Functional exosome production system

Overview

In this system, we applied ODE（ordinary differential equation）- system to predict the production of the exosome and other target proteins. Also, the interactions among them are inclusive in the time-amount model.

Assumptions

1. Production of exosome is mainly associated with booster protein.
2. mRNA and proteins are limited by natural degradation.
3. Amount of proteins stored into exosome is proportional to the size of each exosome, so size of exosome can be ignored.
4. The packaging of protein to exosome and finishing of translation happen simultaneously because the interval is too short to take into account.

Parameters	Definitions	Value	Units	Note
a	Translational rate per amino acid	720	Amino acids residues *min^-1
TCrb	transcription rate of RVG-Lamp2b	0.314	umol *min^-1
TCc3	transcription rate of CX36	0.42	umol *min^-1
TCce	transcription rate of CD63-L7AE	0.37	umol *min^-1
TCdrug	transcription rate of drug mRNA	0.46	umol *min^-1
TCssn	Maximum transcription rate of STEAP3-SCD4-NadB	0.15	umol *min^-1
TCne	transcription rate of nsMase	0.24	umol *min^-1
TCcm2	transcription rate of CD63-MS2	0.41	umol *min^-1
LRB	Length of RVG-lamp2b	469	Amino Acid residues
LC3	Length of Cx36	320	Amino Acid residues
LCE	Length of CD63-L7AE	367	Amino Acid residues
LCM2	Length of CD63-MS2	378	Amino Acid residues
LS3	Length of STEAP3	488	Amino Acid residues
LS4	Length of SCD4	198	Amino Acid residues
LNB	Length of NadB	331	Amino Acid residues
LNE	Length of nsMase	655	Amino Acid residues
dmRNA	Decay rate of mRNA	0.41	umol *min^-1
TCssn	Maximum transcription rate of STEAP3-SCD4-NadB	0.139	min^-1	2016 IGEM imperial college
C	Packaging rate by Exosome	0.6	min^-1
d_prot	Degradation rate of protein	1.39E^-05	min^-1	2016 IGEM imperial college
K11/K12/K13/K14	The rate of booster protein facilitate exosome biogenesis	0.15	min^-1
K15	Combination rate between CD63-L7AE and drug mRNA	0.08	min^-1
K17	transcription rate of CD63-MS2	0.3	min^-1
d_ce-drug	Maximum transcription rate of STEAP3-SCD4-NadB	1.39E^-05	min^-1	Same as degradation rate of other proteins
K18	Disassociation of CD63-L7AE and drug mRNA	3x10^-4
K19	Disassociation of CD63-L7AE and drug mRNA	2x10^-4
K0		7.9x10³

Figure1: state value of target proteins

In this figure, we can see the non-packaging proteins (NadB, nsMase, Steap3) are tend to overexpress, and packaging protein (CX36, CD63-L7AE, SCD4, CD63-L7AE-drug) reached a producing-consuming balance

Figure2: individual demonstration of booster protein

Figure3: individual demonstration of packaging-protein

Figure4: diagram of Exosome production

Figure5：Two version of the functional exosome.

As shown in figure 4, with the help of the booster, our output of functional exosome is significantly increasing. In our design of ExoB1, we add the nsMase as one of our booster members, which genders the amount of exosome into a higher level according to our model results.

In figure5, CM2-loaded exosome which has a low level of toxicity produce relatively less amount of exosome compared to the CE-loaded exosome. That is because L7AE capture the drug-mRNA more efficiently.