Paolo Moi, Italy. Università di Cagliari Ospedale Pediatrico Microcitemico - A. Cao - CAGLIARI

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1 T H E O R E T I C A L A N D P R A C T I C A L T R A I N I N G I N HAEMATOLOGICAL RARE DISEASES: from genetic counselling - through bench - to bed Gene Therapy for Hemoglobinopathies Paolo Moi, Italy Università di Cagliari Ospedale Pediatrico Microcitemico - A. Cao - CAGLIARI

2 Timeline gene therapy 1978 Globin gene cloning 1986 LCR discovery 2000 Gene therapy in murin models Th3/ Human gene therapy in XSCID st Gene therapy of human b-thal 2014 Rapid cure b-thal GT 2017 Gene therapy of SCA

3 Current globin gene therapy relies on the insertion of a normal regulated globin gene into a random chromosome b STOP e Gg Ag d b

4 Major progress from use of lentiviral globin vectors LCR e Gg Ag d b HS LTR GAG POL ENV HS b 5 -LTR

5 Vectors are transferred into HSC by ex-vivo gene therapy procedures Transduction with b- globin lentivctors Genetically corrected and compatible HSC Quality controls Mobilization of HSC into peripheral blood of thalassemia patient from Reinfusion Bone marrow reconstitution

6 Objectives of human GT trials Evaluate safety and efficacy of HSC transduced after myeloablation (complete or partial) Evaluate engraftment and BM reconstitution of genetically modified HSCs Evaluate frequency of blood transfusion after GT Safety issues: Absence of clonal dominance and insertional mutagenesis/oncogenesis Absence of RCL (replication competent virl particles)

7 1 st evidence of GT efficacy in humans in USA_France trial (4/10 pz, 2007) Patients 1 e 2 : not treated, reinfusion of backup BM Patient 3: gene transfer in June 2007 ( transfusion independent after 1 year, 10 years follow-up) Patient 4: Gene transfer 50 months before, still transfusion dependent

8 Features of the Lentiglobin vector HPV569 chs4 R-U5-gag Y env cppt RRE b A-T87Q globin gene prom. HS2 HS3 HS4 R-U3 chs4 * chs4 insulators The variant b A-T87Q has double function label vector derived Hb antagonize sickling b A-T87Q

9 Hb g/dl Trial 1 outcome: Hb concentration in pts 3 e b /be b /be Patient n. 3 stably transfusion independent with 2,7 gr di Hb T87Q a 8 yrs of follow-up Patient n. 4 still transfusion dependent with only 0,4 gr di Hb T87Q at 3 years follow up Months post GT HbA T87Q HbF HbE HbA

10 Cloud on the horizon: clonal dominance M Cavazzana-Calvo et al. Nature 467, (2010) doi: /nature09328

11 Insertional mutagensis of the lentivector into the HMGA2 gene HMGA2 250x HMGA x Lentiviral vector LTR LTR mir let7 ex1 ex2 ex3 ex4 ex5 HMGA2 gene

12 % Modified leukocytes HMGA2 dominance reduction with time Months post GT Cells with vector Cells with HMGA2 IS From the 5 th year integration sites of the SPATS2 and ZZEF1 genes outnumber HMGA2 Hb levels constsant after loss of HMGA2 dominance No adverse hematological or clinical effect of the HMGA2 gene in 7 years F-U follow up

13 ( Northstar study HGB-204, 2014) International, multicenter (USA-AUS-THAI), not randomized study, in adults with b-thal major yrs Primary objectives : Efficacy if >2 gr/hbat87q at mesi post GT Safety: absence of RCL, clone dominance, oncogenesis New vector Lentiglobin BB305

14 Vector modifications in study HBG U3-R-U5 gag Y env cppt RRE b-globin gene prom. HS2 HS3 HS4 R-U3 chs4 pa * CMV R-U5-gag Y env cppt RRE b-globin gene prom. HS2 HS3 HS4 R-U3 pa * HPV BB305 Deletion of chs4 insulator Exchange LTRHIV with CMV promoter Improvement of the structure and vector production process

15 Hb g/dl Trial HGB-204: early high production of globin with almost normal Hb levels and transfusion independence in 2/2 pts b /be Months after GT Subject 1: production of 6.7 gr/dl di b A-T87Q a 4,5 months after GT Subject 1: production of 4.2 gr/dl di b A-T87Q a 2 months after GT 6.7 b /be HbA T87Q HbA 2 HbF HbE HbA

16 Hb g/dl Hb fractions (HGB-205): b /be vs b /b b /be Months after GT b /b HbA T87Q HbA 2 HbF HbE HbA

17 Averages post-gt Hgbs at 1 year FU in b-thalassemia patients 4,9 0,2 1 * 5 0,2 1,3 4 4,7 b0/be b0/b0 HbAT87A HbA2 HbF HbE * *

18 10 8 TIGET-btal (Hb gr/dl after GT) b-tal1 (31 M b 39/b+IVS1-110) 6 4 b-tal4 (35 M b 39/b+IVS1-110) b-tal3 (36 F b 39/b+IVS1-110) b-tal5 (14 M b+ivs1 110/b+IVS1-110)

19 Conclusions Efficacy with some dependence from the b- thalassemia genotype Early efficacy of vector BB305 avoids transfusion requirement soon after transplant Same safety profile as autologous transplant with policlonal reconstitution and adverse events no higher than grade 3 The younger the better

20 Conclusions 2 Success linked to complete myeloablation Exceptional results of study 2 ascribed to better vector preparation and to trasduction conditions

21 Future perspectives: genome editing?

22 From random vector integration to gene correction through genome editing and homologous recombination b STOP e Gg Ag d b Cromosoma 11

23 Molecular tools for genome editing Zn FINGER NUCLEASIs (2001) TALEN (2010) CRISPR-CAS9 (2013) PNAs (2016) Normal Mutant Normal DNA NHEJ HDR Paolo Moi - Cagliari

24 CRISPR-Cas9 (Clustered regulatory interspersed palindromic regions- CRISPR associated system 9) The most recent and innovative tool of genome editing Copyright 2014 Elsevier Inc. Terms and Conditions

25 CRISPR/CAS9: a bacterial immune adaptative system

26 The CRISPR/Cas9 system (non homologous end joining, NHEJ) Indel Indel

27 CRISPR/CAS9 Nickase (Homology directed repair, HDR)

28 Reproduction of an HPFH state by CRISPR-CAS9 editing in human cells b-globin locus at 15p15.5 Gg Ag HPFH

29 Homozygous db -Thalassemia Hb: 13.5 gr/dl HbF: 100%

30 Genome-Editing in β-k562 cells CRISPR allin-one vector β-k562 Limiting dilutions 48 h 48 h FACSsorting

31 PCR screening of genome editing events (small indels) Target site Intergenic region Target site G ɣ prom G ɣ A ɣ prom A ɣ 3 amplicons

32 PCR screening of genome editing events (large indels) Target site Intergenic region Target site G ɣ A ɣ G ɣ prom A ɣ prom 3 amplicons G ɣ / A ɣ prom 1 amplicone

33 Expression of g-globin in edited clones g-globin Gg Ag wt H11 H10 H3 G8 G6 E12 D5 C

34 Globin expression of the edited globin genes (western blot) wt wt C1 D5 E12 G6 G8 H3 H10 H11 β-actina g-globina β-globina

35 Delezione omozigote del gene Gg nel clone H3 b d Ag Gg e

36 Deletion in the short arm of chromosome 11 in clone E11

37 Deletion in the short arm of chromosome 11 in clone E11

38 Traslocazione cromosoma 6: 11 nel clone B5

39 Traslocation chrom 6:11 in clone B5

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