Sustained release of plasmid DNA using lipid microtubules and agarose hydrogel.

Non-viral gene therapy typically results in low transfection efficiencies and transient gene expression. To address these limitations, two sustained delivery systems capable of releasing functional, compacted DNA for over 50 days were designed. A luciferase plasmid was compacted with a polylysine-polyethylene glycol conjugate and released from agarose hydrogel and lipid microtubule-hydrogel delivery systems for over 50 days. The released DNA was characterized structurally using sedimentation, electron microscopy, and serum stability, and functionally using in vitro transfections. The released DNA retained its physical compaction and nuclease resistance and was converted from supercoiled to nicked and linear forms. Released compacted DNA produced significant gene expression in vitro, although at lower levels than freshly compacted DNA. Thus, hydrogels and lipid microtubules successfully provided the slow release of bioactive, compacted DNA.

Pharmacokinetics of plasmid DNA in the rat.

PURPOSE: The pharmacokinetics of plasmid DNA after IV bolus administration in the rat by following supercoiled (SC), open circular (OC), and linear (L) pDNA forms of the plasmid. METHODS: SC, OC, and L pDNA were injected at 2,500, 500, 333, and 250 microg doses. The concentrations in the bloodstream of OC and L pDNA were monitored. RESULTS: SC pDNA was detectable in the bloodstream only after a 2,500 microg dose, and had a clearance of 390(+/-50) ml/min and Vd of 81(+/-8) ml. The pharmacokinetics of OC pDNA exhibited non-linear characteristics with clearance ranging from 8.3(+/-0.8) to 1.3(+/-0.2) ml/min and a Vd of 39(+/-19) ml. L pDNA was cleared at 7.6(+/-2.3) ml/min and had a Vd of 37(+/-17) ml. AUC analysis revealed that 60(+/-10) % of the SC was converted to the OC form, and nearly complete conversion of the OC pDNA to L pDNA. Clearance of SC pDNA was decreased after liposome complexation to 87(+/-30) ml/min. However the clearance of OC and L pDNA was increased relative to naked pDNA at an equivalent dose to 37(+/-9) ml/min and 95(+/-37) ml/min respectively. CONCLUSIONS: SC pDNA is rapidly metabolized and cleared from the circulation. OC pDNA displays non-linear pharmacokinetics. Linear pDNA exhibits first order kinetics. Liposome complexation protects the SC topoform, but the complexes are more rapidly cleared than the naked pDNA.

Kinetic modeling of plasmid DNA degradation in rat plasma.

A major obstacle in gene delivery is the transport of intact plasmid DNA (pDNA) to target sites. We sought to investigate the kinetic processes underlying the degradation of pDNA in a rat plasma model, as this is one of the main components responsible for the clearance of pDNA after intravenous administration. We further sought to construct a complete kinetic model to describe the degradation of all three topoforms (supercoiled, open circular, and linear) of pDNA in a rat plasma model. Supercoiled pDNA was incubated in isolated rat plasma at 37 degrees C in vitro. At various time points, the plasma was assayed by electrophoresis for the amounts of supercoiled, open circular, and full-length linear pDNA remaining. The calculated amounts remaining were fit to linear differential equations describing this process. In this model, pDNA degradation is considered to be a unidirectional process, with supercoiled degrading to open circular and then to the linear topoform. The calculated kinetic parameters suggested that supercoiled pDNA degrades in rat plasma with a half-life of 1.2 minutes, open circular pDNA degrades with a half-life of 21 minutes, and linear pDNA degrades with a half-life of 11 minutes. Complexation of pDNA with liposomes resulted in a portion of the supercoiled plasmid remaining detectable through 5.5 hours.

Supercoil-dependent recognition of specific DNA sites by chromosomal protein HMG 2.

The ability of the chromosomal high mobility group protein HMG 2 to recognize supercoil-dependent structures within the chicken adult beta-globin gene was investigated by examining its ability to protect such sites from digestion by S1 nuclease. Low molar ratios of HMG 2 were found to be sufficient for complete inhibition of S1 cleavage of a supercoiled plasmid containing the globin gene. Furthermore, HMG 2 protected an S1 cleavage site within the 5'-flanking region of the globin gene, in preference to a palindromic S1 site within the plasmid vector.

[Accumulation of DNA structural damages in human lymphocytes during aging]. / Narastanie povrezhdenii struktury DNK v limfotsitakh liudei pri starenii.

Elastoviscosometric parameters of DNA from normal subjects of different age and patients with Down syndrome were assessed. Characteristics of DNA isolated from lymphocytes trisomic for chromosome 21 were studied to compare normal and pathological rates of ageing. Increased elastoviscosity was observed in normal subjects above 60. Similar changes in this parameter were noted in aberrant lymphocytes isolated from patients above 10. The established dependence of elastoviscosity on ethidium bromide concentration led to the assumption that an increase in hydrodynamic DNA volume in human leukocytes during ageing was due to accumulation of spontaneous irreparable DNA lesions.

Detection of an altered DNA conformation at specific sites in chromatin and supercoiled DNA.

We show that bromoacetaldehyde, which reacts selectively at the N-1 and N-6 positions of unpaired adenine and at the N-3 and N-4 positions of unpaired cytosine residues reacts with chromosomal DNA in intact cells at probable regulatory sequences near active genes. A region of about 200 base pairs 5' to the chicken beta A-globin gene, which contains sites sensitive to nuclease S1, to several restriction endonucleases, and to very low levels of DNase I, also contains DNA structures that are preferentially sensitive to bromoacetaldehyde. These altered DNA structures are found at reproducible positions relative to the beta A-globin gene regardless of whether the bromoacetaldehyde is presented to intact erythrocytes, erythrocyte nuclei, or the beta A-globin gene itself carried in pBR322 as purified supercoiled DNA. The unpaired DNA 5' to the adult beta A-globin gene in adult erythrocytes is not detectable in embryonic erythrocytes that express embryonic beta-globin in contrast to adult beta A-globin. Our results suggest that well-defined regions of DNA with effectively unpaired bases occur in intact nuclei and that these structures may be important for specific recognition because they are tissue specific and are found at putative regulatory regions.