Improvement of bone disease by imiglucerase (Cerezyme) therapy in patients with skeletal manifestations of type 1 Gaucher disease: results of a 48-month longitudinal cohort study.

Progressive skeletal disease accounts for some of the most debilitating complications of type 1 Gaucher disease. In this 48-month, prospective, non-randomized, open-label study of the effect of enzyme replacement therapy on bone response, 33 imiglucerase-naïve patients (median age 43 years with one or more skeletal manifestations such as osteopenia, history of bone crisis, or other documented bone pathology) received imiglucerase 60 U/kg/2 weeks. Substantial improvements were observed in bone pain (BP), bone crises (BC), and bone mineral density (BMD). Improvements in BP were observed at 3 months (p < 0.001 vs baseline) and continued progressively throughout the study, with 39% of patients reporting pain at 48 months vs 73% at baseline. Eleven of the 13 patients with a pre-treatment history of BC had no recurrences. Biochemical markers for bone formation increased; markers for bone resorption decreased. Steady improvement of spine and femoral neck BMD, measured using dual-energy X-ray absorptiometry was noted. Mean Z score for spine increased from -0.72 +/- 1.302 at baseline to near-normal levels (-0.09 +/- 1.503) by month 48 (p = 0.042) and for femoral neck from -0.59 +/- 1.352 to -0.17 +/- 1.206 (p = 0.035) at month 36. This increase was sustained at 48 months. With imiglucerase treatment, patients should anticipate resolution of BC, rapid improvement in BP, increases in BMD, and decreased skeletal complications.

Imiglucerase (Cerezyme) improves quality of life in patients with skeletal manifestations of Gaucher disease.

Health-related quality of life (HRQOL) can be diminished in patients with type 1 Gaucher disease (GD) owing to the debilitating clinical manifestations of this chronic disease. This study investigates the impact of imiglucerase treatment on HRQOL of patients with type 1 GD and bone involvement. Thirty-two previously untreated type 1 GD patients with skeletal manifestations including bone pain, medullary infarctions, avascular necrosis, and lytic lesions received biweekly imiglucerase (at 60 U/kg). The Short Form-36 Health Survey (SF-36) was administered at regular intervals to assess HRQOL. Mean baseline SF-36 physical component summary (PCS) scores were diminished relative to US general population norms. Low PCS scores were more common in patients with medullary infarction, lytic lesions, and higher bone pain severity scores. Statistically significant improvements were observed for all eight SF-36 subscales after 2 years of treatment. Mean PCS and mental component summary (MCS) scores increased to within the normal range after 2 years of treatment and were maintained through year 4. Large HRQOL gains were observed even in patients with the most advanced disease and lowest baseline PCS scores. Imiglucerase treatment has a significant positive impact on HRQOL of type 1 GD patients with skeletal disease, including those with bone infarctions, lytic lesions, and avascular necrosis.

Gene therapy for lysosomal storage disorders.

The lysosomal storage disorders (LSD) are monogenic inborn errors of metabolism with heterogeneous pathophysiology and clinical manifestations. In the last decades, these disorders have been models for the development of molecular and cellular therapies for inherited metabolic diseases. Studies in preclinical in vitro systems and animal models have allowed the successful development of bone marrow transplantation (BMT) and enzyme replacement therapy (ERT) as therapeutic options for several LSDs. However, BMT is limited by poor donor availability and high morbidity and mortality, and ERT is not a life-long cure. Moreover, the neuropathology present in many LSDs responded poorly, if at all, to these treatments. Therefore, gene therapy is an attractive therapeutic alternative. Gene therapy strategies for LSDs have employed ex vivo gene transduction of cellular targets with subsequent transplantation of the enzymatically corrected cells, or direct in vivo delivery of the viral vectors. Oncoretroviral vectors and more recently adeno associated vectors (AAV) and lentiviral vectors have been extensively tested, with some success. This review summarises the main gene therapy strategies which have been employed or are under development for both non-neurological and neuronopathic LSDs. Some of the in vitro and in vivo preclinical studies presented herein have provided the rationale for a gene therapy clinical trial for Gaucher disease Type I.

Lessons learned from the development of enzyme therapy for Gaucher disease.

Enzyme replacement therapy for the lysosomal storage disorders derives its impetus from the successes achieved in the treatment of Gaucher disease. After nearly two decades of persistent but unsuccessful efforts, the promise of therapy through enzyme replacement was losing credibility. Then, the fortunate intersection of two different lines of scientific research produced the necessary breakthrough. The dramatic responses to enzyme replacement therapy in patients with Gaucher disease made it immediately clear that this treatment approach was a success. Furthermore, the large number of patients with the disorder guaranteed commercial involvement. The lessons learned from the development of enzyme replacement therapy for Gaucher disease are broadly applicable to other lysosomal storage diseases and will be reviewed in this paper.

Long-term expression and transfer of arylsulfatase A into brain of arylsulfatase A-deficient mice transplanted with bone marrow expressing the arylsulfatase A cDNA from a retroviral vector.

A deficiency of arylsulfatase A (ASA) results in the lysosomal lipid storage disease metachromatic leukodystrophy. The disease mainly affects the central nervous system causing a progressive demyelination. A therapeutic effect will depend on the delivery of the deficient enzyme to the central nervous system. We have transplanted ASA-deficient mice with bone marrow transduced with a retroviral vector expressing the human ASA cDNA. All transplanted animals initially showed high serum levels of human ASA. In 50% of the recipients high ASA serum levels were sustained for 12 months after transplantation. In the remaining mice, serum levels decreased rapidly to low or undetectable levels. ASA activity and immunoreactivity was detectable in all organs of animals with continuous levels of ASA in serum. Most notably, substantial amounts of ASA activity were transferred into the brain, reaching up to 33% of the normal tissue level. In contrast to peripheral organs, the amount of enzyme delivered to the brain did not correlate with ASA serum levels as an indicator of overexpression. This reveals that enzyme transfer to the brain is not due to endocytosis of serum ASA by endothelial cells, but rather to bone marrow-derived cells migrated into the brain. Gene Therapy (2000) 7, 1250-1257.

Retrovirus-mediated transfer of human alpha-galactosidase A gene to human CD34+ hematopoietic progenitor cells.

Fabry disease, caused by a deficiency of lysosomal enzyme alpha-galactosidase A (alpha-gal A), is one of the inherited disorders potentially treatable by gene transfer to hematopoietic stem cells. In this study, a high-titer amphotropic retroviral producer cell line, MFG-alpha-gal A, was established. CD34+ cells from normal umbilical cord blood were transduced by centrifugal enhancement. The alpha-gal A activity in transduced cells increased 3.6-fold above the activity in nontransduced cells. Transduction efficiency measured by PCR for the integrated alpha-gal A cDNA in CFU-GM colonies was in the range of 42-88% (average, 63%). The expression of functional enzyme in TFI erythroleukemia was sustained for as long as cells remained in culture (84 days) and for 28 days in LTC-IC cultures of CD34+ cells. The ability of the transduced CD34+ cells to secrete the enzyme and to correct enzyme-deficient Fabry fibroblasts was assessed by cocultivation of these cells. The enzyme was secreted into the medium from transduced CD34+ cells and taken up by Fabry fibroblasts through mannose 6-phosphate receptors. These findings suggest that genetically corrected hematopoietic stem/progenitor cells can be an enzymatic source for neighboring enzyme-deficient cells, and can potentially be useful for gene therapy of Fabry disease.

Gene transfer approaches to the lysosomal storage disorders.

The work summarized in this paper used animal and cell culture models systems to develop gene therapy approaches for the lysosomal storage disorders. The results have provided the scientific basis for a clinical trial of gene transfer to hematopoietic stem cells (HSC) in Gaucher disease which is now in progress. The clinical experiment is providing evidence of HSC transduction, competitive engraftment of genetically corrected HSC, expression of the GC transgene, and the suggestion of a clinical response. In this paper we will review the progress made in Gaucher disease and include how gene transfer might be studied in other lysosomal storage disorders.

Long-term expression and secretion of human glucocerebrosidase by primary murine and human myoblasts and differentiated myotubes.

Gaucher disease (GD) is caused by a deficiency in glucocerebrosidase (GC). Enzyme replacement for GD disease is effective but expensive and requires life-long treatment. Development of alternative therapeutic strategies is therefore important. One approach is an enzyme delivery system which could supply GC into the circulation continuously. We have previously reported that human GC cDNA in a retroviral vector (MFG-GC) efficiently transduced a murine myoblast line (C2C12) and expressed GC intracellularly and extracellularly. Now we have demonstrated that primary murine and human myoblasts are transduced at very high efficiency by MFG-GC (five to ten copies of human GC gene per cell at a multiplicity of infection of 5-10), 100% of MFG-GC transduced cells expressed human GC. The transduced primary murine and human myoblasts had an intracellular GC activities about five to ten times above nontransduced controls. Furthermore, transduced primary myoblasts secreted human GC extracellularly for up to 35 weeks in vitro. The secreted human GC is specifically taken up by bone marrow derived macrophages, the cell type most important to the pathogenesis of GD. These data suggest that transduced primary myoblasts may be useful in supplying GC as an alternative approach to the treatment of GD.

Long-term expression, systemic delivery, and macrophage uptake of recombinant human glucocerebrosidase in mice transplanted with genetically modified primary myoblasts.

A critical requirement for treatment of Gaucher disease via systemic delivery of recombinant GC is that secreted enzyme be in a form available for specific takeup by macrophages in vivo. In this article we investigated if transplanted primary myoblasts can sustain expression of human GC in vivo and if the secreted transgene product is taken up by macrophages. Transduced primary murine myoblasts were implanted into syngeneic C3H/HeJ mice. The results demonstrated that transplanted mice sustained long-term expression of transferred human GC gene in vivo. Furthermore, human GC is secreted into the circulation of mice transplanted with syngeneic primary myoblasts retrovirally transduced with human GC cDNA. The transplanted primary myoblasts differentiate and fuse with adjacent mature myofibers, and express the transgene product for up to 300 days. Human GC in the circulation reaches levels of 20-280 units/ml of plasma. Immunohistochemical studies of the target organs revealed that the secreted human GC is taken up by macrophages in liver and bone marrow. Immunochemical identification of reisolated myoblasts from transplanted mice showed that MFG-GC-transduced cells also survived as muscle stem cells in the implanted muscle. These results present in encouraging prospect for the treatment of Gaucher disease.

Comparison of methods for retroviral mediated transfer of glucocerebrosidase gene to CD34+ hematopoietic progenitor cells.

Gaucher disease is an excellent candidate for gene therapy by transduction of hematopoitic stem cells. In this study, we compared methods which allow an increase in transfer of the glucocerebrosidase gene to human hematopoietic progenitor cells. Several techniques were employed, including the use of cytokines, bone marrow stroma, fibronectin, centrifugal enhancement and in vitro long-term culture. The effect of prestimulation with cytokines interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF) on transduction of cord blood CD34+ cells was examined. The results suggest that 16-h prestimulation was sufficient for efficient transduction. We examined the effect of bone marrow stroma and fibronectin, both of which increased transduction efficiency up to 36% and 44%, respectively, as measured by PCR for the integrated GC-cDNA in clonogenic cells (9% without any support). Transduction efficiency of 83% was obtained using 2-h centrifugation. Combining centrifugation and in vitro culture in long-term bone marrow culture media containing cytokines (IL-3/IL-6/SCF), CD34+ cells from cord blood and peripheral blood of 3 Gaucher patients were transduced weekly for 21 d. The results of 6 separate experiments consistently demonstrated transduction efficiency of 100% after 7-d in vitro culture. This transduction protocol combining centrifugation and in vitro long-term culture is an attractive method and can be applied to clinical trials.

Detection and isolation of gene-corrected cells in Gaucher disease via a fluorescence-activated cell sorter assay for lysosomal glucocerebrosidase activity.

Gaucher disease type 1 results from the accumulation of glucocerebroside in macrophages of the reticuloendothelial system, as a consequence of a deficiency in glucocerebrosidase (GC) activity. Recent improvements in the methodologies for introducing foreign genes into bone marrow stem cells have prompted several groups to test the efficacy of gene transfer therapy as a curative treatment for Gaucher disease. Limitations of this approach include the potential for insufficient engraftment of gene-corrected cells and incomplete transduction of hematopoietic stem cells using retroviral gene transfer. Overcoming these obstacles may be critical in the case of treatment for Gaucher disease type 1, because GC transduced cells have not been shown to have a growth advantage over noncorrected cells. Here, we describe the development and application of a novel, fluorescence-activated cell sorter based assay that directly quantitates GC activity at the single cell level. In a test of this application, fibroblasts from a Gaucher patient were transduced, and high expressing cells sorted based on GC activity. Reanalysis of cultured sorted fibroblasts reveals that these cells maintain high levels of enzymatic activity, compared with the heterogeneous population from which they were sorted. The assay is sufficiently sensitive to distinguish GC activity found in Gaucher patient monocytes from that in normal controls. Furthermore, preliminary results indicate that increased GC activity can be detected in transduced, CD34+ enriched peripheral blood mononuclear cells isolated from a Gaucher patient. This method should be a useful addition to current gene therapy protocols as a means to quantitatively assess gene correction of relevant cell populations and potentially purify transduced cells for transplantation.

The effect of cationic liposome pretreatment and centrifugation on retrovirus-mediated gene transfer.

Pretreatment of retroviral supernatants with the cationic liposomes DOTMA-DOPE (Lipofectin), DC-Chol-DOPE and DOSPA-DOPE (Lipofectamine) was found to enhance static transductions of TF-1 target cells. The relative effectiveness at increasing transduction efficiencies (TE) was: DOSPA > DC-Chol > DOTMA, resulting in average increases over nontreated controls of 11.9-, 6.2- and 1.2-fold, respectively. This pretreatment was found to be synergistic when combined with centrifugation, having the same order of effectiveness, and resulting in 57-, 35- and 27-fold increases over nontreated controls. For Lipofectamine and DC-Chol-DOPE liposomes, the combined approach yielded 2.2- and 1.3-fold increases over untreated centrifuged samples. Individual colonies picked from colony-forming unit granulocyte-macrophage assays of infected CD34+ cells were screened for the presence of the transgene by polymerase chain reaction (PCR). Colonies from cells infected using centrifugation were positive 27% of the time, while the combined approach had positive colonies 31 and 50% of the time for DC-Chol and Lipofectamine, respectively. The addition of protamine sulfate to the liposome-supernatant mixture during pretreatment was found to be inhibitory. With increasing centrifugal force, the TE of cells infected with Lipofectamine pretreated and untreated supernatants increased proportionally. However, the TE of the cells infected with the pretreated supernatants was significantly higher than the TE of the cells infected with untreated supernatants at all points examined. The increase in TE associated with liposomal pretreatment of retroviral supernatants was not shown to be attributed to a nonreceptor-mediated pathway for viral entry into the cell.

Gaucher's disease: studies of gene transfer to haematopoietic cells.

Transfer of the gene coding for glucocerebrosidase (GC) via a retroviral vector (MFG-GC) to haematopoietic progenitors results in engraftment and life-long expression of the human protein at high levels in transplanted mice. Studies of human CD34 cells were carried out to evaluate their potential use in a gene therapy approach to Gaucher's disease. High transduction efficiency and correction of the enzyme deficiency was possible in CD34 cells obtained from patients with Gaucher's disease. Based on these results, a clinical trial of gene therapy was designed and initiated. Preliminary results of this study indicate the persistence or engraftment of genetically corrected cells in the transplanted patients.

Methods for Retrovirus-Mediated Gene Transfer to CD34(+) Enriched Cells.

Hematopoietic stem cells (HSC) provide for contmuous replenishment of the entire immune and hematopoietic systems, and also replenish themselves in a process termed self-renewal (1).The HSCs can be enriched from hematopoietic tissues using MAbs that bind to the CD34 antigen, a universally recognized marker for hematopoietic progenitors (2-4).Enriched HSC populations are being widely investigated for use in transplantation and gene therapy because they appear to provide rapid hematopoietic reconstitution in myeloablated patients (5-11), and they offer good targets for gene transfer (12-17).

Expression of human lysosomal alpha-mannosidase activity in transfected murine cells and human alpha-mannosidase deficient fibroblasts.

We studied the human lysosomal alpha-mannosidase (MANB) by expressing the putative cDNA in mammalian cells, using the eucaryotic expression vector pCDE. The construct pCDE-MANB and pSV2-Neo were cotransfected into human alpha-mannosidase deficient fibroblasts and into a murine cell line and selected by culture in the presence of G418. Six G418 resistant 3T3 clones had increased alpha-mannosidase activity 2 to 3 times above the controls. Two clones from transfected human fibroblasts showed a 2 fold increase in enzyme activity. The human MANB cDNA gene was demonstrated in the target cells by Southern blot analysis and the expression of the gene was shown by RT-PCR analysis. This study is the first to successfully express the MANB gene in a human and a murine cell line. The results confirm that the putative MANB cDNA encodes the full length of lysosomal alpha-mannosidase. Molecular characterization of mannosidosis and approaches to gene therapy are now possible using this cDNA.