RAC-Clinigene Meeting, Bethesda, Dec 2010 iget SAN RAFFAELE TELETHON INSTITUTE FOR GENE THERAPY Improving the Safety of HSC Gene Therapy by Targeting/De-targeting Gene Transfer Luigi Naldini M.D., Ph.D. San Raffaele Telethon Institute for Gene Therapy, Milan, Italy Improving the Safety of HSC Gene Therapy • Regulate transgene expression – Ectopic or constitutive expression may cause toxicity • Alleviate risk of insertional mutagenesis – Improve vector choice & design • Target integration to specific sites Regulate Transgene Expression • Stable gene transfer requires integration into HSC – Ectopic or constitutive transgene expression may cause toxicity – Target gene expression to mature cells or selected lineage Targeting Gene Transfer • Transcriptional targeting Select lineage-specific transcriptional control elements → Transgene expression directed to desired cell lineage Promoter Transgene mirT • Post-transcriptional targeting Incorporate miRNA target sequences into vector transcript → De-target from unwanted cell types Regulating Transgenes by Endogenous miRNAs RISC Dicer Drosha PGK GFPDNA mirT miRNA X DNAPGKGenomicGFP DNA mirT target sequences for miRNA X Mature Cells Stem/Progenitor Cells miRNAs in Hematopoietic Stem Cells • Little is known of miRNAs expressed in hematopoietic stem/progenitor cells (HSPC) • Expression profile data – bulk vs. pure populations – in rare cell populations • Functional activity – expression level vs. activity – in vivo studies From Landgraf et al. Cell 2007 Bidirectional miRNA-Regulated Vectors GA RRE Amendola et al, polyA ∆LNGFR PGK GFP WPRE Nat Biotech 2005 293T Kidney Cells U937 Monocytes 4 4 10 1% 99% 10 3% 96% x=5 x=4046 x=6 x=630 3 3 10 y=63 y=1503 10 y=191 y=1214 2 2 10 10 1 1 10 10 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 4 4 10 10 142-3pT 142-3pT 11% 83% 77% 1% 142-3pT 142-3pT Bd.LV.miRT x=5 x=235 x=4 x=16 3 3 y=186 y=1139 10 y=1092 y=1433 LNGFR 10 ∆ 2 2 10 10 1 1 10 10 0 0 10 10 0 1 2 3 4 0 1 2 3 4 Brown et al, Nat Med 2006 10 10 10 10 10 10 10 10 10 10 Brown, Gentner et al, Nat Biotech 2007 GFP Profiling miRNAs in Mouse HSPC GA RRE polyA NGFR PGK d4GFP WPRE miRT Ψ 126T 126T 126T 126T SD SA 223T 223T 223T 223T ...T ...T ...T ...T Ex vivo HS/PC Identify HS/PC by immunophenotyping transduction Transplant into conditioned mice Bone Marrow miRNAs Specific for HSC / Early Progenitors miRNAs in Human HSPC and LV Regulation Transduce human HSPC Read miRNA activity NGFR PGK GFP miRT (CD34+ cells from cord In HSPC After DIFF blood or bone marrow) (2 days post (in vitro transduction) culture) Ctr 126T 130aT 223T - l H S CD34+38 P C CD34+38+ + 3 D CD1 I F + 35 F 2 D miRNA activity (Fold repression) C miR-126 in Human HSP & Myeloid Cells NGFR PGK GFP NGFR PGK GFP 126T - 2 repeats CD34+38 GFP G-CSF CD34+38+ NGFR CFU-G/- Myeloblast Promyelocyte Myelocyte Metamyelocyte/Gr GM CD34int13+11b- CD34-13+11b- CD13+11b+16- CD13+11b+16+ CD34hi13+ GFP.126T NGFR Summary: miRNAs in HS/PC • Quantitative functional profile of miRNAs – miRNAs specifically active in human and murine HSPC identified • Exploit them to – Improve the safety of HSC gene therapy Gentner, Visigalli, Hiramatsu, Lechman et al., Science Transl. Med., 2010 Gene Therapy of Leukodystrophies Arylsulfatase A Galactocerebrosidase ARSA GALC SO2 GALACTO CEREBROSIDE SULFATIDE CEREBROSIDE (CERAMIDE) METACHROMATIC GLOBOID LEUKODYSTROPHY LEUKODYSTROPHY Transplantation of autologous LV-transduced, enzyme over-expressing HSC Toxicity of GALC over-expression in HSPC but NOT in differentiated progeny De-target expression from HSPC GALC Expression in HSPC is Limited by Toxicity Galc-/- HSPC Differentiated HC LV. GALC 120 100 0,12 + 80 0,1 /mg) 60 mU 0,08 TUNEL TUNEL 0,06 % 40 0,04 20 0,02 GALC activityGALC ( 0 0 GALC GALC.126T GFP.126T UT GALC GALC.126T GLD Gene Therapy by LV.126T Wild-type HSPC + LV.GFP Transplant into neonate Galc-/- HSPC + LV.GALC.126T GLD mice Prolonged survival Dose-effect relationship 100 200 ** UT -/- GALC-126T 175 75 +/+ GFP 150 125 50 100 75 25 50 25 0 0 0 50 100 150 200 250 <3 >3 Days Brain GALC activity (nmol/mg/h) Summary: miR-126 LV & GLD Gene Therapy • miR-126 regulated LV – Protect human and mouse HSPC from GALC toxicity – Enable HSC-based gene therapy of GLD in mouse model – Can be broadly used to improve safety of HSC gene therapy Gentner, Visigalli, Hiramatsu, Lechman et al., Science Transl. Med., 2010 Site-Specific Gene Insertion • Minimizes risk of insertional mutagenesis • Insertion downstream endogenous promoter – Restores function and endogenous expression control (gene correction) – Most mutations, including insertions and deletions can be corrected • Insertion into a safe genomic site – Robust predictable expression without perturbing endogenous transcription Designed Zinc Finger Nuclease (ZFN) ZFP ZFP . Contains two domains: . Nuclease domain of FokI restriction enzyme . Engineered Zinc Finger Protein (ZFP) . Cleaves DNA as a dimer . Can be engineered to cleave a pre-determined sequence Kim & Chandrasegaran, PNAS USA 1996; Porteus & Baltimore, Science 2003 Bibikova & Carroll, Science 2003; Urnov et al. Nature 2005 Exploiting ZFNs for Gene Targeting DNA DSB at ZFN target site no yes donor template NHEJ HDR Gene Disruption Transgene Insertion Lombardo et al., Nature Biotechnology 2007 Efficiency of Targeted Integration in Human Cells % Transgene Cell Type Target Site Positive Cell Lines (several types) ILR2G, CCR5, AAVS1 up to 60% Primary Fibroblasts CCR5, AAVS1 up to 11% Primary Keratinocytes CCR5 up to 5% Primary Lymphocytes CCR5, AAVS1 up to 5% Hematopoietic ILR2G, CCR5, AAVS1 up to 0.3% Stem/Progenitors ES Cell Lines ILR2G, CCR5 up to 5% iPS Cells CCR5, AAVS1 up to 1% Neuronal Stem Cells AAVS1 up to 15% A. Lombardo, P. Genovese, E. Provasi, C. Bonini, G. Maruggi, F. Mavilio, B. Di Stefano, M. Neri Summary: Gene Correction • Knock-in of IL2RG cDNA downstream endogenous promoter – normal γ-c expression • Gene correction coupled to selectable marker – in primary SCID-X1 patient’s fibroblasts • Reprogramming by single copy excisable LV Vector- and Reprogramming Factors-free, gene corrected iPSC Insertion into a Safe Genomic Site? • Choice of site not obvious – Gene desert vs. inter- or intra-genic – Info available mostly for transcribed genome • Knock-out of target locus must be well tolerated – Insertion may inactivate target gene – ZFNs may disrupt both alleles – Candidate Loci: CCR5, AAVS1 • Robust & predictable transgene expression • Lack of interference with expression of the flanking genes Summary: Assessing Safe Insertions in the Genome • Transcriptional perturbation – Upregulation of flanking genes depends on type - but not strength - of exogenous promoter – varies with the targeted locus – interference with transcription of targeted gene can be prevented by vector engineering • AAVS1 has desirable features – No deregulation of flanking genes even with strong promoter – Stable robust expression Lombardo et al., submitted Contributors: HSC miRNAs & GLD Gene Therapy San Raffaele Telethon Univ. Toronto, Ontario Institute for Gene Therapy Inst. for Cancer Res. John Dick Brian Brown iget Hidefumi Hiramatsu SAN RAFFAELE Bernhard Gentner TELETHON INSTITUTE FOR GENE THERAPY Alice Giustacchini Eric Lechman Massimo Saini Francesco Boccalatte Univ. Perugia Mario Amendola Sabata Martino Giulia Schira Aldo Orlacchio Lucia Sergi Sergi Alessandra Biffi European Research Council Ilaria Visigalli Advanced Grant Silvia Ungari Angelo Quattrini Contributors: Site-Specific Integration San Raffaele Telethon San Raffaele Institute Institute for Gene Therapy Milan, Italy Angelo Lombardo Chiara Bonini iget Pietro Genovese Elena Provasi SAN RAFFAELE TELETHON INSTITUTE FOR GENE THERAPY Pietro Lo Riso Vania Broccoli Daniela Cesana Bruno Distefano Eugenio Montini Sara Maffioletti Angela Gritti European Research Council Margherita Neri Advanced Grant Sangamo BioSciences NCTD, Heidelberg, FRG Richmond, CA Christof von Kalle Philip D. Gregory Manfred Schmidt Michael C. Holmes Richard Gabriel.