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Transient transfection of 293T cells was utilized to produce high-titer murine recombinant retroviral vectors for clinical studies. This system was initially optimized by gene transfer using different retroviral envelope proteins into activated human CD4+ T lymphocytes in vitro. Higher titer and infectivity were obtained than with stable murine producer lines; titers of 0.3-1 x 10(7) infectious units per milliliter for vectors encoding the green fluorescent protein (GFP) were achieved. Virions pseudotyped with envelope proteins from gibbon ape leukemia virus or amphotropic murine leukemia virus resulted in gene transfer of > or = 50% in CD4+ human T lymphocytes with this marker. Gene transfer of Rev M10 with this vector conferred resistance to HIV infection compared with negative controls in the absence of drug selection. Thus, the efficiency of transduction achieved under these conditions obviated the need to include selection to detect biologic effects in T cells. Finally, a protocol for the production of large-scale supernatants using transient transfection was optimized up to titers of 1.9 x 10(7) IU/ml. These packaging cells can be used to generate high-titer virus in sufficient quantities for clinical studies and will facilitate the rapid, cost-effective generation of improved retroviral, lentiviral, or other viral vectors for human gene therapy.

Delivery of genes into hematopoietic progenitor cells offers an attractive means for the introduction of corrective or protective genes into cells of both the myeloid and lymphoid lineage. Previously, investigators have often used murine retroviral vectors for gene delivery which require cells to be cycling for efficient delivery. We describe a nonviral method of gene delivery using particle-mediated gene transfer to obviate many disadvantages of viral vectors related to safety, production costs and the need for cell cycle proliferation. Using a CMV-CAT reporter plasmid, we show transfection of highly purified CD34+ cells isolated from umbilical cord blood. Effective gene transfer was shown in unstimulated and in growth-stimulated cells. Following transfection with a neomycin resistance gene, differentiation into cells of the myeloid lineage was observed, assayed by CFU-GM in the presence of G-418. Both unstimulated and stimulated cells gave rise to CFU-GM in the presence of G-418, indicating that stable expression of the neomycin resistance gene was maintained in early progenitors. These results demonstrate that particle-mediated gene transfer into human hematopoietic cells from umbilical cord blood can be achieved without affecting their CFU-GM differentiation potential. This gene transfer method offers an alternative approach to gene therapy studies involving human hematopoietic progenitor cells.

The immune system confers protection against a variety of pathogens and contributes to the surveillance and destruction of neoplastic cells. Several cell types participate in the recognition and lysis of tumors, and appropriate immune stimulation provides therapeutic effects in malignancy. Foreign major histocompatibility complex (MHC) proteins also serve as a potent stimulus to the immune system. In this report, a foreign MHC gene was introduced directly into malignant tumors in vivo in an effort to stimulate tumor rejection. In contrast to previous attempts to induce tumor immunity by cell-mediated gene transfer, the recombinant gene was introduced directly into tumors in vivo. Expression of the murine class I H-2Ks gene within the CT26 mouse colon adenocarcinoma (H-2Kd) or the MCA 106 fibrosarcoma (H-2Kb) induced a cytotoxic T-cell response to H-2Ks and, more importantly, to other antigens present on unmodified tumor cells. This immune response attenuated tumor growth and caused complete tumor regression in many cases. Direct gene transfer in vivo can therefore induce cell-mediated immunity against specific gene products, which provides an immunotherapeutic effect for malignancy, and potentially can be applied to the treatment of cancer and infectious diseases in man.

Direct gene transfer into localized arterial segments can be performed in vivo by transfection with DNA-liposome complexes. This technique holds promise for the treatment of human diseases, including malignancy and cardiovascular disorders. We have previously characterized the potential toxicity of this form of treatment in mice in vivo (Stewart et al., 1992). In this report, we examined two issues relevant to long-term expression of foreign recombinant genes: (i) the potential for autoimmune damage to major organs and (ii) DNA localization in gonadal tissue. Autoimmunity and toxicity of allogeneic major histocompatibility (MHC) gene transfer was assessed in mice after induction of an immune response to a recombinant murine class I MHC gene by direct gene transfer in vivo. Histological examination of brain, heart, lung, liver, kidney, spleen, and skeletal muscle revealed no clinically significant immunopathology or organ damage. The toxicity of gene delivery by DNA liposomes was also analyzed in pigs and rabbits in vivo. No histopathology was observed following the introduction of plasmids encoding several different gene products, and analysis of serum following DNA liposome delivery revealed no abnormalities of serum biochemical parameters. The potential for transfer of recombinant DNA into testes and ovary in animals was evaluated by the polymerase chain reaction. Although evidence of recombinant plasmid was consistently observed in transfected, but not untransfected, arterial sites and occasionally in lung, kidney, spleen, and liver, no plasmid DNA was detected in testes or ovary. These studies suggest that uptake of recombinant DNA following direct gene transfer by liposomal transfection in major organs is not associated with autoimmunity, toxicity, or gonadal localization.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA can be introduced into a variety of cell types after formation of liposomal complexes with cationic lipids. In this report, conditions have been established to optimize the production of DNA-liposome complexes that efficiently transfect cells. The safety and toxicity of this method of gene delivery have been assessed after in vivo administration, either by intravenous or direct intratumor injection. Nine to eleven days after intravenous injection, DNA was found primarily in heart and lung tissue by PCR analysis. No abnormalities were evident from histologic examination of tissue, examination of tissue-specific serum enzymes, routine biochemical parameters, or electrocardiographic monitoring. DNA-liposome complexes can therefore be used for the delivery of recombinant genes in vivo with minimal toxicity.