Gene and cell therapy
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Gene and Cell Therapy. Sandra Mercier, MD, PhD [email protected] 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

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Gene and cell therapy

Gene and Cell Therapy

Sandra Mercier, MD, PhD

[email protected]

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 viral vectors


Gene and cell therapy

Retrovirus

Adenovirus

RNA

DNA

No integration to the genome

Integration to the genome


Positive criteria for gene therapy

Positive criteria 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

  • 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

  • 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

Overviewon 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

SCIDSevere 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

  • Gene therapy strategy:

    • Severe phenotype

    • Retroviral-mediated transfer of IL2RG gene to autologous cells

  • Success: 19 recoveries out of 24


Scid x11

SCID-X1

  • 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

  • Gene therapy

    • DNA transfert

    • From gene knowledge

  • Cell therapy

    • Cytogenotherapy

    • iPS: induced Pluripotent Stem cells


Gene therapy from gene knowledge

Gene therapy from gene knowledge

Genomics

Epigenomics

Transcriptomics

Proteomics


Transcript manipulation

Transcript manipulation

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

PTC124

not convenient for frameshift mutations

Clinical trial Phase III:

Good safety

but efficacy?


Exon skipping

Exon skipping

  • Antisense oligonucleotides

  • Modified U7 snRNA

     snRNA family


Splicing reminder

Splicing: reminder

Branch point

Donor site

Acceptor site


Spliceosome formation

Spliceosome formation


Gene and cell therapy

Splicing: 1st transesterification


Gene and cell therapy

Splicing: 2nd transesterification


Exon skipping1

Exon skipping


Therapeutic exon skipping

U7snRNA

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 []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 []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

  • 1st step:proof of efficacy

    • Validation on animal model (mouse)

    • Large animals (GRMD dogs)

  • 2nd step:clinical trials in Humans


Rnai therapy

RNAi-therapy

  • 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

  • Candidate disorders:

  • Gain-of-function mutation ++

    • Dominant neurodegenerative disorders

      • Huntington disease

      • Spino-cerebellar ataxia…

    • Cancers

    • Infectious diseases (HIV…)


Gene and cell therapy

Plan

  • Gene therapy

    • DNA transfert

    • From the knowledge of genes

  • Cell therapy

    • Cytogenotherapy

    • iPS: induced Pluripotent Stem cells


Cell therapy cytogenotherapy

Cell therapyCytogenotherapy

  • 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

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

  • 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

  • Gene therapy

    • DNA transfert

    • From the knowledge of genes

  • Cell therapy

    • Cytogenotherapy

    • iPS: induced Pluripotent Stem cells


Induced pluripotent stem cells

Induced Pluripotent Stem Cells


Stem cells in pharmacogenomics

Stem cells in Pharmacogenomics

  • 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

ConclusionsKeys of genetic therapy

Kay, 2011


Conclusions

Conclusions

  • Cell and gene therapy:

    • Therapeutic hope in human genetic disorders

    • Few clinical validation today, but in progress

  • Association of different therapeutic approaches


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