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1.Bba_K1633003
MOR siRNA -1(siRNA for mouse Mu opioid receptor)
INTRODUCTION
This part is an artificially designed RNA strand. It serves as an element of the Team
NJU CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of
Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
software accessible online. This tool can find the best siRNA sequences on target gene
MOR to insure the maximum gene-specificity and silencing efficacy. This tool also
designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
the plasmid. Then we order them through a DNA synthesis company. We got four such RNA.
MOR siRNA-1 is a backup.
Figure 2. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-1
plasmids.
2.Bba_K1633004
MOR siRNA -2(siRNA for mouse Mu opioid receptor)
INTRODUCTION
This part is an artificially designed RNA strand. It serves as an element of the Team
NJU CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of
Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
software accessible online. This tool can find the best siRNA sequences on target gene
MOR to insure the maximum gene-specificity and silencing efficacy. This tool also
designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
the plasmid. Then we order them through a DNA synthesis company.
Figure 3. The sequence of MOR siRNA-2.
USAGE AND BIOLOGY
We package MOR siRNA into exosomes by transfecting HEK293 cells with a plasmid
expressing MOR siRNA and then collect siRNA-encapsulated exosomes. When inject the
modified exosomes into the bloodstream, exosome will specifically recognize
acetylcholine receptors and fuse with neurons under the direction of the RVG peptide.
Once inside neurons, MOR siRNA will degrade MOR mRNA by base-pairing, resulting in sharp
decrease of MOR on neuron membrane. As a consequence, MOR reduction and disturbed
function will result in the inhabitation of the secretion of GABA and the suppression of
the dopaminergic reward pathway, which ultimately have some therapeutic effects on
opioid dependence.
To ensure the interference efficiency of the MOR siRNA, three siRNA sequences targeting
different sites of MOR mRNA were designed and transfected into the mouse neuroblastoma
cell line Neuro2A. Efficient knockdown of MOR in Neuro2A cells is observed, and the
sequence with the best interfering effect was selected for further study. Not showing
the best efficiency, MOR finally functions just as a backup. Showing the best
efficiency, MOR siRNA-2 is used for further study.
Figure 4. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-1
plasmid.
CHARACTERIZATION
` 3.1 Package of MOR siRNA into exosomes
The levels of MOR siRNA in isolated exosomes were assayed by a quantitative RT-PCR
assay. The MOR siRNA concentration in exosomes was calculated to be approximately 0.14
pmol/μg. The results showed that MOR siRNA can be successfully packaged into exosomes,
no matter the exosomes were modified on the outside membrane with or without RVG
peptide.
Figure 5. The concentration of MOR siRNA in unmodified or RVG-modified exosomes.
3.2 TEM photographs of exosomes carrying MOR siRNA inside and RVG on
membranes
We next characterized the RVG exosomes loaded with MOR siRNA using transmission electron
microscopy (TEM). The TEM photographs showed that the exosomes presented normal
morphological characteristics after outside modification and siRNA loading, with a
diameter of approximately 90 nm and a double-layer membrane surrounded. These
characteristics indicate that the exosome properties were not affected by the
modifications.
Figure 6. TEM photographs of the exosomes with outside RVG modification and inside siRNA
loading.
3.3 RVG exosomes specifically deliver MOR siRNA into neuronal cells
Subsequently, MOR siRNA levels were assayed in recipient Neuro2A cells when incubating
with RVG exosomes loaded with MOR siRNA. The siRNAs concentrations were barely detected
in untreated control cells or in cells treated with RVG exosomes or unmodified exosomes
loaded with MOR siRNA. In contrast, a significant amount of siRNAs were detected in
Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. As a control, MOR
siRNA was also barely detected in C2C12 cells treated with RVG-exosome loaded with MOR
siRNA. Taken together, these results clearly demonstrate that the RVG peptide
modification on the exosome membrane specifically guides exosomes to target neuronal
cells bearing the surface acetylcholine receptor, allowing for the efficient delivery of
MOR siRNA into the recipient cells.
Figure 7. Quantitative RT-PCR analysis of MOR siRNA concentration in Neuro2A and C2C12
cells treated with RVG exosomes (RVG exosome), unmodified exosomes loaded with MOR siRNA
(siRNA-exosome) or RVG exosomes loaded with MOR siRNA (siRNA-RVG exosome).
RVG exosomes loaded with MOR siRNA specifically reduce MOR expression
in neuronal cells
We next evaluate the effect of RVG exosome-delivered siRNA on MOR expression in vitro.
MOR expression levels were assayed in Neuro2A cells after treatment with RVG exosomes
loaded with MOR siRNA. Compared with control cells, MOR protein and mRNA levels were
dramatically reduced by RVG exosome-delivered siRNA, while no reduction in the MOR
protein and mRNA levels were observed by exosomes without the RVG peptide on their
surface. The results suggest that the RVG peptide modification on the exosome membrane
can specifically guides exosomes to target neuronal cells, allowing for the delivery of
MOR siRNA into the neuronal cells to reduce MOR expression levels.
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Figure 8. RVG exosome-delivered siRNA specifically enters Neuro2A cells and reduce MOR
expression. (A) Western blot analysis of MOR protein levels in untreated control Neuro2A
cells or cells treated with MOR siRNA loaded in normal exosomes or RVG exosomes. (B)
qRT-PCR analysis of MOR mRNA levels in untreated control Neuro2A cells or cells treated
with MOR siRNA loaded in normal exosomes or RVG exosomes.
3.5 The effects of siRNA delivered by RVG exosomes on morphine-induced
CPP
MOR and its signaling pathway are known to be involved in the dependence and relapse of
opioids such as morphine and heroin. Importantly, relapse always disrupts the process of
opioid withdrawal. Subsequently, we focus on investigating the effect of exosomal siRNA
of MOR on opioid relapse. We evaluate the consequences of MOR knockdown by exosomal
siRNA in the animals by conducting the morphine-induced conditioned place preference
(CPP) test, a mouse model for morphine wanting/liking behaviors. In the CPP paradigm,
mice learned to associate the rewarding effect of morphine with a drug-paired
environment. The CPP test was designed to mimick the process of relapse of morphine
(Fig. 5A). Before conditioning, the mice showed a preference for visiting black chamber.
Then, morphine dependence was developed when mice were place-conditioned by
intraperitoneal injection with morphine in the white chamber on even-numbered days (on
days 2, 4, 6, 8 and 10) and with saline in the black chamber on odd-numbered days (on
days 3, 5, 7, 9 and 11). On day 12, CPP test 1 was conducted by allowing the mice to
freely visit the morphine-paired white chamber or saline-paired black chambers. As
expected, mice showed a significant preference in visiting the morphine-paired white
chamber, suggesting the development of morphine dependence. Then, morphine treatment was
removed for several days. On day 26, CPP test 2 was conducted and mice spent less time
in the morphine-paired white chamber than the saline-paired black chamber, suggesting
the disappearance of morphine dependence. Then, mice were intravenously injected with
saline or with siRNAs loaded in normal exosome or RVG exosome once every two days for a
total of four times, and CPP test 3 was performed on day 32. Mice maintained their
natural preference for the black chamber, suggesting that MOR siRNA had no effect on the
behavior of the mice. Finally, mice were relapsed on morphine on day 33, and CPP test 4
was performed the next day. Interestingly, the mice treated with RVG exosome-delivered
siRNAs maintained their natural preference for the black chamber, while the mice treated
with saline or with siRNAs loaded in normal exosome show preference to morphine-paired
white chamber, suggesting that the MOR siRNAs delivered by RVG exosome restrain drug
addiction when the mice were re-exposed to morphine.
Figure 9. The effects of siRNA delivered by RVG exosomes on morphine-induced CPP. A flow
chart depicting the experimental design is shown. The panel is represented by the value
of the time mice stay in morphine-paired white chamber minus the time mice stay in
saline-paired black chamber.
h3> 3.6 The effects of siRNA delivered by RVG exosomes on MOR expression in
vivo
After the CPP test, mice were sacrificed, and total RNA and protein were extracted from
mouse brain to evaluate the expression levels of MOR in vivo. Both MOR protein and mRNA
levels were reduced in the mice treated with RVG exosome-delivered siRNA. In contrast,
siRNAs delivered by unmodified exosome could not reduce MOR mRNA and protein levels in
mouse brain. Thus, these results clearly demonstrate that exosomes with RVG modification
passed through the BBB and delivered MOR siRNA into the central nervous system to
regulate MOR expression, while natural exosomes without the RVG modification were not
capable of delivering siRNA into the central nervous system or regulating target gene
expression.
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Figure 10. RVG exosomes can transfer MOR siRNA through the BBB and reduce MOR expression
levels in vivo. (A) Western blot analysis of MOR protein levels in the brains of mice
following injection with saline or with MOR siRNA loaded in normal exosomes or RVG
exosomes. (B) qRT-PCR analysis of MOR mRNA levels in the brains of mice following
injection with saline or with MOR siRNA loaded in normal exosomes or RVG exosomes.
3.Bba_K1633005
MOR siRNA-3(siRNA for mouse Mu opioid receptor)
INTRODUCTION
This part is an artificially designed RNA strand. It serves as an element of the Team
NJU CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of
Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
software accessible online. This tool can find the best siRNA sequences on target gene
MOR to insure the maximum gene-specificity and silencing efficacy. This tool also
designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
the plasmid. Then we order them through a DNA synthesis company. We got four such RNA.
MOR siRNA-3 is a backup.
Figure 11. The sequence of MOR siRNA-2.
USAGE AND BIOLOGY
We package MOR siRNA into exosomes by transfecting HEK293 cells with a plasmid
expressing MOR siRNA and then collect siRNA-encapsulated exosomes. When inject the
modified exosomes into the bloodstream, exosome will specifically recognize
acetylcholine receptors and fuse with neurons under the direction of the RVG peptide.
Once inside neurons, MOR siRNA will degrade MOR mRNA by base-pairing, resulting in sharp
decrease of MOR on neuron membrane. As a consequence, MOR reduction and disturbed
function will result in the inhabitation of the secretion of GABA and the suppression of
the dopaminergic reward pathway, which ultimately have some therapeutic effects on
opioid dependence.
To ensure the interference efficiency of the MOR siRNA, three siRNA sequences targeting
different sites of MOR mRNA were designed and transfected into the mouse neuroblastoma
cell line Neuro2A. Efficient knockdown of MOR in Neuro2A cells is observed, and the
sequence with the best interfering effect was selected for further study. Not showing
the best efficiency, MOR siRNA-3 finally functions just as a backup.
Figure 12. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-3
plasmid.
4.Bba_K1633006
MOR siRNA-4(siRNA for mouse Mu opioid receptor)
INTRODUCTION
This part is an artificially designed RNA strand. It serves as an element of the Team
NJU CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of
Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
software accessible online. This tool can find the best siRNA sequences on target gene
MOR to insure the maximum gene-specificity and silencing efficacy. This tool also
designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
the plasmid. Then we order them through a DNA synthesis company. We got four such RNA.
MOR siRNA-4 is a backup.
Figure 13. The sequence of MOR siRNA-4.
USAGE AND BIOLOGY
We package MOR siRNA into exosomes by transfecting HEK293 cells with a plasmid
expressing MOR siRNA and then collect siRNA-encapsulated exosomes. When inject the
modified exosomes into the bloodstream, exosome will specifically recognize
acetylcholine receptors and fuse with neurons under the direction of the RVG peptide.
Once inside neurons, MOR siRNA will degrade MOR mRNA by base-pairing, resulting in sharp
decrease of MOR on neuron membrane. As a consequence, MOR reduction and disturbed
function will result in the inhabitation of the secretion of GABA and the suppression of
the dopaminergic reward pathway, which ultimately have some therapeutic effects on
opioid dependence.
To ensure the interference efficiency of the MOR siRNA, three siRNA sequences targeting
different sites of MOR mRNA were designed and transfected into the mouse neuroblastoma
cell line Neuro2A. Efficient knockdown of MOR in Neuro2A cells is observed, and the
sequence with the best interfering effect was selected for further study. Not showing
the best efficiency, MOR siRNA-4 finally functions just as a backup.
Figure 14. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-4
plasmid.
5.Bba_K1633007
USAGE AND BIOLOGY
It is artificial designed to target and downregulate GFP protein. This part is a short
hairpin RNA (shRNA) sequence. When this shRNA sequence is cut by restriction enzyme and
then integrated into pcDNA 6.2 vector, this shRNA can play a RNAi function in mammalian
cell lines such as HEK293 cell. When the shRNA vector of GFP is transfected into HEK293
cells, the shRNA hairpin structure is cleaved by Dicer into siRNA of MOR and loaded into
the RISC. The siRNA-RISC complex targets at GFP mRNA under the guide of siRNA sequence
and cleave the GFP mRNA.
CHARACTERIZATION
To determine whether siRNA delivered via RVG exosomes can pass through the BBB and
regulate endogenous gene expression, we packaged siRNA against green fluorescent protein
(GFP) into RVG exosomes and injected them into GFP-transgenic mice through the tail
vein. Then, the GFP levels in various tissues were determined by measuring fluorescence
emission using a fluorescence microscope. Compared with control mice, injection of the
RVG exosomes loaded with GFP siRNA dramatically reduced GFP levels in different parts of
the brain of GFP-transgenic mice. In contrast, unmodified exosomes loaded with GFP siRNA
did not induce obvious GFP silencing in mouse brain. On the other hand, while unmodified
exosomes loaded with GFP siRNA had significant effect on GFP levels in lung, liver and
spleen of GFP-transgenic mice, RVG exosomes loaded with GFP siRNA only induced a slight
but non-significant GFP silencing in these tissues. The results successfully demonstrate
that exosome-packaged siRNA can be delivered to various tissues and thus silence
endogenous gene expression. The results also indicate that RVG peptide on the surface of
exosome has some selectivity for neuronal tissues, which may simultaneously prevent
siRNA from spreading to non-neuronal tissues.
Figure 15. Fluorescence confocal microscopy photographs showing sections from different
tissues of GFP-transgenic mice. GFP-transgenic mice were intravenously injected with
saline (control) or with GFP siRNA loaded in normal exosomes (siRNA-exosome) or RVG
exosomes (siRNA-RVG exosome).
6.Bba_K1633008
nSMase2 (coding sequence to express nSMase 2 protein)
INTRODUCTION
This part is a codon-optimized nSMase 2 gene CDS. We express this gene in HEK 293 cell
to increase the amount of exosomes.
USAGE AND BIOLOGY
Neutral sphingomyelinase 2 (nSMase 2) is a key regulatory enzyme generating ceramide
from sphingomyelin and can actively induce exosome secretion from cells and trigger
cellular export of small RNAs. The original sequence of nSMase 2 from Homo Sapiens is
from NCBI. NCBI Gene ID is 55512. The sequence codon of this part was optimized. We
ordered the sequence from a DNA synthesis company. When this part is inserted into pcDNA
3.1 vector and transfected into mammalian cells, it can express nSMase 2 gene in
mammalian cells and stimulate the secretion of exosomal siRNAs from cells.
CHARACTERIZATION
Stimulation of exosome and exosomal siRNA secretion by introduction
of nSMase 2
Extracellular vesicles (EVs) are generated through several different and poorly
understood biogenetic mechanisms, of which neutral sphingomyelinase (nsMase) is involved
in emission of exosomes [1]. nSMase2 was reported to play the vital role in exosome
biogenesis, the inhibition of which markedly reduced the release and loading capability
of exosome [2].
Because nSMase2 can stimulate both exosome production and siRNA loading to exosomes, we
selected nSMase2 as a “molecular pump” to accelerate the amounts of exosomes released
by cells and to promote the generation of exosomal siRNAs. A plasmid expressing nSMase2
was constructed and transfected into HEK293 cells to stimulate the secretion of exosomes
and exosomal siRNA from HEK293 cells. As anticipated, both exosomes and exosomal siRNA
secreted by HEK293 cells were increased after overexpressing nSMase2 in HEK293 cells.
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Figure 16. (A) Total amounts of exosomes (shown as total protein) secreted by HEK293
cells with or without the introduction of nSMase2. (B) Quantitative RT-PCR analysis of
siRNA levels in exosomes secreted by HEK293 cells with or without the introduction of
nSMase2.
We then performed nanoparticle tracking analysis (NTA) to have a more precise
determination of the quantity and size of secreted exosomes [3]. The use of Nanosight
enabled quantification and size determination of the EV, as nanoparticles can be
automatically tracked and sized based on Brownian motion and the diffusion coefficient.
Because exosomes are more homogenous and generally smaller than most EVs with a diameter
size ranging from 40 to 120 nm [4], the percentage of nanoparticles whose size ranges
from 40 to 120 nm could be a good indicator of total amount of exosomes. The shift of
peak of size distribution of EV from 170 nm to 120 nm and the significant raise of the
peak indicated the increase of relative level of exosomes in secreted EVs after
overexpression nSMase2 in HEK293 cells. From what we have discussed above, the
improvement of manufacturing exosomes by overexpressing nSMase2 is proved to be feasible
and effective.
Figure 17. Characterization of secreted exosomes after overexpression of nSMase2 in
HEK293 cells. (A and B) Representative screen shots of the NTA videos for EV from HEK293
cells under normal condition or after transfection with nSMase2 plasmid. (C and D) Size
and intensity of EV from HEK293 cells under normal condition or after transfection with
nSMase2 plasmid. (E) Concentration of different particle sizes of exosomes with (red
line) or without (blue line) transfection with nSMase2 plasmid. The peak of size
distribution of EV shifted from 170 nm to 120 nm after transfection with nsMase2
plasmid, indicating the increase in quantity of secreted exosomes.
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