As introduced in the description page, liposomes offer various advantages in medical treatment including in cancer therapy [2].

Firstly, liposomes enjoy a lower toxicity as compared to other drug delivery vectors. Liposomes are primarily composed of phospholipids, natural or synthetic, that are Generally Recognized As Safe (GRAS), biocompatible, biodegradable, and non-immunogenic [2]. The adverse effects of liposome-based treatments are therefore expected to be minimal. In contrast, non-biocompatible or non-human-inspired drug vectors such as Virus-Like Particules are often highly immunogenic [3]. The safety of using liposomes in medical treatment is further ensured by the fact that liposomes are not GMOs. The encapsulated DNA template can solely be expressed in a cell-free system that contains regulatory elements orthogonal to human cells.

Liposomes are also versatile. They can encapsulate hydrophilic drugs or biomaterials in their aqueous lumen and incorporate membrane proteins or hydrophobic molecules within the bilayer. The liposome's content is protected by the bilayer membrane. This way, the half-life of the encapsulated molecules is increased. Interactions between the encapsulated molecules and the microenvironment of the liposome can still occur depending on various parameters.

In our case, the liposome encapsulates a prodrug, Tegafur, the gene of the Thymidine phosphorylase enzyme to cleave the prodrug into the anticancer drug, 5-Fluorouracil (5-FU), and the transcription-translation machinery PURE system. See the part corresponding to the Thymidine phosphorylase here.

Because most anticancer drugs also affect healthy cells, targeted delivery is often required. The liposomal membrane can easily be modified to display targeting agents. This prevents off-targets and helps spare as many healthy cells as possible.

PURE system was used as the protein factory to synthesize proteins directly inside liposomes starting from encapsulated genes. This is an essential ingredient of CALIPSO, in particular for the in situ production of Thymidine phosphorylase as a response to the oncometabolite 2-HG.

The PURE (Protein synthesis Using Recombinant Elements) system technology is a reconstituted cell-free protein synthesis system developed by Professor Takuya Ueda at the University of Tokyo [4].

Unlike cell lysates (e.g., E. coli extract S30 system), PUREfrex contains highly purified compounds for transcription, translation, aminoacylation, and energy recycling (Fig. 2). Therefore, adverse effects during liposome injection are expected to be minimal due to the strictly defined composition of the kit.

We used the commercial PUREfrex kits available at GeneFrontier. The kit mainly consists of three vials:
• Solution I contains amino acids, NTPs, other small-molecule compounds and tRNAs.
• Solution II contains T7 RNA polymerase, translation factors and other proteins.
• Solution III contains purified ribosomes.
The three solutions are mixed with DNA or RNA templates for in vitro synthesis of the protein(s) of interest. Find more information on GeneFrontier's website.

Figure 2: the PUREfrex transcription-translation system.

We used three different types of PUREfrex kits:
1. PUREfrex2.0
2. PUREfrex2.1
3. A custom kit PUREfrex

Genefrontier recommends using different PUREfrex kits based on the structural features of the protein of interest, particularly the presence of disulfide bridges.

PUREfrex2.0 contains dithiothreitol (DTT) as a reducing agent, which inhibits the formation of disulfide bonds. In contrast, PUREfrex2.1 does not contain DTT and an appropriate reducing agent can be selected.

If the protein of interest requires disulfide bond formation, the suitable kit is PUREfrex2.1 supplemented with DsbC Set (includes oxidized glutathione (GSSG) and E. coli disulfide isomerase DsbC) or PDI Set (includes GSSG, human protein disulfide isomerase (PDI) and human ER oxidoreductin-1 (Ero1alpha)).

Finally, we ordered a PUREfrex custom kit consisting of PUREfrex2.1 without T7 RNA polymerase in the solution II. It is the final PURE system concoction that will be encapsulated inside the liposome if the two biosensing systems are combined, along with the prodrug, SP6 RNA polymerase, and the DNA templates encoding for the split T7 RNA polymerase proteins and Thymidine phosphorylase.

To visualize the production of our different proteins in PURE, we used the co-translational fluorescent marker GreenLys. This system enables fluorescent labeling and detection of newly synthesized proteins, based on a lysine-charged tRNA labeled with the fluorophore BODIPY®-FL at the ε position. Fluorescent lysine residues are thus incorporated exclusively in synthesized proteins during the translation process. After SDS-PAGE, proteins are visualized on a fluorescence gel imager.

In our second design, the anchoring of the liposome to the surface of cancer cells is mediated by folate exposed on the liposome's surface, and also by the antibodies Pertuzumab and Trastuzumab, which are themselves involved in this second biosensing system.

To trigger enzyme expression in the vicinity of the tumor, we sought to engineer a protein that would enable intraliposomal gene expression activation upon recognition of the HER2 receptors. The design complexity lies in the fact that the cancer cell-binding domain must be exposed outside the liposome, while the transcriptional regulator domain must be localized in the lumen. We drew inspiration from the protein family of enzyme-linked receptors that transduce extracellular signals to the cytoplasm across the plasma membrane of eukaryotes. These receptors contain a single transmembrane domain with an extracellular N-terminal ligand binding domain and a cytoplasmic C-terminus with enzymatic (e.g. tyrosine kinase) activity. We decided to engineer a heterodimer consisting of the split-T7 RNA polymerase coupled to anti-HER2 antibodies via a single transmembrane domain. The binding of the two antibodies to distinct epitopes of HER2 not only provides a means to anchor the liposome to the surface of cancer cells, it also enables complementation of the two split T7 RNA polymerase units that are facing the opposite side of the membrane. The functionally assembled T7 RNA polymerase can then trigger the expression of the Thymidine phosphorylase gene.

T7 RNA polymerase is an RNA polymerase derived from the T7 bacteriophage first described in the literature in the 1970s. During purification, a cleaved form of the protein was obtained between amino acids 179 and 180. This cleavage generated a small N-term fragment of 20 kDa and a large C-term fragment of 80 kDA. These two fragments alone lacked catalytic activity, but the polymerase activity of the enzyme was recovered when they were both mixed in vitro [10].
More recent studies demonstrated that the co-transformation of E. coli bacteria with two plasmids carrying the N-term and C-term fragments resulted in the recovery of an active polymerase in bacteria. This property makes it a model of choice as a biosensing platform for studying interactions between proteins of interest.
It is possible to visualize protein interactions by merging candidate proteins onto the ends of the N-term and C-term fragments of the split-T7 RNAP. Transformation of bacteria with expression vectors encoding these constructs and the use of a reporter gene under the control of a T7 promoter showed that in vivo interaction of the candidate proteins enabled functional reassembly of the polymerase [11].

A luciferase-based reporter of HER2 receptor has been described in the literature [11].
Two fragments of a split-Luciferase were fused to antibodies recognizing different epitopes of HER2. Recognition of the target protein by the antibodies reassembles the two fragments of the split-Luciferase into a functional luminescent protein.

Inspired by these articles, our team decided to fuse the N-term and C-term fragments of split-T7 RNA polymerase to the antibodies Omnitarg and Herceptin, also known as Pertuzumab and Trastuzumab. Our aim was to induce the reconstitution of T7 RNA polymerase in the liposome when it is anchored to the surface of HER2+ cancer cells.

Figure 8: Re-functionalization of the two-partite RNA polymerase-linked antibody against HER2.

We engineered two pairs of biosensor proteins that differ in size and in the nature of the linker that fuses a fragment of the split-T7 RNA polymerase with Pertuzumab or Trastuzumab.

Proteins Pertuzumab-SL-T7Nterm and Trastuzumab-SL-T7Cterm were adapted from the article of Bryan C. Dickinson et al. [11], in which the two subunits of the split T7 RNA polymerase were fused to anti-rapamycin antibodies. Here, we substituted the antibodies against rapamycin with Pertuzumab and Trastuzumab for creating an HER2-inducible system. Here, the linker is hydrophilic and short in length. We wanted a proof-of-concept for the production in PURE system and the functional reassembly of the split-T7 RNA polymerase upon HER2 binding. See the part corresponding to Pertuzumab-SL-T7Nterm and Trastuzumab-SL-T7Cterm.

In the second pair of engineered proteins, the N-term and C-term fragments of the split-T7 RNA polymerase were fused to Pertuzumab and Trastuzumab through a transmembrane linker. The goal was to incorporate this hetero dimer into the membrane of our liposomes. The nature and size of the linker were selected by careful molecular mechanic considerations, find more information on our In silico protein design.

We generated these different constructs through cloning, and the experimental results are available on our Cloning results page.

PUREfrex2.1 was used for the production of Pertuzumab and Trastuzumab linked to the subunits of the T7 RNA polymerase in bulk as the two antibodies display disulfide bonds. The two genes are under control of an SP6 promoter to enable constitutive transcription. The PURE system solution was therefore supplied with SP6 RNA polymerase. We chose the SP6 transcriptional element because it is orthogonal to the T7 system.

Figure 9: Ribbon representation of the Trastuzumab structure with the 4 disulfide bonds highlighted in red.

Figure 10: Ribbon representation of the Pertuzumab structure with the 4 disulfide bonds highlighted in red.