**5. Studies using microbubbles and ultrasound for gene delivery**

In this section we summarize studies that have been performed using microbubbles and ultrasound for gene delivery (Klibanov, Liu, Newman).

Over the period of more that a decade a number of different studies have been performed, in vitro and in vivo. In vitro studies showed that ultrasound increased the efficacy of transfection with cationic liposomes in cell culture studies to express reporter genes. Saphenous vein grafts have been transfected with ultrasound and MBs (Kodama) to enhance graft survival after implantation (Akowuah). In vivo studies have been performed with reporter genes showing increased expression of the reporter gene after IV administration of microbubbles either binding or in association with the reporter genes. The zones of highest expression have been in the regions of tissues of insonation (except that cationic microbubbles have also been accumulated by liver, lungs, spleen and phagocytic organs). Most of the in vivo studies Have been performed with reporter genes. A few studies have performed with therapeutic genes. A wide variety of different tissues (Hauff) and cells have shown enhanced transfection with ultrasound including neuronal cells (Fischer) and skeletal muscle (Liang).

A number of groups have studied ultrasound and microbubbles to transfect tumors (Michel, Anwer). With collaborators we performed a study assessing tumor regression with transfection of the IL-12 gene with ultrasound and cationic liposomes (Anwer). In this study the liposomes were lyophilized and may have contained nitrogen gas. The tumors were insonated with 1 MHz ultrasound. Increased expression of IL-2 was observed in the insonated tumors and statistically significant tumor regression.

In addition to phospholipid coated microbubbles, MBs can be stabilizes with denatured serum albumin (e.g. Optison®, GE Healthcare Medical Diagnostics, Princeton, NJ). Albumin binds a variety of molecules and also appears to bind DNA. In one study the plasmid of AdCMV-b-Gal was attached to the microbubbles (Shohet). In these studies the solution of AdCMV-b-Gal

necrosis at high temperatures (Ter Haar). We emphasize, however, that ultrasound power levels within the limits for diagnostic ultrasound are generally safe. Ultrasound power levels can be optimized for gene therapy to maximize gene expression while minimizing

Fig. 12. Drawing of three-dimensional transducer for imaging and treating the heart.

In this section we summarize studies that have been performed using microbubbles and

Over the period of more that a decade a number of different studies have been performed, in vitro and in vivo. In vitro studies showed that ultrasound increased the efficacy of transfection with cationic liposomes in cell culture studies to express reporter genes. Saphenous vein grafts have been transfected with ultrasound and MBs (Kodama) to enhance graft survival after implantation (Akowuah). In vivo studies have been performed with reporter genes showing increased expression of the reporter gene after IV administration of microbubbles either binding or in association with the reporter genes. The zones of highest expression have been in the regions of tissues of insonation (except that cationic microbubbles have also been accumulated by liver, lungs, spleen and phagocytic organs). Most of the in vivo studies Have been performed with reporter genes. A few studies have performed with therapeutic genes. A wide variety of different tissues (Hauff) and cells have shown enhanced transfection with

A number of groups have studied ultrasound and microbubbles to transfect tumors (Michel, Anwer). With collaborators we performed a study assessing tumor regression with transfection of the IL-12 gene with ultrasound and cationic liposomes (Anwer). In this study the liposomes were lyophilized and may have contained nitrogen gas. The tumors were insonated with 1 MHz ultrasound. Increased expression of IL-2 was observed in the

In addition to phospholipid coated microbubbles, MBs can be stabilizes with denatured serum albumin (e.g. Optison®, GE Healthcare Medical Diagnostics, Princeton, NJ). Albumin binds a variety of molecules and also appears to bind DNA. In one study the plasmid of AdCMV-b-Gal was attached to the microbubbles (Shohet). In these studies the solution of AdCMV-b-Gal

**5. Studies using microbubbles and ultrasound for gene delivery** 

ultrasound including neuronal cells (Fischer) and skeletal muscle (Liang).

insonated tumors and statistically significant tumor regression.

ultrasound for gene delivery (Klibanov, Liu, Newman).

unwanted bioeffects (Chen, Rahim).

was added to the microbubble suspension and mixed for 2 hours at 4°C. The mixture was separated into 2 distinct layers. The upper layer consisted of microbubbles with attached virus; the bottom layer, which contained unattached virus, was discarded. The concentration of microbubbles with attached AdCMV-β-Gal was 1.2x109 bubbles/mL; the mean diameter was 3.5 um. The viral titer of these microbubbles was determined. Rats were anesthetized, and MBs were administered IV. Echocardiography was performed at 1.3 MHz with a mechanical index of 1.5. Images were ECG-triggered to deliver a burst of 3 frames of ultrasound every 4 to 6 cardiac cycles. The hearts of all 6 rats in the experimental group showed blue staining with 5 bromo-4-chloro-3-indolyl-β-D-galactopyranoside. None of the control rats showed myocardial staining, which confirmed that the destruction of the microbubbles containing the virus was responsible for the observed β-galactosidase expression in the rat myocardium.

Initiated in 2001, researchers began using gas-filled, albumin-shelled MBs (Optison) and non-viral carriers to increase nascent serum HDL cholesterol in mice and Sprague-Dawley rats. Initial feasibility studies were performed using reporter genes, 5-bromo-4-chloro-3 indolyl-β-D-galactopyranoside and GFP. Subsequent studies now use ApoA-I DNA plasmids in combination with Optison. In a series of rat studies performed with partial support from an NIH SBIR award (44HL095238-01), the results revealed that an average peak response of HDL-C elevation occurred within 24 hours of treatment and 88% of the responses occurred within 48 hours. The control animal group included ultrasound only, apoA-I DNA only, and microbubble only rats. These data demonstrated a rapid incorporation of the plasmid into the cell and efficient ApoA-I protein production that was later incorporated into serum HDL-C.

Summarized data revealed an average increase of 16.8% on day one post treatment. Due to wide normal variation in individual rat HDL cholesterol values, all data is plotted as a percentage from an individual animal's baseline. When examining the overall response to treatment in mg/dL, the average peak response to treatment was 14.0% (p-value <0.0001) as seen in Figure 1 (below). This response was observed uniformly across different animals and ranged from 40mg/dL HDL-C baseline to 100mg/dL HDL-C baseline.

Ongoing studies include additional design of experiments for the following: (1) optimization of sonoporation utility for raising HDL cholesterol, (2) efficient energy delivery algorithms, (3) modes and methods of delivery, and (4) mechanistic analyses of the intracellular plasmid location.

Ongoing studies have been performed with lipid coated MBs binding plasmid DNA with genes to treat diabetes. In vivo studies in rats have shown long-term normalization of blood glucose levels in diabetic rats. Studies are planned to test this system in primates.

Encouraging work has been performed in models of hypercholesterolemia showing the potential to perform gene therapy for H1Alpha to improve the capacity of the liver to produce HDL as treatment for atherosclerosis. Promising work continues to progress in the use of MBs binding genes to treat hemophilia. Cationic MBs binding DNA (similar to Figure 7) have been tested with ultrasound and compared to DefinityR MBs which do not bind DNA. Expression of Factor IX transgenes is more robust with the cationic MBs binding DNA than with MBs not binding DNA. In both the HI1Alpha/HDL and hemophilia treatment applications the target organ is the liver. The goal in both of these programs and in many others is to generate sustained transgene expression in the liver (Guo). Catheter mediated administration may prove attractive to maximize efficiency of delivery to hepatocytes and catheters can be deployed for delivery to other organs such as the kidneys, heart and blood vessels.

Ultrasound can be targeted to any organ for which it is possible to create an acoustic window. Most tissues are readily accessible to ultrasound. Ultrasound can be applied across

Gene Delivery with Ultrasound and Microbubbles 211

Akowuah EF, Gray C, Lawrie A, Sheridan PJ, Su CH, Bettinger T et al. Ultrasound-mediated

Apfel, RE, Holland, CK. Microbubbles Lower Thresh-hold of Ultrasound Energy for

Brujan EA, Ikeda T, Matsumoto Y. Jet formation and shock wave emission during collapse of

Conger AD, Ziskin MC, Wittels H. Ultrasonic effects on mammalian multicellular tumor

Deng CX, Sieling F, Pan H, Cui J. Ultrasound-induced cell membrane porosity. Ultrasound

Feril LB, Kondo T, Zhao QL, Ogawa R, Tachibana K, Kudo N et al. Enhancement of

Forsberg F, Merton DA, Goldberg BB. In vivo destruction of ultrasound contrast microbubbles is independent of the mechanical index. J Ultrasound Med 2006; 25: 143–144 Guo DP, Li XY, Sun P, Tang YB, Chen XY, Chen Q et al. Ultrasound-targeted microbubble

FischerAJ,StankeJJ,OmarG,AskwithCC,BurryRW.Ultrasound- mediated gene transfer into

Hallow DM, Mahajan AD, McCutchen TE, Prausnitz MR. Measurement and correlation of

Hauff P, Seemann S, Reszka R, Schultze-Mosgau M, Reinhardt M, Buzasi T et al. Evaluation

Klibanov AL. Microbubble contrast agents: targeted ultrasound imaging and ultrasoundassisted drug-delivery applications. Invest Radiol 2006; 41: 354–362. Kodama T, Tan PH, Offiah I, Partridge T, Cook T, George AJ et al. Delivery of

ultrasound and microbubbles. Ultrasound Med Biol 2005; 31: 1683–1691. Liang HD, Lu QL, Xue SA, Halliwell M, Kodama T, Cosgrove DO et al. Optimisation of

HepG2 cells. Biochem Biophys Res Commun 2006; 343: 470–474.

study in rodent tumor models. Radiology 2005; 236: 572–578.

of high-intensity focused ultrasound. Phys Med Biol 2005; 50: 4797–4809. Chen S, Shohet RV, Bekeredjian R, Frenkel P, Grayburn PA. Optimization of ultrasound

Cavitation. Ultrasound Med Biol. 1991;17(2):179-85.

spheroids. .I Clin Ultrasound 1981;9:167-174.

neuronal cells. J Biotechnol 2006; 122: 393–411.

Ultrasound Med Biol 2004; 30: 1523–1529.

Med Biol 2004; 30: 527–538.

Med Biol 2003; 29: 331–337.

delivery of TIMP-3 plasmid DNA into saphenous vein leads to increased lumen size in a porcine interposition graft model. Gene Therapy 2005; 12: 1154–1157. Anwer K, Kao G, Proctor B, Anscombe I, Florack V, Earls R, Wilson E, McCreery T, Unger E,

Rolland A, Sullivan SM. Ultrasound enhancement of cationic lipid-mediated gene transfer to primary tumors following systemic administration. Gene Ther. 2000;7

ultrasound-induced cavitation bubbles and their role in the therapeutic applications

parameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid by ultrasound targeted microbubble destruction. J Am Coll Cardiol 2003; 42: 301–308.

Med Biol 2004; 30: 519–526. 15 Zarnitsyn VG, Prausnitz MR. Physical parameters influencing optimization of ultrasound-mediated DNA transfection. Ultrasound

ultrasound-induced apoptosis and cell lysis by echo-contrast agents. Ultrasound

destruction improves the low density lipoprotein receptor gene expression in

acoustic cavitation with cellular bioeffects. Ultrasound Med Biol 2006; 32: 1111–1122.

of gas-filled microparticles and sonoporation as gene delivery system: feasibility

oligodeoxynucleotides into human saphenous veins and the adjunct effect of

ultrasound-mediated gene transfer (sonoporation) in skeletal muscle cells.

**8. References** 

(21):1833-9.

Fig. 13. Baseline to peak response measurement in 30 treated rats recorded in raw data values of mg/dL. A statically significant 14% increase was measured (p-value= 0.0001).

the intact skin or, if need be, via specialized probes. While ultrasound is blocked by air/tissue or air/fluid interfaces, even in the lung, it is possible to create acoustic windows to the bronchi with bronchoscopes and water filled balloons. Ultrasound can be targeted precisely to tissues to treat volumes of tissues ranging to more than 500 ml to less than a ml. Most studies have shown relatively low toxicity due to ultrasound and MBs. It also is possible to repeat treatment, potentially indefinitely.

Despite early studies in ultrasound and gene delivery with MBs being performed more than a decade ago, none of the ultrasound gene delivery programs with MBs, at the time of preparation of this chapter, have advanced to clinical trials. The field is still early in its development, but the tolerability, ease of use and high degrees of expression in the target tissue indicate that this technology merits serious consideration for clinical translation.
