**5.9 Cell therapies:** *Ex vivo* **gene therapy**

In this section, we discuss several examples of US-mediated gene delivery using cell cultures *in vitro*; these may have some mechanistic relevance to the *in vivo* condition. However, some of the results obtained using *in vitro* cells may have direct bearing on cell therapies.

Shock waves and MBs have been used *in vitro* to increase pDNA uptake in cultured HEK cells; the technique worked, but was associated with substantial cell killing {Bekeredjian *et al.* 2007} as might be expected (see, *e.g*., {Brayman *et al.* 1999; Hwang *et al.* 2006; Hwang *et al.* 2003}). As a general rule, only a small fraction (~5 – 10%) of cells insonated with MBs *in vitro* undergo transient poration; these yields are often associated with cell killing rates of ~50% (see, *e.g*. {AIUM 2000; AIUM 2008} and references therein). If those cells can be sorted (*e.g*., by flow cytometry) and cultured to increase cell numbers, this may be acceptable for cell therapies – if the gene vector is integrated into the host genome. An obvious problem for cell therapies based on this approach with naked DNA, however, is that the pDNA does not replicate with the host cell, and so there would be little to be gained by increasing cell numbers of the transfected subpopulation of cells. However, using suspended KHT-C cells, and 3 V% Definity, high levels of *in vitro* permeabilization to 70 kDa fluorescein isothiocyanate (FITC) dextran were achieved using 0.5 MHz US at a Pa of 0.57 MPa, 16 cycle pulses repeated at 3 kHz PRF. About 30% of the cells were permeabilized, with >95% retention of viability {Karshafian *et al.* 2009}. If similar results can be obtained with naked DNA, the prospects for cell therapies using cells transfected using *in vitro* UTMD seem much brighter.

### **5.10 Gene therapies for tumoricidal activity**

Strategies currently under investigation for treatment of cancers include anti-angiogenic therapies, the introduction of 'suicide genes' which either induce apoptosis or sensitize cells to subsequent treatment with drugs {Aoi *et al.* 2008; Azuma *et al.* 2008; Daigeler *et al.* 2010; Kirn *et al.* 2002; Zhou *et al.* 2010}, or down-regulate oncogenes {Wang *et al.* 2009}. Naked DNA bearing the Herpes-derived 'suicide gene' for thymidine kinase has been delivered to murine squamous cell carcinomas by UTMD methods (1.3 MHz, estimated Pr ~1.8 MPa). The DNA was bound to lipid shelled MBs at a rate of about 100 fg DNA/MB. With daily ganciclovir treatments beginning 3 d after UTMD and pDNA treatment, tumor doubling times were significantly reduced (by ~17%) in the UTMD-treated tumors {Carson *et al.* 2011}.

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

There are many papers which indicate that endothelial cells *in vitro* can express transgenes delivered by UTMD methods (see {Su *et al.* 2010} and internal references for recent examples). Su and colleagues found that treatment with 1 MHz US of 1 MPa Pr together with pGL3 pDNA and MBs yielded a 100-fold increase in luciferase expression relative to pDNA treatment alone. It remains to be determined if endothelial cells *in vivo* can be 'recruited' as effective synthesizers and secretors of therapeutic gene expression products.

In this section, we discuss several examples of US-mediated gene delivery using cell cultures *in vitro*; these may have some mechanistic relevance to the *in vivo* condition. However, some of

Shock waves and MBs have been used *in vitro* to increase pDNA uptake in cultured HEK cells; the technique worked, but was associated with substantial cell killing {Bekeredjian *et al.* 2007} as might be expected (see, *e.g*., {Brayman *et al.* 1999; Hwang *et al.* 2006; Hwang *et al.* 2003}). As a general rule, only a small fraction (~5 – 10%) of cells insonated with MBs *in vitro* undergo transient poration; these yields are often associated with cell killing rates of ~50% (see, *e.g*. {AIUM 2000; AIUM 2008} and references therein). If those cells can be sorted (*e.g*., by flow cytometry) and cultured to increase cell numbers, this may be acceptable for cell therapies – if the gene vector is integrated into the host genome. An obvious problem for cell therapies based on this approach with naked DNA, however, is that the pDNA does not replicate with the host cell, and so there would be little to be gained by increasing cell numbers of the transfected subpopulation of cells. However, using suspended KHT-C cells, and 3 V% Definity, high levels of *in vitro* permeabilization to 70 kDa fluorescein isothiocyanate (FITC) dextran were achieved using 0.5 MHz US at a Pa of 0.57 MPa, 16 cycle pulses repeated at 3 kHz PRF. About 30% of the cells were permeabilized, with >95% retention of viability {Karshafian *et al.* 2009}. If similar results can be obtained with naked DNA, the prospects for

the results obtained using *in vitro* cells may have direct bearing on cell therapies.

cell therapies using cells transfected using *in vitro* UTMD seem much brighter.

Strategies currently under investigation for treatment of cancers include anti-angiogenic therapies, the introduction of 'suicide genes' which either induce apoptosis or sensitize cells to subsequent treatment with drugs {Aoi *et al.* 2008; Azuma *et al.* 2008; Daigeler *et al.* 2010; Kirn *et al.* 2002; Zhou *et al.* 2010}, or down-regulate oncogenes {Wang *et al.* 2009}. Naked DNA bearing the Herpes-derived 'suicide gene' for thymidine kinase has been delivered to murine squamous cell carcinomas by UTMD methods (1.3 MHz, estimated Pr ~1.8 MPa). The DNA was bound to lipid shelled MBs at a rate of about 100 fg DNA/MB. With daily ganciclovir treatments beginning 3 d after UTMD and pDNA treatment, tumor doubling times were significantly reduced (by ~17%) in the UTMD-treated tumors {Carson *et al.* 2011}.

in the brain.

**5.8 Endothelium** 

**5.9 Cell therapies:** *Ex vivo* **gene therapy** 

**5.10 Gene therapies for tumoricidal activity** 

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 groups {Luo *et al.* 2010}.
