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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
What is gene therapy?
Introduction of normal genes into an
individual’s cells and tissue to treat a
genetic disease.
Different strategies for delivering a
therapeutic gene into a patient organ
In vivo
Ex vivo
Gene Therapy Vectors
Viral Vectors
•Adeno Virus
•Adeno Associated virus
•RetrovirusLentivirus
•Herpes virus
•…
Non Viral Vectors
•Naked DNA
•Liposome
•Oligonucleotides
Vectors used in gene therapy clinical
trial
Retrovirus
 ssRNA virus
 Infect proliferating cells
 Integrate in the host genome (stable
expression)
 7.5 Kb insert size
Retroviral Vector: production
5’ LTR
5’ LTR
3’ LTR
3’ LTR
Long Terminal
Repeat (LTR):
Regulatory
sequence (promoter
and enhancer)
Retroviral vector: infection
•The virus enter the target cell
• the viral genome is integrated
in the host genome
•The therapeutic protein is produced
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
Number of Gene Therapy Clinical
Trails approved worldwide 1989-2009
Two examples of Gene Therapy for
hematologic diseases.
 ADA-SCID
 b-thalassemia
Replacement of the gene in
Hematopoietic Stem Cells (HSC)
Blood and Tissues
Bone Marrow
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
ADA-SCID : treatment
 Bone Marrow Transplantation
 ADA enzyme therapy
 Gene Therapy
Gene Therapy Clinical Trial for ADASCID in Italy
(Aiuti et al. Science 2002)
Gene Therapy Clinical Trial for ADASCID in Italy: vector
Retroviral vector production
LTR
ADA
Sv40 NeoR
LTR
Gene Therapy Clinical Trial for ADASCID in Italy: protocol.
Blood
Retroviral vector
T-Lymphocyte
NK cells
B-Lymphocyte
Erythrocyte
Platelets
Granulocytes
Monocytes
Bone
Marrow
Macrophages
Bone Marrow Stem Cells
(CD34+)
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
Dendritic Cells
Tissue
Gene Therapy Clinical Trial for ADASCID 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)
ADA-SCID gene therapy
(Aiuti at al. Hematology 2009)
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)
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
Follow up study in ADA-SCID
patients from the italian trial
(Journal of Clinical Investigation, 2007)
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)
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.
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
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
b-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 β-globin gene could have a therapeutic potential in β thalassemia .
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 bglobin gene.
 Latest vector of choice for b-globin gene is
SIN-lentiviral vector
R U5
b-Globin
bP HS2 HS3 HS4
U3
U3
SIN-Lentiviral vector for
b-thalassemia gene therapy
R U5
Lentiviral vector:
retrovirus family
ssRNA
Integrate in the host genome
8Kb insert size
Infect also quiescent cells
Safety features:
SIN=Self 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
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.
(Felsenfeld et al., Science 2001)
I
R U5
b-Globin
bPHS2 HS3 HS4
U3
U3
Use of “Insulator” in a b-globin lentiviral
vector for Gene Therapy of b-Thalassemia
I
R U5
?Oncogene
Insertion of insulator sequences in a Lentiviral Vector to increase
the safety of the vector, blocking the activity of the enhancer towards surrounding
genes.
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