Accurate prediction of busulfan exposure is critical to achieve target myeloablative exposure while minimizing toxicity in pediatric hematopoietic stem cell transplantation. Model-informed precision dosing is recommended for improved accuracy and flexibility; dose adjustments in clinical practice are frequently performed using linear proportionality between dose changes and area under the concentration–time curve (AUC), optionally accounting for decrease of busulfan clearance over time. We retrospectively analyzed 54 consecutive pediatric patients receiving once-daily intravenous busulfan with therapeutic drug monitoring to characterize the relationship between dose adjustments and AUC variations, and to explore determinants of acute toxicity. Correlation between dose adjustments and AUC variations was linear for dose changes up to 1.16-fold, but linearity underestimated AUC increases following larger dose escalations. A quadratic model better described the dose–AUC relationship, particularly for dose increases, and was supported by an inverse correlation between dose variation and clearance change. While cumulative AUC was consistently maintained within target ranges, higher peak single-dose AUCs were associated with increased acute toxicity, including longer duration of mucositis and greater parenteral nutrition requirements. Initial dosing at 80 mg/m2 was associated with lower peak AUCs and reduced need for subsequent dose corrections compared with 120 mg/m2, without differences in engraftment outcomes. These findings highlight the clinical relevance of non-linear dose–exposure relationships and peak exposure in busulfan-related toxicity.

Metachromatic leukodystrophy (MLD) is a rare, autosomal recessive lysosomal storage disorder caused by a deficiency of the enzyme arylsulfatase A (ARSA, MIM #250100), which leads to progressive demyelination in the central and peripheral nervous systems, resulting in severe neurodegeneration and premature death. With the recent approval of the first ex vivo gene therapy for MLD, newborn screening (NBS) programs have begun to implement pilot studies for early detection, as identifying affected individuals during the pre-symptomatic therapeutic window is crucial. We present the analytical workflow and the NBS algorithm developed during the first year of MLD screening in Lombardy. The screening strategy comprised a first-tier test (1TT) using mass spectrometry to quantify sulfatide concentrations, a second-tier test (2TT) measuring ARSA enzymatic activity, and a third-tier test (3TT) based on whole-exome sequencing. A total of 16,130 newborns were screened for MLD. Of these, 6.41% required retesting (1TT), 1.53% underwent duplicate 2TT, and 0.36% proceeded to 3TT. No neonates were recalled, and no cases of MLD were identified through the screening program. The first-year implementation of MLD NBS in Lombardy demonstrates that a mass spectrometry-based sulfatide assay combined with a multi-tiered screening algorithm is feasible and reliable. Incorporating genetic analysis alongside expanded validation of additional sulfatide species may enhance specificity, reduce false positives, and facilitate timely identification of infants affected by MLD.

Background Metachromatic leukodystrophy (MLD) is a rare, progressive neurodegenerative disorder caused by arylsulfatase A deficiency, leading to accumulation of sulfatides and widespread demyelination. Autologous haematopoietic stem and progenitor cell gene therapy (HSPC-GT; atidarsagene autotemcel, arsa-cel) has emerged as an effective treatment for early-onset MLD when administered before or at very early stages of neurological involvement. However, standardized recommendations for pre-treatment evaluation, treatment phase care, and long-term follow-up are lacking. Methods Under the auspices of the INTEGRATE-ATMP project, a European multidisciplinary expert panel conducted a multi-round consensus process. Following a literature review, key clinical questions were addressed during five structured virtual and hybrid meetings. Recommendations were developed for pre-treatment assessment, treatment-phase management, and post-treatment follow-up. Investigations were classified as “mandatory” or “optional” based on expert agreement. Results The panel defined comprehensive, phase-specific recommendations for children undergoing HSPC-GT for MLD. Pre-treatment guidance emphasizes rapid diagnostic confirmation, standardized neurological and developmental assessments, and multidisciplinary eligibility evaluation. Treatment phase recommendations address stem cell collection, busulfan conditioning, supportive care, and monitoring for acute complications. Post-treatment guidance outlines a structured long-term follow-up programme, including neurological, developmental, imaging, and laboratory surveillance. Long-term data collection and systematic biobanking are strongly encouraged for a minimum of 15 years to support safety monitoring and outcome evaluation. Conclusion These European expert recommendations provide a practical framework for standardized care of children treated with HSPC-GT for MLD. Implementation across qualified treatment centres may improve clinical consistency, facilitate real-world data collection, and support sustainable delivery of gene therapy programmes. By detailing the comprehensive monitoring and follow-up required, including assessments beyond current standards of care, this work highlights the resource-intensive nature of gene therapy. It also provides a framework for the multidisciplinary care efforts needed to support planning and appropriate reimbursement by health authorities.

Wiskott-Aldrich Syndrome (WAS) is a rare, X-linked, life-threatening inborn error of immunity and platelet disorder caused by WAS protein (WASP)-encoding gene mutations. Etuvetidigene autotemcel (etu-cel) is an autologous gene therapy (GT) consisting of hematopoietic stem progenitor cell (HSPCs) transduced ex vivo with a lentiviral vector encoding human WAS cDNA. Etu-cel was intravenously infused after rituximab and reduced-intensity conditioning. Data from WAS patients treated in two prospective open-label clinical trials (phase I/II n=8; phase III n=10) and one expanded access program (EAP) (n=9) were integrated to evaluate efficacy and safety of etu-cel. Primary efficacy endpoints were overall survival, rate of severe infections from 6 to 18 months after GT and rate of moderate/severe bleeding episodes in the first 12 months post-treatment compared with 1 year prior to GT. Secondary efficacy endpoints included engraftment of gene-corrected cells, WASP expression, T-cell function, platelet count, autoimmunity and eczema over time. Safety endpoints included adverse events (AEs), immune response to transgene, development of replication-competent lentivirus (RCL) and abnormal clonal proliferation (ACP). Median follow-up was 5.7 years (range: 0.4-13.3). Median age at treatment was 2.6 years (range: 1.0-35.1). Overall survival was 96%; one EAP subject died early post-GT due to deterioration of a pre-existing neurological condition. The rate of severe infections per person-year of observation (PYO) decreased from 2.00 (95% CI: 1.50-2.61) pre-GT to 0.15 (95% CI: 0.04-0.39) in the 6-18 months period post-GT. The rate of moderate and severe bleeding events per PYO decreased from 2.00 (95% CI: 1.50-2.61) to 0.80 (95% CI: 0.49-1.22) in the 12 months after GT. After GT, multilineage engraftment of gene-corrected cells was sustained over time. WASP expression in platelets and lymphocytes increased. Platelet count, T-cell functionality, eczema and autoimmunity improved. The most common adverse event ≥ grade 3 was device related infection. Etu-cel was well-tolerated with no treatment-related adverse events and no evidence of insertional oncogenesis. With up to 13 years follow-up, etu-cel demonstrates a favorable benefit-risk profile, leading to sustained long-term clinical benefit. (Funded by GlaxoSmithKline [GSK], Orchard Therapeutics, Fondazione Telethon; ClinicalTrials.gov numbers: NCT01515462 , NCT03837483 )

Metachromatic leukodystrophy (MLD) is a rare inherited disorder of lysosomal storage, caused by a deficiency in the arylsulfatase A (ARSA) enzyme, leading to toxic accumulation of sulfatides, which progressively impair motor and cognitive function. MLD is a candidate for inclusion in newborn screening (NBS) programs, due to the narrow pre-symptomatic window for effective therapeutic intervention. We set up a prospective pilot NBS program for MLD in Tuscany, based on a two-step approach. The first-tier test quantified four sulfatides; if levels exceeded the cut-off, we performed the second-tier test by measuring ARSA activity on the same neonatal dried blood spot (DBS). We performed the first-tier test on 42,262 newborns over two years and the second-tier test on residual neonatal DBS from 90 of them (0.21%). We recalled 10 newborns (0.02%) for an additional DBS, due to insufficient residual material for a second-tier test (n = 4) or to low ARSA activity (n = 6). We found normal ARSA activity in all new DBS and identified no new cases of MLD. Retrospective analysis of eight neonatal and fifteen non-neonatal DBS from patients with genetically confirmed MLD showed that the algorithm accurately identified MLD patients. This diagnostic algorithm proved feasible and accurate for early detection of MLD in prospective NBS.

This study evaluated the efficacy of atidarsagene autotemcel (arsa-cel) gene therapy in mitigating the severity and progression of peripheral neuropathy as assessed by nerve conduction velocity (NCV) in individuals affected by late-infantile metachromatic leukodystrophy (LI-MLD). A post hoc analysis was conducted on pre-symptomatic patients affected by LI-MLD treated with ex vivo autologous haematopoietic stem cell gene therapy (arsa-cel) in the context of prospective open-label, single-arm, interventional trials and expanded access programmes. All patients were followed longitudinally with nerve conduction studies (NCSs) of peripheral motor (ulnar, deep peroneal) and sensory (median, sural) nerves. These results were compared with those from a control group of untreated patients (NHx) studied with the same standardized protocol. We then analysed the effects of baseline characteristics (age at treatment, severity of neuropathy pre-treatment expressed as age-matched NCV Z-scores) and arylsulphatase A (ARSA) enzyme activity (measured in peripheral blood myeloid CD15+ cells post-treatment) on NCVs of treated patients. The primary end point of this post hoc analysis was NCV, reflecting the severity of demyelinating neuropathy. Changes in dermal nerve histopathology in skin biopsies were used as an exploratory outcome. Fifteen treated and 16 NHx patients were included in the analyses, with a median age (interquartile range) at treatment of 13 (9.1-14.5) months. At 36 months of age, treated patients showed higher estimated NCVs in all nerves compared with age-matched controls (∼15 m/s difference in motor nerves). Peripheral neuropathy was observed in the majority of treated patients at their pre-treatment examination (age range 7.3-17.4 months). Severity of pre-treatment neuropathy in treated patients did not have an effect on NCV values at 2 years post-gene therapy or on the rate of NCV-slowing afterwards. A younger age at treatment was associated with higher NCVs of motor ulnar nerve and sensory medial nerve 2 years post-gene therapy. Overall, ARSA levels in CD15+ cells correlated with NCVs of motor deep peroneal nerve at 2 years post-gene therapy, and ARSA levels were associated with a slower decrease or a slight increase in NCVs of the deep peroneal, ulnar and medial nerves afterwards. In summary, peripheral neuropathy assessed by NCV is significantly ameliorated in LI patients treated with arsa-cel compared with untreated patients of similar age. In addition to the potential role of early age at treatment in the preservation of myelin, supraphysiological ARSA levels may slow demyelination of the deep peroneal and other peripheral nerves. Arsa-cel may exert a stronger effect on NCV than allogeneic haematopoietic stem cell transplantation due to its greater ARSA expression.

BACKGROUND:Metachromatic leukodystrophy (MLD) is an ultrarare, severe lysosomal storage disorder caused by a deficiency of arylsulfatase A (ARSA). METHODS:We treated patients who had MLD with atidarsagene autotemcel (arsa-cel), a hematopoietic stem-cell-based gene therapy, in two prospective open-label clinical studies and expanded-access programs. We compared their outcomes with those of untreated patients (natural history cohort). The primary end point was survival free from severe motor impairment (the time from birth to the first occurrence of loss of locomotion and of sitting without support or death from any cause). RESULTS:A total of 39 treated patients and 49 untreated patients were included. The median follow-up was 6.76 years (range, 0.64 to 12.19). Arsa-cel resulted in a significantly lower risk of severe motor impairment or death than no treatment among patients with presymptomatic late-infantile MLD (P<0.001), those with presymptomatic early-juvenile MLD (P = 0.04), and those with early-symptomatic early-juvenile MLD (P<0.001). The estimated percentage of patients surviving without severe motor impairment at 6 years of age was 0% (95% confidence interval [CI], not evaluable) among untreated patients with late-infantile MLD and 100% (95% CI, 100 to 100) among treated patients with presymptomatic late-infantile MLD. The estimated percentage of patients surviving without severe motor impairment at 10 years of age was 11.2% (95% CI, 0.9 to 36.4) among untreated patients with early-juvenile MLD and 87.5% (95% CI, 38.7 to 98.1) and 80.0% (95% CI, 40.9 to 94.6) among treated patients with presymptomatic and early-symptomatic early-juvenile MLD, respectively. No evidence of insertional oncogenesis was found. The most common grade 3 or higher adverse event was febrile neutropenia. Anti-ARSA antibodies were detected transiently in 6 of 39 patients (15%). Three deaths occurred, all of which were considered by the investigators to be unrelated to arsa-cel. CONCLUSIONS:Among patients with presymptomatic late-infantile or early-juvenile MLD and those with early-symptomatic early-juvenile MLD, the risk of severe motor impairment or death was significantly lower among those who received treatment with arsa-cel than in a natural history cohort that did not receive treatment. (Funded by Orchard Therapeutics and others; ClinicalTrials.gov numbers, NCT01560182 and NCT03392987.).

BACKGROUND Wiskott-Aldrich Syndrome (WAS) is a rare, X-linked, life-threatening inborn error of immunity and platelet (plt) disorder caused by WAS protein (WASP)-encoding gene mutations. WAS is characterized by thrombocytopenia, bleeding events, recurrent and severe infections, eczema, and increased risk of immune dysregulation and malignancy. Allogeneic hematopoietic stem cell transplantation (HSCT) can be curative but is still hampered by limited donor availability and potential complications [Burroughs et al. 2020; Albert et al. 2022]. Etuvetidigene autotemcel (Etu-cel) is an experimental autologous gene therapy (GT) with BLA in the US and MAA in Europe currently under evaluation. It consists of hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a lentiviral vector encoding human WAS cDNA under endogenous promoter control. We previously reported interim safety and efficacy data in 17 WAS subjects treated in a phase I/II clinical trial and in an expanded access program (EAP) [Ferrua et al. 2019]. We now report the results of an integrated efficacy and safety analysis in 27 WAS patients treated with Etu-cel with extended long-term follow-up. METHODS Patients received fresh (phase I/II trial, NCT01515462, n=8; EAP, n=9) or cryopreserved (phase III trial, NCT03837483, n=10) formulation of Etu-cel. Autologous CD34+ HSPCs source for drug product (DP) manufacturing consisted in bone marrow (n=5), mobilized peripheral blood (n=21) or both (n=1). Etu-cel was intravenously infused after rituximab and reduced-intensity conditioning (RIC). Primary efficacy endpoints were overall survival (OS), rate of severe infections from 6 to 18 months (mos) post-GT and rate of moderate/severe bleeding events in the first 12 mos post-GT. Secondary efficacy endpoints included engraftment of gene-corrected cells, WASP expression, T-cell function, plt count, autoimmunity and eczema over time. Safety endpoints included adverse events (AEs), immune response to transgene, development of replication-competent lentivirus (RCL) and abnormal clonal proliferation (ACP). RESULTS Median age at treatment was 2.6 years (range: 1.0-35.1). OS was 96% (95%CI: 82-99%); one EAP subject died 4.5 mos post-GT due to deterioration of a pre-existing neurological condition. Median follow-up in surviving patients was 5.72 years (range: 2.31-13.26). The rate of severe infections per person-year of observation (PYO) decreased from 2.00 (95%CI: 1.50-2.61) in the 12 mos before GT to 0.15 (95%CI: 0.04-0.39) in the 6-18 mos post-GT and 0.05 (95%CI: 0.01-0.16) in the >5-year post-treatment period. The rate of moderate and severe bleeding events per PYO decreased from 2.00 (95%CI: 1.50-2.61) in the 12 mos before GT to 0.80 (95%CI: 0.49-1.22) in the 12 mos after GT and 0.02 (95%CI: 0.00-0.10) in the >5-year post-treatment period. Durable multilineage engraftment of gene-corrected cells led to restoration of WASP expression in lymphocytes and plt, which resulted in ameliorated immune-cell function and plt count. Sustained immunoglobulin replacement could be stopped in all patients at a median of 320.5 days after GT (range: 91–1843) and cessation of sustained antimicrobial treatment was achieved in 25 (92.6%) patients. Plt transfusions were stopped at a median of 46.5 days after treatment (range: 9-261), with no need of thrombopoietin agonist long-term. Median plt count at year 5 post-GT was 68.3 (48.0-228.0)×109/L (n=22).Eczema resolved in all and immune dysregulation improved in most patients post-GT, with no need of prolonged immune suppression. After GT, hospitalization rate declined, and quality of life improved in all. Etu-cel was well tolerated with no treatment-related AEs, no engraftment failures, no evidence of ACP, RCL or immunogenicity. Overall, AEs following treatment with Etu-cel are consistent with those expected in WAS patients undergoing hematological reconstitution after RIC. The most common AE ≥grade 3 was device-related infection. None of the treated patients required any secondary procedures (e.g. splenectomy, HSCT) after GT. Outcome was similar across different ages, disease characteristics, WAS mutations, and DP formulations. Results from this integrated analysis on a larger population and longer follow-up show the efficacy and safety of a single infusion of Etu-cel for the treatment of WAS. With up to 13 years follow-up, Etu-cel demonstrates a favorable benefit-risk profile, with sustained long-term significant clinical benefit.