CRISPR/Cas9 and genome editing for genetic neuromuscular ......• CRISPR/Cas9 enables editing of the genome • Potential for generating model systems • There is therapeutic potential,

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

September 20183 Aartsma-Rus

• 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


• 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


Genome editing


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


DNA damage repair


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?


September 2018Aartsma-Rus10

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


Becker: genetic code maintained


September 2018Aartsma-Rus13

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


It works! In mice….


What did they do in mice?




Local delivery

DNA and RNA analysis

Local delivery

Protein analysis

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



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


Tests in patient-derived cells


Multiexon skipping


What did they do in patient cells?


Multiexon skipping

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

Tests in patient derived cells


Exon skippen

Genome editing

What did they do in patient cells?


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


Media hype


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


“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


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)


Documents

CRISPR/Cas9 and genome editing for genetic neuromuscular ......• CRISPR/Cas9 enables editing of the genome • Potential for generating model systems • There is therapeutic potential,