Aim
Astroglia-based glutamate-grabbing therapy
Since astrocytic dysfunction can dramatically weaken the defenses against neurodegenerative disease, our primary goal is to save astrocytes from the high glutamate level accumulated. When transporters are not present in sufficient quantities, large amounts of glutamate accumulated in the synaptic cleft can triggers a series of cascade in astrocytes and damage them (Kim et al., 2011). However, as long as excitatory amino acid transporter 2 (EAAT2) is present in sufficient quantities on their membrane, astrocytes would have the capacity to absorb all glutamate into their cytoplasm for further processing (Kim et al., 2011). Therefore, we design this theraputic method to rescue the astrocytes by enhancing the glutamate absorbing function with overexpression of EAAT2 on their membrane.
Safety, side effects, duration and productivity are important factors in a drug development. When designing the therapy, we considered a series of questions:
Safety aspect：
How can the therapy be reversible without causing long-term risks?
We use mRNA to overexpress EAAT2 in the target cells. The mRNA vectors do not pose any safety risk such as genomic integration, antibiotic resistance, or strong immunogenic response caused by a replicating vector. Expression of mRNA-encoded proteins is transient and more direct than DNA-based vectors, which requires intermediate steps such as nuclear localization and transcription. In addition, the effect of this overexpression method is totally reversible. Thus, mRNA can be used as vector for the overexpression of membrane proteins like EAAT2 (Sahin et al., 2014)
How to ensure efficient transportation without causing excessive immune response?
The mRNA has a very short half-life without protection, so we need a physical shell to protect the mRNA during transportation. Exosomes, as a mimic of "the nature's delivery systems", carry the role of cell-cell communication in the body to initiate downstream physiological functions. They are naturally stable and have inherent targeting properties depending on the composition of the exosomes. Compared with exosome, most small molecule agents or drugs have low solubility and toxicity due to nonspecific tissue targeting and short half - life, which leads to the poor efficacy of the drugs. Furthermore, the composition of exosomes are based on the parental cell it derived from. Therefore, exosomes with medicinal functions can be produced by gene modification of the production cell lines.
Transport and Targeting：
How to across the blood-brain barrier (BBB)?
Rabies virus glycoprotein (RVG) is a protein that specifically binds the nicotinic acetylcholine receptor (nAchR) (Liu et al., 2009). RVG29 peptide binds cell surface receptors on the brain endothelial cells that form the BBB, which further mediates the transcytosis of nanoparticles across the cell layer. The majority of the presented RVG-29-functionalized drug delivery system clearly show a targeted uptake into the CNS (Liu et al., 2009).
How to achieve precise targeting of astrocytes?
RVG can target acetylcholine receptors on the nerve cells to achieve the initial targeting function (Liu et al., 2009). Connexin 43 (Cx43) is a gap junction (GJ) protein, which is a hemichannel on the membrane and only opens when meet another hemichannel. In the central nerve system (CNS), Cx43 is mainly expressed on the membrane of astrocytes, and mainly distribute on the hippocampus and inferior olivary complex (Nagy et al., 2003). Thus, by loading Cx43 to the membrane, exosomes can achieve the precise astrocyte targeting.
How to ensure more mRNA delivered to astrocytes?
We chose the Cx43 S368A mutant to accelerate the mRNA delivery. Since this S368A mutant can prevent gap junction internalization and degradation of exosomes, it can transport the cargo into the astrocyte by membrane fusion (Kojima et al., 2018), and therefore, more Cx43 hemichannels are loaded onto the membrane of target cells. The result is that there is a higher possibility to bind to the exosomes with Cx43 hemichannels.
Production process：
How to load cargo mRNA into the exosomes？
Without any gene modification, HEK293T cells would randomly envelope the cytoplasmic contents into the exosomes. Due to the random nature of this process, even if we overexpressed the mRNA in the cytoplasm of HEK293T, we still can not ensure efficient cargo mRNA loading. We figured out two methods to solve this problem:
The first approach is to express RNA binding protein on the exosomal membrane and modify the sequence in the untranslated region (UTR) of the mRNA, which enables mRNA to be bound to the exosomal membrane. Here, we selected L7Ae as the RNA binding protein and the C/D box as the 3’-UTR modification (Kojima et al., 2018).
The second method is to directly use the plasmid to express of EAAT2, and conduct in vitro transcription (IVT) to obtain the mRNA of EAAT2. Then, we transfect that mRNA into the exosomes produced by HEK293T (Sahin et al., 2014).
Modularity
Overcoming the difficulties one after another, we collected these parts together to assemble them into an efficient production system. We divided them into two devices, which are the exosome membrane device and mRNA production device:
Exosome membrane device
We have designed two exosome membrane devices, including one for intracellular generation system and one for in vitro system, modification and packaging. Both of the devices includes a pCMV high efficiency promoter, KOZAK sequence for ribosome binding, Lamp2b-RVG for passage through BBB (blood brain barrier) and astrocyte recognition and Cx43 for mRNA release in target cells. The device for intracellular generation system also includes CD63-L7AE for mRNA specific sequence (C/D box) recognition to convey efficient in vivo mRNA packaging to the exosome (Kojima et al., 2018).
mRNA production device
We have designed two mRNA production devices respectively for intracellular generation system and for in vitro system. Both of the devices includes pCMV high efficiency promoter, KOZAK sequence for ribosome binding, and EAAT2 for delivering treatment effect on astrocytes (described down scroll). The one for in vivo system and packaging device (the upper one) also includes a C/D box sequence corresponding to CD63-L7AE for efficient in vivo packaging of the mRNA product (Kojima et al., 2018).
Production system
With combination of the two devices described above, we have designed two production systems for intracellular and for in vitro generation. The intracellular generation one is combined with the corresponding devices from membrane modification and mRNA production. This production system could utilize cyto-transcriptional devices from transfected HEK293 cells for mRNA transcription, post-transcriptional modification, and packaging for mRNA into exosome (Kojima et al., 2018).
The in vitro production system requires only single in vitro exosome membrane device transfected into HEK293 cells purification of modified exosome. The transcription and post-transcriptional modification for product mRNA is conducted with in vitro kits. Then mRNA product is in vitro transfected into purified exosomes with exo-fect kit to load exosome with cargo (Sahin et al., 2014)
We use EAAT2 as our treatment protein coded in delivered mRNA. Endogenous EAAT2 on astrocytes acts as symporters for removing glutamate from synaptic cleft or related internal environments to prevent its potential neurovirulence. As we focused on symptoms of the patients where EAAT2 expression level is reduced and glutamate concentration rises, introduction of EAAT2 coded mRNA will give additional expression of EAAT2 and alleviate excess glutamate relevant symptoms (Kim et al., 2011).
Project advancement
Prototyping by modeling
Before starting the experiments on the EAAT2 function test, we wondered whether EAAT2 overexpression could alleviate the sustained glutamate level increase. Therefore, then we gathered the parameters for modeling to understand the EAAT2 expression level and its transport efficiency to guide the experimental design.
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Validation by experiments
Referring to the feedback from the EAAT2 modeling ,we optimized the protocol we used in the EAAT2- related experiments. By following the standard measurement, we collected our results and feedback to the modeling by comparing with the parameters used in this model.
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Routine debugging
Every weekend, all members would gather together to reflect on our project and to find out the gap between the work of each group. Our PI also offered their enthusiastic help on the debugging process. Then, we used the collected suggestions for the subsequent project design. At the beginning of the project, we attended several conference, introducing and dicussing our projects with professors attending the conferences. With this experience,we gained a deeper understanding on the topic about this field.
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References
Bojar, D. et al. (2018) Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment.’, Nature Communications , 9(1), pp.1305. doi: 10.1038/s41467-018-03733-8.t.
Das, S. K. et al. (2012) ‘Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics’, Journal of Cellular Physiology , 226(10), pp. 2484–2493. doi: 10.1002/jcp.22609.
Han, L. et al. (2009) ‘Brain-targeting gene delivery and cellular internalization mechanisms for modified rabies virus glycoprotein RVG29 nanoparticles.’, Biomaterials , 30(25), pp.4195-4202. doi: 10.1016/j.biomaterials.2009.02.051.
Ionescu, A.V., Lynn, B.D., Nagy, J.I. & Rash, J.E. (2003) ‘Coupling of astrocyte connexins Cx26, Cx30, Cx43 to oligodendrocyte Cx29, Cx32, Cx47: Implications from normal and connexin32 knockout mice.’, Glia , 44(3), pp205-218. DOI: 10.1002/glia.10278.
KARIKó, K., SAHIN, U. & TRECI, Ö. (2014) ‘mRNA-based therapeutics--developing a new class of drugs.’, Nature reviews. Drug discovery , 13(10), pp.759-780. doi: 10.1038/nrd4278.
