pDNA Dose Research Articles

Copy number of naked DNA delivered into nucleus of mammalian cells by electrotransfection

Electrotransfection is a non-viral method for delivery of nucleic acids into cells. In our previous study, we have determined the minimal copy number of plasmid DNA (pDNA) per cell required for transgene expression post electrotransfection, and developed a statistical framework to predict the pDNA copy number in the nucleus. To experimentally verify the prediction, the current study was designed to quantify the average copy number of pDNA per nucleus post electrotransfection. To achieve it, we developed a novel approach to effectively obtain isolated nuclei with minimal contamination by extranuclear pDNA. This sample preparation method enabled us to accurately measure intranuclear pDNA using quantitative real-time PCR. The data showed that the copy number of pDNA per nucleus was dependent on the period of cell culture post pulsing and the pDNA dose for electrotransfection. Additionally, the data were used to improve the statistical framework for understanding kinetics of pDNA transport in cells, and predicting how the kinetics depended on different factors. It is expected that the framework and the methodology developed in the current study will be useful for evaluating factors that may affect kinetics and mechanisms of pDNA transport in cells.

Clinically relevant dosing and pharmacokinetics of DNA-encoded antibody therapeutics in a sheep model

DNA-encoded delivery and in vivo expression of antibody therapeutics presents an innovative alternative to conventional protein production and administration, including for cancer treatment. To support clinical translation, we evaluated this approach in 18 40-45 kg sheep, using a clinical-matched intramuscular electroporation (IM EP) and hyaluronidase-plasmid DNA (pDNA) coformulation setup. Two cohorts of eight sheep received either 1 or 4 mg pDNA encoding an ovine anti-cancer embryonic antigen (CEA) monoclonal antibody (mAb; OVAC). Results showed a dose-response with average maximum serum concentrations of respectively 0.3 and 0.7 µg/ml OVAC, 4-6 weeks after IM EP. OVAC was detected in all 16 sheep throughout the 6-week follow-up, and no anti-OVAC antibodies were observed. Another, more exploratory, cohort of two sheep received a 12 mg pOVAC dose. Both animals displayed a similar dose-dependent mAb increase and expression profile in the first two weeks. However, in one animal, an anti-OVAC antibody response led to loss of mAb detection four weeks after IM EP. In the other animal, no anti-drug antibodies were observed. Serum OVAC concentrations peaked at 4.9 µg/ml 6 weeks after IM EP, after which levels gradually decreased but remained detectable around 0.2 to 0.3 µg/ml throughout a 13-month follow-up. In conclusion, using a delivery protocol that is currently employed in clinical Phase 1 studies of DNA-based antibodies, we achieved robust and prolonged in vivo production of anti-cancer DNA-encoded antibody therapeutics in sheep. The learnings from this large-animal model regarding the impact of pDNA dose and host immune response on the expressed mAb pharmacokinetics can contribute to advancing clinical translation.

GALA-Modified Lipid Nanoparticles for the Targeted Delivery of Plasmid DNA to the Lungs.

This study describes the development of lipid nanoparticles (LNPs) for the efficient and selective delivery of plasmid DNA (pDNA) to the lungs. The GALA peptide was used as a ligand to target the lung endothelium and as an endosomal escape device. Transfection activity in the lungs was significantly improved when pDNA was encapsulated in double-coated LNPs. The inner coat was composed of dioleoylphsophoethanolamine and a stearylated octaarginine (STR-R8) peptide, while the outer coat was largely a cationic lipid, di-octadecenyl-trimethylammonium propane, mixed with YSK05, a pH-sensitive lipid, and cholesterol. Optimized amounts of YSK05 and GALA were used to achieve an efficient and lung-selective system. The optimized system produced a high gene expression level in the lungs (>107 RLU/mg protein) with high lung/liver and lung/spleen ratios. GALA/R8 modification and the double-coating design were indispensable for efficient gene expression in the lungs. Despite the fact that NPs prepared with 1-step or 2-step coating have the same lipid amount and composition and the same pDNA dose, the transfection activity was dramatically higher in the lungs in the case of 2-step coating. Surprisingly, 1-step or 2-step coatings had no effect on the amount of nanoparticles that were delivered to the lungs, suggesting that the double-coating strategy substantially improved the efficiency of gene expression at the intracellular level.

Evaluation of transgene expression characteristics and DNA vaccination against melanoma metastasis of an intravenously injected ternary complex with biodegradable dendrigraft poly-L-lysine in mice

We developed a biocompatible splenic vector for a DNA vaccine against melanoma. The splenic vector is a ternary complex composed of plasmid DNA (pDNA), biodegradable dendrigraft poly-L-lysine (DGL), and γ-polyglutamic acid (γ-PGA), the selective uptake of which by the spleen has already been demonstrated. The ternary complex containing pDNA encoding luciferase (pCMV-Luc) exhibited stronger luciferase activity for RAW264.7 mouse macrophage-like cells than naked pCMV-Luc. Although the ternary complex exhibited strong luciferase activity in the spleen after its tail vein injection, luciferase activity in the liver and spleen was significantly decreased by a pretreatment with clodronate liposomes, which depleted macrophages in the liver and spleen. These results indicate that the ternary complex is mainly transfected in macrophages and is a suitable formulation for DNA vaccination. We applied the ternary complex to a pUb-M melanoma DNA vaccine. The ternary complex containing pUb-M suppressed the growth of melanoma and lung metastasis by B16-F10 mouse melanoma cells. We also examined the acute and liver toxicities of the pUb-M ternary complex at an excess pDNA dose in mice. All mice survived the injection of the excess amount of the ternary complex. Liver toxicity was negligible in mice injected with the excess amount of the ternary complex. In conclusion, we herein confirmed that the ternary complex was mainly transfected into macrophages in the spleen after its tail vein injection. We also showed the prevention of melanoma metastasis by the DNA vaccine and the safety of the ternary complex.

A statistical framework for determination of minimal plasmid copy number required for transgene expression in mammalian cells

Plasmid DNA (pDNA) has been widely used for non-viral gene delivery. After pDNA molecules enter a mammalian cell, they may be trapped in subcellular structures or degraded by nucleases. Only a fraction of them can function as templates for transcription in the nucleus. Thus, an important question is, what is the minimal amount of pDNA molecules that need to be delivered into a cell for transgene expression? At present, it is technically a challenge to experimentally answer the question. To this end, we developed a statistical framework to establish the relationship between two experimentally quantifiable factors — average copy number of pDNA per cell among a group of cells after transfection and percent of the cells with transgene expression. The framework was applied to the analysis of electrotransfection under different experimental conditions in vitro. We experimentally varied the average copy number per cell and the electrotransfection efficiency through changes in extracellular pDNA dose, electric field strength, and pulse number. The experimental data could be explained or predicted quantitatively by the statistical framework. Based on the data and the framework, we could predict that the minimal number of pDNA molecules in the nucleus for transgene expression was on the order of 10. Although the prediction was dependent on the cell and experimental conditions used in the study, the framework may be generally applied to analysis of non-viral gene delivery.

Highly versatile cell-penetrating peptide loaded scaffold for efficient and localised gene delivery to multiple cell types: From development to application in tissue engineering

Gene therapy has recently come of age with seven viral vector-based therapies gaining regulatory approval in recent years. In tissue engineering, non-viral vectors are preferred over viral vectors, however, lower transfection efficiencies and difficulties with delivery remain major limitations hampering clinical translation. This study describes the development of a novel multi-domain cell-penetrating peptide, GET, designed to enhance cell interaction and intracellular translocation of nucleic acids; combined with a series of porous collagen-based scaffolds with proven regenerative potential for different indications. GET was capable of transfecting cell types from all three germ layers, including stem cells, with an efficiency comparable to Lipofectamine® 3000, without inducing cytotoxicity. When implanted in vivo, GET gene-activated scaffolds allowed for host cell infiltration, transfection localized to the implantation site and sustained, but transient, changes in gene expression – demonstrating both the efficacy and safety of the approach. Finally, GET carrying osteogenic (pBMP-2) and angiogenic (pVEGF) genes were incorporated into collagen-hydroxyapatite scaffolds and with a single 2 μg dose of therapeutic pDNA, induced complete repair of critical-sized bone defects within 4 weeks. GET represents an exciting development in gene therapy and by combining it with a scaffold-based delivery system offers tissue engineering solutions for a myriad of regenerative indications.

Bioinspired Star-Shaped Poly(l-lysine) Polypeptides: Efficient Polymeric Nanocarriers for the Delivery of DNA to Mesenchymal Stem Cells.

The field of tissue engineering is increasingly recognizing that gene therapy can be employed for modulating in vivo cellular response thereby guiding tissue regeneration. However, the field lacks a versatile and biocompatible gene delivery platform capable of efficiently delivering transgenes to mesenchymal stem cells (MSCs), a cell type often refractory to transfection. Herein, we describe the extensive and systematic exploration of three architectural variations of star-shaped poly(l-lysine) polypeptide (star-PLL) with varying number and length of poly(l-lysine) arms as potential nonviral gene delivery vectors for MSCs. We demonstrate that star-PLL vectors are capable of self-assembling with pDNA to form stable, cationic nanomedicines. Utilizing high content screening, live cell imaging, and mechanistic uptake studies we confirm the intracellular delivery of pDNA by star-PLLs to MSCs is a rapid process, which likely proceeds via a clathrin-independent mechanism. We identify a star-PLL composition with 64 poly(l-lysine) arms and five l-lysine subunits per arm as a particularly efficient vector that is capable of delivering both reporter genes and the therapeutic transgenes bone morphogenetic protein-2 and vascular endothelial growth factor to MSCs. This composition facilitated a 1000-fold increase in transgene expression in MSCs compared to its linear analogue, linear poly(l-lysine). Furthermore, it demonstrated comparable transgene expression to the widely used vector polyethylenimine using a lower pDNA dose with significantly less cytotoxicity. Overall, this study illustrates the ability of the star-PLL vectors to facilitate efficient, nontoxic nucleic acid delivery to MSCs thereby functioning as an innovative nanomedicine platform for tissue engineering applications.

Ag85A DNA Vaccine Delivery by Nanoparticles: Influence of the Formulation Characteristics on Immune Responses.

The influence of DNA vaccine formulations on immune responses in combination with adjuvants was investigated with the aim to increase cell-mediated immunity against plasmid DNA (pDNA) encoding Mycobacterium tuberculosis antigen 85A. Different ratios of pDNA with cationic trimethyl chitosan (TMC) nanoparticles were characterized for their morphology and physicochemical characteristics (size, zeta potential, loading efficiency and pDNA release profile) applied in vitro for cellular uptake studies and in vivo, to determine the dose-dependent effects of pDNA on immune responses. A selected pDNA/TMC nanoparticle formulation was optimized by the incorporation of muramyl dipeptide (MDP) as an immunostimulatory agent. Cellular uptake investigations in vitro showed saturation to a maximum level upon the increase in the pDNA/TMC nanoparticle ratio, correlating with increasing Th1-related antibody responses up to a definite pDNA dose applied. Moreover, TMC nanoparticles induced clear polarization towards a Th1 response, indicated by IgG2c/IgG1 ratios above unity and enhanced numbers of antigen-specific IFN-γ producing T-cells in the spleen. Remarkably, the incorporation of MDP in TMC nanoparticles provoked a significant additional increase in T-cell-mediated responses induced by pDNA. In conclusion, pDNA-loaded TMC nanoparticles are capable of provoking strong Th1-type cellular and humoral immune responses, with the potential to be further optimized by the incorporation of MDP.

Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro-chemo-gene therapy.

Electroporation improves the anti-tumour efficacy of chemotherapeutic and gene therapies. Combining electroporation-mediated chemotherapeutics with interleukin 12 (IL-12) plasmid DNA produces a strong yet safe anti-tumour effect for treating primary and refractory tumours. A previously published report demonstrated the efficacy of a single cycle of IL-12 plasmid DNA and bleomycin in canines, and, similarly, this study further demonstrates the safety and efficacy of repeated cycles of chemotherapy plus IL-12 gene therapy for long-term management of aggressive tumours. Thirteen canine patients were enrolled in this study and received multiple cycles of electro-chemo-gene therapy (ECGT) with IL-12 pDNA and either bleomycin or gemcitabine. ECGT treatments are very effective for inducing tumour regression via an antitumour immune response in all tested histotypes except for sarcomas, and these treatments can quickly eradicate or debulk large squamous cell carcinomas. The versatility of ECGT allows for response-based modifications which can overcome treatment resistance for affecting refractory lesions. Importantly, not a single severe adverse event was noted even in animals receiving the highest doses of chemotherapeutics and IL12 pDNA over multiple treatment cycles. This report highlights the safety, efficacy and versatility of this treatment strategy. The data reveal the importance of inducing a strong anti-tumour response for successfully affecting not only the treated tumours, but also non-treated metastatic tumours. ECGT with IL12 pDNA plus chemotherapy is an effective strategy for treating multiple types of spontaneous cancers including large, refractory and multiple tumour burdens.

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.

Pharmacokinetics and transgene expression of implanted polyethylenimine-based pDNA complexes.

Locally delivered plasmid DNA (pDNA) is currently pursued as gene-based therapy for regenerative medicine, but important information on in situ pDNA pharmacokinetics and transgene expression is lacking in animal models. To investigate pDNA pharmacokinetics in implants, low molecular weight (2 kDa) polyethylenimine (PEI) and linoleic acid substituted 2 kDa PEI (PEI-LA) were used for pDNA delivery in gelatin sponges. An efficient pDNA extraction method combined with quantitative PCR (qPCR) was found to give equivalent quantitation of naked and polymer-bound pDNA, making it suitable to assess pDNA polyplexes in implants. Naked pDNA implanted in a rat subcutaneous model was >98% lost after 24 hours whereas PEI and PEI-LA delivered pDNA remained intact in implants for 2 and 4 weeks, respectively. Using a plasmid expressing DsRed as a reporter gene, mRNA and protein expression was observed only for PEI-LA despite the extended retention and cellular uptake of PEI complexes. The in vivo data were in agreement with in vitro results showing that only PEI-LA was an effective transfection agent even though both PEI and PEI-LA complexes were internalized by the cells. Dose dependence was observed for mRNA expression, with a 20 μg dose giving faster onset and higher expression levels compared to a 5 μg pDNA dose. The mRNA expression after PEI-LA mediated delivery was sustained for at least 4 weeks and a significant correlation between pDNA retention in sponges and mRNA expression was observed. In addition to establishing a promising gene carrier for gene delivery, these studies provided important information about the retention and transgene expression by implanted non-viral carriers.

Gelatin coating to stabilize the transfection ability of nucleic acid polyplexes

Amphiphilic polymers are effective in complexing and delivering therapeutic nucleic acids, such as plasmid DNA (pDNA) and short interfering RNA (siRNA). However, long-term stability of the complexes is not desirable, as it may have an impact on the transfection efficiency in vivo. To develop a method to preserve complex stability we first showed that pDNA complexes formed with the amphiphilic polymer linoleic acid-substituted polyethylenimine (PEI–LA) and incubated at 37°C lost ∼90% of their transfection efficiency after only 24h of complex formation. Polyethyleneglycol modification of complexes to control the increase in complex size and incubation in scaffolds used for implantation did not preserve the transfection ability of the complexes. Among a variety of approaches explored, gelatin coating of complexes was found to be the best at maintaining the original transfection efficiency. Mechanistic studies suggested that improved complex uptake, not size stability, was responsible for retention of the transfection efficiency. Similarly to the results with pDNA, gelatin coating also prevented the decreases in uptake and silencing efficiency of siRNA complexes observed following incubation at 37°C. Gelatin-stabilized complexes were, furthermore, effective in vivo and led to subcutaneous transgene expression with a low pDNA dose that was otherwise ineffective. We conclude that a simple gelatin coating approach offers an efficient means to preserve the transfection efficiency of polyplexes.

Cationic influenza virosomes as an adjuvanted delivery system for CTL induction by DNA vaccination

DNA vaccines have emerged as an attractive approach to induce CTL responses against cancer and infectious agents in recent years. Although CTL induction by DNA vaccination would be a valuable strategy for controlling viral infections, increasing the potency of DNA vaccines is mandatory before DNA vaccines can make it to the clinic. In this study, we developed and characterized a new and safe adjuvanted delivery system for DNA vaccination using cationic influenza virosomes (CIV). CIV were produced by reconstitution of detergent-solubilized influenza virus membranes in the presence of cationic lipids. Plasmid DNA (pDNA) mixed with these virosomes was efficiently transfected into cells of a mouse macrophage cell line (RAW-Blue cells). Moreover, the cells were effectively activated as demonstrated by production of an NFκB/AP-1-inducible reporter enzyme. Following three intradermal immunizations, CIV-delivered epitope-encoding pDNA induced equal numbers of IFNγ- and granzyme B-producing T cells than a 10-fold higher dose of naked pDNA. Virosomes without cationic lipids also improved induction of cellular immunity by pDNA but to a significantly lower extent than CIV. These findings suggest that pDNA–CIV complexes could be an efficacious delivery system suitable for CTL induction by DNA vaccination.

Vaxfectin-adjuvanted plasmid DNA vaccine improves protection and immunogenicity in a murine model of genital herpes infection

The herpes simplex type 2 (HSV-2) envelope glycoprotein (gD2) was evaluated as a potential antigen candidate for a plasmid DNA (pDNA)-based HSV-2 vaccine. The pDNA was formulated with Vaxfectin, a cationic lipid-based adjuvant, and tested in a murine HSV-2 lethal challenge model. gD2 was expressed as full-length (FL) and secreted (S) gD2 forms. A 0.1 µg pDNA dose was tested to distinguish treatment conditions for survival and a 100 µg pDNA dose was tested to distinguish treatment conditions for reduction in vaginal and latent HSV-2 copies. Vaxfectin-formulated gD2 pDNA significantly increased serum IgG titres and survival for both FL gD2 and S gD2 compared with gD2 pDNA alone. Mice immunized with FL gD2 formulated with Vaxfectin showed reduction in vaginal and dorsal root ganglia (DRG) HSV-2 copies. The stringency of this protection was further evaluated by testing Vaxfectin-formulated FL gD2 pDNA at a high 500 LD(50) inoculum. At this high viral challenge, the 0.1 µg dose of FL gD2 Vaxfectin-formulated pDNA yielded 80 % survival compared with no survival for FL gD2 pDNA alone. Vaxfectin-formulated FL gD2 pDNA, administered at a 100 µg pDNA dose, significantly reduced HSV-2 DNA copy number, compared with FL gD2 DNA alone. In addition, 40 % of mice vaccinated with adjuvanted FL pDNA had no detectable HSV-2 viral genomes in the DRG, whereas all mice vaccinated with gD2 pDNA alone were positive for HSV-2 viral genomes. These results show the potential contribution of Vaxfectin-gD2 pDNA to a future multivalent HSV-2 vaccine.

Role of DNA topology in uptake of polyplex molecules by dendritic cells.

Dendritic cells (DCs) are an attractive target for DNA vaccines as they are potent antigen presenting cells. This study demonstrated how non-viral gene delivery to DCs involving complexes of poly-l-lysine (PLL) and plasmid DNA (pDNA) (polyplexes) showed dependence on DNA vector topology. DNA topology is of importance from both production and regulatory viewpoints. In our previous study with CHO cells we demonstrated that polyplex uptake was dependent on DNA topology whereby complexes containing supercoiled (SC) pDNA were smaller, more resistant to nucleases and more effectively condensed by PLL than open circular (OC) and linear-pDNA complexes. In this study polyplex uptake in DCs was measured qualitatively and quantitatively by confocal microscopy along with gene expression studies and measurement of DC phenotype. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Quantification studies revealed that by 1h following uptake 15% (±2.59% relative standard error [RSE]) of SC-pDNA polyplexes were identified to be associated (fluorescent co-localisation) with the nucleus, in comparison to no nuclear association identified for OC- and linear-pDNA complexes. By 48 h following uptake, 30% (±1.82% RSE) of SC-pDNA complexes associated with the nucleus in comparison to 16% (±4.40% RSE) and 12% (±6.97% RSE) of OC- and linear-pDNA polyplexes respectively. Confocal microscopy images showed how DNA and PLL remained associated following uptake by dual labelling. Polyplex (containing 20 μg pDNA) gene expression (plasmid encoded lacZ [β-galactosidase] reporter gene) in DCs was greatest for SC-pDNA polyplexes at 14.12% unlike that of OC- (9.59%) and linear-pDNA (7.43%). DCs express cell surface markers which contribute towards antigen presentation. Polyplex gene expression did not alter DC phenotype through surface marker expression. This may be due to the pDNA dose employed (20μg) as other studies have used doses as high as 200 μg pDNA to induce DC phenotypic changes. Although no change in DC phenotype occurred, this could be advantageous in terms of biocompatibility. Collectively these results indicate that DNA topology is an important parameter for DC vector design, particularly pDNA in the SC conformation in regards to DNA vaccination studies.

Dose Response in Rodents and Nonhuman Primates After Hydrodynamic Limb Vein Delivery of Naked Plasmid DNA

The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle. Expression from the CMV promoter was highest. For mice, rats, and rhesus monkeys, the linear range for pDNA dose response could be defined by the mass of pDNA relative to the mass of target muscle. Correlation between pDNA dose and expression was linear between a threshold dose of 75 μg/g and maximal expression at approximately 400 μg/g. One HLV injection into rats of a dose of CMV-LacZ yielding maximal expression resulted in an average transfection of 28% of all hind leg muscle and 40% of the gastrocnemius and soleus. Despite an immune reaction to the reporter gene in monkeys, a single injection transfected an average of 10% of all myofibers in the targeted muscle of the arms and legs and an average of 15% of myofibers in the gastrocnemius and soleus.

Reconstitutable charged polymeric (PLGA) 2- b-PEI micelles for gene therapeutics delivery

This study investigated the potential of creating a charged polymeric micelle-based nucleic acid delivery system that could easily be reconstituted by the addition of water. (PLGA 36kDa) 2- b-bPEI 25kDa (PLGA MW 36 kDa, bPEI M w 25 kDa, PLGA:bPEI block ratio = 2) was synthesized and used to prepare cationic micelles. The copolymer retained proton-buffering capability from the bPEI block within the endosomal pH range. Micelle/pDNA complexes retained their particle size (100–150 nm) and surface charge (30–40 mV) following reconstitution. It was found that adding a small amount of low molecular weight bPEI (1.8 kDa) completely shielded pDNA in the micelle/pDNA complexes and enhanced transfection efficiency 50–100 fold for both fresh and reconstituted complexes without affecting complex size. Transfection efficiency for “reconstituted” micelle/pDNA/bPEI 1.8kDa (WR 1) complexes was 16-fold higher than its “fresh” counterpart. Although transfection levels achieved using “reconstituted” micelle/pDNA/bPEI 1.8kDa complexes were 3.6-fold lower than control “fresh” bPEI 25kDa/pDNA (N/P 5) complexes, transfection levels were 39-fold higher than “reconstituted” bPEI 25kDa/pDNA (N/P 5) complexes. The micelle/pDNA/bPEI 1.8kDa system showed very low cytotoxicity in MCF7 cells even with pDNA doses up to 20 μg, and transfection levels increased linearly with increasing pDNA dose. These results indicate that this PLGA- b-bPEI polymeric micelle-based system is well suited as a reconstitutable gene delivery system, and has high potential for use as a delivery system for gene therapy applications.

Functional Efficacy of Dystrophin Expression from Plasmids Delivered to mdx Mice by Hydrodynamic Limb Vein Injection

In these studies we delivered by hydrodynamic limb vein (HLV) injection plasmid DNA (pDNA) expressing the full-length mouse dystrophin gene to skeletal muscles throughout the hind limbs of the mdx mouse model for Duchenne muscular dystrophy (DMD). We evaluated the levels and stability of dystrophin expression and measured the resulting muscle protection, using Evans blue dye (EBD) to mark the damaged myofibers. Plasmid delivery was as efficient in the dystrophic mice as in wild-type mice and equally efficient in young adult and old mice, as long as the dose of pDNA was adjusted for the target muscle weight. The HLV gene delivery procedure was tolerated well by the dystrophic mice and repeat injections could be performed over an extended period of time. Multiple gene deliveries additively increased the amount of dystrophin protein and also increased the percentages of dystrophin-expressing myofibers. Plasmids expressing dystrophin from a cytomegalovirus (CMV) promoter construct containing the HMG1 intron provided stable dystrophin expression for the life of the mouse and provided significant benefit to the limbs. EBD staining showed that dystrophin gene delivery preserved myofibers in the CMV-HMGi-mDys-injected leg by 2.5- to 5-fold in large groups of muscles and by 2.5-fold throughout the injected legs, compared with the contralateral control legs injected with a nonexpressing plasmid. A similar degree of protection was measured in young adult mice evaluated soon after the last gene delivery and in aged mice injected over an extended period of time. This degree of protection resulted from 18 to 20% of the normal level of dystrophin protein, with 11-16% dystrophin-expressing myofibers. These studies show promise for the use of HLV injections to deliver therapeutic doses of full-length dystrophin-expressing plasmids for long-lasting protection of skeletal muscles in patients with DMD.

Transfection of Rat Pancreatic Islet Tissue by Polymeric Gene Vectors

In vitro genetic modification has been regarded as one option to improve the viability and functionality of pancreatic islets when used for transplantation in patients with diabetes, either as naked islets or in a type of bioartificial pancreas. In this approach, vector safety and poor transfection efficiency are major concerns. In this study, the influence of in vitro transfection conditions on polyplexes constructed of polyethyleneimine (PEI) and plasmid DNA (pDNA) on the transfection efficiency was investigated by varying the transfection medium, the pDNA dose, and the amines of polycation/phosphates of pDNA (N/P) ratio. Ca2+-containing Krebs-Ringer-HEPES medium was more effective than RPMI 1640 medium by increasing transfection efficiency (2.5-fold). An increase in pDNA dose slightly reduced the transfection efficiency but had minimal influence on islet loss. However, the N/P ratio had a large effect on islet viability and transfection efficiency. For example, the PEI/pDNA ratio at N/P = 10 caused greater islet loss (56% vs. 28%) and 30-fold less transfection efficiency than at N/P = 5. Even under a set of best conditions selected from this study, mostly a fraction of cells located in the peripheral regions of an islet were transfected, and the viability and insulin secretion from the treated islets were not altered. However, it was found that the extent of apoptosis was noticeably higher (approximately 16%) than in untreated islets (approximately 2%). These results suggest that the gene delivery efficacy to isolated islets can be improved by manipulating the transfection conditions. Polymeric vectors will broaden the options for islet transfection, which is currently limited to viral vectors.

Modified Escherichia coli B (BL21), a superior producer of plasmid DNA compared with Escherichia coli K (DH5α)

Plasmid DNA (pDNA) is an emerging experimental vaccine, produced in E. coli, initially targeted for viral diseases. Unlike traditional protein vaccines whose average dose is micrograms, the average dose of pDNA is on the scale of milligrams. Production yields are, therefore, important for the future development of this vaccine. The E. coli strains currently used for pDNA production, JM109 and DH5alpha, are both suitable for production of stable pDNA due to the deletion of recA and endA, however, these two E. coli K strains are sensitive to growth conditions such as high glucose concentration. On the other hand E. coli BL21 is less sensitive to growth conditions than E. coli JM109 or DH5alpha, this strain grows to higher densities and due to its active glyoxylate shunt and anaplerotic pathways is not sensitive to high glucose concentration. This strain is used for recombinant protein production but not for pDNA production because of its inability to produce stable pDNA. To adapt E. coli BL21 for stable pDNA production, the strain was mutated by deleting both recA and endA, and a proper growth and production strategy was developed. Production values, reaching 2 g/L were obtained using glucose as a carbon source. The produced plasmid, which was constructed for HIV clinical study, was found to have identical properties to the plasmid currently produced by E. coli DH5alpha.