Synthetic cells synthesize therapeutic proteins inside tumors

The existing dogma is that protein medicines need to be produced in large factories, and then injected to the patient. We propose that miniature artificial inert factories can be injected to the patient, to produce a protein of interest directly in the diseased tissue. We engineered artificial cell-like particles with an autonomous capacity to synthesize protein drugs after receiving an external signal. The protein is tuned to the patient\u27s needs based on a predetermined DNA code we incorporate inside the particles. This approach increases treatment efficiency and reduces adverse effects to healthy tissues. We developed a new T7-S30 based cell-free protein synthesis system, which contains all the transcription and translation machines and molecules required for protein production (Krinsky et al., PloS one 2016). This system was used to prepare liposomes that act as artificial cells, capable of producing proteins autonomously in response to a physical trigger. Functional enzymes (luciferase and tyrosinase) and fluorescent proteins (GFP) were successfully produced using the new cell-free protein synthesis system and inside the particles both in vitro and in vivo. In addition, we demonstrated the therapeutic capabilities of the protein producing particles 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 (Krinsky et al. Advanced Healthcare Materials, 2017). Synthetic cells serve as autonomous, trigger-able, artificial particles that produces a variety of proteins. This platform has promise to address a wide range of fundamental questions associated with protein synthesis in nature, as well as applicative protein delivery needs. Please click Additional Files below to see the full abstract

Preparation of S30-T7 lysate.

<p>Preparation of S30-T7 lysate.</p

<i>Pseudomonas</i> exotoxin (PE) productions using the S30-T7 CFPS system originated from two different <i>E</i>. <i>coli</i> strains (BL21 and MRE600).

<p>Reactions were performed with and without the presence of DNA template. (A) Western blot analysis of cell-free reactions demonstrated the production of PE ~ 66 kDa. Purified PE served as positive control (described in Appendix F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165137#pone.0165137.s001" target="_blank">S1 File</a>.). Arrows indicate the position of PE bands. (B) The therapeutic potency of PE was evaluated on 4T1 cell-line. The viability of the cells was determined by MTT assay. Cell viability values obtained without the presence of purified PE or DNA were set as 100%, and the other values were normalized according to them (error bars represent standard deviation from at least three independent samples).</p

A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

<div><p>Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from <i>E</i>. <i>coli</i>. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 <i>E</i>. <i>coli</i> strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and <i>E</i>. <i>coli</i> strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.</p></div

A historical overview of improvements made to CFPS procedures over time.

<p>A historical overview of improvements made to CFPS procedures over time.</p

<i>In vitro</i> protein synthesis using cell-free system based on S30-T7 lysate.

<p><i>In vitro</i> protein synthesis using cell-free system based on S30-T7 lysate.</p

Enzyme productions using S30-T7 CFPS systems sourced from two different <i>E</i>. <i>coli</i> strains (BL21 and MRE600).

<p>(A) The produced <i>Renilla</i> luciferase activity was demonstrated by integrating 10 seconds of luminescence measurements (error bars represent standard deviation from at least three independent samples). (B) & (C) TyrBm production was confirmed by monitoring the conversion of 1mM L-Dopa to dopachrome (error bars represent standard deviation from three independent samples). (C) The observed enzymatic activity of TyrBm, produced by the S30-T7 CFPS in a 96-well plate. The three wells to the right present cell-free reaction in the present of DNA template, while in the three wells to the left no DNA template was incorporated into the reaction. The dark color indicates on the conversion of L-Dopa to dopachrome (followed by polymerization and accumulation of melanin), and thus on the production of TyrBm. (D) Temperature effect on cell-free <i>superfolder</i> GFP production efficiency of the S30-T7 CFPS (error bars represent standard deviation from at least four independent samples). The protein production amount was evaluated according to the fluorescence levels. The fluorescence values obtained at 37°C were set to 100%, and all the other values were normalized according to them. Negative controls (N.C.) were reactions without DNA templates. * Significant difference between lysates from the two E. coli strains, where α<0.05 according to a Student's t-Test with a two-tailed distribution with equal variance.</p

A schematic overview of the cell-free protein production process.

<p>A schematic overview of the cell-free protein production process.</p

Synthetic cells with self-activating optogenetic proteins communicate with natural cells

Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the design and implementation of bioluminescent intercellular and intracellular signaling mechanisms in synthetic cells, dismissing the need for an external light source. First, we engineer light generating SCs with an optimized lipid membrane and internal composition, to maximize luciferase expression levels and enable high-intensity emission. Next, we show these cells’ capacity to trigger bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent intracellular signaling with self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of controlling engineered processes inside tissues