**5. Selected applications of ultrasound-mediated gene delivery**

#### **5.1 Cardiovascular**

Recent reviews of cardiac applications of UTMD are provided elsewhere {Laing & McPherson 2009; Porter 2009}. Desired endpoints may be improved vascular function (*e.g*., following ischemia) or inhibition of vascularization to 'starve' tumors. The following discussion is intended only to provide an indication of some of the exciting avenues of research in the area.

*Cardiovascular graft remodeling*: Tissue remodeling after transplantation surgery is required for long term transplant success. It has been shown that US-mediated gene therapy can improve transplanted vessel patency after surgery. Carotid interposition saphenous vein

The US contrast agent pioneer Thomas Porter recognized early that UTMD delivery of oligonucleotides had the potential to influence vascular tissue remodeling after injury. In a 2001 study, an oligonucleotide which inhibits vascular smooth muscle cell proliferation was bound to albumin-shelled MBs and UTMD effected by transcutaneous application of 20 kHz US to porcine carotid artery walls following balloon catheter injury. Thirty days after treatment, the percent area stenosis in UTMD-treated animals was half that in controls {Porter *et al.* 2001}. The uptake of ODNs into intact *ex vivo* human saphenous veins and isolated smooth muscle cells from the veins was also potentiated by US {Kodama *et al.* 2005}. In addition, UTMD facilitated the delivery of antisense ODN targeting the human androgen receptor (AR) in prostate tumor cells, resulting in 49% transfected cells, associated with a

UTMD-mediated sonoporation (frequency: 1 MHz; intensity: 2 W/cm2; exposure time: 2 min) was capable of enhancing *in vivo* siRNA delivery into salivary gland of rats, leading to significant GAPDH gene silencing by 10-50% for 48 hours {Sakai *et al.* 2009}. No gene silencing was observed with exposure to US only in the absence of Optison MBs. Intraventricular co-injection of siRNA-GFP and MB BR14 with concomitant ultrasonic exposure resulted in a substantial reduction in EGFP expression in the coronary artery in EGFP transgenic mice {Tsunoda *et al.* 2005}. Liposomal MBs combined with US can efficiently delivery siRNA with only 10s of US exposure *in vitro*. siRNA was also efficiently delivered into the tibialis muscles using the same system and the gene-silencing effect could be sustained for more than 3 weeks {Negishi *et al.* 2008}. These results demonstrate that UTMD-mediated delivery of siRNA can serve as a very useful tool for loss-of-function

Cell therapy is a promising strategy for many applications, including genetic diseases, cancer, regenerative medicine, and others. However, it is very difficult to transfect certain cell types and maintain their viability following transfection, including hematopoietic and mesenchymal stem cells, T cells, and others which are important targets for cell therapy using the transfection methods currently available. UTMD has been demonstrated to facilitate the delivery of siRNA into mesenchymal stem cells (MSCs) {Otani *et al.* 2009}, which knocked down mRNA expression of specific genes, leading to the improvement of cellular function and viability. The application of UTMD has high potential to facilitate the delivery of genetic materials into target cells and can be expanded for use in a variety of cell

Recent reviews of cardiac applications of UTMD are provided elsewhere {Laing & McPherson 2009; Porter 2009}. Desired endpoints may be improved vascular function (*e.g*., following ischemia) or inhibition of vascularization to 'starve' tumors. The following discussion is intended only to provide an indication of some of the exciting avenues of

*Cardiovascular graft remodeling*: Tissue remodeling after transplantation surgery is required for long term transplant success. It has been shown that US-mediated gene therapy can improve transplanted vessel patency after surgery. Carotid interposition saphenous vein

**5. Selected applications of ultrasound-mediated gene delivery** 

decrease in AR expression compared to untreated controls {Haag *et al.* 2006}.

genetic engineering both *in vitro* and *in vivo*.

**4.5 Transduced cell therapy** 

therapy protocols.

**5.1 Cardiovascular** 

research in the area.

grafts in pigs were treated *ex vivo* prior to transplantation with 1 MHz US at ~1.8 MPa Pa with both a MB contrast agent and a plasmid encoding for metalloproteinase 3 (TIMP-3; the enzyme inhibits post-graft vessel restriction) present during US exposure. At 4 weeks, luminal diameters in animals receiving the transfected grafts were significantly greater than in controls {Akowuah *et al.* 2005}. Similarly, US treatment enhanced the delivery of an adenoviral vector to the aortic root, yielding a 2.5-fold enhancement in gene delivery {Beeri *et al.* 2002}. A technical issue of note is that a balloon catheter was used to briefly occlude the aortic root above the sinuses to increase the dwell time of the injected MBs and adenoviral vectors.

*Reperfusion therapies*: US-mediated gene therapy to improve myocardial reperfusion following induced myocardial infarcts in mice was studied using Definity MBs, and UTMD achieved using high frequency (8 MHz), relatively high Pa (estimated ~4.5 MPa) US from a diagnostic US machine. Plasmids were either empty (controls) or encoded for Stem Cell Factor (SCF; expected to enhance reperfusion by recruitment of cells during tissue remodeling) or VEGF (expected to stimulate angiogenesis). At 21 days, UTMD with either VEGF or SCF-bearing pDNA increased the microvessel density and blood flow relative to controls {Fujii *et al.* 2009}. Similarly, reperfusion of ischemic rat hind limbs was improved by US-mediated gene therapy. Cationic lipid-shelled MBs and a pDNA encoding for VEGF-165 were used; US frequency and amplitude were 1.3 MHz and ~2 MPa, respectively {Kobulnik *et al.* 2009}. A murine cardiac infarct model treated with UTMD with pDNA encoding for either VEGF or stem cell factor (SCF) has been reported to increase reperfusion; the observation that SCF-encoding pDNA increased perfusion was interpreted as evidence for the recruitment of reparative cells into the area of infarction {Fujii *et al.* 2009}.

Expression of a reporter plasmid gene delivered to the myocardium by UTMD methods was relatively brief (4 d), but was improved by retreatment {Bekeredjian *et al.* 2003}. Under similar exposure conditions, damage to the heart was negligible {Bekeredjian *et al.* 2004}. However, UTMD-enhanced gene delivery to the heart is often attended by at least minimal damage, which can include extravasation of large molecules and red cells {Hernot *et al.* 2010}. It is noteworthy that under diagnostic US exposure conditions, premature ventricular contractions can occur with the use of US contrast agents (see, *e.g.* {Miller *et al.* 2005}), and in contrast with the results of {Hernot *et al.* 2010} these have been unambiguously correlated with cell killing of cardiomycetes. In rats, using 1.7 MHz ultrasound, premature complexes and cardiomycete death were observable at Pas of 2 MPa or greater {Miller *et al.* 2011}. Premature contraction complexes appear to be related directly to extravascular cell killing, so even absent gross side effects, some side effects can be expected in gene therapies involving UTMD. This has important implications not only for safety, but also efficacy; *i.e*., one hopes to transfect the target cells, not kill them. It seems unlikely that transfection to meaningful extents can be achieved without some cell killing, so attempts to optimize UTMD treatments must strive to achieve an acceptable balance between desired effect (transfection) and undesired effect (killing of the target cells).

When treating the heart using reporter genes, MBs and a diagnostic scanner as the acoustic source, superior results were obtained by moving the scan head about to 'paint' a larger volume of tissue {Geis *et al.* 2009}. Another noteworthy finding was that moving the beam relative to the heart did not increase Evans blue dye extravasation, which suggests less microcirculation damage per unit transgene expression when the insonifying beam is moved relative to the target.

Ultrasound-Mediated Gene Delivery 229

Our group has extensively studied gene delivery of reporter and therapeutic genes into the liver. We have demonstrated that UTMD (1 MHz US) can significantly enhance gene transfer of naked pDNA into the mouse liver in the presence of either Optison {Miao 2005} or Definity MBs {Shen *et al.* 2008}. Transgene expression was dependent exponentially on Pr, with an inflection point usually between 1 and 2.5 MPa followed by a plateau above 3 MPa {Song *et al*. 2011a}, consistent with an inertial cavitation mechanism. More than thousand-fold enhancement of gene transfer efficiencies was obtained compared to control experiments in the absence of UTMD. Recently we have gained preliminary success in scaling up pDNA delivery in larger animal models, including rats {Song *et al*. 2011b } and dogs {Noble *et al.* 2011}. Previously we have shown that near therapeutic levels of factor IX were achieved by UTMD-mediated gene delivery in mice. Technical improvements to further enhance gene transfer of factor VIII for treatment of hemophilia A and factor IX for treatment of hemophilia B are currently being pursued in small and large animal models.

A number of kidney diseases could be potentially treated with gene therapies. Hydrodynamic approaches have met with some success. Xing *et al*. attempted to improve on these results by combining hydrodynamic and UTMD approaches to naked DNA reporter gene delivery to surgically-exposed rat kidneys. Combined US (unspecified frequency) from a Sonotron 2000 hand-held diagnostic US machine and hydrodynamic therapy together yielded better reporter gene transfection than hydrodynamic therapy alone, producing an approximately 4-fold increase in reported gene expression when the estimated Pa was in the range of 0.3 – 0.8 MPa and no MBs were used. The effect was intensity-dependent. When Optison MBs were injected with the naked DNA during hydrodynamic therapy and US exposure, the same effect (4-fold increase in gene expression) was observed at an intensity of

The failure of wounds to heal in diabetic patients is a significant clinical problem. Gene therapies which promote angiogenesis represent a promising approach to this problem. VEGF-encoding gene vectors (either minicircle naked DNA; a supercoiled form with a molecular weight estimated as 331 g/mol, or naked DNA borne on the gene carrier branched polyethylinimine) were tested for efficacy in inducing circulating VEGF expression and accelerating wound healing in an induced diabetic mouse model. Wounds were treated by peripheral injection of gene vectors with or without exposure to US (1 MHz, 2 W/cm2, 20% duty cycle; estimated 0.25 - 0.5 MPa). In some treatments, SonoVue MBs were injected with the microcircle DNA prior to sonication. Markedly greater levels of circulating VEGF were observed in mice treated with [VEGF-encoding minicircle DNA + US + MBs] relative to controls, but not as high as those obtained using the polyethylinimine gene carrier. Nonetheless, the [minicircle DNA + US + MBs] treatment produced a significant

*Brain:* Much work has been done on 'opening' (*i.e*., making more permeable) the bloodbrain barrier, which so tightly regulates traffic between the vascular space and the brain that chemotherapeutic agents often cannot cross the barrier {Meairs & Alonso 2007}. Much

only 1 Watt/cm2 (estimated 0.2 – 0.5 MPa Pa) {Xing *et al.* 2009}.

improvement in healing rates of the treated skin wounds {Yoon *et al.* 2009}.

**5.5 Kidney** 

**5.6 Skin (DNA vaccine)** 

**5.7 Other solid organs** 

*Inhibition of neovascularization*: A substantial literature on therapies to inhibit neoangiogenesis exists. Here we mention one recent example: MBs and pDNA encoding for pigment epithelium derived factor, which inhibits neovascularization in the retina, were injected into the vitreous humor of rats having laser-induced choroidal injury, which leads to neovascularization. The eyes were treated immediately with 0.3 MHz US of Pa estimated to be in the range of 0.1 – 0.3 MPa. At 28 days post treatment, choroidal neovascularization was inhibited in the UTMD group relative to untreated controls {Zhou XY *et al.* 2009}. Similarly, pDNA carrying a silencing sequence for the gene coding for survivin were introduced into implanted murine tumors using UTMD methods. Treated tumors were sonicated with 3 MHz US at an intensity of 2 W/cm2 (estimated Pr: 0.2 - 0.5 MPa). Transgene expression was significantly increased in tumors treated with UTMB. It was proposed that the technique could be applied therapeutically to tumors to increase in tumor cell apoptosis *via* the silencing effect on survivin expression in transfected cells {Chen *et al.* 2010}.
