Gene Therapy for Metachromatic Leukodystrophy (TIGET-MLD)

• Conditioning regimen-related safety [ Time Frame: at +60 days after transplantation ] [ Designated as safety issue: Yes ]
  The absence of engraftment failure or delayed hematopoietic reconstitution (prolonged aplasia), defined as Absolute Neutrophil Count (ANC)<500/μl with no evidence of BM recovery, requiring cellular back-up administration.
  
  
• Conditioning regimen-related toxicity [ Time Frame: 3 years ] [ Designated as safety issue: Yes ]
  The absence of regimen related toxicity, as determined by a surveillance of clinical (NCI >2) and laboratory (NCI >3) parameters.
  
  
• The short-term safety and tolerability of lentiviral-transduced cell infusion [ Time Frame: 48 hours after transplant ] [ Designated as safety issue: Yes ]
  It will be evaluated on the basis of adverse events reporting and monitoring of the systemic reactions to cell infusion (fever, tachycardia, nausea and vomiting, joint pain, skin rash).
  
  
• Improvement of GMFM score [ Time Frame: 24 months after treatment ] [ Designated as safety issue: No ]
  An improvement of 10% of the total GMFM score in treated patients, when compared to the GMFM scores in control MLD population. GMFM is a clinically relevant endpoint being a direct measure of motor impairment and there is a causal relationship between the potential beneficial outcome measured with the GMFM and the treatment, being motor impairment consequent to the involvement of both CNS and PNS, and less influenced by other variables. A 10% modification of the total GMFM score is as a clinically relevant change according to the experience of therapists in children with cerebral palsy.
  
  
• Increase of residual ARSA activity [ Time Frame: 24 months after treatment ] [ Designated as safety issue: No ]
  A significant (≥ 2 SD) increase of residual ARSA activity as compared to pretreatment values, measured in PBMC and/or BM progenitors.
  
  
• The long-term safety of lentiviral-transduced cell infusion [ Time Frame: 24 months after the treatment ] [ Designated as safety issue: Yes ]
  • Absence of Replication Competent Lentivirus (RCL): ELISA for HIV p24 antigen (plasma) and DNA PCR for VSV-G env (cells). RT-PCR for HIV-pol RNA, anti HIV p24 antibodies. If of the abovementioned test results are positive, the screening evaluations will be repeated at the next planned follow up visit; in the case two of the three screening tests result positive, a confirmatory RCL culture tests will be performed.
  • Absence of Abnormal Clonal Proliferation (ACP). Abnormal hematopoietic clonal expansion will be monitored by clinical and laboratory surveillance, repertoire study, and bone marrow examination.
  
  

• The absence of immune responses against the transgene [ Time Frame: every three months for the first year, then once a year. ] [ Designated as safety issue: Yes ]
  Even if we do not expect immune responses against the functional ARSA enzyme, according to our preclinical data and having most of MLD patients residual enzymatic activity, treated patients will be monitored for antibodies anti-ARSA (immunoblot analyses).
  
  
• Improvement in the NCV Index for ENG and in the total score for MR [ Time Frame: 24 months after treatment. ] [ Designated as safety issue: No ]
  An improvement in the NCV Index for ENG and in the total score for MR of ≥ 2 SD, when compared to the scores observed in the historical control MLD population. ENG and MR were validated as informative quantitative instrumental read outs during our natural history study.
  
  
• Transduced cell engraftment [ Time Frame: 12 months after treatment ] [ Designated as safety issue: No ]
  Transduced cell engraftment above 4% in PBMCs and CD34+ progenitors in BM (determined as VCN/cell ≥0.04 at quantitative PCR, equivalent to 4% assuming a VCN of 1). This value is based on clinical experience in other gene therapy trials, which has demonstrated 4% average long-term engraftment of autologous hematopoietic stem/progenitor cells transduced with onco-retroviral vectors in the bone marrow of pediatric patients suffering from immunodeficiency (ADA-SCID) and receiving reduced intensity, non-myeloablative conditioning.
  
  
• IQ measurement above 55 [ Time Frame: 24, 30 and 36 months after treatment ] [ Designated as safety issue: No ]
  The measurement of an IQ above 55 (threshold for severe disability) at neuro-psychological testing
  
  

Genetic: Autologous CD34+ stem cells transduced with ARSA encoding lentiviral vector
Autologous hematopoietic stem/progenitor cells collected from the bone marrow and transduced ex vivo with a Lentiviral vector encoding the human ARSA cDNA under the control of the human phosphoglyceratekinase (PGK) promoter. Dose: ≥ 2x10^6 transduced CD34+ cells/Kg (maximum 20x10^6) at bedside for infusion.

Metachromatic Leukodystrophy (MLD) is an autosomal recessive Lysosomal Storage Disorder (LSD) characterized by severe and progressive dysmyelination affecting the central and peripheral nervous system.

Hematopoietic cell transplantation (HCT) is ineffective in ameliorating patients' phenotype or delaying disease evolution. No evidences of efficacy of enzyme replacement strategies are available at the moment. Transplantation of genetically corrected autologous hematopoietic stem cells (HSC) could represent a novel and potentially efficacious treatment for MLD patients.

We are conducting a gene therapy clinical trial based on autologous HSC and advanced generation lentiviral vectors (LV) for patients affected by the most severe, early onset forms of the disease, based on our preclinical efficacy and safety data and on the experience we acquired from the treatment of Adenosine deaminase deficiency (ADA-SCID) patients by a similar approach. Our general goal is to demonstrate safety and efficacy of this gene therapy approach in MLD patients. We have developed and tested a clinically applicable LV containing the ARSA cDNA controlled by a constitutive eukaryotic promoter sequence. Preclinical studies aimed at assessing efficacy and safety of ARSA gene transfer using the selected vector have been conducted in the murine model of the disease and in progenitors and mature hematopoietic cells from MLD patients. The following results were obtained:

1. efficient ARSA gene transfer;
2. long term in vitro and in vivo multilineage ARSA expression;
3. ability to prevent and correct neurological disease manifestations;
4. good tolerance and long-term safety of the treatment in the animal model.

We expect to treat 20 patients, selected according to disease severity and clinical features. A myeloablative conditioning regimen based on iv Busulfan will be administered to the patients to enable engraftment of the transduced stem/progenitor cells in the bone marrow and enhance CNS microglia/macrophage repopulation by the transduced cell progeny. An efficient transduction protocol based on LVs has been optimized to guarantee abundant ARSA expression in the transduced patients' HSC and their progeny. In the treated patients, we will study the short-term and long-term safety of the administration of the autologous transduced HSC, their long-term engraftment, the expression of vector-derived ARSA, and the ability of the transduced cells to provide a clinical benefit to the patients. The treated patients will be followed for 3 years and thereafter monitored for the safety of gene therapy for additional 5 years. If successful, this study will provide key results on the safety and efficacy of gene therapy for MLD patients.