crispr/cas9 and genome editing for genetic neuromuscular ......• crispr/cas9 enables editing of...

CRISPR/Cas 9 and genome editing for

genetic neuromuscular disorders

Annemieke Aartsma-Rus

September 2018

AFDELING HUMANE GENETICS, LUMC, LEIDEN

Disclosures

Sept 2018

• Employed by LUMC, which has patents on exon skipping

technology, some of which has been licensed to BioMarin and

subsequently sublicensed to Sarepta. As co-inventor of some of

these patents I am entitled to a share of royalties

• Ad hoc consultant for PTC Therapeutics, BioMarin

Pharmaceuticals Inc., Alpha Anomeric, Global Guidepoint, GLG

consultancy, Grunenthal, Wave, Sarepta, Eisai and BioClinica

• Member of the scientific advisory boards of ProQR, MirrX

therapeutics and Philae Pharmaceuticals. Remuneration for

these activities paid to LUMC.

• LUMC received speaker honoraria from PTC Therapeutics and

BioMarin Pharmaceuticals.

Muscular dystrophies – shared pathology

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• Connection cytoskeleton muscle fibers and extracellular

matrix is lost/impaired due to genetic mutations

• Less stability during muscle contraction

• Muscle fibers more prone to damage

• Pathology:

• Continuous damage of muscle fibers

• Chronic inflammation

• Fibrosis/adiposis and suppressed regeneration

• Continuous loss of muscle tissue and function

Muscular dystrophies

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• Treatment often is multidisciplinary care

• Research ongoing to restore missing protein with genetic

therapies

• For Duchenne some approved drugs

• Ataluren (EMA) and eteplirsen (FDA)

• Mutation specific

• Not available everywhere in Europe

• Not applicable to all patients

• Genome editing to the rescue

Genome editing for Duchenne

• Use DMD as showcase

• What is CRISPR/Cas9 and how does it work?

• How can it be used as a therapy for DMD?

• Why is everyone so enthusiastic

• What has been done?

• What still needs to be done?

• Misconceptions

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Genome editing

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Cas9

CRISPR system

(“guide RNA”)

Why would you cut DNA?

• DNA contains genes (genetic blueprint)

• DNA damage is NOT good

• Our DNA is damaged continuously

• This is repaired ASAP

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DNA damage repair

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DNA damage

Recombination (dividing cells)

Errorless repair

NHEJ (non dividing cells)

Non homologous end joining

Information is lost

Repair

DNA damage repair in muscle

• Muscles and neurons are postmitotic cells

• For NMDs only the NHEJ system available

• This does not correct mutations, but generates

mistakes in the DNA

• Why would you want this?

• Permanent exon skipping

• Recap: what is exon skipping?

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Gene to protein

Exons Introns

13

5

6

7 Gene (DNA)

RNA copy (pre mRNA)

messenger RNA

1 - - - - - - - - - 79

dystrophin protein

Splicing

2

4

3 4 56 7

1 21 2 3 4 5 6 7 8

Duchenne: genetic code disrupted

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Becker: genetic code maintained

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Restore genetic code

Exon 52Intron 51Intron 47/50Exon 47 Exon 51 Intron 52AON

Exon 46 Exon 47 Exon 52

Code repaired

Partially functional dystrophin

Exon skipping

• Repairs code on transcript (RNA) level

• Temporary effect: weekly treatment

• Repair code on DNA level permanent effect

• This can be done with genome editing

• Advantages over exon skipping

• Single treatment

• Can also skip multiple exons

• Works for duplications

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It works! In mice….

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What did they do in mice?

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What did they do in mice?

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Local delivery

DNA and RNA analysis

Local delivery

Protein analysis

Nelson et al., Science 2016; 351: 403-7

What did they do in mice?

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Systemic delivery

Protein analysis

Long et al., Science 2016; 351: 400-3

So what did they achieve in mice

• Generated the target deletion or skip on DNA level

• Restored dystrophin

• BUT

• Efficiency rather low

• Systemic delivery challenging

• Need viral vector to deliver CAS9 and guideRNAs

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Tests in patient-derived cells

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Multiexon skipping

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What did they do in patient cells?

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Multiexon skipping

Oosterhout et al., Nat Comm 2015; 6: 6244

Tests in patient derived cells

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Exon skippen

Genome editing

What did they do in patient cells?

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Duplication mutation (exon 18-30 duplication)

Wojtal et al., AJHG 2016; 98: 90-101

Why are people so enthusiastic?

• Technique has potential

• Targeted modification of DNA

• Offers opportunities not available before

• Generating disease models (cells and animals)

• Therapeutic options

• Easy (in cell cultures)

• Many examples of ‘it working’ in DMD models

• Media attention

• Recent paper in dogs in science: showed dystrophin

restoration after systemic treatment in 1 dog

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Media hype

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What still needs to be solved?

• Delivery

• Need to deliver Cas9 and guide RNAs to all muscles

• Viral vectors (AAV)

• Tested for gene addition with promising results

• Here: two-component system and two-step process

• Manufacturing….

• Efficiency

• Currently very low

• Safety

• How specific are the Cas9 enzymes?

• Integration of AAV vectors

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“CRISPR Cure”

• For most NMDs: restoring code will not result in

restoring functional protein

• For DMD genetic code is restored

• Becker like proteins, partially functional

• This is not a cure

• This does not halt muscular dystrophy

• Effect depends on time of intervention

• Loss of muscle and muscle function is irreversible

• So need to treat earlySeptember 201828 Aartsma-Rus

Genome editing of embryos

• You need to know that parents are carriers (recessive)

or one parent is a carrier (dominant/X-recessive

• BUT….if you know this, embryo selection is a less risky

and less invasive option

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Summary

• CRISPR/Cas9 enables editing of the genome

• Potential for generating model systems

• There is therapeutic potential, BUT

• For muscle diseases delivery is very challenging as yet

• Outstanding questions on safety

• If it works, it will slow down disease progression

(“CRISPR Cure” is a misnomer)

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