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Gene therapy

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Gene therapy Fabrizia Urbinati 01/12/2010 Outline Gene therapy introduction: Delivery method Vectors Candidate Diseases ADA-SCID clinical trial b-Thalassemia ... – PowerPoint PPT presentation

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Title: Gene therapy


1
Gene therapy
  • Fabrizia Urbinati
  • 01/12/2010

2
Outline
  • Gene therapy introduction
  • Delivery method
  • Vectors
  • Candidate Diseases
  • ADA-SCID clinical trial
  • b-Thalassemia

3
What is gene therapy?
  • Introduction of normal genes into an individuals
    cells and tissue to treat a genetic disease.

4
Different strategies for delivering a therapeutic
gene into a patient organ
In vivo
Ex vivo
5
Gene Therapy Vectors
Non Viral Vectors
Viral Vectors
  • Naked DNA
  • Liposome
  • Oligonucleotides
  • Adeno Virus
  • Adeno Associated virus
  • RetrovirusLentivirus
  • Herpes virus

6
Vectors used in gene therapy clinical trial
7
Retrovirus
  • ssRNA virus
  • Infect proliferating cells
  • Integrate in the host genome (stable expression)
  • 7.5 Kb insert size

8
Retroviral Vector production
Long Terminal Repeat (LTR) Regulatory sequence
(promoter and enhancer)
5 LTR
3 LTR
3 LTR
5 LTR
9
Retroviral vector infection
  • The virus enter the target cell
  • the viral genome is integrated
  • in the host genome
  • The therapeutic protein is produced

10
Diseases addressed by Gene Therapy clinical trials
  • It must be caused by a single gene defect (some
    exceptions apply)
  • Gene causing the disease must be identified and
    cloned
  • The tissue/organ has to be accessible for gene
    delivery
  • No effective conventional treatment is available
    for that disease

11
Number of Gene Therapy Clinical Trails approved
worldwide 1989-2009
12
Two examples of Gene Therapy for hematologic
diseases.
  • ADA-SCID
  • b-thalassemia

13
Replacement of the gene in Hematopoietic Stem
Cells (HSC)
Blood and Tissues
Bone Marrow
14
Adenosine-Deaminase (ADA) Deficiency
  • ADA is an enzyme involved in purine metabolism
    It is needed for the breakdown of adenosine from
    food and for the turnover of nucleic acid in
    tissues.
  • ADA deficiency is an autosomal recessive disorder
  • Lack of B and T cell function
  • Immune system is severely compromised and the
    disease is often fatal, if untreated, due to
    infections

15
ADA-SCID treatment
  • Bone Marrow Transplantation
  • ADA enzyme therapy
  • Gene Therapy

16
Gene Therapy Clinical Trial for ADA-SCID in Italy
(Aiuti et al. Science 2002)
17
Gene Therapy Clinical Trial for ADA- SCID in
Italy vector
Retroviral vector production
LTR
Sv40
NeoR
ADA
LTR
18
Gene Therapy Clinical Trial for ADA- SCID in
Italy protocol.
  • Bone Marrow stem cells collection from 2 patients
  • Infection of BM stem cells with Retroviral vector
  • Busulfan prior to BM infusion (non-myeloablative
    conditioning).
  • Re-infusion of corrected BM cells into the patient

19
Gene Therapy Clinical Trial for ADA- SCID in
Italy results
  • ADA enzyme activity was
  • restored and lymphoid reconstitution was
    shown after gene therapy treatment
  • Immune reconstitution by 6 months.
  • T cells gene-marked at 100

(Aiuti et. Al Science 2002)
20
ADA-SCID gene therapy
(Aiuti at al. Hematology 2009)
21
Setbacks
  • In the French trial for X-SCID gene therapy a
    total of 4 patients from 10 treated developed
    leukemia due to uncontrolled proliferation of
    mature T lymphocytes after gene therapy
    treatment. Three of the patients were treated and
    recovered one unfortunately died.

(Science 2003)
22
Retroviral integration into the host genome
insertional mutagenesis
Leukemia was caused by the retroviral vector
carrying the therapeutic gene (IL2RG)
In the first 2 patients that developed leukemia,
the integration of the retroviral vector close
to the LMO-2 oncogene lead to over-expression of
the gene and uncontrolled proliferation of
T-cells
23
Follow up study in ADA-SCID patients from the
italian trial
(Journal of Clinical Investigation, 2007)
24
Follow up study in ADA-SCID patients from the
italian trial
Retroviral integration site in patient with
ADA-SCID many oncogenes were hit by the provirus
Expression of LMO-2 gene in pt. treated with
gene therapy The expression of the oncogene did
not change
(Aiuti et al. JCI 2007)
25
Results of the follow-up study (Aiuti et al. JCI
2007)
  • the analysis revealed a nonrandom distribution of
    integrated proviruses, with a strong preference
    for gene-dense regions and a tendency to hit
    genes that are highly expressed in CD34 cells at
    the time of transduction.
  • Expression of the oncogenes hit by the viral
    integration did not change insertions in
    potentially dangerous genomic sites are not
    sufficient per se to induce a proliferative
    advantage in T cells in vivo, confirming that
    multiple cooperating events are required to
    promote oncogenic transformation in humans
  • In summary, the data show that transplantation of
    ADA-transduced HSCs does not result in selection
    of expanding or malignant cell clones, despite
    the occurrence of insertions near potentially
    oncogenic loci.

26
Need for improving the safety of viral vectors.
  • Gene therapy of genetic diseases require the
    development of safer gene-transfer such as
  • self-inactivating viral vectors
  • the use of physiologically controlled gene
    expression cassettes.
  • Use of Insulator sequences in viral vectors

27
Improving the safety of viral vectors the
example of b-thalassemia Gene Therapy
  • Thalassemias are hereditary anemias and are the
    most common
  • single gene defects worldwide.
  • b-thalassemia result from mutations in the
    ???-globin gene cluster
  • There is reduced hemoglobin production leading to
    ineffective erythropoiesis
  • Currently, the only curative therapy is
    allogeneic Bone Marrow Transplantation (BMT).
  • However, allogenic BMT is limited by the
    availability of donors and potentially serious
    side effects.
  • Insertion of a normal ß-globin gene could have a
    therapeutic potential in ß -thalassemia .

28
b-thalassemia Gene Therapy
  • There are no current Gene Therapy trials for
    b-thalassemia.
  • Many studies have been focused on the
    optimization of the vectors carrying the b-globin
    gene.
  • Latest vector of choice for b-globin gene is
    SIN-lentiviral vector

29
SIN-Lentiviral vector for b-thalassemia gene
therapy
  • Lentiviral vector
  • retrovirus family
  • ssRNA
  • Integrate in the host genome
  • 8Kb insert size
  • Infect also quiescent cells
  • Safety features
  • SINSelf Inactivating vector a portion of the
    viral LTR has been deleted
  • to prevent transcription of the viral vector
    sequence after integration
  • (increase safety of the vector)
  • The expression of the b-globin gene is driven by
    the b-globin promoter
  • and its enhancer (increase safety of the vector)
    that are lineage specific

30
Use of Insulator in a b-globin lentiviral
vector for Gene Therapy of b-Thalassemia
Insulator is a sequence found in the genome and
it is a genetic boundary element. The need for
them arises where two adjacent genes on a
chromosome have very different transcription
patterns, and it is critical that the inducing or
repressing mechanisms of one do not interfere
with the neighbouring gene.
31
Use of Insulator in a b-globin lentiviral
vector for Gene Therapy of b-Thalassemia
Insertion of insulator sequences in a Lentiviral
Vector to increase the safety of the vector,
blocking the activity of the enhancer towards
surrounding genes.
32
Gene therapy summary
  • Gene therapy overview
  • Different delivery methods, vectors, diseases,
  • 2 Gene Therapy studies
  • ADA-SCID trial successful but need to find
    safer delivery vectors
  • b-Thalassemia Gene Therapy as an example of
    optimization of safer vectors
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