Biomedical subjects

E Gilboa

Publications and source records attributed to E Gilboa.

At least 55 records · Page 3Linked to original sources

PubMed1994

Immunotherapy of bladder cancer with cytokine gene-modified tumor vaccines.

This study explored the use of cytokine gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology and responds to treatment in a manner similar to that of its human counterpart. In a previous study we have shown that interleukin 2 (IL-2)-secreting, irradiated, MBT-2 cell preparations were capable of curing animals from orthotopically established tumors and engendered protective immunological memory in the cured animals. In this study we have compared the effectiveness of several cytokines and found that while IL-1 alpha, IL-1 beta, and gamma-interferon were only weakly effective in the therapeutic vaccination protocol, granulocyte-macrophage colony-stimulating factor was almost as effective as but not superior to IL-2, as reported previously for another tumor model system. Induction of cytotoxic T-lymphocyte correlated only poorly with the therapeutic benefit of the cytokine gene-modified tumor cell preparations, questioning its prognostic value for the development of improved genetically modified tumor vaccines.

Animals↗

PubMed1994

Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction.

A double-copy Moloney leukemia virus-based retroviral construct containing both the NeoR gene and a mutant human dihydrofolate reductase (DHFR) cDNA (Ser31 mutant) was used to transduce NIH 3T3 and mouse bone marrow (BM) progenitor cells. This resulted in increased resistance of these cells to methotrexate (MTX). The transduced BM progenitor cells were returned to lethally irradiated mice. The recipients transplanted with marrow cells infected with the recombinant virus showed protection from lethal MTX toxicity as compared with mock-infected animals. Evidence for integration of the proviral DNA was obtained by amplification of proviral DNA by polymerase chain reaction (PCR) and Southern analysis. Sequencing a portion of the PCR-amplified human DHFR cDNA showed the presence of the mutation. These studies with the human Ser31 mutant DHFR cDNA gave results comparable with those obtained with the mutant murine DHFR cDNA (Leu to Arg22) in developing MTX-resistant BM. The Ser31 mutant human DHFR cDNA is currently being tested for infection of human CD34+ human BM and peripheral blood stem cells in vitro.

3T3 Cells↗

PubMed1994

Immunotherapy of prostate cancer in the Dunning rat model: use of cytokine gene modified tumor vaccines.

Adenocarcinoma of the prostate is the most common cancer in men. The majority of cancers are discovered once they have already metastasized, and there is no effective therapy for prostatic cancer at this stage. The use of cytokine-secreting tumor cell preparations as therapeutic vaccines for the treatment of advanced prostate cancer was investigated in the Dunning rat R3327-MatLyLu prostatic tumor model. IL-2 secreting, irradiated, tumor cell preparations were capable of curing animals with s.c. established tumors, and induced immunological memory that protected animals from subsequent tumor challenge. Immunotherapy was less effective when tumors were induced orthotopically, but nevertheless led to improved outcome, significantly delaying, and occasionally preventing, recurrence of tumors after resection of the cancerous prostate. Granulocyte-macrophage colony stimulating factor secreting tumor cell preparations were less effective, and interferon-gamma secreting cells had only a marginal effect. Induction of a potent immune response in tumor bearing animals against the nonimmunogenic MatLyLu tumor supports the view that active immunotherapy warrants further investigation as a potential therapeutic approach to prostate cancer.

Adenocarcinoma↗

PubMed1994

The role of IL-2 secreted from genetically modified tumor cells in the establishment of antitumor immunity.

Although the importance of CD4+ T cells for the induction of an effective CD8+ cytolytic T cell response is well documented, the mechanism by which MHC class II-negative tumor cells recruit CD4+ T help is not well understood. We have previously shown that IL-2 or IFN-gamma gene-modified CMS-5 tumor cells do not grow in syngeneic mice; however, mice which rejected the cytokine-secreting tumor cells develop a protective immune response against a challenge with parental tumor cells. Here we show that rejection of IL-2-secreting CMS-5 cells is not mediated by T cells. However, establishment of a protective immune response against CMS-5 tumor cells requires the presence of both CD4+ and CD8+ T cell subsets during the period of immunization with IL-2-secreting CMS-5 cells as well as during the effector phase. Extensive histologic analysis has failed to detect the presence of T cells at the site of immunization with either IL-2- or IFN-gamma-secreting CMS-5 cells. The main infiltrate at the site of inoculation with IL-2-secreting CMS-5 cells consisted of NK cells that appeared to play a role in their rejection. The predominant infiltrate at the site of inoculation with IFN-gamma-secreting CMS-5 cells consisted of macrophages. These observations argue against a direct role for the intact tumor cell in presenting either T helper or CTL epitopes to the immune system, and support the view that specialized APC are responsible for the in vivo priming of a T cell response against MHC class I-restricted Ag.

Animals↗

PubMed1994

An experimental model simulating local recurrence and pelvic lymph node metastasis following orthotopic induction of prostate cancer.

In order to develop an animal model that more closely simulates the organ environment and metastatic routes of human prostatic cancer, R3327-MatLyLu tumors were induced by orthotopic implantation in the ventral prostatic lobe of Copenhagen rats. This procedure reproducibly resulted in metastatic spread of the intraprostatic tumor to the pelvic and retroperitoneal lymph nodes, and invariably to the lungs. Further, a tumor recurrence model was established using an approach that combined orthotopic tumor implantation and subsequent surgical resection of the primary tumor. When prostatectomy was carried out 4 days or more after induction, tumors recurred locally in all animals. The surgical procedures described may provide an animal model to test the in vivo response to experimental adjuvant treatment protocols for advanced prostate cancer. Immunological studies are now in progress using cytokine gene-modified prostatic tumor cells as cellular antitumor vaccines in orthotopically established R3327-MatLyLu tumors.

Animals↗

PubMed1994

Gene therapy for infectious diseases: the AIDS model.

Genetic manipulation of somatic cells may be of therapeutic value in a variety of infectious diseases, particularly in human immunodeficiency virus (HIV) infection. Stable insertion of custom-designed 'resistance genes' into cells susceptible to HIV could reduce the viral burden in infected individuals and potentially retard the characteristic progressive immune dysfunction. Alternatively, ectopic expression of genes that encode viral antigens might induce potent antiviral immune responses and form the basis for novel prophylactic and therapeutic vaccines. While laboratory studies have proved that the approach works in principle, preclinical and clinical studies will be necessary to evaluate the therapeutic benefit of such gene-based therapies.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Inhibition of human immunodeficiency virus type 1 in human T cells by a potent Rev response element decoy consisting of the 13-nucleotide minimal Rev-binding domain.

Intracellular immunization is an anti-viral gene therapy strategy based on the introduction of DNA templates into cells to stably express genetic elements which inhibit viral gene expression and replication. We have recently developed an intracellular immunization strategy for human immunodeficiency virus (HIV) infection that uses RNA decoys. RNA decoys are short RNA oligonucleotides corresponding to the HIV trans activation response element (TAR) or Rev response element (RRE) sequences, which function by inhibiting the binding of the HIV regulatory proteins Tat and Rev to the authentic HIV RNA TAR and RRE regions, respectively. In this report we describe the characterization of potent RRE decoys containing the minimal 13-nucleotide primary Rev binding domain of the RRE. Using an improved tRNA cassette to express high levels of RRE transcripts in CEM cells, we found that this new generation of minimal RRE decoys were more potent inhibitors of HIV in isolated cell lines than previously described TAR or RRE decoys. CEM cells expressing RRE decoys exhibited diminished Rev function in cotransfection assays, confirming the specificity of inhibition of HIV by RRE decoys and indicating that the 13-nucleotide minimal Rev binding domain defined by using in vitro binding studies also binds Rev in vivo. Significant differences in the degree of HIV inhibition between individual CEM cell lines transduced with RRE decoy vectors which were not due to sequence alterations in the tRNA-RRE DNA template, differences in RRE decoy expression level, or endogenous variations in the resistance of CEM clonal cell lines to HIV were observed. In order to evaluate the efficacy of RRE decoys in a more realistic fashion than by comparison of individual clonal cell lines, polyclonal populations of transduced CEM cells were infected with HIV. By using a novel flow cytometric method for quantitating intracellular p24 expression, one version of the RRE decoys tested in this study was found to be capable of durably protecting polyclonal populations of CEM cells from HIV.

Bacterial Proteins↗

PubMed1994

Long-term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double-copy vector retrovirus containing a mutant dihydrofolate reductase.

A double-copy Moloney murine leukemia virus-based retroviral construct containing both the NEOr gene and a mutated dihydrofolate reductase cDNA (Leu 22-->Arg) was used to infect mouse bone marrow cells. The infected mouse marrow was returned to lethally irradiated mice. Primary, secondary, and even tertiary recipients transplanted with bone marrow cells infected with the recombinant virus showed protection from lethal methotrexate toxicity. The viral construct containing a SV-40 promoter in the U3 region of the 3' long terminal repeat appeared to be more effective than a similar construct containing the adenosine deaminase promoter, although both afforded protection. Evidence for integration into blood cells of both the NEOr gene and the mutated dihydrofolate reductase gene was obtained by polymerase chain reaction; sequencing of the amplified dihydrofolate reductase cDNA showed the presence of the point mutation. These results indicate that early hematopoietic progenitor cells in the mouse can be successfully transduced with a drug resistance gene.

Animals↗

PubMed1994

Immunotherapy of cancer using cytokine gene-modified tumor vaccines.

Distant metastasis is the major cause for therapeutic failures in clinical oncology. Active immunotherapy of patients with low tumor burden would not only contribute to further reduction of the remaining tumor burden to subclinical levels, but it would also establish a constant state of immunity, i.e. immunological memory, that could protect the patient from recurrence of disease. Studies employing rodent tumor models with little or no intrinsic immunogenicity have shown that genitically modified tumor cell preparations consisting of irradiated tumor cells transduced with and expressing cytokines such as IL-2, IL-6, IFN-gamma or GM-CSF were capable of inducing the regression of a preexisting tumor burden and cure animals from their disease. Moreover, in some instances the cured animals have retained immunological memory, as indicated by the fact that such animals have resisted a second challenge with the parental tumor cells. Induction of potent immune responses in tumor bearing animals against non-immunogenic tumors supports the view that active immunization of cancer patients deserves consideration despite lack of demonstrable immunogenicity in many human tumors.

Animals↗

PubMed1993

Regression of bladder tumors in mice treated with interleukin 2 gene-modified tumor cells.

This study explored the use of interleukin 2 (IL-2) and interferon gamma (IFN-gamma) gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor used is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology, and responds to treatment in a manner similar to its human counterpart. Using retroviral vectors, the human IL-2 and mouse IFN-gamma genes were introduced and expressed in MBT-2 cells. The tumor-forming capacity of the cytokine gene-modified MBT-2 cells was significantly impaired, since no tumors formed in mice injected intradermally with either IL-2- or IFN-gamma-secreting cells, using cell doses far exceeding the minimal tumorigenic dose of parental MBT-2 cells. Furthermore, mice that rejected the IL-2- or IFN-gamma-secreting tumor cells became highly resistant to a subsequent challenge with parental MBT-2 cells, but not to 38C13 cells, a B cell lymphoma of the same genetic background. To approximate the conditions as closely as possible to the conditions prevailing in the cancer patient, inactivated cytokine-secreting cells were used to treat animals bearing tumors established by orthotopic implantation of MBT-2 cells into the bladder wall of the animal. Treatment of mice carrying a significant tumor burden with IL-2-secreting MBT-2 cells had a significant inhibitory effect on tumor progression with extended survival. Moreover, in 60% of the mice the tumor regressed completely and the animals remained alive and free of detectable tumor for the duration of the observation period. Treatment of tumor-bearing animals with IL-2-secreting MBT-2 cells was superior to the use of cisplatin, a chemotherapeutic agent used in the treatment of bladder cancer. The therapeutic effect of IFN-gamma-secreting cells was minimal and treatment with unmodified MBT-2 cells had no effect on tumor growth or survival, showing that the parental MBT-2 cells were nonimmunogenic in this experimental setting. Most importantly, mice that exhibited complete tumor regression after treatment with IL-2-secreting MBT-2 cells became resistant to a subsequent challenge with a highly tumorigenic dose of parental MBT-2 cells, indicating that long-term immunological memory was established in the "cured" mice.

Animals↗

PubMed1993

Antimetastatic vaccination of tumor-bearing mice with two types of IFN-gamma gene-inserted tumor cells.

IFN-gamma genes were introduced through retroviral vectors into the high metastatic, low H-2Kb class I expressor, and poorly immunogenic 3LL-D122 clone. Two types of IFN-gamma infectants were isolated: IFN-gamma high secretors (128 to 256 IU/ml) and IFN-gamma non- or very low secretors (< 2 IU/ml). Both manifested high expression of MHC class I Ag. IFN-gamma secretors showed significant decrease in tumorigenicity and metastatic growth, whereas IFN-gamma nonsecretors retain tumorigenicity and were more metastatic than parental D122 cells. However, both groups, when inoculated in an irradiated form, induced similar high levels of CTL and protected mice to the same degree against a subsequent graft of parental cells. This indicates that enhanced expression of MHC class I and related genes caused by IFN-gamma is the major participant in CTL induction. Immunization of mice carrying an established tumor of parental D122 cells with IFN-gamma infectants is capable of almost completely preventing lung metastasis. Immunotherapy of tumor-bearing hosts is more effective when IFN-gamma secreting cells are used as immunogens, indicating that mechanisms, in addition to CTL, are stimulated by secreted IFN-gamma. Moreover, immunization with IFN-gamma high secretors of postoperative mice, carrying established micrometastases, almost completely cured these mice. Support for the participation of non-T cell effectors in the response to IFN-gamma secretors derives from the reduced tumorigenicity of these cells in CD1 nude mice. These data provide a rationale for the use of IFN-gamma gene-transferred tumor cells as a modality for cancer therapy.

Animals↗

PubMed1993

Anti-metastatic vaccination of tumor-bearing mice with IL-2-gene-inserted tumor cells.

IL-2 gene was introduced through retroviral vectors into the highly malignant and poorly immunogenic 3LL-D122 clone. Both high and low D122-IL-2 secretors showed elimination of tumorigenicity in syngeneic immune-competent mice; however, in nude mice only the high IL-2 secretor showed reduced tumorigenicity as compared with parental D122 cells. Also, following intravenous inoculation, only the high IL-2 secretor showed reduced generation of metastases, whereas the low IL-2 secretors were as highly metastatic as parental cells. These results seem to indicate that low levels of IL-2 secreted by tumor cells are sufficient to activate T cells, while higher levels are needed in order to activate non-T-cell effectors. D122-IL-2 secretors induced high levels of anti-tumor CTL, while their sensitivity to the lytic activity of these CTL was similar to the sensitivity of D122 cells, thus indicating that the elevation of immunogenicity of IL-2 secretors was essentially due to the secreted IL-2. In accordance with CTL induction, pre-immunization with IL-2 secretors protected mice against subsequent challenge of parental cells. Moreover, immunization in an "immunotherapy protocol" i.e., vaccination of mice carrying an established tumor of parental D122 cells with inactivated D122-IL2 infectants, almost completely eliminated the generation of lung metastases by D122 cells, thus providing a rationale for the use of IL-2 gene transferred tumor cells as a modality for treatment of metastasis.

Animals↗

PubMed1993

Transfer of the ADA gene into bone marrow cells and peripheral blood lymphocytes for the treatment of patients affected by ADA-deficient SCID.

Severe combined immunodeficiency (SCID) caused by deficiency of the enzyme adenosine deaminase (ADA) is the first genetic disorder considered for human somatic cell gene therapy. ADA-SCID patients can be cured by HLA-matched sibling donor bone marrow transplantation. Alternative transplantation strategies as well as enzyme replacement are being tested in those patients who do not have a suitable matched sibling donor. Some ADA-SCID patients may not be candidates for cytoablation due to infectious damage to the lung or liver, or may have a milder phenotype that does not justify the risks associated with haploidentical bone marrow transplantation. Replacement therapy with PEG-ADA has resulted in improvement in growth, a variable increase in the number of peripheral blood lymphocytes, and a decrease in the incidence of severe infections. Another approach to the treatment of severe genetic diseases is now represented by somatic cell gene therapy. We and others have conducted experiments in vitro and in vivo that have documented that T-lymphocytes are suitable vehicles for gene transfer. Although the pluripotent stem cell remains the ideal target cell for somatic cell gene therapy of disorders of the hematopoietic system, the use of T-lymphocytes as gene therapy vehicles is specifically indicated for ADA-deficient patients where they represent the affected cells. Furthermore, the selective engraftment of T-cells only, following bone marrow transplantation, has resulted in reconstitution of cellular and humoral immunity. A model for the functional analysis in vivo of the human immune system has been utilized for the preclinical evaluation of this approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Deaminase↗

PubMed1993

Retroviral gene transfer of epidermal growth factor receptor into HL60 cells results in a partial block of retinoic acid-induced granulocytic differentiation.

HL60 cells are devoid of endogenous epidermal growth factor receptor (EGFR). They respond to retinoic acid and undergo terminal granulocytic differentiation. EGFR complementary DNA was introduced into HL60 cells by retroviral gene transfer. Scatchard plot showed that the binding characteristics are identical to those of A431 cells. HL60-EGFR cells were estimated to express 34,000 EGFR/cell (Kd = 5 nM). The tyrosine phosphorylation upon ligand binding is the first step of signal transduction. The dominant phosphotyrosyl proteins in epidermal growth factor-stimulated HL60-EGFR cells include a 170 kDa protein (EGFR itself), and 125 and 53 kDa proteins. The EGFR signal results in the induction of 92 kDa gelatinase/matrix metalloproteinase in HL60-EGFR cells, thereby providing evidence of the function of the exogenous EGFR and a semiquantitative measure of the EGFR signal. These HL60-EGFR cells offer a unique opportunity to examine the potentially important role of EGFR (c-erbB) in maintaining homeostasis between self-renewal and differentiation. c-erbB has been shown to play a physiological role in the self-renewal of the very early avian stem cells which do express EGFR. The v-erbB (double truncated EGFR) has been shown to cause avian erythroblastosis. We found that these HL60-EGFR cells responded to retinoic acid differently from the HL60-control cells. A partial block of only 45% granulocytic differentiation and concomitant proliferation was noted, consistent with a shift of balance between self-renewal and differentiation toward the former.

Cell Differentiation↗

PubMed1993

Development of a lacZ marked WEHI-3B D+ murine leukemic cell line as an in-vivo model of acute non-lymphocytic leukemia.

Established leukemic cell lines have been useful models for studying the biology of leukemia. Analysis of the actions of differentiating agents on leukemic cell lines in vivo has been limited by an inability to unambiguously distinguish host hematopoietic elements from differentiated leukemic cells. In order to identify and quantify leukemic cells during in vivo studies, a derivative of the murine myelomonocytic leukemia cell line WEHI-3B D+, which stably expresses beta-galactosidase, was constructed utilizing retroviral vector gene transfer. This cell line, termed WEHI-3B D+/lacZ 2.8, demonstrated in vitro growth and differentiation properties similar to the parental cell line. WEHI-3B D+/lacZ 2.8 expressed high levels of beta-galactosidase following prolonged in vitro growth and following differentiation in suspension cultures and clonogenic assays. In vivo, WEHI-3B D+/lacZ 2.8 was leukemogenic and high level expression of beta-galactosidase was maintained. Quantification of tissue involvement with WEHI-3B D+/lacZ 2.8 leukemia was performed utilizing staining with the fluorogenic beta-galactosidase substrate fluorescein di-beta-galactoside and fluorescence-activated cell sorting analysis. In vivo differentiation efficiency following granulocyte colony-stimulating factor (G-CSF) administration was determined using a simultaneous nuclear and cytoplasmic staining procedure. Results indicate that treatment of mice inoculated with WEHI-3B D+/lacZ 2.8 cells with G-CSF administration causes detectable but limited differentiation.

Animals↗

PubMed1992

Retroviral gene transfer induced constitutive expression of interleukin-2 or interferon-gamma in irradiated human melanoma cells.

Cytokines are important modulators of host antitumor responses. Two of these cytokines, interleukin-2 (IL-2) and interferon gamma (IFN-gamma), are produced after antigen-induced activation of helper lymphocytes. The cytokines are released into the immediate vicinity where they either interact with the appropriate receptors on effector cell populations or are rapidly degraded. To mimic this physiologic release of cytokines at the effector-target site, we used retroviral vectors to transduce melanoma cells with the IL-2 or IFN-gamma cDNA. Five melanoma cell lines were transduced with IL-2- or IFN-gamma-containing vectors and secreted IL-2 at 1 to 40 U/mL/10(6) cells/24 h or IFN-gamma 1 to 8 U/mL/10(6) cells/24 h, respectively. After gamma irradiation, these cells continued to secrete cytokines for about 3 to 4 weeks. Secretion of IFN-gamma induced upregulation of major histocompatibility complex class I molecules in a subset of melanoma cell lines. IL-2 production by human melanoma xenografts induced tumor rejection in BALB/c nu/nu mice, showing the in vivo effect of this cytokine. This study shows that (1) human melanoma cells can be stably transduced with cytokine-containing retroviral vectors; (2) cytokines are secreted constitutively by the transduced tumor cells and have the expected biologic effects in vitro and in vivo; and (3) after gamma irradiation, cytokines continue to be secreted for several weeks. These data suggest that irradiated cytokine-secreting allogenic or autologous tumor cells can be used in vaccination protocols for cancer patients.

3T3 Cells↗

PubMed1992

Retroviral vector-mediated lymphokine gene transfer into human renal cancer cells.

Effective vaccination against cancer, either for prophylaxis or therapy, has been an elusive goal for years. Cytokine gene therapy offers a novel approach to generate immunogenic tumor cell vaccines. To examine the feasibility of cytokine gene transfer into human renal cancer (RC) cells, we introduced the cDNAs for human interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) into various RC cell lines with retroviral vectors. Using the NIH3T3 amplification assay, no replication competent retroviral particles were detectable in cell culture supernatants taken from gene-modified RC cell lines. Efficient expression of both lymphokines was achieved. Depending on the cell line and the vector construct used, lymphokine gene-modified human RC cell lines released 4 to 29 units/10(6) cells of IL-2, or up to 10 units/10(6) cells of IFN-gamma within 48 h. Fluorescence-activated cell sorter analysis of SK-RC-29 cells releasing IFN-gamma showed increased expression of major histocompatibility complex class I antigen, beta 2-microglobulin, and ICAM-1, as well as induction of major histocompatibility complex class II antigen expression [human leukocyte antigen(HLA)-DR, -DP], but no changes in these cell surface markers were observed with SK-RC-29 cells releasing IL-2. Following in vitro gamma-irradiation with 5,000 or 10,000 rad, growth of lymphokine gene-modified RC cells was abrogated, but their capability to release lymphokine and express lymphokine-induced antigenic determinants, such as HLA-DR, was retained. Tumor formation by the human RC cell line SK-RC-29 in BALB/c nude mice was not affected by IFN-gamma secretion, but was inhibited by in vivo release of IL-2 from s.c. injected tumor cells. These studies demonstrate the feasibility of retroviral mediated lymphokine-gene transfer into human RC cells and suggest a means for generating autologous or HLA-matched allogeneic tumor cell vaccines for the treatment of patients with renal cell carcinoma.

Animals↗

PubMed1992

Retroviral-mediated transfer of the human acid sphingomyelinase cDNA: correction of the metabolic defect in cultured Niemann-Pick disease cells.

Types A and B Niemann-Pick disease (NPD) result from inherited deficiencies of the lysosomal hydrolase, acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.1.4.12). To evaluate the feasibility of somatic gene therapy for the treatment of these disorders, retroviral-mediated gene transfer was used to introduce the full-length ASM cDNA into cultured fibroblasts from two unrelated type A NPD patients. The ASM activities in these cells were less than 4% of mean normal levels, and, consequently, they accumulated approximately 3-fold elevated levels of sphingomyelin. After retroviral-mediated transfer of the ASM cDNA, ASM activities in the NPD cells increased to levels up to 16-fold those found in normal fibroblasts. In addition, the sphingomyelin content was reduced to normal levels, indicating that the vector-encoded enzyme was properly targeted to lysosomes, where it was enzymatically active and able to degrade the accumulated substrate. In situ cell-loading studies also were undertaken to evaluate the effects of retroviral-mediated gene transfer on the pathology of NPD fibroblasts. When a pyrene derivative of sphingomyelin was introduced into the lysosomes of cultured fibroblasts from a type A NPD patient by using apolipoprotein E-mediated endocytosis, only approximately 6% of the delivered substrate was degraded. In contrast, normal cells and NPD cells transduced (i.e., "corrected") by retroviral-mediated gene transfer could degrade approximately 80% of the delivered sphingomyelin. These results provided further evidence that retroviral-mediated gene transfer may be used to correct the pathology of NPD cells. Cell-loading studies were also used to develop a selection system for discriminating between NPD cells and those transduced by retroviral-mediated gene transfer. This selection scheme was based on the fluorescence emission of intact NPD cells, which, when loaded with pyrene-labeled sphingomyelin, was 3- to 5-fold that of normal or transduced cells. As a consequence, the NPD and transduced cells could be efficiently sorted by flow cytometry with a fluorescence-activated cell sorter. In addition, the NPD cells could be selectively killed by photosensitization after irradiation with a long-wavelength UV light. These results should permit direct selection of ASM-expressing cells after retroviral-mediated gene transfer without the need to preselect for a cotransferred marker gene.

Cell Survival↗