Increased gene transfer into human CD34+ progenitor cells using retroviral vectors produced by a canine packaging cell line

Magnetic resonance imaging (MRI) was used in 13 patients with peripheral lymphedema and 2 patients with extensive cavernous lymphangioma of the limb for the purpose of evaluating its role in diagnosis of lymphatic disorders. In chronic lymphedema, MRI showed deformity of lymphatics at different tissue levels. In the subcutis, MRI characteristically displayed diffuse edema or a honeycombed pattern consistent with reticular lymphangiectasis and "lakes" with a marked increase in signal intensity with T2-weighted imaging. In lymphedema hyperplasia and chylous reflux, MRI depicted dilated retroperitoneal lymphatic collectors and lumbar trunks. In cavernous lymphangiomatosis, MRI demonstrated a prominent lattice-like pattern which had lower signal intensity on T1-weighted imaging and higher intensity on T2-weighted imaging. The findings of MRI are valuable not only for accurate assessment of lymphatic dysplasia syndromes but also provide a blueprint for treatment options.     

A packaging cell line for lentivirus vectors

Lentivirus vectors can transduce dividing and nondividing cells. Using three-plasmid transient transfections, high-titer (>10(9) IU/ml) recombinant lentivirus vectors pseudotyped with vesicular stomatitis virus G (VSV-G) protein can be generated (T. Kafri et al., Nat. Genet. 17:314-317, 1997; H. Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319-10323, 1997; L. Naldini et al., Science 272:263-267, 1996). The recombinant lentiviruses can efficiently infect brain, liver, muscle, and retinal tissue in vivo. Furthermore, the transduced tissues demonstrated long-term expression of reporter genes in immunocompetent rodents. We now report the generation of a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 10(6) IU/ml for at least 3 to 4 days. The vector produced by the inducible cell line can be concentrated to titers of 10(9) IU/ml and can efficiently transduce nondividing cells in vitro and in vivo. The availability of a lentivirus packaging cell line will significantly facilitate the production of high-titer lentivirus vectors for gene therapy and study of human immunodeficiency virus biology.     

Gene transfer into human dendritic antigen-presenting cells by vaccinia virus and adenovirus vectors

In a search for means to deliver exogenous gene(s) into human dendritic cells (DCs) from the perspective of tumor-specific vaccination, we have evaluated two recombinant viruses, both of which carry a reporter gene which is namely a modified vaccinia virus Ankara (MVA) and an adenovirus, as possible expression vectors. The recombinant MVA-P11 LZ vector carries the Escherichia coli lacZ gene coding for the enzyme beta-galactosidase, and the recombinant Ad-MFG-AP vector carries a modified membrane-exposed alkaline phosphatase (AP) gene. DCs were generated ex vivo in the presence of tumor necrosis factor-alpha, granulocyte macrophage colony-stimulating factor, stem cell factor, and flk-2/flt-3 ligand taken from CD34+ hematopoietic progenitors that were mobilized into the peripheral blood of cancer patients treated with high-dose cyclophosphamide and filgrastim. The target cells used for gene delivery were either CD34+ cells that had been subsequently induced to differentiate into mature DCs or DCs transduced after ex vivo generation from CD34+ cells. The results showed that: (a) infection of CD34+ cell derived-DCs (mature DCs) with either viral vector resulted in the efficient synthesis of recombinant protein, and (b) CD34+ cells were permissive for the expression of the recombinant reporter gene after infection with Ad-MFG-AP but not after infection with MVA-P11 LZ. In conclusion, these results suggest that vaccinia and adenovirus vectors are candidate to act as vehicles in genetically engineering human DCs.     

Gene transfer into hepatocytes and human liver tissue by baculovirus vectors

Gene therapy of liver diseases requires the development of efficient vectors for gene transfer in vivo. Retroviral and adenoviral vectors have been shown to deliver genes efficiently into hepatocytes in vitro and in vivo. However, these vectors do not allow for exclusive infection of the liver which would be highly advantageous for in vivo gene therapy strategies. We have recently demonstrated that genetically modified baculoviruses (Autographa californica nuclear polyhedrosis virus) efficiently deliver genes into cultured cells and have a strong preference for hepatocytes of different origin. Baculoviral gene transduction efficiency into human hepatocytes was determined to approach 100% and expression levels are high, provided that gene expression is controlled by mammalian promoters. In this report, we present further properties of baculoviruses regarding their use for hepatocyte gene transfer. Baculovirus-mediated gene expression declines rapidly in the hepatocellular carcinoma cell line Huh7 and more slowly in primary cultures of mouse hepatocytes. Direct application of baculoviruses for gene delivery to the liver in vivo is hampered by serum components, presumably by complement. However, we demonstrate here that baculoviral gene transfer is feasible in ex vivo perfused human liver tissue. This result suggests the development of a strategy using baculoviral vectors for liver-directed gene therapy.     

Enhanced retroviral transduction of 5-fluorouracil-resistant human bone marrow (stem) cells using a genetically modified packaging cell line

Pluripotent hematopoietic stem cells (PHSC) are rare cells capable of multilineage differentiation, long-term reconstituting activity and extensive self-renewal. Such cells are the logical targets for many forms of corrective gene therapy, but are poor targets for retroviral mediated gene transfer owing to their quiescence, as retroviral transduction requires that the target cells be cycling. To try and surmount this problem we have constructed a retroviral producer line that expresses the membrane-bound form of human stem cell factor (SCF) on its cell surface. These cells are capable, therefore, of delivering a growth signal concomitant with recombinant retroviral vector particles. In this report we describe the use of this cell line to transduce a highly quiescent population of cells isolated from adult human bone marrow using the 5-fluorouracil (FU) resistance technique of Berardi et al. Quiescent cells selected using this technique were transduced by cocultivation with retroviral producers expressing surface bound SCF or with the parent cell line that does not. Following coculture, the cells were plated in long-term bone marrow culture for a further 5 weeks, before plating the nonadherent cells in semisolid media. Colonies forming in the semisolid media over the next 14 days were analyzed by polymerase chain reaction for the presence of the retroviral vector genome. Over six experiments, the transduction frequency of the quiescent 5-FU resistant cells using the SCF-expressing producer line averaged about 20%, whereas those transduced using the parent producer line showed evidence of reduced levels or no transduction.     

Efficient gene transfer into primary and immortalized human fetal glial cells using adeno-associated virus vectors: establishment of a glial cell line with a functional CD4 receptor

Adeno associated virus (AAV) is a non-pathogenic dependent parvovirus with a broad host range, capable of high levels of transduction and stable integration into the host cell genome. We have investigated the potential for using AAV as a vector for gene transfer into glial cells of the human fetal nervous system. Recombinant AAV vectors expression either the reporter gene beta-galactosidase or a human CD4 receptor were able to transduce both primary glial cells of the human fetal nervous system and an SV40 immortalized human fetal glial cell line (SVG). No difference in transduction efficiency was observed between the primary cells and the cell line which in both cases was as high as 95%. Stable transfectants of the glial cell line expressing the CD4 receptor were selected. An SVG/CD4 expressing line was then established. The presence of the CD4 receptor was confirmed by immunohistochemistry, Westerm immuno-blotting and flow cytometric analysis. The CD4 receptor was shown to be functional by infection of the SVG/CD4 cell line with the human immunodeficiency virus (HIV). Upon infection, the SVG/CD4 cells produced 20-fold higher levels of the HIV intracellular core antigen P24 than the CD4 negative parental cells and in addition formed syncytia. The use of AAV vectors should prove useful in biological investigations of human glial cells and offers promise as a means of ex vivo and in vivo gene delivery.     

An inhibitor of lymphocyte proliferation produced by a human colonic adenocarcinoma cell line in culture

1. The relative ability of the renal and femoral vascular beds to remove infused angiotensin II and noradrenaline was examined in anaesthetized greyhounds. 2. The degree of extraction of infused drug by each vascular bed was expressed as a percentage, calculated by comparing the pressor response to intra-arterial infusion with that obtained when the same dose was administered by the intravenous route. 3. When compared with the same dose given intravenously, the pressor responses after renal artery administration of angiotensin II were reduced by a mean of 77.8 +/- 4.1% (mean +/- SEM, n = 12), whereas those after femoral artery infusions at the same dose were reduced by a mean of only 27.2 +/- 4.9% (n = 12). 4. The pattern of extraction seen with noradrenaline infusions administered in a similar manner was the reverse of that with angiotensin II. There was a 28.9 +/- 6.8% (n = 7) reduction in pressor responses to renal artery infusions; in contrast, femoral artery infusions of the same dose exhibited a 99.0 +/- 1.0% (n = 7) reduction in the pressor responses. 5. Local arterial administration of the angiotensin II competitive antagonist, [Sar1,Ile8]angiotensin II, potentiated the systemic pressor responses to renal artery infusions of angiotensin II, but not those to femoral artery infusions. 6. It is suggested that the marked ability of the renal vascular bed to remove circulating angiotensin II may, in part, involve receptor-binding, although this seems not to be the case in the femoral vascular bed.     

Gene transfer of herpes simplex virus type I thymidine kinase gene as a drug sensitivity gene into human lung cancer cell lines using retroviral vectors

A number of packaging materials are being used not only to contain food during distribution but also to serve as the cooking container. The higher temperatures that these materials reach led the US Food and Drug Administration (FDA) to issue an intent to publish new regulations in 1989. The food and packaging industries responded by conducting extensive research and submitting the results to FDA. The methods used and results obtained are discussed. Most of the data were focused on microwave susceptors and the volatile compounds generated. One project showed that for a specific product, popcorn, there was no transfer into the food. Work is continuing to validate methods to test for non-volatile compounds. In addition to susceptors, various paper and plastic materials are used in dual ovenable (microwave and conventional ovens) applications. Most of the research on these materials has investigated the food contact temperatures on testing for migrants. An update on the current regulatory status of packaging materials intended for high temperature use in the US is discussed.     

Poor transduction efficiency of human hematopoietic progenitor cells by a high-titer amphotropic retrovirus producer cell clone

The transduction efficiency of human bone marrow CD34+ cells with supernatants from the retrovirus producer cell clone PA317/LGSN 16 was only one-fifth of that with supernatants from GP+ envAm12/LGSN 15, even though both producers had similar infection titers on 3T3 cells. PA317/LGSN 16-conditioned medium inhibited the proliferation of the bone marrow CD34+ cells, and this inhibitory effect was partially blocked by anti-transforming growth factor beta antibodies. These studies suggest that cytokine secretion plays a role in the suppression of retrovirus transduction of human CD34+ cells.     

Increased biological activity due to basic isoforms in recombinant human follicle-stimulating hormone produced in a human cell line

FSH has four asparagine-linked oligosaccharides with variable sialic acid contents, so that FSH is not a single molecule, but a heterogeneous group of isoforms. These isoforms differ in their biological properties and their distribution changes in various physiological states, allowing the modulation of FSH activity. Recombinant human (h) FSH has been produced in Chinese hamster ovary cells and has an isoform profile similar to those of both pituitary FSH standard and purified urinary FSH. These FSH preparations, however, do not contain the full spectrum of FSH isoforms found in the circulation. Production of recombinant hFSH in a cell line with a different pattern of glycosylation could broaden its isoform profile and potentially alter its biological activity. Thus, we transfected human embryonal kidney cells (293) with the human alpha and FSH beta genes to produce recombinant hFSH (hFSH-293) and determined its biological activity in a rat granulosa cell bioassay. Although hFSH-293 was immunologically indistinguishable from pituitary FSH standard, its biological potency was 3- to 6-fold higher than those of two different pituitary FSH standards. To investigate this increased potency, we separated the isoforms of hFSH-293 by chromatofocusing and determined their biological potencies in the rat granulosa cell bioassay. The isoform profile of hFSH-293 demonstrated a greater number of basic isoforms than that of pituitary FSH standard. Several of these basic isoforms exhibited enhanced in vitro biological potency, accounting for the increased biological potency of hFSH-293. This pattern of high in vitro biological activity and more basic isoforms is analogous to the FSH circulating during GnRH stimulation, pubertal induction, and ovulation.     

Functional properties of a new line of immortalized human endothelial cells

An immortalized human endothelial cell line was obtained by transfecting umbilical vein endothelial cells in primary culture with plasmid pMK16 containing SV40 replicated origin defective gene. The essential functional properties demonstrated in these immortalized human endothelial cells also retaining the classical phenotypical characteristics of endothelial cells in primary culture are: (1) endothelin-1 secretion; (2) capacity to convert big endothelin-1 into endothelin-1; (3) the capacity to secrete IL1 beta and IL6 interleukins both spontaneously and after lipopolysaccharide (LPS) stimulation; (4) arginine transfer from the extracellular to the intracellular medium. Such stable cell line could facilitate studies of regulation of endothelin-1 production; (5) No-synthase activity; (6) binding and metabolisation of acetylated low-density lipoproteins.     

Infection of human cells by an endogenous retrovirus of pigs

The possible use of pig organs and tissues as xenografts in humans is actively being considered in biomedical research. We therefore examined whether pig endogenous retrovirus (PERV) genomes can be infectiously transmitted to human cells in culture. Two pig kidney cell lines spontaneously produce C-type retrovirus particles. Cell-free retrovirus produced by the PK-15 kidney cell line (PERV-PK) infected pig, mink and human kidney 293 cell lines and co-cultivation of X-irradiated PK-15 cells with human cells resulted in a broader range of human cell infection, including human diploid fibroblasts and B- and T-cell lines. Kidney, heart and spleen tissue obtained from domestic pigs contained multiple copies of integrated PERV genomes and expressed viral RNA. Upon passage in human cells PERV-PK could rescue a Moloney retroviral vector and acquired resistance to lysis by human complement.     

Hybridization of a human myeloma permanent cell line with mouse cells

A population of hybrid cells derived from the fusion of a permanent human myeloma cell line, which secretes complete IgE, and a subline of mouse L cells, did not secrete IgE as evidenced by sensitive immunosorbent tests. Also, the hybrid cells were observed not to contain intracellular IgE (epsilon or lambda chains) in amounts to be detectable by fluorescent antibody techniques. The doubling times and cell cycle parameters of the hybrid cells were found to be similar to those of the slow-growing parental human myeloma cells, in addition, the growth of the hybrid cells was characterized by a higher degree of contact inhibition than the parent mouse cells.     

Gene transfer to the human trabecular meshwork by anterior segment perfusion

PURPOSE: To determine whether adenovirus vectors are capable of transferring a foreign active protein to the perfused anterior segment of the human eye. METHODS: Primary cultures from the human trabecular meshwork tissue were exposed to replication-deficient adenovirus Av1LacZ4 carrying the reporter beta-galactosidase gene driven by the Rous Sarcoma Virus promoter. Anterior segments of six pairs of human eyes from normal donors were placed in organ culture and were perfused with culture medium at 2.5 microl/min constant flow. After 24 hours, one eye was injected once with 8 X 10(8) plaque-forming units (20 microl) of the viral vector, while the paired eye was injected with vehicle. Forty-eight hours (four pairs) and 7 days (two pairs) after injection, tissues were fixed, were assayed histochemically for transferred enzyme activity, and were analyzed morphologically. RESULTS: In monolayers, gene transfer occurs very efficiently in all distinct types of human outflow pathway cells. All human anterior segments injected with the adenovirus vector showed active gene transfer in cells of the outflow pathway: trabecular, juxtacanalicular, and inner wall of Schlemm's canal. Expression of the reporter enzyme was still present at 7 days after treatment. No activity was observed in any of the paired, vehicle-injected controls. Cell morphology and tissue architecture appeared normal in treated and control tissues, although some trabecular cell loss was observed in the corneoscleral and uveal regions of the perfused treated eyes. CONCLUSIONS: Adenoviral vectors were able to transfer active foreign genes into perfused, intact human trabecular meshwork.     

Use of amphotropic retroviral vectors for gene transfer in human colon carcinoma cells

Previous studies in rodent models have demonstrated the feasibility of gene transfer to the stem cells of the intestinal epithelium using ecotropic retroviral vectors delivered luminally. This report represents a next step toward targeting the human intestine as a site for somatic gene therapy. The first experiment assessed the viability of amphotropic retroviral vectors in the luminal environment. It was found that after 4 hr at 37 degrees C in luminal effluent, the loss of titer was no greater than when incubated in control media. Likewise, neither the vector nor the target cells were adversely affected by N-acetylcysteine, which is likely to be used as a preparatory agent for mucus removal. To determine whether human intestinal cells are transducible by these vectors, three colon carcinoma cell lines were studied: HT-29, T84, and Caco-2. All were transduced; however, the expression of the reporter gene was highest in the HT-29 cells. Subsequent studies using these cells showed that with regular stocks of vector, gene transfer peaked at a stock dilution of 1/10 and declined at full strength. This problem could be partially overcome by centrifugal concentration of the retroviral stocks. With this approach, gene transfer increased with increasing particles up to 10x regular stock titers but was inefficient at 100x. Overall, these findings provide encouraging evidence that amphotropic retroviral vectors may eventually be used for in vivo gene transfer into human intestinal epithelium. However, they also point to the need for improved methods of concentrating retroviral vectors.