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

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GENE EDITING WITH CRISPR/CAS9: OPPORTUNITIES FOR HUMAN THERAPEUTICS

Bill Lundberg, MD 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

3 ¡ # publications

The CRISPR-Cas Adaptive Immune System

Baya et al. Ann. Rev. Genet. 2011

4 ¡

CRISPR-Cas9 as a Tool for Gene Editing

5 ¡

Knockout Correct / Insert Cas9 DNA Break Gene Editing Cas9 DNA Binding Gene Regulation Repress Activate

“…RNA programmed Cas9 … could offer considerable potential for gene-targeting and genome-editing applications” Jinek Chylinski… Doudna Charpentier Science (2012) Cas9 programmed with crRNA:tracrRNA duplex or single guide RNA (sgRNA)

Cas9 “Nickase” and dCas9

Jinek et al. Science 2012 Qi et al. Cell 2013

CRISPRs vs. TALENs in hPSCs

Ding et al. Cell Stem Cell 2013

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

CRISPR/Cas TALEN VS.

Efficiency (% of mutant clones) 50-80% 0-30% Homozygous KOs 7-25% 0% Knock-ins >10% ~1%

Ding et al. Cell Stem Cell 2013

Low Incidence of Off-Target Mutations

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

Off Target Frequency - Compared to Control

9 ¡

64.433 91.176 0.001 0.01 0.1 1 10 100 Indel Frequency %

Mock CRISPR

CRISPR Internal Data

Cool Things You Can Do With CRISPR

10 ¡

′

Mali et al. Nat Methods 2013

Gene Editing

• 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

Shalem et al. Science 2014

Genetic Screens Animal Models

Yang et al. Cell 2013

Cool Things You Can Do With CRISPR

Publications

• Gene disrupt or correct
• Genome-scale screens
• Modify cell lines
• Modify primary cells
• Multiplex editing
• Rapid multiplex mouse models
• In vivo editing
• Nickases
• Gene repression/activation
• Targeted epigenetic modification
• Gene or chromosome marking
• Modify other organisims
• Therapeutics for human disease
• Implications for agriculture, crop sciences, foods, and other areas

11 ¡

#CRISPRFacts

7/29/2015

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· Jul 24

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

RETWEET

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@thegeach Happens to everyone! I put a 96-well plate in the Tecan without a lid or sticker the other day and it all evaporated... ;_;

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@thegeach I just have a Google Doc for each project. Saves right to the cloud and I can link other protocols, add pics etc.

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“It Just Works”

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

13 ¡ HSCs

• Monogenic diseases
• Enzyme replacement
• Other indications e.g. HIV

Oncology cell therapies

• CAR-Ts, TCR

Liver

• Metabolic diseases
• Enzyme replacement

iPSC cells

• Regenerative

cell based therapies

Ex vivo In vivo

Eye

• Monogenic diseases
• Complex indications

Other organs e.g. brain, lung, systemic

Delivery Required

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

“Selecting the study dose(s) of a CT product can be challenging.”

16 ¡ Considerations for the Design of Early-Phase Clinical Trials of Cellular and Gene Therapy Products _________________________________ Guidance for Industry

FDA Guidance June 2015

• Guideline on quality, non-clinical and clinical aspects of

medicinal products containing genetically modified cells

• Guideline on the quality, non-clinical and clinical aspects

4

• f gene therapy medicinal products

5

Draft

6

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

DATE

GENE EDITING WITH CRISPR/CAS9: OPPORTUNITIES FOR HUMAN THERAPEUTICS

Bill Lundberg, MD CSO, CRISPR Therapeutics