Electroporation-facilitated delivery of plasmid DNA in skeletal muscle: plasmid dependence of muscle damage and effect of poloxamer 188.

Electroporation has been reported to facilitate naked DNA gene transfer in skeletal muscle, but has also been implicated in the pathogenesis of electrical injuries. To assess the effects of electroporation on gene transfer, mouse quadriceps muscles were injected with the luciferase reporter plasmid VR1255 and electroporated with caliper electrodes. Intramuscular luciferase expression was increased 10- to 70-fold by electroporation, depending on the DNA dose and injection volume used. In the absence of plasmid DNA injection, electroporation of quadriceps muscles resulted in rapid elevations in serum creatine phosphokinase activity, but did not elicit visible muscle damage. However, in muscles injected with plasmid DNA and electroporated, visible lesions consistently developed in the areas proximal to electrode placement when field strengths optimal for gene transfer (300 volts/cm) were applied. The development of muscle lesions was independent of plasmid transgene expression and required the presence of plasmid in the muscle during electroporation. Co-injection of poloxamer 188 (pluronic F68) with VR1255 substantially reduced elevations in serum creatine phosphokinase activity following electroporation, but did not inhibit the development of muscle lesions. In non-electroporated muscles, co-injection of poloxamer 188 increased luciferase expression threefold. Poloxamer 188 may thus constitute a useful excipient for intramuscular delivery of naked DNA.

Vaxfectin enhances antigen specific antibody titers and maintains Th1 type immune responses to plasmid DNA immunization.

Antigen specific immune responses were characterized after intramuscular immunization of BALB/c mice with 5 antigen encoding plasmid DNAs (pDNAs) complexed with Vaxfectin, a cationic lipid formulation. Vaxfectin increased IgG titers for all of the antigens with no effect on the CTL responses to the 2 antigens for which CTL assays were performed. Both antigen specific IgG1 and IgG2a were increased, although IgG2a remained greater than IgG1. Furthermore, Vaxfectin had no effect on IFN-gamma or IL-4 production by splenocytes re-stimulated with antigen, suggesting that the Th1 type responses typical of intramuscular pDNA immunization were not altered. Studies with IL-6 -/- mice suggest that the antibody enhancement is IL-6 dependent and results in a correlative increase in antigen specific antibody secreting cells.

Vaxfectin enhances the humoral immune response to plasmid DNA-encoded antigens.

This report characterizes Vaxfectin, a novel cationic and neutral lipid formulation which enhances antibody responses when complexed with an antigen-encoding plasmid DNA (pDNA). In mice, intramuscular injection of Vaxfectin formulated with pDNA encoding influenza nucleoprotein (NP) increased antibody titers up to 20-fold, to levels that could not be reached with pDNA alone. As little as 1 microg of pDNA formulated with Vaxfectin per muscle resulted in higher anti-NP titers than that obtained with 25 microg naked pDNA. The antibody titers in animals injected with Vaxfectin-pDNA remained higher than in the naked pDNA controls for at least 9 months. The enhancement in antibody titers was dependent on the Vaxfectin dose and was accomplished without diminishing the strong anti-NP cytolytic T cell response typical of pDNA-based vaccines. In rabbits, complexing pDNA with Vaxfectin enhanced antibody titers up to 50-fold with needle and syringe injections and also augmented humoral responses when combined with a needle-free injection device. Vaxfectin did not facilitate transfection and/or increase synthesis of beta-galactosidase reporter protein in muscle tissue. ELISPOT assays performed on bone marrow cells from vaccinated mice showed that Vaxfectin produced a three- to five-fold increase in the number of NP-specific plasma cells. Thus, Vaxfectin should be a useful adjuvant for enhancing pDNA-based vaccinations.

Sodium phosphate enhances plasmid DNA expression in vivo.

Intramuscular injection of plasmid DNA results in myofiber cell expression of proteins encoded by the DNA. The preferred vehicle for plasmid DNA injections has been saline (154 mM sodium chloride) or PBS (154 mM NaCl plus 10 mM sodium phosphate). Here, it is shown that injection of luciferase or beta-galactosidase encoding plasmid DNA in a 150 mM sodium phosphate vehicle into murine muscle resulted in a two- to seven-fold increase in transgene expression compared with DNA injected in saline or PBS. When the DNA encoded secreted alkaline phosphatase, preproinsulin or interferon, sodium phosphate vehicle increased their serum levels by two- to four-fold. When the DNA encoded mouse erythropoietin, sodium phosphate vehicle increased hematocrits by two-fold compared with DNA injected in saline. When the DNA encoded influenza nucleoprotein, sodium phosphate increased anti-nucleoprotein antibody titers by two-fold. The expression of luciferase from plasmid DNA instilled into lung was increased five-fold compared with that in vehicle without sodium phosphate. Incubation of plasmid DNA with muscle extract or serum showed that sodium phosphate protected the DNA from degradation. Thus, a change from sodium chloride to sodium phosphate vehicle can enhance the expression of plasmid DNA in a tissue, possibly by inhibiting DNA degradation. Gene Therapy (2000) 7, 1171-1182.

A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung.

Effective gene therapy for lung tissue requires the use of efficient vehicles to deliver the gene of interest into lung cells. When plasmid DNA encoding chloramphenicol acetyltransferase (CAT) was administered intranasally to BALB/c mice without carrier lipids, CAT activity was detected in mouse lung extracts. Plasmid DNA delivered with optimally formulated commercially available transfection reagents expressed up to 10-fold more CAT activity in lung than observed with naked DNA alone. Liposome formulations consisting of (+/-)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis (dodecyloxy)-1-propanaminium bromide (GAP-DLRIE) plus the neutral colipid dioleoylphosphatidylethanolamine (DOPE) enhanced CAT expression by more than 100-fold relative to plasmid DNA alone. A single administration of GAP-DLRIE liposome-CAT DNA complexes to mouse lung elicited peak expression at days 1-4 posttransfection, followed by a gradual return to baseline by day 21 postadministration. Readministration of GAP-DLRIE liposome CAT complexes at day 21 led to another transient peak of reporter gene expression. Histological examination of lungs treated with GAP-DLRIE complexed beta-galactosidase DNA revealed that alveolar epithelial cells were the primary locus of expression and that up to 1% of all alveoli contained epithelial cells expressing the transgene.

Novel, high expressing and antibiotic-controlled plasmid vectors designed for use in gene therapy.

The promise of effective gene therapy can only be accomplished by high-level expression and regulatable delivery of gene products. To achieve this end, a eukaryotic expression plasmid was modified to make transcription dependent on a tetracycline(Tc)-regulated chimeric transactivator. Mouse muscle injected with this two plasmid cis/trans control system expressed reporter proteins at levels five- to 10-fold greater than the cytomegalovirus immediate-early promoter-controlled parental plasmid. Tetracycline could be useful to either repress or activate transactivator-controlled expression based on the position of the tetO control sequences within the reporter plasmid. Finally, a prototype single plasmid construct was made and shown to express a self-regulating bicistronic transcript containing both the reporter and the transactivator. These Tc-controlled plasmids, termed maximum expression and regulated vectors (MERVs), have the potential to target a variety of gene therapy applications.

Converting an alcohol to an amine in a cationic lipid dramatically alters the co-lipid requirement, cellular transfection activity and the ultrastructure of DNA-cytofectin complexes.

Cytofectins are positively charged lipophilic molecules that readily form complexes with DNA and other anionic polynucleotides. Normally, cytofectins are combined with an activity-augmenting phospholipid such as dioleoylphosphatidylethanolamine (DOPE), and a film of dried, mixed lipid is prepared and hydrated to form cationic liposomes. The liposome solution is then mixed with a plasmid DNA solution to afford cytofectin-DNA complexes which, when presented to living cells, are internalized and the transgene is expressed. One of the most potent cytofectins, dimyristoyl Rosenthal inhibitor ether (DMRIE), is presently being used to deliver transcriptionally active DNA into human tumor tissues. Here we report the remarkable consequences of replacing the alcohol moiety of DMRIE with a primary amine. The resulting cytofectin, called beta-aminoethyl-DMRIE (betaAE-DMRIE), promoted high level transfection over a broad range of DNA and cationic lipid concentrations. A comparison of in vitro transfection activity between DMRIE and betaAE-DMRIE in 10 cell types revealed that betaAE-DMRIE was more active than DMRIE, and that betaAE-DMRIE, unlike DMRIE, was maximally effective in the absence of colipid. The consequences of the alcohol-to-amine conversion on the structure of the cytofectin/DNA complex was also examined by Atomic Force Microscopy. Strikingly dissimilar images were found for plasmid DNA alone and for the plasmid complexes of betaAE-DMRIE and DMRIE/DOPE.

Improved cationic lipid formulations for in vivo gene therapy.

The problem of assessing in vivo activity of gene delivery systems is complex. The reporter gene must be carefully chosen depending on the application. Plasmids with strong promoters, enhancers and other elements that optimize transcription and translation should be employed, such as the CMVint and pCIS-CAT constructs. Formulation aspects of cationic lipid-DNA complexes are being studied in several laboratories, and the physical properties and molecular organization of the complexes are being elucidated. Likewise, studies on the mechanism of DNA delivery with cationic lipids are accumulating which support the basic concept that the complexes fuse with biological membranes leading to the entry of intact DNA into the cytoplasm. Naked plasmid DNA administered by various routes is expressed at significant levels in vivo. This observation is not restricted to skeletal and heart muscle, but has been observed in lung, dermis, and in undefined tissues following intravenous administration. Most of the widely available cationic lipids, including Lipofectin, Lipofectamine and DC-cholesterol have a very poor ability to enhance DNA expression above the baseline naked DNA level, at least in lung. In this report we have revealed a novel cationic lipid, DLRIE, which can significantly enhance CAT expression in mouse lung by 25-fold above the naked DNA level. Other compounds are currently being evaluated which can enhance the naked DNA expression even higher. Plasmid vector improvements have led to further increase in in vivo lung expression, so that the net improvement is > 5,000-fold. Results of this nature are advancing the pharmaceutical gene therapy opportunities for synthetic cationic lipid based gene delivery systems.

Quantitative methods for scoring cell migration and invasion in filter-based assays.

The ability of cells to traverse pores in a biocompatible filter provides means for examining cell chemoattraction. Filter-based assays also permit rapid, quantitative assessment of the in vitro migratory and invasive potential of tumor cells. Scoring migration has relied on visual counting of stained cells which appear on the underside of the filter and determining a true percentage score involves arduous counting of cells on both filter surfaces. Visual counting of random fields may be unreliable, and counting all fields is laborious. In the present study we developed and compared two alternative methods for scoring cell numbers in filter-based assays, a colorimetric assay of toluidine blue binding, and a radioassay of cells prelabeled with [3H]thymidine. Each method was evaluated for sensitivity, variability, ease of use and efficiency, and suitability for use in assays of cell migration and invasion. The radiolabeling method proved to be sensitive and reliable and was the most efficient technique. Although less sensitive and specific, the colorimetric dye method offered a rapid and reliable, nonradioactive alternative with the distinct advantage of preserving intact cultures for follow-up visual assessments. We conclude that colorimetric and radiolabel scoring of filter-based assays are reliable and efficient semiautomated methods which provide means to obtain more complete assessments of cell migration and invasion.

Crown of microfilaments in the extending cytoplasmic processes of medulloblastoma glial progenitors.

Microfilaments and microtubules play a part in the extension of neuronal processes but their roles in the formation of glial processes have not yet been determined. The objectives of this study were to determine the organization of microfilaments in differentiating glial progenitors (RB2 cells) and to study the effects of microfilament or microtubule disruption on process extension. Intense F-actin staining (crown of microfilaments) was observed at the leading edge of a small extending conical tip in differentiating RB2 cells, but was absent in process-bearing TE671 rhabdomyosarcoma cells. No significant difference was noted in the mean number of TE671 cells with processes treated with a microfilament disrupter from that of similarly treated controls. In contrast, a significant difference was noted in the mean number of RB2 cells with processes after microfilament disruption treatment from that of similarly treated controls. Microtubule disruption arrested extension and caused process retraction in both cell types. The results of this study demonstrate that microtubules play an equally important part in the extension and stabilization of the RB2 and TE671 processes. Moreover, the crown of microfilaments concentrated in the glial RB2 process (and not in the TE671 process) may be critical to its extension during differentiation.