Robert G. Hawley

A GFP reporter system to assess gene transfer and expression in human hematopoietic progenitor cells

Hematopoietic stem cells are widely recognized as attractive targets for gene therapy but current protocols to transduce these cells using recombinant retroviral vectors are inefficient. To evaluate optimization of retroviral transduction of hematopoietic stem cells and stability of gene expression in their progeny, the green fluorescent protein (GFP) was explored as a reporter. We first improved sensitivity of detection >100-fold over that achieved previously by using a novel retroviral vector (termed MGIN) expressing a high level of an enhanced GFP gene. Primitive human hematopoietic cells bearing the CD34 surface antigen and lacking lineage differentiation markers (CD34+Lin−) were transduced with the MGIN vector using a clinically applicable supernatant procedure. Under the conditions employed, >75% of the target cells retained the CD34+Lin− primitive phenotype after 4–5 days in culture; of those ⩾25% expressed a high level of GFP detectable by both flow cytometric analysis and fluorescence microscopy. When transduced cells were cultured in clonogenic progenitor assays, GFP fluorescence was readily detected in situ, indicating that GFP expression was stable and not detrimental to the differentiative potential of the transduced CD34+Lin− cells. We conclude that GFP is effective as a vital marker to quantify retrovirus-mediated gene transfer into human hematopoietic and perhaps other types of stem/progenitor cells, and monitor gene expression during their subsequent cell lineage determinations.

Double-copy bicistronic retroviral vector platform for gene therapy and tissue engineering: application to melanoma vaccine development

The efficient genetic modification of solid tumors in situ to stimulate therapeutic immune responses against them is currently under active investigation, but is not yet possible using existing gene transfer technologies. Thus, ex vivo/in vivo vaccination strategies have been proposed in which the patient’s tumor is surgically excised, single cell suspensions are prepared, the therapeutic genes are introduced and then the gene-modified cells, after being γ-irradiated, are injected back into the patient. However, even with high- efficiency gene delivery systems, this is a labor-intensive process. Moreover, it is often difficult to obtain sufficient numbers of gene-modified primary tumor cells during short-term culturing. On the other hand, extended in vitro passaging of primary tumor explants may alter their immunophenotypic properties. One approach to overcome these limitations would be to design universal vaccines consisting of standardized gene-transduced neoplastic cell lines or mixtures of gene-transduced cell lines, to be combined with autologous tumor samples if available. Melanoma, which is notable for being one of the most immunogenic human malignancies, represents a cancer where shared tumor- associated antigens have been identified. We developed and analyzed several different retroviral vectors for their ability to stably express exogenous genes at high levels in a panel of melanoma cell lines. All vectors contained a reporter gene (nlslacZ) encoding β-galactosidase with a nuclear localization signal and the neomycin phosphotransferase (neo) gene as selectable marker. One vector, DCCMV, which carried a bicistronic nlslacZ-neo transcriptional unit under the control of the human cytomegalovirus immediate–early promoter in the U3 region of its 3′ LTR, was found to perform consistently better than the other vectors. The DCCMV vector, which is an extreme example of the double-copy class of retroviral vectors, was subsequently used to generate melanoma cell lines constitutively secreting human interleukin-6 or a soluble form of the human interleukin-6 receptor for potential use in a phase II clinical vaccine trial for the treatment of melanoma patients. The DCCMV vector design may also be useful in gene therapy applications where the intent is to implant polymer-encapsulated cell lines genetically engineered to stably express high levels of bioactive proteins.

Development of a Double-Copy Bicistronic Retroviral Vector for Human Gene Therapy

The efficient genetic modification of solid tumors in situ to stimulate therapeutic immune responses against them is currently under active investigation but is not yet possible using existing gene transfer technologies. Thus, ex vivo/in vivo vaccination strategies have been proposed in which the patient’s tumor is surgically excised, single cell suspensions are prepared, the therapeutic genes are introduced and then the gene-modified cells, after being γ-irradiated, are injected back into the patient. However, even with high efficiency gene delivery systems, this is a labor-intensive process. Moreover, it is often difficult to obtain sufficient numbers of gene-modified primary tumor cells during short-term culturing. On the other hand, extended in vitro passaging of primary tumor expiants may alter their immunophenotypic properties. One approach to overcome these limitations would be to design universal vaccines consisting of standardized gene-transduced neoplastic cell lines or mixtures of gene-transduced cell lines, to be combined with autologous tumor samples if available.

Immunoglobulin synthesis in non-B cell lines

A recombinant vector carrying rearranged immunoglobulin heavy (mu) and light (kappa) chain genes was introduced into several cell lines of non-B lineage including T cell (EL4) and two fibroblast lines (CV-1 and L cells). Our results indicated that intracytoplasmic mu-chains, both secretory and membrane types, were expressed in the recipient cell lines. Small amounts of kappa-chain transcripts, corresponding in size to authentic kappa-chain mRNA, were detected in EL4 and L cell transformants, whereas CV-1 transformants did not express the authentic kappa-chain RNA. EL4 and L cell transformants produced kappa-chain. This system will be quite useful for the analysis of regulatory mechanisms involved in immunoglobulin synthesis in non-B cells.