Long-Term Follow-Up of Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy

Abstract

In 2009, cerebral adrenoleukodystrophy (c-ALD) became the first brain disease to be treated with lentiviral (LV)-based hematopoietic stem cell gene therapy with the ABCD1 gene in four boys (P1–P4) who had demyelinating lesions expected to be lethal in the short term and no bone marrow donor. We report the clinical and magnetic resonance imaging (MRI) follow-up over a mean of 8.8 years posttransplant. In parallel, vector genome copies, expression of transgenic ALD protein (ALDP), and viral integration sites were determined in peripheral blood cells. Prior to transplant, the four patients had a normal or near normal neurocognitive status but gadolinium-enhanced demyelination in various brain regions. Gadolinium diffusion disappeared during the first year posttransplant. P3 kept a near normal status until 8.3 years of follow-up, but P1, P2, and P4 showed major cognitive degradation around 9, 28, and 60 months posttransplant. Neurological status and demyelination stabilized until last evaluation in P2, but deteriorated in both P1 at 10 years and P4 at 3 years posttransplant. The proportion of myeloid and lymphoid cells expressing transgenic ALDP decreased by half within 5 years then stabilized around 5% to 10%. Integration site analysis revealed a durable polyclonal distribution of genetically corrected hematopoietic cells. No adverse effects were observed. The long-term arrest of demyelination at MRI and persistence of transduced hematopoietic progenitors support that LV gene therapy may be a safe and durable treatment of c-ALD. However, the neurological degradation observed in three out of four patients mitigates the benefit of this therapy, calling for an earlier intervention, more potent vectors, and additional therapeutic strategies.

Introduction

Cerebral adrenoleukodystrophy (c-ALD) is caused by mutations of the ABCD1 gene encoding the ALD protein (ALDP).¹ If left untreated, boys develop a rapidly progressive multifocal brain demyelination resulting in a devastating neurological deterioration leading most of them to die before adolescence.^2,3 Until 2009, allogeneic hematopoietic stem cell transplantation (allo-HSCT), thought to result in the cerebral engraftment of microglial cells derived from donor bone marrow myelomonocytic cells,^4,5 was the only effective therapy if performed at an early stage of c-ALD.^6
–8 However, allo-HSCT is limited by donor-related constraints and carries a major risk of mortality.^6
–8 In 2009, our group reported that lentiviral-based HSC (LV-HSC) gene therapy with the ALD gene was a therapeutic alternative to allo-HSCT, based on two patients with c-ALD who had no bone marrow donor and showed a favorable evolution within 2 years of gene therapy.⁹ Two additional cases were included in this pioneer trial but have not been published yet. In a more recent trial, seventeen patients received gene therapy for c-ALD using lenti-D, a more potent LV vector, and 16 were reported after a follow-up averaging 2.4 years (one patient died from transplantation complications).¹⁰ In 15 out of these 16 patients, LV-HSC gene therapy appeared to be an effective treatment of c-ALD. In order for these short-term results to be extended into lasting success, a durable renewal of microglial cells supplied to the brain from efficiently transduced hematopoietic progenitors is required.

Metachromatic leukodystrophy,^11,12 severe combined immunodeficiencies,¹³ Wiskott–Aldrich syndrome (WAS),^14
–16 β-thalassemia,^17,18 and sickle cell disease¹⁹ were also successfully treated with LV vectors. Because the transgene integrates into the genome of hematopoietic progenitors, it was thought that it will remain expressed in the reconstituted hematopoiesis for many years or even for lifetime,^20,21 but the only data available are still limited in terms of follow-up time. Indeed, the longest follow-up, yet, reported average of 3 years (1.5–4.5 years) in the Italian metachromatic leukodystrophy trial,¹² 5.6 years in the Italian WAS trial,¹⁶ and 12 years in the first French patient with β-thalassemia.¹⁷

Since the number of patients treated with LV-HSC gene therapy is growing,^20,21 long-term safety data have also become critical.

For all these reasons, we report herein the evolution of clinical and neuroimaging data over a median of 8.8 years (5.5–12 years) in the first patients treated with LV-HSC gene therapy for c-ALD, as well as the follow-up of ALDP expression in myeloid and lymphoid cells, and the evolution of the vector integration pattern in blood cell genome.

Methods

Patients

c-ALD was diagnosed at the age of 3.5 (P1), 6.3 (P2), 3.6 (P3), and 6 years (P4) (current P1 is P2 in Cartier et al.⁹ and vice versa). Having found no HLA-matched donors for allo-HSCT, patients received gene therapy at 7 (P1), 7.5 (P2), 4.4 (P3), and 7 years (P4) in a pilot trial using a LV vector (CG1711 hALD) expressing ABCD1 cDNA controlled by the MND promoter.⁹ This gene therapy trial was approved by AFSSAPS on November 15, 2005 and CCPPRB Paris-Cochin (local Institutional Review Board) on September 7, 2004. Informed consent was obtained from the parents of the patients after the nature and possible consequences of the studies were explained.

After a fully myeloablative conditioning regimen with cyclophosphamide and busulfan, cryopreserved transduced CD34⁺ cells were infused into the four patients: P1 received 7.2 × 10⁶, P2 4.6 × 10⁶, P3 5.6 × 10⁶, and P4 8.2 × 10⁶ cells per kilogram (mean 6.4 × 10⁶ cells per kilogram). The details of our protocol can be found in Cartier et al.⁹

The same neurologists (P. Aubourg and C.B.) rated the neurologic status according to c-ALD Neurologic Function Scale (NFS), a 25-point scale (0–25) with higher scores indicating more severe deficits.^6,22 Cognitive functions were assessed by the same neuropsychologist (C.C.) using verbal intelligence quotient (VIQ) and nonverbal performance intelligence quotient (PIQ) scores.²² Loss of communication, cortical blindness, tube feeding dependence, total incontinence, wheelchair dependence, and loss of voluntary movement were classified as Major Functional Disabilities (MFDs).⁸ The Loes score, a 34-point scale (0–34), was used to quantify demyelination on magnetic resonance imaging (MRI).²³ Stabilization of Loes score was defined as an increase of <6 from baseline or a score ≤9.¹⁰

Laboratory assessments

ALDP expression was assessed with immunofluorescence in peripheral-blood mononuclear cells, and CD3⁺, CD14⁺, CD15⁺, and CD19⁺ cells.⁹ Vector copy number (VCN) was determined with quantitative PCR.⁹ For analysis of vector integration sites, DNA was purified from peripheral blood cells and the total number of unique integration sites was determined. The relative contribution of the 10 most common integration sites was determined by means of Shearing-Extension Primer Tag Selection Ligation-Mediated Polymerase Chain Reaction and next-generation sequencing^24,25 processed through GENE-IS pipeline²⁶ (see Methods paragraph in Supplementary Data).

Statistics

We used Spearman's nonparametric correlation to test associations across clinical, MRI, and biological parameters.

Results

Cognitive functions and neurological status

Before gene therapy, the four patients had normal or near normal neurocognitive functions. Given the individual variability of the subsequent cognitive and neurological evolution, each of them is described in Fig. 1 and Supplementary Data.

Figure 1.

Evolution of the neurological and cognitive parameters in the four patients. (A) NFS. (B) VIQ. (C) PIQ. Correlation between VIQ and PIQ (R = 0.71, p < 0.001). The gray band indicates the normal mean ± SD of VIQ and PIQ in age-matched boys.²⁸ NFS, neurologic function scale; PIQ, performance intelligence quotient; VIQ, verbal intelligence quotient.

Only P3 has evolved in a remarkably stable way, with a near normal neurological and cognitive status up to 8.3 years posttransplant. In contrast, cognitive functions have begun to decline at various rates in P1, P2, and P4 around 60, 9 and 16 months posttransplant, respectively (Fig. 1B, C). In P2 and P4, this was due to the aggravation of their initially mild frontal syndrome. Finally, at age 16 years, P2 had become unable to make sentences or dress alone, had poor intellectual performance, and disordered behavior. After 33 months, due to major cognitive degradation, P4 could no longer be tested. Over the whole survey in the four patients, both cognitive indices VIQ and PIQ were correlated (R = 0.71, p < 0.001).

As for neurological functions, NFS did not correlate with cognitive functions, as reported in allo-HSCT²⁷ and evolution was also variable. P1 started having visual field cut at 20 months and then started rapid deterioration of NFS (cortical blindness and severe ataxia) soon after 5 years posttransplant. At 8.4 years posttransplant, P2 still had NFS at 1 in contrast with his severe cognitive worsening. As for P4, he added seizures, loss of spontaneous speech, and total urinary incontinence (MFDs) to his severe cognitive deficits. At his last follow-up at age 11.5 years, P4 could only repeat single words or execute simple orders, but could not eat or dress alone, with a NFS at 7.

Neuroimaging

Before gene therapy, enhanced gadolinium contrast showed active inflammatory lesions disrupting the blood–brain barrier in all patients. Gadolinium enhancement disappeared in P1, P2, and P3 at 9, 12, and 1.5 months, respectively, then remained undetectable. In P4, gadolinium enhancement disappeared 4 months posttransplant, reappeared slightly at 16 months, then persisted up to 4.5 years at a low level, before disappearing again at 5.5 years. In P2, P3, and P4, demyelination increased with appearance of new lesions within 16–30 months posttransplant (Supplementary Data), after which the Loes score showed remarkable stability (Fig. 2). P1 kept the same Loes score over the whole survey. Overall, the Loes score showed no relationship with cognitive or neurological status; notably P2 and P4 had a severe evolution of their frontal syndrome without any visible change in frontal demyelination.

Figure 2.

Evolution of brain demyelination (A) Long-term evolution of the Loes score.²³ Maximum score is 34; stability is defined as an increase from baseline of less than 6 points or a score of 9 or less.^6,10 (B) Brain T2/FLAIR MRIs from P2. White arrows indicate the demyelination lesions. Just before transplant (2007), demyelination involved the auditory tracts within the cerebral peduncles and the periventricular parieto-occipital white matter; Loes score was 7. Soon after 16 months, the abnormal hypersignal in the auditory pathways disappeared completely. No further worsening of the parieto-occipital lesions was observed, but dilatation of posterior horns of the ventricles and subcortical atrophy of the occipital lobe white matter occurred between 20 and 30 months, resulting in a Loes score of 9 at 12 years posttransplant (2019). MRI, magnetic resonance imaging.

Vector genome and ALDP expression in blood cells

Vector copy number averaged 0.62 (0.6–0.7) in the infused CD34⁺ cells. VCN in peripheral blood mononuclear cells (PBMC) decreased from a mean value of 0.31 at 2 months to 0.20 at 6 months, then stabilized at 0.18 at 2 years, and then 0.15 until 5.5–12 years posttransplant (Fig. 3A).

Figure 3.

Posttransplant gene marking and ALDP expression in blood cells. Red color signals P3, the only patient who had full long-term success of gene therapy. (A) VCN in bone marrow CD34⁺ cells infused to the patient. Decrease of VCN in circulating PBMC after the transplant; Correlation between VCN in PBMC and percentage of CD14⁺ monocytes expressing ALDP (R = 0.72, p < 0.001) (B) Percentage of ALDP⁺ cells among CD34⁺ bone marrow cells infused to the patients. Percentage of ALDP⁺ cells among bone marrow cells harvested from patients at 1 and 2 years. Lack of relationship between ALDP⁺ cells in bone marrow and in blood (averaged across all studied blood cell subsets) (C1) Percentage of ALDP⁺ cells in CD3⁺ T lymphocytes (C2) Percentage of ALDP⁺ cells in CD19⁺ B lymphocytes and correlation with CD3⁺ T lymphocytes (R = 0.79 p < 0.001); (C3) Percentage of ALDP⁺ cells in CD14⁺ monocytes and correlation with ALDP⁺ CD3⁺ T lymphocytes (R = 0.79, p < 0.001) and CD19⁺ B lymphocytes (R = 0.92, p < 0.001) (C4) Percentage of ALDP⁺ cells in CD15⁺ granulocytes. ALDP, ALD protein; PBMC, peripheral blood mononuclear cells; VCN, vector copy number.

At 1 and 2 years posttransplant, ALDP expression in blood cells and bone marrow CD34⁺ cells were not correlated (Fig. 3B). Within a few months posttransplant, the percentage of cells expressing ALDP decreased rapidly in all blood cell lineages. At 5 years, the percentage of blood cell lineages expressing ALDP averaged half of that observed at 1 year and stabilized around 5–10% from then on (Fig. 3C1–C4). The percentage of ALDP expressing monocytes correlated with VCN (R = 0.72, p < 0.001) (Fig. 3A). A strong correlation was observed for ALDP expression across blood cell lineages: between CD3⁺ T and CD19⁺ B lymphocytes (R = 0.62, p < 0.001), between CD14⁺ monocytes and T lymphocytes (R = 0.79 p < 0.001), and between monocytes and B lymphocytes (R = 0.82, p < 0.001) (Fig. 3C2–C3). ALDP expression in blood cells did not correlate with clinical status or Loes score. Notably P3, the only patient with a long-term true success of gene therapy, had no more ALDP expression in blood cells than the other patients (Fig. 3C1–C4).

Integration site analyses

Integration sites analyses on PBMC, B cells, T cells, monocytes, and neutrophils revealed the persistence of a consistent polyclonal multilineage hematopoietic reconstitution at 6 to 10 years posttransplant, without any sign of clonal outgrowth (Fig. 4A–B and Supplementary Figures S1–S3). Clustering of integration sites was found in subgenomic regions that were previously described as typical of LV-based gene therapy (Fig. 4C and Table 1).^10,11,14,16 Apart from eminent KDM2A and PACS1, the most prominent clusters included CCND2-AS1, MECOM, and SMG6 (Fig. 4C and Table 1). None of the clones, including the above mentioned ones, represented more than 7% of the whole blood cell population at a given time. MECOM and SMG6 were related to the myeloid compartment, CCND2-AS1 to the lymphoid one (Supplementary Fig. S2). Overall, the long-term landscape of vector integration was similar across the four patients and in line with the report in P1 and P2 at 2 years posttransplant.⁹

Figure 4.

ISs profiles. (A) ISs profile identified in blood of four patients at latest available time point. Cumulative retrieval frequencies of the ten most prominent ISs detected in whole blood samples of P1–P4, at last available follow-up visit in years (yr). Total unique ISs count represents number of distinct ISs that could be mapped to a definite (unique) position in the genome. RefSeq names of genes closest to the respective ISs are given in the table. The IS frequency (Freq. %) represents relative sequence count calculated in relationship to all sequences in respective sample. (B) ISs found in distinct sorted cell lineages of P1 and P3. Venn diagram presents counts of identical ISs being detected in distinct lineages. (C) Word cloud of 20 most prominent genes detected in ISs analysis. Details of the applied methods are provided in the Supplementary Data. IS, integration sites.

Table 1.

Common integration sites

	IS Count	Chr.	Dimension [nt]	Gene^a
Top 1	87	11	236023	ADRBK1, ANKRD13D, KDM2A , LOC100130987
Top 2	86	11	208353	KLC2, PACS1 , RAB1B, SF3B2
Top 3	75	11	463826	DPF2, EHBP1L1, FRMD8, KAT5, KCNK7, LTBP3, MALAT1, MAP3K11, MIR612 , NEAT1, PCNXL3, POLA2, RELA, RNASEH2C, SCYL1, SIPA1, SSSCA1-AS1
Top 4	64	17	248352	AFMID, C17orf99, TK1, TMC6, TNRC6C
Top 5	63	16	391112	C16orf91, CCDC154, CLCN7, CRAMP1L, HN1L, IFT140 , MAPK8IP3 , PTX4, TELO2, UNKL
Top 6	62	17	242826	SGSM2, SMG6 , SRR, TSR1
Top 7	57	17	271513	ATP6V0A1, PTRF, STAT3, STAT5B
Top 8	56	1	221806	ASH1L
Top 9	53	17	244207	ANKFY1, SPNS3, UBE2G1
Top 10	52	9	346912	AGPAT2, EDF1, EGFL7, FAM69B, FBXW5, LCN10, LCN15, LINC01573, MAMDC4, RABL6, SNHG7, TMEM141, TRAF2
Top 11	51	19	401176	ASNA1, C19orf43, MAN2B1, MIR5684, TNPO2, ZNF443, ZNF490 , ZNF564, ZNF709, ZNF791, ZNF799
Top 12	50	19	264611	EMR2 , OR7A10, OR7A17 , OR7A5, OR7C1, OR7C2, SLC1A6, ZNF333
Top 13	49	22	341926	ARSA, CHKB-AS1, KLHDC7B, LMF2, NCAPH2, PPP6R2 , SBF1, SHANK3, SYCE3
Top 14	46	17	301066	RPTOR
Top 15	45	17	299848	MIR4733, NF1
Top 16	43	1	313508	EIF4G3
Top 17	42	19	437197	LOC100289333, ZNF136, ZNF433, ZNF439, ZNF440, ZNF441, ZNF625-ZNF20, ZNF69, ZNF700, ZNF763, ZNF788 , ZNF844
Top 18	40	12	399293	ATN1, C1RL, C1S , CD4, CLSTN3, COPS7A, DSTNP2, ENO2, LEPREL2, LPCAT3, LRRC23, MLF2, PTPN6, RPL13P5, SPSB2, USP5
Top 19	39	3	137297	SETD2
Top 20	38	11	228656	FCHSD2

Integration hotspots were constructed using an approach based on graphs theory applying threshold distance of 10 kbp. Common ISs are sorted based on ISs count (number of ISs included in cluster) and 20 leading clusters are shown listing Chromosome, Dimension (span of most distant integration locations), and gene names representing cluster. Bolded gene name indicates IS in/near gene that is the highest contributor of the respective cluster.

Bolded gene name indicates highest contributor of the respective cluster.

IS, integration sites; Chr, chromosome.

Discussion

The clinical outcomes of LV-mediated gene transfer into HSCs, as well as the evolution of vector transduced cells and associated risks, are still unknown in the long term of c-ALD. We can draw two lessons from the long-term follow-up of our four patients over 8.8 years, one optimistic and the other pessimistic. The long-term observations we report herein reinforce the hard truth that gene therapy for c-ALD “is not a miracle cure, at least not in its current form.”²⁹

On one side, we observed that LV-HSC gene therapy was able to obtain several years of pause in the neurological deterioration and demyelination visible on MRI, a remarkable result compared with untreated patients who tend to progress inexorably and rapidly toward devastating degradation and death.^8,22,30,31 Indeed, among 14 boys with untreated c-ALD who showed gadolinium enhancement and, yet, no MFD like our patients, 12 developed MFD scores up to 4–6 within only 2 years, and 8 patients died within 5 years.^8,22 Starting in 1990,⁶ allo-HSCT allowed MFD-free survival for patients who found a donor; 76% had a NFS still at 0 at 5 years, although 67% developed a major neurocognitive impairment.²⁷ As for the gene therapy of c-ALD in patients who did not find a bone marrow donor, the only results yet available indicate a favorable neurological evolution and MFD-free survival in 17 of 18 patients at 2–3 years posttransplant, but have not yet been studied beyond that time.^9,10

At 8.8 years after transplant, two out of our four patients still have MFD-free survival. Gadolinium diffusion, the hallmark of inflammatory lesions in c-ALD, was durably reversed in the four patients soon after gene therapy, as reported for allo-HSCT.⁸ Still on the positive side, it is remarkable that P3, who underwent gene therapy at the age of 4.4 years, earlier than the other three patients, and only 9 months after c-ALD was diagnosed, kept a preserved near-normal clinical status, although he received only 5.6 × 10⁶ transduced CD34⁺ cells per kilogram and had a level of ALDP expression in his blood cells comparable to that observed in the three aggravated patients (Fig. 3C1–C4).

One does not know what is going on at the cellular level in the brain of patients having undergone LV-gene therapy. Transduced progenitor cells, once differentiated locally into microglia,⁴ create a long-term microglial chimerism that could eventually be maintained in the long term at a different level than the one observed in blood cells.³² Since gadolinium diffusion is arrested within a few months of gene therapy, with almost no recurrence afterward, it is likely that the long-term engraftment of a fraction of the transduced progenitor cells has successfully allowed microglia replacement and transgene expression to reach a level capable of halting inflammation.^12,32

Although short-term results were definitely promising in both our patients and the larger group studied by Eichler et al.,¹⁰ optimism must be tempered by the severe neurological degradation that occurred in three of our four patients. Indeed, the 19-year-old P1 showed clinical deterioration, both for cognitive functions and later on for neurological status. The 16-year-old P2 has stopped making sentences or dressing alone. The 11.5-year-old P4 has lost spontaneous speech and developed epilepsy and urinary incontinence. The frontal syndrome that was mild in P2 and P4 before transplant showed marked aggravation despite the durable disappearance of gadolinium diffusion and apparent stability of lesions observed at MRI.

No patient showed any improvement in demyelinated areas, which may reflect the fact that myelin-making oligodendrocytes and astrocytes remain uncorrected by LV-HSC gene therapy.³³ The lack of correlation between MRI demyelination and clinical manifestations, already observed in multiple sclerosis,³⁴ is likely due to an insufficient sensitivity of routine MRI to detect small demyelination lesions or progression of deleterious glia pathology at the cell level³⁵ that can nevertheless have a strong clinical impact.

Our observations also made clear that the NFS or MFD scales, as well as the Loes score at MRI, do not have sufficient sensitivity to accurately assess the effects of gene therapy on neurological degradation in c-ALD.

At 2 years posttransplant, the average level of vector genome copies in bone marrow and peripheral blood cells was ∼2.5-times lower and ALDP expression in CD14⁺ cells was also approximately two times lower than in the gene therapy trial using the lenti-D vector.¹⁰ This reflected a less efficient transduction in our patients treated 10 years before, who received 1.6-times less transduced CD34⁺ cells than in the lenti-D vector study (10.5 × 10⁶ cells per kilogram, range 6–19.4 × 10⁶) and consistently showed a 1.7-times lower VCN level at 2 years.¹⁰ Most of the decrease in vector copies and ALDP expression in blood cells occurred within 2 years posttransplant in our patients, in contrast with LV-HSC trials for other diseases^12,15 and the more recent c-ALD trial.¹⁰

Regarding safety, it is reassuring that none of the four patients showed any adverse effect in the long term. Moreover, the diversity of the gene corrected cell pool and the multilineage hematopoietic recovery were both maintained. Genome-wide integration sites profiles were in line with previous reports of LV-HSC gene therapy.^10,11,13,15 None of the identified clones presented overtime growth indicating potential clonal dominance or insertional oncogenesis. Among most targeted subgenomic regions were CCND2-AS1, SMG6, and MECOM. While MECOM is listed as oncogene in cancer gene database, both CCND2-AS1 and SMG6 are not. SMG6 was previously reported in ALD gene therapy at 2 years^9,10 and in other LV gene therapy studies.¹³ SMG6 plays a role in the nonsense-mediated mRNA decay pathway and is expressed in HSC, monocytes, and neurons, but not related to neoplasia.

In conclusion, LV-HSC gene therapy has maintained in the long term in our four patients its positive effects on brain inflammation and demyelination visible at MRI, despite a rather low level of transduced peripheral blood cells. In this respect, our data, as well as the positive evolution of patient P3, are certainly encouraging, but should not mask that three out of four patients developed severe cognitive and neurological aggravation. This relative failure could be due to a low level of transduced microglial cells and/or to an ongoing long-term progression of brain lesions not seen on MRI. It is also possible that HSC gene therapy was not attempted early enough in our four patients, except for P3, although all had normal or near normal cognitive and neurological status just before the transplant. Since the 40% of boys with ABCD1 mutations who will develop c-ALD cannot be identified based on genotype, family history, or any other parameter² early and vigilant neuroimaging is certainly key. While early stages of c-ALD can be diagnosed by scheduled brain MRIs in presymptomatic patients who have a genetic or biochemical diagnosis of ALD,^2,36,37 the detection of demyelination yet needs to be accelerated by neonatal screening and by novel and more sensitive neuroimaging methods.

One looks forward to the long-term results of the trial by Eichler et al. ¹⁰ to see whether a higher transduction achieves better results than those reported herein, or whether the disease continues to progress despite microglia correction. Progress may also come from an optimization of transduction rates by new routes of administration of transduced cells. Indeed, the intracerebroventricular injection of HSPCs into mouse models allows a more rapid and robust microglia-like cell engraftment compared to intravenous administration.³⁸ Also, we believe that progress may also come from novel gene therapy approaches able to target other cell types in the central nervous system and promote the remyelination of existing lesions.³⁷

Footnotes

Authors' Contributions

P.B. was involved in the preclinical and clinical gene therapy program with P. Aubourg, and in the coordination, data assembly, interpretation, and writing. S.H.-B.-A. was involved in the HSC gene therapy transplant, data collection, and writing. M.S. and I.L. were involved in gene integration studies and article writing. C.A. did the MRI studies, and C.C. and C.B. the cognitive and neurological examination.

Acknowledgments

Without the initial involvement of the ALD-Gene Therapy group (listed below), the current study would not have been carried out. P. Aubourg could not participate in the current article, but organized the whole study and follow-up as principal investigator. We thank P. Hantraye for support of the MIRCen Institute, A.J. Valleron and A. Fischer for critical reading of the ms, L. Etcheverry for access to crucial data. We thank the contributors to the long-term study: C. von Kalle and C. Bartholomae (Integration sites studies), G. Dufayet-Chaffaud, Y. Gunes (ALDP expression studies), I. Laurendeau, (Vector copy number), and S. Valtat (Statistics). The ALD Long-Term Study RCAOM03043 was supported by Direction de la Recherche Clinique de l'Assistance Publique and by the Neuratris program. We are grateful to J.-F. Dhainaut, P. Ravaud, D. Sicard, and M. Schumacher for providing their expertise to organize the authorship. We thank A. Dewynter and nurses for their care dedicated of the patients during the follow-up in St-Vincent de Paul and Bicêtre hospitals. Most of all, we thank the patients and their parents.

The ALD-Gene Therapy trial was supported until 2 years posttransplant by INSERM, the European Leukodystrophy Association, L'Association Française contre les Myopathies, La Fondation de France, the sixth Framework European Economic Community Program (LSHM-CT2004-502987) and the Programme Hospitalier de Recherche Clinique (AOM 3043, French Health Ministry). Clinical investigators were P. Aubourg (Principal Investigator), A. Fischer (Head of transplant), S. Blanche (Clinical care of transplant), M. Cavazzana, (Gene therapy transplant), C. Castaignède (Neuropsychology), C. Adamsbaum (MRI studies). Laboratory studies were performed by N. Cartier (Preclinical studies), C. von Kalle, M Schmidt, C. Bartholomae (Integration sites).

Author Disclosure

P.B. discloses founding two companies (Adrenas Therapeutics, USA, and TherapyDesignConsulting, France). M.S. discloses founding one company (GeneWerk) GmbH. The remaining authors declare that they have no conflicts of interest to disclose.

Funding Information

The ALD Long-Term Study was supported by Direction de la Recherche Clinique de l'Assistance Publique. (RCAOM03043 grant) and by the NEURATRIS Program (AO-2017-2019-TGALD).

Supplementary Material

Supplementary Data

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

References

Mosser

, Douar

, Sarde

, et al. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature, 1993; 361:726–730.

Engelen

, Kemp

, De Visser

, et al. X-linked adrenoleukodystrophy (X-ALD): clinical presentation and guidelines for diagnosis, follow-up and management. Orphanet J Rare Dis, 2012; 7:51.

Zhu

, Eichler

, Biffi

, et al. The changing face of adrenoleukodystrophy. Endocr Rev, 2020; 41:bnaa013.

Eglitis

, Mezey

. Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc Natl Acad Sci U S A, 1997; 94:4080–4085.

Priller

, Flügel

, Wehner

, et al. Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment. Nat Med, 2001; 7:1356–1361.

Aubourg

, Blanche

, Jambaqué

, et al. Reversal of early neurologic and neuroradiologic manifestations of X-linked adrenoleukodystrophy by bone marrow transplantation. N Engl J Med, 1990; 322:1860–1866.

Shapiro

, Krivit

, Lockman, et al. L Long-term effect of bone-marrow transplantation for childhood-onset cerebral X-linked adrenoleukodystrophy. Lancet, 2000; 356:713–718.

Raymond

, Aubourg

, Paker

, et al. Survival and functional outcomes in boys with cerebral adrenoleukodystrophy with and without hematopoietic stem cell transplantation. Biol Blood Marrow Transplant, 2018; 25:538–548.

Cartier

, Hacein-Bey-Abina

, Bartholomae

, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science, 2009; 326:818–823.

10.

Eichler

, Duncan

, Musolino

, et al. Hematopoietic stem-cell gene therapy for cerebral adrenoleukodystrophy. N Engl J Med, 2017; 377:1630–1638.

11.

Biffi

, Montini

, Lorioli

, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science, 2013; 341:1233158.

12.

Sessa

, Lorioli

, Fumagalli

, et al. Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial. Lancet, 2016; 388:476–487.

13.

Fischer

, Hacein-Bey-Abina

. Gene therapy for severe combined immunodeficiencies and beyond. J Exp Med, 2020; 217:e20190607.

14.

Aiuti

, Biasco

, Scaramuzza

, et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science, 2013; 341:1233151.

15.

Salima

Hacein-Bey

, et al. Outcomes following gene therapy in patients with severe Wiskott-Aldrich syndrome. JAMA, 2015; 313:1550–1563.

16.

Ferrua

, Cicalese

, Galimberti

, et al. Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical study. Lancet Haematol, 2019; 6:e239–e253.

17.

Cavazzana-Calvo

, Payen

, Negre

, et al. Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia. Nature, 2010; 467:318–322.

18.

Thompson

, Walters

, Kwiatkowski

, et al. Gene therapy in patients with transfusion-dependent β-thalassemia. N Engl J Med, 2018; 378:1479–1493.

19.

Ribeil

, Hacein-Bey-Abina

, Payen

, et al. Gene therapy in a patient with sickle cell disease. N Engl J Med, 2017; 376:848–855.

20.

Dunbar

, High

, Joung

, et al. Gene therapy comes of age. Science, 2018; 359:eaan4672.

21.

Naldini

, Trono

, Verma

. Lentiviral vectors, two decades later. Science, 2016; 353:1101–1102.

22.

Moser

, Loes

, Melhem

, et al. X-linked adrenoleukodystrophy: overview and prognosis as a function of age and brain magnetic resonance imaging abnormality. A study involving 372 patients. Neuropediatrics, 2000; 31:227–239.

23.

Loes

, Hite

, Moser

, et al. Adrenoleukodystrophy: a scoring method for brain MR observations. Am J Neuroradiol, 1994; 15:1761–1766.

24.

Schmidt

, Hoffmann

, Wissler

, et al. Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples. Hum Gene Ther, 2001; 12:743–749.

25.

Paruzynski

, Arens

, Gabriel

, et al. Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing. Nat Protoc, 2010; 5:1379–1395.

26.

Afzal

, Wilkening

, von Kalle

, et al. GENE-IS: time-efficient and accurate analysis of viral integration events in large-scale gene therapy data. Mol Ther Nucl Acids, 2017; 6:133–139.

27.

Pierpont

, Eisengart

, Shanley

, et al. Neurocognitive trajectory of boys who received a hematopoietic stem cell transplant at an early stage of childhood cerebral adrenoleukodystrophy. JAMA Neurol, 2017; 74.

28.

Ramsden

, Richardson

, Josse

, et al. Verbal and non-verbal intelligence changes in the teenage brain. Nature, 2011; 479:113–116.

29.

Van Haren

, Engelen

. Decision making in adrenoleukodystrophy: when is a good outcome really a good outcome?. JAMA Neurol, 2017; 74:641–642.

30.

Suzuki

, Takemoto

, Shimozawa

, et al. Natural history of X-linked adrenoleukodystrophy in Japan. Brain Dev, 2005; 27:353–357.

31.

Mallack

, van de Stadt

, Caruso

, et al. Clinical and radiographic course of arrested cerebral adrenoleukodystrophy. Neurology, 2020; 94:e2499–e2507.

32.

Capotondo

, Milazzo

, Politi

, et al. Brain conditioning is instrumental for successful microglia reconstitution following hematopoietic stem cell transplantation. Proc Natl Acad Sci U S A, 2012; 109:15018–15023.

33.

Franklin

, Frisén

, Lyons

. Revisiting remyelination: towards a consensus on the regeneration of CNS myelin. In: Seminars in Cell & Developmental Biology. https://doi.org/10.1016/j.semcdb.2020.09.009.

34.

Goodin

DS.

Magnetic resonance imaging as a surrogate outcome measure of disability in multiple sclerosis: have we been overly harsh in our assessment?. Ann Neurol, 2006; 59:597–605.

35.

Bergner

, Van Der Meer

, Winkler

, et al. Microglia damage precedes major myelin breakdown in X-linked adrenoleukodystrophy and metachromatic leukodystrophy. Glia, 2019; 67:1196–1209.

36.

Aubourg

, Sellier

, Chaussain

, et al. MRI detects cerebral involvement in neurologically asymptomatic patients with adrenoleukodystrophy. Neurology, 1989; 39:1619–1621.

37.

van Haren

, Engelen

, Wolf

Measuring early lesion growth in boys with cerebral demyelinating adrenoleukodystrophy. Neurology 2019.

38.

Capotondo

, Milazzo

, Garcia-Manteiga

, et al. Intracerebroventricular delivery of hematopoietic progenitors results in rapid and robust engraftment of microglia-like cells. Sci Adv, 2017; 3:e1701211.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.04 MB

0.55 MB

0.20 MB

0.11 MB