Overview

Capsule degradation - at the end of the therapy production of the secretory alginate lyase is induced which degrades the microcapsules, allowing immune cells to reach and clean up the therapeutic cells.

Termination tag - our therapeutic cells constitutively produce thymidine kinase. At the end of the therapy the prodrug ganciclovir is added which is transformed into a toxic compound that initiates cell apoptosis only in therapeutic cells that constitutively produce thymidine kinase.

Escape tag - therapeutic cells express MICA protein tag at their surface that has strong affinity for the receptor NKG2D expressed by natural killer cells. In case the therapeutic cells escape form the alginate capsule the MICA protein at their surface recruits the natural killer cells that induce their killing.

Figure 1. Overview of the designed safety mechanisms for therapeutic use of microencapsulated engineered cells.

Inactivation and elimination of the cellular devices

In addition to the ability to regulate production of therapeutic proteins the synthetic biological device has to be completely safe. Physicians must be able to control the implanted system from the outside and have the ability to turn it off at any time, particularly at the end of the therapy, by using a simple remote control, such as ingestion of a pill containing a harmless signalling compound (chemical inducer).

We concluded that the regulated induction of apoptosis would be the best solution since apoptotic cells are resorbed by the surrounding cells and do not cause inflammation. Apoptosis can be triggered through many different paths. The challenge was to decide what type of inducer would be appropriate to trigger apoptosis since even small leakage might in the long term kill the majority of encapsulated cells. Rather than trying to reduce the leakiness of promoters we decided that we can avoid this problem by modifying the therapeutic cells to produce an enzyme that converts a prodrug into a toxic compound. Therefore the addition of a prodrug initiates only apoptosis of the therapeutic cells.

Although cells are encased in the alginate matrix, isolating them from the cells of the immune system, it is possible that some of the capsules might get damaged, enabling the modified therapeutic cells to escape and in the worst case continue to multiply if the host immune system does not recognize them as foreign. The device driving therapeutic cells, both encapsulated and released, into apoptosis solves only part of the problem. The physician or patient does not know whether any cells have escaped from the capsule during therapy.

Therefore we need to modify the therapeutic cells in a way that would enable their destruction if they are released from the capsules. We decided to employ the host's immune system to remove the escaped cells by expressing protein tags on transformed cells for the recognition and destruction by the body's innate immune system.

Additionally we want our system to be traceless, ideally leaving no remains after the end of the therapy. Therefore we also want to degrade the alginate capsules. Alginate is a biocompatible and nonimmunogenic compound, however mammalian cells do not produce the enzymes to degrade alginate. On the other hand several marine bacteria produce alginate lyase, which we planned to introduce into the therapeutic cells with an added secretory signal peptide and under the control of an inducible system to initiate the degradation at any selected time.