Systemic administration of exosomal siRNA inhibited huntingtinexpression in transgenic mice of Huntington's disease

PEI, Zhong, MD & PhD Department of NeurologyThe First Affiliated Hospital Sun Yat-Sen UniversityGuangzhou, China

Peter Como HD Research Symposium at HSG 2019: Navigating HD

Sacramento, CANov 7, 2019

Outline

p HD in China

Chinese Huntington’s Disease Network (CHDN)

p Exosomal siRNA-mediated inhibition of HTT

2009-20103 exploratory workshops

2011Official Launch

2013 Established 11 Centers in China

2014Published First Collaborative Article

ShenyangBeijing(2)

SuzhouShanghaiHangzhouFuzhou

Guangzhou

ChangshaChengdu Wuhan

Urumqi Lanzhou

Nanning

ShenyangBeijing(2)

SuzhouShanghaiHangzhouFuzhou

Guangzhou

ChangshaChengdu Wuhan

Urumqi Lanzhou

Nanning

Committed CentersPotential Centers

2011 Launch Meeting

Prof. Jean-marc Burgunder

Huntingtin gene CAG repeat numbers in Chinese patients with Huntington’s disease and controls

Chinese Huntington’s Disease network (CHDN)

Year年份

Pedigree家系

Patient Total确诊患者

2013 457 660

2014 497 733

2015 575 896

2016 634 955

2019 891 1454

The number of patients registered in CHDNCHDN中心收录患者总人数统计

Category Patient Total确诊患者

Persons at risk (blood relative)潜在患者（有血缘关系的亲属）

No. Approx.4000~7000

Approx.15000~30000

Estimated HD Patients in China中国HD患者预估人数

*Estimation based on prevalence rate in published articles from Hongkong and Taiwan (2~4/106), actual number may be higher

Steering Committee• Call for PI Meeting• Decision making• Drawing up project proposal

Central Coordination• Information gathering and distribution

• Communication central• Meeting and training organization

• Project coordination

Local CentersDaily patient care

• Conduct individual and collaborative research

Patient Advocacy• Bridge between

HD patients and researchers

• Promoting awareness of HD

Patients & Families

Health Professionals

Honorary Members• Advisory to SC• Esteemed Researches

in the field

CHDN: Administration and Management

Multi-center clinical observation study

• General Clinical Forms

Stroop Test, Symbol Digit Modality Test, Trail-making Test MMSE, Verbal Fluency Test

UHDRS: Total Function Capacity, Functional Assessment ScaleIndependence Scale

UHDRS Behavior, PBA-s, BDI II, HAD, SIS

History (medical, disease, psychiatric, family)Scocio-demographic (Fixed & Variable)Pseudonymisation, Informed ConsentCurrent TherapiesHDCC (On-set assessment)General neurological assessments (for normal control)

• Functional

• Behavioral

• Cognitive

CHDN Clinical Activity

CHDN Physician Training

No. of people累积培训人数

Year年份

Local Training中心培训

Training Session

CHDN培训

PBA training by EHDN

EHDN培训

Chinese-PBA training

PBAs中文培训

EHDN assoc.member

EHDN附属会员2010-2012

15~20 each time - - - 3

2013

-

30 - - 11

2014 40 2 6 44

2015 50 2 18 51

2016 57 2 25 53

2017 65 2 33 53

CHDN Clinical Activity

Training & Certification

On-site Training

Certification

2012 Local Training in Guangzhou Center

EHDNCHDN

Exchange fellowship

CertificationTwo way academic exchange

Ensure only qualified physicians and researchers can participate

CHDN Activity

Training & CertificationCHDN Clinical Activity

Annual PI meetings

Guangzhou广州

Hangzhou杭州

Chengdu成都

Beijing北京

Fuzhou福州

CHDN Clinical Activity

Chinese HD Association

President :Xi Cao

Contact us:

cao@hdcare.org.cn

Provide Support Network

for Patients and Families

Advocate for Better Policies for HD Community

Vice President

international huntington association

Since 2013

CHDN Activity

Survey on HD Patients' Living Conditions in China

1. Lack of HD knowledge and disease management

37.25% of the patients never received any treatment for HD.Only 1.96% of the patients had surgical treatment for HD and 0.98% had physiotherapyDBS procedure is not covered by local medical insurance inmost cities

2. Inadequate medical insurance system

17.96% of the patients in the survey don't have any medical insuranceHD is not covered by any commercial insurance plans Many HD families have more than one HD patient, resulting in decreasing family income, a lot of families have to live on social benefits, which barely cover their expenses.

Chinese HD Association CHDN Activity

CHDN Translational Research Collaboration

IPS Cells

Animals

Isolating epithelial cells

Reprogramming the cells into UiPSCs

Patient-specific cell model

Getting urine from patients

PotentialTreatment

Drug Testing

Understand pathology

Guangzhou center in collaboration with South China Institute for Stem Cell Biology and Regenerative Medicine

HD Transgenic Pig HD Knock-In Pig(Unpublished)

PIG

MICE Novel Full-length Htt Mouse ModelBy Prof. Li Chenjian’s team at Beijing University

By Prof. Li Xiaojiang & Prof. Li Shihua’s team& Prof. Lai Liangxue’s team at Chinese Academy of Science

Potential Imaging

Studies

Future Collaborations

test IPS treatment etc.

CHDN Activity

Preclinical model: Huntingtin knockin pig (Cell 173, 1-14, May 3, 2018)Li Xiao-Jiang Lai Liangxue

CHDN Translational Research Collaboration

Exosomal siRNA-mediated inhibition of HTT in vivo

Chen Xi Department of Life sciences Nanjing University

PEI, ZhongDepartment of Neurology The First Affiliated Hospital Sun Yet-Sen University

CHDN Translational Research Collaboration

Wild EJ et al, Lancet Neurol, 2017

siRNA and Huntingtin-lowering therapeutics

Systemic siRNA delivery and tissue targeting

Hartmann G, et al Nature Biotechnology 2011

Barriers:q Unfavorable chemical properties

Negative charges etc

q Instability

q Plasma half-lives (10 min)

Big challenges for siRNA therapeutics

p Exosomes:

ü Extracellular membrane vesicles (30-150 nm).ü Transmission of macromolecules between cells.

Advantages as drug vehicles:ü Small sizeü Low immunogenicityü Long half-life in the circulation ü Ability to load various cargoes ü Easiness to cross Brain-blood barrier

Exosomes: a novel biocarrier for siRNA Delivery to the brain

Zhang M. et al, J. Mater. Chem. 2019

Efficient scalable production of clinical-grade exosomes

Brain specific delivery

Bottleneck 1 Bottleneck 2

Challenges for exosome-based therapeutics in CNS diseases

Liver: the endogenous exosome factory for siRNA

q siRNA accumulated in liver following I.V. injection of plasmids

q The liver is a primary site of exosome uptake

Chen XNanjing University

Liu et al in preparation

Large production of exosomes

Self-assembly exosomal siRNA delivery system

RVG-Lamp2b modified exosomes: Brain specific delivery

q RVG (rabies virus glycoprotein) CNS-specific uptake of exosomes

q Lamb2b (lysosomal associated membrane protein-2b)Link RVG to the external exosomal surface

RVG

RVGLamb2b

Brain cells

RVG/LAMP2b significantly increased the delivery of siRNA to the brain

b rain

live r

s p lee n

lun g

k idn e y

02 0 04 0 06 0 08 0 0

1 0 0 03 0 0 0

3 5 0 0

4 0 0 0

siR

NA

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l(c

op

y/c

ell

)

P B SS c ra m b les iR N AR V G -s iR N A

UD

UD

UD

UD

UD

UD

UD

UD

UD

UD

UD

siRNAScramble RVG-siRNA

Liver

Brain

Lung

In situ hybridization

Chen XNanjing University

DAPI

eGFP

RVG-siRNA

Liver Brain

siRNAScramble RVG-siRNAScramble siRNA

Cre eGFP reporter mouse

Exosomal siRNA2 and 5 exhibited potent inhibition of huntingtin expression in SHSY5Y cells and BACHD mice

SHSY5Y cells

Intravenous administration of exosomal siRNA for two weeks reduced huntingtin expression in N171-82Q mice

YAC 1286weeks

Plasmid injection10mg/kg iv. Twice a week.

RotarodOpen Field

1week

Exosomal siRNA for 8 weeks significantly suppressed huntingtin expression in both cortex and striatum in YAC128 mice

YAC 1286weeks

Plasmid injection10mg/kg iv. Twice a week.

RotarodOpen Field

1week

Intravenous administration of exosomal siRNA for 8 weeks significantly improved motor function in YAC128 mice

Exosomal siRNA reduced HTT aggregates in YAC128 mice

Normal levels of Liver enzymes

Preliminary data: Systemic exosomal siRNA did not induced toxicity in mice

Normal morphology of major organs

Summary

• Systemic exosomal siRNA lowers HTT and improves disease-phenotypes.

• Great potential for an affordable, non-invasive solution for HTT lowering therapy.

AcknowledgmentsHD clinic

Chen DBYou HQWu QLiang XL

Exosomal siRNAChen X’s lab Liu JYu MJNanjing University

Pei’ labWu TT Chinese Huntington’s Disease Network

Jean-Marc BurgunderShang HuiFangCHDI foundationEHDN

Christopher A. RossWanli W. SmithJohns Hopkins University

Thanks