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.

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.

Efficient retroviral mediated transfer of the glucocerebrosidase gene in CD34+ enriched umbilical cord blood human hematopoietic progenitors.

Obtaining efficient transfer of a normal gene and its sustained expression in self-renewing hematopoietic stem cell populations is a central concern for gene therapy initiatives. Potentially, 10(8) to 10(9) CD34+ enriched cells per patient will be required for transduction and subsequent reimplantation. These studies present an efficient method for the transduction of human CD34+ cells that can be used in a clinical study of gene transfer. The method uses a centrifugation-enhanced technique for the retroviral-mediated transfer of the normal human glucocerebrosidase (GC) gene to human CD34+ enriched umbilical cord blood cells (CB). Previous studies had described high expression of GC in CD34+ enriched cells but had not reported transduction efficiency in the progenitor population specifically. The data demonstrate an average transduction efficiency in the progenitor cell population of 50% as measured by polymerase chain reaction (PCR) for the integrated GC-cDNA in clonogenic cells. Measurements of enzyme activity comparing transduced and nontransduced fractions at 6 days posttransduction indicate an average enzyme increase of six-fold over normal background levels. PCR of colony forming units-granulocyte/macrophage (CFU-GM) plated at 6 weeks from long-term culture-initiating cell (LTC-IC) cultures also indicates transfer of the transgene to early progenitor cells. Finally, experiments were carried out with the human erythroleukemia cell line, TF-1, to estimate the durable expression of the transgene. Enzymatic activities in transduced TF-1 cultures remained at 30-fold above the activity of nontransduced controls. The expression persisted for 6 weeks in culture. These studies demonstrate efficient transduction of early progenitor cells and sustained expression of the transgene in cell cultures.

Cytokine mobilization of peripheral blood stem cells in patients with Gaucher disease with a view to gene therapy.

As clinical trials for gene therapy in Gaucher disease (GD) begin, questions regarding the biology of the hematopoietic stem cell in this disease remain unanswered. This study demonstrates the ability to mobilize and collect CD34+ cells in three patients with the disorder. Our RAC/FDA-approved clinical trial utilizes mobilized peripheral blood stem cells (PBSC) as the target cells for gene transfer. In this approach, a white blood cell fraction is collected by apheresis, enriched for CD34+ cells, and transduced with a retroviral vector carrying the glucocerebrosidase (GC) gene. Transduced cells from the patient with activity corrected to at least normal levels will be returned to the patient without myelosuppressive therapy. We report here the effect of cytokines in mobilizing PBSC in three patients with GD. Two (patients 1 and 2) were given granulocyte colony-stimulating factor (G-CSF) at a dose of 5 micrograms/kg/d and one (patient 3) was given 10 micrograms/kg/d for 10 days. Leukaphereses were done daily for 5 days and the products enriched for CD34+ cells using the clinical Ceprate (CellPro) column. The CD34+ cells in all fractions were monitored daily during mobilization and leukaphereses. Subset analysis for the expression of Thy-1, CD38, HLA-DR, and CD33 on the CD34+ cells was performed. An increase in CD34+ cells in the peripheral blood was noted from day 5 onward (up to a six-fold increase). Up to a 625-fold enrichment in CD34+ cells in the apheresis product was noted using the clinical Ceprate column. Totals of 1.2, 3.5, and 2.1 x 10(6) CD34+ cells/kg were collected in the three patients. A diminution in the percent of CD34+/Thy-1+ cells was noted with enrichment. In vitro retroviral transduction of the CD34-enriched cells using centrifugation promoted transduction protocol previously described (Bahnson AB et al., Centrifugal enhancement of retroviral-mediated gene transfer. Journal of Virology Methods 54:131, 1995) and modified for clinical use, demonstrated a mean transduction efficiency of 37% (range 8.3-87.1%) in clonogenic cells and up to 50% in long-term culture-initiating cells (LTC-IC) at week 6. Significantly, we have been able to achieve up to a 50-fold increase in the level of GC above deficient levels in the patients' CD34+ enriched cells when maintained in vitro in culture. The study demonstrates that up to a six-fold increase in CD34+ cells in the PB can be achieved with cytokines in patients with GD. CD34+ cells can be collected in numbers sufficient for conventional transplantation and transduced efficiently in vitro. In gene therapy trials for genetic disorders to date, myelosuppressive therapy is not advocated. The clinical trial will demonstrate whether this number of transduced CD34+ cells will be adequate for competitive engraftment of genetically corrected PBSC.

Long-term expression of the glucocerebrosidase gene in mouse and human hematopoietic progenitors.

Gaucher disease (GD), one of the most common inherited metabolic disorders, is an excellent candidate for gene therapy using hematopoietic stem cells as targets. Animal models have demonstrated the feasibility of introducing the human glucocerebrosidase (GC) gene into hematopoietic progenitors with long term expression using a variety of retroviral vectors. We have previously demonstrated the expression and integration of the human GC gene in mouse hematopoietic progenitors and their progeny 4-8 months post transplant in primary recipients using the retroviral vector MFG-GC. We now demonstrate enzyme expression in peripheral blood lymphocytes of secondary recipients more than 12 months post transplantation. We also show a transduction efficiency of up to 95% in colony forming unit-granulocyte macrophage (CFU-GM) colonies generated from transduced CD34+ cells from a variety of sources, using a centrifugation promoted infection protocol. Transduction has also been documented in long term culture initiating cells (LTCIC) from the same transduced CD34+ cells. These data indicate efficient transduction of mouse hematopoietic progenitors as well as human CD34+ cells using the retroviral vector MFG-GC.

Centrifugal enhancement of retroviral mediated gene transfer.

Centrifugation has been used for many years to enhance infection of cultured cells with a variety of different types of viruses, but it has only recently been demonstrated to be effective for retroviruses (Ho et al. (1993) J. Leukocyte Biol. 53, 208-212; Kotani et al. (1994) Hum. Gene Ther. 5, 19-28). Centrifugation was investigated as a means of increasing the transduction of a retroviral vector for gene transfer into cells with the potential for transplantation and engraftment in human patients suffering from genetic disease, i.e., gene therapy. It was found that centrifugation significantly increased the rate of transduction into adherent murine fibroblasts and into non-adherent human hematopoietic cells, including primary CD34+ enriched cells. The latter samples include cells capable of reconstitution of hematopoiesis in myeloablated patients. As a step toward optimization of this method, it was shown that effective transduction is: (1) achieved at room temperature; (2) directly related to time of centrifugation and to relative centrifugal force up to 10,000 g; (3) independent of volume of supernatant for volumes > or = 0.5 ml using non-adherent cell targets in test tubes, but dependent upon volume for coverage of adherent cell targets in flat bottom plates; and (4) inversely related to cell numbers per tube using non-adherent cells. The results support the proposal that centrifugation increases the reversible binding of virus to the cells, and together with results reported by Hodgkin et al. (Hodgkin et al. (1988) J. Virol. Methods 22, 215-230), these data support a model in which the centrifugal field counteracts forces of diffusion which lead to dissociation during the reversible phase of binding.

Exon scanning for mutation of the NF2 gene in schwannomas.

Family studies and tumor analyses have combined to indicate that neurofibromatosis 2 (NF2), a disorder characterized by multiple benign tumors of the nervous system, and sporadic non-inherited forms of the same tumor types are both caused by inactivation of a tumor suppressor gene located in 22q12. Recently, the gene encoding merlin, a novel member of a family of cytoskeleton-associated proteins, was identified as the NF2 tumor suppressor. To facilitate the search for merlin mutations, we have defined the exon-intron boundaries for all 17 NF2 exons, including one subject to alternative splicing. We have developed polymerase chain reaction assays to amplify each exon from genomic DNA, and used these assays to perform single-strand conformation polymorphism analysis of DNA from 30 sporadic and eight NF2-derived schwannomas, the hallmark tumor type in this disorder. Of a maximum of 60 alleles scanned, 32 showed mutations affecting expression of the merlin protein. Thirty of these mutations are predicted to lead to a truncated protein due to frameshift, creation of a stop codon, or interference with normal splicing, while two are missense mutations. Thus, inactivation of merlin is a common feature underlying both inherited and sporadic forms of schwannoma.

DNA diagnosis of neurofibromatosis 2. Altered coding sequence of the merlin tumor suppressor in an extended pedigree.

OBJECTIVE: To define the DNA mutation causing neurofibromatosis 2 (NF2), a severe genetic disorder involving the development of multiple nervous system tumors in adulthood, in a large, well-studied NF2 pedigree previously used to chromosomally map and to isolate the disease gene. DESIGN: Single-strand conformational polymorphism (SSCP) and DNA sequence analysis of the NF2 gene amplified from affected and unaffected family members. PARTICIPANTS: Affected, unaffected, and at-risk members of a large pedigree segregating NF2, an autosomal dominant disorder caused by inactivation of the merlin tumor suppressor encoded in chromosome band 22q12. RESULTS: A DNA alteration in the merlin coding sequence caused a shift on SSCP gels that was characteristic of the disease chromosome in this NF2 pedigree, being transmitted with the disorder, present only in affected members of the pedigree, absent in unaffected members of the family, and absent from 158 unrelated individuals. The alteration caused substitution of a tyrosine for an asparagine at position 220 of the merlin protein, in a region highly conserved in closely related members of the family of cytoskeletal-associated proteins. The DNA change could also be detected by restriction enzyme digestion with Rsa I. CONCLUSION: Current practice dictates screening of all those "at risk" for NF2 with magnetic resonance imaging, but the frequency and duration of screening are problematic because of the variable course of the disease. The identification of a DNA alteration in the NF2 gene will permit predictive molecular testing of individuals at risk in this specific family, sparing the expense and emotional burden of protracted screening programs. This information, by providing diagnostic certainty, should also reduce psychological and financial burdens and improve medical care for affected family members. A similar approach to defining the underlying lesion and developing a predictive test is applicable in any documented NF2 family.