CD4+ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice. Disease development is prevented by cotransfer of purified CD4+ T cells.

Purified CD4+ lymph node T cells were sorted into two populations on the basis of their expression of CD45RB (CD45RBhi and CD45RBlo) and injected into congenic severe combined immunodeficient (SCID) mice. After a period of time that was dependent on the number of cells injected, the SCID mice that received CD45RBhi/CD4+ T cells developed a wasting disease that was not seen in SCID mice that received the CD4+/CD45RBlo cells or whole lymph node cells. At death, SCID mice that received the CD4+/CD45RBhi cells had increased spleen and lymph node cellularity compared with normal SCID mice and SCID mice that received the CD4+/CD45RBlo T cells. The spleen and lymph node contained CD4+ cells and neither CD8+ nor surface immunoglobulin M-positive cells, plus a population of cells that did not express any of those markers. At necropsy, the SCID mice that received the CD4+/CD45RBhi cells had significant hyperplasia of the intestinal mucosa with significant lymphoid cell accumulation in the lamina propria. Interestingly, mice that received mixtures of whole lymph node or purified CD4+ cells with CD4+/CD45RBhi cells did not develop weight loss, indicating that the unseparated CD4+ population contained cells that were capable of regulating the reactivity of the CD4+/CD45RBhi cells.

A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) characterized by plaques of infiltrating CD4(+) and CD8(+) T cells. Studies of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS, focus on the contribution of CD4(+) myelin-specific T cells. The role of CD8(+) myelin-specific T cells in mediating EAE or MS has not been described previously. Here, we demonstrate that myelin-specific CD8(+) T cells induce severe CNS autoimmunity in mice. The pathology and clinical symptoms in CD8(+) T cell-mediated CNS autoimmunity demonstrate similarities to MS not seen in myelin-specific CD4(+) T cell-mediated EAE. These data suggest that myelin-specific CD8(+) T cells could function as effector cells in the pathogenesis of MS.

Interleukin 4 expressed in situ selectively alters thymocyte development.

Using a transgenic mouse model we show that increased intrathymic expression of interleukin 4 (IL-4) significantly perturbs the development of thymocytes. Transgenic double-positive (CD4+CD8+) thymocytes, which are present in dramatically reduced numbers, exhibit increased T cell receptor (TCR) expression and increased mobilization of calcium mediated by these receptors. In contrast, transgenic single-positive (CD4+CD8- and CD4-CD8+) thymocytes and peripheral T cells exhibit decreased TCR-mediated calcium mobilization. The development of CD4-CD8+ thymocytes is significantly perturbed by IL-4 expressed in vivo; only peripheral CD4+ T cells are found in significant numbers in transgenic mice, while CD4-CD8+ thymocytes are present in increased numbers, apparently because of their failure to emigrate to the periphery. In contrast to these selective effects on T cell development, no significant differences in the numbers of B cells or mast cells, or in the plasma levels of IgE and IgG1 are observed between transgenic and control mice. These observations suggest that IL-4 in vivo exerts its major effects locally rather than systemically, even when its expression is constitutively increased.

Humoral immune responses in CD40 ligand-deficient mice.

Individuals with X-linked hyper-IgM syndrome fail to express functional CD40 ligand (CD40L) and, as a consequence, are incapable of mounting protective antibody responses to opportunistic bacterial infections. To address the role of CD40L in humoral immunity, we created, through homologous recombination, mice deficient in CD40L expression. These mice exhibited no gross developmental deficiencies or health abnormalities and contained normal percentages of B and T cell subpopulations. CD40L-deficient mice did display selective deficiencies in humoral immunity; basal serum isotype levels were significantly lower than observed in normal mice, and IgE was undetectable. Furthermore, the CD40L-deficient mice failed to mount secondary antigen-specific responses to immunization with a thymus-dependent antigen, trinitrophenol-conjugated keyhole limpet hemocyanin (TNP-KLH). By contrast, the CD40L-deficient mice produced antigen-specific antibody of all isotypes except IgE in response to the thymus-independent antigen, DNP-Ficoll. These results underscore the requirement of CD40L for T cell-dependent antibody responses. Moreover, Ig class switching to isotypes other than IgE can occur in vivo in the absence of CD40L, supporting the notion that alternative B cell signaling pathways regulate responses to thymus-independent antigens.

Systemic gene expression after intravenous DNA delivery into adult mice.

Direct gene transfer into adult animals resulting in generalized or tissue-specific expression would facilitate rapid analysis of transgene effects and allow precise in vivo manipulation of biologic processes at the molecular level. A single intravenous injection of expression plasmid:cationic liposome complexes into adult mice efficiently transfected virtually all tissues. In addition to vascular endothelial cells, most of the extravascular parenchymal cells present in many tissues including the lung, spleen, lymph nodes, and bone marrow expressed the transgene without any apparent treatment-related toxicity. The transgene was still expressed in large numbers of cells in multiple tissues for at least 9 weeks after a single injection. Expression could be targeted to specific tissues and cell types, depending on the promoter element used.

Development of partial tolerance to the gastrointestinal effects of high doses of recombinant tumor necrosis factor-alpha in rodents.

Treatment of healthy rats and mice with a single intravenous injection of recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) caused a dose-dependent gastrointestinal inflammation. Within 30 min gastric emptying was blocked and tissue edema occurred in the small and large intestine. In the cecum hemorrhage occurred after 4 h at doses greater than or equal to 250 micrograms/kg. The cecum exhibited an acute inflammatory response following rHuTNF-alpha treatment similar to that seen in tumor necrosis at the same dose. The vascular endothelium became swollen, increased numbers of neutrophils and other leukocytes attached to and penetrated the endothelium, and finally hemorrhage occurred. Treatment of rats with daily injections of rHuTNF-alpha (250 micrograms/kg per d) for 3 wk failed to produce cachexia. Within 24-48 h rats became resistant to the hemorrhagic effect of rHuTNF-alpha, however, the cytokine still caused a transitory block of gastric emptying after 10 d of treatment. Treatment at 5- or 10-d intervals produced results similar to the initial injection. These results suggest that maximum hemorrhagic response will occur when rHuTNF-alpha is administered at intervals of 5-10 d rather than daily.

Anti-P-selectin monoclonal antibody attenuates reperfusion injury to the rabbit ear.

Neutrophil adherence and/or aggregation has been implicated in ischemia reperfusion injuries. We examined the role of P-selectin in PMN-mediated injury after reperfusion of the rabbit ear. The ear was partially amputated, and then reattached leaving the central artery and vein intact. To induce ischemia the central artery was then occluded. Treatment was at reperfusion with either saline or one of two murine P-selectin mAbs, designated PB1.3 and PNB1.6 mAb PB1.3 cross-reacts with rabbit P-selectin and prevents histamine-induced leukocyte rolling, whereas PNB1.6 does not. Using a peroxidase-antiperoxidase system P-selectin was detected in the ischemic ear, but not in the nonischemic ear. Ear volume increased to 5.3 times baseline in the saline-treated animals (n = 8), 6.6 times baseline in the nonblocking mAb PNB1.6-treated animals (n = 2), and 3.7 times baseline in the blocking mAb PB1.3-treated animals (n = 8). Estimated tissue necrosis of the combined saline- and PNB1.6-treated animals was 46 vs. 2.7% for the mAb PB1.3-treated animals. We conclude that: (a) P-selectin is expressed in ischemia reperfusion; (b) P-selectin participates in PMN-endothelial cell interactions in ischemia reperfusion; and (c) inhibiting P-selectin adhesion significantly reduces reperfusion injury.

Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes.

In utero injection of cationic liposome-DNA complexes (CLDCs) containing chloramphenicol acetyltransferase, beta-galactosidase (beta-gal), or human granulocyte colony-stimulating factor (hG-CSF) expression plasmids produced high-level gene expression in fetal rats. Tissues adjacent to the injection site exhibited the highest levels of gene expression. Chloramphenicol acetyltransferase expression persisted for at least 14 days and was reexpressed following postnatal reinjection of CLDCs. Intraperitoneal administration of the hG-CSF gene produced high serum hG-CSF levels. X-gal staining demonstrated widespread beta-gal expression in multiple fetal tissues and cell types. No toxic or inflammatory responses were observed, nor was there evidence of fetal-maternal or maternal-fetal gene transfer, suggesting that CLDCs may provide a useful alternative to viral vectors for in utero gene transfer.

Liposome-DNA complexes infused intravenously inhibit tumor angiogenesis and elicit antitumor activity in dogs with soft tissue sarcoma.

Intravenous gene delivery using liposome-DNA complexes (LDC) has previously been shown to elicit antitumor activity, but only in rodent tumor models. Therefore, we conducted a study to determine in a large animal spontaneous tumor model whether intravenous infusions of LDC could target gene expression to cutaneous tumor tissues and whether repeated treatments had an effect on tumor growth or angiogenesis. A total of 13 dogs with cutaneous soft tissue sarcomas were enrolled in the study and were randomized to receive a series of 6 weekly infusions of LDC containing either canine endostatin DNA or DNA encoding an irrelevant gene (luciferase). Serial tumor biopsies were obtained to assess transgene expression, tumor microvessel density (MVD), and intratumoral leukocyte inflammatory responses. We found that intravenous infusion of LDC did not result in detectable gene expression in cutaneous tumor tissues. However, two of 13 treated dogs had objective tumor responses and eight dogs had stable disease during the treatment period. In addition, a significant decrease in tumor MVD was noted in six of 12 treated dogs at the completion of six treatments. These results suggest that intravenous infusions of LDC may elicit nonspecific antitumor activity and inhibit tumor angiogenesis.

Immunomodulatory and toxic effects of free and liposome-encapsulated tumor necrosis factor alpha in rats.

Tumor necrosis factor alpha has potent immunomodulatory and antitumor activity, but its therapeutic applications may be limited by its significant host toxicity. We showed that liposome-encapsulated recombinant human tumor necrosis factor alpha (rHuTNF-alpha) retained full anticellular activity in vitro. We then assessed the immunomodulatory and toxic effects of two different doses of i.v. free or liposome-encapsulated rHuTNF-alpha in normal rats. Both free and liposome-encapsulated rHuTNF-alpha significantly enhanced alveolar macrophage- and blood monocyte-mediated interleukin 1 release and tumor cell lysis, as well as natural killer cell cytotoxicity, when compared to buffer-treated controls. However, administration of rHuTNF-alpha in liposomes substantially reduced tumor necrosis factor alpha-mediated toxicity. Animals receiving liposome-encapsulated rHuTNF-alpha showed significantly less tissue injury, gastric retention, and circulating leukocyte shifts than animals receiving free rHuTNF-alpha. In addition, liposome-based delivery significantly increased lung and liver uptake of rHuTNF-alpha. Therefore, liposome-encapsulated rHuTNF-alpha retains immunomodulatory activity, significantly reduces toxic inflammatory effects, and may allow targeting of tumor necrosis factor alpha to selected organs after i.v. administration.

Characterization of vascular leak syndrome induced by the toxin component of Pseudomonas exotoxin-based immunotoxins and its potential inhibition with nonsteroidal anti-inflammatory drugs.

Clinical trials of immunotoxins in cancer patients have been limited in many cases by vascular leak syndrome (VLS). Recently, rats were identified as a model for VLS induced by BR96 sFv-PE40, a carcinoma-reactive single-chain immunotoxin. In this study, the toxin component of this immunotoxin, PE40, was found to be responsible for inducing hydrothorax in rats, thereby demonstrating that direct binding to the BR96 antigen was not essential to the onset of VLS. Mutational analysis of PE40 determined that both ADP ribosylation and proteolytic processing functions innate to Pseudomonas exotoxin A (PE) were necessary for PE40 to induce hydrothorax in rats; however, neither function by itself was sufficient for VLS induction. Additionally, nonsteroidal anti-inflammatory agents were found to block VLS in rats receiving BR96 sFv-PE40. These results demonstrate that the toxin component of PE-based immunotoxins induce VLS and suggest agents for clinical management of the toxicity.

In situ histochemical detection of beta-galactosidase activity in lung: assessment of X-Gal reagent in distinguishing lacZ gene expression and endogenous beta-galactosidase activity.

Bacterial lacZ is one of the most commonly used reporter genes for assessing gene transfer to lung. However, lung contains endogenous beta-galactosidase (beta-Gal), which can confound estimation of exogenous lacZ expression by histochemical techniques (i.e., X-Gal) for in situ demonstration of enzyme activity. We investigated several parameters of the X-Gal reaction, including time and temperature of X-Gal exposure as well as lung tissue processing and fixation techniques, and found that none of these could be used to distinguish between endogenous and exogenous beta-Gal activities. The mammalian and bacterial beta-Gal enzymes, however, have pH optima in the acidic and neutral ranges, respectively. Exposing whole lung, lung minces, or mounted frozen sections of lung to X-Gal at mildly alkaline pH (pH 8.0-8.5), minimized detection of endogenous activity in lungs from a variety of species while preserving that resulting from bacterial enzyme activity in a transgenic mouse expressing lacZ. This technique was also useful in distinguishing endogenous activity from that resulting from adenovirus-mediated lacZ gene transfer to diploid lung fibroblasts in primary culture. An appropriate buffer that maintains the desired pH throughout the duration of X-Gal exposure must be used.

Perflubron enhances adenovirus-mediated gene expression in lungs of transgenic mice with chronic alveolar filling.

Perfluorochemical (PFC) liquids have both low surface tension and a high capacity to dissolve O2 and CO2, and have been shown to improve gas exchange and lung compliance in animal models of lung injury. We have previously demonstrated that perflubron and other PFC liquids enhance transgene expression in lungs of spontaneously breathing normal rodents after intratracheal instillation of either adenoviral or liposomal vectors followed by a single instillation of PFC liquid. We reasoned that PFC liquids may also be useful for enhancing transgene expression in abnormal lungs. GM-CSF knockout mice develop chronic accumulation of surfactant lipids and proteinaceous material in alveolar spaces and serve as a useful model of chronic alveolar filling. Intratracheal instillation of the adenoviral vector Adlac-Z resulted in patchy in situ distribution of beta-Gal activity, predominantly in larger proximal airways. In contrast, in mice instilled with Adlac-Z followed by instillation of a single dose of perflubron (10 ml/kg body weight), increased expression was observed in distal airway and alveolar epithelial cells. In particular, expression was observed in epithelial cells of debris-filled alveoli. Spectrophotometric measure of quantitative beta-Gal activity in lung homogenates demonstrated increased activity in lungs of mice receiving Adlac-Z plus perflubron compared with lungs of animals receiving Adlac-Z alone. These studies demonstrate that use of perflubron enhances transgene expression in lungs of animals with a chronic alveolar filling process. This approach may be applicable for gene delivery in diseases marked by chronic airway or alveolar filling such as cystic fibrosis.

Intravenous cytokine gene delivery by lipid-DNA complexes controls the growth of established lung metastases.

Local expression of cytokine genes by ex vivo transfection or intratumoral gene delivery can control the growth of cutaneous tumors. However, control of tumor metastases by conventional nonviral gene therapy approaches is more difficult. Intravenous injection of lipid-DNA complexes containing noncoding plasmid DNA can significantly inhibit the growth of early metastatic lung tumors. Therefore, we hypothesized that delivery of a cytokine gene by lipid-plasmid DNA complexes could induce even greater antitumor activity in mice with established lung metastases. The effectiveness of treatment with lipid-DNA complexes containing the IL-2 or IL-12 gene was compared with the effectiveness of treatment with complexes containing noncoding (empty vector) DNA. Treatment effects were evaluated in mice with either early (day 3) or late (day 6) established lung tumors. Lung tumor burdens and local intrapulmonary immune responses were assessed. Treatment with either noncoding plasmid DNA or with the IL-2 or IL-12 gene significantly inhibited the growth of early tumors. However, only treatment with the IL-2 or IL-12 gene induced a significant reduction in lung tumor burden in mice with more advanced metastases. Furthermore, the reduction in tumor burden was substantially greater than that achieved by treatment with recombinant cytokines. Treatment with the IL-2 or IL-12 gene was accompanied by increased numbers of NK cells and CD8+ T cells within lung tissues, increased cytotoxic activity, and increased local production of IFN-gamma by lung tissues, compared with treatment with noncoding DNA. Thus, cytokine gene delivery to the lungs by means of intravenously administered lipid-DNA complexes may be an effective method of controlling lung tumor metastases.

Gene expression along the cerebral-spinal axis after regional gene delivery.

We demonstrate here that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or cationic liposome: DNA complexes (CLDCs) produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Gene expression was identified both within the spinal cord and the brain after intracerebroventricular or intrathecal injection of either CLDCs or plasmid DNA alone. Intracerebroventricular or intrathecal injection of CLDCs containing the beta-galactosidase (beta-Gal) gene produced patchy, widely scattered areas of beta-Gal expression. The chloramphenicol acetyltransferase (CAT) reporter gene product reached peak levels between 24 hr and 1 week postinjection, and was still present at significant levels 3 weeks after a single intracerebroventricular or intrathecal injection. Intrathecal injection of the human granulocyte colony-stimulating factor (G-CSF) gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine nerve growth factor (NGF) gene increased mNGF levels in the hippocampus, a target region for cholinergic neurons in the medial septum, and increased cholinergic neurotransmitter synthetic enzyme choline acetyltransferase (ChAT) activity within the brain, a well-characterized effect of both purified and recombinant NGF protein. These findings indicate that intracerebroventricular or intrathecal injection of CLDCs can produce significant levels of expression of biologically and therapeutically relevant genes within the CNS. Efficient gene transfer into the CNS will facilitate the evaluation of gene function and regulation within the brain and spinal cord. We attempted to transfer and express genes within the brain and spinal cord by direct CNS injection of either DNA alone or CLDCs into the intraventricular and subarachnoid compartments. We show that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or CLDCs produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Intrathecal injection of the hG-CSF gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine NGF gene increased mNGF levels in the hippocampus, and increased cholinergic neurotransmitter synthetic enzyme ChAT activity within the brain. Locoregional diffusion of gene products expressed by transfected meningeal lining cells into brain and spinal cord parenchyma could potentially target secreted proteins within brain and spinal cord regions relevant to neuropathological states while limiting peripheral side effects.

Topical gene delivery to murine skin.

We topically applied naked plasmid DNA containing the luciferase or chloramphenicol acetyltransferase cDNA directly to mouse skin. Gene expression was detected in skin samples as early as 4 h after DNA application, plateaued from 16 to 72 h post-application, and had decreased significantly by 7 d post-application. Reporter gene activity following topical DNA delivery was comparable with that produced by intradermal injection of DNA. Plasmid DNA at concentrations > or =0.25 microg per microl were required to achieve maximal expression levels. Reporter gene expression following topical administration was largely confined to the superficial layers of the epidermis and to hair follicles. Surprisingly, certain cationic liposomes inhibited the efficiency of cutaneous gene transfer. This technique provides a simple, clinically relevant approach to deliver genes to the skin, with potential application in treating a variety of cutaneous disorders.

Efficient gene expression in skin wound sites following local plasmid injection.

Transfection of the skin by local gene delivery, as well as widespread transfection of systemic tissues following intravenous injection of cationic liposome/DNA complexes have been reported. Here, we show that surgically wounded mouse skin can be transfected either by local injection of DNA alone or by intravenous injection of optimized cationic liposome/DNA complexes; however, direct cutaneous injection produces much higher levels of gene expression in the skin, which is targeted to dermal and subdermal layers. High levels of chloramphenicol acetyltransferase activity were present from 3 h to 2 wk following direct injection of a gene expression plasmid into wounded skin and were maintained at detectable levels up to 8 wk after injection. Expression of transferred chloramphenicol acetyltransferase as well as beta-GAL genes was localized to fibroblasts, macrophages, and adipocytes as determined by histochemistry and immunohistochemistry. Further- more, local injection of a human granulocyte- colony-stimulating factor gene expression plasmid produced high levels of the biologically relevant human granulocyte-colony-stimulating factor protein in wounded mouse skin. This efficient and simple method of site-specific gene transfer into wounds may lead to the development of cutaneous gene therapy directed against disorders of abnormal cutaneous wound healing.

An evaluation of the functions of the 22-kilodalton (kDa), the 20-kDa, and the N-terminal polypeptide forms of human growth hormone using transgenic mice.

Multiple peptide hormones can be derived from the single human GH gene. In addition to the full-length 191-amino acid 22-kilodalton (kDa) form, a 20-kDa variant can be produced by alternative splicing, and a 5-kDa variant can be produced by posttranslational cleavage. To more fully appreciate the physiological roles of these proteins, we have made a comparison of transgenic mice that constitutively overexpress one or another of these variants. We have found that both the 22-kDa and the 20-kDa forms of human GH stimulate linear growth and liver hypertrophy. The increase in linear growth in 22-kDa transgenic mice does not, however, correlate with an increase in circulating IGF-I; rather, the increase in IGF-I that does finally occur correlates with marked liver pathology. Both groups of mice also develop glomerulosclerosis and suffer from hyperinsulinemia. Although there are histologically obvious lesions in the livers of both the 22-kDa and the 20-kDa transgenic mice, only the former exhibit hyperalbuminemia and hypercholesterolemia. Both forms of GH lead to anemia, which is normocytic in the 20-kDa transgenic mice and macrocytic in the 22-kDa transgenic mice. Despite the presence of high levels of the 5-kDa N-terminal form of human GH, the transgenic mice that express this protein are indistinguishable from their nontransgenic littermates.

Modulation of radiation-induced cytokine elevation associated with esophagitis and esophageal stricture by manganese superoxide dismutase-plasmid/liposome (SOD2-PL) gene therapy.

Radiation of the esophagus of C3H/HeNsd mice with 35 or 37 Gy of 6 MV X rays induces significantly increased RNA transcription for interleukin 1 (Il1), tumor necrosis factor alpha (Tnf), interferon gamma inducing factor (Ifngr), and interferon gamma (Ifng). These elevations are associated with DNA damage that is detectable by a comet assay of explanted esophageal cells, apoptosis of the esophageal basal lining layer cells in situ, and micro-ulceration leading to dehydration and death. The histopathology and time sequence of events are comparable to the esophagitis in humans that is associated with chemoradiotherapy of non-small cell lung carcinoma (NSCLC). Intraesophageal injection of clinical-grade manganese superoxide dismutase-plasmid/liposome (SOD2-PL) 24 h prior to irradiation produced an increase in SOD2 biochemical activity in explanted esophagus. An equivalent therapeutic plasmid weight of 10 microgram ALP plasmid in the same 500 microliter of liposomes, correlated to around 52-60% of alkaline phosphatase-positive cells in the squamous layer of the esophagus at 24 h. Administration of SOD2-PL prior to irradiation mediated a significant decrease in induction of cytokine mRNA by radiation and decreased apoptosis of squamous lining cells, micro-ulceration, and esophagitis. Groups of mice receiving 35 or 37 Gy esophageal irradiation by a technique protecting the lungs and treating only the central mediastinal area were followed to assess the long-term effects of radiation. SOD2-PL-treated irradiated mice demonstrated a significant decrease in esophageal wall thickness at day 100 compared to irradiated controls. Mice with orthotopic thoracic tumors composed of 32D-v-abl cells that received intraesophageal SOD2-PL treatment showed transgenic mRNA in the esophagus at 24 h, but no detectable human SOD2 transgene mRNA in explanted tumors by nested RT-PCR. These data provide support for translation of this strategy of SOD2-PL gene therapy to studies leading to a clinical trial in fractionated irradiation to decrease the acute and chronic side effects of radiation-induced damage to the esophagus.

In vivo studies of gene expression via transient transgenesis using lipid-DNA delivery.

As the sequencing of the human genome proceeds, the need for a new screen for in vivo function is becoming apparent. Many investigators are turning to various transgenic models as a means of studying function. However, these approaches are very time consuming, with a transgene-expressing mouse model often taking months to establish. We have developed an efficient system for delivering genes in vivo, which allows the gene product to be studied as early as 24 h after introduction into the mouse model. The delivery system employs a novel cationic lipid, 1-[2-(9-(Z)-octadecenoyloxy)ethyl]-2-(8-(Z)-heptadecenyl)-3- (hydroxyethyl)imidazolinium chloride (DOTIM), and a neutral lipid, cholesterol, complexed with an expression vector containing the reporter gene chloramphenicol acetyl transferase (CAT). After a single intravenous injection of these complexes, several tissues were seen to express the transgene. High, persistent expression in the vascular endothelial cells in the mouse lung was obtained. Delivery of DNA in vivo has been evaluated by quantitative polymerase chain reaction and protein expression by CAT activity assays. In vivo studies showed reproducible expression in more than 500 mice injected via the tail vein. An early peak of expression was followed by lower, but sustained, expression for > 50 days. Transgene expression of CAT could also be identified by immunohistochemistry staining in mouse lung and appeared to be located within the capillaries. The pattern of in vivo expression could be modulated and targeted to specific organs by altering the lipid-DNA formulation. New expression vectors with altered introns and polyadenylation sites further improved expression. The expression reported here may be sufficient in magnitude, duration, and flexibility to be an attractive alternative, in some cases, to establishing transgenic animals by stable gene transfer.