GENE EDITING WITH CRISPR/CAS9: OPPORTUNITIES FOR HUMAN THERAPEUTICS - PowerPoint PPT Presentation

GENE EDITING WITH CRISPR/CAS9: OPPORTUNITIES FOR HUMAN THERAPEUTICS Bill Lundberg, MD DATE CSO, CRISPR Therapeutics September 9, 2015

Disclosures § I ¡am ¡a ¡paid ¡employee ¡of ¡and ¡own ¡shares ¡in ¡CRISPR ¡Therapeu8cs ¡ § The ¡opinions ¡expressed ¡here ¡are ¡my ¡own ¡ 2 ¡

The CRISPR Craze “Now you can essentially change a genome at will to almost anything you want. The sky's the limit.” - Craig Mello Nobel Laureate 2006 and Scientific Founder CRISPR Therapeutics # publications 3 ¡

The CRISPR-Cas Adaptive Immune System 4 ¡ Baya et al. Ann. Rev. Genet. 2011

CRISPR-Cas9 as a Tool for Gene Editing Cas9 programmed with crRNA:tracrRNA duplex or single guide RNA ( sgRNA ) “…RNA programmed Cas9 … could offer considerable potential for gene-targeting and genome-editing applications” Jinek Chylinski… Doudna Charpentier Science (2012) Cas9 “Nickase” and dCas9 Cas9 DNA Break Cas9 DNA Binding Gene Editing Gene Regulation Knockout Correct / Insert Repress Activate Jinek et al. Science 2012 5 ¡ Qi et al. Cell 2013

CRISPRs vs. TALENs in hPSCs TALENs CRISPRs Chr ll Efficiency Efficiency Efficiency of Gene Line b (Start (Mutants/Clones (Mutants/Clones Homozygous Screened) c Screened) c Mutants AKT2 ES 9 chr19:40762 8.9% (17/192) AKT2 ES 9 chr19:40762 60.6% (86/142) 12.7% (18/142) CELSR2 HUES 1 c 3.5% (18/506) CELSR2 HUES 1 c 66.2% (45/68) 7.4% (5/68) CIITA iPS chr16:10989 12.7% (37/292) CIITA iPS chr16:10989 78.7% (96/122) 11.5% (14/122) GLUT4 HUES 9 chr17:71866 33.5% (52/155) GLUT4 HUES 9 chr17:71866 66.5% (123/185) 24.9% (46/185) LDLR ES 9 chr19:11210 0% (0/568) LDLR ES 9 chr19:11210 51.1% (90/176) 8.0% (14/176) LINC00116 HUES 9 chr2:110970 29.5% (26/88) LINC00116 HUES 9 chr2:110970 57.4% (93/162) 8.6% (14/162) SORT1 exon 2 ES 1 chr1:109912 22.2% (128/576) SORT1 exon 2 ES 1 chr1:109912 68.5% (100/146) 13.0% (19/146) SORT1 exon 3 HUES 9 chr1:109910 10.9% (21/192) SORT1 exon 3 HUES 9 c 75.9% (148/195) 10.3% (20/195) 1.6% (3/192) d AKT2 E17K ES 9 chr19:40762 10.6% (10/94) d 1.1% (1/94) d AKT2 E17K ES 9 chr19:40762 AKT2 off-target chr5:226839 Ding et al. Cell Stem Cell 2013

CRISPR/Cas TALEN VS. Efficiency 50-80% 0-30% (% of mutant clones) 7-25% Homozygous KOs 0% >10% Knock-ins ~1% Ding et al. Cell Stem Cell 2013

Low Incidence of Off-Target Mutations Off-Target Sites N-Fold Enrichment of Reads with Variants (vs. Control) crCCR5 Significant Off-Target On-Target Total Sites Off-Target InDel Off-Target Other Sites* Combined Combined** A 19 0 0.83 1.16 1.06 39.87 B 30 1* 1.14* 1.28 1.22 63 C 23 0 1.07 0.94 0.96 23.78 D 18 0 1.08 0.84 0.88 40.35 Q 36 0 0.98 0.78 0.85 57.66 Mandal et al. Cell Stem Cell 2014

Off Target Frequency - Compared to Control 64.433 91.176 100 Mock 10 CRISPR Indel Frequency % 1 0.1 0.01 0.001 9 ¡ CRISPR Internal Data

Cool Things You Can Do With CRISPR Genetic Screens Shalem et al. Science 2014 Gene Editing Animal Models Fig. 2. GeCKO library design and application for genome-scale negative selection screening ( A ) Design ofsgRNA library for genome-scale knockout of coding sequences in human cells Mali et al. Nat Methods 2013 Yang et al. Cell 2013 10 ¡ ′

7/29/2015 Dr. Someone (@Splinky2112) | Twitter Cool Things You Can Do With CRISPR “It Just Works” New to Twitter? Sign up Publications #CRISPRFacts Have an account? Log in Search Twitter Gene disrupt or correct • Genome-scale screens • TWEETS FOLLOWING FOLLOWERS FAVORITES Follow 94 81 18 170 Modify cell lines • Modify primary cells • Tweets Tweets & replies Photos & videos Dr. Someone Multiplex editing • @Splinky2112 Popular now Rapid multiplex mouse models • Introvert, anime nerd, grad student. This is a non-judgemental space to both Dr. Someone @Splinky2112 · Jul 24 In vivo editing • complain and celebrate CRISPR can find you a 1-bdrm in Berkeley for under $1000/mnth! Jk, you still need to share with 4 other people #crisprfacts #berkeley Berkeley, California Nickases • RETWEET FAVORITE Gene repression/activation 1 1 • 8 Photos and videos Targeted epigenetic modification • 7:19 AM - 24 Jul 2015 · Details Gene or chromosome marking • Modify other organisims • Dr. Someone @Splinky2112 · Jul 23 @thegeach Happens to everyone! I put a 96-well plate in the Tecan Therapeutics for human disease • without a lid or sticker the other day and it all evaporated... ;_; View conversation --------------- Implications for agriculture, crop sciences, foods, and other areas • Dr. Someone @Splinky2112 · Jul 21 New to Twitter? @thegeach I just have a Google Doc for each project. Saves right to the cloud and I can link other protocols, add pics etc. Sign up now to get your own personalized timeline! View conversation Sign up Dr. Someone @Splinky2112 · 2h 11 ¡ Late to my first Safety Committee Meeting You may also like · Refresh #toocoolforgradschool Ellen DeGeneres @TheEllenShow Dr. Someone retweeted Washington Nationals @Nationals NIH Bear @NIH_Bear · 12h Did you know they let Canadians in space? NFL_IRONMAN @LFletcher59 2 6 https://twitter.com/Splinky2112 1/4

Gene Editing - Principles • Guide design matters - Highly selective guides can be identified • Cell type matters – Editing efficiency varies across different cell lines/cell types • Cannot predict relative ability to delete intervening DNA between to guide sites from individual guide NHEJ disruption rates • For moderate deletion, size does not correlate with deletion efficiency • No bioinformatics tool is good enough to use alone to select optimal guides • OPTIMIZING GUIDES IS LARGELY EMPIRIC AND SYSTEM DEPENDENT 12 ¡

Delivery - Key Challenge For In Vivo Applications Ex vivo In vivo Liver HSCs • Metabolic diseases • Monogenic diseases • Enzyme replacement • Enzyme replacement • Other indications e.g. HIV Eye Delivery Oncology cell • Monogenic diseases Required therapies • Complex indications • CAR-Ts, TCR Other organs e.g. brain, lung, systemic iPSC cells • Regenerative cell based therapies 13 ¡

Making a “CRISPR” Medicine: Some of the Challenges • Pharmacodynamics • Regulatory framework and Product Development: Selecting the Dose as an example • Safety – off-target effects • Manufacturing and controls 14 ¡

Pharmacodynamics (Proof-of-Principle) • Animal Models – Do they need to be “Humanized” ? • Assess Pharmacology: “Amount” of gene edit • Functional tests (restoration of function) • Marker of biological activity • Impact on ‘clinical’ endpoint • • Comparability to product candidate 15 ¡

Regulatory Framework and Product Development: Selecting the Dose as an Example Considerations for the Design of Guideline on quality, non-clinical and clinical aspects of Early-Phase Clinical Trials of medicinal products containing genetically modified cells Cellular and Gene Therapy Products _________________________________ Guideline on the quality, non-clinical and clinical aspects 4 of gene therapy medicinal products 5 Guidance for Industry Draft 6 FDA Guidance June 2015 “Selecting the study dose(s) of a CT product can be challenging.” 16 ¡ ● ● ● ● ● ● ● ● ●

Manufacturing Process and Quality Ex-vivo CD34+ HSC as an Example 17 ¡

Creating CRISPR-Based Therapeutics CRISPR-based gene editing is efficient and can be highly specific • Optimizing guides is largely empiric and system dependent • Creating a new medicine requires good drug development • CRISPR Therapeutics research operations established in Cambridge MA – • expanding to 19,000 SF Accelerating programs towards the clinic • Hiring! Come join us: www.crisprtx.com/careers • 18 ¡

CRISPR-Cas9 Mediated Genome Engineering 19 ¡ Doudna and Charpentier Science 2014

GENE EDITING WITH CRISPR/CAS9: OPPORTUNITIES FOR HUMAN THERAPEUTICS Bill Lundberg, MD DATE CSO, CRISPR Therapeutics