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Edición genética con CRISPR: del deseo a la realidad

@LluisMontoliuCNB-CSIC y CIBERER-ISCIII

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www.albinismo.es

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He Jiankui (Shenzhen, China)

Monday, 26 November 2018

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Definitions

• Moral/morality: what people believe to be right/good or wrong/bad. Set of codes of conductadopted governing human behavior.

• Ethics: is a critical reflection about morality. Providesa systematic way to work through dilemas to determine the best course of action amongconflicting choices, a generalized conceptual framework for decision making.

• Bioethics: the study of ethical issues arising fromhealth care, in biological and medical sciences.

Basic principles in Bioethics

• Principle of non‐maleficence

• Principle of beneficence

• Principle of respect for autonomy

• Principle of justice

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Genome editing tools (CRISPR)

• Great tools for basic research

• Have become instrumental to investigate on human congenitaldiseases

• We do not control the output

• Still associated with uncertainty

• Not yet ready for clinical use (unsafe forin vivo, potentially useful for ex-vivo)

Mojica and Montoliu, Trends in Microbiology 2016

Prokaryotes Eukaryotes

Francis J. M. MojicaUniversity of Alicante (Spain)

1993 First CRISPR described in archaea2001 Coins the name CRISPR2005 CRISPR as inmune systems2009 Coins the name PAM

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Mojica and Montoliu, Trends in Microbiology 2016

Prokaryotes Eukaryotes

Fixing the DSB: NHEJ vs HDR

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The CRISPR-CasSystem in 2012

The CRISPR-CasSystem in 2019

RNP

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Off-target effects

On-targeteffects

Mosaicism

HDR isnot the

preferredrepairingpathway

Related toCRISPR

Unrelated toCRISPR

Current limitations of CRISPR-Cas9 tools

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We have not foundoff-target sites withaltered sequences in genome-edited mice

Confirmed by NGS

What about off-targets?

Mostly observed in vitro

Seruggia et al. 2015 NAR

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A1 B1

A2

A10

B2

B10

A1 – AAGTTAGGATTAGAGGATCCATACCCTAGCGG||||||||||||||||||||||||||||||||

B1 - AAGTTAGGATTAGAGGATCCATACCCTAGCGG

A10 – AAGCTAGGATAAGAGGATCCATACCCTACCGG|*|*||||||*|||*|||||*|||||||*|||

B10 - ATGTTAGGATTAGACGATCCTTACCCTAGCGG

Spontaneous Mutations + Genetic Drift Divergent Mouse Subcolonies

Montoliu & Whitelaw, Transgenic Res. 2018

• Founder animals are nearly always complexmosaic

• Many different allelescan be present

• Not all of them mighttransmit throughgermline

One 8-cell embryo = 16 possible alleles

On-targets: the real problem

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Albinism Facts

Genetic condition, recessive trait1:17.000 (rare disease)

Visually impaired, lack foveaReduced visual acuityAbnormal quiasmatic connectionsPhotofobia, Nystagmus

Several types, syndromic or notMost (but not all) associated withhypopigmentation in skin & eyes, OCA (or eyes only, OA)

Do not tan, sunburnCan have increased skin cancerGenetically heterogeneousAt least 20 genes associated

Mouse Human Albinism Mutations (HGMD)Tyr TYR OCA1 395Oca2 OCA2 OCA2 235Tyrp1 TYRP1 OCA3 37Slc45a2 SLC45A2 OCA4 116?? 4q24 OCA5 1slc24A5 SLC24A5 OCA6 13Lrmda LRMDA OCA7 8Gpr143 GPR143 OA1 148Sl38a8 SLC38A8 FHONDA 11Lyst LYST CHS1 88Hps1 HPS1 HPS1 45Ap3b1 AP3B1 HPS2 31Hps3 HPS3 HPS3 14Hps4 HPS4 HPS4 18Hps5 HPS5 HPS5 27Hps6 HPS6 HPS6 26Dtnbp1 DTNBP1 HPS7 3Bloc1s3 BLOC1S3 HPS8 2Bloc1s6 BLOC1S6 HPS9 1Ap3d1 AP3D1 HPS10 2

There are at least 20 genes associated with albinism

Montoliu et al. PCMR 2014; Montoliu & Marks PCMR 2016

mel

ano

cyte

sM

elan

oso

mes

/lyso

som

es

HGMD ® 2017.4

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Diversity of pigmentations but all are visually impaired

OCA1OCA4HPS

OCA4 avatar mouse models

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Reference: AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACGGTAGGGTTGATTTCAGGAAATGTAAB9040.1 AACATTGGAGGAGCTGC ACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAG-----------GTAGGGTTGATTTCAGGAAATGTAAB9040.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGGTAGGTAGG-TTGATTTCAGGAAATGTAAB9040.3 AACACTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAG-----------GTAGGGTTGATTTCAGGAAATGTAAB9040.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGC-----AGGGTTGATTTCAGGAAATGTAAB9040.5 CAAGCTCCTGCCCCACTTTCAAAGCTGTACTGAACTGCAGTTTCTTCTCCACCCAGATTCCTGCAAGACCTTGCACCGGGG------------(437bp)------------------B9040.6 --------------------------------------------(561bp)-------------------------------------------------------------------B9041.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCTGTTACCTAGGGTTGATTTCAGGAAATGB9041.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAAC--------AGGGTTGATTTCAGGAAATGTAAB9041.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAGTGTAAB9041.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACG----GGTAGGGTTGATTTCAGGAAATGTAAB9041.5 AACATTGGAGGAGCTGCCACTGCTATTTGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAATGTAAB9042.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACGGTAGGGTTGATTTCAGGAAATGTAAB9042.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAAT---------------GGTAGGGTTGATTTCAGGAAATGTAAB9042.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAA-TGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACGGTAGGGTTGATTTCAGGAAATGTAAB9042.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAAT---------------GGTAGGGTTGATTTCAGGAAGTGTAAB9043.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAAT---------------GGTAGGGTTGATTTCAGGAAGTGTAAB9043.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACACTGGTAGGGTTGATTTCAGGAAGTGB9043.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTCCCAATCAGGAGTTGAGAAAAAT---------------GGTAGGGTTGATTTCAGGAAGTGTAAB9043.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGATAGACATGTCGAGGA------(134bp)--------------------B9044.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAAT---------------GGTAGGGTTGATTTCAGGAAATGTAAB9044.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCGATCAGGAGTTGAGAAAAATGGTAGGTAAC--------AGGGTTGATTTCAGGAAATGTAAB9045.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTTGTTTAT-------------------------------CAAGGTAGGGTTGATTTCAGGAAATGTAAB9045.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAAT--------------------GGTTGATTTCAGGAAATGTAAB9045.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTTGTTTAT-------------------------------CAAGGTAGGGTTGATTTCGGGAAATGTAAB9046.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACCTAGGTAGGGTTGATTTCAGGAAATGB9046.2 AACATTG--------------------------------(87bp)-------------------------------------------------TAGGGTTGATTTCAGGAAATGTAAB9046.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCAATGGTAGGGTTGATTTCAGGAAATGTAB9046.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACCTAGGTAGGGTTGATTACAGGAAATGB9060.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAATGTAAB9060.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAGATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAATGTAAB9060.2 GACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAATGTAAB9060.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCT-------GTTGATTTCAGGAAATGTAAB9060.4 AACATCGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAATGTAAB9064.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCAAAAATGGTAGGTAGGGTTGATTTCAGGB9064.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTA-----------GGTAGGGTTGATTTCAGGAAATGTAAB9064.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAGTGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCAAAAATGGTAGGTAGGGTTGATTTCAGGB9064.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGC-----AGGGTTGATTTCAGGAAATGTAAB9064.5 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCA------------------------------GGTAGGGTTGATTTCAGGAAATGTAA

sgRNA‐A476

ssDNA

Multiple alleles present in CRISPR founder gene-edited mice

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Doudna & Charpentier (2014) Science

CRISPR-Cas is the future

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IN VIVO EX VIVO

Ex-vivo immunotherapy: targeting PD-1 gene with CRISPR tools

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Ex-vivo CRISPR targeting of BCL11A enhancer forSickle cell anemia and beta-thalassemia

Bauer Lab (Dana Farber-Boston Children’s)

First ex-vivo CRISPR therapyapproved in Europe

Associated Press, 15 Nov 2017

• UCSF Benioff Children's Hospital in Oakland, California

• IV injection of viral particles with ZFNs• Approved by NIH• Sangamo• Hunter’s syndrome (I2S gene)

Mucopolysaccharidosis II (MPS II)• Lysosomal storage disease• Injected on 13 Nov 2017• No therapeutic effect seen• No toxicity detected

First genome editing (driven by ZFN) somatic gene therapy in a patientIN VIVO

IN VIVO

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CRISPR‐Cas9 and in vivo somatic gene therapy

Amoasii et al. Science 20183-92% correction

Increasing number of animal models of raremonogenic diseases corrected via CRISPR

• Duchenne muscular distrophy (DMD)• Ornithine transcarbamylase (OTC) deficiency• Hereditary tyrosinemia I (FAH deficiency)• Congenital cataract (CRYGC)• Chronic granulomatous disease (CGD)• Retinitis pigmentosa (RP)• Leber congenital amaurosis (LCA)• Hungtinton Disease (HD)• …• Alsomany iPS cells models correcting gene mutations via

CRISPR strategies

Preclinical animal models

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Cas9: Bang Wong, Broad Institute of Harvard and MIT, Cambridge, MA

• Cas9 antibodiesfound in human serum

• Anti‐Cas9 T lymphocites found in human blood

• 79% individuals haveantibodies againstSaCas9

• 65% individuals haveantibodies againstSpCas9

• 46% individuals haveanti‐Cas9 T cells

• Immunosupression oralternative Cas proteins

5 Jan 2018 – 28 Jan 2019 Nature Med.

Cas9Streptococcus pyogenesStaphylococcus aureus

Human retina

Correcting a point mutation in CEP290 gene with NHEJ CRISPR

Leber’s congenital amaurosis type 10

In vivoDecember 2018

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Correcting a point mutation in CEP290 gene with NHEJ CRISPR

Leber’s congenital amaurosis type 10

December 2018

CRISPR and human embryos

• Several studies from China using 3n/2n embryos

• Many different alleles are produced

• Most edited embryos are mosaic

• Anticipate potential off-target effects

• Need for careful risk/benefit analysis

• Consider alternative technologies (PGD: preimplantationgenetic diagnosis)

• Need to be cautious before applying

• Poses Ethics dilemas and contravenes current norms

(art 13 and art. 18, Asturias Convention, 1997)

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2 August 2017

CRISPR-Cas9

Large deletions after DSB induced by CRISPR-Cas9

Kosicki et al. 2018 Nature Biotech.Adikusuma et al. 2018 Nature Comm.

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♀

♂

*CRISPR-Cas9

Alternative interpretations for Mitalipov experiments

Gene therapy on human embryoswith CRISPR

Illegal, Unnecessary & Unwise

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20 September 2017

CRISPR Oct4 in mouse embryos Blastocyst OK, related genes alteredCRISPR Oct4 in human embryos Blastocyst compromised, related genes OK

Gene research on human embryoswith CRISPR

Legal, Necessary & Wise

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He Jiankui (Shenzhen, China)

7 couples 31 embryos 2 pregnancies 1 delivered (twin girls: Lulu y Nana) +1

Gene improvement on human embryoswith CRISPR

Illegal, Unnecessary, Unwise& Irresponsible

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He Jiankui (Shenzhen, China)

The red lines crossed

• Without knowledge, without authorization, withoutethics review, without ethics approval, secrecy

• Implanting genome-edited human embryos and lettingthem develop to term (two newborns)

• Genome-editing on unaffected human embryos not fortherapeutical purposes but for enhancement, addingthem a new trait (resistance to HIV infection)

He Jiankui (Shenzhen, China)

Technical issues

• The CCR5 gene

• Off-target mutations

• Mosaicism

• Uncertainty / unpredictable risks

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The CCR5 gene / co‐receptor

• Used by HIV to access T lymphocytes

• Has numerous other functions in theimmune system

• A deletion of 32 bp was found in 1996 in people resistant to HIV infection(CCR5‐Δ32)

• Loss of CCR5 function associated with:

• Increased infectivity by flavivirus like West Nile virus and thosecausing Dengue and Yellow Fewer

• Increased mortality after infectionby Influenza virus

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Jing‐Bao Nie

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He Jiankui (Shenzhen, China)

The lessons

• This should have NOT occurred

• But this was likely to occur, sooner or later

• Many reports and recommendations published

• No universal codes / rules / laws yet available

• Need to promote global governance rules

Numerous reports on genome editing

• US NAS/NAM

• EASAC

• INSERM

• EGE

• ASHG

• Deutscher Ethikrat

• Fed. Eur. Acad. Med.

• HUGO

• Leopoldina

• Schweizer Ethikrat

• Nuffield Bioethics Council

• …

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NAS‐2017 (USA)

• Clinical trials using heritable genome editing should be permitted only within a robust and effective regulatory framework that encompasses:

– the absence of reasonable alternatives;

– restriction to preventing a serious disease or condition;

– restriction to editing genes that have been convincingly demonstrated to cause or to strongly predispose to that disease or condition;

– restriction to converting such genes to versions that are prevalent in the population and are known to be associated with ordinary health with little or no evidence of adverse effects;

– the availability of credible preclinical and/or clinical data on risks and potential health benefits of the procedures;

– ongoing, rigorous oversight during clinical trials of the effects of the procedure on the health and safety of the research participants;

– comprehensive plans for long-term, multigenerational followup that still respect personal autonomy;

– maximum transparency consistent with patient privacy;

– continued reassessment of both health and societal benefits and risks, with broad ongoing participation and input by the public; and

– reliable oversight mechanisms to prevent extension to uses other than preventing a serious disease or condition.

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NCB‐2018 (UK)

• Reproductive cells that have been subject to heritable genome editing interventions should only be used for purposes that are consistent with the welfare of the future person.

• The use of heritable genome editing interventions would only be ethically acceptable if carried out in accordance with principles of social justice and solidarity.

• If heritable genome editing were to become feasible, those whose genomes have been edited should be entitled to the same enjoyment of human rights as everyone else.

Initial pre‐ARRIGE 2017 publications in Nature and Transgenic Research

Nature (2017) Hirsch et al.

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Association for Responsible Research and Innovation in Genome Editing

ARRIGE Kick‐Off meeting, Paris, 23 March 2018

ARRIGE featuredin Science

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ARRIGE featured in NatureBiotechnology

ARRIGE presented in The CRISPR Journal

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http://arrige.org• Fostering the development of genome editing technologies within a safe and ethical framework for individuals and for our societies.

• Fostering an inclusive debate with a risk‐management approach, taking into account the human, environmental, animal and economic issues

• Promoting a global governance of genome editing through a comprehensive setting for all stakeholders

• Creating an ethical tool box and informal guidance for genome editing technology users, regulators, governance and the civil society at large

• Developing a robust particular reflection on the role of the lay public in this debate and the necessity for improved public engagement

Aims:

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Genome editing (CRISPR)

• We should not be using genomeediting on human beings until thesetechniques are deemed safe and effective, with precise therapeutic applications justified after a broad and open debate.

UNESCO is the United Nations Educational, Scientific and Cultural Organization

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Association for Responsible Research and Innovation in Genome Editing

https://arrige.org

join@arrige.org

a

Marion AbecassisBernard BaertschiHervé ChneiweissFrançois HirschPierre JouannetChristine LemaitreJennifer MerchantLluis MontoliuCyril Sarrauste de Menthière

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Lluís Montoliu’s Lab at CNB

http://www.cnb.csic.es/~montoliu

www.cnb.csic.es/~montoliu/CRISPR/

Google CNB + CRISPR

Para más información sobre CRISPR

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