Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an autosomal recessive neurodegenerative disease caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). Hematopoietic stem cell transplantation (HSCT) provides modest benefit in presymptomatic patients but is well short of a cure. Gene transfer experiments using viral vectors have shown some success in extending the survival in the mouse model of GLD, twitcher mice. The present study compares three single-stranded (ss) AAV serotypes, two natural and one engineered (with oligodendrocyte tropism), and a self-complementary (sc) AAV vector, all packaged with a codon-optimized murine GALC gene. The vectors were delivered via a lumbar intrathecal route for global CNS distribution on PND10-11 at a dose of 2 × 10(11) vector genomes (vg) per mouse. The results showed a similar significant extension of life span of the twitcher mice for all three serotypes (AAV9, AAVrh10, and AAV-Olig001) as well as the scAAV9 vector, compared to control cohorts. The rAAV gene transfer facilitated GALC biodistribution and detectable enzymatic activity throughout the CNS as well as in sciatic nerve and liver. When combined with BMT from syngeneic wild-type mice, there was significant improvement in survival for ssAAV9. Histopathological analysis of brain, spinal cord, and sciatic nerve showed significant improvement in preservation of myelin, with ssAAV9 providing the greatest benefit. In summary, we demonstrate that lumbar intrathecal delivery of rAAV/mGALCopt can significantly enhance the life span of twitcher mice treated at PND10-11 and that BMT synergizes with this treatment to improve the survival further. © 2016 Wiley Periodicals, Inc.

Extended normal life after AAVrh10-mediated gene therapy in the mouse model of Krabbe disease.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). This deficiency results in accumulation of certain galactolipids including psychosine which is cytotoxic for myelin-producing cells. Treatment of human patients at this time is limited to hematopoietic stem cell transplantation (HSCT) that appears to slow the progression of the disease when performed in presymptomatic patients. In this study, adeno-associated virus (AAV) serotype rh10-(AAVrh10) expressing mouse GALC was used in treating twitcher (twi) mice, the mouse model of GLD. The combination of intracerebroventricular, intracerebellar, and intravenous (iv) injection of viral particles in neonate twi mice resulted in high GALC activity in brain and cerebellum and moderate to high GALC activity in spinal cord, sciatic nerve, and some peripheral organs. Successfully treated mice maintained their weight with no or very little twitching, living up to 8 months. The physical activities of the long-lived treated mice were comparable to wild type for most of their lives. Treated mice showed normal abilities to mate, to deliver pups, to nurse and to care for the newborns. This strategy alone or in combination with other therapeutic options may be applicable to treatment of human patients.

Bone marrow transplantation augments the effect of brain- and spinal cord-directed adeno-associated virus 2/5 gene therapy by altering inflammation in the murine model of globoid-cell leukodystrophy.

Globoid-cell leukodystrophy (GLD) is an inherited demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC). A previous study in the murine model of GLD (twitcher) demonstrated a dramatic synergy between CNS-directed adeno-associated virus 2/5 (AAV2/5) gene therapy and myeloreductive bone marrow transplantation (BMT). However, the mechanism by which these two disparate therapeutic approaches synergize is not clear. In addition, the therapeutic efficacy may have been limited since the CNS-directed gene therapy was restricted to the forebrain and thalamus. In the current study, intrathecal and intracerebellar injections were added to the therapeutic regimen and the mechanism of synergy between BMT and gene therapy was determined. Although AAV2/5 alone provided supraphysiological levels of GALC activity and reduced psychosine levels in both the brain and spinal cord, it significantly increased CNS inflammation. Bone marrow transplantation alone provided essentially no GALC activity to the CNS and did not reduce psychosine levels. When AAV2/5 is combined with BMT, there are sustained improvements in motor function and the median life span is increased to 123 d (range, 92-282 d) compared with 41 d in the untreated twitcher mice. Interestingly, addition of BMT virtually eliminates both the disease and AAV2/5-associated inflammatory response. These data suggest that the efficacy of AAV2/5-mediated gene therapy is limited by the associated inflammatory response and BMT synergizes with AAV2/5 by modulating inflammation.

Regulation of glioblastoma multiforme stem-like cells by inhibitor of DNA binding proteins and oligodendroglial lineage-associated transcription factors.

Tumor-initiating stem cells (also referred to as cancer stem cells, CSCs) are a subpopulation of cancer cells that play unique roles in tumor propagation, therapeutic resistance and tumor recurrence. It is increasingly important to understand how molecular signaling regulates the self-renewal and differentiation of CSCs. Basic helix-loop-helix (bHLH) transcription factors are critical for the differentiation of normal stem cells, yet their roles in neoplastic stem cells are not well understood. In glioblastoma neurosphere cultures that contain cancer stem cells (GBM-CSCs), the bHLH family member inhibitors of DNA binding protein 2 and 4 (Id2 and Id4) were found to be upregulated during the differentiation of GBM-CSCs in response to histone deacetylase inhibitors. In this study, we examined the functions of Id2 and Id4 in GBM neurosphere cells and identified Id proteins as efficient differentiation regulators of GBM-CSCs. Overexpression of Id2 and Id4 promoted the lineage-specific differentiation of GBM neurosphere cells as evidenced by the induction of neuronal/astroglial differentiation markers Tuj1 and GFAP and the inhibition of the oligodendroglial marker GalC. Id protein overexpression also reduced both stem cell marker expression and neurosphere formation potential, a biological marker of cancer cell "stemness." We further showed that Id2 and Id4 regulated GBM neurosphere differentiation through downregulating of another bHLH family member, the oligodendroglial lineage-associated transcription factors (Olig) 1 and 2. Our results provide evidence for distinct functions of Id proteins in neoplastic stem cells, which supports Id proteins and their downstream targets as potential candidates for differentiation therapy in CSCs.

Differentiation of AFS cells derived from the EGFP gene transgenic porcine fetuses.

We have obtained the EGFP (enhanced green fluorescence protein) gene transgenic porcine fetuses before. The aims of this study were (i) to determine whether stem cells could be isolated from amniotic fluid of the transgenic porcine fetuses, and (ii) to determine if these stem cells could express EGFP and differentiate in vitro. The results demonstrated that stem cells could be isolated from amniotic fluid of the EGFP gene transgenic porcine fetuses and could express EGFP and differentiate in vitro. Undifferentiated AFSs (amniotic fluid-derived stem cells) expressed POU5F1, THY1 and SOX2, while the following differentiation cells expressed markers for chondrogenic (COL2A1), osteogenic (osteocalcin and osteonectin) and neurogenic cells such as astrocyte (GFAP), oligodendrocyte (GALC) and neuron (NF, ENO2 and MAP).

Krabbe disease successfully treated via monotherapy of intrathecal gene therapy.

Globoid cell leukodystrophy (GLD; Krabbe disease) is a progressive, incurable neurodegenerative disease caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). The ensuing cytotoxic accumulation of psychosine results in diffuse central and peripheral nervous system (CNS, PNS) demyelination. Presymptomatic hematopoietic stem cell transplantation (HSCT) is the only treatment for infantile-onset GLD; however, clinical outcomes of HSCT recipients often remain poor, and procedure-related morbidity is high. There are no effective therapies for symptomatic patients. Herein, we demonstrate in the naturally occurring canine model of GLD that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, normalized psychosine concentration, improved myelination, and attenuated inflammation in both the CNS and PNS. Moreover, AAV-mediated therapy successfully prevented clinical neurological dysfunction, allowing treated dogs to live beyond 2.5 years of age, more than 7 times longer than untreated dogs. Furthermore, we found that a 5-fold lower dose resulted in an attenuated form of disease, indicating that sufficient dosing is critical. Finally, postsymptomatic therapy with high-dose AAV9 also significantly extended lifespan, signifying a treatment option for patients for whom HSCT is not applicable. If translatable to patients, these findings would improve the outcomes of patients treated either pre- or postsymptomatically.

Effect of bone marrow transplantation on enzyme levels and clinical course in the neurologically affected twitcher mouse.

The effect of allogeneic bone marrow transplantation (BMT) was investigated in the neurologically affected twitcher mouse, a model for human Krabbe's disease. Twitcher mice have a hereditary deficiency of the lysosomal enzyme galactosylceramidase, which causes growth delay, tremor, and paralysis of the hind legs. Death occurs at 30-40 d of age. After BMT galactosylceramidase activity increased to donor levels in hemopoietic organs. In lung, heart, and liver, galactosylceramidase activity rose to levels intermediate between those of twitcher and normal mice. Increased galactosylceramidase activity in liver parenchymal cells indicated uptake of the donor enzyme by recipient cells of nonhemopoietic origin. Enzyme activity also increased in kidney tissue. BMT resulted in a gradual increase in galactosylceramidase activity in the central nervous system to 15% of normal donor levels. A 5-6-fold increase in galactosylceramidase activity was found in the peripheral nervous system. This increase in enzyme activity was accompanied by a partial alleviation of neurological symptoms. In particular, paralysis of the hind legs was prevented by BMT. BMT led to a modest restoration of growth and prolonged survival. In several cases, the mice survived for more than 100 d, but eventually all animals died with severe neurological disease.

DNA promoter hypermethylation contributes to down-regulation of galactocerebrosidase gene in lung and head and neck cancers.

Galactocerebrosidase (GALC) is a lysosomal enzyme responsible for glycosphingolipids degradation byproducts of which are important for synthesis of apoptosis mediator ceramide. Reduced expression of GALC has been identified in human malignancies; however, molecular mechanisms underlying down-regulation of GALC expression in cancer remain unknown. We performed methylation and expression analysis on GALC gene in a panel of head and neck cancer (HNC) and lung cancer cell lines, attempting to understand the regulation of GALC in human cancer. QRT-PCR and western blot analysis were performed to detect the expression of GALC in HNC. Bisulfite DNA sequencing and real-time qMSP were used to detect the methylation of GALC in HNC and lung cancer cell lines. 5aza-dC treatment assay was used to analysis the functional effect of GALC methylation on GALC expression in HNC. Reduction or complete absence of GALC expression was observed in more than a half of the tested HNC cell lines (8/14). 7 out of 8 cell lines with down-regulated expression harbored heavy CpG island methylation, while all cell lines with abundant expression of the gene contained no methylation. Hypermethylation was also found in primary HNC tumor tissues and lung cancer cell lines whereas absent in normal oral mucosa tissues. Demethylating treatment demonstrated that 5aza-dC significantly restored GALC expression in cell lines with methylated promoter while showed no effect on cell lines without promoter hypermethylation. Our findings for the first time demonstrated that promoter hypermethylation contributed to down-regulation of GALC Gene, implicating epigenetic inactivation of GALC may play a role in tumorigenesis of cancer.

GALC gene is downregulated by promoter hypermethylation in Epstein-Barr virus-associated nasopharyngeal carcinoma.

The aim of the study was to investigate the tumor-suppressor effect of GALC in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) and determine whether GALC was downregulated by promoter hypermethy-lation in the NPC cell line, CNE-2Z. Forty-one archival NPC biopsy specimens were compared with 15 chronic nasopharyngitis specimens. EBV-encoded RNA (EBER) was verified by in situ hybridization and GALC protein expression was analyzed by immunohistochemistry. Promoter methylation in CNE-2Z cells was analyzed by bisulfite sequencing polymerase chain reaction. The functional role of GALC in NPC was investigated by restoring GALC expression in CNE-2Z cells via treatment with the DNA-deme-thylating agent 5-Aza-2'-deoxycytidine (5-Aza­dC). EBER was expressed in 92.68% NPC specimens but no chronic nasopharyngitis specimens (P<0.01). GALC protein was present in 60% of chronic nasopharyngitis specimens and 24.39% NPC specimens (P<0.05). GALC protein expression was present significantly more frequently in tumors without lymph node metastasis than in those with metastasis (P<0.05). Logistic regression showed that GALC protein expression protected against lymph node metastasis (P<0.05). GALC protein expression was not correlated with age, gender and TNM stage (P>0.05). Treatment of GALC-negative CNE-2Z cells with 5-Aza-dC reduced GALC promoter methylation and restored GALC expression in a dose-dependent manner (P<0.05). The re-expression of GALC in CNE-2Z cells reduced cell proliferation and migration compared to the controls (P<0.05). GALC was downregulated by promoter hypermethylation and contributed to the pathogenesis of EBV-associated NPC. The findings showed the putative tumor-suppressor effect of GALC in NPC.

Retinoic acid and human olfactory ensheathing cells cooperate to promote neural induction from human bone marrow stromal stem cells.

The generation of induced neuronal cells from human bone marrow stromal stem cells (hBMSCs) provides new avenues for basic research and potential transplantation therapies for nerve injury and neurological disorders. However, clinical application must seriously consider the risk of tumor formation by hBMSCs, neural differentiation efficiency and biofunctions resembling neurons. Here, we co-cultured hBMSCs exposed to retinoic acid (RA) with human olfactory ensheathing cells (hOECs) to stimulate its differentiation into neural cells, and found that hBMSCs following 1 and 2 weeks of stimulation promptly lost their immunophenotypical profiles, and gradually acquired neural cell characteristics, as shown by a remarkable up-regulation of expression of neural-specific markers (Tuj-1, GFAP and Galc) and down-regulation of typical hBMSCs markers (CD44 and CD90), as well as a rapid morphological change. Concomitantly, in addition to a drastic decrease in the number of BrdU incorporated cells, there was a more elevated synapse formation (a hallmark for functional neurons) in the differentiated hBMSCs. Compared with OECs alone, this specific combination of RA and hOECs was significantly potentiated neuronal differentiation of hBMSCs. The results suggest that RA can enhance and orchestrate hOECs to neural differentiation of hBMSCs. Therefore, these findings may provide an alternative strategy for the repair of traumatic nerve injury and neurological diseases with application of the optimal combination of RA and OECs for neuronal differentiation of hBMSCs.

Intrinsic resistance of neural stem cells to toxic metabolites may make them well suited for cell non-autonomous disorders: evidence from a mouse model of Krabbe leukodystrophy.

While transplanted neural stem cells (NSCs) have been shown to hold promise for cell replacement in models of a number of neurological disorders, these examples have typically been under conditions where the host cells become dysfunctional due to a cell autonomous etiology, i.e. a 'sick' cell within a relatively supportive environment. It has long been held that cell replacement in a toxic milieu would not likely be possible; donor cells would succumb in much the same way as endogenous cells had. Many metabolic diseases are characterized by this situation, suggesting that they would be poor targets for cell replacement therapies. On the other hand, models of such diseases could prove ideal for testing the capacity for cell replacement under such challenging conditions. In the twitcher (twi ) mouse -- as in patients with Krabbe or globoid cell leukodystrophy (GLD), for which it serves as an authentic model -- loss of galactocerebrosidase (GalC) activity results in the accumulation of psychosine, a toxic glycolipid. Twi mice, like children with GLD, exhibit inexorable neurological deterioration presumably as a result of dysfunctional and ultimately degenerated oligodendrocytes with loss of myelin. It is believed that GLD pathophysiology is related to a psychosine-filled environment that kills not only host oligodendrocytes but theoretically any new cells placed into that milieu. Through the implantation of NSCs into the brains of both neonatal and juvenile/young adult twi mice, we have determined that widespread oligodendrocyte replacement and remyelination is feasible. NSCs appear to be intrinsically resistant to psychosine -- more so in their undifferentiated state than when directed ex vivo to become oligodendrocytes. This resistance can be enhanced by engineering the NSCs to over-express GalC. Some twi mice grafted with such engineered NSCs had thicker white tracts and lived 2-3 times longer than expected. While their brains had detectable levels of GalC, it was probably more significant that their psychosine levels were lower than in twi mice that died at a younger age. This concept of resistance based on differentiation state extended to human NSCs which could similarly survive within the twi brain. Taken together, these results suggest a number of points regarding cellular therapies against degenerative diseases with a prominent cell non-autonomous component: Cell replacement is possible if cells resistant to the toxic environment are employed. Furthermore, an important aspect of successful treatment will likely be not only cell replacement but also cross-correction of host cells to provide them with enzyme activity and hence resistance. While oligodendrocyte replacement alone was not a sufficient treatment for GLD (even when extensive), the replacement of both cells and molecules -- e.g. with NSCs that could both become oligodendrocytes and 'pumps' for GalC -- emerges as a promising basis for a multidisciplinary strategy. Most neurological disease are complex in this way and will likely require multifaceted approaches, perhaps with NSCs serving as the 'glue'.

Cloning and expression of cDNA encoding human galactocerebrosidase, the enzyme deficient in globoid cell leukodystrophy.

Globoid cell leukodystrophy (Krabbe disease) is an autosomal recessive disorder resulting from the deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of galactosylceramide, a major lipid in myelin, kidney and epithelial cells of small intestine and colon. We describe the molecular cloning of human GALC cDNA and its expression in COS-1 cells. Degenerate PCR primers, derived from N-terminal amino acid sequence from the 51 kDa band from human brain, were used to amplify cat testes RNA, and the resulting product was used to screen human testes and brain libraries. Two overlapping clones contained the total protein coding region, while additional clones and PCR amplification were needed to obtain the complete 3' end of the cDNA. The 3795 bp obtained include 47 bp 5' to the initiation start site, 2007 bp of open reading frame (coding for 669 amino acids), and 1741 bp of 3' untranslated sequence. Modification of the sequence surrounding the initiation codon to one more favorable for expression, resulted in a 6-fold increase in GALC activity in transfected COS-1 cells. The isolation of this clone will permit investigations into the causes for GALC deficiency in humans and available animal models, development of more accurate tests for patient and carrier identification, and evaluation of methods for effectively treating GALC deficiency, initially using the animal models.

Molecular defects in Krabbe disease.

Krabbe disease (globoid cell leukodystrophy) is an autosomal recessive neurodegenerative disorder that affects both the central and peripheral nervous systems due to an enzymatic defect of the galactocerebrosidase. In this study, molecular defects in Krabbe disease were investigated in 11 patients (seven Japanese and four non-Japanese) using cultured skin fibroblasts. A Japanese late infantile patient had a missense mutation of Pro at codon 302 to Ala and a non-Japanese patient had a missense mutation of Val at codon 550 to Gly. The reduced enzymatic activities expressed from the cDNAs with these missense mutations and from the previously reported nonsense mutation (E369X, Glu at codon 369 to stop codon) were confirmed. Genomic DNA analyses revealed that the P302A and E369X mutations were heterozygous and the V550G mutation was homozygous in these patients. A 12 base deletion with a 3 base insertion was found in three unrelated Japanese infantile patients, but not in 30 controls. The mutation was homozygous in two patients and heterozygous in one patient. We could not find any confirmed mutation in the coding region in the other six patients. These findings suggest that mutations in infantile and late infantile patients are relatively heterogeneous.

Retroviral vector-mediated transfer of the galactocerebrosidase (GALC) cDNA leads to overexpression and transfer of GALC activity to neighboring cells.

Galactocerebrosidase (GALC) is responsible for the lysosomal catabolism of certain galactolipids, including galactosylceramide and psychosine. Patients with GALC deficiency have an autosomal recessive disorder known as globoid cell leukodystrophy (GLD) or Krabbe disease. Storage of undegraded glycolipids results in defective myelin and the characteristic globoid cells observed on pathological examination of the central and peripheral nervous systems. Most patients have the infantile form of GLD, although older individuals are also diagnosed. Recently the human, mouse, and canine GALC genes were cloned, and mutations causing GLD have been identified. We now describe the construction of a vector containing human GALC cDNA (MFG-GALC), and the transduction of cultured skin fibroblasts from molecularly characterized Krabbe disease patients, as well as rat brain astrocytes and human CD34(+) hematopoietic cells, using retrovirus produced by the psi-CRIP amphotropic packaging cell line. The transduced fibroblasts showed extremely high GALC activity (up to 20,000 times pretreatment levels, about 100 times normal). GALC was secreted into the media and was taken up by untransduced fibroblasts from the same or a different patient. Mannose-6-phosphate receptor-mediated uptake was only partially responsible for the efficient transfer of GALC to neighboring cells. Additional studies confirmed the presence of normal GALC cDNA and mRNA in the transduced cells. The GALC produced by the transduced cells and donated to neighboring untransduced cells was localized to lysosomes as demonstrated by the normal metabolism of [14C]stearic acid-labeled galactosylceramide produced from endocytosed [14C]sulfatide.

Cloning of the canine GALC cDNA and identification of the mutation causing globoid cell leukodystrophy in West Highland White and Cairn terriers.

Globoid cell leukodystrophy, or Krabbe disease, is a severe, autosomal recessive disorder resulting from a deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of certain galactolipids, including galactosylceramide and psychosine. In addition to the human patients, there are several naturally occurring animal models for this disease, including the twitcher mouse, West Highland White terriers (WHWT), and Cairn terriers. All species have deficient GALC activity and have the characteristic pathological findings in the nervous system. We now describe the cloning of the canine GALC cDNA and the identification of the disease-causing mutation in both terrier breeds. The 2007-bp open reading frame is 88% identical to that in human, and the deduced amino acid sequence is about 90% identical. However, the 3'-untranslated region is about 1 kb shorter than that in the human. Two nucleotide changes were found in affected dogs, an A to C transversion at cDNA position 473 (Y158S) and a C to T transition at position 1915 (P639S). Expression studies in COS-1 cells demonstrated that the A to C change at 473 is the disease-causing mutation. A rapid test for the identification of the genotype at that position has been developed, and over 100 WHWT and Cairn terriers have been screened. This will allow breeders to mate their dogs selectively and will permit the establishment of a colony of dogs for use in therapy trials.

Transcriptional repression of the human galactocerebrosidase gene in squamous cell carcinomas of the larynx.

Alterations of gene expression in squamous cell carcinoma (SCC) cell lines derived from the larynx and keratinocytes derived from adjacent normal mucosa of the larynx have been studied using the mRNA differential display technique. Lane-to-lane comparison of reverse transcribed mRNA showed a strong repression of a 148 bp fragment in SCC cells. The fragment was reamplified and cloned. Sequencing revealed a 99.3% homology with a region in exon 17 of the human galactocerebrosidase (GALC) gene. Northern blot analysis confirmed the differential expression of this gene in both carcinoma cell lines and laryngeal SCC biopsies in contrast with corresponding normal mucosa. To provide further evidence for the differential expression rate, both types of cells were transiently transfected with a 152 bp (-176 to -24) high regulatory promoter element of the 5' flanking region of the GALC gene. Results of 3 independent transfection experiments indicated a 16-fold repression of the GALC gene expression in SCC cells compared with benign keratinocytes. However, neither mutation nor other alterations of the promoter sequence were detected. Expression of the GALC gene is thus greatly affected in SCCs of the larynx.