Physical fitness and psychological health in overweight/obese children: A cross-sectional study from the ActiveBrains project.

OBJECTIVES: To examine the associations of physical fitness (i.e. cardiorespiratory fitness, muscular strength, and speed/agility) with psychological distress and psychological well-being in overweight/obese pre-adolescent children. DESIGN: 110 overweight/obese children (10.0±1.1years old, 61 boys) from the ActiveBrains project (http://profith.ugr.es/activebrains) participated in this cross-sectional study. METHODS: Physical fitness was evaluated by the ALPHA battery test. Cardiorespiratory fitness was additionally evaluated by a maximal incremental treadmill. Stress was assessed by the Children's Daily Stress Inventory, anxiety by the State-Trait Anxiety Inventory, depression by the Children Depression Inventory, positive affect and negative affect by the Positive and Negative Affect Scale for Children, happiness by the Subjective Happiness Scale, optimism by the Life Orientation Test, and self-esteem by the Rosenberg Self-Esteem questionnaire. Linear regression adjusted for sex and peak height velocity was used to examine associations. RESULTS: Absolute upper-body muscular strength was negatively associated with stress and negative affect (ß=-0.246, p=0.047; ß=-0.329, p=0.010, respectively). Furthermore, absolute lower-body muscular strength was negatively associated with negative affect (ß=-0.301, p=0.029). Cardiorespiratory fitness, expressed by the last completed lap, and relative upper-body muscular strength were positively associated with optimism (ß=0.220, p=0.042; ß=0.240, p=0.017, respectively). Finally, absolute upper-body muscular strength was positively associated with self-esteem (ß=0.362, p=0.003) independently of sex and weight status (p for interactions >0.3), and absolute lower-body muscular strength was also positively associated with self-esteem (ß=0.352, p=0.008). CONCLUSIONS: Muscular strength was associated with psychological distress (i.e. stress and negative affect) and psychological well-being (i.e. optimism and self-esteem) as well as cardiorespiratory fitness was associated with optimism. Therefore, increased levels of physical fitness, specifically muscular strength, could have significant benefits for overweight/obese children psychological health.

Selective siRNA-mediated suppression of 5-HT1A autoreceptors evokes strong anti-depressant-like effects.

Depression is a major health problem worldwide. Most prescribed anti-depressants, the selective serotonin reuptake inhibitors (SSRI) show limited efficacy and delayed onset of action, partly due to the activation of somatodendritic 5-HT(1A)-autoreceptors by the excess extracellular serotonin (5-HT) produced by SSRI in the raphe nuclei. Likewise, 5-HT(1A) receptor (5-HT(1A)R) gene polymorphisms leading to high 5-HT(1A)-autoreceptor expression increase depression susceptibility and decrease treatment response. In this study, we report on a new treatment strategy based on the administration of small-interfering RNA (siRNA) to acutely suppress 5-HT(1A)-autoreceptor-mediated negative feedback mechanisms. We developed a conjugated siRNA (C-1A-siRNA) by covalently binding siRNA targeting 5-HT(1A) receptor mRNA with the SSRI sertraline in order to concentrate it in serotonin axons, rich in serotonin transporter (SERT) sites. The intracerebroventricular (i.c.v.) infusion of C-1A-siRNA to mice resulted in its selective accumulation in serotonin neurons. This evoked marked anti-depressant-like effects in the forced swim and tail suspension tests, but did not affect anxiety-like behaviors in the elevated plus-maze. In parallel, C-1A-siRNA administration markedly decreased 5-HT(1A)-autoreceptor expression and suppressed 8-OH-DPAT-induced hypothermia (a pre-synaptic 5-HT(1A)R effect in mice) without affecting post-synaptic 5-HT(1A)R expression in hippocampus and prefrontal cortex. Moreover, i.c.v. C-1A-siRNA infusion augmented the increase in extracellular serotonin evoked by fluoxetine in prefrontal cortex to the level seen in 5-HT(1A)R knockout mice. Interestingly, intranasal C-1A-siRNA administration produced the same effects, thus opening the way to the therapeutic use of C-1A-siRNA. Hence, C-1A-siRNA represents a new approach to treat mood disorders as monotherapy or in combination with SSRI.

PFKFB3 gene silencing decreases glycolysis, induces cell-cycle delay and inhibits anchorage-independent growth in HeLa cells.

The high rate of glycolysis despite the presence of oxygen in tumor cells (Warburg effect) suggests an important role for this process in cell division. The glycolytic rate is dependent on the cellular concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), which, in turn, is controlled by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). The ubiquitous PFK-2 isoenzyme (uPFK-2, alternatively named UBI2K5 or ACG) coded by the pfkfb3 gene is induced by different stimuli (serum, progesterone, insulin, hypoxia, etc.) and has the highest kinase/phosphatase activity ratio amongst all PFK-2 isoenzymes discovered to date, which is consistent with its role as a powerful activator of glycolysis. uPFK-2 is expressed in brain, placenta, transformed cells and proliferating cells. In the present work, we analyze the impact of small interfering RNA (siRNA)-induced silencing of uPFK-2 on the inhibition of cell proliferation. HeLa cells treated with uPFK-2 siRNA showed a decrease in uPFK-2 RNA levels measured at 24h. uPFK-2 protein levels were severely depleted at 48-72h when compared with cells treated with an unrelated siRNA, correlating with decreased glycolytic activity, Fru-2,6-P2, lactate and ATP concentrations. These metabolic changes led to reduced viability, cell-cycle delay and an increase in the population of apoptotic cells. Moreover, uPFK-2 suppression inhibited anchorage-independent growth. The results obtained highlight the importance of uPFK-2 on the regulation of glycolysis, on cell viability and proliferation and also on anchorage-independent growth. These data underscore the potential for uPFK-2 as an effective tumor therapeutic target.

Copolymers of poly-L-lysine with serine and tryptophan form stable DNA vectors: implications for receptor-mediated gene transfer.

Inefficient gene transfer and poor stability in physiological medium are important shortcomings for receptor-mediated gene transfer vectors. Here, we evaluate vectors formulated with random copolymers of L-lysine/L-serine (3:1) and L-lysine/L-tryptophan (4:1), focusing on both their biophysical and functional characterization. By means of dynamic light scattering (DLS) and transmission electron microscopy (TEM), we demonstrate that poly-L-lysine (pK), poly-L-lysine-L-tryptophan (pKW) and poly-L-lysine-L-serine (pKS) are able to form compacted, small particles when mixed with plasmid DNA in the absence of salt. Upon dilution in physiological medium, copolymers of both lys/ser and lys/trp do not aggregate, in contrast with poly-L-lysine DNA complexes as determined by scattering, DLS and TEM measurements. Tight packing, as demonstrated by resistance to heparin, SDS and trypsin treatments, is also featured in tryptophan-containing complexes. Successful receptor-mediated endocytosis gene transfer using galactosylated copolymers into cells expressing the asiagloglycoprotein receptor correlated with lack of aggregation. Particles obtained using galactosylated poly-L-lysine-L-tryptophan (Gal-pKW) copolymer demonstrated specific receptor-mediated gene transfer since reporter gene activity dropped in the presence of an excess ligand in the culture medium during transfection. Although copolymers of galactosylated poly-L-lysine-L-serine (Gal-pKS) do not aggregate in the presence of salt, they are not able to internalize in a specific receptor-mediated endocytosis fashion. The introduction of bulky aromatic/hydrophobic (tryptophan) or hydrophillic (serine) moieties into the positively charged vectors allows the compacted particles to disperse into salt-containing medium avoiding salt-induced aggregation. Moreover, tryptophan-containing particles are able to mediate specific gene transfer via receptor-mediated endocytosis.

Receptor-mediated gene transfer vectors: progress towards genetic pharmaceuticals.

Although specific delivery to tissues and unique cell types in vivo has been demonstrated for many non-viral vectors, current methods are still inadequate for human applications, mainly because of limitations on their efficiencies. All the steps required for an efficient receptor-mediated gene transfer process may in principle be exploited to enhance targeted gene delivery. These steps are: DNA/vector binding, internalization, subcellular trafficking, vesicular escape, nuclear import, and unpacking either for transcription or other functions (i.e., antisense, RNA interference, etc.). The large variety of vector designs that are currently available, usually aimed at improving the efficiency of these steps, has complicated the evaluation of data obtained from specific derivatives of such vectors. The importance of the structure of the final vector and the consequences of design decisions at specific steps on the overall efficiency of the vector will be discussed in detail. We emphasize in this review that stability in serum and thus, proper bioavailability of vectors to their specific receptors may be the single greatest limiting factor on the overall gene transfer efficiency in vivo. We discuss current approaches to overcome the intrinsic instability of synthetic vectors in the blood. In this regard, a summary of the structural features of the vectors obtained from current protocols will be presented and their functional characteristics evaluated. Dissecting information on molecular conjugates obtained by such methodologies, when carefully evaluated, should provide important guidelines for the creation of effective, targeted and safe DNA therapeutics.

Single-stranded DNA condensed with poly-L-lysine results in nanometric particles that are significantly smaller, more stable in physiological ionic strength fluids and afford higher efficiency of gene delivery than their double-stranded counterparts.

Nonviral gene transfer vectors have been actively studied in the past years in order to obtain structural entities with minimum size and defined shape. The final size of a gene transfer vector, which is compacted into unimolecular complexes, is directly proportional to the mass of the nucleic acid to be compacted. Thus, the purpose of this study was to assess the possibility of producing ssDNA vectors and their biophysical and biological characterization. We have obtained ssDNA/poly-L-lysine complexes that are significantly smaller than their double-stranded counterparts. We have also identified a lesser aggregative behavior of compacted single-stranded vs. double-stranded DNA vectors in the presence of physiological NaCl concentrations. Expression of compacted ssDNA is observed in hepatoma cell lines. Moreover, we have successfully delivered galactosylated ssDNA complexes into cells that express the asialoglycoprotein receptor via receptor-mediated endocytosis. The reduced size and biophysical behavior of ssDNA vectors may provide an advantage for transfection of eukaryotic cells.

Biological properties of poly-L-lysine-DNA complexes generated by cooperative binding of the polycation.

We have evaluated the effect of NaCl concentration on the mode of binding of poly-L-lysine to DNA and the resulting structural and functional features of the condensed DNA particles using DNA precipitation, DNase I resistance, electron microscopy, and receptor-mediated gene transfer assays. At a high concentration of NaCl and in the presence of excess DNA, poly-L-lysine interacted with DNA cooperatively, fully condensing some of the DNA and leaving the rest of the DNA unbound. At low NaCl concentrations, poly-L-lysine molecules interacted with DNA in a noncooperative fashion, i.e. they bind randomly to the whole population of DNA molecules. Cooperative binding of poly-L-lysine to DNA occurred over a narrow range of NaCl concentrations, and the specific salt concentration depended on the length of the poly-L-lysine. The ability of condensed DNA to withstand digestion by DNase I was correlated with the structural features of the condensed DNA as determined by electron microscopy. Using our condensation procedure, cooperative binding of poly-L-lysine to DNA is a necessary prerequisite for the preparation of condensed DNA having a spherical shape and a diameter of 15-30 nm. Condensed DNA, containing galactosylated poly-L-lysine, was evaluated further for the extent and specificity of receptor-mediated gene transfer into HuH-7 human hepatoma cells via the asialoglycoprotein receptor. Efficient receptor-mediated transfection occurred only when condensed DNA complexes had a spherical shape with a diameter of 15-30 nm; asialofetuin, a natural ligand for the asialoglycoprotein receptor, inhibited this process by up to 90%. Our results support the importance of appropriate DNA condensation for the uptake and ultimate expression of DNA in hepatic cells.

The influence of maternal erythrocyte deformability on fetal growth, gestational age and birthweight.

The increase in blood viscosity during pregnancy reduces maternal-fetal blood flow, which can lead to fetal hypoxia and acidosis. These factors have been related to a reduction in fetal growth and to premature births. We carried out a longitudinal study of 36 normal-term gestations at different stages of the pregnancy. We analyzed the erythocyte deformability, the intraerythocyte viscosity and the plasma viscosity in the mother, as well as the relation of these parameters to fetal growth (biparietral diameter (BPD) and length of the femur), birthweight, gestational age at birth and the Agpar score. The results obtained were as follows: from weeks 25 to 36 of pregnancy (30.9 (SD 2 weeks)) there occurs a significant increase in maternal erythocyte rigidity (p < 0.05) (despite the compensatory decrease in intracellular viscosity). This increase is very significantly related to the fetal biparietral diameter (r = -0.50, p < 0.01), the length of the fetal femur (r = -0.48, p < 0.02), gestational age at birth (r = -0.73, p < 0.0001, birthweight (r = -0.63, p < 0.001) and the Agpar score 5 minutes after birth (r = 0.67, p < 0.001). Our conclusions are that the reduction in erythocyte deformability (which we attribute to alterations in the fluidity or elasticity of its membrane) and the factors that increase the aggregation capacity of the red cells (modulators of blood viscosity and of blood flow in the placental intervillous space) are risk factors for reduced fetal growth, lower birthweight and lower gestational age at birth. By avoiding maternal hematocrit levels higher than 36% we could improve uteroplacental perfusion, fetal growth and perinatal results.

Transfer of the human Alpha1-antitrypsin gene into pulmonary macrophages in vivo.

Several viral and nonviral methods have introduced functional genes into the lungs. An alternative strategy, receptor-mediated gene transfer, exploits the ability of receptors on the surface of cells to bind and internalize DNA complexes and could potentially be used to deliver genes to specific cells in the lung. The gene encoding human alpha1-antitrypsin (A1AT) was delivered to macrophages in vitro and in vivo by targeting the mannose receptor with mannose-terminal molecular conjugates. The human A1AT transcript was detected 2 d after transfection of macrophages in culture, but transgene expression was transient. Human A1AT protein was secreted into the culture medium, and Western blot hybridization revealed the mature human antiprotease. In addition, Sprague-Dawley rats underwent intravenous injections of increasing doses of plasmid DNA (0.2 mg, 1.0 mg, and 2.0 mg) complexed to the molecular conjugate. Four days after transfection, human A1AT mRNA was found in lungs from six of the 13 rats (46%) that received the higher doses of plasmid. Transgene expression was limited to cells in perivascular and alveolar regions, which conformed to the distribution of pulmonary macrophages. Human A1AT was measured in the epithelial lining fluid of rats treated with transfection complexes. Animals that received 1.0 mg of plasmid had human A1AT levels of 7.4 +/- 3.4 pM, which was significantly different from nontransfected and mock-transfected controls. Thus the mannose receptor permitted direct delivery of genes to pulmonary macrophages, though transgene expression was detected in the lung only at low levels.

Gene transfer into hepatoma cell lines via the serpin enzyme complex receptor.

The serpin enzyme complex receptor (SECR) expressed on hepatocytes binds to a conserved sequence in alpha 1-antitrypain (alpha 1-AT) and other serpins. A molecular conjugate consisting of a synthetic peptide (C1315) based on the SECR binding motif of human alpha 1-AT covalently coupled to poly-L-lysine was used to introduce reporter genes into hepatoma cell lines in culture. This conjugate condensed DNA into spheroidal particles 18-25 nm in diameter. When transfected with the SECR-directed complex containing pGL3, Hep G2 cells that express the receptor, but not Hep G2 cells that do not, expressed a peak luciferase activity of 538,731 +/- 144,346 integrated light units/mg protein 4 days after transfection. Free peptide inhibited uptake and expression in a dose-dependent manner. Complexes of DNA condensed with polylysine or LC-sulfo-N-succinimidyl-3-(2-pyridyldithio)propionate-substituted polylysine were ineffective. Transfection with a plasmid encoding human factor IX produced expression in Hep G2 (high) and HuH7 cells that express SECR but not Hep G2 (low) cells that lack the receptor. Fluorescein-labeled C1315 peptide labeled 9-31% of Hep G2 (high), 10-14% of HuH7, and 0.6-3.4% of Hep G2 (low) cells, and when the lac Z gene was transfected, only these cells expressed beta-galactosidase. SECR-mediated gene transfer gives efficient, specific uptake and high-level expression of three reporter genes, and the system merits further study for gene therapy.

Safety-modified episomal vectors for human gene therapy.

The effectiveness of ongoing gene therapy trials may be limited by the expression characteristics of viral and plasmid-based vectors. To enhance levels of heterologous gene expression, we have developed a safety-modified episomal expression vector that replicates extrachromosomally in human cells. This vector system employs a simian virus 40 (SV40) large T antigen mutant (107/402-T) that is deficient in binding to human tumor suppressor gene products, including p53, retinoblastoma, and p107, yet retains replication competence. These SV40-based episomes replicate to thousands of copies by 2-4 days after gene transfer in multiple types of human cell lines, with lower activity in hamster cells, and no detectable activity in dog, rat, and murine cell lines. Importantly, 107/402-T has enhanced replication activity compared with wild-type T antigen; this finding may be due, in part, to the inability of p53 and retinoblastoma to inactivate 107/402-T function. We demonstrate that the level and duration of 107/402-T expression regulates the observed episomal copy number per cell. Compared with standard plasmid constructs, episomes encoding 107/402-T yield approximately 10- to 100-fold enhanced levels of gene expression in unselected populations of transient transfectants. To determine if 107/402-T-based episomes replicate extrachromosomally in vivo, tumor explants in nude mice were directly injected with liposome/DNA complexes. Using a PCR-based assay, we demonstrate that SV40-based episomes replicate in human cells after direct in vivo gene transfer. These data suggest that safety-modified SV40-based episomes will be effective for cancer gene therapy because high level expression of therapeutic genes in transient transfectants should yield enhanced tumor elimination.

Biochemical and functional characterization of DNA complexes capable of targeting genes to hepatocytes via the asialoglycoprotein receptor.

Electrostatic binding of polycations or basic polypeptides to the DNA phosphate backbone has been previously described as a one-step process which results in uncontrolled aggregation and precipitation of the DNA in solution. We describe here a multistep process in which the condensation of DNA in the presence of poly-L-lysine can be controlled to produce particles of discrete size and shape suitable for receptor-mediated gene transfer in vivo and in vitro. The first step in this process involves the gradual accretion of poly-L-lysine onto the DNA phosphate backbone, until charges are neutralized. The addition of poly-L-lysine to a concentrated solution of DNA in this fashion prevents intermolecular aggregation of the DNA, presumably by promoting the formation of a nucleus of condensation along the length of each DNA molecule. The second stage of the process involves adjusting the ionic strength of the solvent to facilitate the solubilization of compact DNA.poly-L-lysine complexes. Several physical and biochemical parameters have been studied and correlated with the efficacy of DNA/ligand-poly-L-lysine particles in transferring genes to the liver of adult animals by receptor-mediated endocytosis.

Axon-mediated gene transfer of retinal ganglion cells in vivo.

Modification of the intracellular functions of mature neurons through specific gene transfer has many potential applications. Here we present a new methodology for the successful transfection of retinal ganglion cells by administration of plasmid at the cut end of the optic nerve, or at their intact axon terminals; the latter is significantly more efficient. Plasmids contained either the SV40 promoter linked to the luciferase gene, or the CMV or RSV promoter linked to the lacZ gene. Assays for both reporter genes demonstrated significant expression of exogenous DNA in the retina for at least 10 days after retrograde transport. Duration of expression was extended to 20 days or more (duration of the experiment) when plasmid DNA was condensed with poly(L-lysine). beta-Galactosidase analysis revealed transfection of ganglion cells in high numbers. Such an approach for gene delivery to specific subpopulations of neurons might be useful in studies of molecular functions in vivo and as an experimental therapeutic strategy to extend survival and restore their function.

Immunologic responses to gene transfer into mice via the polymeric immunoglobulin receptor.

The respiratory epithelium is the primary target tissue for gene therapy of cystic fibrosis, and several methods of gene transfer permit the introduction of the gene encoding the normal cystic fibrosis transmembrane conductance regulator into cells of the respiratory tract in animals. DNA complexes based on Fab antibodies to secretory component have been used to mediate the delivery and uptake of expression plasmids into the respiratory tract via the polymeric immunoglobulin receptor both in vitro and in vivo. We evaluated the efficacy of gene transfer after several administrations of the DNA complexes, and examined the immunogenicity and toxicity of repetitive administration of anti-secretory component Fab-based complexes. Mice received single or multiple injections of the DNA complexes containing the plasmid pGL2 every 21 days after the initial treatment, and lysates from the lung and liver were assayed for luciferase expression. Luciferase activity was detected in the lungs of mice that received a single injection of the DNA complexes, whereas transgene expression was significantly lower in the mice that received three injections of the DNA complexes (17338 +/- 5469 integrated light units/mg and 3771 +/- 1778 integrated light units/mg, respectively). Serum samples from animals that underwent single or multiple injections were analyzed for a serologic response against the conjugate-DNA complexes by ELISA. No anticomplex antibodies were detected in the mice after a single injection. An escalating antibody response was noted with increasing number of treatments with the conjugate-DNA complexes. This serologic response was directed exclusively against the rabbit-derived, anti-secretory component (anti-SC) Fab antibody, and not against either the plasmid DNA or poly-L-lysine. Single injection of the conjugate-DNA complexes did not result in the consumption of circulating complement. Using direct immunofluorescence, perivascular deposits of immunoglobulin G were found in the liver of animals that received three treatments; no such deposition was detected in the lungs or kidneys. No increase in inflammatory cell infiltrates was observed in tissues after single and repeated injections of the DNA complexes. Thus, we conclude that repeated injections of the anti-SC Fab-based complexes evoked a humoral immune response against the heterologous Fab portion of the complex that was associated with reduced efficiency of gene transfer.

Receptor-mediated gene transfer into macrophages.

Gene transfer systems targeting various receptors have been developed to introduce functional genes into cells in culture and into intact animals. A synthetic molecular conjugate, consisting of mannosylated polylysine that exploits endocytosis via the macrophage mannose receptor, was constructed and complexed to expression plasmids containing either the Photinus pyralis luciferase or Escherichia coli beta-galactosidase (lacZ) reporter genes. The DNA complexes were used to transfect murine macrophages isolated from peritoneal exudates in vitro. Luciferase and beta-galactosidase activity was found in transfected cells in culture, whereas complexes consisting of an irrelevant plasmid bound to mannosylated polylysine or the expression plasmid bound to galactosylated polylysine resulted in no detectable transgene expression. Gene transfer was inhibited by the addition of excess mannosylated bovine serum albumin to the culture medium before transfection. Reporter genes were also transferred into macrophages residing in the spleen and liver of adult animals using this system. Luciferase activity was maximal at 4 days after transfection and decreased to lower levels by 16 days. Transgene expression conformed to the distribution of cells that had nonspecific esterase, a cytochemical marker for macrophages. Thus, this system can be used to introduce functional genes into macrophages and may be an approach to the treatment of storage diseases that affect the reticuloendothelial system.

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.

An evaluation of receptor-mediated gene transfer using synthetic DNA-ligand complexes.

Receptor-mediated gene transfer is an attractive method for therapeutically correcting human genetic diseases since it permits the targeting of DNA to cellular receptors in specific tissues of adult animals. Genes introduced by this technique have been shown to be expressed in the target tissue for varying periods. However, to be useful for gene therapy, it is critical that both the chemical properties and physical interactions of the reagents involved in the design of the DNA delivery vehicle be rigorously characterized. In this review, we discuss the critical steps in the preparation of the DNA-ligand complex and the factors involved in the delivery and regulated expression of a transgene in animal tissues. The feasibility of using this technique for the therapeutic delivery of genes to mammalian tissues will also be evaluated.

Expression of the neomycin-resistance (neo) gene induces alterations in gene expression and metabolism.

The amino 3'-glycosyl phosphotransferase (neo) gene is the selectable marker most widely used in stable transfection or infection protocols. Because the neo gene product has phosphotransferase activity, it might modify the phosphorylation state when introduced in mammalian cells. NIH-3T3 fibroblast cells expressing the neo gene, after either infection with retroviral vectors or transfection with plasmids, showed a 50% reduction in both fructose 2,6-bisphosphate (Fru 2,6-P2) concentration and lactate production compared with control NIH-3T3 cells, indicating that these neo-expressing cells are less glycolytic. In addition, a marked decrease in the levels of mRNA for the procollagen 1 alpha and fibronectin genes was also observed in neo-expressing NIH-3T3 cells. This decrease was concomitant with an increase in the mRNA concentration of the endogenous c-myc gene. FTO-2B rat hepatoma cells also showed modifications in gene expression when the neo gene was introduced by stable transfection or infection. In these cells an increase in both P-enolpyruvate carboxykinase (PEPCK) and tyrosine aminotransferase (TAT) mRNA was observed. These results suggest that neo gene expression may induce changes in the cells, which should be considered when neo-selected cells are used to deliver specific genes in different therapy approaches and in embryo manipulation.

Gene transfer in vivo: sustained expression and regulation of genes introduced into the liver by receptor-targeted uptake.

Receptor-mediated gene transfer has been used to introduce genes into tissues of animals in vivo. The genes introduced by this approach have been transiently expressed at low levels in animal tissues. High levels of expression, for longer periods, have been attained by the induction of cell division (i.e., partial hepatectomy) or disruption of lysosomal degradation of the DNA. We have studied the correlation of specific structural features on the DNA/ligand complexes with their ability to efficiently introduce DNA into the livers of intact animals. A chimeric gene containing the phosphoenolpyruvate carboxykinase gene promoter (nucleotides -460 to +73) linked to the structural gene for human factor IX (PEPCK-hFIX gene) was condensed with galactosylated poly(L-lysine) by titration with NaCl, resulting in complexes of defined size (10-12 nm in diameter) and shape. The PEPCK-hFIX gene complex was injected into the caudal vena cava of adult rats and the conjugated DNA was specifically targeted to the livers of the animals; no detectable DNA was noted in other tissues. The plasmid containing the PEPCK-hFIX gene was found as an episome in the livers of the rats 32 days after injection of the DNA complex. Human factor IX DNA, mRNA, and functional protein were detected up to 140 days after administration of the DNA complex (the duration of the experiment). Transcription from the PEPCK promoter could be induced over the entire course of the experiment by feeding the rats a high-protein, carbohydrate-free diet. We conclude that the structure of the DNA/ligand complexes is of key importance for the successful introduction of genes into the tissues of animals by receptor-mediated endocytosis.