Metachromatic leukodystrophy (MLD) is a neurodegenerative lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. The disease primarily affects children and invariably leads to premature death. In previous work with a mouse model of MLD, we used a lentiviral vector (LV) to introduce a functional ARSA gene into hematopoietic stem cells (HSCs) ex vivo and showed that reinfusion of the engineered HSCs prevented and corrected disease manifestations in the animals. To determine whether this gene therapy strategy is safe and can offer therapeutic benefit to patients with early-onset MLD, we designed a phase I/II trial.

HSC gene therapy can prevent progression of metachromatic leukodystrophy. Magnetic resonance (MR) images of the brain of a patient (MLD01) before and after gene therapy. The brain of this patient appeared largely normal 2 years after treatment. In contrast, the brain of an untreated, age-matched late infantile MLD patient (UT LI MLD) showed severe demyelination associated with diffuse atrophy. (Top) Axial T2 weighted fast spin-echo MR images. (Bottom) Fluid-attenuated inversion recovery (FLAIR) MR images.

We optimized LV manufacturing and HSC transduction in clinical grade conditions. Three children with ARSA deficiency and mutations associated with early-onset MLD were treated at the presymptomatic stage; all had at least one older sibling affected by the same disease variant. HSCs from the patients were transduced ex vivo with a LV carrying the ARSA gene.

Patients were treated with a myeloablative busulfan conditioning regimen before reinfusion of the engineered HSCs. Clinical and objective evaluations were collected up to 24 months after treatment. Molecular follow-up of vector integration site distribution was performed on hematopoietic cells derived from bone marrow and peripheral blood.

There was high-level stable engraftment of the transduced HSCs in the bone marrow and peripheral blood of all patients at all times tested, with 45 to 80% of bone marrow–derived hematopoietic colonies harboring the vector. With these high gene marking levels, ARSA activity was reconstituted to above normal values in the hematopoietic lineages and in the cerebrospinal fluid. Analysis of >36,000 different LV integration sites showed that the high gene marking was sustained by highly polyclonal engraftment of transduced cells without evidence of aberrant clonal behavior. Several integration sites were shared among progenitors and mature myeloid, B- and T-cells sampled at long intervals after treatment, indicating efficient transduction and engraftment of HSCs. These findings were associated with a clear therapeutic benefit because the disease did not progress in any of the treated patients, even after the time of onset projected from sibling cases.

Our gene therapy protocol allows stable engraftment of transduced HSCs at high levels and without evidence of vector-induced genotoxicity. The reconstitution of ARSA activity in the cerebrospinal fluid and the arrested progression of neurodegenerative disease in the three treated patients demonstrate that the transplanted cells, or their progeny, can seed the nervous system and deliver therapeutic levels of active enzyme. Although our data are promising, long-term follow-up of the patients is needed in order to establish the full therapeutic potential of this gene therapy strategy for MLD. In addition, our data position LV gene transfer as a feasible means to engineer human hematopoiesis to its near entirety—an approach that could be exploited for treatment of other diseases.