In this project, liver cells were used as the chassis, and exosomes produced by liver cells were used to deliver siRNA targeting to lung cancer cells, so as to complete the targeting of lung cancer cells.To ensure security, we added the Tet-On control system and the TRE-lox2272-loxP control system as switches. In order to ensure that enough exosomes were produced to perform the therapeutic effect, we added the KIBRA part as a biological generator to promote exosome release to enhance the therapeutic effect.
Target patients and application characteristics
The outbreak of COVID-19 has prevented many cancer patients from going to hospitals for treatment, which puts them at greater risk of worsening their disease. There is an urgent need for an alternative therapy that can be administered for long term. The ExosomeBomb SiRNA therapy that we designed is an excellent complement. The treatment with AAV as the carrier ensures that a single dose can form a long-term treatment, and intravenous injection provides a fast and convenient treatment means, which can avoid the pain caused by surgery and repeated chemoradiotherapy.
Doxycycline can be taken orally as an on/off switch when treatment is needed. At the same time, siRNA therapy designed for NSCLC caused by KRAS mutation combined with siRNA inhibited by CD47 innate immune checkpoint and PD-L1 acquired immune checkpoint can activate the immune system to remove tumor cells under the condition of inhibiting tumor proliferation.
After consulting the experts of biomedical engineering from GenScript company, we learned the manufacturing procedure of AAV. The drug development potential of AAV has long been noticed by biomedical industry. To meet the requirements for scale production and biosafety, standardized procedures of AAV production have been developed, which included the establishment of working cell bank, the expansion and transfection in bioreactor, the purification and concentration of AAV, and the formulation and filling process (as shown in Figure below). All these procedures were operated following strict standards approved by authorities which will guarantee the safety of products.
Safety Concern
However, when we discussed with GenScript experts, we learned that the use of AAV is likely to cause a strong immune response in the host, which is likely to cause an immune storm in the host and hinder the following treatment. This is also one of the difficulties recognized by the academic community at present, which has become one of the difficulties impediments to the success of our project. The dose selection of AAV for treatment will also be difficult. In our discussion with the professor, the professor once told us that AAV can cause body immunity function, so the same AAV therapy can only be treated with one injection, if the treatment effect is not good, it can't be treated again. If the dose is too high, it may cause unpredictable side effects. Therefore, we need to seriously consider how to use and implement our therapies.
In addition, the off-target effect of RNA interference therapy is also a potential safety concern. We will verify the off-target effect of our designed siRNA in future experiments. And present it in a timely manner.
Challenges
In some cancer patients, there is a high risk of cancer metastases. For example, lung cancer has brain metastasis, liver metastasis and so on. In this situation, our targeting of lung cancer cells is likely to lose therapeutic effect, which is extremely negative for treatment. In addition, there is a special class of patients with autoimmune diseases, and we may not be able to use their own immune system to kill tumor cells by inhibiting CD47 and PD-L1, which is also one of the problems we need to improve.¸
Summarize
In terms of therapeutic effect and targeting, the therapy we adopted has a prominent effect compared with the existing chemotherapy, radiotherapy and other therapies. At the same time, its targeting is reflected in the targeting of genes, rather than tissues. Meanwhile, it can target Undruggable KRAS, which is a huge advantage compared with other targeted drugs.However, the safety and dose-response limitations of this therapy are likely to be used as a supplement to existing therapies, and we will further explore the possibility and effectiveness of this therapy in the future.