ESGCT XXV Anniversary Congress in Collaboration with the German Society for Gene Therapy

125 Pages·2017·1.32 MB·English

ESGCT XXV Anniversary Congress
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HUMANGENETHERAPY28:A2–A125(2017)
ªMaryAnnLiebert,Inc.
DOI:10.1089/hum.2017.29055.abstracts
Selected Oral Presentations
OR01 Institute, Woolloongabba, Queensland 4102, Australia.
3: Leibniz Research Laboratories for Biotechnology and
Generation of three-dimensional human artificial skeletal
Artificial Organs (LEBAO), Department of Cardiac, Thoracic,
muscle tissue from iPS cells enables complex disease
Transplantation, and Vascular Surgery; Hannover Medical
modelling for muscular dystrophy
School,D-30625Hannover,Germany 4:DepartmentofHuman
S M Maffioletti1 S Sarcar1 A Henderson1 I Mannhardt2 DNA Variability, GENYO, PTS Granada, 18016 Granada,
L Pinton1 L A Moyle1 H Steele-Stallard1 O Cappellari4 Spain. 5: Max Delbru¨ck Center for Molecular Medicine,
K E Wells4 M Ragazzi1 W Wang1 P Zammit3 D J Wells4 D-13125 Berlin, Germany 6: Fachbereich Mathematik,
T Eschenhagen2 F S Tedesco1 Naturwissenschaften & Informatik, Technische Hochschule
Mittelhessen, D-35390 Gießen, Germany.
1: Department of Cell andDevelopmental Biology,University
y. College London,WC1E 6DELondon,UK 2:Department of Human induced pluripotent stem cells (hiPSCs) hold substan-
nl ExperimentalPharmacologyandToxicology,UniversityMedical
o tialpromiseforregenerativemedicine.However,reprogramming
use CenterHamburgEppendorf(UKE),20246HamburgandDZHK and subsequent hiPSC cultivation can result in genetic and epi-
nal (GermanCentrefor CardiovascularResearch), partner site geneticabnormalities,andmutationsthatcanunderminetheiruse
o
ers Hamburg/Kiel/Lu¨beck,Germany3:RandallDivisionofCelland in regenerative medicine, because they compromise biosafety of
or p MolecularBiophysics,King’sCollegeLondon,SE11ULLondon, hiPSC-derived differentiated cells. Activation of endogenous
7. F UK 4: Department of Comparative Biomedical Sciences,Royal mobile retrotransposons LINE-1 (Long Interspersed Element-1,
1/1 Veterinary College, NW10TU London,UK L1), Alu and SVA can cause such mutations. In differentiated
1
12/ cells, L1issuppressedbymethylationofitsCpG-richpromoter.
m at Generating artificial human skeletal muscle would be instru- We show that reprogramming triggers transcription of L1 ele-
co mental for investigating skeletal muscle pathology and therapy. ments via demethylation and that reprogramming factors further
ub. However, current bioengineering platforms are challenged by the upregulate L1 promoter activity. By applying retrotransposon
p
ert limited expansion potential and differentiation ability of tissue- capture-sequencing to 8 hiPSC lines and their parental cells, we
online.lieb addniemdriveinendsdioumncyeadolgapretlniufiirccipicaoeltleslsnk.teHlesettearmel,mwcueeslcldslee,sticinsrcsibluueedtfihnroegmgceehnlulesmraaftrinoomnemopbfartythioerneneitcs- fgnoeounvnoodmrtiehctariotntsLrea1rntsitopranonsss:ictWiroinpetiieodvneeannltitafiscettdhivanatutimoocnecrucoraurusesdLes1d,nuAeriwlnugLan1red-pmSroeVdgAiraatmdede-
om with Duchenne, limb-girdle and congenital muscular dystrophies. ming and hiPSC cultivation. We estimated *1 L1 de novo in-
97 fr Skeletal myogenic differentiation of pluripotent cells was induced sertion per hiPSC, constituting a mutagenic load per cell as
9.1 within hydrogels under tension to provide alignment. Artificial represented by exogenously applied integrating gene therapy
2.11 musclesrecapitulatedcharacteristicsofhumanskeletalmuscletis- vectors. *50% of all new retrotransposition events occurred in
86. sue and could be implanted into immunodeficient mice. Im- transcribed protein-coding genes, including genes involved in
ded by poofrtsaonmtley,ftohremysenoafblmedusincuvliatrrodmysotdroepllhinygwofitphathhioglhoegricfiadlehlaitlylmtahrakns oinntceorfgeernenescies,odfevneelwopmL1enitnoserrstiigonnaslwtraitnhsdhuocsttiogne.nWeeexdpermesosniostnraitne
a
nlo standard bi-dimensional cultures. Finally, we show generation of hiPSCs. Consistently, analysis of 1800 marked L1 insertions re-
ow complex multicellularconstructs, allhumaniPScell-derived, con- sulting from mobilization of genetically engineered L1 reporter
D
tainingisogeniccelltypespresentinnormalskeletalmuscle,such elements during hiPSC cultivation, confirmed that *50% of all
as vascular endothelial cells and pericytes. These results lay the insertions accumulate in host genes. L1 de novo insertions were
foundationforahumanskeletalmuscleorganoid-likeplatformfor
alsoenrichedatactivetranscriptionalstartsiteswheretheymight
regenerativemedicine,diseasemodellingandtherapydevelopment.
affecthostgeneexpression.Ourfindingsimplyconsequencesfor
thebiological safety ofhiPSC-derived cell therapies.
OR02
Reprogramming triggers mobilisation of endogenous
OR03
retrotransposons in human induced pluripotent stem cells
with genotoxic effects on host gene expression Dynamic remodelling of neural cellular and extracellular
signatures depicted in 3D in vitro differentiation
S Jung-Klawitter1 N V Fuchs1 5 K R Upton2 A Fro¨mmrich3
of human iPSC-derived NSC
C Miskey1 M Mun˜oz-Lopez4 R Shukla2 J Wang5 A Sebe1
S Merkert3 A Bock1 U Held1 M Menzel6 A Gogol-Do¨ring6 D Sima˜o1 A P Terrasso1 M M Silva1 F Arez1 M F Sousa1
A Haase3 Z Izsva´k5 Z Ivics1 U Martin3 J Garcia-Perez4 NRaimundo2PGomes-Alves1EJKremer3PMAlves1CBrito1
G J Faulkner2 G G Schumann1
1: iBET, Instituto de Biologia Experimental e Tecnolo´gica,
1: Division of Medical Biotechnology, Paul-Ehrlich-Institute, Oeiras, Portugal and Instituto de Tecnologia Qu´ımica e
D-63225 Langen, Germany. 2: Mater Medical Research Biolo´gicaAnto´nioXavier,UniversidadeNovadeLisboa,Oeiras,
A2
SELECTED ORAL PRESENTATIONS A3
Portugal 2: Universita¨tsmedizin Go¨ttingen, Institut fu¨r photoreceptor genesis. Immunohistological examination of the
Zellbiochemie, Go¨ttingen, Germany 3: Institut de Ge´ne´tique organoids at different culture time points revealed that the or-
Mole´culaire de Montpellier, CNRS UMR 5535, Montpellier, ganoids recapitulate the different stages of retinogenesis and
France and Universite´ de Montpellier, Montpellier, France confirmed the production of a large number of photorecep-
tors.Thiscelllinecannowservetostudyhumanphotoreceptor
Brain microenvironment plays an important role in devel- development and pathologies as well as to produce a large
opment and function. Disruption of its homeostasis is often photoreceptor number for drug screening and transplantation
related to pathological conditions, as the lysosomal storage studies.
disease mucopolysaccharidosis type VII (MPS VII), caused
by deficient b-glucuronidase activity. We hypothesized that
3D differentiation of human neural stem cells (hNSC) neu-
rospheres in perfusion stirred-tank bioreactors could sustain
microenvironment remodeling, recapitulating key cell-ECM
OR05
interactions. Differentiation of hNSC derived from induced
pluripotent stem cells (hiPSC-NSC), both from healthy do- AAV-mediated CYP46A1 gene therapy
nors and a MPS VII patient, were shown to recapitulate for Huntington’s disease
neurogenic developmental pathways, generating tissue-like
N Cartier1 S Alves2 W Christaller3 E Subashi1 R Kacher4
3D structures with neuronal, astroglial and oligodendroglial
A Lamazie`re5 G Despres5 F Saudou3 J Caboche4 S Betuing4
cells. Changes in neural microenvironment during differen-
nly. tiation,namelyatcellmembraneandECMcomposition,were 1: INSERM/CEA UMR1169, MIRCEN CEA and Universite’
o
use addressed using quantitative transcriptomic (NGS) and pro- Paris-Sud, Universite’ Paris Saclay, 91400 Orsay, France
al teomic data (SWATH-MS). Data revealed a significant enrich- 2: BrainVectis Therapeutics, 18 route du Panorama 92 265
n
2/11/17. For perso mbmIlanrteeeemnMvdti,bcPiraaSnansnVsatehtnrIecduIocatcnteceusncltraluiastsmul,ceuipinnmlrtaosCpttioe,(oeorant.glagolon.yn,ftcglgaadwnlmiyssiecit,nhaoisssndueasocm,whhciannolarlolsmelgaglanguyreelckaunastrsnioowsacn.naeGondref,lfiibavrbaelerscrideaslilmipficnfieaetsunnr)--t,. FUGRPoeanINgnriviut,seel-3narSs8ateii0yotiyn-0nae0,Gu,NGxrSe-eorRnureoobrnbosooelsnbescnlieAeCe,nleUpFcdeneressaix,vn,PeGcrFaersrreiia4tnsen:o’ScbsNe,eleeiUn3uIPe:rno,MIsnNItinCatSsulEtUtiSRtdinugMeitnveseaNUorlisfe1niuB2tger1i’ooa6sPlncoadiigeneyrGdnrceeeneset,
at 1 entiation was increased and alterations in neuronal activity and MarieCurie-Paris6,INSERM/UMR-S1130,CNRS/UMR8246,
m connectivity were observed. In summary, we demonstrated that 75005 Paris, France 5: Laboratory of Mass Spectrometry,
o
b.c neural cellular and extracellular developmental features are re- INSERM ERL 1157, CNRS UMR 7203 LBM, Sorbonne
ebertpu ccaapnitbuelatveadluianblheiPinSCv-itNroSCm-doedreivlsedtonaedudrarelssmimcroolteicssuulaers.dTefheecstes USaniinvte-rAsnitteo’isn-e,U7n5iv0e1r2siPtea’rPisi,eFrrreanecteMarie Curie-Paris 6, CHU
e.li associated with neurological disorders that affect the microen-
n
nli vironment homeostasis, as MPS VII. Braincholesterolhomeostasisdefectsintheadultbrainare
o
m linked to neurodegenerative diseases, such as Niemann-Pick
o
7 fr C, Alzheimer and Huntington’s diseases (HD). In HD, cho-
9
9.1 lesterolhomeostasisdefectsinvolveageneralperturbationin
2.11 OR04 the expression of cholesterol biosynthesis enzymes. 24S-
6. hydroxycholesterol (24OH-Chol), the catabolite of choles-
8
by Endogenous CRX activity monitoring by CRISPR/Cas9 terolmetabolism,isdecreasedinHDpatientsplasma.Levels
ed engineering of human iPS cells allows rapid detection of CYP46A1, the rate-limiting enzyme, which catalyses the
d
oa of photoreceptor genesis production of 24OH-Chol in neuronal cells, are decreased in
nl
ow Y Arsenijevic1 R Moser1 S Decembrini1 D Gamm2 the striatum of HD patients and HD mice models. CYP46A1
D
V Ponce de Leone1 plays major roles in activating brain cholesterol turnover
and thus increasing the mevalonate pathway, with benefi-
1: Universite´ de Lausanne 2: University of Winsconsin cial effects on synaptic plasticity and function. Restoring
CYP46A1expressioninvivobyadeno-virus-mediated(AAV-
Retinal degenerative diseases resulting in the loss of photo- CYP46A1)deliveryinthestriatumoftwoHDmousemodels
receptorsareoneofthemajorcausesofblindness.Photoreceptor (R6/2andZQ175)resultsinsignificantimprovementinmotor
replacement therapy is still in development to demonstrate the behaviour associated with decreased huntingtin-positive ag-
proof of concept of this approach, necessitating thus a large gregates, increased neuroprotection and synaptic plasticity.
amount of retinal cells to conduct such studies. In parallel, the Furthermore, cholesterol biosynthesis pathway is restored in
generation of retina organoids is a promising unlimited cell the targeted brain regions, leading to a normalization of
source. We and others have demonstrated the possibility to cholesterol, desmosterol, lanosteroland 24OH-Chollevelsin
produce a large quantity of mouse photoreceptors and to iso- these two HD mouse models. In addition, we show that
late them with cell specific reporter genes. To generate a large CYP46A1 overexpression corrects the BDNF/TrkB pathway
amount of human photoreceptors and to trace them, we used a that is dramatically impaired in HD, as well as, vesicular
geneeditingapproachtoinsertacomete-GFPattheUTRsiteof transport. Towards clinical evaluation in patients with HD,
the CRX gene which is specifically expressed in the photore- dose-responsive studies in mice and translational steps in
ceptors.ThehCas9_D10Awaschosentoreduceoff-targetsand non-human primates were performed, demonstrating the
gRNAs were screened for their efficacy and specificity in 293T feasibility and efficacy of AAV-CYP46A1 gene delivery in
cells, and then used in a human iPSC line. In these cells, three the striatum. We propose a phase I/II clinical application to
out of 26 clones showed correct integration of the transgene. evaluate the efficacy and safety of a single administration of
Differentprotocolsweretestedforretinaorganoidinductionand AAV-CYP46A1 in the striatum of HD patients at an early
theappearanceoffluorescencehelpedtodeterminetheperiodof stage of disease progression.
A4 SELECTED ORAL PRESENTATIONS
OR06 sured by multielectrode array (MEA) and patch clamp tech-
niques, demonstrated sustainable 6 months expression of
Gradual improvements in the motor and cognitive function
GS030-DP and its ability to convert RGCs into photo-
after gene therapy for patients with AADC deficiency
activatablecellsinnormalmacaqueretina,andallowedactive
K Kojima1 A Miyauchi1 T Nakajima1 N Taga1 S A Ono1 dose-range determination. A complete GLP safety and toler-
HMizukami1MKato3HOsaka1SMuramatsu12TYamagata1 ability program, including toxicity, biodistribution, and im-
munologicalassessmentinnormalmacaques,indicatedagood
1: Jichi Medical University 2: Tokyo University 3: Showa
tolerance profile of GS030-DP in preliminary interim results.
University
Taken together, these preclinical data allowed to frame the
dose-rangetoevaluateintheplannedphaseI/IIclinicaltrial.A
Patientswitharomaticl-aminoaciddecarboxylase(AADC) visualinterfaceconsistingofstimulatingglasses(GS030-MD)
cannot produce catecholamines and serotonin in the brain.
necessary to complement GS030-DP was developed. These
They exhibit oculogyric crises, dystonia, impaired voluntary
stimulating glasses deliver tailored light amplified visual
movements and intellectual disability. We report the clinical
stimuli onto the genetically engineered retina at intensities
courseoffourseverepatientswithAADCdeficiencyandone
abovenaturallightingconditions.Wethereforeassessedinthe
milder patient whose cognitive and motor function improved
rd1 mouse model the safety of GS030-DP upon light stimu-
markedly after gene therapy. Patients and Methods: Three
lation in conditions mimicking those of the stimulating glas-
male (10, 15 and 19 years) and 1 female (12 years) patient
ses. The results allowed to determine the safe lighting
with severe phenotypes were bedridden with no voluntary
stimulation conditions to be evaluated in the clinical trial.
nly. movements nor speech. One female patient (5 years) could Updatednon-clinicaldataandoverviewofthemedicaldevice
e o walk with support and speak several words after MAO-B supportingthefirst-in-humanstudyofGS030combinationwill
al us inhibitor treatment. They received 2·1011 vector genomes be presented.
n of AAV2 vector harboring AADC gene via bilateral in-
o
ers traputaminal infusions. Results: By two years after gene
p
or therapy, all patients showed an improved motor function and
F
7. no dystonia. Two severe patients walked with a walker, and
1
1/ the milder patient could run and ride a bicycle. Regarding OR08
1
m at 12/ m12e-nyteaalr-doelvdelfoepmmaelentr,etshpeonmdielddeqrupiactkileynttocosuplodkecnonovredresres..TPhae- Lcoognngi-ttievremdeecxlpinreesisnioangeodf saencirmetaelds-klotho protects against
co tients only exhibited transient chorea as adverse events. Po-
ub. sitron emission tomography with 6-[18F]fluoro-l-m-tyrosine, A Masso´2 A Sa´nchez2 A Bosch2 3 R Blanch1 J F Espinosa1
p
ert a tracer of AADC, showed persistent AADC expression. L Gime´nez-Llort2 M Chillo´n1 2 4
b
e.lie Discussion:Sincetheputamenisthemainoutputstructureof 1: Vall d’Hebron Research Institute (VHIR) 2: Universitat
nlin the motor network in the basal ganglia, improved motor Auto`noma de Barcelona 3: CIBERNED 4: ICREA
m o performance and amelioration of dystonia can be expected
o after putaminal AADC gene delivery. The motor, cognitive
97 fr and speech functions remarkably improved in the milder pa- BACKGROUND: Klotho is a gene regulator of aging, in-
9.1 tient. The restoration of dopamine production in the putamen creasinglifeexpectancywhenoverexpressedandaccelerating
2.11 may have broader effects depending on the patient age at the development of aging phenotypes, including cognitive
86. treatment and genetic severity. deficits, when inhibited. We have previously demonstrated
by that: (a) normal brain aging is associated with a significant
ed downregulation of Klotho expression, and (b) klotho expres-
d
oa sion in the CNS was negatively influenced by Alzheimer’s
nl
w disease, but positively influenced by healthy lifestyle, since
o
D OR07 moderate continuous exercise in adulthood prevents the de-
cline in expression of the klotho transcripts. It has been re-
Efficacy and safety of ocular AAV mediated optogenetic ported that Klotho is neuroprotective to both neurons and
therapy for retinitis pigmentosa in rd1 mice oligodendrocytes. Thus, identifying ways to increase Klotho
and non-human primates support the first-in-human expression may prove to be a cognition enhancer in normal
clinical trial of GS030 aging. Here we study, the functional relevance at behavioural
A Douar1 C Bouquet1 D Pruneau1 N Thomasson1 J Chavas1 level,ofmodifyingthelevelsofthesecretedKlothoisoformin
FGalluppi1DDalkara2RBesnosman2GChenegros2SPicaud2 the aging brain. METHODS: We used AAVrh10 vectors to
J Sahel2 deliver and sustain expression of Klotho in the CNS of adult
and middle-aged wild type C57BL/6J males, to determine
1: GenSight Biologics, Paris, France 2: Vision Institute, Paris, effects of Klotho overexpression on cognitive performance.
France RESULTS: This study demonstrates for the first time in vivo,
that 6 months after a single injection of an AAV vector ex-
We have shown previously that visual restoration through pressing the Klotho gene into the CNS, long-lasting and
retinal ganglion cells (RGCs) engineered to express the algae quantifiableenhancementoflearningandmemorycapabilities
red-shifted modified opsin ChrimsonR-tdTomato using an are found. More importantly, cognitive improvement is also
AAV2.7m8 vector (GS030-DP) was an effective strategy in observablein18-months-oldmicetreatedonce,atmiddle-age.
rd1miceandnon-humanprimates.Atranslationalprogramto CONCLUSIONS: Long-term expression of AAV-Klotho
support GS030 first-in-human evaluation in patients with ret- vectors specifically in the CNS enhances cognitive perfor-
initispigmentosa(RP)wasconductedtoconfirmsustainability mance in aged naive animals. These findings demonstrate the
of opsin expression and safety of GS030-DP, as well as to therapeutic potential of Klotho as a treatment for cognitive
define the starting clinical doses. Pharmacodynamics, mea- decline associated with aging.
SELECTED ORAL PRESENTATIONS A5
OR09 cytometry,andfoundthatitwasexpressedonthesurfaceof75%
of the analyzed cell lines. By inoculating the cells with rMV-
Oncolyticmeaslesvirusesastherapeuticvectorsfortargeted
SLAMblind expressing EGFP, rMV-SLAMblind infected the
BiTE expression in solid tumours
nectin-4-expressing TNBC cell lines, and showed cytotoxicity
T Speck1 J Heidbu¨chel1 R Veinalde1 D Ja¨ger2 C von Kalle1 in vitro. Furthermore, we examined anti-tumor effect of rMV-
C R Ball1 G Ungerechts1 2 3 C E Engeland1 2 SLAMblind in vivo by using mouse xenograft models. In-
tratumoral injection of the virus suppressed tumor growth. To
1: Department of Translational Oncology (G100), German
further assess the effectiveness of rMV-SLAMblind treatment
Cancer Research Center (DKFZ) and National Centre for
for metastatic cancer, we administered rMV-SLAMblind ex-
Tumor Diseases (NCT), Heidelberg, Germany 2: Department
pressing luciferase via intravenous route. In vivo imaging indi-
of Medical Oncology, National Centre for Tumor Diseases
cated that the virus replicated selectively in the tumor. In
(NCT) and Heidelberg University Hospital, Heidelberg,
addition, the tumor growth was significantly suppressed. These
Germany 3: Centre for Innovative Cancer Research, Ottawa
results suggest that rMV-SLAMblind is a promising candidate
Hospital Research Institute, Ottawa, Ontario, Canada
as a therapeutic agent for metastatic TNBC. We are advancing
translational research of rMV-SLAMblind in Japan.
BispecificTcellengagers(BiTEs)comprisetwosingle-chain
variable fragments translated in tandem, one directed against
CD3 on T cells and the other against a tumor antigen. Thus,
BiTEs crosslink T cells to tumor cells and thereby mediate OR11
y. tumor-specificTcellcytotoxicityirrespectiveofTcellreceptor
nl Efficacy, safety, and covariates of outcomes with
use o shpaescibfieceintydaenmdoannsttirgaetendpfroesrentrteaatitomne.nTthoefirBthecrealplemutaicligpnoatnecniteiasl. axicabtagene ciloleucel (axi-cel; KTE-C19) from ZUMA-1,
al a pivotal trial in patients with refractory, aggressive non-
n However, treatment of solid tumors remains challenging. On-
erso colyticmeaslesvirusvectorsareaversatileplatformforsafeand Hodgkin lymphoma (NHL)
p
or targeted immunomodulation. We aimed to limit toxicity and Y Lin1 F L Locke2 S S Neelapu3 N L Bartlett4 L J Lekakis5
F
7. increase efficacy of BiTEs against solid tumors by targeted ex- D Miklos6 C A Jacobson7 I Braunschweig8 O Oluwole9
1
1/ pression. We successfully generated oncolytic measles viruses T Siddiqi10 J Timmerman11 P M Reagan12 A Bot13 J Rossi13
1
12/ (MV) encoding bispecific T cell engagers (MV-BiTEs) to en- L Navale13 Y Jiang13 J S Aycock13 M Elias13 J Wiezorek13
m at hance cellular immunity in cancer virotherapy. MV-BiTE in- W Y Go13
co fectedcellsexpresssecretableBiTEswhichspecificallybindthe
ub. relevanttargetantigensandmediateTcellcytotoxicityinvitro. 1:MayoClinic2:MoffittCancerCenterandResearchInstitute
ertp Viral replication and oncolytic activity are not impaired by the 3: MD Anderson Cancer Center 4: Washington University in
e.lieb additional transgene, compared to the parental vector. Ther- S7t:.DLaonuais-F5a:rUbenrivCerasnitcyerofInMstiiatumtei 68::SMtaonnftoerfidorUenMiveedrsicitayl Center
nlin apeutic efficacy of MV-BiTE was demonstrated in unique xe- 9: Vanderbilt University Medical Center 10: City of Hope
m o nograft models of colorectal carcinoma with CEA-expressing 11:UniversityofCaliforniaLosAngeles(UCLA)12:University
o human primary tumor spheres and the transfer of human
97 fr PBMCs. MV-BiTE with the transfer of PBMCs significantly of Rochester School of Medicine 13: Kite Pharma
1
9. prolonged survival, compared to survival of MV-BiTE only or
2.11 PBMCs only treated control mice. Conclusively, targeted ex- Outcomes for patients with refractory, aggressive NHL are
86. pressionofBiTEsbyoncolyticMVenhancesantitumorefficacy. poorwithcurrentlyavailabletherapies.Axi-cel,ananti-CD19
ded by Taghaiisnssttusdoylidpcroavnicdeerss.proof of concept for MV-BiTE efficacy cinhitmheisricpoapnutilgaetinonrecinepZtoUrM(CAA-R1.)TElcigeilblltehepraaptiye,nwtsas(‡e1v8alyu)atreed-
oa ceivedatargetdoseof2·106anti-CD19CARTcells/kgafter
nl
w low-dose conditioning chemotherapy. The primary endpoint
o
D
wasORR.Keysecondaryendpointsweredurationofresponse
OR10 (DOR),overallsurvival(OS),andadverseevents(AEs).Asof
January 27, 2017, 111 patients were enrolled; 101 (91%) re-
An oncolytic recombinant measles virus is a candidate of a ceivedaxi-cel.Medianagewas58years,77%wererefractory
novel therapeutic agent for triple negative breast cancer to ‡second-line therapy, and 21% relapsed £12 months of
T Fujiyuki1 Y Amagai1 K Shoji1 A Sugai1 M Awano1 H Sato1 ASCT. The study met the primary endpoint of ORR (82%;
M Yoneda1 C Kai n=92; P<.001). In the modified intent-to-treat population
(n=101), ORR was 82% (CR, 54%; PR, 28%). At a median
1: The University of Tokyo follow up of 8.7 months, 44% of patients were in response;
39% in CR. Median DOR was 8.2 months overall and not
Triple-negative breast cancer (TNBC) is known to be ag- reached for patients with CR. Median OS was not reached;
gressiveandoftenrelapseswithpoorprognosismorethanother 80%ofpatientsremainedaliveat6months.Themostcommon
typesofbreastcancer.Owingtoresistancetobothhormoneand grade ‡3 AEs were anemia (43%), neutropenia (39%), febrile
trastuzumab treatments, TNBC often causes recurrence and neutropenia (31%), thrombocytopenia (24%), and encepha-
metastasis.Thus,noveleffectivetherapiesforTNBCareneeded. lopathy (21%). Grade ‡3 cytokine release syndrome (CRS)
Measlesvirus(MV)hasoncolyticactivity. Wehavepreviously and neurologic events (NE) occurred in 13% and 28% of pa-
demonstrated that a recombinant MV (rMV-SLAMblind) in- tients, respectively, and resolved except 1 grade 1 memory
fected several breast cancer cell lines using Nectin-4 as a re- impairment. There were 3 (3%) grade 5 AEs. Expansion of
ceptor and showed antitumor activity. In this study, we CAR T cells was associated with ORR and Grade ‡3 NE, but
examinedwhetherrMV-SLAMblindiseffectiveforTNBC.We notCRS.Axi-celdemonstratedsignificantclinicalbenefitwith
examined expression level of Nectin-4, a receptor of rMV- a manageable safety profile in patients lacking curative treat-
SLAMblind, on the surface of 12 TNBC cell lines with flow ment options.
A6 SELECTED ORAL PRESENTATIONS
OR12
N
W
Intraputaminal AADC gene therapy for advanced A
R
Parkinson’s disease: interim results of a phase 1b Trial D
H
T
B Ravina1 C Christine2 K Bankiewicz2 A Van Laar3 W I
M Richardson3 A Kells1 B Boot1 A Martin2 M Thompson2 C T
A
P Larson2 R
T
S
B
1: Voyager Therapeutics 2: University of California A
San Francisco 3: University of Pittsburgh
Objective:Toevaluatethesafetyofgenetherapywithhuman
aromatic L-amino acid decarboxylase (AADC) using large in- OR14
fusion volumes and intraoperative MRI to improve delivery.
Correction of sensory ataxia in a novel mouse model
Background: Levodopa therapy for Parkinson’s disease (PD)
of Friedreich’s ataxia using gene therapy approach
becomeslesseffectiveovertime,possiblyduetoprogressiveloss
of AADC, which converts levodopa into dopamine. Methods: F Piguet1 N Vaucamps1 C de Montigny1 A Eisenmann1
Ten subjects with advanced PD received bilateral infusions of L Reutenauer1 H Puccio1
AAV-hAADCvector(0.83·1012vg/ml)withgadoteridol.Five
y. subjectsincohort1receivedupto450ll/putamenand5subjects 1: IGBMC
nl
e o in cohort 2 received up to 900ll/putamen. 18F-dopa PET was
us obtained at baseline and 6 months to assess gene expression. Friedreich’s ataxia (FA)is characterized by a sensory andspi-
nal Results: Twenty one percent of the putamen was covered by nocerebellar ataxia, hypertrophic cardiomyopathy and increase
o
ers vector in cohort 1 and 34% in cohort 2. Treatment was well incidenceofdiabetes.FAiscausedbyreducedlevelsoffrataxin,a
p
or tolerated with no vector related SAEs. There was one post- mitochondrial protein involved in the biosynthesis of iron-sulfur
F
7. procedure pulmonary embolus which resolved. 18F-dopa PET clusters.Proprioceptiveneuronswithinthedorsalrootgangliaand
1/1 increasedby13%incohort1and56%incohort2.Fivesubjects cardiomyocytes are the most affected tissues in FA patients. To
1
12/ in cohort 1 and 3 in cohort 2 have completed the 12-month datetherearenoteffectivetreatmentforFA.Wehavepreviously
m at evaluation. On-time without troublesome dyskinesias, measured established the primary proof-of-concept for developing gene
co bymotordiaries,increasedby1.6hoursincohort1and4.1hours therapyofFAcardiomyopathyandshowedthatAAVrh.10vector
ub. in cohort 2 at 12 months. UPDRS motor scores on medication expressing FXN injected intravenously rapidly and completely
p
ert and PDQ-39 scores showed similar dose-dependent trends. Due reversedthecardiacdisease. Werecently generateda conditional
b
m online.lie trCoeodniumcclepudrsoiaovnne:daTvmhereoastgeoerinoftfuenr1icm0t%iornef,sourdltacsiolsyhhoolrewtv1othdaaontpdAa3Ae5Vq%u-ihvfAoarlAecnDotCshowgretenr2ee. mcexiaoptueresdesstmionogFdcAeelllttsho,aitndcreellucedatepinitfguraltahtateexspinrfoapsitprhieofcucilefilpyctaitlvhleyensieneunrstohoneryspoaaftratvxhaielabDuaRsmsGoin-.
o therapy using intraoperative MRI is well-tolerated, provides Using this model, we have developed a gene therapy approach
97 fr dose-dependentgene expression andpotentialclinical efficacy. basedonanintravenousdeliveryofAAV9-CAG-hFXN-HAvec-
9.1 tor and shown at an early symptomatic stage of the disease a
1
1 complete prevention of the ataxic phenotype and electrophysio-
2.
6. logicalanalysisshowedmaintenanceofthesensorywave.Wethen
8
by OR13 evaluated the therapeutical approach at a post symptomatic stage
d
ade Dynorphin-based ‘‘drug on demand’’ gene therapy ofthediseasewithacombinationofintraveinousandintracerebral
ownlo suppresses seizures and restores lost brain functions dcoelmivpelreiteesroefveArsAioVn-CoAf tGh-ehFprXoNpr-iHocAe.ptTivreeaptehdenaontiympaelsevdaislupalateydedbya
D in drug-resistant temporal lobe epilepsy
gaitanalysis,coordinationtestandEMGaswellatthehistological
levels with a full preservation of neurons within DRG and a
completeregenerationofaxonswithinperipheralnerves.Together,
ourresultsprovideaproof-of-conceptfordevelopinggenetherapy
N
forthesensoryataxiainFA.
W
A
R
D OR15
H
T Gene therapy with AAV-CDKL5 vectors in models
W I of CDKL5 disorder
T Y Gao1 E E Irvine1 I Eleftheriadou1 L Bosch1 L Murdoch1
C A Czerniak1 J A Glegola1 R James1 I Meloni2 A Renieri2
A M Kinali3 N D Mazarakis1
R
T
1: Imperial College London 2: University of Siena 3: Chelsea
S
B and Westminster NHS Foundation Trust
A
CDKL5 disorder is a severe neurodevelopmental disorder
caused by mutations in the X-linked cyclin-dependent kinase-
like5 (CDKL5) gene. It predominantly affects females that typi-
cally present with severe epileptic encephalopathy, intellectual
SELECTED ORAL PRESENTATIONS A7
disability, microcephaly, autistic features, sleep disturbances and of in vivogenome editing and can potentially offer a new treat-
motor dysfunction. Currently, there is no therapy apart from an- ment modalityformonogenic diseases.
tiepilepticdrugsforseizuremanagement.Wesetouttodevelopa
gene replacement therapy, by first characterising the CDKL5
OR17
transcript and protein isoforms expressed in human brain, human
neuronal cell lines and hESC-derived cortical interneurons. We CRISPR/Cas9-mediated editing for dominant genetic
found that the hCDKL5_1 and to a lesser extent the hCDKL5_2 disorders:efficientexcisionoftrinucleotiderepeatexpansion
isoforms were expressed in these and cloned their coding region in myotonic dystrophy
downstream of the CBh promoter in ssAAV2 transfer plasmids.
High titre rAAV vectors pseudotyped with AAV9, the variant S Dastidar1 S Ardui7 K Singh1 N Nair1 Y Fu2 3 10 D Reyon2 3
capsid PHP.B orthe hybrid capsid DJ were produced. We found E Samara1 M F M Gerli4 11 A F Klein5 W DeSchrijver1
that AAV-DJ-CDKL5 vectors were the most efficient in trans- D Majumdar1 J Tipanee1 S Seneca6 W Tulalamba1 H Wang1
ducing CDKL5-mutantiPSC-derived neural progenitorsand their YCChai1PIn’tVeld8DFurling5FSTedesco4JRVermeesch7
isogenic controls, which were subsequently differentiated into J K Joung2 3 M K Chuah1 9 T VandenDriessche1 9
mature neurons. Intrajugular delivery of 1·1012 vg of AAV-
1:DepartmentofGeneTherapy&RegenerativeMedicine,Free
PHP.B-GFPvectorsinwildtypemicetransducedneuronsandglia
University of Brussels, Brussels, 1090, Belgium 2: Molecular
inbrain,spinalcord,DRGsandretinamoreefficientlythanAAV9.
Pathology Unit, Center for Cancer Research and Center for
CDKL5 KO mice weretreated with 1·1012 vg ofAAV-PHP.B-
Computational and Integrative Biology, Massachusetts General
CDKL5 vectors via the intrajugular route at 28-30 days of age.
nly. Behavioural testing was conducted at 1-2 months after vector HPaotshpoitlaolg,yC, hHaarrlveastrodwMn,eMdicAal02S1c2h9o,olU,SBAos3to:nD,eMpaArt0m2e1n1t5o,fUSA
o
al use dCeDliKveLr5y-traenadtedbraKinOs mwiecreeetxahkiebnitefdorsigannaifilycsainst amftoetror3immpornotvhes-. 4C:oDlleegpearLtmonednotno,fLCoenldloann,dWDCe1vEel6oDpEm,enUtKal.B5:ioSloogrby,onUnneiversity
n
7. For perso mtChDeernKtasLn5caolypmsaiptsiaetrnoetdsa.stcoerGtaFinP-itfrethaitsedgecnoenttrhoelrsa.pWyecoaurledcboentdraunctsilnagtedfutro- URSaenlsipeveˆaetrrrsciieh`tre´eisn,UPMPayrMoislC,ogFUy-,7n5Iivn0s1Pt3iat,urFtisrda0en6Mc,eIy.No6Slo:EgRRieeM,se,GaCHrNchPRiSGt,ire´Co-uepnter for
1/1 Reproduction and Genetics (REGE), Center for Medical
2/1 OR16 Genetics, UZ Brussels, Free University of Brussels, Brussels,
1
at 1090, Belgium. 7: Department of Human Genetics, University
m In vivo genome editing via non-viral delivery of zinc finger ofLeuven,Leuven,3000,Belgium.8:DepartmentofPathology,
o
b.c nucleases enables supraphysiological levels of therapeutic Free University of Brussels, Brussels, 1090, Belgium.
u
ertp proteins and greater than 90% protein knockdown of 9: Center for Molecular & Vascular Biology, Department of
eb multiple therapeutic gene targets via targeted integration Cardiovascular Sciences, University of Leuven, Leuven, 3000,
e.li and NHEJ, respectively, in wild type mice Belgium 10: Cell and Gene Therapy, Biogen, Cambridge, MA,
n
97 from onli ARC CBCaoDrnbweoKasaye2l1vTMer1RMeDdeenEldmePlea1isecJrh2GoMnar1nCBesH1LoAMlmBueois2y1kPGoL1LiKne2eK1YimK1TLaZmh2ang1 UR02eS1gA1e.4n1,e1rUa:StMiAvea.sMsaecdhiucsineett,sHGaernvearradlMHeodspiciatallSCchenotoelr, Bfoorston, MA
1
9.
11 1: Sangamo Therapeutics 2: Acuitas Therapeutics CRISPR/Cas9 is an attractive platform to potentially correct
2.
6. dominantgeneticdiseasesbygeneediting.Inthecurrentproof-
8
by Zinc finger nucleases (ZFNs) are sequence-specific nucleases of-principle study, we explored the use of CRISPR/Cas9 for
ed that can be engineered to target virtually any sequence in the geneeditinginmyotonicdystrophytype1(DM1),anautosomal
d
oa genome.WehavepreviouslyshownZFNscanbepackagedinto dominantdisorderassociatedwithseveremyotoniaandskeletal
nl
w adeno-associatedviruses(AAV)anddeliveredintravenouslyinto muscle dysfunction. The DM1 pathology is caused by trinu-
o
D miceandnon-humanprimatestoinducehighlyefficientgenome cleotide CTG repeat expansion in the 3¢ untranslated region
editing in the liver. Lipid nanoparticles (LNP) are a novel de- (UTR) of the human myotonic dystrophy protein kinase
livery vehicle that enables repeat administration and are not (DMPK) gene. We designed a CRISPR/Cas9-based strategy
limitedbythepresenceofpre-existingneutralizingantibodiesin using dual guide RNAs and S. pyogenes Cas9 to excise this
theserumoftreatedsubjects.HereweshowthatLNPpackaged pathogenic CTG repeat expansion in the DMPK 3¢ UTR. We
with mRNA encoding ZFNs can induce high levels of genome firstgeneratedDM1 patient-specificiPScellsandsubsequently
editing(upto47%indels)attheAlbuminlocuswithinthemurine inducedthemtodifferentiateintomyogeniccellsandmyotubes.
liver after a single intravenous dose. Co-delivering the mRNA- CRISPR/Cas9-mediated excision of the triplet repeats expan-
LNPwithAAVcomprisingeitherapromoterlesshumanIDSor sion resulted in the disappearance of the characteristic ribo-
FIX transgene donor results in therapeutically-relevant levels of nuclearfocithatsequesterMBNL1splicingfactorsintheDM1-
enzymatic activity (1950nmol/hour/mL) and protein expression iPSC-derived myogenic cells. This was consistent with the
(1015ng/mL),respectively,withintheplasma(*7700-foldwild normalization of the splicing pattern of quintessential DM1
type levels). In addition, repeat administration of the mRNA- markergenessuchSERCA1.Therepeatexpansionwasexcised
LNPafterasingleAAVdonordosesignificantlyincreasedlevels with high efficiency (50-60%) and was depending on both
of genomeediting andtransgeneexpression (*double after 2-3 gRNA and Cas9 expression. State-of the-art single molecule
doses). For gene knockout applications, ZFNs were designed real-time (SMRT) sequencing on the target site provided mo-
against the murine TTR and PCSK9 coding regions, which are lecularevidenceofthecompleteexcisionoflargetrinucleotide
clinically-validatedgeneknockout/knockdowntargets.After2-3 repeatexpansion(1371-1600repeats).Noevidenceofoff-target
mRNA-LNP doses (0.8mg/kg), 91% and 92% protein knock- effects were apparent by deep sequencing. This proof-of-
downinplasmawasobservedforTTRandPCSK9,respectively, concept study validates the use of CRISPR/Cas9 to efficiently
whichwasstableoutto77days.TheseresultsdemonstrateLNP- correct nucleotide repeat expansions associated with dominant
mediated ZFN mRNA delivery can drive highly efficient levels genetic disorders that cause severe human pathologies.
A8 SELECTED ORAL PRESENTATIONS
OR18 ficiency(SCID)toautoimmunephenotypes.Thetreatmentremains
criticalsincehaematopoieticstemcells(HSCs)transplantationfrom
Direct correction of Fanconi anaemia associated mutations
partiallyHLA-matcheddonorisoftenassociatedwithunsatisfactory
in haematopoietic stem and progenitor cells by a novel
outcomes.However,safetyconcernsrelatedtoectopicandunregu-
NHEJ-mediated gene editing approach
lated Rag1 expression has so far hampered conventional gene re-
F J Roman-Rodriguez1 2 B Diez1 2 L Alvarez1 2 C Risuen˜o1 2 placement.Weproposeaspecificgenomeeditingstrategy,basedon
R Torres-Ruiz3 M Corton2 C Ayuso2 J A Bueren1 2 P Rio1 2 thedeliveryofengineerednucleasesandDNAtemplate,tocorrect
RAG1 mutations and restore both gene function and expression
1: Division of Haematopoietic Innovative Therapies, Centro de
control. We optimized the protocol to deliver RAG1 gene donor
Investigaciones Energe´ticas Medioambientales y Tecnolo´gicas
vector and Zinc Finger Nucleases (ZFN) in human cell lines and
(CIEMAT) / Centro de Investigacio´n Biome´dica en Red de
CD34+cells.PreliminaryresultsshowedhighlevelsofZFNactivity
Enfermedades Raras (CIBERER-ISCIII). Madrid, 28040, Spain
andtargetingefficiencytogetherwithareducedtoxicityonCD34+
2:AdvancedTherapiesUnit,InstitutodeInvestigacio´nSanitaria
cells,that maintainedengraftmentcapabilityinNSGmicecompa-
Fundacio´nJime´nezD´ıaz(IIS-FJD/UAM).Madrid,28040,Spain
rabletonon-editedcells.CompetitivetransplantsinRAG1-/-mice
3: Molecular Cytogenetics Group, Human Cancer Genetics
showedthatasmallportionofwildtypeHSCs(5-10%)issufficient
Program, Centro Nacional de Investigaciones Oncolo´gicas.
topartiallyrescueBandTcells,restoringIgMandIgGresponses
Madrid, 28029, Spain
after TNP/KLH immunization, suggesting that our targeting fre-
quencymightrescueRag1deficiency.Inparallel,toobtainoptimal
Nowadays gene therapy is considered a promising alternative
repopulationofgenecorrectedcells,wetesteddifferentconditioning
nly. forFanconiAnaemia(FA)patientswithoutawell-matcheddonor regimens:classicaltotalbodyirradiation,cytotoxicchemotherapeu-
e o for allogenic haematopoietic stem and progenitor cell (HSPC) ticsandnewlydevelopedimmunotoxins.Thesepreliminaryresults
al us transplantation, due to the proliferative advantage evidenced by areinstrumentaltofurtheroptimizethegenomeeditingprotocolfor
n gene-corrected FA-cells.Incontrasttoconventional homologous
o RAG1correction,whichwillbeusedonCD34+cellsderivedfrom
pers recombination-mediatedgeneediting,wheretheprecisecorrection patients.
or ofthemutatedgeneispursued,theapproachproposedinthisstudy
F
7. exploitstheerror-pronenatureofthenon-homologousendjoining
1
1/ (NHEJ) to directly remove/compensate FA-associated mutations OR20
1
m at 12/ adsouablecosntsreanqduenbcreeakof(DthSeB)inrseeprtaiiorn,sm/diemleitciokninsggseonmereateredvedrusiroinngs ‘s‘ySsitleemncefoarntdheRetrpelaactme’e’n:tDoefvoelcouplompehnatroyfnagesainlgmleuAscAuVlarvector
ub.co dtwesocrFibAe-dAinpaFtAie-nmt-odseariicvepdatliyenmtsp.hOobulrahstyipcocthelelsilsinwesascfiarrrsytitnegstebdiailn- dystrophy (OPMD)
p
ert lelic c.3558insG (p.R1187EfsX28)orc.295C>T (p.Q99X)muta- V Strings1 A Malerba2 S Harbaran1 O Cappellari2 F Roth3 4 5
b
nline.lie twioitnhs.eTffihceasceiemsuutpatitoon2s0w%e.reMtaorrgeeotveedr,usNinegxtCGReInSePrRat/iConasS9enquucelnecaisnegs HP RCohealyvtionwk12DGSCuohryd1ova3 4 5 C Trollet3 4 5 G Dickson2
m o identifiedcorrectiveNHEJ-repaireventsthatconferredamarked 1:BenitecBiopharma2:RoyalHolloway,UniversityofLondon
o in vitro proliferative advantage. Interestingly, these cells re-
97 fr expressedFANCAprotein;revertedthemitomycinChypersen- 3: Inserm U974 4: UPMC - Paris 6 5: Institut de Myologie
1
9. sitivityandtheexacerbatedROSproductioncharacteristicofFA
2.11 cells; and restored FANCD2 foci formation. Finally, the use of OPMD is an autosomal dominant, late-onset disorder that
86. this strategy in primary CD34+ cells from FA-A patients har- initiallyimpactseyelidandpharyngealmusclesleadingtoptosis,
ded by bgoenuerirnagtintghecocr.r2e9c5tiCv>eTNHmEutJa-trieopnaircoenvfiernmtsedinthtehesfeeascilbiniliictyallyo-f dnyesssp.hTaghiea,diasnedaseeviesnctuaaulsleydpbroygarbesnsoersmtaol epxropxainmsiaolnliomfbalawneinake--
oa encoding trinucleotide repeats in the poly(A) binding protein
nl relevantcells,evidencedbyasignificantproliferativeadvantage
w nuclear-1 (PABPN1) gene. Previous preclinical studies in the
o of NHEJ-corrected HSPCs in vitro. Importantly, the absence of
D A17 mouse model used a two-vector system: one AAV ex-
mutations in the top-five off-target loci confirmed the safety of
pressed shRNAs to silence mutant PABPN1, a second AAV
thisapproachinFA-patientHSPCs.Theseresultsdemonstratefor
vector expressed a codon optimized version of the wild type
thefirsttimethefeasibilitytocorrectthephenotypeofFA-HSPCs
protein. A17 mice display many of the hallmarks of OPMD
byanefficientNHEJ-mediatedgeneeditingapproach.
including intranuclear inclusions (INIs), fibrosis, and loss of
muscle strength. Application of both vectors resulted in im-
provement of many of these phenotypes including a restoration
OR19 ofmusclestrengthtowildtypelevels.Herewedescribeasingle
vector system, termed BB-301, in which both ‘‘silence and re-
Targetedgenomeeditingofrecombinationactivatinggene1 place’’modalities arecombinedintoasingletherapeutic vector
to potentially treat severe combined immunodeficiency and tested in A17 mice. Driven by a muscle specific promoter,
N Sacchetti1 2 M C Castiello1 A Jacob1 S Ferrari1 G Schiroli1 BB-301 produces a single RNA that encodes two anti-mutant-
M Holmes4 A Conway4 P Genovese1 L Naldini1 2 A Villa1 3 PABPN1 shRNAs as well as the coding sequences for the wild
typePABPN1.Inadose-dependentandtime-dependentmanner,
1: Telethon Institute of Gene Therapy (HSR-TIGET) groupaveragesofBB-301treatmentsyieldrobustinhibition,up
2: University Vita-Salute San Raffaele 3: Istituto di Ricerca to 87%, knockdown of mutant PABPN1 and results in restora-
Genetica e Biomedica, CNR 4: Sangamo BioSciences, Inc. tion of wild type PABPN1 levels up to 53%.BB-301 treatment
results in correction of INIs, fibrosis as well as muscle strength
Lymphoid-specificrecombination-activatinggene1(RAG1),ex- asassessedbymaximalforce.Together,theseresultssupportthe
pressed during T cell and B cell development, initiates V(D)J re- use of a single vector ‘‘silence and replace’’ based approach to
combination. Mutations in RAG1 are associated with a broad treat OPMD. A first in man study is anticipated second half
spectrumofclinicalphenotypes,fromseverecombinedimmunode- 2018.
SELECTED ORAL PRESENTATIONS A9
OR21 independentofAAVR.Invivo,rh32.33alsoshowedsimilarlevels
of transduction in AAVR KO vs wild type mice. In order to
Combined Notch and PDGF signalling in muscle satellite
identify evolutionarilyhowAAVgainedorlosttheability to use
cells induces a pericyte-like phenotype with potential cell
AAVR,areconstructedputativeAAVlineagewasinterrogatedfor
therapy relevance
AAVR dependency. Lastly, a combination of site-directed muta-
LAMoyle1MFMGerli1EUcuncu1ILouca1CConstantinou1 genesis and chimeric capsids were used to narrow down the
H Sakai2 M Ragazzi1 S Tajbakhsh2 G Cossu3 F S Tedesco1 AAVR binding site on the viral capsid. Our results demonstrate
that AAVR usage is not universal, as previously thought, and
1: University College London 2: Institut Pasteur 3: University
suggest that AAVR independent serotypes may be a valuable
of Manchester
optionforgenetherapyintissueswithlowAAVRexpression.
Satellite stem cells are responsible for post-natal skeletal
muscle maintenance and regeneration; upon activation they
proliferateastransientamplifyingmyoblasts,mostofwhichfuse OR23
intomyofibres.Despitetheencouragingoutcomesobtainedfrom
transplantingmyoblastsintodystrophicanimalsandpatientswith Ad3.0: An engineered adenovirus library to significantly
localised forms of muscular dystrophy, results achieved in clin- expand the spectrum of available vector types and their
ical trials with more severe forms of muscle diseases, such as applications
Duchennemuscular dystrophy(DMD),showed limitedefficacy. W Zhang1 E Hage2 J Liu1 A Heim A Ehrhardt1
y. In addition to satellite cells, pericyte-derived mesoangioblasts
nl
e o can contribute to muscle regeneration and give rise to satellite 1: University Witten/Herdecke 2: Hannover Medical School
us cells. When injected systemically they migrate through the vas-
nal cularendothelium,circumventingtherequirementforlocalintra- So far >70 types of human adenoviruses (Ads) have been
o
ers muscularinjections.Thesecellshaverecentlyundergoneclinical identified, with virus type-dependent tissue tropisms and patho-
p
or experimentation in a phase I/II first-in-human DMD trial. We genicities.Previousresearchhashighlightedaseriesofapplications
F
7. hypothesised that by modulating Notch and PDFG signaling, using different adenovirus types as promising tools to develop
1
1/ involvedinpericytespecificationintheembryo,wemightinduce disease-specific therapeutic approaches. In our recent work we
1
12/ pericyte-like features in adult satellite cells. Here we show that generatedaclonedadenovirus(Ad)librarycomprisingof32natural
m at Dll4 and PDGF-BB-treated satellite cells acquire perivascular occurringwildtypehumanAdsrepresentingallknownspeciesand
co markers and functional properties, including stabilisation of 20 recombinant adenoviruses (rAds) labeled with measurable
ub. capillary-like networks and enhanced migration ability in vitro. markers (Zhang et al., Cell Reports 2017). This reporter-labeled
p
ert Interestingly, treated satellite cells also show up-regulation of rAdVlibraryenableshigh-throughputscreening(HTS)ofdisease-
b
e.lie Pax7, a marker normally found in quiescent satellite cells. This specific cell lines to identify better candidates for gene therapy,
nlin implies that treated cells may acquire an intermediate stem cell oncolytic virotherapy and vaccination. Here we report significant
m o phenotype between satellite cells and pericytes. Importantly, extension of this library including novel adenovirus types 70, 73,
o preliminary in vivo data shows increased engraftment of ‘‘re- 74, 75, and 80 and we applied advanced technologies to convert
97 fr programmed’’ cells upon intramuscular delivery in dystrophic clonedwildtypevirusesintoreplication-competentandreplication-
1
9. mice.Theseresultsextendourunderstandingofsmooth/skeletal deficient vectors. Methods to convert these cloned viruses into
1
2.1 muscle lineage choice and provide evidence of a druggable vectors will be provided and rely on linear-linear homologous
86. pathway with potentialclinicalrelevance. recombination (LLHR) and linear-circular homologous recombi-
y
b nation (LCHR).We exemplified this conceptforadenovirus type
d
de 17 (AdV17). After construction of a vector based on AdV17
a
nlo (replication-deficient, deleted for the adenoviral gene E1) we
w
o OR22 foundthat this vectorresultsin enhanced transductionofimmor-
D
talized and primary endothelial cells in vitro and in vivo. There-
Divergent AAV serotypes demonstrate an alternate, AAVR
fore,webelievethatthisvectormayprovideanovelplatformfor
independent, entry pathway
gene therapy, especially for cardiovascular diseases. Taken to-
A M Dudek1 S Pillay2 A S Puschnik2 J E Carette2 gether,theexplorationofnaturalhumanadenovirusdiversitywill
L H Vandenberghe1 help us to identify a number of novel vectors which can be ex-
ploredinnovel therapeuticapplicationsandvaccinationstudies.
1:GrousbeckGeneTherapyCenter,MassachusettsEyeandEar
Infirmary, Harvard University, Boston MA, 02114, USA
2: Department of Microbiology and Immunology, Stanford
University, Stanford CA, 94305, USA
OR24
Understanding the mechanism of Adeno-Associated Virus Generation of highly variably AAV capsid libraries
(AAV)entryiscrucialforitssuccessfulimplementationasagene using a novel DNA shuffling platform
therapy vector. Recently a universal AAV receptor (AAVR) was MCabanes-Creus123AFilip1SHYLiao1JWard1SLGinn1
identifiedtoberequiredforentryofalltestedAAVserotypes,and G Santilli2 3 A J Thrasher2 3 I E Alexander1 4 L Lisowski1 5
required for AAV9 transduction in vivo. We have demonstrated
that the highly divergent serotypes AAV4 and AAVrh32.33 are 1: Children’s Medical Research Institute 2: Institute of Child
abletobothbindandtransduceAAVRKOcellsasefficientlyas Health 3: University College London 4: University of Sydney
AAVR expressing cells. By using dose response experiments in 5: Military Institute of Hygiene and Epidemiology
AAVR knock-out cells and re-introduction of AAVR in non-
permissive cells, we were able to demonstrate that AAV4 and Vectorselectionstrategiesbasedondirectedevolutionrelyon
AAVrh32.33 use a distinct non-redundant entry pathway that is generationofhighlyvariableAAVcapsidlibraries.Acommonly
A10 SELECTED ORAL PRESENTATIONS
usedmethodforcreatingsuchlibrariesisDNA-familyshuffling, amount of full length viral vector genomic RNA by Northern
atechniquereliantonrandomfragmentationofcapsidgenesand blotanalysis,whichincreasedinDroshaKOcells.Thefindingis
PCR-based reassembly dependent to partial sequence homolo- anticipated to have important implications in future production
gies. One limitation of this method is inherent dependence on of viral shRNAmiRs for RNAi-based therapy.
homology between parental cap donors at the DNA level,
leading to decreased shuffling efficiency as the phylogenetic
OR26
distance between parental AAVs increases. To overcome this
limitation, we have developed a custom codon optimization Phase I/IIa clinical trial for recessive dystrophic
method for improving homology between serotypes while epidermolysis bullosa using EB-101 (COL7A1 gene-
maintainingcapsidfunctionality,whichallowsthegenerationof corrected autologous keratinocytes)
highly variable shuffled AAV libraries containing distant sero-
Z Siprashvili1 N T Nguyen1 E S Gorell1 K Loutit1
types. We have validated this shuffling platform by creating a
YDutt-Singkh1JNazaroff1PKhuu1LFurukawa1HPLorenz1
library based on human AAV serotypes belonging to Clade-A
THLeung2D RKeene3KE Rieger1PAKhavari1ATLane1
(AAV6), Clade-B (AAV2)and adistant clonalisolate (AAV5).
J Y Tang1 M P Marinkovich1
Modification of capsid sequences using our codon optimization
method increased the average homology between capsids by 1:StanfordUniversity2:UniversityofPennsylvania3:Shriners
10.2% when compared with wild type counterparts. Further- Hospital for Crippled Children 4: VA Palo Alto, Stanford CA
more, our optimized shuffling platform led to higher shuffling
nly. ebfefitwcieeenncyin,deivviiddeunatlbcyapinsicdrsea(s5e–d3nuamndbe2r3o–f6sefqouretnhceeccornotsrosolvaenrds inhRereicteedssigveeneDtiycstsrkoipnhdicisoErpdiedrercmhaorlaycstiesriBzeudllobsyac(hRroDnEicB)bliisstearn-
o
al use ofrpatgimmieznetdscloibnrtarribyu,triensgpteoctsihvueflyfl)edancdapdsiedcsre(afrsoemd a3v5e8rbapgetos9iz5ebpo)f. ilnifge,soppaenn.RanDdEpBaipnaftuilenwtsoulancdks,fjuonicnttiocnoanltrtaycpteurVesI,Iacnodllasgheonrte(Cne7d)
n
For perso EvananrdcioaKnutr5sa6ga2enddcebslylusc,icnwecsersefaausrleesdenlcoeowcmtibopunleilxodifitnytghaistmtnohoreevelcelovlmeiblproarefryhienondnsiivvHieduuhlai7-l omdweariimnngacl-oteompmipdouenrtaemtniatolnobsfasianenmcthheeonrtgimnegneemfibCbrrOialnsLe7(.AAAF1P)thhwaahsteiceIhn/IcIsotcdalbeinsiliicCzae7ls,trttihhaeel
17. braries based on fourteen codon-optimized AAV serotypes for evaluating woundclosure after EB-101(ex vivo gene corrected
2/11/ directed evolution applications on human CD34+ cells and keratinocytes) treatment was evaluated in patients with severe
at 1 human primary hepatocytes. RDEB subjects (n=6, 36 total treatments). Autologous RDEB
m keratinocytes isolated from skin biopsies were transduced with
o
ub.c OR25 retrovirus encoding full-length COL7A1 and applied onto non-
ertp healing chronic wounds with a mean time open prior to treat-
eb Enhancementoftitres forvectorsencodingmiRNAadapted mentof8.5years.Noseriousadverseeventshavebeenreported
e.li shRNAs (shRNAmiRs) associated with increased viral and replication competent retroviruses have not been detected
n
nli genomic transcripts via Drosha microprocessor knockout for up to 3 years. Wound healing defined as >50% healing
o
m (KO) in producer cells compared over baseline has been observed in 100% (36/36
o
197 fr HPark12RTriboulet12MBentler3SGuda12PDu12HXu12 g1r2afmtso)natth3s.mWonotuhnsd,8h9e%ali(n3g2/3d6efi)nated6mason>t7h5s%,anhdea8l3in%g(h2a0s/2b4e)eant
9. R Gregory1 2 C Brendel1 2 4 D A. Williams1 2 4
11 observed in 83% (30/36 grafts) at 3 months, 61% (22/36) at
6.2. 1: Boston Children’s Hospital 2: Harvard Medical School 6 months, and 50% (12/24) at 12 months. By comparison, un-
8
y 3: Institute of Experimental Haematology, Hannover, Germany treatedcontrolwoundsremainedunhealed(0%).Inaddition,C7
b
ed 4: Dana-Farber Cancer Institute expression was observed in biopsies of EB-101 treated wounds
d
oa upto2yearspost-treatment.TheongoingPhaseI/IItrialresults
wnl RNA interference (RNAi)-based gene therapy using miRNA demonstrate COL7A1 ex-vivo gene transfer has a favorable
o
D adapted shRNA (shRNAmiR) is a powerful approach to modu- safety profile and significant wound healing efficacy that cor-
late gene expression because of its high knockdown efficiency relates with C7 molecular correction.
and reduced cytotoxicity. We recently developed a lentiviral
vector containing a shRNAmiR targeting BCL11A for the gene
OR27
therapy of sickle cell disease (SCD) and preparations for a
clinical trial are ongoing. However, we have observed low in- Geneediting-mediatedexcisionofmutation-bearingexon80
fectious viral titers with microRNA-containing vectors and hy- of COL7A1 gene for the efficient correction of recessive
pothesized that this could be due to cleavage of viral genomic dystrophic epidermolysis bullosa patient derived-epidermal
RNA by the endogenous miRNA processing machinery, spe- stem cells
cifically by the microprocessor complex during virus assembly J Bonafont1 2 C Chamorro1 2 A Mencia1 2 3 B Duarte1 2 3 4
in producer cells. To test this hypothesis, we designed several D Ortega-Cruz1 R Torres5 M del Rio1 2 3 4 S Llames2 3 6
gRNACRISPR/Cas9constructstargetingexon4andexon30of F Larcher1 2 3 4 R Murillas2 3 4
Drosha,thecorecomponentofthemicroprocessorcomplex,and
successfully generated single and biallelic Drosha knockout 1:DepartmentofBiomedicalEngineering,CarlosIIIUniversity
(KO) cell lines in HEK 293T cells. Drosha KO was verified by (UC3M), Madrid, Spain 2: Instituto de Investigacio´n Sanitaria
sequencing and Western blot analysis. We produced lentiviral de la Fundacio´n Jime´nez D´ıaz, Madrid, Spain 3: Centro de
vectorswithorwithouthairpinsusingDroshaKOproducercells. Investigacio´n Biome´dica en Red en Enfermedades Raras
We observed a concomitant increase in the fluorescence inten- (CIBERER) U714, Madrid, Spain 4: Epithelial Biomedicine
sityofhairpincontainingVenustranscripts,andalmostcomplete Division, Centro de Investigaciones Energe´ticas
recovery in the viral titer of hairpin-containing vectors. We Medioambientales y Tecnolo´gicas (CIEMAT), Madrid, Spain
confirmed the absence of shRNAmiR processing by Northern 5: Molecular Cytogenetics Group. Centro Nacional
blot analysis and showed that this directly correlated with the Investigaciones Oncolo´gicas (CNIO), Madrid, Spain