**4. The vectors: Packaging desired genes for ultrasound-mediated delivery**

#### **4.1 Naked DNA: Plasmids**

Naked plasmid DNA provides many advantages as a nonviral gene transfer vector, including: (1) ease of preparation, (2) cost-effectiveness, (3) minimum toxicity, and (4) it is least immunogenic of the vectors. Indeed, the immunogenic CpG moiety can be modified easily if needed. Most importantly, quality control is quite easy compared to other pDNA complexes containing synthetic vehicles. However, it has been a very challenging problem to deliver naked pDNA into specific cells due to its large size and negative charge. *In vivo* delivery of naked pDNA is especially difficult with additional impediments of instability of pDNA in blood serum as well as in cellular sub-compartments such as cytosol, endosome, and nucleus after cell entry. Recently a hydrodynamic approach has been developed to drive efficient gene delivery into liver {Liu *et al.* 1999} and muscle {Danko *et al.* 1997} resulting in therapeutic levels of transgene expression in animal disease models, including hemophilia B {Miao *et al.* 2003} and others {Zhang *et al.* 2000}. This method in its current form is not suitable for clinical use; however, notable recent advances have been made in large animal models (*vide, e.g*., {Fabre *et al.* 2008; Kamimura *et al.* 2009; Suda *et al.* 2008}. Alternatively, innovative US and MB technology to facilitate delivery of naked pDNA is a potential clinically feasible nonviral gene therapy approach {Miao *et al.* 2005; Shen *et al.* 2008; Song *et al.* 2011}.

The potential for sonoporation to increase pDNA loading of cells was recognized years ago {Fechheimer *et al.* 1987}. Sonoporation-enhanced transport of nanoparticles into cells is dependent on molecular size; uptake of particles ≤ 37 nm diameter was enhanced by sonoporation without gross damage, but particle uptake generally declined as particle size increased {Mehier-Humbert *et al.* 2005a}. pDNA gene expression is faster with sonoporation than with liposome-based methods which depend on endocytosis {Mehier-Humbert *et al.* 2005}, indicating that pDNA enters cells through transient pores. Moreover, due to the risk of pDNA degradation by serum nucleases and removal by phagocytes {Niidome & Huang

Ultrasound-Mediated Gene Delivery 225

these with home-made phospholipid mixtures, agitating under a perflutren atmosphere to generate MBs, and then repeatedly washing the bubbles to remove unassociated DNA, a per bubble loading of 50 x 10-15 gram/MB was achieved {Chen *et al.* 2006}. Similar binding rates (approximately 100 fg/MB) were reported by others {Carson *et al.* 2011}. Using a layer-bylayer approach, DNA loading of pre-existing cationic MBs was increased 10-fold by first bringing anionic DNA to the surface of the cationic bubble, followed by binding a coating of poly-cationic polylysine to the DNA, followed by more DNA, *etc*. Loading as high as ~2500 fg DNA/MB was achieved, without apparent impact on the bubbles' dynamical response to acoustic excitation {Borden *et al.* 2007}. However, others have found that *in vitro* transfection rates are not enhanced by conjugation of reporter pDNA to MB shells {Tlaxca *et al.* 2010}, as

found in the *in vivo* canine myocardium example discussed earlier in this section.

which the adenoviral vectors were believed to be attached.

efficiencies and restrict targeted sites of viral gene transfer.

**4.4 Small RNAs and oligonucleotides** 

There have been many reports that US treatment alone, or in combination with MBs, can increase transgene uptake by cells {Miller *et al.* 2002; Newman & Bettinger 2007}. Even with the difficulty of translating UTMD-mediated results obtained under tissue culture conditions (*e.g*., infinite media for suspended cells *vs*. nearby noncompliant boundaries for monolayers) to the fully 3-D, viscoelastic intravascular environment *in vivo*, *in vitro* experiments often have the advantage of being better controlled and the results therefore more easily understood. This is not always the case, however. Zheng and colleagues exposed two different endothelial pigment cell lines to adenoviral gene vectors in combination with US or US and MBs (1 – 3 W/cm2; Pr appears to have been ~0.17 – 0.30 MPa at 1 MHz; SonoVue MBs were used when bubbles were employed). They found that treatment with US and MBs increased adenoviral gene transfer in human retinal pigment epithelium cells, but was without effect on rat retinal pigment epithelial cells under otherwise identical conditions {Zheng *et al.* 2009}. The differences were speculated to arise in consequence to differing ability of the two cell lines to phagocytize the SonoVue MBs, to

It is difficult to limit the specificity of delivery of viral vectors, which are usually delivered systemically. By using retrovirus-loaded MBs, UTMD facilitated the delivery of viral vectors in a restricted area of cells exposed to Pr of 0.4 MPa or greater, despite uniform dispersion of the vector {Taylor *et al.* 2007}. An envelope-deficient retroviral vector was combined with cationic MBs and added to target cells. Transduction efficiencies and sites can thus be controlled by means of US exposure. These results emphasize that UTMD can not only facilitate the delivery of nonviral vectors, but also has the potential to enhance

Small RNAs and oligonucleotides (ODN) have recently been developed as promising therapeutics to treat diseases like viral infections, cancer, and several genetic disorders. Among these, small inhibitory RNA (RNAi or siRNA) based therapeutics have been investigated for treating a number of different diseases, including viral infections (*e.g*., hepatitis, HIV, influenza), cancer, Huntington's disease, and others. Other therapeutic agents including microRNA (miRNA), transfer RNA (tRNA), and antisense ODN are also being developed to regulate gene or cell functions as treatment regimens. Since these agents are small, it is expected that they will be good candidates for augmented delivery by UTMD.

**4.3 Viral vectors** 

2002}, injection of a nonviral gene transfer vector long before US treatment occurs is unlikely to be effective. With simultaneous delivery of pDNA and US exposure, UTMD can significantly facilitate the transfer of naked pDNA up to several hundred fold *in vitro* {Miller *et al.* 2002; Newman & Bettinger 2007} and up to several thousand fold *in vivo*, depending on different US and transfection systems {Chen *et al.* 2010; Miao *et al.* 2005; Shen *et al.* 2008; Song *et al.* 2011}. Furthermore, with introduction of specific cis-acting elements in the gene transfer constructs, persistent expression of near-therapeutic levels of proteins can be achieved from episomal plasmids following UTMD-mediated gene therapy {Miao *et al.* 2005}. These results demonstrate that development of UTMD has high potential to achieve a therapeutic effect for treating specific diseases.

### **4.2 Encapsulated or compacted DNA**

Polyanionic solutes can be complexed to phospholipid polar head groups *via* Ca2+ bridges {Huster *et al.* 1999}. Thus, anionic DNA molecules can be compacted onto cationic MB lipid shells. Polylysine has also been used to link naked DNA to phospholipid MB shells {Wang *et al.* 2009}. It appears to be widely believed that enhanced efficiency of gene delivery can be obtained with pDNA in close proximity to MBs and any cell membrane 'defects' they may create, thereby increasing the probability that pDNA will be available to enter through these pores prior to their closure. Thus far, however, there has been inconsistent support for this idea. For example, pDNA coupled electrostatically with cationic MBs were used for local delivery of DNA to vascular muscle cells. One percent of cells were transduced with 40% of the cells remaining viable {Phillips *et al.* 2010}. However, reporter plasmid bound to the cationic MB preparation MRX-225 was used to transfect canine myocardium which was exposed to diagnostic US. Reporter gene activity was only observed in the myocardium of those animals that received MB-linked DNA and were exposed to US but not in control untreated animals {Vannan *et al.* 2002}. It is not clear if the pDNA-MB linkage influenced the experimental outcome.

Due to large size of DNA molecules and the concerns of enzymatic degradation of the injected pDNA as well as the low pDNA concentration in the vicinity of sonoporated cell membranes, polymer-coated MBs that can bind and protect the pDNA have been developed for UTMD-mediated gene delivery. Coating albumin-shelled MBs with poly(allylamine hydrochloride) (PAH) makes the surface charge of the MBs positive, but did not affect the size distribution of the MBs. The cationic coating allowed the MBs to bind to 100 fg of pDNA per MB and protected the bound DNA against nucleases. The PAH coating also significantly increased the lifetime of MBs (half-life ~7 h), making them more convenient for *in vivo* applications {Lentacker *et al.* 2006}.

Another approach to compacting DNA onto MBs is to first incubate the pDNA with a cationic lipid such as GL67 or a cationic polymer such as polyethylenimine (PEI), followed by mixing with MBs. PEI/DNA mixed with SonoVue MBs were injected intravenously in tumor–bearing mice. Following US exposure, reporter gene expression in tumor xenografts was significantly enhanced without causing any apparently adverse effect. Furthermore, with UTMD and PEI complex, vectors carrying a short hairpin RNA (shRNA) targeting human survivin were efficiently delivered into the tumor site, leading to inhibition of surviving gene expression and apoptosis of the tumor cells {Chen *et al.* 2010}.

DNA loading of MB shells can be impressive, but may also be limiting to high yield gene therapies using UTMD. By first forming pDNA-Lipofectamine 2000 complexes, mixing

2002}, injection of a nonviral gene transfer vector long before US treatment occurs is unlikely to be effective. With simultaneous delivery of pDNA and US exposure, UTMD can significantly facilitate the transfer of naked pDNA up to several hundred fold *in vitro* {Miller *et al.* 2002; Newman & Bettinger 2007} and up to several thousand fold *in vivo*, depending on different US and transfection systems {Chen *et al.* 2010; Miao *et al.* 2005; Shen *et al.* 2008; Song *et al.* 2011}. Furthermore, with introduction of specific cis-acting elements in the gene transfer constructs, persistent expression of near-therapeutic levels of proteins can be achieved from episomal plasmids following UTMD-mediated gene therapy {Miao *et al.* 2005}. These results demonstrate that development of UTMD has high potential to achieve a

Polyanionic solutes can be complexed to phospholipid polar head groups *via* Ca2+ bridges {Huster *et al.* 1999}. Thus, anionic DNA molecules can be compacted onto cationic MB lipid shells. Polylysine has also been used to link naked DNA to phospholipid MB shells {Wang *et al.* 2009}. It appears to be widely believed that enhanced efficiency of gene delivery can be obtained with pDNA in close proximity to MBs and any cell membrane 'defects' they may create, thereby increasing the probability that pDNA will be available to enter through these pores prior to their closure. Thus far, however, there has been inconsistent support for this idea. For example, pDNA coupled electrostatically with cationic MBs were used for local delivery of DNA to vascular muscle cells. One percent of cells were transduced with 40% of the cells remaining viable {Phillips *et al.* 2010}. However, reporter plasmid bound to the cationic MB preparation MRX-225 was used to transfect canine myocardium which was exposed to diagnostic US. Reporter gene activity was only observed in the myocardium of those animals that received MB-linked DNA and were exposed to US but not in control untreated animals {Vannan *et al.* 2002}. It is not clear if the pDNA-MB linkage influenced the

Due to large size of DNA molecules and the concerns of enzymatic degradation of the injected pDNA as well as the low pDNA concentration in the vicinity of sonoporated cell membranes, polymer-coated MBs that can bind and protect the pDNA have been developed for UTMD-mediated gene delivery. Coating albumin-shelled MBs with poly(allylamine hydrochloride) (PAH) makes the surface charge of the MBs positive, but did not affect the size distribution of the MBs. The cationic coating allowed the MBs to bind to 100 fg of pDNA per MB and protected the bound DNA against nucleases. The PAH coating also significantly increased the lifetime of MBs (half-life ~7 h), making them more convenient for

Another approach to compacting DNA onto MBs is to first incubate the pDNA with a cationic lipid such as GL67 or a cationic polymer such as polyethylenimine (PEI), followed by mixing with MBs. PEI/DNA mixed with SonoVue MBs were injected intravenously in tumor–bearing mice. Following US exposure, reporter gene expression in tumor xenografts was significantly enhanced without causing any apparently adverse effect. Furthermore, with UTMD and PEI complex, vectors carrying a short hairpin RNA (shRNA) targeting human survivin were efficiently delivered into the tumor site, leading to inhibition of

DNA loading of MB shells can be impressive, but may also be limiting to high yield gene therapies using UTMD. By first forming pDNA-Lipofectamine 2000 complexes, mixing

surviving gene expression and apoptosis of the tumor cells {Chen *et al.* 2010}.

therapeutic effect for treating specific diseases.

**4.2 Encapsulated or compacted DNA** 

experimental outcome.

*in vivo* applications {Lentacker *et al.* 2006}.

these with home-made phospholipid mixtures, agitating under a perflutren atmosphere to generate MBs, and then repeatedly washing the bubbles to remove unassociated DNA, a per bubble loading of 50 x 10-15 gram/MB was achieved {Chen *et al.* 2006}. Similar binding rates (approximately 100 fg/MB) were reported by others {Carson *et al.* 2011}. Using a layer-bylayer approach, DNA loading of pre-existing cationic MBs was increased 10-fold by first bringing anionic DNA to the surface of the cationic bubble, followed by binding a coating of poly-cationic polylysine to the DNA, followed by more DNA, *etc*. Loading as high as ~2500 fg DNA/MB was achieved, without apparent impact on the bubbles' dynamical response to acoustic excitation {Borden *et al.* 2007}. However, others have found that *in vitro* transfection rates are not enhanced by conjugation of reporter pDNA to MB shells {Tlaxca *et al.* 2010}, as found in the *in vivo* canine myocardium example discussed earlier in this section.

#### **4.3 Viral vectors**

There have been many reports that US treatment alone, or in combination with MBs, can increase transgene uptake by cells {Miller *et al.* 2002; Newman & Bettinger 2007}. Even with the difficulty of translating UTMD-mediated results obtained under tissue culture conditions (*e.g*., infinite media for suspended cells *vs*. nearby noncompliant boundaries for monolayers) to the fully 3-D, viscoelastic intravascular environment *in vivo*, *in vitro* experiments often have the advantage of being better controlled and the results therefore more easily understood. This is not always the case, however. Zheng and colleagues exposed two different endothelial pigment cell lines to adenoviral gene vectors in combination with US or US and MBs (1 – 3 W/cm2; Pr appears to have been ~0.17 – 0.30 MPa at 1 MHz; SonoVue MBs were used when bubbles were employed). They found that treatment with US and MBs increased adenoviral gene transfer in human retinal pigment epithelium cells, but was without effect on rat retinal pigment epithelial cells under otherwise identical conditions {Zheng *et al.* 2009}. The differences were speculated to arise in consequence to differing ability of the two cell lines to phagocytize the SonoVue MBs, to which the adenoviral vectors were believed to be attached.

It is difficult to limit the specificity of delivery of viral vectors, which are usually delivered systemically. By using retrovirus-loaded MBs, UTMD facilitated the delivery of viral vectors in a restricted area of cells exposed to Pr of 0.4 MPa or greater, despite uniform dispersion of the vector {Taylor *et al.* 2007}. An envelope-deficient retroviral vector was combined with cationic MBs and added to target cells. Transduction efficiencies and sites can thus be controlled by means of US exposure. These results emphasize that UTMD can not only facilitate the delivery of nonviral vectors, but also has the potential to enhance efficiencies and restrict targeted sites of viral gene transfer.

#### **4.4 Small RNAs and oligonucleotides**

Small RNAs and oligonucleotides (ODN) have recently been developed as promising therapeutics to treat diseases like viral infections, cancer, and several genetic disorders. Among these, small inhibitory RNA (RNAi or siRNA) based therapeutics have been investigated for treating a number of different diseases, including viral infections (*e.g*., hepatitis, HIV, influenza), cancer, Huntington's disease, and others. Other therapeutic agents including microRNA (miRNA), transfer RNA (tRNA), and antisense ODN are also being developed to regulate gene or cell functions as treatment regimens. Since these agents are small, it is expected that they will be good candidates for augmented delivery by UTMD.

Ultrasound-Mediated Gene Delivery 227

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

*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}.

(transfection) and undesired effect (killing of the target cells).

moved relative to the target.

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

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

adenoviral vectors.

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

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

#### **4.5 Transduced cell therapy**

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 therapy protocols.
