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Future work
The successful trial of our ideas on mice is only the beginning of fighting a tough battle against drug addiction. Iterative trials and improvements should be performed before ultimate clinical application. We have carefully examined the perspective of our trial and have become aware of several potential drawbacks that might cause serious problems if not dealt with properly.
1. Potential drawbacks
We first examined the safety of our project. Exosomes, because of their biological nature, have considerable advantages—direct cytosolic delivery without causing genomic insertion or inflammatory response—over other biomaterials such as viruses and synthetic nanoparticles. However, a subset of complicated safety issues must be considered in developing a novel gene therapy. In our project, we modified human embryonic kidney 293 (HEK293) cells to produce exosomes. HEK293 cells are a specific cell line originally derived from an aborted human embryo. The widely application of HEK293 cells to produce exosomes may cause ethical controversial. Moreover, exosomes derived from HEK293 cells may not be optimal and may cause a severe immune response when applied to different individuals due to different histocompatibilities.
Second, constantly performing complicated experiments to produce therapeutic exosomes is not feasible in terms of cost. The expense of exosome isolation cannot be overlooked at present. Similarly, plasmid purification and cell transfection are expensive and labor-intensive processes.
Third, our therapeutic strategy based on exosomal siRNA still heavily relies on the personal willingness of patients to overcome drug addiction. Regular surveillance of exosome injections is indispensable because the drug effect is not permanent. Thus, problems may still arise if patients fail or refuse to receive regular injections to help them avoid relapse.
2. Future directions
To solve the above-mentioned problems, we have come up with two possible future directions. The first direction is aimed at solving the first problem regarding safety and ethnics, also giving insights into devising clinical trials. The second direction may shed light on methods of addressing issues concerning the expense and applicability of this therapy, integrating all considerations to develop an ultimate and automated machine.
2.1 Direction 1 (primary goals)
Safety issues regarding ethnics, histocompatibility and immune response are not uncommon in personalized medicine. Cancer immunotherapy is promising and addresses this problem in a proper manner; thus, we will learn and apply some core ideas when devising our therapy.
In a typical adoptive immunotherapy for cancer (ACT), tumor-infiltrating lymphocytes (TILs) are generated from patients and cultured ex vivo before being genetically modified and introduced back into the human body. Individual-derived cells share the same major histocompatibility complex (MHC) and may not cause considerable immunological rejection if injected back into the same individual. Furthermore, cells used for modification are completely derived from the individual and will not cause any ethical problems. The principle method by which ACT deals with safety and ethical issues also applies to our project because exosomes, as endogenous and natural membrane vesicles, share similar biological properties with the cells of donor individuals. Thus, we have developed the following reference protocol for future clinical trials.
The first step is to isolate certain types of cells from human blood. The isolated cells should have the ability to secrete an adequate amount of exosomes and can be readily modified at the genetic level. Dendritic cells are optimal for producing exosomes. Moreover, human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) could be an efficient mass producer of exosomes for downstream modification. Furthermore, generating stem cells using techniques such as induced pluripotent stem (IPS) cells may be used to set up a robust and scalable manufacturing process.
The next step is to genetically modify the derived cells to endow the exosomes with enhanced therapeutic capacity. Based on our project, the modifications include, but are not limited to, the following list:
i) RVG modification of exosomes to ensure that the siRNA can pass through the BBB and arrive at neuronal cells,
ii) encapsulating MOR siRNA into exosomes, and
iii) facilitating the loading of siRNA into exosomes and export of exosomes to increase the production efficiency of siRNA packaged in exosomes.
These modifications can be performed by direct ex vivo reprogramming at the genetic level so that permanently modified cells can produce therapeutic exosomes efficiently and persistently.
The last step is to harvest exosomes and inject them back into the patient body. We expect that these exosomes can pass through the BBB and deliver siRNA into neuronal cells and that the siRNA can then knock down the expression of the MOR gene and finally block the reward pathway as observed in mice in our project.
Figure 1. Primary protocols for exosome-mediated detoxification therapy (EMDT), edition one. Exosome-mediated detoxification therapy (EMDT) requires the generation of highly avid blood cells. A subgroup of immune cells derived directly from human blood can be efficiently isolated and modified ex vivo to function as a stable cell factory to produce an abundant quantity of exosomes. Engineering certain groups of cells by incorporating a specific set of transgenes will equip exosomes with precise targeting and therapeutic potential for drug detoxification. Patients who receive exosome injections are expected to show significantly attenuated reinforcement and consequently have less chance of relapsing or developing drug addiction owing to blockage of the reward pathway.
2.2 Direction 2 (ultimate goals)
The principal drawback of all current therapies for detoxification is the regular administration of drugs. Patients need surveillance to ensure that they have taken the medicine at the correct time. The high rate of relapse indicates that there is a much room for improvement. We believe that this improvement can be performed by directly injecting modified cells into the human body. To accomplish this goal, we should first develop a sensor device that can detect the change in morphine levels in the blood.
The first step is to isolate immune cells from human blood and subsequently incorporate both fundamental genes for therapeutic potential and sensory function. This reprogramming process can also be performed ex vivo. After successful selection and culture of modified cells, we need to directly inject these cells into the patient body and allow the machine run automatically. Within the patient body, the sensor device tells the modified cells when to activate the expression of certain genes to arm exosomes with weapons and guarantees that these genes are silenced unless the level of morphine is increased in the blood after opioid drug administration. Once the morphine level exceeds a certain threshold in the blood of patient, the sensor device is activated and triggers the cells to release controlled amount of exosomes into patient’s body. Then exosomes circulate through the body and deliver siRNA to neuronal cells to block the rewarding pathway. Theoretically, our new approach employing RVG exosome loaded with MOR siRNA may functions as both monitor and curer for addicts.
Integrating delivery, silencing and sensory devices into one machine will replace human surveillance at the molecular level. Patients would not need regular injections because the machine can function for a long period. Patients are also under constant protection from opioids, which allows the convalescent to move on from opioids. We hope that with this ultimate automated device, we could finally win the opioid war.
Figure 2. Primary protocols for exosome-mediated detoxification therapy (EMDT), edition two. Immune cells are isolated and genetically modified as described in the previous protocol. Apart from fundamental modifications to equip exosomes with therapeutic potential, an extra sensor device is added to monitor the instant change in morphine molecules in the blood in real-time. The modified cells are implanted into the human body and function as an automated machine. Under normal circumstances, the machine is turned off and secreted exosomes play physiological roles in cell communication as usual. The machine is activated upon the detection of an abrupt increase in morphine and begins to turn on the burst mode to produce a large amount of therapeutic exosomes. These exosomes then circulate through the body and deliver siRNA to neuronal cells to block the reward pathway.
However, our designed therapeutics is not a miracle medicine. The success of treatment also depends on person’s level of commitment to treatment and the level of support available to him. Because our approach does not cure craving for drugs, diverse treatment options are needed to integrate both pharmacological treatment and psychosocial approaches. Besides medication, there are some equally important parts for drug addiction treatment, such as counseling and family support. The patient is recommended to take part in a treatment program that deals with both the physical and psychological aspects of drug addiction. It is also recommended to stay out of the environment in which drugs are readily available. Detoxification and subsequent psychosocial treatment are essential components of an effective treatment system for people with opioid dependence.
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