Genome Editing In the Clinic Fyodor D. Urnov, PhD Project Leader and Senior Scientist, Sangamo...
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![Page 1: Genome Editing In the Clinic Fyodor D. Urnov, PhD Project Leader and Senior Scientist, Sangamo BioSciences, Inc. NAS International Summit on Human Gene.](https://reader036.fdocuments.in/reader036/viewer/2022062519/5697bfa91a28abf838c99c2c/html5/thumbnails/1.jpg)
Genome Editing In the Clinic
Fyodor D. Urnov, PhDProject Leader and Senior Scientist,
Sangamo BioSciences, Inc.
NAS International Summit on Human Gene EditingDecember 3, 2015
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Genome Editing Is In the Clinic
Fyodor D. Urnov, PhDProject Leader and Senior Scientist,
Sangamo BioSciences, Inc.
NAS International Summit on Human Gene EditingDecember 3, 2015
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Genome editing has expanded the definition of the term “druggable target”:
If it’s in the DNA, it’s a druggable target
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Bedside to Bench to Bedside
The use of somatic cell genome editing to create a disease-protective genetic state:
1. (T cells and HSPCs) CCR5 editing HIV2. (HSPCs) BCL11A enhancer editing β-thalassemia / SCD3. (Liver) In vivo targeted gene addition monogenic disease
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Pharmaceutical Drug Development Applied toGenome Editing
• Generate therapeutic reagents maximized for potency / specificity (the “medicinal chemistry” of genome editing)
• Using established regulatory frameworks to characterize the product appropriately for safety:– Tailored to the nature of the technology– Appropriate for the intended product and indication (risk-benefit)– In vitro (using multiple distinct assays / readouts)– In vivo (using appropriate models)
3’5’ C C A A C G C G A A T T A T G G C G G C G T G C G C T T A A C G C A T G G G T
G G T T G C G C T T A A T A C C G C C G C A C G C G A A T T G C G T A C C C AT A CA T G
3’5’
module module module
module module module
module linker
Fok linker
Fok linkerNH2
NH2module linker
module linker
module linker
T T G AA A C T
T T G AA A C T
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Human Genome Editing With Engineered ZFNs:Targeted Disruption
Urnov, Rebar, Holmes, Gregory Nature Reviews Genetics 2010
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Genome Editing for HIV
CCR5
CCR5 gene disruption
NHEJ error-prone repair
T cells
Cells returned to patient
Preclinical efficacy: Perez et al Nature Biotechnology 2008Manufacturing: Maier et al Human Gene Therapy 2013Clinical data from first cohort: Tebas et al NEJM 2014
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Subject 04-046
0 30 60 90 120 150 180 210 240 270 300 330
Cel
ls /
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tam
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pie
s/ m
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Absolute CD4 CountAbsolute CD8 CountPentamerVIral Load
Treatment Interruption
Days from Baseline
In-clinic Efficacy Readouts:Genome Modification, CD4/8 Count, Viral Load
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Editing Hematopoietic Stem Cells Endows All Their Progeny With the Desired Genomic Signature
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Disease-Protective Genetic Variation in β-thalassemia
BCL11A-
2008:
BCL11A ↓ = fetal globin ↑ = disease ameliorated or eliminated
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Genome Editing for β-thalassemia and Sickle Cell Disease:a “Simple” Plan
BCL11A
BCL11A disruption
NHEJ error-prone repair
HSPCs
Cells returned to patient
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The Achilles Heel of the BCL11A Erythroid Enhancer
Bauer/Orkin – Science Oct 2013“We propose the GWAS-identified enhancer of BCL11A as a particularly promising therapeutic target for genome engineering in the β-hemoglobinopathies.”
Vierstra et al Nat Meth 2015A genome editing-based “walk” across the enhancer using two orthogonal nuclease platforms illuminated a single, specific binding site for GATA1 as key to enhancer function in erythroid-specific BCL11A activation and fetal globin suppression. (also see Canver et al Nature 2015)
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Initial Hit Yielded Enhanced γ-globin Levels
Vierstra et al Nature Methods 2015
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Fine-tuning Cut Site Further Enhances γ-globin Levels
Vierstra et al Nature Methods 2015
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BCL11A Knockout vs Enhancer Disruption:Equivalent Effects on Fetal Globin in PB-derived HSPCs
• erythropoiesis proceeds indistinguishably to control cells (Vierstra et al 2015)• equivalent elevation of gamma globin in RBCs obtained from PB-derived HSPCs
Coding KO enhancer KO
Editing Level of fetal globin mRNA
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85% On-Target Editing in HSPCs Using Clinical Lead ZFNs
ZFN mRNA
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Genome Editing En Route to the Clinic:CD34 GMP Cell Manufacturing Process
CliniMACSCD34 enrichment
Day 0
Platelet depletion
Day 0
Day 0-1
Rest cells in cultureFormulate CD34 cells
Day 4
Gene modified CD34 cellsFinal Product after QC testing and release
Maxcyte ZFN mRNA electroporation
Day 2Day 2-4
Static culture step to allow ZFN gene expression
Apheresis from G-CSF/Plerixafor mobilized subjects
Day -2 & -1
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From GWAS to the Clinic
Proposal (Bauer et al (2013)): “We propose the GWAS-identified enhancer of BCL11A as a particularly promising therapeutic target for genome engineering in the β-hemoglobinopathies”
Findings:• An editable “Achilles Heel” in the enhancer• Enhancer KO elevates disease-protective fetal globin• Efficient knockout at clinical scale• On-track for IND filing H1 2016 for our beta-thalassemia program
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Readouts of Genome Editing Safety: Charted Path Based on Two Open INDs For ZFN-Driven HSPC Editing
Nuclease + cell/organ
Genome
Overall
Sequence-directed
Cell
In vitro
In vivo
DSB enumerationKaryotyping
Genome-wide, end-capture-guidedBioinformatics/biochemistry- guided
Soft agar transformationCell growthErythropoietic potential
NSG mouse engraftmentNSG mouse tumorigenicity
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Human Genome Editing With Engineered ZFNs:Targeted Gene Addition
Urnov, Rebar, Holmes, Gregory Nature Reviews Genetics 2010
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Targeted Addition of Therapeutic Gene to a “Safe Harbor”
Hockemeyer et al Nature Biotechnology 2009DeKelver et al Genome Research 2010Lombardo et al Nature Methods 2011Wang et al Nature Biotechnology 2015; NAR 015
40% targeted integration in HSPCs50% targeted integration in T cells
Safe harbor locus
ZFN (mRNA)Therapeutic gene
donor (AAV6)
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Targeted Addition of Therapeutic Gene to a “Safe Harbor”
40% targeted integration in HSPCs50% targeted integration in T cells
Safe harbor locus
ZFN (mRNA)Therapeutic gene
donor (AAV6)
Hockemeyer et al Nature Biotechnology 2009DeKelver et al Genome Research 2010Lombardo et al Nature Methods 2011Wang et al Nature Biotechnology 2015; NAR 015
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Human albuminZFN Pair and
Corrective Gene
Nucleic Acids Coding for ZFNs and Corrective Gene
in AAV Vectors
Peripheral IV Administration
AAV Traffics to LiverAnd ZFNs PermanentlyModify the Genome
AAV2/6 Vectors
Liver Cells SecreteCorrected Protein
In Vivo Protein Replacement (e.g. Factor IX)
ZFN
ZFN
Homology Corrective Gene
Targeted Gene Integration via Systemic AAV Delivery of ZFP Therapeutics: In Vivo Treatment of Monogenic Disease
Homology
Also see Li, Holmes, High et al Nature 2011
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In Vivo Genome Editing: Robust Circulating hF.IX Levels Following Its ZFN-Driven Addition to Albumin – at 60 wks
hF.IX Levels ALT Levels
Therapeutic hF.IX levels observed for 60 weeks (longest time point assayed)No change in ALT levels
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NHP Genome Editing:Therapeutic hF.IX Levels Following Its Addition to ALB
Low Medium High1:1:8 1:1:2 1:1:8 1:1:2 1:1:4 1:1:8 Ratio: ZFN : ZFN : Donor
ZFN Dose
3% Normal hF.IX
IND reviewed by FDA and is open – first study of in vivo genome editing
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Sangamo’s In Vivo Protein Replacement PlatformBroadly Leverageable
Hemophilia A Hemophilia B Gaucher Fabry MPS II (Hunter) Other…
Very strong promoter ZFN
ZFN
Donor: Homology Gene of Interest Homology
Albumin Gene:
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A quote from Sydney Brenner, and a look to the future
“Progress in science depends on new technologies, new ideas, and new discoveries – in that order” Sydney Brenner
Medicalconditions
Medicalconditions
HIV
β-thal/SCD Hem B
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A quote from Sydney Brenner, and a look to the future
“Progress in science depends on new technologies, new ideas, and new discoveries – in that order” Sydney Brenner
“Pharmacological-grade” genome editing:
• Potency• Specificity• Delivery• Regulatory path
Medicalconditions
Gene targets Clinical gradenucleases
Clinical-gradedelivery (CMC)
Safety assays
Regulatory path
CCR5
BCL11ASafe harbor
Potency Specificity
Ease Scalability
GMP
Medicalconditions
HIV
β-thal/SCD Hem B
In vitro Animal model
FDA RAC
IRB
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Debt of gratitude: “academic” research
Zinc finger designRebar and Pabo Science 1994Isalan Klug Nature Biotech 2001
EditingRouet and Jasin MCB, PNAS 1994Bibikova and Carroll MCB 2001, Genetics 2002, Science 2003Porteus and Baltimore Science 2003
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Debt of gratitude: collaborators and colleagues
CollaboratorsHIV: Carl June, Pablo Tebas, Bruce Levine (Penn); G. Blick, J. Lalezari.β-thalassemia: George Stamatoyannopoulos, Thalia Papayannopoulou, Kai-Hsin Chang (UW)Jeff Vierstra, John Stamatoyannopoulos (UW)Stuart Orkin, Daniel Bauer (Boston Children’s/DFCI)Haiyan Jiang, Siyuan Tan, Kasra Kasraian (Biogen)
SangamoEdward Rebar, Jeffrey Miller, Lei ZhangAndreas ReikLisa FoxMichael Holmes, Thomas WechslerPhilip GregoryDale Ando, Geoff Nichol
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