Failure of intrathecal allogeneic mesenchymal stem cells to halt progressive demyelination in two boys with cerebral adrenoleukodystrophy.

Abstract Cerebral adrenoleukodystrophy is an inflammatory demyelinating condition that is the result of a mutation in the X‐linked ABCD1 gene, a peroxisomal very long chain fatty acid transporter. Although mutations in this gene result in adrenal insufficiency in the majority of affected individuals, 40% of those affected develop the demyelinating cerebral form, cerebral adrenoleukodystrophy (CALD). CALD is characterized by imaging findings of demyelination and contrast enhancement on magnetic resonance imaging (MRI). Although allogeneic hematopoietic cell transplantation can arrest progression of CALD early in its course, there is no accepted therapy for patients with advanced CALD. Mesenchymal stem cells (MSCs) have been used in a variety of clinical trials to capitalize on their anti‐inflammatory properties as well as promote tissue repair. We delivered MSCs via intrathecal (IT) route to two boys with rapidly advancing CALD. The first boy received three doses 1 week apart, whereas the second boy received a single dose of IT MSCs. We note delivery of IT MSCs was feasible and without complication. Follow‐up MRI scans after IT MSC delivery showed progressive demyelination in the first patient and no change in demyelination or contrast enhancement in the second patient. Although the infusion of IT MSCs was safe, it did not halt CALD progression in this setting, and future studies should focus on patient selection and dose optimization.

enchymal stem cells (MSCs) have been used in a variety of clinical trials to capitalize on their anti-inflammatory properties as well as promote tissue repair. We delivered MSCs via intrathecal (IT) route to two boys with rapidly advancing CALD. The first boy received three doses 1 week apart, whereas the second boy received a single dose of IT MSCs.
We note delivery of IT MSCs was feasible and without complication. Follow-up MRI scans after IT MSC delivery showed progressive demyelination in the first patient and no change in demyelination or contrast enhancement in the second patient. Although the infusion of IT MSCs was safe, it did not halt CALD progression in this setting, and future studies should focus on patient selection and dose optimization. In 30%-40% of boys with ALD, a neuroinflammatory process known as cerebral ALD (CALD) is initiated between the ages of 4 and 10 years. The trigger and pathophysiological mechanism surrounding this process are largely unknown. On magnetic resonance imaging (MRI), CALD is denoted by signs of demyelination (changes in signal on T2-weighted imaging) accompanied by blood-brain barrier (BBB) disruption defined by intravenous contrast enhancement positivity indicating an active disease process. Both microglial cell death and endothelial disruption contribute to the pathophysiology of active disease. 4,5 The origination of the inflammatory process that accompanies CALD is unclear, as is how inflammation contributes to the overall neuropathology. 6 The neuroinflammation is characterized by activated microglial cells and invading immune cells such T cells and macrophages, with the occasional B cells and immunoglobulin G-expressing plasma cells. 7 Like other inflammatory conditions, the presence of a humoral system response and auto-antibody production has also been reported. 8 Only early hematopoietic cell transplant (HCT) can arrest the cerebral disease process in CALD via unclear mechanisms but may involve immune modulation and healthy donor macrophage/microglial engraftment. 9 Mesenchymal stem cells are a stroma cell type isolated most commonly from the bone marrow, although similar cell types are found associated with many organ systems. 10 MSCs have been demonstrated to have a wide variety of tissue repair-and cell growth-promoting properties and have also been extensively studied clinically for their anti-inflammatory properties in a wide variety of conditions, with improvements observed in select patients. 10 Given that CALD has a significant neuroinflammatory component, we reasoned that MSCs may be able to counter cerebral inflammation and slow disease progression. Although prior studies have used both auto and allogeneic MSCs to treat neurological disease, 11 we chose an allogeneic approach being that CALD is caused by a single gene defect that would be present in an autologous MSC product and perhaps dampen its therapeutic potential. In this study, we present two clinical cases in which MSCs were delivered intrathecally to boys with advanced CALD and thus not eligible for HCT, with the goal of arresting disease progression as assessed radiologically.

| MATERIALS AND METHODS
Enrichment of the mononuclear cell fraction of the marrow was accomplished using a semiautomated separation method involving ficoll hypaque density gradient medium, specific gravity 1.077 g/dL (Isolymph; Gallard-Schlesinger Industries, Carle Place, NY) followed by washing with Hank's Balanced Salt Solution (without phenol red, calcium, or magnesium). Cells were seeded at 1.0-1.5 × 10 5 cells/cm 2 at a media depth of 1.6 mm in an appropriately sized culture vessel and placed in a 5% CO 2 incubator at 37 C. Growth media consisted of alpha-minimal essential medium, 16.5% fetal bovine serum, and L-glutamine (2 mM). On days 1 and 2 after plating, nonadherent cells were removed and the media  Follow-up CSF studies for the following 2 weeks and sampling at 2 months after MSC therapy showed no change in any parameter.
A.S. was a 7-year-old boy who had normal growth, development, and cognitive skills until 6 months prior to his diagnosis when he presented with difficulty completing math assignments (that had previously been easy for him) and developed difficulties following instructions in school.
He also had spatial disorientation and balance and gait disturbances. Further assessments included an MRI that showed extensive demyelination within the white matter including the ventral pons along the corticospinal tracts and corpus callosum, accompanied by abnormal gadolinium contrast enhancement; the ALD MRI severity score was 17 ( Figure 2B). Elevated VLCFAs and a mutation in the ABCD1 gene confirmed the diagnosis of Xlinked CALD. His NFS was 7 (losses in vision, processing, and mobility; supplemental online Table 1). The patient received 5 × 10 6 /kg third-party MSCs by IT route. For this patient, MSCs were thawed and cultured for 48 hours prior to IT delivery owing to a recent report that freshly thawed MSCs used in treatment may have impaired immunosuppressive activity compared with MSCs growing in the log phase used as therapy. 14 Premedications were given prior to MSC infusion, similar to the previous patient. Additional cytogenetic analysis of the re-expanding MSCs indicated a new clonal chromosomal abnormality had been acquired during the brief expansion, and although the first dose of MSCs was given, the latter two treatments were withheld. An MRI was performed 1 month after the IT MSC injection and showed unchanged demyelination signal and no change in the abnormal gadolinium contrast enhancement, indicating that his cerebral disease was not in remission ( Figure 2B). In both of these cases, further treatment was not offered.

| DISCUSSION
Multiple characteristics of MSCs include abilities to support hematopoiesis, 15,16 differentiate into numerous cell types for tissue regeneration, 17 and modulate the immune response. 18,19 The effects Although the patients in our study showed disease progression after MSCs, we cannot rule out the possibility that an inadequate MSC dose was used (and one patient only received a single dose), although anywhere from 0.1 to 10 × 10 6 cells/kg are commonly found as doses in clinical trials. 10 This potential variability in dosing limits conclusive interpretation of the optimal effect. An additional possibility is that these patients were far too advanced to receive benefit from intervention. We have previously shown that patients with very involved cerebral disease (MRI score > 10) do poorly after HCT. 25 In any event, further novel therapies need to be identified for severely affected boys with CALD.

| CONCLUSION
The delivery of MSCs intrathecally was safe and well tolerated. We did not find toxicity related to the delivery of the MSCs. Unfortunately, neither patient showed any reversal or arrest in the progression of disease as assessed by MRI studies. Whether this was because of the rapidly progressive nature of their disease or other factors such as lack of adequate cell dosing is unknown, and future studies should focus on these factors.

ACKNOWLEDGMENTS
We gratefully acknowledge the staff at the University of Minnesota Molecular and Cellular Therapeutics Facility for their assistance in cell processing. The data that support the findings of this study are available from the corresponding author upon reasonable request.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author.