mRNA delivery simulation

Assessment of delivery helper: CX43

Background

Gap junction proteins are composed of two hemichannels in the plasma membrane of two neighboring cell They are controlled by the voltage difference between the two cellular interiors, called transjunctional voltage (Vj)-thus are voltage-gated. They are sites where molecules exchange between adjacent cells. The voltage sensing mechanism of a hemichannel failed to switch into open state if the other hemichannel was in the inactive state.

CX43, a family member of gap junction proteins, can only help us to deliver exosome if it is open and exchange signal with neuron cell to trigger membrane fusion process. Here, we present a model demonstrating a macroscopic situation in gap junctions, and thereby ensuring the functionality of CX43 as a cytosolic delivery helper.

Splitting Gap Junctions into Hemi-Channels:

Assumption

1. The whole CX43 can be regarded as the serial combination of two dissimilar hemichannels[8]. So, we apply the modular approach to model the sensitivity of the entire CX43.

2. Here we aim to characterize CX43 protein for its voltage sensing mechanism, so we do not consider the other factors influencing CX43 channel state.

3. Conductance(G) is calculated to represent the gate state of the whole channel. The channel will be at open state if it is staying on the high level of condutance.

Process and Results

Firstly, we split the whole CX43 assembly into two hemichannels. Whole channel conductance changing with transjuctional voltage is then simulated. The scope of the transjunctional potential during exosome-neuron contact is between 45.51-54.09mV. [13, 14] So if the channel is staying on the high level of conductance when the Vj is between 45.51-54.09mV, then we could expect CX43 to open and exosome membrane is fused with neuron cell membrane.

Conductance of two hemi-channels:

Figure1: Conductance of Left-Hemi Channel changes with voltage.

Figure2: Conductance of Right-Hemi Channel changes with voltage

The total gap junctional conductance, Gj, can now be evaluated as:

And then we could characterize CX43 as a whole voltage-gated channel.

Figure3: Bifurcation of voltage sensitivity of an entire gap junction into its left and right hemi-channel

Discussion

As figure 3 shown above, the range of voltage rendering channel activated is falling around -60—60mV, which matches the scope of the transjunctional potential during exosome-neuron contact (45.51-54.09mV). [13, 14] So the CX43 can be theoretically proved as a good delivery helper to trigger exosome to fuse with neuron. Sequentially, the mRNA is then released and expressed.

LEFT/RIGHT	Gmax	Gmin	S	V₀
Left	122^[9]	17.30^[9]	8.94^[9]	55.99^[9]
Right	122^[9]	16.64^[9]	9.67^[9]	56.75^[9]
concept	Maximum conductance of Left/Right hemi-channel	Minimum conductance of Left/Right hemi-channel	relative change in the slope factor	shift in half-maximal point

mRNA expression simulation

Process

After validating the availability of our delivery helper, the expression of therapeutic mRNA can be predicted. The initial amount of delivered mRNA is set as 11 umol.

Assumptions

1. Exosome elimination by endosome can be neglected.
2. Proteins and mRNA is limited by natural degradation.

CE-drug	CD63-L7AE-drug on membrane
Mdrug	Drug mRNA in neuron cell
Drug	Therapeutic protein

Parameters	Definitions	Value	Units	Note
D1	Dissociation rate of CE-drug	1.34	min^-1
d_prot(1)	Degradation rate of membrane protein	0.23E-05	min^-1	[4]
d_prot(2)	Degradation rate of cytoplasmic protein	0.23E-05	min^-1	[4]
d_mRNA	Degradation rate of mRNA	300E-4	min^-1	Estimated from[3]
T	Translational rate per amino acid in neuron cell	300	Amino acids residues *min^-1	Estimated from[3]
L_Mdrug	Length of therapeutic mRNA	956	Amino Acid residues	Cited from Pubmed

Results

Discussion

a. production of drug in neuron cells. Our therapeutic mRNA is expressed around 120umol in neuron cells given that the final amount of mRNA entering into blood is 11 umol. b. amount of therapeutic mRNA in neuron after delivery. Delivered mRNA will all be degraded within 90 minutes c. Amount of CE-drug complex on membrane after exosome fuse with neuron. The remaining CE-drug degrade very fast and almost disappear within five minutes. So we could expect our exosome to execute its function without harming neuron cells.