such as hyaluronidase [33], and by adding drugs such as lidocaine and adjusting temperature [14]; and electro-sonoporation [34–36]. Electro-sonoporation is a particularly interesting procedure in which electroporation and sonoporation are applied simultaneously to facilitate cell membrane pore formation. In a proof of concept investigation, Longsine-Parker et al. found that cell viability was 97.3% and membrane pore formation efficiency was 95.6% in mammalian cells after application [36]. These promising results were further explored with

in vitro

and

in vivo studies that found that electro-sonoporation performed significantly better in both transfection efficiency and cell viability compared to electroporation alone^[34][35]. While more work is needed to confirm these results and elaborate on the effectiveness of electro-sonoporation, it appears to be a highly promising technique. While all these possible adaptations would add more conditions to consider in the standardization procedure, they could greatly enhance the efficacy of sonodelivery for gene therapy.

studies that found that electro-sonoporation performed significantly better in both transfection efficiency and cell viability compared to electroporation alone [34,35]. While more work is needed to confirm these results and elaborate on the effectiveness of electro-sonoporation, it appears to be a highly promising technique. While all these possible adaptations would add more conditions to consider in the standardization procedure, they could greatly enhance the efficacy of sonodelivery for gene therapy.

Another direction of high importance and interest for intramuscular sonoporation will be the delivery of therapeutic genes, as opposed to reporter genes (i.e., luciferase). Although sonoporation has been used to deliver therapeutic genes intratumorally^[10][37], the majority of the intramuscular sonodelivery completed to date has utilized reporter genes to test sonoporation conditions and efficiency. It could be argued that testing the delivery of therapeutic genes via sonoporation is of greater importance than standardization, as having a standardized method will only be clinically relevant when the conditions are optimized for therapeutic expression. To help mitigate the potential for non-standard conditions to be a confounding factor of a lack of therapeutic expression, this work could be conducted in tandem with the above-mentioned standardization work. Of particular interest would be the delivery of therapeutic proteins tagged or fused with a reporter gene, such as luciferase, which would allow for simultaneous testing of therapeutic expression and optimization of sonodelivery conditions.

Another direction of high importance and interest for intramuscular sonoporation will be the delivery of therapeutic genes, as opposed to reporter genes (i.e., luciferase). Although sonoporation has been used to deliver therapeutic genes intratumorally [10,37], the majority of the intramuscular sonodelivery completed to date has utilized reporter genes to test sonoporation conditions and efficiency. It could be argued that testing the delivery of therapeutic genes via sonoporation is of greater importance than standardization, as having a standardized method will only be clinically relevant when the conditions are optimized for therapeutic expression. To help mitigate the potential for non-standard conditions to be a confounding factor of a lack of therapeutic expression, this work could be conducted in tandem with the above-mentioned standardization work. Of particular interest would be the delivery of therapeutic proteins tagged or fused with a reporter gene, such as luciferase, which would allow for simultaneous testing of therapeutic expression and optimization of sonodelivery conditions.

Further improvements for intramuscular sonodelivery and gene therapy might include the incorporation of muscle-specific refinements of payload, such as microbubbles designed to target skeletal muscle or the extracellular matrix (ECM) or vectors containing skeletal muscle-specific promoters to increase the efficiency of transgene expression^[38].

Further improvements for intramuscular sonodelivery and gene therapy might include the incorporation of muscle-specific refinements of payload, such as microbubbles designed to target skeletal muscle or the extracellular matrix (ECM) or vectors containing skeletal muscle-specific promoters to increase the efficiency of transgene expression [38].

In light of all these considerations, it can easily be seen that there is additional optimization needed for these approaches and there would be great benefit to the scientific community in implementing more extensive testing of therapeutic delivery to skeletal muscle using sonoporation.

References

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