Gene Therapy

Ultrasound-Mediated Molecular Gene Delivery for hemophilia: Gene therapy can be described as the introduction of a foreign gene into a host, with subsequent expression of the gene product to overcome inherent deficiencies that produce disease. Examples of genetic diseases which may someday be treatable by gene therapies are hemophilia A and B, diabetes, muscular dystrophy, cystic fibrosis, chronic myocardial ischemia, mitochondrial disorders, and some cancers. The programmatic goal of this project is to develop ultrasound mediated nonviral gene delivery to the liver for the treatment of hemophilia. Our current expertise in this area involves collaboration between APL and Dr. Carol Miao (Seattle Children's Hospital; molecular biology). Dr. Miao's gene therapy program has many facets, including, e.g., generating cutting-edge gene constructs. uWAMIT would catalyze our acoustically enhanced gene delivery collaboration on a more formal setting, providing industrial and clinical guidance and support.

Aim: We have previously shown that ultrasound and microbubble (MB)-assisted nonviral gene delivery can be achieved to high levels in a mouse model.  The principal mechanism suggests ultrasonic induction of intrasinusoid inertial cavitation in tissues 'seeded' with cavitation nuclei (contrast agent microbubbles), which creates small-scale liver damage and greatly enhances gene transfer. Plasmids, microbubbles, and a high pressure acoustic signal must be present simultaneously for effect (Figure 7). 

Figure. 7. (a) Effect of Ultrasound on gene delivery efficiency via intraportal injection of the pDNA+ microbubble mixture in mice. Gene expression was maximized with acoustic peak negative pressures of about 3 MPa. (b) The effect of microbubble concentration of ultrasound-enhanced gene delivery in mice. It is clear that there is an optimum concentration.

The principal goal of this project is to leverage our existing program to develop treatment devices suitable for large animals, permitting much more rapid iterative development and evaluation of preclinical US therapy devices than would be possible under a typical (e.g., NIH -supported) program, and to then merge the output of these efforts with on-going research intended to improve efficacy from a more biological perspective. This project will greatly accelerate transition of the combined technologies from small animals toward the clinic.

Background and Significance: Nonviral gene transfer has several advantages over viral gene transfer, including (1) reduced immunogenicity, (2) low toxicity, (3) ease and low cost of vector production, (4) avoiding recombination events, (5) avoiding possible oncogenic events by random integration into the host genome, and (6) ability to deliver large genes. However, the low efficiency of non-viral gene delivery remains a significant challenge. Therapeutic ultrasound has a particularly promising potential for nonviral gene transfer using plasmid DNA (pDNA). Problems of toxicity and targeting specific organs can be overcome with the combination of ultrasound, microbubbles, and pDNA vectors; in addition, there is the potential for repeated application. Ultrasound exposures involving microbubbles can create transient openings in cell membranes – called sonoporation, allowing gene transfer, or delivery. A key feature of US-enhanced gene delivery is that DNA delivery can be targeted. 

Sonoporation is often associated with microbubbles [Kamaev 2004, Bao 1997, Greenleaf 1998, Ward 1999, 2000]. Even large molecules can enter cells through transient pores [Mehier-Humbert 2005]. With targeting agents such as microbubbles, effects on ultrasound-enhanced gene transfer can be dramatic [Lawrie 2000]. Ultrasound-enhanced gene transfer in vivo or ex vivo has been explored in many tissues or organs [Sonoda 2006; Shimamura 2004, 2005; Manome 2005, Hynynen 2001, 2003a, 2003b; Hou 2005; Chen 2006; Miao 2005, Shen 2008; Ohta 2003; Nishida 2006; Nakashima 2003].  Three areas have attracted the most attention: treatment of (1) tumors, (2) cardiovascular tissues, and (3) skeletal muscle.

We have shown that ultrasound with microbubbles enhances gene delivery of a liver-specific, high-expressing factor IX (FIX) plasmid into mice. Human FIX (hFIX) levels approaching the therapeutic range for treating hemophilia patients were achieved by transducing one liver lobe, a 66-fold increment relative naked DNA alone [Miao 2005, Shen 2008]. (Most studies show increases in gene expression by factors <10. Our studies show enhancements of 100-fold.) Ultrasound with microbubbles therefore has the potential to promote safe and efficient nonviral gene transfer of hFIX to treat hemophilia. The next steps are to adapt and develop the biological and engineering components of the therapy to ever larger animal models as necessary milestones toward clinical application. Current efforts are to further optimize US parameters in rats. This application addresses the need for therapy devices suitable for large animals.

Approach: Much larger tissue volumes to be treated impose practical constraints on the treatment for two principal reasons: (1) Linear scaling of injected amounts of plasmid and microbubble would be prohibitively expensive (we are now exploring various protocols to address this issue);  (2) Because intravenous Microbubbles wash in and out of the liver fairly quickly, in large animals a large tissue volume must be treated acoustically while the plasmids and microbubbles remain in the tissues; i.e., the tissues must be treated on a time scale comparable to the wash-in/wash-out times; another approach is needed. Our current therapy device development efforts concentrate on the use of unfocused, planar transducers that can be driven impulsively at high instantaneous electrical powers to produce high acoustic pressures.  The idea is to create therapy transducers with a wide 'footprint' and a large 'focal' depth to treat large tissue volumes with each acoustic pulse. Transducer output is limited by size, transduction efficiency, robustness, and the amount of power available to drive it, among other factors.  There is no overall off-the-shelf solution to this multifaceted problem, which will require iterative development and assessments of efficacy, ergonomics, etc. The Brayman/Miao team has the requisite expertise in acoustic biophysics and gene therapies, respectively, for iterative input to instrumentation design and device testing, while simultaneously evaluating other strategies to improve gene delivery in large animals.  We have an established working relationship with Sonic Concepts, Inc., a local small high-tech business which engineers and fabricates custom ultrasound transducers, with whom we will work closely as device development progresses. Our approach in larger animals will concentrate on generating large surface area devices capable of producing high acoustic pressures in low duty factor pulse mode (to avoid heating effects on tissues and self-heating related destruction of the transducers), and to apply the device first directly to the surface of surgically-exposed liver, and later (as devices and treatment protocols improve), with transcutaneous application to treat the liver without surgical exposure.

uWAMIT would help facilitate translation of this research into the clinic in several ways. To facilitate translation to humans, we must optimize ultrasound gene delivery, and test various therapies for the most effective regimen. Optimization can be facilitated with consultations with our clinical and industrial partners, providing an efficient mechanism for translating this promising science into the clinic.