ASGCT 2010 Emerging Field Review: Regulatory RNA iget SAN RAFFAELE TELETHON INSTITUTE FOR GENE THERAPY How to Study and Exploit microRNAs for Gene Therapy Bernhard Gentner, M.D. Naldini Lab San Raffaele Telethon Institute for Gene Therapy, Milan, Italy Presenter Disclosure Bernhard R. Gentner The following relationships exist related to this presentation: No Relationships to Disclose microRNA: an abundant class of non-coding RNA • >700 miRNA species known in human, most are highly conserved in mammals • negatively regulate HALF of ALL mRNA transcripts by • directing mRNA destruction • inhibiting translation • extent of regulation of single targets may seem minor (in general 2-3 fold reduction at the protein level); BUT: combinatorial regulation of multiple mRNAs • „It may prove difficult to find a biological function or process that is not influenced, at least to some degree, in some cell type, by miRNAs“ (David Bartel) From Filipowicz et al, Nat Rev Genetics 2008 Some microRNAs are expressed in a tissue, lineage and differentiation state specific manner Lineagespecific miRNAs Differentiationstate specific miRs /Prog. Tissue specific microRNAs Pan-Hem Lymphoid Myeloid Adapted from Baskerville and Bartel, RNA (2005) Adapted from Landgraf et al., Cell (2007) Gentner et al., unpublished Impact of RNAi on Vector Technology Specific gene knock-down Regulating vector expression Stable microRNA knock-down shRNA/artificial miRNA platforms to stably express siRNA Artificial miRNA TARGET sequences coupled to a… Artificial miRNA DECOY sequences coupled to a strong promoter Marker gene: miRNA reporter Therapeutic cDNA: Regulated transgene expression Viral Genome: Control Tropism Regulating Transgenes by Endogenous miRNAs RISC Dicer Drosha PGK GFP DNA mirT miRNA X DNA PGK GFP Genomic DNA mirT Cell not expressing miRNA Cell expressing miRNA “Target” “Off Target” target sequences for miRNA X (I) microRNA reporter vectors Identify pluripotent cells and their differentiated progeny • Purge the culture to avoid in vivo tumor formation Hematopoietic • Establish differentiation protocol Modified from Kiskinis et al, J. Clin. Invest. 120:51–59 (2010). • Identify karyotypically normal, reprogrammed cells and establish line miRNA reporter: pluripotent vs. non-pluripotent GFP GFP.ES-specific miRT Thymidine kinase.ES.miRT GFP.non-ES miRT Puromycin. non-ES miRT Enrich for pluripotent cells (ES/iPS) ES-specific miRT reporter to identify cells which have lost pluripotency upon ES/iPS differentiation GFP 302aT 302aT 302aT 302aT GFP direct fluorescence Tuj1 TO-PRO3Merge Embryoid Body (30d) ES ES-derived Neurons Brown, Gentner et al, Nat Biotech 25(12):1457-67 (2007) GFP direct fluorescence Tuj1 TO-PRO3Merge Potential applications of miRNA reporters for ES/iPS cell therapy miRNA regulated vector Transgene on e.g. GFP reporter FACS sort Transgene off e.g. suicide gene kill contaminants 124aT neurons 125bT Patientspecific iPS Differentiation 302aT 122T liver 375T pancreatic islets Select let7T 133aT muscle 206T Differentiation-induced miRNAs: induction of the miRNA switches transgene off 142T hematopoietic Identify neurons in ES differentiation cultures by a miRNA-124 reporter GFP 124aT 124aT 124aT 124aT GFP direct fluorescence Tuj1 TO-PRO3Merge Embryoid Body (30d) ES Embryoid Body (30d) Brown, Gentner et al, Nat Biotech 25(12):1457-67 (2007) GFP direct fluorescence Tuj1 Merge “miR-ON” designs Pro Repressor miRT TetO7 Pro Reporter Double transduction miRNA absent Pro Repressor miRT miRNA present TetO7 Pro Reporter Pro Repressor miRT TetO7 Pro Reporter Bidirectional miRNA reporter vectors (BdLV) Artificial microRNA target sequences miRT Reporter 1 BdPro Reporter 2 RISC Dicer Drosha miRNA Brown et al, Nat Med 12(5):585-91 (2006) miRT Reporter 1 BdPro Reporter 2 Brown, Gentner et al, Nat Biotech 25(12):1457-67 (2007) Reporter BdLV to report and measure miRNA activity in complex cell populations by FACS GA RRE polyA NGFR PGK GFP WPRE Ψ SD SA Ex vivo BM transduction (C57 mice) Bd.LV.ctrl 3‘UTR Bd.LV.miRT no modification in miRT miRT Bone Marrow Transplantation into congenic mice miRT miRT MFI(x) : MFI(x ) MFI(y) : MFI(y) BdLV.223T MFI(y) PB Leuko BdLV.GFP : Fold Repression (miR-223) miR-223 marks the myeloid lineage 40 30 20 10 0 Granu Mono B cells T cells MFI(x) Mono T cells GFP GFP Granu B cells GFP BdLV.GFP NGFR NGFR BdLV.223T Summary: miRNA reporter vectors • Easy to use reporter system to identify/select iPS/ES, monitor lineage-specific differentiation and purge grafts for cell therapy Brown et al, Nat. Biotech 2007 – Enhanced designs: miR-ON vector: presence of miRNA positive signal; Bidirectional reporter vector • Quantify miRNA activity by FACS at the single cell level, in rare and incompletely defined cell types – miRNA footprinting in the hematopoietic system Brown et al, Nat. Biotech 2007; Papapetrou et al J.Clin.Invest 2008; Gentner et al, under consideration – Hematopoietic stem cell-specific miRNAs see Abstr#337 Friday 21/5 2pm (Maryland ABC) (II) Regulating transgene expression Next Generation Gene Therapy • Transcriptional targeting Promoter Transgene mirT • Detargeting from unwanted cell types • ON/OFF switch responsive to cell activation, differentiation • Combinatorial arrangement Application I: avoid immune clearance/induce tolerance Potential solution: de-target the hematopoietic tissue Transgene expression in antigen presenting cells might trigger immune response PGK GFP TO-PRO PGK GFP 142T F4/80 PGK GFP 142/122T CD31 Brown et al, Nature Med 2006 Brown et al, Nature Biotech 2007 Brown et al, Blood 2007 Annoni & Brown, Blood, 2009 Application II: avoid stem/progenitor cell toxicity in hematopoietic stem cell-based gene therapy Sustained myeloid or multilineage gene addition requires HSC transduction with integrating vectors Ubiquitous transgene may reduce HSPC fitness Counterselection Selection for non-neutral integrations Transcriptional regulation not sufficient to overcome this issue: • Weak transcriptional activity • Limited specificity De-Targeting HSPC • HSPC- and Myeloid Precursor- specific miRNAs with high activity identified – Detarget transgene expression from HSPC • miRNA downregulation during myeloid and lymphoid differentiation switches transgene ON – Transduce the stem cell, express the transgene only in the diseased progeny • miRNA regulation can overcome GALC toxicity in HSPC while maintaining transgene expression in monocytes/macrophages see Abstr#337: Gentner et al, Friday 21/5 2pm (Maryland ABC) see Abstract #313: Visigalli et al, Friday 21/5 2pm (Delaware AB) Application III: avoid negative thymic selection for T cell engineering Application IV: limit hepatotoxicity in suicide gene therapy of liver tumors Application V: Change virus tropism VSV (-RNA) Let7 ubiquitously expressed (except pluripotent cells and some cancer), preventing VSV replication in normal tissues 3x let7T COXSACKIE A21 (+RNA) 2x 133aT 2x 206T miR-133a and miR-206 are expressed in muscle, preventing CAV21 induced lethal myositis while retaining its oncolytic activity POLIO( +RNA) 124T miR-124a expressed in neurons. Attenuation of polio neurovirulence, while permitting replication in non-neuronal tissues induction of protective immunity strategy for live vaccine development • Strategy also applicable to control DNA virus replication (Adeno): Ylösmäki et al, J Virol 2008; Cawood et al, PLOS Pathogens 2009; Leja et al, PLOS One 2010 • Viral breakthrough can occur: Cawood et al, Leia et al, Kelly et al, PLOS Pathogens 2010 Stable Knock-down of microRNA Competitive microRNA inhibition August 2007 • Strong promoter to overexpress tandem binding sites for miRNA • “soak up” miRNA with a complementary seed region, i.e. the whole miRNA family • In cell lines as effective as chemically modified antagomirs Combine a “sponge” construct with a lentiviral delivery platform stable miRNA knockdown (submitted: June 2008) Regulated targets vs. miRNA knockdown Vectors can be rationally designed to be regulated by miRNA or to knock down miRNA Regulated targets Knockdown targets • express at physiologic levels •Perfectly complementary to the miRNA • ≤ 4 closely spaced miRT copies • miRT needs to be overexpressed •mispaired with central region of the miRNA (around nt. 10) • 6-8 miRT copies •may combine sites for co-expressed miRNAs • kd easier for low-expressed miRNAs • need highly expressed miRNAs •avoid miRNA which are members of a • whole miRNA family is knocked down family Gentner et al, Nat. Methods 2009; Brown&Naldini, Nat.Rev.Gen. 2009 miRNA knockdown: a matter or miRT dose? 10000 CTRL LV 1000 MFI (GFP) Wpre GFP Prom LV.PGK.dGFP LV.SFFV.dGFP 1058_1e6 TUpML.fcs 1038_1e6 TUpML.fcs SFFV.223T Count 1 296 UT 148 443 148 M1 10 100 VCN 0 1 10 10 296 2 3 10 FL 1 Log 10 0 4 10 10 LV.223T Ctrl.LV 1058_1e7 TUpML.f cs 1 10 2 10 FL 1 Log 3 10 4 10 10000 1038_1e7 TUpML.fcs 591 591 M1 M1 1000 443 443 Count Count 148 296 296 148 148 0 0 0 10 0 0 101 10 2 10 FL 1 Log 13 1010 4 10 2100 10 FL 1 Log 1058_3e7 TUpML.f cs 1 10 32 10 FL 1 Log 10 3 10 4 10 4 10 MFI (GFP) Count 10 0 443 296 0 moi: 100 SFFV.dGFP 1 M1 M1 591 443 0 PGK.dGFP 10 PGK.223T 1 M1 M1 100 1038_3e7 TUpML.fcs 591 591 443 443 Count GFP Count moi: 300 100 591 1058_1e6 TUpML.fcs 591 Count moi: 10 223T LV moi: 10 Saturation of miR-223? 0 296 296 148 148 GFP Reporter regulated by miR-223. 1 10 0 0 0 10 1 10 2 10 FL 1 Log GFP 3 10 4 10 0 10 1 10 2 10 FL 1 Log GFP 3 10 4 VCN 10 U937 cells 100 Loss of reporter regulation coincides with upregulation of natural miRNA targets Scrambled miRT 223T or or 223BT Western Blot Analysis SFFV Wpre GFP UT U937 cells 100000 SFFV.GFP SFFV.142BT 223BT SFFV.142T 223T scrT 223T NFI-A 60 kD 55 kD GAPDH 36 kD 1000 p=0.02 p=0.02 Counts MFI (GFP) 10000 100 10 GFP 1 1 10 100 moi (LV.SFFV) 1000 223BT In vivo Skewing of Hematopoietic Lineage Proportions by SFFV.miRT Vectors LV.SFFV.dGFP.223T or Control.SFFV RSV GA RRE SFFV GFP Wpre Ψ Mice transplanted with SFFV.223Ttransduced cells show • Neutrophilic Leukocytosis in the PB • and.... Gentner et al, Nat.Methods 2009 Peripheral Blood of Transplanted Mice Inflammatory Lung Pathology in Mice transplanted with HSC overexpressing SFFV.223T SFFV.223T Control SFFV Phenotype of mice transplanted with SFFV.223T-transduced cells closely resembles the phenotype of miR-223 knock-out mice (Johnnidis et al, Nature 451: 1125-30, 2008) Strict dose-dependance of miRNA knockdown in vivo n= 7 mice: SFFV.223T RSV GA RRE SFFV GFP Wpre n= 8 mice: PGK.223T RSV Ψ GA RRE PGK Ψ p < 0.05 GFP Wpre Take home messages: Regulation/Saturation • Need to find dose window for each single miRT sequence – Most of the highly expressed miRNAs (1 log above let7a) will have a therapeutic window for regulating vectors – Knowledge on k/d phenotype required to assess a safe dose window • Stable miRNA knockdown by miRT overexpression – Works! tool to study miRNA biology; allows long term steady state analysis, can be targeted to specific cell types; “cleaner” and cheaper than antagomirs in vivo – Carefully optimize promoter, miRT sequence, transduction conditions crucial to avoid non-specific toxicity, especially in primary cells Contributors & Collaborators Fondazione San Raffaele, Milan, Italy iget SAN RAFFAELE TELETHON INSTITUTE FOR GENE THERAPY LUIGI NALDINI Giulia Schira Alice Giustacchini Erika Zonari Francesco Boccalatte Massimo Saini Mario Amendola Alessio Cantore Lucia Sergi Sergi Angela Gritti Annalisa Lattanzi Alessandra Biffi Ilaria Visigalli Silvia Ungari M.G. RONCAROLO Brian Brown John Dick Eric Lechman Hidefumi Hiramatsu H.P. Kiem Brian Beard Maurilio Ponzoni Vania Broccoli Bruno Di Stefano Cesare Galli Silvia Colleoni Anders Björklund Johan Jakobsen