**5.2 Skeletal muscle**

Among the applications for gene therapy in skeletal muscle is the treatment of muscular dystrophy (see, e.g., {Alter *et al.* 2009} and references therein). As a specific recent example, Kodama and colleagues used various means to attempt delivery of PGL3 luciferase pDNA into murine skeletal muscle. Optison, human albumin MBs or acoustic liposomes (all formulated with C3F8 gas) were compared, using a 1 MHz US source; Pa was 0.2 MPa. Treatment with plasmid, US and either Optison or liposomes increased luciferase expression relative to controls. The ~200 nm diameter acoustic liposomes produced the highest expression rates, presumably because of their very high concentration {Kodama *et al.* 2010}.

#### **5.3 Pancreas**

Pancreatic gene therapies have focused principally on treatments for diabetes types I and II. The feasibility of using pDNA and UTMD methods to deliver transgenes to the pancreas has been described by Grayburn and colleagues. Following injection of reporter gene pDNA attached to the MB phospholipid shells, the reporter gene was expressed with fairly high spatial specificity, declining after 4 days, but with measurable expression persisting for as long as 21 days ({Chen *et al.* 2006}; see internal citations for a discussion of viral approaches). US of ~3 MHz was used, with an apparent Pa of ~1 MPa at the pancreas. When plasmid vectors carrying human insulin and hexokinase I gene were delivered using UTMD, transgene expression was obtained in islets and decreased circulating glucose levels were observed in treated rats. These data indicate that UTMD allows relatively noninvasive delivery of genes to pancreatic islets to modulate beta cell function. More recently, the same group reported that delivery of NeuroD1 gene driven by a rat insulin promoter (RAP3.1) into rats by UTMD-mediated gene delivery targeting pancreas *in vivo* promoted islet regeneration from surviving beta-cells, with normalization of glucose, insulin and C-peptide levels at 30 days {Chen *et al.* 2010}.

#### **5.4 Liver**

Liver is a major organ for protein synthesis, and therefore represents an important target for gene therapy. We will discuss here UTMD-mediated gene therapy targeting liver for treating non-tumoral diseases.

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.

### **5.5 Kidney**

228 Non-Viral Gene Therapy

*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

Among the applications for gene therapy in skeletal muscle is the treatment of muscular dystrophy (see, e.g., {Alter *et al.* 2009} and references therein). As a specific recent example, Kodama and colleagues used various means to attempt delivery of PGL3 luciferase pDNA into murine skeletal muscle. Optison, human albumin MBs or acoustic liposomes (all formulated with C3F8 gas) were compared, using a 1 MHz US source; Pa was 0.2 MPa. Treatment with plasmid, US and either Optison or liposomes increased luciferase expression relative to controls. The ~200 nm diameter acoustic liposomes produced the highest expression rates, presumably because of their very high concentration {Kodama *et al.* 2010}.

Pancreatic gene therapies have focused principally on treatments for diabetes types I and II. The feasibility of using pDNA and UTMD methods to deliver transgenes to the pancreas has been described by Grayburn and colleagues. Following injection of reporter gene pDNA attached to the MB phospholipid shells, the reporter gene was expressed with fairly high spatial specificity, declining after 4 days, but with measurable expression persisting for as long as 21 days ({Chen *et al.* 2006}; see internal citations for a discussion of viral approaches). US of ~3 MHz was used, with an apparent Pa of ~1 MPa at the pancreas. When plasmid vectors carrying human insulin and hexokinase I gene were delivered using UTMD, transgene expression was obtained in islets and decreased circulating glucose levels were observed in treated rats. These data indicate that UTMD allows relatively noninvasive delivery of genes to pancreatic islets to modulate beta cell function. More recently, the same group reported that delivery of NeuroD1 gene driven by a rat insulin promoter (RAP3.1) into rats by UTMD-mediated gene delivery targeting pancreas *in vivo* promoted islet regeneration from surviving beta-cells, with normalization of glucose, insulin and C-peptide

Liver is a major organ for protein synthesis, and therefore represents an important target for gene therapy. We will discuss here UTMD-mediated gene therapy targeting liver for

*et al.* 2010}.

**5.3 Pancreas** 

**5.4 Liver** 

levels at 30 days {Chen *et al.* 2010}.

treating non-tumoral diseases.

**5.2 Skeletal muscle** 

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

#### **5.6 Skin (DNA vaccine)**

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

#### **5.7 Other solid organs**

*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

Ultrasound-Mediated Gene Delivery 231

In tumors as in other systems, extravasation of gene vectors seems to be a constraint for delivery of antitumoral therapeutics which are delivered intravascularly. Here, too, UTMD appears to aid in increasing vascular permeability. For example, in implanted subcutaneous hepatomas, extravasation of Evans blue dye was 5-fold higher in the UTMD and plasmidtreated tumors than in untreated control tumors; there was no increase in Evans blue extravasation when US was applied without MBs. In this case, however, there was no significant transfection by the pDNA {Bekeredjian *et al.* 2007}. UTMD techniques have been used successfully to introduce the gene for tumor suppression protein p53 into murine retinoblastoma xenografts; when insonated in the presence of MBs or liposomes, significant expression of p53 resulted; there was no expression in the plasmid-only or plasmid + US

Much exciting work on ultrasound-mediated gene delivery has already been done, but the field remains young and even preclinical applications involving therapeutic genes are relatively few. A few generalities may be put forth: In most (but not all) cases, ultrasoundmediated gene delivery is dependent on, or greatly facilitated by, exogenous microbubbles. Where this is the case, the acoustic exposures are usually such that the bubbles can be expected to behave in a nonlinear way. Given the intense effort being devoted to the topic, we can anticipate with some confidence that successful gene therapies in large animals and

The authors acknowledge gratefully the support of NIH grants 5R01HL069049 (C.H.M., A.A.B), 5R21/R33HL089038 (C.H.M., A.A.B); 5R01HL082600 (C.H.M.); 1R01CA917794 (A.A.B.), and 1RO1CA109557 (A.A.B.), 5RO1EB000350 (A.A.B.) and Bayer Hemophilia Award (C.H.M.). We thank Drs. Thomas J. Matula, Wayne Kreider, Michael R. Bailey, Peter J. Kaczkowski, Mark Borden, Misty Noble, Sophia Song and Ms. Hong Chen for helpful

AIUM. (2000) Mechanical Bioeffects from Diagnostic Ultrasound: AIUM Consensus

AIUM. (2008) American Institute of Ultrasound in Medicine Consensus Report on Potential

Akowuah EF, Gray C, Lawrie A, Sheridan PJ, Su CH, Bettinger T, Brisken AF, Gunn J,

Alter J, Sennoga CA, Lopes DM, Eckersley RJ, Wells DJ. (2009) Microbubble stability is a

size in a porcine interposition graft model. *Gene Ther* 12: 1154-1157.

ultrasound contrast agents. *J Ultrasound Med* 19: 120 - 142.

vivo gene transfer. *Ultrasound Med Biol* 35: 976-984.

Statements. Section 6 - Mechanical bioeffects in the presence of gas-carrier

Bioeffects of Diagnostic Ultrasound: Executive Summary. *J Ultrasound Med* 27: 503-

Crossman DC, Francis SE, Baker AH, Newman CM. (2005) Ultrasound-mediated delivery of TIMP-3 plasmid DNA into saphenous vein leads to increased lumen

major determinant of the efficiency of ultrasound and microbubble mediated in

groups {Luo *et al.* 2010}.

**6. Concluding remarks** 

**7. Acknowledgements** 

**8. References** 

515.

early human trials will be achieved in the near future.

discussions during preparation of this manuscript.

success with tracer molecules (*e.g*., Evans blue dye, gadolinium MRI contrast agents, *etc*.) has been achieved using UTMD methods, principally in small animal models. However, low energy US applied through the temporal bone of swine produced short-term permeabilization of the blood-brain barrier with exogenous MBs (see {Xie *et al.* 2008} and citations within). However, we have found no reports of UTMD gene therapies attempted in the brain.
