Gene therapy

Fabrizia Urbinati01/12/2010

Outline

Gene therapy introduction:

Delivery method Vectors Candidate Diseases

ADA-SCID clinical trial -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 Non Viral Vectors

•Adeno Virus•Adeno Associated virus•RetrovirusLentivirus•Herpes virus•…

•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

-thalassemia

Replacement of the gene in Hematopoietic Stem Cells (HSC)

Bone Marrow

Blood and Tissues

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

(Aiuti et al. Science 2002)

Gene Therapy Clinical Trial for ADA-SCID in Italy

LTRSv40 NeoRADALTR

Retroviral vector production

Gene Therapy Clinical Trial for ADA- SCID in Italy: vector

T-Lymphocyte

B-Lymphocyte

NK cells

Bone Marrow

Blood

Dendritic Cells

Macrophages

Monocytes

Erythrocyte

Granulocytes

Tissue

Platelets

Retroviral vector

Bone Marrow Stem Cells(CD34+)

Gene Therapy Clinical Trial for ADA- SCID in Italy: protocol.

Bone Marrow stem cells collection from 2 patientsInfection of BM stem cells with Retroviral vectorBusulfan prior to BM infusion (“non-myeloablative conditioning”). Re-infusion of corrected BM cells into the patient

(Aiuti et. Al Science 2002)

ADA enzyme activity wasrestored and lymphoid reconstitution was shown after gene therapy treatment

Immune reconstitution by 6 months.

T cells gene-marked at 100%

Gene Therapy Clinical Trial for ADA- SCID in Italy: results

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

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

Leukemia was caused by the retroviral vector carrying the therapeutic gene (IL2RG)

Follow up study in ADA-SCID patients from the italian trial

(Journal of Clinical Investigation, 2007)

Expression of LMO-2 gene in pt. treated with gene therapy:The expression of the oncogene did not change

Retroviral integration site in patient with ADA-SCID: many oncogenes were hit by the provirus

Follow up study in ADA-SCID patients from the italian trial

(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 -thalassemia Gene Therapy

Thalassemias are hereditary anemias and are the most common single gene defects worldwide.

-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 .

-thalassemia Gene Therapy

There are no current Gene Therapy trials for -thalassemia.

Many studies have been focused on the optimization of the vectors carrying the -globin gene.

Latest vector of choice for -globin gene is SIN-lentiviral vector

SIN-Lentiviral vector for -thalassemia gene therapy

U3

R U5HS2 HS3 HS4-GlobinU3

R U5 P

Lentiviral vector:

retrovirus familyssRNAIntegrate in the host genome8Kb insert sizeInfect 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 -globin gene is driven by the -globin promoterand its enhancer (increase safety of the vector) that are lineage specific

Use of “Insulator” in a -globin lentiviral vector for Gene Therapy of -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)

R U5IU

3 ?OncogeneHS2 HS3 HS4-Globin PR U5IU

3

Use of “Insulator” in a -globin lentiviral vector for Gene Therapy of -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.

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

-Thalassemia Gene Therapy as an example of optimization of safer vectors