1. Gene therapy
Fabrizia Urbinati
01/12/2010

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

3. What is gene therapy?
Introduction of normal genes into an individual’s 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
ADA
Sv40
NeoR
LTR

18. Gene Therapy Clinical Trial for ADA- SCID in Italy: protocol.
T-Lymphocyte
B-Lymphocyte
NK cells
Bone Marrow
Blood
Dendritic Cells
Macrophages
Monocytes
Erythrocyte
Granulocytes
Tissue
Platelets
Retroviral vector
Bone Marrow Stem Cells
(CD34+)
But long-term engraftment of a multiuponte stem cell capable of reconstituting the whole hematopoietic system would be highky desirable to fully correct the metabolic defect of the disease.
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 β-globin gene could have a therapeutic potential in β -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
U3
R U5
HS2 HS3 HS4
b-Globin bP
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

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.
(Felsenfeld et al., Science 2001)

31. Use of “Insulator” in a b-globin lentiviral vector for Gene Therapy of b-Thalassemia
R U5 I
U3
?Oncogene
HS2 HS3 HS4
b-Globin bP
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 2 Gene Therapy studies:
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