Precise Genome Editing (PGE): Pathway to the Clinic

Mick Fellows EBE Regulatory Conference on ATMP 5 December 2017

Clustered regularly interspaced short palindromic repeats. A Brief History of CRISPR

1993 - 2005 Discovery of CRISPR and its function. Mojica 2005 Discovery of Cas9 and PAM. Bolotin 2006 Hypothetical scheme of adaptive immunity. Koonin 2008 Spacer sequences are transcribed into guide RNAs. van der Oost CRISPR acts on DNA targets. Marraffini and Sontheimer

2010 Cas9 cleaves target DNA. Moineau. 50 Publications 2011 Discovery of tracrRNA for Cas9 system. Charpentier 2011 – 2012 CRISPR systems can function heterologously in other species and Biochemical characterization of Cas9-mediated cleavage. Siksnys 2012 Similar and Single guide RNA. Charpentier and Doudna 150 Publications 2013 CRISPR-Cas9 harnessed for genome editing in mammalian cells. Zhang 2013, 300 Publications. 2016, nearly 3000!!

Cas9 double strand break repair promotes gene editing Precise Genome Editing (PGE)

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•  RNA-guided endonuclease, occurring in nature (bacteria)

•  Specificity programmed by short guide RNA (sgRNA)

•  Easy to design and use system. multiplexing possible, engineering possible

•  Most insertion/deletion (INDEL) Repair by NHEJ (esp in none dividing cells)

•  Homology directed repair (HDR) required for gene insertion (low non-dividing cells). Still a major challenge

•  CRISPR Cas9 has most press but other technologies already in the clinic (Zinc Finger Nucleases) and/or progressing fast TALEN, Meganuclease

Cas9

DNA HNH

RuvC/RNaseH-like Template DNA

Stop Codon INDEL

PAM

gRNA

NHEJ: non-homologous

end joining

HDR: homology directed repair

Key safety concerns

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Relevance for PGE •  No safe dose of a DNA reactive mutagen

•  Limits its use to unmet need in life threatening condition?

•  Standard genetox tests: •  Often use single gene targets •  Low sensitivity for DNA strand breakage

•  What is needed to ensure PGE is safe for use as a therapeutic?

•  Bioinformatics to predict off targets? •  NGS to determine mutation sites, what method, how sensitive?

•  But: quote George Church Harvard Medical School ‘..... CRISPR seems capable of less than 1 error (off-target cut) per 300 trillion base pairs’ •  Equates to one off-target hit per 100,000 cells •  Human cell: >100 endogenous DNA strand breaks per day

The debate will continue Safety considerations for PGE

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Recent(ish) paper, Schaefer et al •  Unexpected mutations after CRISPR–Cas9 editing in vivo

Nature Methods 14 547–548 (2017) doi:10.1038/nmeth.4293

•  Co-injection into FVB/J mouse zygotes of Cas9 plasmid, single guide RNA (Pde6b gene correction for blindness) and 3 ng/µL Cas9 protein

•  Unusual dual Cas9 administration

•  Somewhat less optimistic than Church. Schaefer’s group found significant indels (>100) but also >1000 single-nucleotide variants (SNVs), including some in exons

•  This has caused a major stir in the CRISPR community

•  Initially, shares in several Genome Editing Therapeutic companies fell by over 10% (later recovered)

Safety considerations for PGE

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http://arep.med.harvard.edu/pdf/Schaefer_Opinion_2Jun2017.pdf

•  Genetic differences likely present prior to editing: •  Two treated transgenic mice, compared against a single control •  Control of single ‘co-housed mouse rather than parent or litter

mate was inappropriate, no sham controls •  Previous data shown reported nearly different 1000 single

nucleotide variants (SNV) between untreated litter mates. •  Other groups since reported SNV’s commonly seen •  Unusual ‘dual’ Cas9 delivery and guide used had propensity for

off target effects that would not be selected for therapeutic use. However, the actual predicted off-targets were not seen!

•  Mutations in the two mice were often in identical positions, strongly suggesting they were pre-existing

•  Probably need 50+ mice to be tested to generate real data •  A follow-up study is required with appropriate controls.

Anyone's guess of how many labs are now doing this!

•  We are in a world when bad news travels fast •  Would a negative study have been published? •  Remember ASO’s and triplex formation?

Including from George Church and Harvard compatriots

The debate will continue

Ex vivo now

• Ex vivo PGE safety concerns are monitorable in target cells • Sangamo zinc finger precedent: HIV T-cell CD5 in Phase II trials (>70 patients treated) • Off-target measurable with NGS and in vitro cell transformation models can be used • Low risk of Cas9 & vector toxicity / immunogenicity • Follow current EMA and FDA cell therapy guidance. Updates required?

Translation to systemic

•  In vivo PGE safety translation activities • In vivo off target analysis. CircleSeq, SiteSeq etc. On target site analysis for rearrangements • Investigations into DNA repair & novel in vitro / in vivo oncogenicity assays. Functional consequences • Research into novel delivery vectors • Early regulatory interactions

3-5 Years?

• Systemic PGE safety concerns need to be addressed • Off-target: potential in any tissue, germline exposure, contribution from human SNP’s? • Cas9 & delivery vector toxicity / immunogenicity? • No current regulatory guidance • Translation of animal safety model data to man is difficult (different DNA coding)

Key CRISPR Safety Considerations Lessons From Gene Therapy Safety Issues •  Leukaemia on early clinical trials (gamma-retroviral vectors) •  Jesse Gelsinger: death from Ad vector induced shock syndrome •  Potential for regulatory caution with new gene editing technology •  Ensure similar mistakes are not made on early CRISPR clinical trials

Jesse Gelsinger died aged 19, gene therapy for ‘mild’ OTC deficiency

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Recent EMA Expert Meeting

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•  Nearly all the major PGE companies represented •  Editas Medicine, CRISPR Therapeutics, Intellia, Sangamo

Therapeutics, Cellectis, Bluebird Bio •  Highlights

•  All are working on more specific editing technologies (e.g. new versions of Cas9)

•  Most companies following ex vivo approaches (especially blood disorders) or liver specific targets

•  All companies using guide screening in silico (easy but biased) •  Most using versions of Joung labs off target analysis methods (Gude-

seq/Circle-seq) or similar (e.g Digenome-seq) •  Most investigating potential chromosomal rearrangements around on-

target site (significance to be better understood) •  Cas9 toxicity as move towards in vivo delivery:

•  Immunogenicity? •  Stem and germ cell lineage? •  Influence of chromatin modification? •  Individual SNPs?

•  EMA very positive that with appropriate Risk/Benefit analysis PGE holds great promise.

•  Early regulatory interactions will be key

Summary London Oct 2017

Conclusions

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•  Pathway to the clinic for ex vivo approaches is being established:

•  In silico analysis •  Unbiased next generation screening (NGS) •  Cell screening: e.g. karyotype analysis (on target re-

arrangements) •  Function of sites more relevant than number of off-target sites •  Cell transformation: e.g. Loss of anchorage independence •  Tumourogenicity testing in mice (humanised models?) •  Still significant challenge for translation of animal model to

human (i.e we have different DNA!) •  Need to define what is the actual product, reagent or edited cells

•  What additional work is needed for in vivo dosing •  Chromatin modification? •  Single Nucleotide Polymorphisms (SNPs)? •  Potential germ cell exposure? •  Immunogenicity (esp. if editing requires multiple treatments)? •  Cas9 expression and toxicity? •  Appropriate delivery vectors?

Outlook Prospects from Industry for therapeutic PGE

Cas9 DNA

HNH

RuvC/RNaseH-like

Template DNA

Stop Codon INDEL

PAM

gRNA

NHEJ: non-homologous

end joining

HDR: homology directed repair

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