Ph.D Thesis


Ph.D StudentTom Haber
SubjectUltrasound as a Gene Delivery Technology for Mesenchymal
Stem Cell-Based Therapy
DepartmentDepartment of Biotechnology
Supervisor Full Professors Machluf Marcelle


Abstract

Gene therapy offers a treatment for an array of inherited, malignant and infectious disorders. A key factor in gene therapy is the delivery of the gene into a wide variety of cells and tissues using safe and effective vectors with minimal side-effects.

Mesenchymal stem cells (MSC) have attracted much attention as a source for cancer cell-based therapy. The rational for using bone-marrow derived MSCs as a cell-based delivery platform is based on their natural homing ability to cancer sites, their hypo-immunogenicity and immunosuppressive properties.

In the current study we aimed to safely transfect MSC, using a non-viral method for gene delivery: therapeutic ultrasound (TUS) and deliver a cDNA that encodes for PEX, a c-terminally-derived fragment of matrix-metalloproteinase-2 (MMP2) that is known to inhibit angiogenesis in-vitro and in-vivo.

We showed that TUS with the appropriate optimized parameters and the addition of ultrasound contrast agent (USCA), can be used as a powerful method to deliver genes into MSCs while maintaining their viability.

In order to use the transfected-MSC for future in-vivo applications, we needed to study the long-term effects of the TUS on the transfected-MSC such as cells' morphology and  stemness. We also studied the effect of TUS on the homing ability of MSC in-vivo and their bio-distribution.

We demonstrated that a single i.v administration of TUS-transfected MSCs with pPEX and Optison led to a 70% inhibition in tumor growth after 21 days. Most importantly, TUS-pPEX transfected-MSC can be repeatedly administrated, leading to a significant inhibition of tumor growth by 84% and the angiogenic indices.

The mechanistic studies revealed that transfection via pore-formation and the TUS mechanical force contribute to the TUS transfection, however, not in the order of magnitude it is believed to be. Endocytosis seems to be more meaningful than the other mechanisms.


In conclusion, this study demonstrates, for the first time, that TUS with Optison is a powerful technique to deliver therapeutically active genes into MSCs in-vitro, and the efficacy of TUS-transfected MSC as a combination of cell-based therapy and a non-viral gene delivery approach for the treatment of cancer in-vivo. Such approach allows better control over protein localized expression duration, leading to the inhibition of prostate cancer tumors. Furthermore, and most importantly, this approach can overcome the difficulties associated with the vectors today, and by further understanding the mechanism and the bio-effects of TUS-mediate transfection to expand the use of cell-based gene delivery in the clinic.