Jukka Hartikka - Academia.edu (original) (raw)
Papers by Jukka Hartikka
Molecular Therapy, 2001
Electroporation has been reported to facilitate naked DNA gene transfer in skeletal muscle, but h... more 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.
Journal of Neuroscience Research, 1992
We studied how stimulation of protein kinase C and CAMP-dependent protein kinases affect the deve... more We studied how stimulation of protein kinase C and CAMP-dependent protein kinases affect the development of mesencephalic dopaminergic neurons in primary cell cultures derived from fetal rats at embryonic day E14. The effects of compounds which activate these second messenger systems were compared to those of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I). In mesencephalic cultures, there was a continuous loss of dopaminergic neurons. Despite this decline in cell number, neurotransmitter uptake per neuron increased with time, indicating that the surviving dopaminergic neurons continued their biochemical differen tiation while others degenerated. IGF-I and bFGF did not affect the number of dopaminergic neurons. However, dopamine uptake per neuron was significantly higher in bFGF and IGF-I treated cultures, suggesting that these factors stimulated differentiation. Protein kinase C and CAMP-dependent protein kinases were not involved in mediating the effects of bFGF and IGF-I. Treatment of cultures with phorbol esters did not affect dopamine uptake, whereas elevated levels of intracellular CAMP resulted in an increase in dopamine uptake which was additive to that elicited by bFGF or IGF-I. Further analysis revealed that exposure of mesencephalic cultures to dibutyryl CAMP (dbcAMP) during the first 3 days after plating increased the survival of dopaminergic neurons, whereas prolonged treatment attenuated the development of the dopamine uptake system. Moreover, cyclic AMP, but not bFGF, was able to prevent the degeneration of dopaminergic neurons induced by 1-methyl-4-phenyl-pyridinium ion (MPP+), the active metabolite of l-methyl-reveal novel possibilities for the treatment of Parkinson's disease.
European Journal of Neuroscience, 1995
The neuropathology of Parkinson's disease is characterized by the degeneration of dopaminergic ne... more The neuropathology of Parkinson's disease is characterized by the degeneration of dopaminergic neurons in the substantia nigra. We have recently shown that the activation of protein kinase A improves the survival of dopaminergic neurons in culture and, furthermore, protects them from the dopaminergic neurotoxin, l-methyl-4phenylpyridinium ion (MPP+) in vitro. We have now analysed the potential of phosphodiesterase inhibitors to increase cAMP levels in dopaminergic neurons, to improve their survival in culture and to protect them from the toxicity of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) in vivo. Increasing intracellular cAMP with phosphodiesterase type IV-specific inhibitors enhanced the survival of dopaminergic neurons in culture. Inhibitors of other phosphodiesterase types were not active. In vivo, phosphodiesterase type IV inhibitors reduced the MPTP-induced dopamine depletion in the striatum of C57BU6 mice. Furthermore, the loss of tyrosine hydroxylase-immunopositive neurons in the substantia nigra of these animals was diminished. After Nissl staining, a similar reduction of the MPTP-induced loss of neurons was observed in the substantia nigra. The protective effect of protein kinase A activation did not appear to be due to the blocking of MPP' uptake into dopaminergic neurons. This was not decreased after treatment with forskolin or 8-(4-~hlorophenylthio)-cAMP. Thus, protein kinase A regulates the survival and differentiation of dopaminergic substantia nigra neurons in vivo, implicating a therapeutic potential for substances which regulate cAMP turnover in these neurons.
Gene Quantification, 1996
... INJECTION J* g VR12231 DAY AFTER pgLUX PER UNIT n TISSUE SITE INJ. UNIT Skeletal Tibialis 50 ... more ... INJECTION J* g VR12231 DAY AFTER pgLUX PER UNIT n TISSUE SITE INJ. UNIT Skeletal Tibialis 50 7 1400000 20 Tibialis ant. ... Anal Biochem 188: 310-16. Conry RM, LoBuglio AF, andCuriel DT (1996): Polynucleotide-mediated immunization therapy of cancer. ...
Vaccine, 2001
This report characterizes Vaxfectin, a novel cationic and neutral lipid formulation which enhance... more 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 mg of pDNA formulated with Vaxfectin per muscle resulted in higher anti-NP titers than that obtained with 25 mg 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 b-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.
Vaccine, 2009
Mice were immunized either with unadjuvanted seasonal trivalent influenza vaccine (TIV) or TIV fo... more Mice were immunized either with unadjuvanted seasonal trivalent influenza vaccine (TIV) or TIV formulated with Vaxfectin ® , a cationic lipid-based adjuvant. Increasing doses of Vaxfectin ® resulted in increased hemagglutination-inhibition or anti-TIV ELISA antibody titers, with up to a 200-fold increase obtained with 900 g of Vaxfectin ®. A ≥10-fold dose-sparing effect was demonstrated with Vaxfectin ® formulations. Vaxfectin ® preferentially increased IgG2 titers compared to IgG1 titers, resulting in a balanced IgG isotype distribution. Lower doses of Vaxfectin ® (30 g) did not enhance antibody responses, but increased the number of IFN-␥ secreting T-cells by up to 18-fold. The data demonstrate that Vaxfectin ® enhances Th1 responses with protein-based seasonal influenza vaccine, and suggest that cellular or humoral immune responses may be preferentially induced by modifying the Vaxfectin ® :antigen ratio in the vaccine formulation.
The Journal of Gene Medicine, 2008
Background Plasmid DNA (pDNA) vaccines have generated significant interest for the prevention or ... more Background Plasmid DNA (pDNA) vaccines have generated significant interest for the prevention or treatment of infectious diseases. Broader applications may benefit from the identification of safe and potent vaccine adjuvants. This report describes the development of a novel polymer-based formulation to enhance the immunogenicity of pDNA-based vaccines. Methods Plasmid DNA was formulated with a nonionic block copolymer, poloxamer CRL1005, and the cationic surfactant benzalkonium chloride (BAK) to produce a thermodynamically stable, self-assembling system. The influence of parameters such as polymer concentration and BAK composition on the immune responses was evaluated in mice vaccinated with pDNA encoding influenza nucleoprotein. Results At concentrations of 7.5 mg/ml CRL1005, 0.3 mM BAK and 5 mg/ml pDNA, CRL1005/BAK/pDNA particles had a mean diameter of 261 ± 0.2 nm and a surface charge of −11.6 ± 0.9 mV. The negative surface charge and atomic force microscopy images suggested that pDNA binds to BAK adsorbed to the surface of poloxamer particles. The CRL1005/BAK/pDNA formulation significantly enhanced antigen-specific cellular and humoral immune responses, and increased transgene levels in muscle and serum. The complexity of the formulation was reduced by replacing the commercial BAK, which is a mixture of four alkyl chains, with a C14 BAK homolog. The substitution yielded an analytically preferable formulation with equivalent physical characteristics and immunogenicity. Conclusions The results suggest that the CRL1005/BAK/pDNA formulation may enhance immunogenicity by improving the delivery of pDNA-based vaccines. This formulation is currently being evaluated for the prevention of CMV-associated disease in a phase 2 clinical trial.
Human Gene Therapy, 1993
Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expre... more Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expression of the DNA in myofiber cells. We have examined the expression of firefly luciferase DNA constructs injected into adult murine skeletal muscle. Considerable variation in luciferase enzyme expression was noted among constructs with different regulatory elements, among different batches ofthe same DNA construct, and among similar transfection experiments performed at different times. This variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitative experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct containing the human cytomegalovirus immediate-early gene promoter plus intron A (a construct termed "p-CMVint-lux") showed the highest expression among several constructs tested. Doseresponse and time course analyses of p-CMVint-lux DNA injections showed that maximal luciferase expression was achieved with 25 ixg of DNA at 7-14 days post-injection. Selected manipulations ofthe transfection process were examined for their influence on luciferase expression. Variations in the rate of DNA injection, needle size, injection volume, and vehicle temperature had no signiflcant effect on luciferase expression. The presence of endotoxin, cationic peptide, muscle stimulants or relaxants, vasoconstrictors, metal chelators, or lysosomal lytic reagents had no signiflcant effect on expression. However, linearization ofthe DNA, injection ofthe DNA in water rather than saline, or inclusion of a DNA intercalating agent nearly abolished luciferase expression. And flnally, increasing the injection dose by giving multiple injections over a lO-day period increased expression proportionally to the number of injections.
Human Gene Therapy, 2005
Several formulated plasmid DNA (pDNA)-based vaccines are being evaluated for safety and efficacy ... more Several formulated plasmid DNA (pDNA)-based vaccines are being evaluated for safety and efficacy in healthy human subjects. A safety concern for any vaccine that contains genetic material, be it whole organism, liveattenuated, or gene-based, is the potential for integration into genomic DNA (gDNA). To address this concern, a preclinical pDNA persistence/integration study was conducted in rabbits to determine the level of pDNA in muscle 2, 28, and 64 days after intramuscular injection of DMRIE:DOPE-formulated pDNAs encoding Bacillus anthracis detoxified LF and PA proteins (VCL-AB01 vaccine). Total DNA was extracted from day 64 muscle tissue and fractionated by column agarose gel electrophoresis (CAGE). Plasmid copy number (PCN) in muscle 64 days after injection (geometric mean, 2808 PCN/g of total DNA or 150,000 diploid genomes) was determined by quantitative polymerase chain reaction. Analysis of total DNA from five VCL-AB01-injected rabbits revealed that two of five samples had no detectable PCN in the high molecular weight fraction after one round of CAGE, two samples had PCN under the lower limit of quantitation, and the remaining sample had 123 PCN/g. All PCN in the latter sample cleared after an additional round of CAGE. It appears, therefore, that persisting PCN fractionate as low molecular weight material and are most likely not integrated into gDNA. Even if the worst-case assumption is made that the highest PCN found associated with gDNA represented covalently integrated pDNA inserts, the frequency of mutation would still be 500-fold lower than the autosomal spontaneous mutation rate.
Human Gene Therapy, 2005
Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence an... more Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.
Human Gene Therapy, 1998
Enhancers and promoters from various muscle-specific genes were substituted for or combined with ... more Enhancers and promoters from various muscle-specific genes were substituted for or combined with the enhancer/promoter of the human cytomegalovirus (CMV) IE gene in a luciferase reporter gene plasmid in an effort to identify new promoter chimeras with increased expression activity after direct intramuscular injection. The regulatory sequence substitutions or additions varied in content, location, and orientation relative to the CMV regulatory sequences. The expression activities of the derivative and parent plasmids were compared quantitatively in vivo using a standard mouse intramuscular injection assay, and in vitro by transfection of differentiated C2C12 mouse myoblasts and BHK hamster kidney cells, to test whether cultured cell transfection could substitute for at least some animal experimentation. In vivo, 1 of 19 of the enhancer/promoter chimeras increased expression levels. In vitro, some chimeras showed significant expression augmentation in C2C12 cells, but not in BHK cells. We conclude that because of differences in plasmid expression profiles, these cell culture systems cannot readily substitute for in vivo testing of new plasmid constructs.
Gene Therapy, 2000
Intramuscular injection of plasmid DNA results in myofiber cell expression of proteins encoded by... more 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 -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 hema
Gene Therapy, 2001
MuStDO 5 is a multivalent plasmid DNA vaccine for malaria comprised of five plasmid DNAs encoding... more MuStDO 5 is a multivalent plasmid DNA vaccine for malaria comprised of five plasmid DNAs encoding five proteins from Plasmodium falciparum and one plasmid DNA encoding human GM-CSF. To evaluate the safety of MuStDO 5, a series of pre-clinical studies were conducted in mice and rabbits. In pharmacology studies in mice, GM-CSF could not be detected in the serum following either intramuscular or a combined intramuscular/intradermal administration of the vaccine, but was readily detected in the muscle following
Gene Therapy, 1997
Gene therapy for muscular diseases requires the efficient Soon after injection of DNA encoding cy... more Gene therapy for muscular diseases requires the efficient Soon after injection of DNA encoding cytoplasmic or transfection of a large proportion of myofiber cells within a nuclear-targeted -galactosidase, expression was noted given muscle. In the present experiments, patterns of predominantly in the myotendinous junction areas, after galactosidase expression were examined in mouse rectus which -galactosidase activity progressed toward the cenfemoris muscles at various time-points after a single injec-tral parts of the myofibers. This preferential transgene tion of lacZ encoded plasmid DNA. -Galactosidase expression at the myotendinous junction may result from expression was detected 3 h after injection and rose to some unique, local property of the myofiber cells and/or peak levels at 3-14 days, and then stabilized at lower lev-from a restricted diffusion or binding of the injected plasmid els. -Galactosidase staining was detected in an average DNA at tendinous surfaces. A better understanding of the of about 6% (up to 15%) of the total 4000 myofiber cells, reasons for this pattern of reporter gene expression in and in about 70% of those myofibers located in the discrete muscle may suggest procedures for increasing the number area containing the greatest proportion of transfected cells. of myofiber cells transfected by direct DNA injections.
Vaccine, 2001
Antigen specific immune responses were characterized after intramuscular immunization of BALB/c m... more 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-g 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.
Fibroblast growth factor-5 (FGF-5) is a member of the fibroblast growth factor gene family, which... more Fibroblast growth factor-5 (FGF-5) is a member of the fibroblast growth factor gene family, which has a signal sequence characteristic of secretory proteins. FGF-5 mRNA has previously been shown to be present in the adult mouse brain. Here we demonstrate that recombinant FGF-5 has neurotrophic activity on cultured rat septal cholinergic and raphe serotonergic neurons. The effect of FGF-5 on serotonin uptake was stronger than that evoked with either brain-derived neurotrophic factor or neurotrophin-3. FGF-5 also increased the choline acetyltransferase activity of cultured rat septal cholinergic neurons, the effect being additive to that of nerve growth factor. In situ hybridization experiments and irnmunohistochemistry using a specific anti-FGF-5 antibody demonstrated that FGF-5 is expressed in rat hippocampal neurons. Like nerve growth factor mRNA, the levels of FGF-5 mRNA in the rat hippocampus increased substantially during early postnatal development. In addition, injection of the rnuscarinic receptor agonist pilocarpine elevated FGF-5 mRNA. The presence of the secretory FGF-5 in the rat hippocampus, a target field of septal cholinergic and raphe serotonergic neurons, suggests that FGF-5 acts as a trophic factor for these neuroris also in vivo.
The Embo Journal, Apr 1, 1993
Serotonergic neurons located at the base of the mammalian brain innervate practically every regio... more Serotonergic neurons located at the base of the mammalian brain innervate practically every region of the brain and the spinal cord. These neurons exhibit spontaneous electrical discharges in a rhythmical way. Their firing frequency is modulated by serotonin autoreceptors which also regulate intracellular cAMP levels. We have investigated how elevated levels of cAMP alter the development and the functional properties of serotonergic neurons in culture. To study the influence of cAMP on the expression of genes underlying serotonergic activity, a quantitative RT-PCR approach using internal standards was developed. Cultures of embryonic rat brain serotonergic neurons were continuously treated with cAMP analogues. Increased cAMP levels had three effects. First, the neuronal morphology was changed towards that typical for mature serotonergic neurons. Second, the expression of tryptophan hydroxylase, the rate-limiting enzyme in serotonin production, was increased in dibutyryl-cAMP treated cultures. Third, the expression of the inhibitory autoreceptor (5-HT1A) was down-regulated. These results suggest the existence of a mechanism by which the neurons react to synaptic input regulating intracellular cAMP levels. Increased cAMP concentrations affect the development and cause a prolonged activation of serotonergic transmission. Since 5-HT1A receptors inhibit cAMP formation, their down-regulation argues against a negative feedback control in this system, consistent with observations in vivo.
Molecular Therapy, 2001
Electroporation has been reported to facilitate naked DNA gene transfer in skeletal muscle, but h... more 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.
Journal of Neuroscience Research, 1992
We studied how stimulation of protein kinase C and CAMP-dependent protein kinases affect the deve... more We studied how stimulation of protein kinase C and CAMP-dependent protein kinases affect the development of mesencephalic dopaminergic neurons in primary cell cultures derived from fetal rats at embryonic day E14. The effects of compounds which activate these second messenger systems were compared to those of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I). In mesencephalic cultures, there was a continuous loss of dopaminergic neurons. Despite this decline in cell number, neurotransmitter uptake per neuron increased with time, indicating that the surviving dopaminergic neurons continued their biochemical differen tiation while others degenerated. IGF-I and bFGF did not affect the number of dopaminergic neurons. However, dopamine uptake per neuron was significantly higher in bFGF and IGF-I treated cultures, suggesting that these factors stimulated differentiation. Protein kinase C and CAMP-dependent protein kinases were not involved in mediating the effects of bFGF and IGF-I. Treatment of cultures with phorbol esters did not affect dopamine uptake, whereas elevated levels of intracellular CAMP resulted in an increase in dopamine uptake which was additive to that elicited by bFGF or IGF-I. Further analysis revealed that exposure of mesencephalic cultures to dibutyryl CAMP (dbcAMP) during the first 3 days after plating increased the survival of dopaminergic neurons, whereas prolonged treatment attenuated the development of the dopamine uptake system. Moreover, cyclic AMP, but not bFGF, was able to prevent the degeneration of dopaminergic neurons induced by 1-methyl-4-phenyl-pyridinium ion (MPP+), the active metabolite of l-methyl-reveal novel possibilities for the treatment of Parkinson's disease.
European Journal of Neuroscience, 1995
The neuropathology of Parkinson's disease is characterized by the degeneration of dopaminergic ne... more The neuropathology of Parkinson's disease is characterized by the degeneration of dopaminergic neurons in the substantia nigra. We have recently shown that the activation of protein kinase A improves the survival of dopaminergic neurons in culture and, furthermore, protects them from the dopaminergic neurotoxin, l-methyl-4phenylpyridinium ion (MPP+) in vitro. We have now analysed the potential of phosphodiesterase inhibitors to increase cAMP levels in dopaminergic neurons, to improve their survival in culture and to protect them from the toxicity of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) in vivo. Increasing intracellular cAMP with phosphodiesterase type IV-specific inhibitors enhanced the survival of dopaminergic neurons in culture. Inhibitors of other phosphodiesterase types were not active. In vivo, phosphodiesterase type IV inhibitors reduced the MPTP-induced dopamine depletion in the striatum of C57BU6 mice. Furthermore, the loss of tyrosine hydroxylase-immunopositive neurons in the substantia nigra of these animals was diminished. After Nissl staining, a similar reduction of the MPTP-induced loss of neurons was observed in the substantia nigra. The protective effect of protein kinase A activation did not appear to be due to the blocking of MPP' uptake into dopaminergic neurons. This was not decreased after treatment with forskolin or 8-(4-~hlorophenylthio)-cAMP. Thus, protein kinase A regulates the survival and differentiation of dopaminergic substantia nigra neurons in vivo, implicating a therapeutic potential for substances which regulate cAMP turnover in these neurons.
Gene Quantification, 1996
... INJECTION J* g VR12231 DAY AFTER pgLUX PER UNIT n TISSUE SITE INJ. UNIT Skeletal Tibialis 50 ... more ... INJECTION J* g VR12231 DAY AFTER pgLUX PER UNIT n TISSUE SITE INJ. UNIT Skeletal Tibialis 50 7 1400000 20 Tibialis ant. ... Anal Biochem 188: 310-16. Conry RM, LoBuglio AF, andCuriel DT (1996): Polynucleotide-mediated immunization therapy of cancer. ...
Vaccine, 2001
This report characterizes Vaxfectin, a novel cationic and neutral lipid formulation which enhance... more 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 mg of pDNA formulated with Vaxfectin per muscle resulted in higher anti-NP titers than that obtained with 25 mg 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 b-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.
Vaccine, 2009
Mice were immunized either with unadjuvanted seasonal trivalent influenza vaccine (TIV) or TIV fo... more Mice were immunized either with unadjuvanted seasonal trivalent influenza vaccine (TIV) or TIV formulated with Vaxfectin ® , a cationic lipid-based adjuvant. Increasing doses of Vaxfectin ® resulted in increased hemagglutination-inhibition or anti-TIV ELISA antibody titers, with up to a 200-fold increase obtained with 900 g of Vaxfectin ®. A ≥10-fold dose-sparing effect was demonstrated with Vaxfectin ® formulations. Vaxfectin ® preferentially increased IgG2 titers compared to IgG1 titers, resulting in a balanced IgG isotype distribution. Lower doses of Vaxfectin ® (30 g) did not enhance antibody responses, but increased the number of IFN-␥ secreting T-cells by up to 18-fold. The data demonstrate that Vaxfectin ® enhances Th1 responses with protein-based seasonal influenza vaccine, and suggest that cellular or humoral immune responses may be preferentially induced by modifying the Vaxfectin ® :antigen ratio in the vaccine formulation.
The Journal of Gene Medicine, 2008
Background Plasmid DNA (pDNA) vaccines have generated significant interest for the prevention or ... more Background Plasmid DNA (pDNA) vaccines have generated significant interest for the prevention or treatment of infectious diseases. Broader applications may benefit from the identification of safe and potent vaccine adjuvants. This report describes the development of a novel polymer-based formulation to enhance the immunogenicity of pDNA-based vaccines. Methods Plasmid DNA was formulated with a nonionic block copolymer, poloxamer CRL1005, and the cationic surfactant benzalkonium chloride (BAK) to produce a thermodynamically stable, self-assembling system. The influence of parameters such as polymer concentration and BAK composition on the immune responses was evaluated in mice vaccinated with pDNA encoding influenza nucleoprotein. Results At concentrations of 7.5 mg/ml CRL1005, 0.3 mM BAK and 5 mg/ml pDNA, CRL1005/BAK/pDNA particles had a mean diameter of 261 ± 0.2 nm and a surface charge of −11.6 ± 0.9 mV. The negative surface charge and atomic force microscopy images suggested that pDNA binds to BAK adsorbed to the surface of poloxamer particles. The CRL1005/BAK/pDNA formulation significantly enhanced antigen-specific cellular and humoral immune responses, and increased transgene levels in muscle and serum. The complexity of the formulation was reduced by replacing the commercial BAK, which is a mixture of four alkyl chains, with a C14 BAK homolog. The substitution yielded an analytically preferable formulation with equivalent physical characteristics and immunogenicity. Conclusions The results suggest that the CRL1005/BAK/pDNA formulation may enhance immunogenicity by improving the delivery of pDNA-based vaccines. This formulation is currently being evaluated for the prevention of CMV-associated disease in a phase 2 clinical trial.
Human Gene Therapy, 1993
Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expre... more Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expression of the DNA in myofiber cells. We have examined the expression of firefly luciferase DNA constructs injected into adult murine skeletal muscle. Considerable variation in luciferase enzyme expression was noted among constructs with different regulatory elements, among different batches ofthe same DNA construct, and among similar transfection experiments performed at different times. This variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitative experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct containing the human cytomegalovirus immediate-early gene promoter plus intron A (a construct termed "p-CMVint-lux") showed the highest expression among several constructs tested. Doseresponse and time course analyses of p-CMVint-lux DNA injections showed that maximal luciferase expression was achieved with 25 ixg of DNA at 7-14 days post-injection. Selected manipulations ofthe transfection process were examined for their influence on luciferase expression. Variations in the rate of DNA injection, needle size, injection volume, and vehicle temperature had no signiflcant effect on luciferase expression. The presence of endotoxin, cationic peptide, muscle stimulants or relaxants, vasoconstrictors, metal chelators, or lysosomal lytic reagents had no signiflcant effect on expression. However, linearization ofthe DNA, injection ofthe DNA in water rather than saline, or inclusion of a DNA intercalating agent nearly abolished luciferase expression. And flnally, increasing the injection dose by giving multiple injections over a lO-day period increased expression proportionally to the number of injections.
Human Gene Therapy, 2005
Several formulated plasmid DNA (pDNA)-based vaccines are being evaluated for safety and efficacy ... more Several formulated plasmid DNA (pDNA)-based vaccines are being evaluated for safety and efficacy in healthy human subjects. A safety concern for any vaccine that contains genetic material, be it whole organism, liveattenuated, or gene-based, is the potential for integration into genomic DNA (gDNA). To address this concern, a preclinical pDNA persistence/integration study was conducted in rabbits to determine the level of pDNA in muscle 2, 28, and 64 days after intramuscular injection of DMRIE:DOPE-formulated pDNAs encoding Bacillus anthracis detoxified LF and PA proteins (VCL-AB01 vaccine). Total DNA was extracted from day 64 muscle tissue and fractionated by column agarose gel electrophoresis (CAGE). Plasmid copy number (PCN) in muscle 64 days after injection (geometric mean, 2808 PCN/g of total DNA or 150,000 diploid genomes) was determined by quantitative polymerase chain reaction. Analysis of total DNA from five VCL-AB01-injected rabbits revealed that two of five samples had no detectable PCN in the high molecular weight fraction after one round of CAGE, two samples had PCN under the lower limit of quantitation, and the remaining sample had 123 PCN/g. All PCN in the latter sample cleared after an additional round of CAGE. It appears, therefore, that persisting PCN fractionate as low molecular weight material and are most likely not integrated into gDNA. Even if the worst-case assumption is made that the highest PCN found associated with gDNA represented covalently integrated pDNA inserts, the frequency of mutation would still be 500-fold lower than the autosomal spontaneous mutation rate.
Human Gene Therapy, 2005
Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence an... more Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.
Human Gene Therapy, 1998
Enhancers and promoters from various muscle-specific genes were substituted for or combined with ... more Enhancers and promoters from various muscle-specific genes were substituted for or combined with the enhancer/promoter of the human cytomegalovirus (CMV) IE gene in a luciferase reporter gene plasmid in an effort to identify new promoter chimeras with increased expression activity after direct intramuscular injection. The regulatory sequence substitutions or additions varied in content, location, and orientation relative to the CMV regulatory sequences. The expression activities of the derivative and parent plasmids were compared quantitatively in vivo using a standard mouse intramuscular injection assay, and in vitro by transfection of differentiated C2C12 mouse myoblasts and BHK hamster kidney cells, to test whether cultured cell transfection could substitute for at least some animal experimentation. In vivo, 1 of 19 of the enhancer/promoter chimeras increased expression levels. In vitro, some chimeras showed significant expression augmentation in C2C12 cells, but not in BHK cells. We conclude that because of differences in plasmid expression profiles, these cell culture systems cannot readily substitute for in vivo testing of new plasmid constructs.
Gene Therapy, 2000
Intramuscular injection of plasmid DNA results in myofiber cell expression of proteins encoded by... more 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 -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 hema
Gene Therapy, 2001
MuStDO 5 is a multivalent plasmid DNA vaccine for malaria comprised of five plasmid DNAs encoding... more MuStDO 5 is a multivalent plasmid DNA vaccine for malaria comprised of five plasmid DNAs encoding five proteins from Plasmodium falciparum and one plasmid DNA encoding human GM-CSF. To evaluate the safety of MuStDO 5, a series of pre-clinical studies were conducted in mice and rabbits. In pharmacology studies in mice, GM-CSF could not be detected in the serum following either intramuscular or a combined intramuscular/intradermal administration of the vaccine, but was readily detected in the muscle following
Gene Therapy, 1997
Gene therapy for muscular diseases requires the efficient Soon after injection of DNA encoding cy... more Gene therapy for muscular diseases requires the efficient Soon after injection of DNA encoding cytoplasmic or transfection of a large proportion of myofiber cells within a nuclear-targeted -galactosidase, expression was noted given muscle. In the present experiments, patterns of predominantly in the myotendinous junction areas, after galactosidase expression were examined in mouse rectus which -galactosidase activity progressed toward the cenfemoris muscles at various time-points after a single injec-tral parts of the myofibers. This preferential transgene tion of lacZ encoded plasmid DNA. -Galactosidase expression at the myotendinous junction may result from expression was detected 3 h after injection and rose to some unique, local property of the myofiber cells and/or peak levels at 3-14 days, and then stabilized at lower lev-from a restricted diffusion or binding of the injected plasmid els. -Galactosidase staining was detected in an average DNA at tendinous surfaces. A better understanding of the of about 6% (up to 15%) of the total 4000 myofiber cells, reasons for this pattern of reporter gene expression in and in about 70% of those myofibers located in the discrete muscle may suggest procedures for increasing the number area containing the greatest proportion of transfected cells. of myofiber cells transfected by direct DNA injections.
Vaccine, 2001
Antigen specific immune responses were characterized after intramuscular immunization of BALB/c m... more 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-g 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.
Fibroblast growth factor-5 (FGF-5) is a member of the fibroblast growth factor gene family, which... more Fibroblast growth factor-5 (FGF-5) is a member of the fibroblast growth factor gene family, which has a signal sequence characteristic of secretory proteins. FGF-5 mRNA has previously been shown to be present in the adult mouse brain. Here we demonstrate that recombinant FGF-5 has neurotrophic activity on cultured rat septal cholinergic and raphe serotonergic neurons. The effect of FGF-5 on serotonin uptake was stronger than that evoked with either brain-derived neurotrophic factor or neurotrophin-3. FGF-5 also increased the choline acetyltransferase activity of cultured rat septal cholinergic neurons, the effect being additive to that of nerve growth factor. In situ hybridization experiments and irnmunohistochemistry using a specific anti-FGF-5 antibody demonstrated that FGF-5 is expressed in rat hippocampal neurons. Like nerve growth factor mRNA, the levels of FGF-5 mRNA in the rat hippocampus increased substantially during early postnatal development. In addition, injection of the rnuscarinic receptor agonist pilocarpine elevated FGF-5 mRNA. The presence of the secretory FGF-5 in the rat hippocampus, a target field of septal cholinergic and raphe serotonergic neurons, suggests that FGF-5 acts as a trophic factor for these neuroris also in vivo.
The Embo Journal, Apr 1, 1993
Serotonergic neurons located at the base of the mammalian brain innervate practically every regio... more Serotonergic neurons located at the base of the mammalian brain innervate practically every region of the brain and the spinal cord. These neurons exhibit spontaneous electrical discharges in a rhythmical way. Their firing frequency is modulated by serotonin autoreceptors which also regulate intracellular cAMP levels. We have investigated how elevated levels of cAMP alter the development and the functional properties of serotonergic neurons in culture. To study the influence of cAMP on the expression of genes underlying serotonergic activity, a quantitative RT-PCR approach using internal standards was developed. Cultures of embryonic rat brain serotonergic neurons were continuously treated with cAMP analogues. Increased cAMP levels had three effects. First, the neuronal morphology was changed towards that typical for mature serotonergic neurons. Second, the expression of tryptophan hydroxylase, the rate-limiting enzyme in serotonin production, was increased in dibutyryl-cAMP treated cultures. Third, the expression of the inhibitory autoreceptor (5-HT1A) was down-regulated. These results suggest the existence of a mechanism by which the neurons react to synaptic input regulating intracellular cAMP levels. Increased cAMP concentrations affect the development and cause a prolonged activation of serotonergic transmission. Since 5-HT1A receptors inhibit cAMP formation, their down-regulation argues against a negative feedback control in this system, consistent with observations in vivo.