Comparative Analysis of Polyethyleneimine Efficiency for Improvement of Plasmid DNA Bioavailability.

We studied the influence of the type and structure of polyethyleneimine on bioavailability and expression of plasmid DNA carrying IGF-1 gene. Polymers with different molecular weights (2.5, 10, 25, and 60 kDa) of linear and branching structure were studied. It was found that the time of polyplex circulation in the blood did not exceed 24 h and the maximum concentration of plasmid DNA was attained with complexes with a molecular weight of 60 kDa. Analysis of liver samples showed that administration of 60-kDa branched polyethyleneimine complex provides DNA protection from degradation for 4 h; in 24 h from the start of the experiment, its concentration was significantly higher than the concentration of other studied polyethyleneimines. The expression of plasmid IGF-1 DNA for this complex attained maximum in 4 h and was equal to 15.50 (7.98; 21.98) arb. units/ml. These results allow us to recommend using polyethyleneimines with branched structure and a molecular weight of 60 kDa for improving plasmid DNA protection and bioavailability.

Infectious Agents in Bovine Red Meat and Milk and Their Potential Role in Cancer and Other Chronic Diseases.

Red meat and dairy products have frequently been suggested to represent risk factors for certain cancers, chronic neurodegenerative diseases, and autoimmune and cardiovascular disorders. This review summarizes the evidence and investigates the possible involvement of infectious factors in these diseases. The isolation of small circular single-stranded DNA molecules from serum and dairy products of Eurasian Aurochs (Bos taurus)-derived cattle, obviously persisting as episomes in infected cells, provides the basis for further investigations. Gene expression of these agents in human cells has been demonstrated, and frequent infection of humans is implicated by the detection of antibodies in a high percentage of healthy individuals. Epidemiological observations suggest their relationship to the development multiple sclerosis, to heterophile antibodies, and to N-glycolylneuraminic acid (Neu5Gc) containing cell surface receptors.

Phase 1 Trial of Bi-shRNA STMN1 BIV in Refractory Cancer.

Stathmin1 (STMN1) is a microtubule modulator that is expressed in multiple cancers and correlates with poor survival. We previously demonstrated in vivo safety of bifunctional (bi) shRNA STMN1 bilamellar invaginated vesicle (BIV) and that systemic delivery correlated with antitumor activity. Patients with superficial advanced refractory cancer with no other standard options were entered into trial. Study design involved dose escalation (four patients/cohort) using a modified Fibonacci schema starting at 0.7 mg DNA administered via single intratumoral injection. Biopsy at baseline, 24/48 hours and resection 8 days after injection provided tissue for determination of cleavage product using next-generation sequencing (NGS) and reverse transcription quantitative polymerase chain reaction (RT-qPCR), 5' RLM rapid amplification of cDNA ends (RACE) assay. Serum pharmacokinetics of circulating plasmid was done. Twelve patients were entered into three dose levels (0.7, 1.4, 7.0 mg DNA). No ≥ grade 3 toxic effects to drug were observed. Maximum circulating plasmid was detected at 30 seconds with less than 10% detectable in all subjects at 24 hours. No toxic effects were observed. Predicted cleavage product was detected by both NGS (n = 7/7 patients analyzed, cohorts 1, 2) and RLM RACE (n = 1/1 patients analyzed cohort 3). In conclusion, bi-shRNA STMN1 BIV is well tolerated and detection of mRNA target sequence-specific cleavage product confirmed bi-shRNA BIV mechanism of action.

Application of polyglycerol coating to plasmid DNA lipoplex for the evasion of the accelerated blood clearance phenomenon in nucleic acid delivery.

Cationic liposomes (CLs) have shown promise as nonviral delivery systems. To achieve in vivo stability and long circulation, most liposomes are modified with hydrophilic polymer polyethylene glycol (PEG). However, we have reported that repeated administration of PEG-coated CLs containing plasmid DNA (pDNA; PEGylated lipoplexes) induces what is referred to as "the accelerated blood clearance (ABC) phenomenon" and, consequently, subsequently administered lipoplexes lose their prolonged circulation characteristics. Anti-PEG IgM produced in response to the first dose of PEG-coated pDNA-lipoplexes (PEG-DCL) has proven to be a major cause of the ABC phenomenon. In this study, to evade and/or attenuate this unexpected immune response, we modified the surface of a lipoplex with polyglycerol (PG)-derived lipid. The PG-coated pDNA-lipoplex (PG-DCL) attenuated the production of anti-polymer IgM, whereas PEG-coated pDNA-lipoplex (PEG-DCL) did not. In addition, a second dose of PG-DCL maintained the accumulation level in the tumor tissue of a tumor-bearing mouse model, comparable to that of the first dose, whereas the tumor accumulation level of a second dose of PEG-DCL was significantly compromised, compared with the first dose of PEG-DCL. Our results indicate that surface modification of lipoplex with PG represents a viable means for the attenuation, and/or evasion, of the ABC phenomenon that is encountered upon repeated administrations of nucleic acids containing PEG-coated nanocarriers.

Improved detection of transgene and nonviral vectors in blood.

Vector biodistribution and clearance studies are essential in the development of gene transfer medicine. To provide reliable and accurate data, protocols for vector analysis must be optimized and validated. We addressed several parameters affecting the detection of gene therapy vectors in blood. Using an in vitro system based on plasmid DNA incorporating, as a transgene, complementary DNA for human erythropoietin gene, we developed and validated a suite of real-time PCR assays for the transgene splicing sites. The most sensitive assays detected the transgene present at 0.011% of the copy number of the endogenous erythropoietin gene in human genomic DNA at 100% specificity. Plasmid linearization incorporated with PCR resulted in an increase in assay sensitivity up to 4.5-fold without compromising analysis workflow. This allowed detection of five copies of transgene in a background of 0.4 µg of genomic DNA (or 0.0035% detectable transgene copies relevant to copies of the endogenous gene). Finally, desktop assessment of 18 DNA extraction protocols was undertaken and 5 kits were evaluated experimentally for extraction of nonviral vectors from blood. Three kits reliably detected 80 copies of the transgene in a milliliter of blood. Adoption of the described protocols will enable more reliable vector analysis in gene therapy and will assist in accurate interlaboratory comparison. The methodology will also facilitate detection of gene doping in sport, a potential new form of misuse of gene transfer technology.

High-affinity PEGylated polyacridine peptide polyplexes mediate potent in vivo gene expression.

Polyethylene glycol (PEG)ylated polyacridine peptides bind to plasmid DNA with high affinity to form unique polyplexes that possess a long circulatory half-life and are hydrodynamically (HD)-stimulated to produce efficient gene expression in the liver of mice. We previously demonstrated that acridine-modified lysine (Acr) in (Acr-Lys)(6)-Cys-PEG(5kDa) stabilizes a 1-µg pGL3 dose for up to 1 h in the circulation, resulting in HD-stimulated (saline only) gene expression in the liver, equivalent in magnitude to direct-HD dosing of 1 µg of pGL3. In this study, we report that increasing the spacing of Acr with either four or five Lys residues markedly increases the stability of PEGylated polyacridine peptide polyplexes in the circulation allowing maximal HD-stimulated expression for up to 5 h post DNA administration. Co-administration of a decoy dose of 9 µg of non-expressing DNA polyplex with 1 µg of pGL3 polyplex further extended the HD-stimulated expression to 9 h. This structure-activity relationship study defines the PEGylated polyacridine peptide requirements for maintaining fully transfection competent plasmid DNA in the circulation for 5 h and provides an understanding as to why polyplexes or lipoplexes prepared with polyethylenimine, chitosan or Lipofectamine are inactive within 5 min following intravenous dosing.

Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping.

Legitimate uses of gene transfer technology can benefit from sensitive detection methods to determine vector biodistribution in pre-clinical studies and in human clinical trials, and similar methods can detect illegitimate gene transfer to provide sports-governing bodies with the ability to maintain fairness. Real-time PCR assays were developed to detect a performance-enhancing transgene (erythropoietin, EPO) and backbone sequences in the presence of endogenous cellular sequences. In addition to developing real-time PCR assays, the steps involved in DNA extraction, storage and transport were investigated. By real-time PCR, the vector transgene is distinguishable from the genomic DNA sequence because of the absence of introns, and the vector backbone can be identified by heterologous gene expression control elements. After performance of the assays was optimized, cynomolgus macaques received a single dose by intramuscular (IM) injection of plasmid DNA, a recombinant adeno-associated viral vector serotype 1 (rAAV1) or a rAAV8 vector expressing cynomolgus macaque EPO. Macaques received a high plasmid dose intended to achieve a significant, but not life-threatening, increase in hematocrit. rAAV vectors were used at low doses to achieve a small increase in hematocrit and to determine the limit of sensitivity for detecting rAAV sequences by single-step PCR. DNA extracted from white blood cells (WBCs) was tested to determine whether WBCs can be collaterally transfected by plasmid or transduced by rAAV vectors in this context, and can be used as a surrogate marker for gene doping. We demonstrate that IM injection of a conventional plasmid and rAAV vectors results in the presence of DNA that can be detected at high levels in blood before rapid elimination, and that rAAV genomes can persist for several months in WBCs.

Retinoic acid and dimethyl sulfoxide promote efficient delivery of transgenes to mouse skin by topically transdermal penetration.

Simple and efficient gene transfer to the skin would facilitate many local and systemic gene therapy applications. This study reports a novel approach that allows expression of plasmid DNA in epidermis and hair follicle cells with dimethyl sulfoxide (DMSO) after pre-treatment with depilation and retinoic acid (RA) for the purposes of gene therapy. This study investigated the transdermal efficacy of gene to mouse skin when utilizing DMSO after RA pre-treatment. Retinoic acid pre-treatment can increase the efficiency of transfection. This finding indicates that one can more effectively and much less expensively make use of genes therapy to treat diseases of the hair and skin.

The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro.

Previous research has suggested that human herpesvirus-6 (HHV-6) may integrate into host cell chromosomes and be vertically transmitted in the germ line, but the evidence--primarily fluorescence in situ hybridization (FISH)--is indirect. We sought, first, to definitively test these two hypotheses. Peripheral blood mononuclear cells (PBMCs) were isolated from families in which several members, including at least one parent and child, had unusually high copy numbers of HHV-6 DNA per milliliter of blood. FISH confirmed that HHV-6 DNA colocalized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, and that the integration site was identical among members of the same family. Integration of the HHV-6 genome into TTAGGG telomere repeats was confirmed by additional methods and sequencing of the integration site. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome. We next asked whether HHV-6A infection of naïve cell lines could lead to integration. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6, the virus integrated into telomeres. Reactivation of integrated HHV-6A virus from individuals' PBMCs as well as cell lines was successfully accomplished by compounds known to induce latent herpesvirus replication. Finally, no circular episomal forms were detected even by PCR. Taken together, the data suggest that HHV-6 is unique among human herpesviruses: it specifically and efficiently integrates into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.

Specific uptake of plasmid DNA without reporter gene expression in Atlantic salmon (Salmo salar L.) kidney after intramuscular administration.

In this study we investigated tissue distribution of pDNA after intramuscular and intravenous administration, cellular localisation, receptor-specific uptake, integrity of pDNA and transgene expression in Atlantic salmon (Salmo salar L). Anatomical distribution of plasmid DNA was determined using both radiotracing and fluorescence microscopy. Cellular uptake was studied in cultures of adherent anterior kidney leucocytes. The integrity of the pDNA in vivo was investigated by Southern blot analysis. Transcription of plasmid DNA encoded luciferase gene and protein synthesis were investigated in salmon tissues by means of real-time reverse transcription-polymerase chain reaction and enzyme activity measurements, respectively. Approximately 50% of the total recovered radioactivity was redistributed from the carcass 168h after intramuscular administration and accumulated mainly in the kidneys (37% of total). The majority of radiolabelled plasmid DNA administered intravenously was taken up within the first 15min mainly by the kidney. Intravenous co-administration of trace amounts of radiolabelled plasmid DNA with excess amounts of unlabelled plasmid DNA or formaldehyde treated albumin (a ligand for the scavenger receptors) significantly inhibited accumulation of the radiotracer in the kidney. Fluorescence microscopy demonstrated that fluorescence was localised intracellularly in cells lining the sinusoids of the kidney after intravenous administration of rhodamine-labelled plasmid DNA. Southern blot analysis demonstrated presence of supercoiled plasmid DNA in all organs and tissue samples 168h after intramuscular administration, but degradation products were only revealed at the administration site. Luciferase transcript and activity were only detectable at the administration site 24-168h after intramuscular administration of plasmid DNA. After incubation with trace amounts of radiolabelled plasmid DNA, only minor amounts of radiolabelled plasmid DNA were cell associated in cultures of adherent anterior kidney leucocytes. These results suggested that a substantial portion of radiolabelled plasmid DNA was redistributed from the carcass and was mainly cleared by a receptor-specific uptake in the kidney. Although intact plasmid DNA was detected in the kidney and other tissues, no luciferase transcripts or activity were detected in these samples at any time points investigated (24-168h), except for the administration site following intramuscular administration.

[Long-fragment DNA of blood plasma as one of the criteria of individual sensitivity to emotional stress and to cerebral ischemia].

Intravenous injection ofpolyethylenoxide WSR-301 reducing hydrodynamic blood resistance (Toms effect) improves gas exchange in the lungs and halved lethality of the animals with cerebral ischemia. The aim of the study was to establish whether free plasma DNA influences blood gases and lethality of the animals with brain ischemia. Common carotid arteries were ligated for 15 min in intact stressed and tested in the open field Wistar male rats, then some of the rats received intravenous solution of homologous long-fragment DNA (20x10(-6) g/ml of blood). Cerebral circulation, acid-base equilibrium, paO2, paCO2, asymptotic blood viscosity, plasmic concentration and length of DNA fragments in plasma, lethality and neurological status of the survivors were studied. It was found that long fragments of rat DNA show hydrodynamic Toms effect. In normal passive rats sensitive to cerebral ischemia part of plasm DNA is fragmented, gas composition and blood viscosity of blood is worse (p < 0.05) than in active animals. There is a direct correlation between the level of long-fragment DNA in plasm and paO2 (r = 0.55) and inverse--with paCO2 (r = -0.84). Intravenous injection of long-fragment DNA improved the course and reduced lethality of brain ischemia 2-3-fold. Thus, qualitative and quantitative characteristics of plasma circulating DNA are responsible for differences in blood gases in rats differently tolerable to cerebral ischemia and can serve as one of the criteria of individual sensitivity to it being essential in pathogenesis of ischemic stroke.

Mechanistic studies of sequential injection of cationic liposome and plasmid DNA.

We previously reported that sequential injection of cationic liposome and plasmid DNA leads to notably reduced inflammatory toxicity and improved transfection in the lung (Y. Tan et al., 2001, Mol. Ther. 3, 673-682). The purpose of the current study was to explore the mechanism involved in sequential injection. It was observed that sequential injection resulted in dramatically lower DNA uptake by the liver and higher DNA levels in the lung than the lipoplex injection. In vitro experiments with macrophage cells further showed that sequential addition of liposomes and DNA could diminish the cellular uptake of DNA by these cells. The contributions of serum to the enhanced bioactivity and decreased toxicity were examined by injecting mice with samples of premixed liposome with serum and then DNA (LSD sample), and the resulting activities were compared to those obtained with injection of lipoplex-serum mixtures (LDS sample). LSD yielded 80% lower TNF-alpha levels and over 10-fold higher transfection than lipoplex, which is consistent with the reported findings with sequential injection. In contrast, LDS resulted in the same TNF-alpha levels and comparable transfection with lipoplex. Thus, the results suggest that the primary interaction of serum with liposome is a critical factor contributing to the superior activity and reduced toxicity of sequential injection. Studies on the interaction between mouse serum, liposomes, and DNA showed that DNA could bind negatively charged liposome-serum complex to form a ternary complex, which has a density similar to that of the ternary complex formed between lipoplex with serum. Further in vitro tests showed that LSD and LDS were similar in particle size and protein content, but different in protein composition as observed by 2-D gel electrophoresis. In addition, DNA in LSD was more readily displaced by dextran sulfate, an anionic polymer, than in LDS. The above findings suggest that the inhibition of opsonin protein binding on the particle surface with the sequential injection may contribute to the reduced macrophage uptake and cytokine induction and that the high ability of DNA release from the particles formed after sequential injection may contribute to the improved lung gene transfection.

Hepatocyte-targeted gene transfer by combination of vascularly delivered plasmid DNA and in vivo electroporation.

To increase transgene expression in the liver, electric pulses were applied to the left lateral lobe after intravenous injection of naked plasmid DNA (pDNA) or pDNA/liver targeting vector complex prepared with galactosylated poly(L-lysine) or galactosylated polyethyleneimine. Electroporation (250 V/cm, 5 ms/pulse, 12 pulses, 4 Hz) after naked pDNA injection dramatically increased the expression up to 200,000-fold; the expression level obtained was significantly greater than that achieved by the combination of pDNA/vector complex and electroporation. We clearly demonstrated that the expression was dependent on the plasma concentration of pDNA at the time when the electric pulses were applied. Separation of liver cells revealed that the distribution of naked pDNA as well as transgene expression was largely selective to hepatocytes in the electroporated lobe. The number of cells expressing transgene product using vascularly administered naked pDNA followed by electroporation was significantly (P<0.01) greater and more widespread than that obtained by local injection of naked pDNA. These results indicate that the application of in vivo electroporation to vascularly administered naked pDNA is a useful gene transfer approach to a large number of hepatocytes.

Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests.

Parvovirus B19 causes a variety of diseases in humans, with outcomes ranging from asymptomatic to severe, such as chronic anemia in immunocompromised patients or fetal hydrops and death after maternal infection during pregnancy. The virus may be transmitted via plasma-derived products. According to the results of solvent-detergent safety studies, an upper limit of B19 DNA in plasma pools was recently defined. To restrict the input of B19 virus into production pools, a quantitative nucleic acid test is a prerequisite. We examined the suitability of the two commercial quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus) for detection, quantification, and differentiation of the three known B19 genotypes, including the newly described erythrovirus variants (genotypes 2 and 3). The former kit was highly sensitive for genotype 1 but was not suitable for detection of genotype 2 or one of two genotype 3 strains. The latter kit detected and differentiated all three genotypes, albeit with lower sensitivity for one of the genotype-3 strains. We furthermore assessed the prevalence of the three B19 virus genotypes in blood donors, by screening pooled plasma samples derived from 140,160 Finnish blood-donor units. None of the pools contained detectable levels of B19 virus genotypes 2 or 3. The origin, mode of transmission, and clinical significance of these genotypes are unknown and deserve further study. The RealArt Parvo B19 LC PCR is suitable for detection, quantification, and differentiation of all three B19 virus genotypes in molecular and clinical research.

Preparation and characteristics of DNA-nanoparticles targeting to hepatocarcinoma cells.

AIM: To prepare thymidine kinase gene (TK gene) nanoparticles and to investigate the expression of TK gene. METHODS: Poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer, was used to prepare recombinant plasmid P(EGFP-AFP) nanoparticles by a double-emulsion evaporation technique. Characteristics of the nanoparticles were investigated in this study, including morphology, entrapment efficiency, and tissue distribution. The expression of TK gene was also investigated by MTT assay, by which the viable cells were determined after the addition of ganciclovir (GCV). The enhanced green fluorescent protein (EGFP) expression in human hepatocellular carcinoma SMMC-7721 cells and normal parenchymal Chang liver cells were assessed by flow cytometry. RESULTS: The prepared plasmid-nanoparticles had regular spherical surface and narrow particle size span with a mean diameter of 72+/-12 nm. The mean entrapment efficiency was 91.25%. A total of 80.14% DNA was found to be localized in the livers after 1-h injection with (32)P-DNA-PLGA nanoparticles in mouse caudal vein. The expression of DNA encapsulated in nanoparticles was much higher than that in naked DNA, and human hepatocellular carcinoma SMMC-7721 cells were more sensitive to GCV than human normal parenchymal Chang liver cells. CONCLUSION: The enhanced transfection efficiency and stronger ability to protect plasmid DNA from being degraded by nucleases are due to nanoparticles encapsulation.

Use of horseradish peroxidase- and fluorescein-modified cisplatin derivatives for simultaneous labeling of nucleic acids and proteins.

BACKGROUND: Microarray platforms will change immunochemical and nucleic acid-based analysis of cell homogenates and body fluids compared with classic analyses. Microarrays use labeled target and immobilized probes, rather than fixed targets and labeled probes. We describe a method for simultaneous labeling of nucleic acids and proteins. METHODS: Horseradish peroxidase- and fluorescein-modified cisplatin derivatives were used for labeling of nucleic acids and proteins. These reagents, called the Universal Linkage System (ULS), bind to sulfur- and nitrogen-donor ligands present in amino acids and nucleotides. For automated screening of proteins and nucleic acids on microarrays, it is advantageous to label these biomolecules without pre- or postpurification procedures. The labeling of antibodies and nucleic acids in whole serum was therefore pursued. RESULTS: Immunoglobulins in nonpurified serum were labeled efficiently enough to be used for immunochemistry. To investigate whether protein-adapted labeling allowed nucleic acid labeling as well, 1 microg of plasmid DNA was added to 1 microL of serum. DNA and serum proteins were simultaneously labeled, and this labeled DNA could be used as a probe for direct fluorescence in situ hybridization. CONCLUSION: ULS provides a direct labeling tool for the (simultaneous) modification of proteins and nucleic acids even in unpurified samples.

Myocardial Doppler tissue velocity improves following myocardial gene therapy with VEGF-A165 plasmid in patients with inoperable angina pectoris.

BACKGROUND: Myocardial tissue velocity and perfusion were studied in patients with severe angina pectoris following gene therapy by intramyocardial injection of phVEGF-A165 via thoracotomy. Plasma concentrations of VEGF-A increased postoperatively. Two months after treatment anginal status and myocardial tissue velocity improved and perfusion showed a tendency to improve. Tissue velocity imaging appears to be a sensitive, objective method for detecting changes in myocardial function following gene therapy. OBJECTIVE: To study effects on myocardial tissue velocity and perfusion in patients with angina pectoris following intramyocardial injection of phVEGF-A165 via thoracotomy. DESIGN: Open label, phase I/II. METHODS: Six patients with Canadian Cardiovascular Society (CCS) angina pectoris functional class III - IV and with major defects at adenosine stress single-photon emission computerized tomography (SPECT) were studied. In addition to SPECT, coronary angiography and dobutamine stress echocardiography with tissue Doppler velocity imaging were performed before and two months after gene transfer. RESULTS: Plasma concentrations of VEGF-A increased 2 to 3 times (P < 0.04) over baseline from 2 to 14 days after injection with normalization after 4 weeks. The CCS class improved about 40%, from 3.3 +/- 0.2 to 2.0 +/- 0.3 (P < 0.02) and nitroglycerine consumption decreased 30 - 40%, from 44 +/- 17 to 15 +/- 5 tablets per week (P < 0.05). The maximal systolic myocardial tissue velocity increased in all patients about 25% (P < 0.02) but did not reach the reference range. Myocardial perfusion at SPECT improved in four of the six patients. CONCLUSIONS: Anginal status, myocardial tissue velocity and perfusion can be improved by phVEGF-A165 intramyocardial injection. Tissue velocity imaging appears to be a sensitive, objective method for detecting changes in myocardial function following gene therapy.

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.

Determination of protection from serum nuclease activity by DNA-polyelectrolyte complexes using an electrophoretic method.

Polyelectrolyte complexes between cationic polymers and DNA have emerged as potential nonviral vectors for DNA delivery. For successful in vivo delivery, methods for analyzing their ability to prevent digestion of the DNA payload by serum nucleases are essential. We report here a simple assay to determine degradation of DNA in these complexes using standard electrophoretic techniques. The assay is based on a high pH buffer which can dissociate the complexes under standard electrophoretic conditions. This assay can be used qualitatively to determine the time taken for degradation to occur. Alternatively, with a standard gel analysis program it can be used quantitatively to investigate rates of DNA degradation from complexes in the presence of serum nucleases. We have shown that it can distinguish between different formulations with the same polymer, and also to distinguish between the time taken to degradation and the rates of degradation of DNA in complexes formed with two structurally related, linear polyamidoamine polymers. The assay could also distinguish between the time to degradation using poly-l-lysine complexes, although these were less well dissociated by the electrophoresis buffer, and could not be analyzed quantitatively. This assay will be of value in investigating and developing polyelectrolyte formulations for parenteral administration.

Characterization of the maturation of human pro-apolipoprotein A-I in an in vitro model.

The reaction conditions and the protein structural features involved in the maturation of pro-apolipoprotein A-I (cleavage of pro-peptide) were investigated in an in vitro model. ProapoA-I, mutants and wild type, were expressed in the PGEX/E. coli expression system as fusion proteins with glutathione S-transferase (GST). Use of GST-proapoA-I and truncated forms of proapoA-I enabled quantitation of the amount of GST and apoA-I formed as a result of cleavage following incubation with human serum. Deletion of the pro-peptide (GST-apoA-I) resulted in complete inhibition of the reaction. Truncation of proapoA-I to residues 222, 150, 135, and 25 as well as substitution of residues -6, -5, and -4 with alanine did not affect the reaction. Substitution of residues -1, -2, 1, 3, and 4 with alanine either completely blocked or substantially inhibited cleavage of the pro-peptide. The reaction was inhibited by addition of EDTA, o-phenanthroline, dithiothreitol, and beta-mercaptoethanol and to a lesser extent by p-chloromercuriphenylsulfonic acid, but not by leupeptin, N-ethylmaleimide, PMSF, pepstatin A, or trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane. Calcium was essential for the activation of the cleavage enzyme, but it had a biphasic effect on the cleavage, activating it at concentrations below 1.5 mM and inhibiting at concentrations above 1.75 mM. Manganese alone was not essential for activation of the enzyme nor did it modify the effect of low concentration of calcium. However, a high concentration of manganese partially reverted the inhibitory effect of a high calcium concentration. Thus, residues within -2 to +4 are involved in forming the cleavage site for the maturation enzyme. The reaction of maturation is inhibited by metalloprotease inhibitors and is dependent upon calcium.