Gene therapy Fabrizia Urbinati 01/12/2010

Outline Gene therapy introduction: ADA-SCID clinical trial 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 Non Viral Vectors Viral Vectors Naked DNA Liposome Oligonucleotides Adeno Virus Adeno Associated virus RetrovirusLentivirus Herpes virus …

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 Long Terminal Repeat (LTR): Regulatory sequence (promoter and enhancer) 5’ LTR 3’ LTR 3’ LTR 5’ LTR

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 ADA-SCID in Italy (Aiuti et al. Science 2002)

Gene Therapy Clinical Trial for ADA- SCID in Italy: vector Retroviral vector production LTR ADA Sv40 NeoR LTR

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

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)

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 -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 b-globin gene. Latest vector of choice for b-globin gene is SIN-lentiviral vector

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

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)

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.

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