Gene Therapy and Gene Editing

Topics

• Background in technology• Where are we to date?• What applications• What ‘issues’

Rob Hoeben

Molecular Cell Biology; LUMC

Impact of genetics

How to modify a cell with a faulty gene?

• Very efficient• Very stable• Safety concerns

- Patient- Environment

Result of random gene therapy

Severe Combined Immune Deficiency

~80 SCID variants:

• X-linked SCID

• ADA-SCID

• Rag1 en Rag2

• Wiskott-Aldrich syndrome

• Chronic granulomatous syndrome

• Artemis

• XLA

SCID gene therapy

• ~ 75 children with SCID treated

• 94% alive and thrivingN.B. non-matched BM transplantation: 60% success rate

• But SAEs16 patients developed leukemia

5 / 19 SCID gamma-c receptor7 / 9 WAS WAS-protein4 / 4 CGD NADPH oxidase0 / 40 ADA adenosine deaminase

Vector improvement

Fisher et al., Clin.Genet. (2015) 88:507-15

repair

The Next Frontier: Therapeutic Genome Editing?

The Next Frontier: Therapeutic Genome Editing?

Efficient gene knock-out with targeted nucleases

Double-stranded breaks are detrimental and need repair

Efficient knock-out with nuclease vectors: Cas9/CRISPR

NHEJ

Some numbers

Targeted indel formation: up to 70 %Off-target indels: ?

targeted integrations: up to 2 – 10%Off-target integrations: < 2 % of total integrations

Reducing the occurrence of off-target indels

• Pairs of nicking nucleases

• Reduce the affinity of Cas9 for DNA

Gene Editing is Feasible

Somatic-Cell Gene-Editing Therapy: Targets I

Low hanging fruits:

(1) Recessive disorders where ‘indels’ could be ‘therapeutic’

• Thalassemia• Hemophilia• Pompe disease• (Duchenne muscular dystrophy)

(2) Infectious disease• AIDS - CCR5 deletion

(3) Cancer• CAR –T cells - endogenous TCR deletion

Mut 1Mut 2

Somatic-Cell Gene-Editing Therapy: Targets II

(4) Dominant disorders where ‘indels’ could be ‘therapeutic’• Huntington’s disease

(5) Recessive disorders requiring gene replacement or correctionMost recessive disorders• Hemophilia • SCID

NB Indels 2-component systemReplacement 3-component system

Somatic-Cell Gene-Editing Therapy: Requirements

• Basic technology• Preclinical models• Proof of principle• Vector production

• viral vectors• protein transduction

Prospect of ‘Personalized treatment’

Somatic-Cell Gene-Editing Therapy: Requirements

Evolution of the fieldVaccinesOncolytic virusesDefective viral gene vectors (classic gene therapy)Targeted‐nuclease vectors  (therapeutic indels)Targeted‐nuclease vectors for gene replacement

Key issues: 1) Limited Expertise in Translation to Clinic2) Limited Capacity in GMP Vector Production3) Uncertainty on Safety /Quality Control Requirements4) Uncertainty on Financial Paradigm