Duration Of Transgene Expression Research Articles

Rubbing Gastric Serosal Surface Enhances Naked Plasmid DNA Transfer in Rats and Mice

We have developed in vivo gene transfer to mesothelial cells on the peritoneal organs, including the stomach. Simple instillation of naked plasmid DNA onto the gastric serosal surface in mice resulted in effective but transient transgene expression. Here, we developed a simple method to improve not only the transfection efficiency but also the duration of transgene expression. Rubbing the gastric serosal surface using a medical spoon immediately after instillation of naked plasmid DNA onto the gastric serosal surface resulted in 59-fold higher transgene expression 24 h after administration in rats. Without rubbing, transgene expression decreased under the detection limit 7 d after administration. On the other hand, rubbing the gastric serosal surface with a medical spoon after instillation of plasmid DNA prolonged transgene expression for one month. Mechanistic study in mice revealed that improved transfection should not be due to stimulation of cell function such as macropinocytosis by rubbing because rubbing before instillation of plasmid DNA did not improve transfection. Plasmid DNA should enter effectively into cells during rubbing. These findings are valuable to develop an effective method of in vivo gene transfer into peritoneal organs.

An Update on Canine Adenovirus Type 2 and Its Vectors

Adenovirus vectors have significant potential for long- or short-term gene transfer. Preclinical and clinical studies using human derived adenoviruses (HAd) have demonstrated the feasibility of flexible hybrid vector designs, robust expression and induction of protective immunity. However, clinical use of HAd vectors can, under some conditions, be limited by pre-existing vector immunity. Pre-existing humoral and cellular anti-capsid immunity limits the efficacy and duration of transgene expression and is poorly circumvented by injections of larger doses and immuno-suppressing drugs. This review updates canine adenovirus serotype 2 (CAV-2, also known as CAdV-2) biology and gives an overview of the generation of early region 1 (E1)-deleted to helper-dependent (HD) CAV-2 vectors. We also summarize the essential characteristics concerning their interaction with the anti-HAd memory immune responses in humans, the preferential transduction of neurons, and its high level of retrograde axonal transport in the central and peripheral nervous system. CAV-2 vectors are particularly interesting tools to study the pathophysiology and potential treatment of neurodegenerative diseases, as anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, oncolytic virus and as a platform to generate chimeric vectors.

Gene Transfer to the Cerebellum

There are several diseases for which gene transfer therapy to the cerebellum might be practicable. In these studies, we used recombinant Tag-deleted SV40-derived vectors (rSV40s) to study gene delivery targeting the cerebellum. These vectors transduce neurons and microglia very effectively in vitro and in vivo, and so we tested them to evaluate gene transfer to the cerebellum in vivo. Using a rSV40 vector carrying human immunodeficiency virus (HIV)-Nef with a C-terminal FLAG epitope, we characterized the distribution, duration, and cell types transduced. Rats received test and control vectors by stereotaxic injection into the cerebellum. Transgene expression was assessed 1, 2, and 4weeks later by immunostaining of serial brain sections. FLAG epitope-expressing cells were seen, at all times after vector administration, principally detected in the Purkinje cells of the cerebellum, identified as immunopositive for calbindin. Occasional microglial cells were tranduced; transgene expression was not detected in astrocytes or oligodendrocytes. No inflammatory or other reaction was detected at any time. Thus, SV40-derived vectors can deliver effective, safe, and durable transgene expression to the cerebellum.

Use of MGMT Resistance to Enrich for Transduced Hematopoietic Cells in Gene Therapy for Fabry Disease.

Abstract 3584Poster Board III-521Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase A (α-gal A) activity that results in the widespread accumulation of neutral glycosphingolipids. Renal failure, neuropathy, premature myocardial infarction, and stroke occur in patients with this condition due to deposition of globotriaosylceramide (Gb3) in vascular endothelial cells. Gb3 primarily originates from the breakdown of red blood cells. Currently, Fabry disease is mainly treated by enzyme replacement therapy (ERT). This treatment represents only a short-term therapeutic strategy and does not solve the basic genetic defect of the disorder. Fabry patients have also shown variable and muted response to ERT. Genetically-modified hematopoietic stem cells (HSCs) are attractive targets for durable transgene expression, however lower transduction efficiencies may be an obstacle to attainment of successful therapy. Therefore, our goal is to design and implement a recombinant lentiviral vector (LV) to confer drug tolerance to modified Fabry patient HSCs in order to enrich them in vivo. O6-methylguanine-DNA-methyltransferase (MGMT) overexpression provides cellular resistance to alkylating agents, which can be administered to kill residual untransduced HSCs while modified cells are protected. To this end, we have developed two different LV constructs. The first iteration was represented by a bicistronic LV encoding the human α-gal A cDNA followed by an EMCV-IRES element and a mutated, but functional, form of the selectable marker (MGMT-P140K). Next, a second iteration has been developed with the order of the transgenes reversed and carrying a codon-optimized copy of the human α-gal A cDNA. Expression and functionality of both cassettes were tested in the human erythroleukaemic cell line (K562) by intracellular MGMT staining and α-gal A activity assays, before and after selection with O6-benzylguanine (BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). We found that the second construct allowed sufficient MGMT expression for selection of transduced cells while maintaining α-gal A expression at appreciable levels. Flow cytometry analysis revealed that one drug treatment was sufficient to increase the MGMT-positive cell population from 15% to 90%, which resulted in a 2-fold increase in the enzyme activity. However, two treatments were required for achieving a similar result with the LV of the first iteration. The second LV also facilitated enrichment of transduced Fabry B cells in vitro. Future directions on this project include the modification of Fabry patient bone marrow cells, transplantation into a novel xenograft mouse model that we have recently created, and selection and amplification of the modified cells in vivo. Considering that clinical evidence suggests that even a slight increase in α-gal A activity may help to correct the disease, these results suggest that the MGMT-based drug selection system holds promise for providing a cure for Fabry Disease with a single gene therapy treatment. Disclosures:No relevant conflicts of interest to declare.

The contribution of plasmid design and release to in vivo gene expression following delivery from cationic polymer modified scaffolds

Tissue engineering scaffolds capable of gene delivery can provide a structure that supports tissue formation while also inducing the expression of inductive factors. Sustained release strategies are hypothesized to maintain elevated plasmid concentrations locally that can enhance gene transfer. In this report, we investigate the relationship between plasmid release kinetics and the extent and duration of transgene expression. Scaffolds were fabricated from polymer microspheres modified with cationic polymers (polyethylenimine, poly( l-lysine), poly(allylamine hydrochloride), polydiallyldimethylammonium) or polydopamine (PD), with PD enhancing incorporation and slowing release. In vivo implantation of scaffolds into the peritoneal fat pad had no significant changes in the level and duration of transgene expression between PD and unmodified scaffolds. Control studies with plasmid dried onto scaffolds, which exhibited a rapid release, and scaffolds with extended leaching to reduce initial quantities released had similar levels and duration of expression. Changing the plasmid design, from a cytomegalovirus (CMV) to an ubiquitin C (UbC) promoter substantially altered the duration of expression. These studies suggest that the initial dose released and vector design affect the extent and duration of transgene expression, which may be sustained over several weeks, potentially leading to numerous applications in cell transplantation and regenerative medicine.

Novel Minicircle Vector for Gene Therapy in Murine Myocardial Infarction

Conventional plasmids for gene therapy produce low-level and short-term gene expression. In this study, we develop a novel nonviral vector that robustly and persistently expresses the hypoxia-inducible factor-1 alpha (HIF-1alpha) therapeutic gene in the heart, leading to functional benefits after myocardial infarction. We first created minicircles (MC) carrying double-fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) for noninvasive measurement of transfection efficiency. Mouse C2C12 myoblasts and normal FVB/N mice were used for in vitro and in vivo confirmation, respectively. Bioluminescence imaging showed stable MC gene expression in the heart for >12 weeks and the activity level was 5.6+/-1.2-fold stronger than regular plasmid at day 4 (P<0.01). Next, we created MC carrying HIF-1alpha (MC-HIF-1alpha) therapeutic gene for treatment of myocardial infarction. Adult FVB/N mice underwent left anterior descending ligation and were injected intramyocardially with: (1) MC-HIF-1alpha; (2) regular plasmid carrying HIF-1alpha (PL-HIF-1alpha) as positive control; and (3) PBS as negative control (n=10/group). Echocardiographic study showed a significantly greater improvement of left ventricular ejection fraction in the MC group (51.3%+/-3.6%) compared to regular plasmid group (42.3%+/-4.1%) and saline group (30.5%+/-2.8%) at week 4 (P<0.05 for both). Histology demonstrated increased neoangiogenesis in both treatment groups. Finally, Western blot showed MC express >50% higher HIF-1alpha level than regular plasmid. Taken together, this is the first study to our knowledge to demonstrate that MC can significantly improve transfection efficiency, duration of transgene expression, and cardiac contractility. Given the serious drawbacks associated with most viral vectors, we believe this novel nonviral vector can be of great value for cardiac gene therapy protocols.

VEGF therapy: a timely retreat

The formation of new blood vessels is required for the onset and progression of many biological processes during embryonic development and into adulthood. Excessive neovascularization contributes to the pathogenesis of disorders such as tumour growth, chronic inflammation, and vascular retinopathies. On the other hand, an inadequate blood supply is a determining factor in the progression of tissue damage in ischaemic processes.1 In spite of the continuous efforts at prevention, brain and heart ischaemia as an end consequence of atherosclerosis is still a major cause of morbidity and mortality in western countries. A major challenge in cardiovascular research today is to improve revascularization of ischaemic tissues through the development of new treatments that can either substitute high-risk surgical procedures or be used as an adjuvant treatment. The increasing knowledge on the intricate mechanisms that regulate neovascularization has allowed the design of new therapies based on the administration of proangiogenic factors such as VEGF-A, FGF-1, -2 and -4, HGF, and Del-1 (therapeutic angiogenesis).2 A variety of approaches have been used to deliver proangiogenic factors to ischaemic tissues. Direct administration of recombinant factors (protein therapy) allows strict dose control; however, the maintenance of effective concentrations over prolonged periods is hard to achieve. Gene therapy with non-viral vectors, such as naked plasmid DNA, has a low transduction efficiency and the duration of transgene expression is limited. An alternative option is gene therapy with viral vectors, which provide higher levels of transduction and protein expression. The choice of viral vector will depend on the particular application; nevertheless, vector-specific characteristics related to transduction efficiency and biological safety … *Corresponding author. Tel: +34 91 4531200; fax: +34 91 453 1265. E-mail address : aalfranca{at}cnic.es

Gene Therapy in Thyroid Cancer

A variety of promising gene therapy approaches have been examined for treatment of follicular cell-derived and medullary thyroid cancer, including corrective gene therapy, cytoreductive gene therapy as well as immunomodulatory gene therapy. In addition, cloning of the NIS gene has provided us with a powerful cytoreductive gene therapy strategy based on targeted NIS gene transfer followed by radionculide ((131)I, (188)Re, (211)At) therapy. The data summarized in this article clearly demonstrate the high potential of currently available gene therapy approaches for future therapy of advanced dedifferentiated and medullary thyroid cancer, in particular as part of a multimodality approach. One of the major hurdles on the way to clinical application of gene therapy approaches in metastasized thyroid cancer is optimal tumor-specific targeting in the presence of low toxicity. Replication-selective viral vectors and novel biodegradable polymers as highly efficient nonviral vectors seem to be most promising candidates for the development of efficient and safe systemic gene therapy strategies. The bystander effect that is associated with some of the above listed gene therapy strategies provides a powerful means to compensate for the limited tumor spread of viral and nonviral vectors. Based on its dual function as therapy and reporter gene allowing noninvasive imaging by (123)I-scintigraphy and (124)I-PET imaging, NIS gene therapy offers the advantage of detailed characterization of in vivo vector biodistribution as well as localization, level, and duration of transgene expression - an essential prerequisite for exact planning and monitoring of clinical gene therapy trials with the aim of individualized therapy.

Long-term reduction of jaundice in Gunn rats by nonviral liver-targeted delivery of Sleeping Beauty transposon #

Asialoglycoprotein receptor (ASGPR)-mediated endocytosis has been used to target genes to hepatocytes in vivo. However, the level and duration of transgene expression have been low because of lysosomal translocation and degradation of the DNA and lack of its integration into the host genome. In this study we packaged the DNA of interest in proteoliposomes containing the fusogenic galactose-terminated F-glycoprotein of the Sendai virus (FPL) for targeted delivery to hepatocytes. After the FPL binds to ASGPR on the hepatocyte surface, fusogenic activity of the F-protein delivers the DNA into the cytosol, bypassing the endosomal pathway. For transgene integration we designed plasmids containing one transcription unit expressing the Sleeping Beauty transposase (SB) and another expressing human uridinediphosphoglucuronate glucuronosyltransferase-1A1 (pSB-hUGT1A1). The latter was flanked by inverted/direct repeats that are substrates of SB. In cell culture, FPL-mediated delivery of the E. coli beta-galactosidase gene (LacZ) resulted in transduction of ASGPR-positive cells (rat hepatocytes or Hepa1 cell line), but not of ASGPR-negative 293 cells. Intravenous injection of the FPL-entrapped pSB-hUGT1A1 (4-8 microg/day, 1-4 doses) into UGT1A1-deficient hyperbilirubinemic Gunn rats (model of Crigler-Najjar syndrome type 1) resulted in hUGT1A1 expression in 5%-10% of hepatocytes, but not in other cell types. Serum bilirubin levels declined by 30% +/- 4% in 2 weeks and remained at that level throughout the 7-month study duration. With histidine containing FPL, serum bilirubin was reduced by 40% +/- 5%, and bilirubin glucuronides were excreted into bile. No antibodies were detectable in the recipient rats against the F-protein or human UGT1A1. FPL is an efficient hepatocyte-targeted gene delivery platform in vivo that warrants further exploration toward clinical application.

872. Durable Transgene Expression by the Artificial Transcription Factor GAL4-VP16

872. Durable Transgene Expression by the Artificial Transcription Factor GAL4-VP16

647. Increasing the Duration of Transgene Expression of Interferon γ for Amelioration of Atopic Dermatitis in NC/Nga Mice

647. Increasing the Duration of Transgene Expression of Interferon γ for Amelioration of Atopic Dermatitis in NC/Nga Mice

Effect of the content of unmethylated CpG dinucleotides in plasmid DNA on the sustainability of transgene expression

Nonviral gene transfer generally suffers from short-term expression of transgenes. We have previously demonstrated that plasmids with reduced CpG content exhibited a more prolonged expression of murine interferon (IFN)-beta or IFN-gamma, which was effective in inhibiting metastatic tumor growth. A further extension of the duration of transgene expression could be achieved by controlling the number and location of CpG motifs in plasmid DNA. Luciferase-expressing plasmids with differing CpG content were injected into the tail vein of mice by the hydrodynamic injection method. The effects of CpG content on the duration of transgene expression were examined, focusing on cytosine methylation and pro-inflammatory cytokines. Based on the findings, IFN-gamma-expressing plasmids were constructed and their transgene expression and inhibitory effect on pulmonary metastasis were evaluated. Plasmids with a few CpG motifs showed a prolonged luciferase activity in the liver. Methylation of CpG motifs in plasmids reduced the expression and the extent of this reduction was greater for plasmids with a high CpG content. Pro-inflammatory cytokines hardly affected the expression. pCpG-Mu gamma, the IFN-gamma-expressing plasmid, which contains 20 CpG motifs only in the cDNA region, exhibited a sustained IFN-gamma concentration at therapeutic levels, and had a great inhibitory effect on the pulmonary metastasis of tumor cells. The duration of transgene expression of IFN-gamma was successfully increased by reducing the CpG content of IFN-expressing plasmid vector, which resulted in an increased anticancer activity of IFN gene transfer.

Comparative Study of Anti-hepatitis B Virus RNA Interference by Double-stranded Adeno-associated Virus Serotypes 7, 8, and 9

Using a hepatitis B virus (HBV) transgenic mouse model, we previously showed that a single dose of double-stranded adeno-associated virus (dsAAV) vector serotype 8 carrying a small hairpin RNA (shRNA) effectively reduces HBV replication and gene expression, but the effect gradually decreases with time. In this report, we compared the anti-HBV RNA interference (RNAi) effect of dsAAV8 with those of dsAAV7 and dsAAV9, two other hepatotropic AAV vectors, and examined whether the sequential use of these heterologous AAV vectors could prolong the anti-HBV effect. Our results showed that shRNA delivered by each of the three dsAAV vectors profoundly reduced the serum HBV titer and liver HBV mRNA and DNA levels in the transgenic mice for up to 22 weeks, with dsAAV8 having the greatest inhibitory effect, followed by dsAAV9 and dsAAV7. The potency of dsAAV8 correlated with the presence of higher levels of vector DNA and anti-HBV shRNA in the liver. An in vivo cross-administration experiment showed that preexisting anti-AAV8 antibody completely blocked the anti-HBV RNAi effect of dsAAV8, but had no effect on the potency of dsAAV7 and dsAAV9. Moreover, we demonstrated that a longer anti-HBV effect could be achieved by the sequential use of dsAAV8 and dsAAV9. These results indicate that effective and persistent HBV suppression might be achieved by a combination of the power of RNAi silencing effect and multiple treatments with different AAV serotypes.Molecular Therapy (2009) 17 2, 352-359 doi:10.1038/mt.2008.245.

Cytotoxic T lymphocyte responses to transgene product, not adeno-associated viral capsid protein, limit transgene expression in mice.

The use of adeno-associated viral (AAV) vectors for gene replacement therapy is currently being explored in several clinical indications. However, reports have suggested that input capsid proteins from AAV-2 vector particles may result in the stimulation of cytotoxic T lymphocyte (CTL) responses that can result in a loss of transduced cells. To explore the impact of anti-AAV CTLs on AAV-mediated transgene expression, both immunocompetent C57BL=6 mice and B cell-deficient muMT mice were immunized against the AAV2 capsid protein (Cap) and were injected intravenously with an AAV-2 vector encoding alpha-galactosidase (alpha-Gal). C57BL=6 mice, which developed both CTL and neutralizing antibody responses against Cap, failed to show any detectable alpha-Gal expression. In contrast, serum alpha-Gal levels comparable to those of naive mice were observed in muMT mice despite the presence of robust CTL activity against Cap, indicating that preexisting Cap-specific CTLs did not have any effect on the magnitude and duration of transgene expression. The same strategy was used to assess the impact of CTLs against the alpha-Gal transgene product on AAV-mediated gene delivery and persistence of transgene expression. Preimmunization of muMT mice with an Ad=alpha-Gal vector induced a robust CTL response to alpha-Gal. When these mice were injected with AAV2=alpha-Gal vector, initial levels of alpha-Gal expression were reduced by more than 1 log and became undetectable by 2 weeks postinjection. Overall, our results indicate that CTLs against the transgene product as opposed to AAV capsid protein are more likely to interfere with AAV transgene expression.

Safety and in vivo Expression of a GNE-Transgene: A Novel Treatment Approach for Hereditary Inclusion Body Myopathy-2

Hereditary inclusion body myopathy-2 (HIBM2) is an adult-onset, muscular disease caused by mutations in the GNE gene. HIBM2-associated GNE mutations causing hyposialyation have been proposed to contribute to reduced muscle function in patients with HIBM2, though the exact cause of this disease is unknown. In the current studies we examined pre-clinical in vivo toxicity, and expression of the plasmid-based, CMV driven wild-type GNE plasmid vector. The plasmid vector was injected intramuscularly (IM) or systemically (IV) into BALB/c mice, following encapsulation in a cationic liposome (DOTAP:Cholesterol). Single IM injections of the GNE-lipoplex at 40 microg did not produce overt toxicity or deaths, indicating that the no observable adverse effect level (NOAEL) dose for IM injection was >or=40 microg. Single intravenous (IV) infusion of GNE-lipoplex was lethal in 33% of animals at 100 microg dose, with a small proportion of animals in the 40 microg cohort demonstrating transient toxicity. Thus the NOAEL dose by the IV route was greater than 10 microg and less than or equal to 40 microg. Real-time RT-qPCR analysis demonstrated recombinant human GNE mRNA expression in 100% of muscle tissues that received IM injection of 40 microg GNE-lipoplex, at 2 weeks. These results indicate that GNE-lipoplex gene transfer is safe and can produce durable transgene expression in treated muscles. Our findings support future exploration of the clinical efficacy of GNE-lipoplex for experimental gene therapy of HIBM2.

Liposome-mediated gene therapy in the kidney.

Gene therapy directed to the kidney has been attempted to improve renal disorders such as inherited kidney diseases and common renal diseases that cause interstitial fibrosis, tubular atrophy, and glomerulosclerosis. Viral and non-viral vectors have been tried and been modulated to obtain sufficient transgene expression. However, gene delivery to the kidney is usually difficult because of characteristics of renal cell biology. Among non-viral vectors, the liposome system is a promising procedure for kidney-targeted gene therapy. Using cationic liposome, tubular cells were effectively transduced by retrograde injection of liposome/cDNA complex. Although transgene expression was reportedly modest using cationic liposomes, this method improved renal disease models such as carbonic anhydrase II deficiency and unilateral ureteral obstruction. In contrast, HVJ-liposome system is an effective transfection method to glomerular cells using intra-renal arterial infusion and improved glomerular disease models such as glomerulonephritis and glomerulosclerosis. In addition, intra-renal pelvic injection of DNA by HVJ-liposome system showed transgene expression in interstitial fibroblasts. In kidney-targeted gene therapy, liposome-mediated gene transfer is an attractive method because of its simplicity and reduced toxicity. In spite of modest transgene expression, several renal disease models were successfully modulated by liposome system. Although one limitation of liposome-mediated gene delivery is the duration of transgene expression, the liposome/cDNA complex can be repeatedly administered due to the absence of an immune response.

903. Pulmonary Transduction in Nonhuman Primates by HDAd: Duration of Transgene Expression and Vector Re-Administration

903. Pulmonary Transduction in Nonhuman Primates by HDAd: Duration of Transgene Expression and Vector Re-Administration

CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression

Pulmonary delivery of plasmid DNA (pDNA)/cationic liposome complexes is associated with an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response and brief duration of transgene expression. We demonstrate that retention of even a single CpG in pDNA is sufficient to elicit an inflammatory response, whereas CpG-free pDNA vectors do not. Using a CpG-free pDNA expression vector, we achieved sustained (>or=56 d) in vivo transgene expression in the absence of lung inflammation.

Characterization of the transgene expression generated by branched and linear polyethylenimine-plasmid DNA nanoparticles in vitro and after intraperitoneal injection in vivo

Polyethylenimine (PEI) is a cationic polymer that has shown significant potential for delivering genes in vitro and in vivo. Mixing cationic PEI with negatively charged plasmid DNA (pDNA) results in the spontaneous electrostatic formation of stable nanoparticle complexes. The structure of PEI can be branched or linear. In this study, we show that branched PEI has a stronger electrostatic interaction with pDNA than linear PEI, which accounts for greater compaction, higher zeta potentials and smaller nanoparticle sizes at equivalent pDNA concentrations. For both linear and branched PEI, increasing the concentration of pDNA mixed in the same volume and at the same nitrogen to phosphate (N:P) ratio results in larger average particle sizes. Increasing the N:P ratio increases luciferase activity generated by branched PEI-pDNA nanoparticles and linear PEI-pDNA nanoparticles in HEK293, COS7 and HeLa cell lines. Increasing the N:P ratio at which branched PEI-pDNA nanoparticles are prepared also increases luciferase expression in HepG2 cells but does not increase luciferase expression generated by linear PEI-pDNA nanoparticles. In all of the cell lines, branched PEI-pDNA nanoparticles prepared at N:P ratios of 10 and above generated significantly higher luciferase activity than linear PEI-pDNA nanoparticles. Luciferase activity was highest in the HEK293 cells and luciferase expression in each of the cell lines followed the order of HEK293>COS7>HepG2>HeLa. Intraperitoneal (IP) injection of PEI-pDNA nanoparticles is attractive because it is simple, reproducible and often leads to a depot effect of nanoparticle complexes residing in the peritoneum. The IP route of administration avoids PEI-pDNA nanoparticle accumulation in the lung and the nanoparticles do not pass through the blood-brain barrier. In this study, using bioluminescent imaging (BLI), we show that changing the PEI structure and dose of the PEI-pDNA nanoparticles has a significant impact on the strength and duration of transgene expression after IP injection in vivo but increasing the N:P ratio does not. Increasing the dose and N:P ratio for all the PEI-pDNA nanoparticle formulations injected IP did not reduce mice survival and all mice remained in good health as determined by the Body Condition Scoring (BCS) technique.

Pharmacokinetic considerations regarding non‐viral cancer gene therapy

Cancer gene therapy, in which pharmacologically active compounds are administered to cancer patients in a genetic form, has been examined not only in animals but also in cancer patients. Viral vector-induced severe side effects in patients have greatly underscored the importance of non-viral gene transfer methods. Even though the importance of pharmacokinetics is undoubtedly understood in the development of anticancer therapies, its importance has been less well recognized in non-viral cancer gene therapy. When transgene products express their activity within transduced cells, such as herpes simplex virus type 1 thymidine kinase and short hairpin RNA, the pharmacokinetics of the vectors and the expression profiles of the transgenes will determine the efficacy of gene transfer. The percentage of cells transduced is highly important if few by-stander effects are expected. If transgene products are secreted from cells into the blood circulation, such as interferons and interleukins, the pharmacokinetics of transgenes becomes a matter of significant importance. Then, any approach to increasing the level and duration of transgene expression will increase the therapeutic effects of cancer gene therapy. Here we review the pharmacokinetics of both non-viral vectors and transgene products, and discuss what should be done to achieve safer and more effective non-viral cancer gene therapy.