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Gene and Cell Therapy. Sandra Mercier, MD, PhD sandra.mercier@chu-nantes.fr Service de génétique clinique HME- CHU Nantes. Plan. Gene therapy DNA transfert From the knowledge of genes Cell therapy Cytogenotherapy iPS: induced Pluripotent Stem cells. Plan. Gene therapy DNA transfert

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Gene and cell therapy

Gene and Cell Therapy

Sandra Mercier, MD, PhD

sandra.mercier@chu-nantes.fr

Service de génétique clinique

HME- CHU Nantes


Gene and cell therapy
Plan

  • Gene therapy

    • DNA transfert

    • From the knowledge of genes

  • Cell therapy

    • Cytogenotherapy

    • iPS: induced Pluripotent Stem cells


Gene and cell therapy
Plan

  • Gene therapy

    • DNA transfert

    • From the knowledge of genes

  • Cell therapy

    • Cytogenotherapy

    • iPS: induced Pluripotent Stem cells


Gene therapy by dna transfert
Gene therapy by DNA transfert

Definition:

Genetic material transfert into cells

to obtain a transient or permanent

therapeutic modification


Gene therapy by dna transfert1
Gene therapy by DNA transfert

  • « Gene therapy »:

    • Phenotype correction by repairing the genetic anomaly

  • « Therapy by the genes »:

    • Phenotype correction by addition of a new normal gene copy


Methodological problems
Methodological problems

  • What? Which quantity?

  • Which target(s)?

  • When?

  • How? (safety problem)

  • What about the stability?


Transgene
Transgene

Construction

Minigene

Cis-enhancer

sequences

Cis-enhancer

sequences

Vector

sequences

Vector

sequences

+/- « insulators » sequences

From Kaplan, Biologie moléculaire et Médecine


Vectors

Modification of the viral genome to remove its pathogenicity:

Replication-defective

Liposome

Peptide-gene delivery system

Polyethylene-imine (PEI)

Low rate of transduction

Transduction ++

Non-sufficient transduction for clinical trials

Vectors

Naked DNA

Replication-defective

viral vectors

Non viral vectors


Replication defective viral vectors
Replication-defective pathogenicity:viral vectors


Gene and cell therapy

Retrovirus pathogenicity:

Adenovirus

RNA

DNA

No integration to the genome

Integration to the genome


Positive criteria for gene therapy
Positive criteria pathogenicity:for gene therapy

  • Pathology:

    • No irreversible damages

    • Limited area or easily-accessible cells

    • Animal model

  • Protein:

    • Well-defined function

    • Active in low quantity

    • Diffusible or selective advantage for modified cells


Preclinical studies
Preclinical studies pathogenicity:

  • Biological efficacy

  • Safety (local / systemic injection)

  • Animal models:

    • Spontaneous / Site directed mutagenesis

    • Importance of large animal models: murin model is not sufficient for gene therapy (vector quantity, safety…)


Clinical trials
Clinical trials pathogenicity:

  • Phase I: safety

    • On healthy volunteers (or affected individuals sometimes)

  • Phase II: therapeutic efficacy

    • On small group of patients

  • Phase III:

    • Randomized controlled analysis

    • On large group of patients

    • Dosage variation


Overview on gene therapy by dna transfert
Overview pathogenicity:on gene therapy by DNA transfert

  • Cancers: 67%

  • Monogenic disorders : 8%

    • SCID, DMD,-thalassemia, Leber retinopathy…

  • Viral diseases (HIV): 6%

  • Other acquired disorders : 12%

  • Non therapeutic trials : 7%


Scid severe combined immunodeficiencies
SCID pathogenicity:Severe Combined ImmunoDeficiencies

  • SCID = Severe Combined ImmunoDeficiencies (B and T)

  • AR, 8 genes

  • Clinical trial SCID-X1: « Bubble boy  »

    • Gene: IL2RG (Xq13.1)

    • Absence of T, NK and dysfunction of B

    • Symptoms: rapidely after birth, high mortality ++

    • Ttt: bone marrow transplant (1/3 of the children)


Scid x1
SCID-X1 pathogenicity:

  • Gene therapy strategy:

    • Severe phenotype

    • Retroviral-mediated transfer of IL2RG gene to autologous cells

  • Success: 19 recoveries out of 24


Scid x11
SCID-X1 pathogenicity:

  • BUT follow-up:

    • T leukemia in 5 cases!

    • 1 lethal case

  • Lentivirus: transgene integration :

    • Either transgene extinction

    • Either insertional oncogenesis if insertion near a proto-oncogene


Gene and cell therapy
Plan pathogenicity:

  • Gene therapy

    • DNA transfert

    • From gene knowledge

  • Cell therapy

    • Cytogenotherapy

    • iPS: induced Pluripotent Stem cells


Gene therapy from gene knowledge
Gene therapy pathogenicity:from gene knowledge

Genomics

Epigenomics

Transcriptomics

Proteomics


Transcript manipulation
Transcript manipulation pathogenicity:

Examples in Duchenne muscular dystrophy:

  • Premature STOP codon read-through

  • Exon skipping

    Example in neurodegenerative disorders:

  • RNA interference-therapy


Stop codon read through
STOP codon read-through pathogenicity:

PTC124

not convenient for frameshift mutations

Clinical trial Phase III:

Good safety

but efficacy?


Exon skipping
Exon skipping pathogenicity:

  • Antisense oligonucleotides

  • Modified U7 snRNA

     snRNA family


Splicing reminder
Splicing: reminder pathogenicity:

Branch point

Donor site

Acceptor site


Spliceosome formation
Spliceosome formation pathogenicity:


Gene and cell therapy

Splicing: pathogenicity:1st transesterification


Gene and cell therapy

Splicing: pathogenicity:2nd transesterification


Exon skipping1
Exon skipping pathogenicity:


Therapeutic exon skipping

U7snRNA pathogenicity:

modifié

AS

DS

AS

DS

Therapeutic exon skipping

BP

DMD

ESE

AS

DS

AS

DS

AS

DS

Targets of exon skipping:

BP: Branch point

ESE: Exonic Splicing Enhancer

AS: Acceptor site

DS: Donor site


Gene and cell therapy

You can lead a h pathogenicity:[]terb ut youca n'tm ake himd rink.

Exon skipping: explanation

Out-of-frame Deletion

You can lead a h [orse to wa]ter, but you can't make him drink.


Gene and cell therapy

You can lead a h pathogenicity:[]but you can't make him drink.

In frame!

Exon skipping: explanation

Out-of-frame Deletion

+ Exon skipping

You can lead a h[orse to wa]ter], but you can't make him drink.


Exon skipping in dmd
Exon skipping in DMD pathogenicity:

  • 1st step:proof of efficacy

    • Validation on animal model (mouse)

    • Large animals (GRMD dogs)

  • 2nd step:clinical trials in Humans


Rnai therapy
RNAi-therapy pathogenicity:

  • Therapy by RNA interference

  • Principle:

    • Use of siRNAs (21-22 nucleotides, double strand)

    • To abolish the targeted gene expression

    • By mRNA destruction or translation stop


Rnai therapy1
RNAi-therapy pathogenicity:

  • Candidate disorders:

  • Gain-of-function mutation ++

    • Dominant neurodegenerative disorders

      • Huntington disease

      • Spino-cerebellar ataxia…

    • Cancers

    • Infectious diseases (HIV…)


Gene and cell therapy
Plan pathogenicity:

  • Gene therapy

    • DNA transfert

    • From the knowledge of genes

  • Cell therapy

    • Cytogenotherapy

    • iPS: induced Pluripotent Stem cells


Cell therapy cytogenotherapy
Cell therapy pathogenicity:Cytogenotherapy

  • Combination between cell and gene therapy

  • Aim: « autologous transplant»

  • Cell types :

    • Embryonic stem cells (ES): totipotentes

    • Post-natal multipotent cells (blood cord, adult tissue)


Research on stem cells
Research on Stem cells pathogenicity:

Allowed in France,

but strictly restricted by the law :

  • Established scientific relevance

  • Medical issue, impossibility to use an alternative as well as efficient method

  • Respect of ethical principles


Origins of stem cells
Origins of stem cells pathogenicity:

  • Embryonic stem cells:

    • IVF (in vitro fertilization): supernumerary embryos or miscarriage products

    • With genitors consent form

  • Adult stem cells:

    • Skin, blood, liver, muscle

    • Ex: hematopoietic cells for leukemia treatment


Gene and cell therapy
Plan pathogenicity:

  • Gene therapy

    • DNA transfert

    • From the knowledge of genes

  • Cell therapy

    • Cytogenotherapy

    • iPS: induced Pluripotent Stem cells



Stem cells in pharmacogenomics
Stem cells in Pharmacogenomics pathogenicity:

  • High Throughput Screening (HTS) activity :

    • is part of the drug discovery process

    • consists in selecting among thousands of molecules the ones that could have a pharmaceutical use

    • to find new targets of already labellised-drugs

I-STEM: Institute for Stem cell Therapy and Exploration of Monogenic diseases


Conclusions keys of genetic therapy
Conclusions pathogenicity:Keys of genetic therapy

Kay, 2011


Conclusions
Conclusions pathogenicity:

  • Cell and gene therapy:

    • Therapeutic hope in human genetic disorders

    • Few clinical validation today, but in progress

  • Association of different therapeutic approaches