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Enzyme replacement therapy results in substantial improvements in early clinical phenotype in a mouse model of globoid cell leukodystrophy

Mayo Clinic College of Medicine, Departments of Pharmacology and Neuroscience, Jacksonville, Florida, USA

¹ Correspondence: E-mail: Eckman{at}mayo.edu

SPECIFIC AIMS

In this study, we tested whether enzyme replacement therapy (ERT) would be clinically beneficial in Globoid cell leukodystrophy (GLD) by examining the consequences of peripheral administration of recombinant galactocerebrosidase (GALC) on the phenotype in twitcher mice, a mouse model of the disease.

PRINCIPAL FINDINGS

1. Intraperipherally administered recombinant GALC results in substantial improvements in failure to thrive, gait, and survival in the twitcher mouse model of GLD
Improvement in survival
Intraperitoneal injection of recombinant GALC showed a significant increase in the life span of twitcher mice from approximately postnatal day (PND) 40 to PND 47. Interestingly, this improvement was similar regardless of whether treatment was begun at PND 10 or PND 20 (after CNS and PNS demyelination is typically apparent).

Improvement in early failure to thrive
As shown in Fig. 1A , twitcher mice normally show a significant failure to thrive in early life when compared with their wild-type littermates. GALC-treated animals, however, gained weight at a rate essentially indistinguishable from the unaffected wild-type mice up until approximately PND 32 resulting in a 70% difference in weight between the treated and control twitcher mice at this age. At day 32, the weight of the GALC-treated animals began to decline precipitously at a rate similar to the saline injected twitcher mice. The mechanism behind this phenomenon that occurs almost invariably at approximately PND 32 in both the treated and control twitcher mice is currently not clear but is obviously independent of the treatment regimen.

View larger version (42K): [in this window] [in a new window]   Figure 1. GALC enzyme replacement therapy significantly improves the early clinical phenotype apparent in twitcher mice. A) Analysis of body weight. Recombinant GALC was administered every other day at 6 mg/kg in 0.9% saline beginning at PND 20. Values presented are the % change in body weight that occurred in each animal relative to that animal’s weight at the beginning of the experiment (PND 20). Each point represents the mean ± SEfrom 3 mice. B) Gait analysis performed at PND 40 showed marked improvement in GALC-treated animals as compared with saline-treated animals also analyzed at PND 40. Imprints from wild-type mice at PND 40 are included as a comparison. Blue and red imprints labeled the front and back paws, respectively.

Improvement in gait
Twitcher mice normally begin to show significant gait abnormalities beginning at approximately PND 35. As shown in Fig. 1B , this is characterized by an irregular dragging pattern due, at least in part, to hind limb paralysis. While not completely corrected, GALC-treated mice showed a substantial improvement in gait. Thus, GALC-treated twitcher animals showed a significant increase in life span, an almost complete attenuation of their early failure to thrive and a noticeable gait improvement.

2. Bio-distribution of peripherally administered GALC and reduction in the level of brain psychosine in treated animals
Table 1 shows an analysis of GALC enzymatic activity in both treated and untreated twitcher mice along with activity normally present in wild-type animals in various tissues. The largest percent elevation in GALC enzymatic activity was observed in the liver. We did, however, also detect an increase in GALC substrate cleavage in brain and kidney homogenates isolated from the treated animals. To determine whether this increase in enzymatic activity was physiologically relevant, we examined the amount of psychosine, a cytotoxic GALC substrate and a major biochemical marker in GLD, that accumulated in the brains of twitcher and control animals. Consistent with an increase in GALC activity in the brain, mass spectral analysis of psychosine levels showed a significant 18% decrease (P=0.0008) in psychosine accumulation in the brain at PND 29.

3. GALC immunohistochemistry and histopathology studies reveal significant improvement in the pathology normally present in the twitcher mice following peripheral administration of recombinant GALC
Upon intraperitoneal injection of GALC, immunohistochemical analysis revealed the presence of the enzyme in Schwann cells, hepatocytes, and kidney cuboidal cells in subcellular localizations that appeared similar to that of the endogenous GALC in wild-type mice (online text, Fig. 3). LFB/ PAS staining of the longitudinal sections of sciatic nerve showed significant improvements in myelination and an apparent decrease in globoid cells (see figures, online text).

CONCLUSIONS AND SIGNIFICANCE

In this study, we found a substantial improvement in several major aspects of early pathology in the twitcher mouse model of GLD following peripheral enzyme replacement therapy. Although we were not able to prevent the later precipitous decline occurring just prior to death, we did observe an increase in life span, an attenuation of the early failure to thrive and an improvement in the gait of GALC-treated animals. It is perhaps worth noting that the pathological changes in the twitcher mice are particularly aggressive and that changes in the life span of these animals due to therapeutic interventions can potentially result in even greater improvements in human patients.

While it is likely that much of the clinical benefit we observed in the treated animals is due to peripheral improvements, we did observe a relatively small amount of enzyme in the brain and a resultant reduction in psychosine levels. Notably, at least two ERT studies for other LSDs have shown evidence of storage reduction in the brain following peripheral injection of other enzymes. While no follow-up biochemical or histological analysis of the brain was performed in either of those studies to examine enzyme levels or activity to determine if the enzyme itself reached the brain, it is tempting to speculate that small amounts of enzyme that effectively crossed the BBB may be the underlying mechanism in those studies as well.

The most effective current treatment for these patients is a bone marrow transplant (BMT). Unfortunately, BMT needs to be initiated very early for it to be effective and the procedure carries a relatively high mortality rate. Unless there is a family history of the disease, many patients with the infantile and most aggressive form of the disease will miss the window of opportunity for an effective transplant. We are particularly intrigued that the increase in life span we observed in the twitcher mice that had been given recombinant GALC was independent of whether the treatment started at PND10 or PND20 as by PND20 signs of both peripheral and CNS demyelination are typically observed in this model. Collectively, our data suggest that peripheral enzyme replacement therapy is likely to be beneficial in patients with GLD and perhaps may even increase the effective therapeutic window for effective bone marrow transplantation.

View larger version (19K): [in this window] [in a new window]   Figure 2. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3826fje;

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This Article Full Text (PDF) All Versions of this Article: 19/11/1549 05-3826fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, W. C. Articles by Eckman, C. B. Search for Related Content PubMed PubMed Citation Articles by Lee, W. C. Articles by Eckman, C. B.