Nucleases for Genome Engineering Philippe Duchateau

2 Zinc-Finger Nucleases Chemical endonucleases Natural proteins 1 st endonucleases used for genome engineering Low apparent modularity (2 separable domains) Artificial protein : zinc finger protein (DNA bindingdomain) fused with a catalytic domain (FokI) 1st engineered endonuclease used to edit a human gene High modularity (6-8 separable domains “polydactyls”) Chemical DNA binding domain (TFO, polyamine) fused to effector (chemical or restriction enzyme) High modularity TALE Nucleases Meganucleases (homing endonucleases) DNA binding domain from Transcription Activator Like Effectors from Xanthomonas Very high modularity (potential code) Early stage technology Rare cutting endonucleases for genome engineering

The four major families of nuclease-mediated genome engineering methods 3 Gene correction Targeted insertion (knock- in) Gene inactivation Homologous Recombination Non-Homologous End Joining (NHEJ) Gene conversion Targeted deletion (knock- out)

Meganucleases induce high frequency gene targeting  : First meganuclease-induced targeting experiments by the Jasin (gene correction) and Dujon’s (gene insertion) laboratories  1995-present: technology proves to be robust and reproducible in several cell types, enables highly efficient gene knock-in, gene knock-out, mutagenesis, gene replacement and gene correction x x Classical gene targeting: of total meganuclease-induced gene targeting: of total

Meganuclease engineering

I-CreI (dimer) I-SceI (monomer) DmoCre or E-DreI (artificial hybrid) I-SceI I-DmoI I-CreI Chevalier et al., Mol. Cell 2002 Epinat et al., NAR 2003 I-CreI The choice of the scaffold: meganucleases from the LAGLIDADG family

Exploring a subdomain, using a semi-rational approach  Mutagenize 3 residues on protein (44, 68 and 70, complexity: 5832)  Mutagenize 3 bp on targets (complexity: 64)  Screen variants from the protein library against the 64 targets (about 1,000,000 combinations)  About 400 novel functional endonucleases cleaving 38 out of 64 targets Arnould et al. (2006) J. Mol. Biol. 355: I-CreI with target DNA

Characterization in extrachromosomal assays Yeast ( poins/week) CHO (10 4 points/week)

Meganucleases with tailored specificity can be generated using a combinatorial process  OMEGABASE, a collection of “building blocks” used in our combinatorial process: >48,000 proteins today  hit frequency:1/250 bp  Success rate: >=40% (>=25% for 1hit/100 bp)  Timeline: 10 weeks-9 month depending on difficulty (deliverable: meganuclease characterized in a cell-based assay) possible further refinement for therapeutic grade  capacity: 200 last year (200 different targets) I-CreI (dimer) Arnould et al. (2006) J. Mol. Biol. Smith et al. (2006) Nucleic Acids Res. Arnould et al. (2007) J. Mol. Biol. Grizot et al. (2009) Nucleic Acids Res.

Meganuclease efficacy and position effect

Sample: 37 MNs (and 39 targets) X Y % 38.5% 30.8% exon intergenic intron intron/exon ( <35% 35-45% >45% 41.0% 18.0% 53.3% 25.2% 21.5% > % 22.4% 8.6% Type of sequenceGC contentCpG number sample genome 61.5% 17.9% 20.5% 5.1% 2.4% 39.8% 57.8%

Efficacy is subject to position effect: A same meganuclease cleaves three cognate sites with different efficacies

Correlation between efficacy and micrococcal nuclease accessibility

TALENs OVERVIEW

TALEs and TALENs FOK AADAAD Nterm Cterm DNA binding domain PromoterR or S genes Disease or resistence promoting genes HDHD (RVD) C Moscou & Bogdanove, Science 2009 Cterm Nterm Cterm Nterm FOK First generation of TALENs DNA sequence to edit Christian & al, Genetics 2010 TALEs

An optimal TALEN TM architecture for efficient nuclease activity TALENs containing from 10 to 16 TALE repeats display high nuclease activity Nuclease activity (Yeast) We have defined an optimal DNA spacer length (15 bp) DNA spacer Binding domain Cterm Number of TALE repeat Nuclease activity (Yeast) We have defined an optimal scaffold design that displays high nuclease activity Nterm

Overcoming TALEN TM sensitivity to cytosine methylation Moscou & Bogdanove Science 2009 XPC1 DNA target TCCGAGATGTCACACAGAGGTACGACCC AGTCTGGATGACAGTGA Left targetSpacer NINI HDHD NINI HDHD NINI NGNG NGNGN HDHD NININ NINI HDHDN NGNG AGGCTCTACAGTGTGTCTCCATGCTGGG TCAGACCTACTGTCACT NGNGN NGNG HDHD NINI HDHD HDHD NGNG HDHDN NINI HDHD NGNG HDHD NINI NINI N*N* Right target or XPCT1L_HD or XPCT1L_N* XPCT1R HDHD Me

Optimization of TALEN TM activity toward the methylated XPC locus TALEN TM can be specifically designed to successfully process the methylated XPC locus TALEN TM XG or X* variants are active toward the endogenous methylated XPC locus whereas HD variant was almost inactive Targeted mutagenesis at the endogenous methyled XPC locus (293H cells) Nuclease activities toward unmethylated XPC target (CHO-KI cells)

APPLICATIONS

Endogenous gene tagging ATG GATXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTAA GFP ORF GATXXXXXXXXXXXXXXX ATG Left homology Right homology ORF of interest GFP ORF GATXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTAA ATG GFP tagged Gene of interest Integration matrix Principle

TALENs TM enable endogenous gene tagging in HeLa & U-2OS cells GFP-ACTB (U-2OS) GFP-ACTB (HeLa) GFP-TUBA1B (U-2OS) C RFP-ACTB (U-2OS) GFP-TUBA1B (U-2OS) Merge Mono gene tagging of Actine and Tubuline using ACTB and TUBA1B TALEN TM Double gene tagging ACTB and TUBA1B TALEN TM

TALENs TM enable endogenous gene tagging in iPS cells Mono gene tagging ACTB TALEN TM ACTB and TUBA1 TALEN TM allows for highly efficient gene tagging in iPS cells, U-2OS or Hela cells

Gene Knock Out: Principle ATGXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTAA ORF of interest to inactivate XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTAA YYYYYYYYYYYY ATGXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTAA Double strand break Non homologous end joining (NHEJ) pathway repair machinery Gene of interest Knock out

TALENs TM enable receptor Knock Out in primary T cells High level of receptor genedisruption obtained in primary T cells Phenotype is stable

TALENs TM enable triple gene Knock Out in primary T cells Efficient simultaneous triple KO with TALENs TM (mRNA) in primary T cells

Gene Correction: XPC gene therapy in patient cell lines Principle ATG XXXX--XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTAA ATGXXXXTGXXXXXXXXXXXXXXXXXXXXXYXXXYXXXYXXYXYXXXYXYXXXXXXXTAA Left homology ~1600 bp Right homology ~1800 bp ∆TG XPC ORF Integration matrix containing the missing TG dinucleotide ∆TG XPCT1 TALEN TM Gene correction can be assessed genotycally and phenotypically Me

Gene Correction: XPC gene therapy in patient cell lines Genotypic assessment of XPC gene correction G T A T G T G T A G A C T G T G T G C A G G G T G T G G G G C C C C C C C C T G T A T G T G T A G A C T G T G C A G G G T G T G G G G C C C C C C C C T DNA Sequencing of KI positive clones TALEN -+ Matrix + + % KI events KI specific PCR screening 75 % of KI positive clones were TG corrected TALEN TM efficiently generate KI positive clones (up to 8% without selection) A majority of KI positive clones are corrected for ∆TG deletion

XPCT1-N* TALEN TM treatment enables re-expression of XPC full length protein Expression of XPC is stable over 3 months (125 PD) XPCT1-N* TALEN TM treatment promotes full recovery of UV-C protection Targeted correction of XPC gene in XPC deficient cell lines XPCT1-N* TALEN TM treatment promotes genotypic and phenotypic correction of XPC and enables XPC deficient patient cell lines to recover their full UV- C resistance

TALEN TM NEXT GENERATIONS FOR IN VIVO APPLICATIONS

Next generations of TALENs TM First generation: TALENs TM Second generation: Compact TALENs TM -Size ~ 5.4 Kb -Two molecules to transfect -Size ~ 2.7 Kb -One single molecule to transfect -Can be inserted into AAV along with a repair matrix for in vivo therapeutic applications

Nuclease activity (Arbitrary Units) - 90% of the compact TALEN TM showed high activity in yeast (extrachromosomal) - 50% of the compact TALEN TM tested induced mutagenesis at endogenous locus - High frequencies of targeted gene insertion (TGI) are obtained at endogenous loci in 293H cells (18 days) - No toxicity detected Extrachromosomal assay (yeast)Activity at endogenous loci (293H) Homologous Gene Insertion (%) Activity of Compact TALEN