Sayoko Iizuka

Reduced α-Gal A enzyme activity in Fabry fibroblast cells and Fabry mice tissues induced by serum from antibody positive patients with Fabry disease

Fabry disease is a progressive, life-threatening lysosomal storage disorder which is characterized by deficient activity of the lysosomal enzyme alpha-galactosidase A. Studies have demonstrated that both enzyme preparations currently available for treatment of Fabry disease (i.e., agalsidase beta and agalsidase alpha) elicit immune responses in the majority of patients which negatively influences the reduction of urinary globotriaosylceramide concentration. In the current study, agalsidase beta antibodies were found to be associated with inhibition of alpha-Gal A enzyme activity in cultured Fabry fibroblast and tissues from Fabry mice. However, the negative effect of antibody formation could be overcome by increasing the dose of enzyme administered to mice. In conclusion, antibody titers and the dose of enzyme influenced alpha-Gal A enzyme activities in vivo. Further studies are required to investigate to what extend antibody formation impacts on therapeutic responses in antibody positive Fabry patients receiving enzyme replacement therapy and if negative effects can be overcome by adjusting the dose of enzyme.

Neonatal gene transfer using lentiviral vector for murine Pompe disease: long-term expression and glycogen reduction

Pompe disease results from the deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to accumulated glycogen in the heart and the skeletal muscles, which causes cardiomyopathy and muscle weakness. In this study, we tested the feasibility of gene therapy for Pompe disease using a lentivirus vector (LV). Newborn GAA knockout mice were treated with intravenous injection of LV encoding human GAA (hGAA) through the facial superficial temporal vein. The transgene expression in the tissues was analyzed up to 24 weeks after treatment. Our results showed that the recombinant LV was efficient not only in increasing the GAA activity in tissues but also in decreasing their glycogen content. The examination of histological sections showed clearence of the glycogen storage in skeletal and cardiac muscles 16 and 24 weeks after a single vector injection. Levels of expressed hGAA could be detected in serum of treated animals until 24 weeks. No significant immune reaction to transgene was detected in most treated animals. Therefore, we show that LV-mediated delivery system was effective in correcting the biochemical abnormalities and that this gene transfer system might be suitable for further studies on delivering GAA to Pompe disease mouse models.

The production of human glucocerebrosidase in glyco-engineered Nicotiana benthamiana plants.

Summary For the production of therapeutic proteins in plants, the presence of β1,2‐xylose and core α1,3‐fucose on plants’ N‐glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down‐regulate the endogenous N‐acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco‐engineered line (Nb GNTI‐RNAi) showed a strong reduction of plant‐specific N‐glycans, with the result that as much as 90.9% of the total N‐glycans were of high‐mannose type. Therefore, this Nb GNTI‐RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The Nb GNTI‐RNAi plant was cross‐pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gauchers disease, requires terminal mannose for its therapeutic efficacy. The N‐glycan structures that were presented on all of the four occupied N‐glycosylation sites of recombinant GC in Nb GNTI‐RNAi plants (GC gnt1) showed that the majority (ranging from 73.3% up to 85.5%) of the N‐glycans had mannose‐type structures lacking potential immunogenic β1,2‐xylose and α1,3‐fucose epitopes. Moreover, GC gnt1 could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gauchers disease. Notably, the Nb GNTI‐RNAi line, producing GC, was stable and the Nb GNTI‐RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N‐glycan structures.

A Japanese patient homozygous for the H1085R mutation in the CFTR gene presents with a severe form of cystic fibrosis

To the Editor: Cystic fibrosis (CF), the most common autosomal recessive disorder in Caucasians, has long been believed to be extremely rare in Orientals (1, 2). In Japan, there have been approximately 130 cases of CF reported in the literature during the last 5 decades, with the estimated incidence of one in 350000 live births (3). Besides, very limited information is available about the spectrum of CF transmembrane conductance regulator (CFTR) gene mutations among Japanese CF patients, although more than 800 mutations of the CFTR gene, including the most common DF508, have been accumulated worldwide through the CF Genetic Analysis Consortium [(4), personal communication]. Here we describe a 15-year-old female Japanese patient who exhibited typical manifestations of CF and proved to be a homozygote for a very rare missense mutation H1085R. She was born to consanguineous parents at full term. From 1 year old, she repeatedly suffered from respiratory symptoms associated with Pseudomonas aeruginosa infection and experienced occasional steatorrhea. At 8 years old, she developed fecal ileus and underwent surgical operation. At 9 years old, she was diagnosed as CF based on the elevated sweat chloride level at 201 mmol/l. She also had chronic sinusitis, and abdominal ultrasonography revealed fibrotic changes of the pancreas. The patient has recently developed shortness of breath on exertion, and room-air arterial blood gas analysis showed moderate hypoxemia. For evaluation of the CFTR gene mutations, her DNA sample was evaluated for 32 major CFTR gene mutations responsible for more than 90% of CF chromosomes in the Caucasian population of North America (5). However, none of these mutations were detected in her CFTR alleles. Next, all 27 exons of the CFTR gene, including both the 5%and 3%-intron–exon junctions, were polymerase chain reaction (PCR) amplified and studied by single strand DNA conformation polymorphism (SSCP) analysis (6, 7). When the PCR products were electrophoresed and silver-stained, the DNA bands with different mobility from those of the control were only detected in exon 17b. Direct sequencing of the band clearly demonstrated an alteration of the nucleotide residue at 3386 from A to G in a homozygous fashion, resulting in His to Arg change at the amino acid 1085, a missense mutation H1085R. The CFTR genotype analysis of her family by PCR amplification of exon 17b and subsequent RsaI restriction digestion revealed that both parents and her three siblings were heterozygous for the mutation (Fig. 1). Further evaluation for three polymorphic loci proved that the H1085R mutation allele was associated with a haplotype of (GATT)6 in intron 6a (8), (TG)12T7 in the TG repeat and polythymidine tract at the splice acceptor site in intron 8 (9, 10), and M470 (1540A) in exon 10 (11, 12), respectively. The mutation H1085R, first described by Mercier et al. (13, 14), was detected on one of the CFTR alleles of a French CF patient who was pancreatic insufficient and a compound heterozygote with DF508 on the other chromosome [(13), personal communication]. However, no other CF patients with H1085R have been reported since then, implying that it is an extremely rare mutation (14). Consistent with the previous report that other mutations located in exon 17b, such as R1066L and M1101R, were usually associated with pancreatic insufficiency, the case presented here and the French case had pancreatic insufficiency, suggesting that the H1085R is also a severe allele [(13), personal communication]. Since the amino acid residue 1085 is located between the fourth and the fifth transmembrane segments of the second membrane-spanning domain and resides at the gate of the channel in the cytoplasm, it is conceivable that a His to Arg alteration would profoundly affect Cl conductance by either reduced Cl flow through a single open channel or by shortened duration for the channel opening (1, 15). One important point is that the H1085R alleles of these 2 patients are most likely to be recurrent

Administration of anti-CD3 antibodies modulates the immune response to an infusion of α-glucosidase in mice.

Animal and human studies of enzyme replacement therapy (ERT) for Pompe disease (PD) have indicated that antibodies (Abs) generated against infused recombinant human α-glucosidase (rhGAA) can have a negative impact on the therapeutic outcome and cause hypersensitivity reactions. We showed that parenteral administration of anti-CD3 Abs into mice can reduce the titer of anti-human GAA Abs in wild-type mice administered the enzyme. Mice that had been treated with anti-CD3 Abs and then subjected to a secondary challenge with rhGAA showed a lower increase in Ab titers than control mice. Moreover, the administration of anti-CD3 Abs also reduced the levels of pre-existing Abs. Treatment with anti-CD3 Abs also prevented a lethal hypersensitivity reaction and reduced the Ab titers in a mouse model of PD. Mice treated with anti-CD3 Abs showed reduced numbers of CD4(+) and CD8(+) cells, and an increased ratio of CD4(+)CD25(+)/CD4(+) and CD4(+)CD25(+)FoxP3(+)/CD4(+) cells. When the CD4(+)CD25(+) cells were depleted using anti-CD25 Abs, the observed reduction in Abs against the enzyme by anti-CD3 Abs was abrogated. This suggests that CD4(+)CD25(+) cells are important for the immune suppressive activity of anti-CD3 Abs. In summary, anti- CD3 Abs are useful for inducing immune tolerance to ERT for PD.

Oral administration of recombinant human acid α-glucosidase reduces specific antibody formation against enzyme in mouse

Animal and human studies of enzyme replacement therapy for Pompe disease have indicated that antibodies generated against the infused recombinant human acid α-glucosidase (rhGAA) can negatively impact therapeutic outcome. In this study, we show that oral administration of rhGAA into mice can reduce the titer of anti-rhGAA antibody following immunization with rhGAA. Oral administration of rhGAA is safe and antigen specific, it offers advantages over other immunosuppressive drugs.

Efficient and persistent expression of β-glucuronidase gene in CD34+ cells from human umbilical cord blood by retroviral vector

Abstract:  We succeeded in efficiently transferring the β‐glucuronidase gene in a retroviral vector to human hematopoietic progenitor cells using a centrifugation enhancement protocol. The transduction efficiency in CFU–GM was highly variable (23–100%) with an average of 66.8%. In the case of BFU–E, efficiency was 83% and 76% in 2 separate experiments. In LTCIC (long‐term culture‐initiating cell), transduction efficiency were 20% and 50% in 2 experiments. The enzymatic activity of β‐glucuronidase in transduced cells were increased above the control level up to 5 wk. Considering that correction of the enzyme deficiency in a small number of hematopoietic cells can be therapeutic for the Sly disease mouse, our data provide encouragement that human trials of gene therapy based on transferring β‐glucuronidase gene to hematopoietic cells may be efficacious.

Frequency of de novo mutations in Japanese patients with Fabry disease.

We examined alpha-galactosidase A (GLA) gene mutations in 74 Japanese families with Fabry disease (FD) to determine the frequency of de novo mutations. In 5 of 74 families (6.8%), the probands had no positive family histories and were diagnosed as de novo because their parents had no mutations in GLA gene. The parents of Fabry patients do not necessarily have mutations in GLA gene which is an important consideration in genetic counseling for FD.

858. Correction of the CNS Pathology and Function of MPS VII Mice by Intraventricular Transplantation of Genetically Modified Bone Marrow Stromal Cells

Mucopolysaccharidosis type VII (MPSVII) is one of lysosomal storage diseases (LSDs) and caused by a genetic deficiency of beta-glucuronidase (GUSB) resulting in the progressive accumulation of glycosaminoglycans(GAGs) in the brain and other tissues. Although current therapies for LSDs are enzyme replacement therapy and bone marrow transplantation, both therapies have various limitations especially in the brain. As an alternative approach, we transplanted genetically modified bone marrow stromal (BMS) cells into the lateral ventricle of neonatal MPSVII mice. The murine BMS cells transduced by a retroviral vector expressing human GUSB gene was injected into the lateral ventricle of newborn MPSVII mice. Two weeks after transplantation, the GUSB positive cells were identified in the olfactory bulb, cerebral cortex and striatum and the enzymatic activities in the various brain areas of treated mice increased about 50 times higher than untreated mice (0.15nmol/hr/mg protein to 7.81nmol/hr/mg protein). It is well known that alfa-Galactosidase A and Hexosaminidase activity in the brain for MPSVII is elevated. In treated mice, these enzymatic activities decreased to those of heterozygote mouse level. Although lysosomal distention in many neural cells and endothelial cells of untreated mice was observed, there was no lysosomal distention in the various brain regions of treated mice at 4 weeks of age. The amount of chondroitine sulfate and hyarulonic acid of GAGs which is known as storage material of MPSVII mice, were decreased significantly in treated mice. This reduction persisted at 16 weeks after transplantation. We performed the Morris Water Maze test (MWM) to evaluate the brain function of treated mice 6 weeks after transplantation. On day 6 of MWM, the platform was removed and the amount of time spent in each quadrant was monitored. Average of the time spent in the target quadrant by treated mice was 22.4 (sec), whereas average of the time spent by untreated mice was 18.2 (sec). Treated mice spent significantly more time in the target quadrant than in the mean of other three non-target quadrants (P=0.0028), whereas in untreated mice the difference of time spent in the target quadrant and the mean of the other three non-target quadrants was not statistically significant (P=0.3201). BMS cells have various advantages, including easy accessibility, minimum ethical issues and minimum immuno-reaction from the host. These data suggest that BMS cells are an excellent vehicle by which to transfer therapeutic protein to the brain in multiple lysosomal storage diseases.