Alfred B. Bahnson

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.

Retroviral vectors for use in human gene therapy for cancer, Gaucher disease, and arthritis.

In the past several years, s igdcant advances have been made in the development of gene therapy for the treatment of certain human diseases such as severe combined immunodeficiency’ and cystic f ibr~s is .~ .~ Given these recent successes in gene therapy, researchers are now trying gene transfer as a therapy modality for a wide variety of genetic and acquired diseases. To this end, both viral and nonviral delivery systems have been developed, including retroviruses, adenoviruses, adenoassociated viruses, herpes simplex viruses, picornaviruses, and liposomes, some of which have been approved for use in clinical mals. Of these viral vector systems, retroviral vectors currently represent the best characterized vector and the most utilized delivery system for human gene therapy applications. This communication will discuss the use of retroviral vectors for gene therapy of neoplastic diseases, focusing on the use of a novel heterodimeric cytokine, interleukin (IL,)-12, for immunotherapy of cancer. In addition, the application of retroviral vectors for gene therapy for Gaucher disease, a well-characterized autosomal recessive lysosomal storage disease, and for an acquired autoimmune disease, rheumatoid arthritis, will also be discussed.

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.

9 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 Gauchers disease. High transduction efficiency and correction of the enzyme deficiency was possible in CD34 cells obtained from patients with Gauchers 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.

Addition of serum to electroporated cells enhances survival and transfection efficiency

Optimal electroporation efficiency of many cell types is associated with poor survival. We show that serum rapidly reseals the membranes of electroporated cells and that timely addition of serum following electroporation can improve cell survival and transfection efficiency.

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

Abstract: 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.

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.

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).