טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentNitzan Krinsky
SubjectStudying Protein-Producing Particles for Onsite Drug
Delivery
DepartmentDepartment of Biotechnology
Supervisor Professor Schroeder Avi


Abstract

Synthetic cells are units which contain biological materials, confined by an artificial or biological membrane and are designed to perform a specific task. They can vary in their content, membrane composition and with dimensions ranging from the nanometric to macrometric scale. One of the common method to design synthetic cells is based on the “bottom-up” approach, where non-living building blocks are assembled into a “living” cell.

Synthetic cells are used as a cell model for evolutionary research and are studied as a biochemical microreactors. In this research synthetic cells were investigated as autonomous drug delivery systems. While the existing dogma is that protein medicines are produced in large-scale, and then injected to the patient body, I propose to produce a therapeutic protein directly in the disease site, by developing synthetic cells with anti-cancerous activity. This approach can increase treatment efficiency and reduce toxicity to non-targeted organs.

In this research, synthetic cells were designed as artificial particles composed of lipids, which provide cell-like characters, a Cell-Free Protein Synthesis (CFPS) system and a modular DNA template. The CFPS component, which includes all the transcription and translation machinery required for protein synthesis such as ribosomes, RNA polymerase, amino acids and energy, provides the particle’s protein production capability. The DNA template adjusts the produced RNA and protein sequence, thus enabling personalized treatment to each individual according to his/her medical needs. By engineering the particles with an autonomous capacity to synthesize protein drugs after receiving an external signal, the required therapeutic protein can be administered only to the diseased tissue.

During my research I developed a new T7-S30 based CFPS system, which can be produced by a rapid and affordable lab-scale protocol. This system has the ability to transcribe an artificial DNA code into RNA, and subsequently translate it into functional proteins. This CPFS system was further used to prepare liposomes (lipid particles) that act as artificial cells, capable of producing proteins autonomously in response to a physical trigger. Functional enzymes (Renilla luciferase and tyrosinase) and fluorescent proteins (super folder green fluorescent protein, sfGFP) were successfully produced using the new CFPS system inside the particles both in vitro and in vivo. In addition, the therapeutic capabilities of the synthetic cells were demonstrated by producing Pseudomonas exotoxin A, an extremely potent protein, for treating cancer. Applying the particles on 4T1 cells (a triple-negative breast cancer cell-line) in vitro or injecting them into a 4T1-induced tumor in vivo, resulted in high cytotoxicity due to the effective production of the therapeutic protein inside the vesicles.

Synthetic cells can serve as autonomous, artificial particles that produces a variety of proteins. I believe this platform can be applicative as a protein delivery system addressing the patients’ need, as well as to address a wide range of fundamental questions associated with protein synthesis in nature.