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Biomedical subjects

E D Ball

Publications and source records attributed to E D Ball.

At least 55 records · Page 3Linked to original sources
PubMed1995

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.

3T3 Cells↗
PubMed1995

The role of natural killer cells in the treatment of chronic myeloid leukemia.

The success of chemotherapy in patients with leukemia whose marrow appears to be replaced by leukemia cells must be due to the persistence of normal stem cells. In this normal population are the progenitors of the cells of the immune system. Natural killer (NK) cells originate in the bone marrow. On maturation and activation with interleukin 2 (IL-2) or other cytokines, NK cells develop cytotoxic activity against a variety of leukemic blasts, including those from patients with chronic myeloid leukemia (CML). In the past few years, bone marrow transplantation (BMT) and alpha-interferon (IFN-alpha) have proved to be the most promising therapies for the treatment of CML. In both these therapies, NK cells may play a prominent role. In this article, we discuss the antitumor/antileukemia activity of human NK cells, the presence of benign NK cell precursors in the different stages of CML, the role of NK cells in BMT and IFN-alpha treatment, and the potential therapeutic applications of NK cells in patients with hematologic malignancies.

Bone Marrow↗
PubMed1995

Lysis of small cell carcinoma of the lung (SCCL) cells by cytokine-activated monocytes and natural killer cells in the presence of bispecific immunoconjugates containing a gastrin-releasing peptide (GRP) analog or a GRP antagonist.

Lung cancer remains the leading cause of cancer deaths in the United States. We have developed a new immunotherapeutic approach to the treatment of small cell carcinoma of the lung (SCCL) by targeting the gastrin-releasing peptide receptor (GRP-R) expressed on the surface of these cells. Bispecific immunoconjugates were constructed by chemical fusion of a GRP analog or a GRP antagonist with monoclonal antibodies directed to the cytotoxic trigger molecules Fc gamma RI and Fc gamma RIII on various immune effector cells. We demonstrated that these bispecific immunoconjugates bound to target SCCL cells in a dose-dependent manner. In the presence of these immunoconjugates, more than 80% of SCCL cells were lysed by cytokine-activated monocytes and natural killer (NK) cells measured by a 51Cr-release assay. These data indicate that bifunctional antibodies targeting GRP may have clinical use.

Antibodies, Bispecific↗
PubMed1995

Homologous restriction of complement-mediated cell lysis can be markedly enhanced by blocking decay-accelerating factor.

Regulation of complement (C') dependent lysis of cells is attributed to certain membrane proteins. One of these is decay-accelerating factor (DAF), CD55, a 70kD glycosylated protein which functions to protect host cells from damage by autologous C'. We hypothesized that blockade of DAF function by a monoclonal antibody (mAb) could augment C'-dependent lysis mediated by another mAb to a cell surface antigen expressed on leukaemia cells. Thus, we tested the effects of the anti-DAF mAb 1C6 on the ability of both rabbit and human C' to lyse human leukaemia cells through activation by complement-fixing murine mAb. DAF antigen was highly expressed on most leukaemia cell lines and primary acute leukaemia blast cells tested. Murine mAb to CD15 (PM-81) and to gp 120 (AML-1-99), both IgM, also bound to the majority of myeloid and lymphoid leukaemia cells. Using human serum as a source of C', the addition of mAb 1C6 reduced by an additional 85-94% the numbers of clonogenic HL-60 (myeloid leukaemia) cells lysed by mAb PM-81 alone. Similarly, the addition of mAb 1C6 reduced by an additional 87% the numbers of HL-60 colonies eliminated by mAb AML-1-99 alone. Similar results were observed in an experimental purging model using the myeloid leukaemia cell lines HL-60, U937 or NB4 cells as targets. These results show that mAb 1C6 can effectively block the actions of DAF. In the presence of mAb 1C6, the cytotoxic activity mediated by human C' was similar to that of rabbit C'. These results show that increased tumour cell killing can be achieved through DAF blockade. This finding has relevance to clinical trials using C'-fixing mAb for purging bone marrow of occult tumour cells prior to autologous transplantation.

Acute Disease↗
PubMed1995

Potentiation of lysis of leukaemia cells by a bispecific antibody to CD33 and CD16 (Fc gamma RIII) expressed by human natural killer (NK) cells.

Bispecific antibodies recognizing tumour-associated antigens and trigger molecules expressed on immune effector cells have been shown to redirect cytotoxicity of several types of peripheral blood cells against relevant tumour targets. Among various effector cells, natural killer (NK) cells appear to play a role in defence against leukaemia. Here we report the successful chemical conjugation of monoclonal antibodies to CD33 and CD16 to create a bispecific antibody (BsAb 251 x 3G8). This bispecific antibody is capable of augmenting the killing of otherwise resistant leukaemia cells by peripheral blood lymphocytes (PBL), purified resting NK (R-NK) cells, and activated NK (A-NK) cells. BsAb 251 x 3G8 may play a role in the therapy of acute myeloid leukaemia (AML) through redirecting the cytotoxic activity of endogenous or adoptively transferred NK cells.

Acute Disease↗
PubMed1995

Introduction: workshop summary of the CD15 monoclonal antibody panel from the Fifth International Workshop on Leukocyte Antigens.

CD15 is expressed on a wide variety of tumor cells including myeloid leukemia, breast, colorectal, lung cancer cells. It is probable that the core proteins and lipids differ on the different cell types since there is marked variability in the binding of the mAb within and among the different cell types. Previously, there had only been a single mAb of the IgG class directed to CD15. The present study demonstrates the existence of one more IgG mAb (an IgG3). Unfortunately, a third mAb submitted as an IgG1, MA14, did not appear to be an IgG in our studies and a fourth mAb, MA9, submitted as an IgG1 did not have significant binding activity and also could not be confirmed as anti-CD15. An interesting finding was that all of the mAb could mediate complement-dependent cytotoxicity with rabbit serum. However, the two IgG3 mAb, MA63 (MCS-1) and MA88 (7C3), could not mediate lysis with human complement, while all of the IgM mAb could do so. It is not clear why this difference exists. CD15 is known to be expressed on mature myeloid cells in the hematopoietic lineage. The expression of CD15 on progenitor cells has been mapped by the indirect method of C'-dependent lysis and measurement of colony-forming cells. These studies have estimated that about 50% of normal CFU-GM express CD15. In the present workshop, we examined the expression of CD15 on a quantifiable cell population, the CD34 positive population.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

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.

Antigens, CD34↗
PubMed1995

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.

Adult↗
PubMed1995

Monocyte-mediated lysis of acute myeloid leukemia cells in the presence of the bispecific antibody 251 x 22 (anti-CD33 x anti-CD64).

Immunotherapy using bispecific antibodies (BsAb) to direct immune effector cells toward target tumor cells has been shown to be effective in a number of studies. Several immune trigger molecules have been characterized. Among them, FcgammaRI appears to play an important role in antibody-dependent cellular cytotoxicity. It is expressed mainly on monocytes, macrophages, and neutrophils under certain clinical situations. The expression of FcgammaRI can be regulated by a variety of cytokines, primarily by IFN-gamma. Recent studies have shown that granulocyte-colony-stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) can increase the number of the FcgammaRI-positive monocytes, increase the expression of FcgammaRI on circulating neutrophils after in vivo infusion, and greatly enhance the cytotoxic activity of circulating neutrophils. CD33 is a glycoprotein expressed on the cell surface of mature monocytes, myeloid progenitor cells, and myeloid leukemic blasts, but not on the earliest hematopoietic progenitor cells and other normal tissues. Herein, we report the construction of a BsAb, 251 x 22, by conjugating an anti-CD33 mAb (mAb 251) to an anti-FcgammaRI mAb (mAb 22). The BsAb 251 x 22 is capable of enhancing the cytotoxicity of several leukemia cell lines by cytokine-activated monocytes. Our data also show that G-CSF- and GM-CSF-stimulated monocytes can mediate cytotoxicity of target leukemia cells comparable to that of IFN-gamma-stimulated monocytes. The expression of FcgammaRI on monocytes after 24-h in vitro incubation with G-CSF and GM-CSF was increased, although not significantly. Prolonged incubation of monocytes with G-CSF for 48 h significantly increased the FcgammaRI expression. Because humanized anti-CD33 and anti-FcgammaRI mAb are available, and because GM-CSF and G-CSF have been used widely for patients after chemotherapy to stimulate the recovery of myeloid hematopoiesis, additional clinical development of this project is feasible. A BsAb comprised of humanized anti-CD33 and anti-FcgammaRI could have clinical application in the treatment of myeloid leukemia, especially in the management of minimal residual disease.

Acute Disease↗
PubMed1995

Autologous peripheral blood stem cell transplantation and adoptive immunotherapy with activated natural killer cells in the immediate posttransplant period.

Relapse after high-dose chemotherapy supported by peripheral blood stem cell transplantation (HDC-PBSCT) is the main cause of therapeutic failure in patients with lymphoma and breast cancer. Adoptive immunotherapy with activated natural killer (A-NK) cells and interleukin 2 might eliminate surviving residual tumor without adding to toxicity. Eleven patients with relapsed lymphoma and one with metastatic breast cancer were entered on a pilot clinical trial of HDC-PBSCT followed on day 2 after transplant by infusion of cultured autologous A-NK cells. Simultaneously, recombinant human interleukin 2 (rhIL-2) was initiated as a 4-day continuous i.v. infusion at 2 x 10(6) IU/m2/day, referred to as high-dose rhIL-2. Therapy with high-dose rhIL-2 was followed by a 90-day continuous i. v. infusion at 3 x 10(5) IU/m2/day, referred to as low-dose rhIL-2. All patients engrafted and nine completed treatment. Posttransplant days to a neutrophil count of 500/microliter and to a platelet count of 50,000/microliter were similar to comparable patients treated with HDC-PBSCT alone. Generation of A-NK cells for therapy was feasible in all patients except the three patients with Hodgkin's disease, whose cells did not proliferate in culture. Overall toxicity associated with early posttransplant transfer of A-NK cells and interleukin 2 did not differ from that observed with peripheral blood stem cell transplantation alone in comparable patients. There was early amplification of natural killer cell activity in the peripheral blood of four patients that appeared to result from the transfused A-NK cells. Adoptive transfer of A-NK cells and rhIL-2 during the pancytopenic phase after HDC-PBSCT was feasible and well tolerated, did not adversely affect engraftment, and resulted in amplified natural killer activity in the peripheral blood during the immediate posttransplantation period.

Adoptive Transfer↗
PubMed1995

Phase I clinical trial of serotherapy in patients with acute myeloid leukemia with an immunoglobulin M monoclonal antibody to CD15.

Sixteen patients with acute myeloid leukemia (AML) were treated with a continuous i.v. infusion of mAb PM-81, an IgM mAb directed against the cellular differentiation antigen CD15, which is expressed on leukemia cells of >95% of patients with AML. MAb PM-81, also referred to as MDX-11, is capable of activating human and rabbit complement and lysing CD15-positive AML cells. In this Phase I study, patients were treated with 0.5, 1.0, or 1.5 mg/kg MDX-11 delivered over a 24-h period followed by conventional chemotherapy. Transient decreases in circulating blast cells postinfusion (prior to chemotherapy) were observed at all doses. We were able to show MDX-11 binding to bone marrow blasts in those patients who achieved stable serum levels of MDX-11. Serum MDX-11 was detectable at the 1. 0- and 1.5-mg/kg doses. Doses of 0.5 and 1.0 mg/kg were generally well tolerated, with no toxicities greater than grade II (Eastern Cooperative Oncology Group) reported. However, two of five patients receiving the 1.5-mg/kg dose experienced grade IV toxicities that resolved with treatment (one of these patients completed the infusion). Common toxicities reported included fever, chills, and hypotension. Only one patient developed human antimouse antibodies at 4 weeks posttreatment. This study determined that 1.0 mg/kg is a biologically effective dose that can be administered safely with little toxicity. Based on these results, we are pursuing a Phase I/II study of MDX-11 infusion following chemotherapy for patients with relapsed AML.

Acute Disease↗
PubMed1994

Differential effect of 4-hydroperoxycyclophosphamide and antimyeloid monoclonal antibodies on T and natural killer cells during bone marrow purging.

Autologous bone marrow (BM) transplantation after high dose therapy is widely used to treat acute leukemia, lymphoma, and selected solid tumors. In studies of BM purging with chemical agents, monoclonal antibodies (MoAbs), or other agents, the emphasis has been on the efficacy of tumor cell removal and sparing of hematopoietic progenitor cells. Two commonly used methods of BM purging for patients with acute myeloid leukemia have been the drug 4-hydroperoxycyclophosphamide (4-HC) and (MoAbs) directed to myeloid antigens such as CD14, CD15, and CD33. Although both methods of BM purging have potent activity against leukemia cells, 4-HC is also quite toxic to normal hematopoietic progenitor cells in the same concentrations that are used to deplete leukemia cells. To further characterize the cellular composition of BM after purging, we examined the effects of MoAbs plus complement and 4-HC on cells of the lymphoid lineage in the BM. 4-HC exerted a concentration-dependent cytotoxicity on clonogenic T lymphocytes, natural killer (NK) cells, and lymphokine (interleukin-2)-activated killer (LAK) cells, whereas the anti-CD14 and anti-CD15 MoAbs had little effect. At a concentration of 4-HC commonly used for BM purging (60 micrograms/mL), there were 4 to 5 logs of T-cell depletion and almost complete elimination of NK- and LAK-cell activity. In contrast, 4-HC at low concentrations (eg, 3 micrograms/mL) spared the majority of lymphoid cells suggesting that low concentration 4-HC combined with MoAb purging may be a desirable alternative to higher concentration 4-HC. These data indicate that purging with antimyeloid MoAbs, but not with 4-HC, spares the function of mature graft lymphocytes. Infusion of viable lymphocytes may be important for the transfer of immune memory against microbial and neoplastic antigens and may hasten immune reconstitution. In addition, mature graft lymphocytes may also be selectively activated and expanded in conjunction with interleukin-2 administration after BM transplantation.

Antibodies, Monoclonal↗
PubMed1994

Prognostic importance of mutations in the ras proto-oncogenes in de novo acute myeloid leukemia.

Mutations of the N- and K-ras genes are the most frequent genetic aberrations in acute myeloid leukemia (AML) and their detection in preleukemic conditions such as the myelodysplastic syndrome (MDS) suggests a role in the earliest phases of leukemogenesis. Despite these observations, little is known about the clinical importance of ras mutations in AML. We studied the clinical impact of ras mutations in 99 patients with de novo AML. All patients were treated in two prospective multicenter trials. The polymerase chain reaction was used to amplify areas surrounding the codons 12, 13, and 61 of the three ras genes N-, K-, and H-ras from DNA from bone marrow cells, ras mutations were detected by an algorithm based on allele-specific oligonucleotide hybridization. Eighteen of 99 (18%) patients harbored mutations in either N- or K-ras. All of the observed mutations occurred in N-ras (N = 10) and K-ras (N = 5) or concurrently in both N- and K-ras (N = 3). There were no significant differences between ras-negative and ras-positive patients according to age, sex, blood counts, cytogenetic abnormalities, or French-American-British classification. However, univariate analysis suggested a longer survival in ras-positive patients (P = .11). When adjusted for age, which was the most important factor affecting outcome, the presence of a ras mutation emerged as a significant predictor for improved survival (P = .03) and along with lower bone marrow blast counts (P = .02) and better cytogenetic category (P = .01). However, the presence of an aberrant ras allele was strongly correlated with lower bone marrow blast counts (P = .007). Thus, whether a mutation in the N-ras or K-ras proto-oncogenes directly affects treatment outcome or indirectly through an association with lower leukemic burden remains to be determined. Nevertheless, these findings counter the prevailing bias that oncogene mutations lead to more aggressive behavior in human malignancies.

Adult↗
PubMed1994

A combination of anti-CD15 monoclonal antibody PM-81 and 4-hydroperoxycyclophosphamide augments tumor cytotoxicity while sparing normal progenitor cells.

A cyclophosphamide congener, 4-hydroperoxycyclophosphamide (4HC), has been used to purge bone marrow (BM) of residual leukemia cells ex vivo for use in support of high-dose chemotherapy for patients with acute myeloid leukemia (AML) undergoing autologous BM transplantation (ABMT). The efficacy and toxicity of 4HC are dose-related. The maximally tolerated concentration, 60-100 micrograms/ml, is toxic to tumor cells but also to normal committed hematopoietic progenitor cells. The anti-CD15 monoclonal antibody (mAb) PM-81 has also been employed for purging BM in patients with AML. In some patients, all tumor cells may not be lysed due to antigenic heterogeneity. Because the two agents used individually are associated with potential limitations in terms of toxicity to normal cells and efficacy of tumor cell purging, using these agents together might have advantages. In fact, in this study the use of these two agents together in subtherapeutic concentration ranges as single agents revealed killing of cells from the HL60 and NB4 promyelocytic leukemia cell lines in addition to cells from patients with AML while sparing normal progenitor cells. Surprisingly, not only did the combination enhance killing of tumor cells, but the order of addition of the two agents was important in maximizing toxicity to tumor cells. Adding mAb+complement (C') first or simultaneously to 4HC was less effective than adding 4HC first followed by mAb + C'. This combination regimen was toxic to HL60 and NB4 leukemia cells that may not be killed by the mAb alone due to antigen-negative tumor cells or by low concentrations of 4HC.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1994

Transduction of CD34+ enriched cord blood and Gaucher bone marrow cells by a retroviral vector carrying the glucocerebrosidase gene.

One promising strategy for gene therapy of Gaucher disease involves ex vivo retroviral transduction of autologous hematopoietic stem cells. Studies in small animals have demonstrated that this approach provides a life-long supply of the glucocerebrosidase (GC) enzyme. Human application has developed to the stage of a clinical trial. In this study, we describe development of a high titer amphotropic producer line for the vector, MFG-GC, and explore transduction of CD34+ cells from various human sources. Higher than three times the normal levels of glucocerebrosidase activity in non-transduced cells were achieved following transduction of CD34+ cells obtained from bone marrow or cord blood from normal donors. The improvement in enzyme activity in Gaucher marrow was about 40-fold above deficient levels. We examined the timing and stepwise effect of multiple rounds of infection and evaluated post-infection expansion of cells in two different cytokine mixtures. Transduction efficiency was determined using immunocytochemistry and Southern blot hybridization.

Antigens, CD34↗
PubMed1994

Transduction of mobilized peripheral blood CD34+ cells with the glucocerebrosidase cDNA.

Gaucher disease (GD), the most common human lysosomal storage disorder, results from a genetic deficiency of the enzyme glucocerebrosidase (GC). The cloning of human GC cDNA, the benefits of allogeneic bone marrow transplantation and the success of enzyme replacement therapy support the feasibility of gene therapy as an approach to a cure for GD. We report the transfer of the GC gene to mobilized human peripheral blood (PB) CD34+ cells obtained from patients primed with granulocyte colony-stimulating factor and/or chemotherapy. A tenfold enrichment of CD34+ cells was achieved using an avidin-biotin immunoadsorption technique. Prestimulation of the CD34+ cells with cytokines, followed by infection for 5 days with a supernatant containing the MFG-GC retroviral vector, resulted in enzyme activity up to 2.5-times greater than non-infected and lac-Z infected controls. Southern blot hybridization of DNA from these cells demonstrated a transduction efficiency of 10-30%. These studies show that the GC gene is transferred efficiently to mobilized PB CD34+ cells by the MFG-GC retroviral vector and results in expression of enzyme activity in the population of cells capable of bone marrow reconstitution. These results advance the development of gene therapy for GD.

Antigens, CD34↗