TERC overexpression using AAV9-CRISPR in Dyskeratosis Congenita

PALCAU ALINA

PELLEGRINI FLAMINIA

PERCIBALLI ELISA

SCALZITTI SILVIA

GENETICS AND MOLECULAR BIOLOGY

GENE THERAPY

PROF. ISABELLA SAGGIO

A.Y. 2018/2019

Adaptedfrom:Holohan Betal.JCellBiol.(2014)

TELOMERASE DEFECTS CAUSE TELOMEROPATHIES

• Telomerasemaintainstelomerelengthinproliferatingcells.

• Telomeraseisaribonucleoproteincomplex.

• Mutationsinoneofitscomponentsaffectitsactivity.

• Overactivationoftelomeraseisanhallmarkofcancer.

TERC HAPLOINSUFFICIENCY IN DYSKERATOSIS CONGENITA

Adaptedfrom:DuHYetal.AgingCell(2007)

TERC

TERCHAPLOINSU

FFICIENCY

W

T

3p

3p

3q

3q

CURRENT CLINICAL TRIALS

STUDYTITLE CONDITIONS INTERVENTIONS LOCATIONS LASTUPDATEPOSTED

InvestigationoftheGeneticsof

HematologicDiseases

• BoneMarrow FailureSyndromes

• Erythrocyte Disorder• Leukocyte Disorder

St.JudeChildren'sResearchHospitalMemphis,Tennessee,UnitedStates

October 23,2018

Treosulfan andFludarabine PhosphateBeforeDonorStemCellTransplantinTreating

PatientsWithNonmalignantInherited

Disorders

• Hematopoietic Cell• Transplantation

Recipient• Non-Malignant

• Procedure:AllogeneicBoneMarrowTransplantation

• Biological:Anti-ThymocyteGlobulin

• Drug:Cyclosporine

• Children's HospitalColoradoAurora,Colorado,UnitedStates

• OregonHealthandScienceUniversityPortland,Oregon,UnitedStateS

• Vanderbilt University

April 25,2018

Hematopoietic StemCell Transplant for

DyskeratosisCongenitaorSevereAplasticAnemia

• DyskeratosisCongenita

• Aplastic Anemia

• Drug:Alemtuzumab• Drug:Fludarabin• Drug:

Cyclophosphamide

UniversityofMinnesotaMedicalCenter,FairviewMinneapolis,Minnesota,UnitedStates

September 25,2018

Rescueoftelomeraseactivityusing

anovelgenetherapyapproach:

CRISPR/dSaCas9mediatedTERC

overexpression.

GOALS

• Increasetelomerelength• Increasetelomeraseactivity• Increasesurvival• Avoidtumorigenesis

FrancescaFioredda etal.July (2018)

Timesincetransplant(months)

Survival

EXPERIMENTAL PLAN

AAV9

HEK293T

HSC Mousemodel

Vectors building

Amplification

Preliminaryexperiments

Invitro

Invivo

VP64

RelA

RtaAD

ITR

ITR

PromoterSV40NLS

dSaCas9

ori

AmpR

Promoter

AAV9-dSaCas9-VPR AAV9-helper

ITR

ori

AmpR

PromoterRep

Cap

Promoter

VECTORS BUILDING

Modified from:https://www.addgene.org/68495/ Modified from:https://www.addgene.org/81070/

ITR

E1

AAV9-sgRNAà TERC

VECTORS BUILDING

ITR

ori

AmpR

PromotersgRNA

Promoter

ITR

AMPLIFICATION

AAV9-dSaCas9-VPR AAV9-helper AAV9-sgRNA

HEK293T

Purification inCsCl gradient

Viral genome titer:qRT-PCR

Selection withAmpicillin

AAV9-dSaCas9-VPR

AAV9-sgRNA

PRELIMINARY EXPERIMENTS: USE OF AAV9 SYEROTYPE AND SPECIFICITY OF dSaCas9

AAV9AAV1 AAV2 AAV5

ChIP-seq

peak

height

Genomic position(bp)

0

25

50

96x106

à ChIP-seqanalysis:thehighestpeakcorrespondstothemostfrequentbindingofdCas9,thatisTERCsequence(chromosome3).

à Analysisoffluorescence:AAV9producesahighGFPexpression,maintainedovertime.

Adaptedfrom:KevinS.Myersetal.Methods(2015)

PRELIMINARY EXPERIMENTS: CO-TRANSFECTION AND VECTOR EXPRESSION

ASSAYS

(dSaCas9)

(sgR

NA)

à FACS:cotransfected cells areinthetoprightsideofthegraph andthey are88,52%ofthetotal amount ofHSC.

TERC

foldchan

ge

2

5 AAV9-empty

AAV9-dSaCas9+AAV9-sgRNA

2weeksPI

à qRT-PCR:TERC expression inHSCis significantly increased afterAAV9-dSaCas9+AAV9-sgRNA

à Pulldownassay:exogenous TERCisable tointeract withTERT

AAV9-empty AAV9-mTERC

TERTTubulin

HSC

IN VITRO: TERC-TERT INTERACTION

Telomeraseactiv

ity

5

10AAV9-empty

AAV9-dSaCas9+AAV9-sgRNA

HSC

à TRAPassay: telomerase activityisincreased intreated cells

HSCTERC+/-

Adaptedfrom:ChristianB.etal.Bloodjournal (2018).

à FlowFish analysis:TERCoverexpression rescues telomere length.

HSCTERC+/- HSCAAV9-TERC

WTHSC

Telomeres

leng

th

IN VITRO: TELOMERE LENGTH ANALYSIS

BrodyH.etal.BMCGenomics(2016).

à qFISH:AAV9-dSaCas9+AAV9-sgRNAincrease telomere length,compaired totheempty vector.

IN VITRO: TELOMERE LENGTH ANALYSIS

AAV9-dSaCas9+AAV9-sgRNAAAV-empty

Adaptedfrom:ChristianB.etal.Bloodjournal (2018).

1234

AAV9-dSaCas9+AAV9-sgRNA

AAV9-empty

Telomerelength

Time(month)

IN VIVO: LOCALIZATION AND TRANSDUCTION

à Immunoistochemistry:theanalisys withAAV9-GFPreportershowedthehighesttransductionlevelinbonemiddleregions.

IHCanti-GFP

Bone

MarrowTibia

AAV9

-emptyA

AV9-GF

P

GFP-po

sitivecells

(%)

5

10

Jointadjacent

Bonemiddle

Intramidollar injection

MOUSETERC+/-

Adaptedfrom:Blasco etal.,Blood,2016

IN VIVO: TERC EXPRESSION AND TELOMERASE ACTIVITY

à qRT-PCR:TERC expression issignificantly increased intreated mice

TERC

foldchan

ge

350

200

1

2weeksPI 8weeksPI

Telomeraseactiv

ity

5

10

2weeksPI 8weeksPI

à TRAPassay: telomerase activityisincreased intreated mice

AAV9-dSaCas9+AAV9-sgRNA

AAV9-empty

IN VIVO: TERC OVEREXPRESSION INCREASES SURVIVAL WITHOUT INCREASING CANCER

à Survivalcurves:thetreatmentsignificantlyrescuesmousesurvival.

à Pathologicalanalysis:ofallmiceundertreatmentattheirtimeofdeath.Treatedmiceofbothagegroupsdidnotshowincreasedcancerincidencecomparedtothecontrols.

7 9

31 34

21 14

AAV9-emptyAAV9-vectorMiceaffected

(%)

AdenocarcinomaLymphomaAdenomaHistiocyticsarcoma

AAV-vector

AAV9-empty

Survival(%

)

DaysPI

Adaptedfrom “Blasco etal.,Blood,2016.

AAV9-dSaCas9+AAV9-sgRNA

AAV9-empty

IN VIVO: HUMAN ENDPOINT BLOOD COUNTS

à Count:AAV9treatmentimprovesblood counts inmice

Erythrocytes

(101

2 /L)

5

10AAV9-empty

AAV9-dSaCas9+AAV9-sgRNA

Haem

oglobin(g/dL)

5

15

10

Platelets(10

9 /L)

400

1200

800

Leukocytes

(109/L)

2

6

4

PITFALLS AND SOLUTIONS

Conditionalgenomeeditingwithlight-inducibleorchemical-inducible

OFFTARGETSEFFECTS

GuideRNAtargetwith2Gat 5’

Codelivery ofDNAdecoy

AAV-9IMMUNOGENICITY

Emptycapsidmutantscanbeusedasdecoystoovercomepre-existinghumoralimmunitybyadsorbingantibodiesinthebloodstreamuponsystemicdeliveryofbothemptyandfunctionalAAVs1

TheAAVcapsidscanbegeneticallyengineeredormutatedtoreducethebindingaffinityandtheneutralizingeffectsofAAVantibodies

IMMUNERESPONSEDUETOPROLONGEDEXPRESSIONOFCAS9

MATERIAL AND COSTSMATERIAL COST

293(E1)CELLS(CellBiolab) 350$(every 10alla6cells)

AAV-GFPCONTROLVECTOR(CellBiolabs) 395$(every 10μg+deliverycosts)

AAVtrasduction kit50reactions (Antiboies-online.com) 1005$

AAV-CMV-Null Titer :1x1013GC/ml- (Vector Biolabs) 5662$

Stemline hematopoietic stem cell expansion medium(SigmaAldrich) 268$

FiSH TagDNAmulticolor kit,Alexa Fluor dye combination(Thermo Fisher) 752$

RT-PCR,WesternBlot,IF,IP,Biochemical assays (e.g.Abcam Ab200390$every 100μl,Ab66601380$every 100μl)

PCRpurification +sequencing (Biofab research) 15.11$(persample)

Plastic 1500$(peryear)

Stabulation 800$(permonth)

TRAPkit 300$

(Weexcludedinstrumentsandmaterialsthatcanbepossiblycollectedthankstocollaborationwithmedicaldepartment)

TOTALCOST:6821,11$

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