What can we do? 01 02 03 Analyze Seek For Study Literature - - PowerPoint PPT Presentation

▶
what can we do
SMART_READER_LITE
LIVE PREVIEW

What can we do? 01 02 03 Analyze Seek For Study Literature - - PowerPoint PPT Presentation

Feb 14, 2024 328 likes •904 views

What can we do? 01 02 03 Analyze Seek For Study Literature Drug Reports Professional Advice Opioids opium morphine heroin POPPY Opioids top the list of illicit drugs and cause the most burden of disease and drug-related deaths

slide-1
SLIDE 1
slide-2
SLIDE 2
slide-3
SLIDE 3

What can we do?

01 02

Seek For

Professional Advice

Study

Drug Reports

Analyze

Literature

03

slide-4
SLIDE 4

Opioids

‘Opioids top the list of illicit drugs and cause the most burden of disease and drug-related deaths worldwide.’

—— The United Nations World Drug Report (2014)

morphine

  • pium

heroin

POPPY

slide-5
SLIDE 5

Abuse of Drug

Opioids Opiates Cocaine ATS “Ecstasy”

20 40 Global estimate of users of different drugs，2012 According to World drug report 2014

Millions of users

60 Heroin Cocaine Alcohol Ecstasy

Mean Pleasure Psychological dependence Physical dependence

Drug hazard index

Harm of opioids

ATS

slide-6
SLIDE 6

Opioid receptor agonist

Methadone

Current treatments

Naltrexone

  • pioid receptor antagonist
slide-7
SLIDE 7

Target—Mu-opioid receptor

Morphine

μ Opioid Recetor

Gi/c Adenylate Cyclase

CREB CREB CREB PKA PKA Altered gene expression

Opioid receptors

Opioids act primarily as agonists of Mu-opioid receptor (MOR) MOR is an ideal therapeutic target for opioid addiction

slide-8
SLIDE 8

Action of heroin on MOR

slide-9
SLIDE 9

Medicine—siRNA

A tiny but powerful gene silencing tool

slide-10
SLIDE 10

Technical barriers：

  • 1. tissue non-specificity
  • 2. immune activation and toxicity
  • 3. brain impermeability

Limitations

slide-11
SLIDE 11

Exosome

Exosome

Borrowing the body’s own RNA transport service Natural, endogenous nano-sized vesicles

slide-12
SLIDE 12

03

Advantages

01

Specificity 02 Safety Brain accessibility

slide-13
SLIDE 13

Experimental

design

slide-14
SLIDE 14

Experimental design

Silencing device Targeting device Pumping device

μ receptor

Rewarding

siRNA exosome

Target——MOR Medicine——siRNA Agent——exosome

  • pioid
slide-15
SLIDE 15

Silencing device

Plasmid skeleton

5’ 3’

slide-16
SLIDE 16

HEK293 cell siRNA exosomes siRNA plasmid siRNA

Silencing device

slide-17
SLIDE 17

Targeting device

RVG： Rabies Virus Glycoprotein Lamp2b：lysosomal-associated membrane protein 2b

Neuron targeting

Lamp2b

RVG

slide-18
SLIDE 18

Pumping device

nsMase2 Molecular pump HEK293 cell HEK293 cell

slide-19
SLIDE 19

targeting plasmid siRNA plasmid HEK293 cell siRNA Lamp2b-RVG exosomes

neuron

mRNA

Assembly of RNAi-Targeting-Pumping devices

nsMase2 Molecular pump targeting plasmid siRNA plasmid

degradation

Acetyl-choline receptor

slide-20
SLIDE 20

Experimental

results

slide-21
SLIDE 21

Silencing capability validation

Experimental results

Safety val alidation Exosome modification and characterization Targeting capabil ility vali lidation

slide-22
SLIDE 22

Interference efficiency of MOR siRNA

Relative levels of MOR mRNA in Neuro2A cells

0.5 1.0 1.5

1.0 0.47 0.22 0.45 0.42

slide-23
SLIDE 23

siRNA is successfully packaged into exosomes

60 80 100

siRNA concentration in exosome (pmol/μg) siRNA-RVG exosome control RVG exosome siRNA exosome

UD UD

siRNA concentration in exosomes

TEM image of modified exosomes 40 20

slide-24
SLIDE 24

nSMase2 as a molecular pump

Quantitative RT-PCR analysis

  • f siRNA loaded into exosomes

1 2 3

Relative siRNA level

4

Nanoparticle tracking analysis of exosome production

control nSMase2 control nSMase2

slide-25
SLIDE 25

Experimental results

Safety val alidation Targeting capabil ility vali lidation Silencing capability validation Exosome modification and characterization

slide-26
SLIDE 26

MCF-7 A5 A549 contr trol siRNA exosome siRNA-RVG exosome contr trol siRNA exosome siRNA-RVG exosome

(n (non-neuro ronal) (n (non-neuro ronal)

Targeting capability validation（in vitro）

Ne Neuro ro2A C2 C2C1 C12

(n (neuronal) (n (non-neuro ronal)

slide-27
SLIDE 27

0.0 0.1 0.2 0.3 0.4

siRNA concentration (siRNA/U6) UD Neuro2A cell C2C12 cell UD UD

Targeting capability validation（in vitro）

slide-28
SLIDE 28

brainstem cortex

epencephalon

Olfactorius bulbus Olfactory medulla

liver lung spleen contr trol siRNA exosome siRNA-RVG exosome

Targeting capability validation（in vivo）

Neuronal tissues Non-neuronal tissues

slide-29
SLIDE 29

Experimental results

Safety val alidation Targeting capabil ility vali lidation Silencing capability validation Exosome modification and characterization

slide-30
SLIDE 30

control

siRNA-RVG exosome

MOR mRNA and protein levels become

Silencing capability （in vitro）

0.0 0.5 1.0 1.5

Relative level of MOR mRNA

siRNA exosome

lower Neuro2A cell

siRNA-RVG exosomes

MOR GAPDH

siRNA exosome

control

slide-31
SLIDE 31

Conditional Place Preference Test

Morphine Injection

Silencing capability （in vivo）

Natural preference Morphine-paired preference

slide-32
SLIDE 32

Day

1 2 4 3 6 8 5 7 9 11 10 12 26 28 30 32 33 34 1 12 26 32 34

CPP SCORE (sec)

  • 500
  • 1000

control

siRNA exosome siRNA-RVG exosome

Day1 Pr

Pre te test

Day12 12 te

test-1

Day26 26 te

test-2

Day34 34 te

test-3

siRNA-RVG exosome siRNA exosome control

Morphine injection Exosome injection

Mobile heatmap

Flow chart

slide-33
SLIDE 33

control siRNA exosome

siRNA-RVG exosome

MOR expression after CPP test

control

0.0 0.5 1.0 1.5

Relative level of MOR mRNA

siRNA exosome siRNA-RVG exosome

MOR GAPDH

RVG exosome-delivered siRNA pass through BBB to down-regulate MOR expression

slide-34
SLIDE 34

Experimental results

Safety val alidation Targeting capabil ility vali lidation Silencing capability validation Exosome modification and characterization

slide-35
SLIDE 35

Safety validation

Forced Swimming Test

100 200 300 400

Inject exosomes

Record the video and analyze the activation

  • f the mice

Mobile time（s）

slide-36
SLIDE 36

Model Model

slide-37
SLIDE 37

Modeling Overview

Delivery Module Silencing Module Signaling Module

slide-38
SLIDE 38

𝑒 𝐶𝑑𝑔 𝑒𝑢 = 𝐿𝑐𝑚𝑝𝑝𝑒𝑒𝑗𝑡 ∙ 𝐶𝑑𝑐 − 𝐿𝑐𝑚𝑝𝑝𝑒𝑐𝑗𝑜𝑒 ∙ 𝐶𝑑𝑔 −

𝑢𝑗𝑡𝑡𝑣𝑓

𝐹𝑢 𝑒 𝐶𝑑𝑐 𝑒𝑢 = −𝐿𝑐𝑚𝑝𝑝𝑒𝑒𝑗𝑡 ∙ 𝐶𝑑𝑐 + 𝐿𝑐𝑚𝑝𝑝𝑒𝑐𝑗𝑜𝑒 ∙ 𝐶𝑑𝑔

𝑒 𝐹𝑢 𝑒𝑢 = 𝐿𝑢𝑠𝑏𝑜𝑡𝑐𝑚𝑝𝑝𝑒 ∙ 𝑞𝑏𝑠𝑗𝑢𝑗𝑝𝑜𝑢𝑗𝑡𝑡𝑣𝑓 ∙ 𝑅𝑢𝑗𝑡𝑡𝑣𝑓∙𝐶𝑑𝑔 𝑅𝑑

− 𝐿𝑐𝑗𝑜𝑒𝑢𝑗𝑡𝑡𝑣𝑓 ∙ 𝐹𝑢 − 𝐿𝑛 ∙ 𝐵𝑆 ∙ 𝐹𝑢

d Tb dt = 𝐿𝑐𝑗𝑜𝑒𝑢𝑗𝑡𝑡𝑣𝑓 ∙ E𝑢 + 𝐿𝑛 ∙ AR ∙ Et − 𝐿𝑗𝑜𝑢𝑢𝑗𝑡𝑡𝑣𝑓 ∙ Tb

d Tissue dt = 𝐿𝑗𝑜𝑢𝑢𝑗𝑡𝑡𝑣𝑓 ∙ Tb − 𝐿𝑓𝑚𝑗𝑛𝑢 ∙ Tissue d Enc dt = 𝐿𝑑 ∙ 𝐿𝑗𝑜𝑢 ∙ Tbbrain − 𝐿𝑓𝑡𝑑𝑓𝑜𝑒𝑤𝑓𝑑 ∙ Enc

exosome binding internalization endosome Endosome escape RISC complex Lysosome degradation

Delivery Module Methods

Pharmarcokinetics of exosome Cellular Trafficking of exosome

slide-39
SLIDE 39

Delivery Module Results

RVG modification helps exosomes pass through the BBB and target into brain

Control siRNA-RVG exosome Control siRNA-RVG exosome

slide-40
SLIDE 40 d R dt = 𝐿𝑓𝑡𝑓𝑜𝑒𝑤𝑓𝑑 ∙ 𝐹𝑜𝑑 − 𝐿𝑒𝑗𝑡RISC ∙ R + 𝐿 𝑔𝑝𝑠𝑛𝑆𝐽𝑇𝐷 · (𝑠𝑢𝑝𝑢 + 𝐿𝑒𝑗𝑡RISC ·

𝑆 −C)·Can+𝐿𝑑𝑚𝑓𝑏𝑤𝑏𝑕𝑓 ·C −𝐿𝑒𝑓𝑕RISC·(R + C) − 𝐿

𝑔𝑝𝑠𝑛RISCm·R·M d 𝐷na dt

= −𝐿𝑒𝑗𝑡RISC ∙ R + 𝐿formRISC ∙ (rtot+𝐿𝑒𝑗𝑡RISC·R −R −C)·Cna −𝐿𝑒𝑓𝑕inna·Cna

d 𝐷 dt = 𝐿formRISC ∙ R ∙ M − 𝐿dis𝑆𝐽𝑇𝐷m ∙ C − 𝐿𝑒𝑓𝑕RISC·(R + C) − 𝐿𝑑𝑚𝑓𝑏𝑤𝑏𝑕𝑓 ·C d M dt = 𝐿 𝑔𝑝𝑠𝑛m𝑆𝑂𝐵 + 𝐿dis𝑆𝐽𝑇𝐷m ∙ C − 𝐿𝑒𝑓𝑕m𝑆𝑂𝐵·M −𝐿 𝑔𝑝𝑠𝑛RISCm·R·M d P dt = 𝐿 𝑔𝑝𝑠𝑛prot·M − 𝐿degprot ∙P

Silencing Module Methods

Deterministic Model of Silencing Process

slide-41
SLIDE 41

Silencing Module Results

Injection of exosomes significantly reduce the level of MOR mRNA and protein

MOR mRNA MOR protein

slide-42
SLIDE 42 d Cabg_R dt

= −ReactionFlux1 + ReactionFlux5

d Cabg_R_𝑛𝑝𝑠𝑞ℎ𝑗𝑜𝑓 dt

= ReactionFlux1 −ReactionFlux2 + ReactionFlux8

d 𝑛𝑝𝑠𝑞ℎ𝑗𝑜𝑓_𝑆 dt

=ReactionFlux2 + ReactionFlux6 −ReactionFlux8

d 𝐻𝑏_𝐻𝑈𝑄 dt

=ReactionFlux2 − ReactionFlux3 + ReactionFlux7

d 𝐻𝑏_𝐻𝐸𝑄 dt

= ReactionFlux3 − ReactionFlux4

d 𝐻𝑏𝑐𝑕 dt

=ReactionFlux4 − ReactionFlux5 + ReactionFlux7 − ReactionFlux

d 𝑆 dt = − ReactionFlux5 − ReactionFlux6 d 𝐻𝑐𝑕 dt

=ReactionFlux2 − ReactionFlux4 + ReactionFlux7

Gαβγ-R Gαβγ + R Gα-GDP Opiates Opiates- R Gαβγ Gα-GTP Gαβγ GTP Gβγ GDP GTP Gαβγ-Opiates-R Gαβγ Opiates

Signaling Module Methods—Activation of MOR

Diagrams and ODEs are created using Matlab Simbiology

slide-43
SLIDE 43

GABA docked

Signaling Module

Stochastic methods using Gillespie's Algorithm GABA release GABA inhibited GABA

synthesized

slide-44
SLIDE 44

Signaling module results

On activation of MOR

Counteractive impacts

On inhibit of GABA release On decrease of cAMP level

slide-45
SLIDE 45

Human practice Human practice

slide-46
SLIDE 46

medical workers

addicts

  • bject

Human Practice

Professor Jing: “I have to praise you for creativity and novelty of the project and for your brilliant job done in terms of difficulty of this project.” Patients: “I hate drug because it ruins my life. to withdraw heroin, I even turn to methamphetamine. No matter whatever methods, as long as it’s safe, I am willing to have a try. ” Doctor Geng: “Your project provides a brand new approach to dealing with treatment of drug addiction and has a potential for further application.” Policeman: “Even a new method is expensive, the government will still support. So the therapy will be totally free for the addicts.”

slide-47
SLIDE 47

Human practice

GuLou District XuanWu District QiXia District

slide-48
SLIDE 48

Human practice

GuLou District XuanWu District QiXia District

slide-49
SLIDE 49

Human practice

GuLou District XuanWu District QiXia District

slide-50
SLIDE 50

Potential drawback

Use of HEK293 as chassis causes ethical problems Therapy relies on patient’s

  • wn willingness and requires

surveillance Exosome production process is expensive and labor-intensive

Potential drawback

01 02 03

slide-51
SLIDE 51

Future work

Replace HEK293 chassis with patient’s own cells

slide-52
SLIDE 52

Future work

Integrate monitor and curer together and replace human surveillance at molecular level

slide-53
SLIDE 53

Acknowledgment

HELP：

slide-54
SLIDE 54

We will win the opioid war

slide-55
SLIDE 55

1. Alvarez-Erviti, L. et al. Nat. Biotechnol. 29, 341–345 (2011). 2. Kosaka N, Iguchi H, Yoshioka Y, Takeshita F, Matsuki Y, Ochiya T. Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells[J]. The Journal of Biological Chemistry. 2010,285(23):17442-52. 3. UN World Drug Report, 2014. United Nations Office on Drugs and Crime. 4. Smyth BP1, Barry J, Keenan E, Ducray K. Lapse and relapse following inpatient treatment of opiate dependence. Ir Med J. 2010 Jun;103(6):176-9. 5. Al-Hasani R, Bruchas MR. Molecular mechanisms of opioid receptor-dependent signaling and behavior. Anesthesiology. 2011 Dec;115(6):1363-81. 6. Matthes HW, Maldonado R, Simonin F et al. Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the µ-opioid-receptor gene. Nature383(6603),819–823 (1996). 7. Sora I, Takahashi N, Funada M et al. Opiate receptor knockout mice define µ receptor roles in endogenous nociceptive responses and morphine-induced analgesia. Proc. Natl Acad. Sci. USA94(4),1544–1549 (1997). 8. Joa ˜o Conde, Natalie Artzi, Are RNAi and miRNA therapeutics truly dead, Trends in Biotechnology 33(2015)141-144. 9. Stoorvogel W. Functional transfer of microRNA by exosomes[J]. Blood. 2012,119(3):646-8.

  • 10. Kaplitt MG, Leone P, Samulski RJ, Xiao X, Pfaff DW, O'Malley KL, et al. Long-term gene expression and phenotypic correction

using adeno-associated virus vectors in the mammalian brain[J]. Nature genetics. 1994,8(2):148-54.

References

slide-56
SLIDE 56

Poster &Media WIKI

Modeling

Human practice

Experiment

Poster &Media WIKI

Modeling

Human practice

Experiment

slide-57
SLIDE 57

Q&A