Ultrasound-mediated gene delivery to achieve therapeutic correction of hemophilia A

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Project Summary The goal of this proposal is to achieve long-term therapeutic correction of hemophilia A (HemA) via a noninvasive protocol of ultrasound (US) mediated gene delivery (UMGD) of factor FVIII (FVIII) plasmids in the dog model. HemA is a genetic disorder characterized by a deficiency of the blood clotting FVIII. Patients are treated acutely or prophylactically by protein replacement therapy, which is very costly and inconvenient. Gene therapy is highly promising for treating HemA patients by delivering hFVIII transgene into targeted cells to persistently produce therapeutic levels of FVIII protein. Recent clinical trials for HemA gene therapy using recombinant adeno- associated viral (rAAV) vectors have shown very promising results. However, significant obstacles remain to prevent treatment to a significant portion of patients especially patients who have high-titer anti-AAV antibodies. Repeated treatment is also prohibited. UMGD has emerged as an effective gene transfer approach with great clinical relevancy and translational potential. In comparison to viral gene transfer, UMGD transfers plasmid vectors that are easier to prepare and more cost-effective; it also elicits less immune response and toxicity due to specific tissue targeting, prevents random integration, and allows for repeated delivery of the vectors. Other nonviral gene delivery method such as DNA-packaged nanoparticle encounters the challenge of crossing the nuclear envelope for DNA transcription. We have established a minimally invasive, transhepatic venous approach to efficiently deliver plasmid DNA (pDNA)/microbubble (MB) mixture into the target liver lobe combined with transcutaneous US applications in large animal models. We showed that high levels of luciferase reporter gene expression were achieved in swine and therapeutic levels of FVIII expression was detected in canine0 using the clinically feasible protocol. Only transient tissue damages were observed and repaired quickly and returned to normal within short time. However, in order to translate this novel technology to clinics, we recognize that several major problems need to be solved, (i) higher FVIII expression levels are needed to achieve a long- term therapeutic effect, (ii) persistence of therapeutic FVIII expression needs to be evaluated and maintained, (iii) consistently high efficiency of US treatment on targeted liver tissue is needed to achieve reproducible and efficient transfection. (iv) Better functional FVIII expression and reduced liver damage are desired. This may be achieved by targeting FVIII transfection in liver sinusoidal endothelial cells (LSECs), the native site of FVIII synthesis or by using newly synthesized nanobubbles (NBs). Thus, we propose to continue improving the transcutaneous UMGD instrument, transducers, US protocols, FVIII plasmid constructs, and MBs/NBs in mice and swine. Furthermore, we will deliver FVIII gene using the best transcutaneous UMGD protocol combined with optimal plasmid constructs and MBs/NBs to achieve persistent and therapeutic levels of FVIII expression in normal and HemA dogs. These progresses will promote the eventual translation of this novel technology into human application, bringing significant benefit for treating HemA patients, and potentially other genetic diseases.