Gene Therapy for Wiskott-Aldrich Syndrome (TIGET-WAS)

• Conditioning regimen-related safety [ Time Frame: two months after gene therapy ] [ Designated as safety issue: Yes ]
  Absence of engraftment failure or prolonged aplasia (<500/ul ANC with no evidence of bone marrow recovery) and surveillance of non haematological regimen related toxicity (for clinical features NCI >2, for metabolic/laboratory NCI >3)
  
  
• Safety of lentivirus gene transfer into HSC [ Time Frame: 3 years ] [ Designated as safety issue: Yes ]

  short-term safety and tolerability of lentiviral-transduced cell infusion

  -long-term safety of lentiviral-transduced cell infusion (absence of Replication Competent Lentivirus (RCL) and abnormal clonal proliferation).

  
  
• Sustained engraftment of genetically corrected haematopoietic stem cells in peripheral blood and/or in bone marrow [ Time Frame: 1 year ] [ Designated as safety issue: No ]
  ≥ 0.04 Vector copy number (VCN)/cell in bone marrow CD34+ or ≥0.1 VCN/cell in peripheral blood T lymphocytes
  
  
• Expression of vector-derived WASP [ Time Frame: 1 year ] [ Designated as safety issue: No ]
  Detection of vector-derived WASP expression by FACS analyses and/or Western Blot
  
  
• Improved T-cell functions [ Time Frame: 3 years ] [ Designated as safety issue: No ]
  Improvement in in vitro T cell proliferation and/or IL-2 secretion upon stimulation with anti-CD3i as compared to pre-gene therapy values.
  
  
• Antigen-specific responses to vaccination [ Time Frame: 1year ] [ Designated as safety issue: No ]
  Ability to mount a humoral response to nominal antigens including antibodies to T cell dependent antigens (Tetanus Toxoid) and unconjugated polysaccharide antigens (Peumococcus, Meningococcus), measured after vaccination (foreseen >1 year after gene therapy). Positive cellular response to Tetanus Toxoid after vaccination measured by in vitro proliferative response >1 year after gene therapy.
  
  
• Improved platelet count [ Time Frame: 3 years ] [ Designated as safety issue: No ]
  Sustained increase in platelet count compared to baseline, analysing the individual longitudinal profile
  
  

• Lack of immune response to transgene [ Time Frame: 3 years ] [ Designated as safety issue: Yes ]
  Immunoblot analysis
  
  
• Reduced frequency of severe infections [ Time Frame: 3 years ] [ Designated as safety issue: No ]
  Decrease in number of severe infections as evaluated in the second and third year after the treatment by clinical history, complete physical examinations, hematological and microbiological tests.
  
  
• Reduced bruising and bleeding episodes [ Time Frame: 3 years ] [ Designated as safety issue: No ]
  Reduction in bruising and/or bleeding manifestations when present, as assessed by clinical monitoring, compared to clinical history
  
  
• Reduced autoimmunity phenomena and eczema [ Time Frame: 3 years ] [ Designated as safety issue: No ]
  Reduction in laboratory markers (number and titer of antibody when available) and/or clinical manifestations of autoimmunity, as evaluated by organ-specific and systemic autoantibodies, imaging and clinical follow-up, compared to clinical history. Reduction in eczema as evaluated by clinical score
  
  
• Improved quality of life [ Time Frame: 3 year ] [ Designated as safety issue: No ]
  Improved quality of life, measured after the first year of treatment by reduced hospitalization, reduced requirement of drugs, school attendance, social activities.
  
  

Genetic: Autologous CD34 positive cells transduced with WAS encoding lentiviral vector.
The Medicinal Product consists of autologous CD34+ cells collected from the bone marrow and/or peripheral blood and transduced with a lentiviral vector encoding WASP controlled by WAS promoter sequences (w1.6W). Dosage indications: a minimum dose of 2x10^6 CD34+ cells/Kg (maximum 20x10^6 CD34+ cells/Kg) and an optimal dose of 5-10x10^6 CD34+ cells/Kg will be infused i.v.

We expect to treat 6 patients with autologous cells collected from bone marrow and/or mobilized from peripheral blood, transduced with a lentiviral vector encoding for WAS and reinfused. The patients are selected according to disease severity (genetic mutation, WASP expression and clinical score), absence of an HLA-identical sibling or adequate unrelated donor, age and clinical features. A conditioning regimen based on low doses of iv Busulfan, Fludarabine and anti-CD20 antibody will be administered to the patients to make space in the bone marrow for gene corrected stem cells, deplete the lymphoid compartment of potentially autoreactive lymphocytes and prevent lymphoproliferative disorder. Patients with autoimmune manifestations will also receive ATG Thymoglobuline to target auto-reactive memory T lymphocytes. This reduced intensity conditioning schedule will provide less toxicity than current preparatory regimens, used in standard allogeneic HSC transplantation. The treated patients will be followed for 3 years and thereafter monitored for the safety for additional 5 years.