Biomedical subjects

V Budker

Publications and source records attributed to V Budker.

12 recordsLinked to original sources

PubMed2001

Efficient expression of naked dna delivered intraarterially to limb muscles of nonhuman primates.

We have previously shown that the intraarterial delivery of naked plasmid DNA (pDNA) into the femoral artery of rats leads to high levels of foreign gene expression throughout the muscles of the hindlimb. The present study shows that the procedure can also enable high levels of foreign gene expression throughout the limb skeletal muscles in rhesus monkeys. The average luciferase expression in the target muscle was 991.5 +/- 187 ng/g for the arm and 1692 +/- 768 ng/g for the leg; compared with 780 ng/g in rat hindlimb. Large numbers of beta-galactosidase-positive myofibers were found in both leg and arm muscles, ranging from less than 1% to more than 30% in various muscles, with an average of 6.9%. The nonhuman primates tolerated the procedure without significant adverse effects in skeletal muscles, arteries, or other organs. Other studies in immunosuppressed rats indicated that stable expression is possible. These results suggest that the procedure is likely to enable efficient and stable gene expression in human muscle without substantial toxicity.

Animals↗

PubMed1999

High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.

We have previously shown that the intramuscular injection of naked plasmid DNA enables foreign gene expression in muscle. Further studies showed that the intravascular delivery of naked plasmid DNA enables high levels of expression not only in muscle but also in hepatocytes. For the liver, this technique required injection directly into the liver vessels (portal vein, hepatic vein, or bile duct) and occlusion of outflow. The present study now demonstrates that high levels of plasmid DNA expression in hepatocytes can be easily obtained by tail vein injections. The highest levels of expression are achieved by rapidly injecting the plasmid DNA in large volumes, approximately 2.5 ml. This technique has great potential for a wide variety of laboratory studies.

Animals↗

PubMed1998

DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA.

The nuclear entry of exogenous DNA in mammalian cells is critical for efficient gene transfer. A novel technique was developed for the covalent attachment of cationic peptides to double-stranded DNA using a cyclo-propapyrroloindole cross-linker. The attachment of the SV40 large T antigen nuclear localization signal peptide induced the nuclear accumulation of the conjugated DNA in digitonin-permeabilized cells via the classical pathway for the nuclear transport of karyophilic proteins. Increased nuclear uptake of the modified DNA, however, did not occur after it was microinjected into the cytoplasm of cultured cells. This demonstration that the covalent modification of DNA with a signal peptide alters its behavior and interaction with other cellular factors portends the potential of DNA vector chemistry to enhance the efficiency of cellular gene transfer.

Antigens, Polyomavirus Transforming↗

PubMed1998

The efficient expression of intravascularly delivered DNA in rat muscle.

Previous studies have demonstrated that muscle can take up and express naked DNA or RNA. This study demonstrates that the pDNA can be delivered to and expressed within skeletal muscle when injected rapidly, in a large volume and when all blood vessels leading into and out of the hindlimb are occluded. The additional use of collagenase, papaverine and ischemia raised expression moderately but was not critical. These results demonstrate that a nonviral method can lead to high levels of expression in the muscles of adult animals large than mice.

Animals↗

PubMed1997

Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers.

A variety of reporter genes within plasmid constructs were injected into the afferent and efferent vessels of the liver in mice, rats, and dogs. Efficient plasmid expression was obtained following delivery via the portal vein, the hepatic vein, and the bile duct. The use of hyperosmotic injection solutions and occlusion of the blood outflow from the liver substantially increased the expression levels. Combining these surgical approaches with improved plasmid vectors enabled uncommonly high levels of foreign gene expression in which over 15 microg of luciferase protein/liver was produced in mice and over 50 microg in rats. Equally high levels of beta-galactosidase (beta-Gal) expression were obtained, in that over 5% of the hepatocytes had intense blue staining. Expression of luciferase or beta-Gal was evenly distributed in hepatocytes throughout the entire liver when either of the three routes were injected. Peri-acinar hepatocytes were preferentially transfected when the portal vein was injected in rats. These levels of foreign gene expression are among the highest levels obtained with nonviral vectors. Repetitive plasmid administration through the bile duct led to successive events of foreign gene expression. The integration of these findings into laboratory and clinical protocols is discussed.

Animals↗

PubMed1997

Protein/amphipathic polyamine complexes enable highly efficient transfection with minimal toxicity.

Ternary complexes of plasmid DNA, histone H1 protein and amphipathic polyamines (PAPA) were able to mediate the efficient transfection of 3T3. HeLa and COS cells in culture. Using both the beta-galactosidase and luciferase reporter gene systems, the transfection efficiency of PAPA complexes was comparable to that of DOSPA/PE cationic liposomes, considered to be a highly-efficient transfection reagent. Using three different assays of cellular toxicity (propidium iodide, BCECF-AM and Trypan Blue), the PAPA complexes caused minimal cellular toxicity. These results indicate that PAPA complexes are useful transfection reagents for the study of gene expression and function in cultured cells.

3T3 Cells↗

PubMed1996

Complexes of non-cationic liposomes and histone H1 mediate efficient transfection of DNA without encapsulation.

Transfection competent complexes were assembled using a three component system. The constituents of the basic system were plasmid DNA, cationic DNA binding protein (NLS-H1) and anionic liposomes (dioleoyl phosphatidylethanolamine (DOPE) or phosphatidylserine (PS)). In contrast to cationic liposome/DNA binary complexes, all of the DNA in these ternary complexes was sensitive to DNase I degradation and ethidium bromide intercalation. Transmission electron microscopy revealed that these ternary complexes formed unique structures in which the DNA was located either on the outside of individual liposomes or bridging two or more liposomes. This provides evidence that plasmid DNA encapsulation is not essential for transfection competency.

3T3 Cells↗

PubMed1996

pH-sensitive, cationic liposomes: a new synthetic virus-like vector.

We describe the use of cationic, pH-sensitive liposomes to mediate the efficient transfer of DNA into a variety of cells in culture. Cationic lipids, containing an amine with a pK within the physiologic range of 4.5 to 8, were synthesized and incorporated with dioleoylphosphatidylethanolamine into liposomes. Acid conditions promoted DNA-binding, DNA-incorporation, and DNA-induced fusion by these cationic, pH-sensitive liposomes. Transfection efficiency in cultured cells was dependent on endosomal acidification in a manner akin to acidic-induced endosomal release of viruses. These liposomes constitute a promising new class of reagents for gene therapy.

3T3 Cells↗

PubMed1996

Naked DNA delivered intraportally expresses efficiently in hepatocytes.

Naked plasmid DNA in hypertonic solutions was injected intraportally in mice whose hepatic veins were transiently occluded. High levels of luciferase expression and beta-galactosidase expression in 1% of the hepatocytes throughout the entire liver were achieved using 100 micrograms of the respective plasmid vector. Two days after the intraportal injection of 100 micrograms of pCMVGH, the mean hGH serum concentration was 65 ng/ml +/- 26 (n = 7) which is approximately 50-fold above normal baseline levels. These unprecedented levels of foreign gene expression from naked plasmid DNA document the ability of parenchymal cells in vivo to take up naked DNA following intravascular delivery.

Animals↗

PubMed1995

Electron-electron spin-spin interaction in spin-labeled low-spin methemoglobin.

Nitroxyl free radical electron spin relaxation times for spin-labeled low-spin methemoglobins were measured between 6 and 120 K by two-pulse electron spin echo spectroscopy and by saturation recovery electron paramagnetic resonance (EPR). Spin-lattice relaxation times for cyano-methemoglobin and imidazole-methemoglobin were measured between 8 and 25 K by saturation recovery and between 4.2 and 20 K by electron spin echo. At low temperature the iron electron spin relaxation rates are slow relative to the iron-nitroxyl electron-electron spin-spin splitting. As temperature is increased, the relaxation rates for the Fe(III) become comparable to and then greater than the spin-spin splitting, which collapses the splitting in the continuous wave EPR spectra and causes an increase and then a decrease in the nitroxyl electron spin echo decay rate. Throughout the temperature range examined, interaction with the Fe(III) increases the spin lattice relaxation rate (1/T1) for the nitroxyl. The measured relaxation times for the Fe(III) were used to analyze the temperature-dependent changes in the spin echo decays and in the saturation recovery (T1) data for the interacting nitroxyl and to determine the interspin distance, r. The values of r for three spin-labeled methemoglobins were between 15 and 15.5 A, with good agreement between values obtained by electron spin echo and saturation recovery. Analysis of the nitroxyl spin echo and saturation recovery data also provides values of the iron relaxation rates at temperatures where the iron relaxation rates are too fast to measure directly by saturation recovery or electron spin echo spectroscopy. These results demonstrate the power of using time-domain EPR measurements to probe the distance between a slowly relaxing spin and a relatively rapidly relaxing metal in a protein.

Cyclic N-Oxides↗

PubMedDate not supplied

Hypothesis: naked plasmid DNA is taken up by cells in vivo by a receptor-mediated process.

Following the initial demonstration that intramuscularly-injected naked plasmid DNA (pDNA) is expressed in myofibers, it was shown that pDNA can be used for vaccination purposes. More recent studies have indicated that naked pDNA can also achieve high levels of transgene expression in vivo. This efficiency of naked pDNA expression, especially via intravascular route, is truly astounding. In this prospective review, we examine the possible mechanisms of naked pDNA uptake. The possible mechanisms; (a) large membrane disruption, (b) small membrane pores, and (c) receptor-mediated endocytosis, are considered in turn. Some recent original laboratory data relevant to these hypotheses are also presented.

Animals↗

PubMedDate not supplied

Time course of gene expression after plasmid DNA gene transfer to the liver.

BACKGROUND: High levels of expression in hepatocytes can be achieved after intraportal delivery of plasmid DNA vectors with up to 10% of all liver cells transfected. CMV promoter-driven expression is very high on Day 1 after injection, but is diminished strongly by Day 2. Expression slowly declines after 1 week. We describe experiments aimed at elucidating the reasons for this rapid decline in transgene expression. METHODS: Histological methods were used to determine the presence and extent of liver damage and hepatocyte proliferation. Viral and liver-specific promoters were tested to study promoter shut-off, Southern blotting was performed to determine the loss of the pDNA vector over time, and several mouse models were used to study the host immunological response. RESULTS: pDNA is lost rapidly early after injection, but remains at a relatively stable copy number after Day 4. Southern blotting experiments showed that plasmid DNA could be detected for at least 12 weeks after injection (0.2 copies per genome). The early rapid decline of expression is promoter dependent. A liver-specific albumin promoter resulted in similar levels of expression on Days 1 and 7, suggesting that promoter inactivation may be responsible for the instability of CMV promoter-driven expression. The slow decline in expression levels after 1 week appears to be the result of an immune response directed against the expressed transgene. Expression was much prolonged in immunosuppressed, immunodeficient, or antigen-tolerized mice. CONCLUSION: The present data suggest that if promoter inactivation can be overcome, intravascular delivery of plasmid DNA could be a highly efficient, simple and non-toxic liver gene therapy approach. Intravascular delivery of pDNA allows for the rapid screening of novel expression vectors in vivo.

Animals↗