Gene therapy for diabetic peripheral neuropathy: A randomized, placebo-controlled phase III study of VM202, a plasmid DNA encoding human hepatocyte growth factor.

VM202 is a plasmid DNA encoding two isoforms of hepatocyte growth factor (HGF). A previous phase II study in subjects with painful diabetic peripheral neuropathy (DPN) showed significant reductions in pain. A phase III study was conducted to evaluate the safety and efficacy of VM202 in DPN. The trial was conducted in two parts, one for 9 months (DPN 3-1) with 500 subjects (VM202: 336 subjects; and placebo: 164) and a preplanned subset of 101 subjects (VM202: 65 subjects; and placebo: 36) with a noninterventional extension to 12 months (DPN 3-1b). VM202 or placebo was administered to calf muscles on days 0 and 14, and on days 90 and 104. The primary end point in DPN 3-1 was change from baseline in the mean 24-h Numerical Rating Scale (NRS) pain score. In DPN 3-1b, the primary end point was safety, whereas the secondary efficacy end point was change in the mean pain score. VM202 was well-tolerated in both studies without significant adverse events. VM202 failed to meet its efficacy end points in DPN 3-1. In DPN 3-1b, however, VM202 showed significant and clinically meaningful pain reduction versus placebo. Pain reduction in DPN 3-1b was even greater in subjects not receiving gabapentin or pregabalin, confirming an observation noted in the phase II study. In DPN 3-1b, symptomatic relief was maintained for 8 months after the last injection suggesting that VM202 treatment might change disease progression. Despite the perplexing discrepancy between the two studies, the safety and long-lasting pain-relieving effects of VM202 observed in DPN 3-1b warrant another rigorous phase III study. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Current therapies for painful diabetic peripheral neuropathy (DPN) are palliative and do not target the underlying mechanisms. Moreover, symptomatic relief is often limited with existing neuropathic pain drugs. Thus, there is a great medical need for safer and effective treatments for DPN. WHAT QUESTION DID THIS STUDY ADDRESS? Can nonviral gene delivery of hepatocyte growth factor reduce pain in patients with DPN and potentially modify progression of the disorder? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? Nonviral gene therapy can be used safely and practically to treat DPN. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? As the first gene medicine to enter advanced clinical trials for the treatment of DPN, this study provides the proof of concept of an entirely new potential approach to the disorder.

A phase 1/2a, dose-escalation, safety, pharmacokinetic, and preliminary efficacy study of intraperitoneal administration of BC-819 (H19-DTA) in subjects with recurrent ovarian/peritoneal cancer.

BACKGROUND: H19 is a paternally imprinted, oncofetal gene expressed in various embryonic tissues and in 85% of the ovarian tumors. H19-DTA (BC-819) is a DNA plasmid that drives the expression of the diphtheria toxin gene under the regulation of the H19 promoter sequence and therefore is a potential treatment for various tumors that overexpress the H19 gene, among them-ovarian cancer. OBJECTIVE: To assess the safety and efficacy of intra-peritoneal (IP) instillations of H19-DTA (BC-819) plasmid in treating ovarian/peritoneal cancer patients with advanced recurrent disease. METHODS: A phase 1-2A multi-centric trial included 14 eligible patients who were either platinum-refractory or platinum-resistant with positive H19 expression. Patients were treated IP with escalating weekly doses of BC-819 for a maximum of 6-9 weeks. Dose-limiting toxicities (DLT) were assessed after the first course of treatment for each patient and each subsequent cohort was enrolled once each subject had completed the first course of treatment and its 4-week follow-up period. The occurrence of adverse events (AEs) and response to treatment were assessed after the induction course and then periodically. RESULTS: During the study, no DLTs were observed. Only 5 grade 1 and 2 AEs, which occurred in 4 patients were considered as possibly related to BC-819. The best tumor response seen was stable disease. Median survivals of 3.2, 5.3 and 6.5 months were observed for the 60, 120 and 240 mg cohorts, respectively. CONCLUSIONS: BC-819 can be considered safe and well tolerated in intraperitoneal doses up to 240 mg. Hybridization of intraperitoneal chemotherapy with the biological treatment of BC-819 should be further evaluated in phase 2 and 3 studies.

A Phase l Study of a Tumor-targeted Systemic Nanodelivery System, SGT-94, in Genitourinary Cancers.

Gene therapy development has been limited by our inability to target multifocal cancer with systemic delivery. We developed a systemically administered, tumor-targeted liposomal nanodelivery complex (SGT-94) carrying a plasmid encoding RB94, a truncated form of the RB gene. In preclinical studies, RB94 showed marked cytotoxicity against tumor but not normal cells. SGT-94 was administered intravenously in a first-in-man study in metastatic genitourinary cancer. Minimal side effects were observed; dose-limiting toxicity (DLT) has not been reached in 11 evaluable patients. There was evidence of clinical activity at the 2.4 mg dose with one complete remission (CR) and one partial remission (PR). The patient in CR was retreated upon progression and had a second PR. Furthermore, there was tumor-specific targeting of the SGT-94 complex. One patient had wedge resections of two lung metastases which demonstrated RB94 expression at the DNA level by polymerase chain reaction (PCR) and at the protein level by Western blotting, with no RB94 present in normal contiguous lung. In conclusion, systemically delivered SGT-94 showed evidence of selective tumor targeting and was well tolerated with evidence of clinical activity. Additional studies are warranted to explore the activity of this drug as a single agent and in combination therapy.

Receptor-mediated gene delivery into human mesenchymal stem cells using hyaluronic acid-shielded polyethylenimine/pDNA nanogels.

Polyethylenimine (PEI) has been used as a vehicle to deliver genes to cancer cells and somatic cells. In this study, cationic polymers of PEI were shielded with anionic polymers of hyaluronic acid (HA) to safely and effectively deliver genes into human mesenchymal stem cells (hMSCs). HA interacted with CD44 in the plasma membranes of hMSCs to facilitate the internalization of HA-shielded PEI/pDNA complexes. The HA-shielded PEI/pDNA nanogels were confirmed by size changes, ζ-potential, and gel retardation assays. HA-shielded nanogels were easily internalized by hMSCs, and this was reduced by pretreatment with a specific monoclonal antibody that blocked CD44. By shielding PEI/pDNA complexes with HA, nanogels were easily internalized to hMSCs when it did not blocked by anti-CD44. These shielded nanogels were also easily internalized by HeLa cells, and this was reduced by pretreatment with an anti-CD44 monoclonal antibody. Following internalization of the SOX9 gene, chondrogenesis of hMSCs was increased, as determined by RT-PCR, real-time quantitative PCR, and histological analyses.

Safety and efficacy of plasmid DNA expressing two isoforms of hepatocyte growth factor in patients with critical limb ischemia.

VM202, a plasmid DNA that expresses two isoforms of hepatocyte growth factor, may elicit angiogenic effects that could benefit patients with critical limb ischemia (CLI). In a phase 2, double-blind trial in 52 CLI patients, we examined the safety and potential efficacy of intramuscular injections of low-dose (n=21) or high-dose (n=20) VM202 or placebo (n=11) in the affected limb (days 0, 14, 28 and 42). Adverse events and serious adverse events were similar among the groups; no malignancy or proliferative retinopathy was seen. In exploratory efficacy analyses, we found no differences in ankle or toe-brachial index, VAS, VascuQuol or amputation rate among the groups. Complete ulcer healing was significantly better in high-dose (8/13 ulcers; P<0.01) versus placebo (1/9) patients. Clinically meaningful reductions (>50%) in ulcer area occurred in high-dose (9/13 ulcers) and low-dose (19/27) groups versus placebo (1/9; P<0.05 and P<0.005, respectively). At 12 months, significant differences were seen in TcPO2 between the high-dose and placebo groups (47.5 ± 17.8 versus 36.6 ± 24.0 mm Hg, respectively; P<0.05) and in the change from baseline among the groups (P<0.05). These data suggest that VM202 is safe and may provide therapeutic bioactivity in CLI patients.

Pharmacokinetics and risk evaluation of DNA vaccine against Schistosoma japonicum.

DNA plasmid immunization is a novel approach of preventive and therapeutic vaccine. More than 100 DNA vaccines have been on preclinical or clinical phase trials, and four kinds of DNA vaccines for livestock have been approved by USDA, CFIA, and APVMA. Schistosomiasis is a worldwide parasitic disease, and vaccine immunization is supposed to be a promising approach to control the health crisis. On the basis of former preclinical studies, we further focused on the pharmacokinetics and risk evaluation of DNA vaccine in vivo. In the present study, enhanced green fluorescent protein (EGFP) report gene was fused with Schistosoma japonicum 23 kDa transmembrane protein antigen gene (Sj23) and constructed into DNA vaccine pVIVO2-Sj23.EGFP. After intramuscularly injecting 100 µg of purified DNA vaccine plasmid to immunizate BALB/c mice, we studied the tissue distribution of DNA plasmid and expressed Sj23.EGFP antigen, the persistence time of elicited antibodies, and the risk of DNA vaccine transferred into intestinal microorganisms. The results showed that DNA vaccine plasmid could be distributed into all tissues of the body after injection; however, only few organs including the injected muscle were detected DNA vaccine at postimmunization until the 100 days by PCR technology; the detection of green fluorescence protein displayed that DNA vaccine could be expressed in almost every tissue and organs; the ELISA assay indicated the immune antibody against Sj23 could persist over 70 days; and the DNA vaccine transferring intestinal flora results was negative. The results indicated that the DNA vaccine has systemic protection and long-lasting effectivity and is safe to intestinal flora.

Phase 1/2a, dose-escalation, safety, pharmacokinetic and preliminary efficacy study of intratumoral administration of BC-819 in patients with unresectable pancreatic cancer.

BC-819 is a DNA plasmid that was developed to target the expression of diphtheria-toxin gene under the control of H19 regulatory sequences. BC-819 has the potential to treat pancreatic cancer that overexpresses the H19 gene. The objectives were to assess the safety, tolerability, pharmacokinetics and preliminary efficacy of BC-819 administered intratumorally in subjects with unresectable, locally advanced, non-metastatic pancreatic cancer. Nine patients with unresectable pancreatic adenocarcinoma were enrolled in an open-label, dose-escalation trial. Subjects were entered into one out of two cohorts with escalating doses of BC-819. Each cohort received 2 weeks of twice weekly intratumoral injection of BC-819 under computerized tomography (CT) (n = 3) or endoscopic ultrasound (EUS) (n = 6) guidance. Patients were assessed by CT or positron emission tomography (PET)/CT during week 4 for tumor response. The maximum tolerated dose of BC-819 was not reached in this study at the highest dose. Asymptomatic elevation of lipase, which was considered as an adverse event with dose-limiting toxicity, occurred in only one subject in the high-dose group and was resolved spontaneously. The tumors did not increase in size 4 weeks after initiating treatment. Two weeks after completing the treatment, the two subjects who went on to receive subsequent chemotherapy or chemoradiation therapy, pancreatic tumors were downstaged and considered surgically resectable. Remarkably, three of the six subjects in cohort no. 2 evaluated at month 3 had a partial response. BC-819 can be safely administered intratumorally via EUS- or CT-guided injection at a dose of at least 8 mg per injection weekly twice. BC-819 given locally in combination with systemic chemotherapy may provide an additional therapeutic benefit for the treatment of pancreatic cancer.

Expression of MUC5AC in ocular surface epithelial cells using cationized gelatin nanoparticles.

Decreased production of the mucin MUC5AC in the eye is related to several pathological conditions, including dry eye syndrome. A specific strategy for increasing the ocular levels of MUC5AC is not yet available. Using a plasmid specially designed to encode human MUC5AC, we evaluated the ability of hybrid cationized gelatin nanoparticles (NPs) containing polyanions (chondroitin sulfate or dextran sulfate) to transfect ocular epithelial cells. NPs were developed using the ionic gelation technique and characterized by a small size (<200 nm), positive zeta potential (+20/+30 mV), and high plasmid association efficiency (>95%). MUC5AC mRNA and protein were detected in conjunctival cells after in vitro transfection of the NPs. The in vivo administration of the NPs resulted in significantly higher MUC5AC expression in the conjunctiva compared to untreated control and naked plasmid. These results provide a proof-of-concept that these NPs are effective vehicles for gene therapy and candidates for restoring the MUC5AC concentration in the ocular surface.

Local application of BMP-2 specific plasmids in fibrin glue does not promote implant fixation.

BACKGROUND: BMP-2 is known to accelerate fracture healing and might also enhance osseointegration and implant fixation. Application of recombinant BMP-2 has a time-limited effect. Therefore, a gene transfer approach with a steady production of BMP-2 appears to be attractive. The aim of this study was to examine the effect of locally applied BMP-2 plasmids on the bone-implant integration in a non-weight bearing rabbit tibia model using a comparatively new non-viral copolymer-protected gene vector (COPROG). METHODS: Sixty rabbits were divided into 4 groups. All of them received nailing of both tibiae. The verum group had the nails inserted with the COPROG vector and BMP-2 plasmids using fibrin glue as a carrier. Controls were a group with fibrin glue only and a blank group. After 28 and 56 days, these three groups were sacrificed and one tibia was randomly chosen for biomechanical testing, while the other tibia underwent histomorphometrical examination. In a fourth group, a reporter-gene was incorporated in the fibrin glue instead of the BMP-2 formula to prove that transfection was successful. RESULTS: Implant fixation strength was significantly lower after 28 and 56 days in the verum group. Histomorphometry supported the findings after 28 days, showing less bone-implant contact.In the fourth group, successful transfection could be confirmed by detection of the reporter-gene in 20 of 22 tibiae. But, also systemic reporter-gene expression was found in heterotopic locations, showing an undesired spreading of the locally applied gene formula. CONCLUSION: Our results underline the transfecting capability of this vector and support the idea that BMP-2 might diminish osseointegration. Further studies are necessary to specify the exact mechanisms and the systemic effects.

Hydrodynamics-based transfection of the combination of betacellulin and neurogenic differentiation 1 DNA ameliorates hyperglycemia in mice with streptozotocin-induced diabetes.

BACKGROUND: The biohazards caused by the viral delivery of pancreatic transcription factors, including neurogenic differentiation 1 (Neurod1) and Betacellulin (Btc), to the murine liver limit application of this procedure in reversing diabetes. We aimed to evaluate the feasibility of hydrodynamics-based transfection (HBT) with Neurod1 and Btc in improving hyperglycemia. METHODS: Murine hepatocellular carcinoma (Hepa1-6) cells were transfected with the combination of Neurod1-expressing plasmid, pcDNA3.1/V5-His A (pcDNA)-Neurod1, and Btc-expressing plasmid, pcDNA3.1/V5-His A (pcDNA)-Btc. Hepatic delivery of a combination of pcDNA-Neurod1 and pcDNA-Btc (experimental group) or pcDNA (control group) to mice with streptozocin-induced diabetes was achieved by HBT. The sequential serum glucose and alanine aminotransferase (ALT) levels were assessed. RESULTS: On day 3 after transfection, the transfection efficiencies of pcDNA-Btc and pcDNA-Neurod1 in the Hepa1-6 cells were 20% and 8%, respectively; respective values in the mouse livers were 30% and 10%. At 1 week after HBT, aside from hepatic expression of insulin, the experimental mice had a significantly lower sugar level (8-14 days after HBT, P values ranged from 0.034 to <0.001) than the control mice; the difference remained for 1 week but diminished afterward. The ALT levels and the body weight change were not different between the two groups. No mortality was noted in both groups. CONCLUSIONS: The hypoglycemic effect of Neurod1 and Btc delivered by HBT was transient and associated with negligible complications. In studies on the short-term hypoglycemic effects of Neurod1 and Btc in vivo, HBT is a potential alternative to viral delivery of Neurod1 and Btc to the murine liver.

Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.

Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 µg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

Efficient in vivo delivery of plasmids and oligonucleotides using hemagglutinating virus of Japan envelope (HVJ-E) vector in immunological disease models.

This unit describes a method for in vivo delivery of oligonucleotides or plasmids using the hemagglutinating virus of Japan envelope (HVJ-E), an inactivated Sendai virus particle, as a delivery system. Viral transfection methods generally show a higher transfection efficiency than nonviral methods for the delivery of genes to cells. However, in using these methods one must bear in mind that the introduction of a virus particle into a host carries a risk for leukemia induction and for creation of disturbances in immune function due to cytotoxicity.

Electroporation gene therapy preclinical and clinical trials for melanoma.

In vivo electroporation (EP) is a versatile delivery method for gene transfer which can be applied to any accessible tissue. Delivery of plasmid DNA encoding therapeutic genes or cDNAs with in vivo EP has been tested extensively in preclinical melanoma models. Direct delivery to the tumor has been shown to generate a direct antitumor effect. Delivery to alternative sites may generate additional therapeutic options, for example the production of cancer vaccines, the reduction of tumor angiogenesis, or the induction of tumor cell apoptosis. Several of the preclinical therapies tested have a demonstrated therapeutic effect against melanomas. Two immunotherapies have advanced to melanoma clinical trials. Delivery of a plasmid DNA encoding interleukin-12 (IL-12) or interleukin-2 (IL-2) using electroporation was demonstrated to be a safe with no grade 3 or 4 toxicities reported. Delivery of IL-12 with electroporation resulted in significant necrosis of melanoma cells in the majority of treated tumors and significant lymphocytic infiltrate in biopsies from patients in several cohorts. In addition, clinical evidence of responses in untreated lesions suggested the induction of a systemic response following therapy. This review discusses preclinically tested electroporation gene therapies for melanoma with clinical potential and the conversion of these therapies to clinical trials.

Biotin-directed assembly of targeted modular lipoplexes and their transfection of human hepatoma cells in vitro.

The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.

Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.

PURPOSE: Gene-based immunotherapy for cancer is limited by the lack of safe, efficient, reproducible, and titratable delivery methods. Direct injection of DNA into tissue, although safer than viral vectors, suffers from low gene transfer efficiency. In vivo electroporation, in preclinical models, significantly enhances gene transfer efficiency while retaining the safety advantages of plasmid DNA. PATIENTS AND METHODS: A phase I dose escalation trial of plasmid interleukin (IL)-12 electroporation was carried out in patients with metastatic melanoma. Patients received electroporation on days 1, 5, and 8 during a single 39-day cycle, into metastatic melanoma lesions with six 100-mus pulses at a 1,300-V/cm electric field through a penetrating six-electrode array immediately after DNA injection. Pre- and post-treatment biopsies were obtained at defined time points for detailed histologic evaluation and determination of IL-12 protein levels. RESULTS: Twenty-four patients were treated at seven dose levels, with minimal systemic toxicity. Transient pain after electroporation was the major adverse effect. Post-treatment biopsies showed plasmid dose proportional increases in IL-12 protein levels as well as marked tumor necrosis and lymphocytic infiltrate. Two (10%) of 19 patients with nonelectroporated distant lesions and no other systemic therapy showed complete regression of all metastases, whereas eight additional patients (42%) showed disease stabilization or partial response. CONCLUSION: This report describes the first human trial, to our knowledge, of gene transfer utilizing in vivo DNA electroporation. The results indicated this modality to be safe, effective, reproducible, and titratable.

Results of a double-blind, placebo-controlled study to assess the safety of intramuscular injection of hepatocyte growth factor plasmid to improve limb perfusion in patients with critical limb ischemia.

BACKGROUND: The Study to Assess the Safety of Intramuscular Injection of Hepatocyte Growth Factor Plasmid to Improve Limb Perfusion in Patients With Critical Limb Ischemia (HGF-STAT trial) determined the effect of hepatocyte growth factor (HGF) plasmid on safety and limb tissue perfusion as measured by transcutaneous oxygen tension (TcPo(2)) in patients with critical limb ischemia (CLI). METHODS AND RESULTS: Randomized patients with rest pain or ischemic ulcers and TcPo(2) <40 mm Hg and/or toe pressure <50 mm Hg received placebo or HGF-plasmid intramuscular injection as follows: 0.4 mg at days 0, 14, and 28 (low dose); 4.0 mg at days 0 and 28 (middle dose); or 4.0 mg at days 0, 14, and 28 (high dose). Patients were evaluated for safety, changes in TcPo(2) and ankle and toe pressure, amputation, and wound healing. Ninety-three of 104 treated patients were evaluated for safety (mean age 70 years, 63% male, 53% diabetic, 64% with tissue loss, mean ankle-brachial index 0.41, and mean toe pressure 26 mm Hg). Adverse events occurred in 86% of the patients, most of which were related to CLI or comorbid conditions and were not different between groups. TcPo(2) (mean+/-SE) increased at 6 months in the high-dose group (24.0+/-4.2 mm Hg, 95% CI 15.5 to 32.4 mm Hg) compared with the placebo (9.4+/-4.2 mm Hg, 95% CI 0.9 to 17.8), low-dose (11.1+/-3.7 mm Hg, CI 3.7 to 18.7 mm Hg), and middle-dose (7.3+/-4.8 mm Hg, CI -2.2 to 17.0 mm Hg) groups (ANCOVA P=0.0015). There was no difference between groups in secondary end points, including ankle-brachial index, toe-brachial index, pain relief, wound healing, or major amputation. CONCLUSIONS: Intramuscular injection of HGF plasmid was safe and well tolerated. Larger studies to determine whether HGF plasmid can improve wound healing and limb salvage in patients with CLI are warranted.

Safety and immunogenicity, after nasal application of HIV-1 DNA gagp37 plasmid vaccine in young mice.

BACKGROUND: There is a need for safe and potent adjuvants capable of delivering vaccine candidates over the mucosal barrier, with good capacity to stimulate both mucosal and systemic cell-mediated and humoral immunity. An adjuvant aimed for intranasal delivery should preferably deliver the antigen and minimize the transfer into the close proximity of the central nervous system, thus avoiding damage on the olfactory tissues. Advantages with a mucosal delivery route would be to provide mucosal and systemic immunity, requiring lower vaccine doses then when given parentally. The aim of this study was to study if the N3 adjuvant intranasally administered with HIV DNA plasmids would be transferred into the olfactory tissues and cause local inflammation and tissue damage. RESULTS: The N3 adjuvant alone and when combined with HIV-1 DNA gag plasmid and delivered intranasally did not cause detectable damage to the nasal epithelium or the olfactory epithelium or bulb over a period of 3 days after delivery. The intranasal administration of HIV-1 gagp37 DNA induced both a humoral and a cell-mediated immunity against the gag antigen. Significantly higher HIV-1-specific humoral, but not cell-mediated immune responses were seen in DNA/N3-immunized mice in comparison with HIV-1 DNA/saline-immunized animals. CONCLUSIONS: A safe and convenient intranasal mode of HIV-1 DNA plasmid and adjuvant delivery was shown not to interfere with the tissues in close proximity to the central nervous system. The N3 adjuvant combined with HIV-1 plasmids enhances the HIV-1-specific immunogenicity and merits to be clinically tested.

Myodys, a full-length dystrophin plasmid vector for Duchenne and Becker muscular dystrophy gene therapy.

Transgene SA is developing Myodys, a non-viral plasmid gene therapy for the potential treatment of Duchenne muscular dystrophy and Becker muscular dystrophy. Phase I clinical trials have been completed, and a phase II clinical trial is planned.

Evaluation of polyamidoamine dendrimer/alpha-cyclodextrin conjugate (generation 3, G3) as a novel carrier for small interfering RNA (siRNA).

As the first step toward an evaluation of the potential use of the PAMAM dendrimer (G3) conjugate with alpha-cyclodextrin (alpha-CDE) for a small interfering RNA (siRNA) carrier, the ternary complexes of alpha-CDE or the transfection reagents such as Lipofactamine 2000 (L2), TransFast (TF) and Lipofectin (LF) with plasmid DNA (pDNA) and siRNA were prepared, and their RNAi effects, cytotoxicity, physicochemical properties and intracellular distribution were compared. Here the pGL2 control vector (pGL2) and pGL3 control vector (pGL3) encoding the firefly luciferase gene and the two corresponding siRNAs (siGL2 and siGL3) were used. The ternary complexes of pGL3/siGL3/alpha-CDE showed the potent RNAi effects with negligible cytotoxicity compared to those of the transfection reagents in various cells. alpha-CDE strongly interacted with both pDNA and siRNA, and suppressed siRNA degradation by serum, compared to those of the transfection reagents. alpha-CDE allowed fluorescent labeled siRNA to distribute in cytoplasm, whereas the transfection reagents resided in both nucleus and cytoplasm in NIH3T3 cells. Furthermore, the binary complex of siRNA/alpha-CDE provided the significant RNAi effect in NIH3T3 cells transiently and stably expressing luciferase gene. These results suggest that alpha-CDE may be utilized as a novel carrier for siRNA.