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.

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.

Religion and the psychotherapeutic relationship. Transferential and countertransferential dimensions.

The salience of religion in society and health care has received increased attention. Recent developments in psychiatry reflect a broader view of religion that includes an appreciation of its adaptive and maladaptive dimensions. An examination of religious countertransferential and transferential reactions provides a framework for examining religious themes. Case examples illustrate the following critical factors that increase therapists' skill in working with religious themes: 1) monitoring the therapist's own attitude toward religious content, 2) attending to religious content, 3) seeking consultation, and 4) using religious content in interpretations.

A combination of CD34 selection and complement-mediated immunopurging (anti-CD15 monoclonal antibody) eliminates tumor cells while sparing normal progenitor cells.

Autologous bone marrow transplantation (ABMT) for acute myeloid leukemia (AML) in first complete remission (CR) results in a prolonged disease-free survival (DFS) of 34%-57%. Relapse of the underlying disease is the major cause for failure of ABMT. Relapse can result fom tumor cells either surviving in the patient or reinfused in the autograft. Genetic marking of autografted cells has demonstrated that transplanted cells contribute to relapse. This finding supports the use of purged autografts. Several purging techniques have been used. Immunologic purging using the monoclonal antibody (mAb) PM-81 (anti-CD15) has been used by our center with a long-term DFS in 50% of AML patients. PM-81 reacts with 90% of AML patients, and we have used it for over 10 years. We have investigated a two-stage purging technique involving initial selection for CD34+ cells followed by mAb purging in bone marrow (BM) and peripheral blood stem cell (PBSC) harvests. This method achieved up to a 7 log diminution in leukemic cells and 1-4 log reduction in CD15+ cells, without a significant loss of hematopoietic progenitor cells. This double-purging technique has the advantages of cytoreduction, elimination of CD34- leukemic cells, and possible improvement in the clinical efficacy of purging by concentrating for CD34+ cells. Cytoreduction by CD34 enrichment followed by purging may facilitate the use of PBSC transplants in AML.

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.