Antibiotic-Free Gene Vectors: A 25-Year Journey to Clinical Trials.

Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors' production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical delivery techniques and to the refinement of their structure, non-viral plasmid DNA are now under intensive consideration as pharmaceutical drugs. Plasmids traditionally carry an antibiotic resistance gene for providing the selection pressure necessary for maintenance in a bacterial host. Nearly a dozen different antibiotic-free gene vectors have now been developed and are currently assessed in preclinical assays and phase I/II clinical trials. Their reduced size leads to increased transfection efficiency and prolonged transgene expression. In addition, associating non-viral gene vectors and DNA transposons, which mediate transgene integration into the host genome, circumvents plasmid dilution in dividing eukaryotic cells which generate a loss of the therapeutic gene. Combining these novel molecular tools allowed a significantly higher yield of genetically engineered T and Natural Killer cells for adoptive immunotherapies due to a reduced cytotoxicity and increased transposition rate. This review describes the main progresses accomplished for safer, more efficient and cost-effective gene and cell therapies using non-viral approaches and antibiotic-free gene vectors.

Predictors of Clinical Failure after Endoscopic Lumbar Spine Surgery During the Initial Learning Curve.

OBJECTIVE: This study aims to identify clinical factors that may predict failed endoscopic lumbar spine surgery to guide surgeons with patient selection during the initial learning curve. METHODS: This is an Australasian prospective analysis of the first 105 patients to undergo lumbar endoscopic spine decompression by 3 surgeons. Modified MacNab outcomes, visual analog scale (VAS) and Oswestry Disability Index (ODI) scores were utilized to evaluate clinical outcomes at 6 months postoperatively. Descriptive statistics and ANOVA t tests were performed to measure statistically significant (P < 0.05) associations between variables using GraphPad Prism v10. RESULTS: Patients undergoing endoscopic lumbar surgery via an interlaminar or transforaminal approach have overall good/excellent modified MacNab outcomes and a significant reduction in postoperative VAS and ODI scores. Regardless of the anatomic location of disc herniations, good/excellent modified MacNab outcomes and significant reductions in VAS and ODI were reported post-operatively, however, not in patients with calcified disc herniations. Patients with central and foraminal stenosis overall reported poor/fair modified MacNab outcomes, however, there were significant reductions in VAS and ODI scores postoperatively. Patients with subarticular stenosis or an associated spondylolisthesis reported good/excellent modified MacNab outcomes and significant reductions in VAS and ODI scores postoperatively. Patients with disc herniation and concurrent degenerative stenosis had generally poor/fair modified MacNab outcomes. CONCLUSIONS: The outcomes of endoscopic spine surgery are encouraging with low complication and reoperation rates. However, patients with calcified disc herniations, central canal stenosis, or disc herniation with concurrent degenerative stenosis present challenges during the initial learning curve and may benefit from traditional open or other minimally invasive techniques.

H2 O2 -Induced Persistent Luminescence Signal Enhancement Applied to Biosensing.

Persistent luminescence nanoparticles (PLNPs) are innovative materials able to emit light for a long time after the end of their excitation. Thanks to this property, their detection can be separated in time from the excitation, making it possible to obtain images with a high signal-to-noise ratio. This optical property can be of particular interest for the development of in vitro biosensors. Here, we report the unexpected effect of hydrogen peroxide (H2 O2 ) on the signal intensity of ZnGa2 O4 :Cr3+ (ZGO) nanoparticles. In the presence of H2 O2 , the signal intensity of ZGO can be amplified. This signal amplification can be used to detect and quantify H2 O2 in various media, using non-functionalized ZGO nanoparticles. This small molecule can be produced by several oxidases when they react with their substrate. Indeed, the quantification of glucose, lactic acid, and uric acid is possible. The limit of detection could be lowered by modifying the nanoparticles synthesis route. These optimized nanoparticles can also be used as new biosensor to detect larger molecules such as antigen, using the appropriate antibody. This unique property, i.e., persistent luminescence signal enhancement induced by H2 O2 , represents a new way to detect biomolecules which could lead to a very large number of bioassay applications.

Carbopol dispersed PAA-modified UIO-66 with high colloidal stability as a combination nano-adjuvant boosts immune response and protection against pseudorabies virus in mice and pigs.

Although inactivated vaccines have higher safety than live-attenuated vaccines in the control of pseudorabies virus (PRV), their protection efficacy is limited due to insufficient immunogenicity when used alone. High-performance adjuvants that can potentiate immune responses are highly desirable to improve the protection efficacy of inactivated vaccines. In this work, we have developed U@PAA-Car, a Carbopol dispersed zirconium-based metal-organic framework UIO-66 modified by polyacrylic acid (PAA), as a promising adjuvant for inactivated PRV vaccines. The U@PAA-Car has good biocompatibility, high colloidal stability, and antigen (vaccine) loading capacity. It significantly potentiates humoral and cellular immune responses over either U@PAA, Carbopol, or commercial adjuvants such as Alum and biphasic 201 by inducing a higher specific antibody titer, IgG2a/IgG1 ratio, cell cytokine secretion, and splenocyte proliferation. A protection rate of over 90% was observed in challenge tests in the model animal mice and the host animal pigs, which is much higher than that observed with commercial adjuvants. The high performance of the U@PAA-Car is attributed to antigen sustainable release at the injection site and highly efficient antigen internalization and presentation. In conclusion, this work not only demonstrates a great potential of the developed U@PAA-Car nano-adjuvant for the inactivated PRV vaccine but also gives a preliminary explanation of its action mechanism. STATEMENT OF SIGNIFICANCE: We have developed a Carbopol dispersed PAA-modified zirconium-based metal-organic framework UIO-66 (U@PAA-Car) as a promising combination nano-adjuvant for the inactivated PRV vaccine. The U@PAA-Car induced higher specific antibody titers and IgG2a/IgG1 ratio, increased cell cytokines secretion, and better splenocyte proliferation than U@PAA, Carbopol, and the commercial adjuvants Alum and biphasic 201, indicating that it induces a significant potentiation of humoral and cellular immune response. In addition, much higher protection rates were achieved with the U@PAA-Car-adjuvanted PRV vaccine in mice and pigs challenge than those observed from the commercial adjuvant groups. This work not only demonstrates the great potential of the U@PAA-Car nano-adjuvant in an inactivated PRV vaccine but also gives a preliminary explanation of its action mechanism.

Liposome Biodistribution via Europium Complexes.

Vector biodistribution is a requirement prior pharmaceutical development. Radioactive tracers allow the most sensitive and quantitative assessment of biodistribution, and conventional fluorophores are widely used in academic laboratories. We propose here to use europium complexes as a label for nanoparticles or biotherapeutics taking liposomes as models. Time-resolved fluorimetry (TRF) has the tremendous advantage of taking into accounts the fluorescence decay time of the lanthanide chelates, resulting in an improved sensitivity in biological media. The work described aimed following liposome biodistribution by TRF. An octadecyl-DTPA.Eu compound has been prepared and incorporated into liposomes without altering its fluorescence signal. The method has been validated through a comparison with fluorophore-labeled liposomes. The way to proceed when using this method for liposome biodistribution assessment is detailed. It could obviously be applied to other nanosystems, such as lipid nanoparticles.

GMP-Grade Manufacturing and Quality Control of a Non-Virally Engineered Advanced Therapy Medicinal Product for Personalized Treatment of Age-Related Macular Degeneration.

The introduction of new therapeutics requires validation of Good Manufacturing Practice (GMP)-grade manufacturing including suitable quality controls. This is challenging for Advanced Therapy Medicinal Products (ATMP) with personalized batches. We have developed a person-alized, cell-based gene therapy to treat age-related macular degeneration and established a vali-dation strategy of the GMP-grade manufacture for the ATMP; manufacturing and quality control were challenging due to a low cell number, batch-to-batch variability and short production duration. Instead of patient iris pigment epithelial cells, human donor tissue was used to produce the transfected cell product ("tIPE"). We implemented an extended validation of 104 tIPE productions. Procedure, operators and devices have been validated and qualified by determining cell number, viability, extracellular DNA, sterility, duration, temperature and volume. Transfected autologous cells were transplanted to rabbits verifying feasibility of the treatment. A container has been engineered to ensure a safe transport from the production to the surgery site. Criteria for successful validation and qualification were based on tIPE's Critical Quality Attributes and Process Parameters, its manufacture and release criteria. The validated process and qualified operators are essential to bring the ATMP into clinic and offer a general strategy for the transfer to other manufacture centers and personalized ATMPs.

Fate and biological impact of persistent luminescence nanoparticles after injection in mice: a one-year follow-up.

Persistent luminescence nanoparticles (PLNPs) are attracting growing interest for non-invasive optical imaging of tissues with a high signal to noise ratio. PLNPs can emit a persistent luminescence signal through the tissue transparency window for several minutes, after UV light excitation before systemic administration or directly in vivo through visible irradiation, allowing us to get rid of the autofluorescence signal of tissues. PLNPs constitute a promising alternative to the commercially available optical near infrared probes thanks to their versatile functionalization capabilities for improvement of the circulation time in the blood stream. Nevertheless, while biodistribution for a short time is well known, the long-term fate and toxicity of the PLNP's inorganic core after injection have not been dealt with in depth. Here we extend the current knowledge on ZnGa1.995O4Cr0.005 NPs (or ZGO) with a one-year follow-up of their fate after a single systemic administration in mice. We investigated the organ tissue uptake of ZGO with two different coatings and determined their intracellular processing up to one year after injection. The biopersistence of ZGO was assessed, with a long-term retention, quantified by ICP-MS, mostly in the liver and spleen, parallel with a loss of their luminescence properties. The analysis of the toxicity related to combining an animal's weight, key hematological and metabolic markers, histological observations of liver tissues and quantification of the expression of 31 genes linked to different metabolic reactions did not reveal any signs of noxiousness, from the macro scale to the molecular level. Therefore, the ZGO imaging probe has been proven to be a safe and relevant candidate for preclinical studies, allowing its long term use without any in vivo disturbance of the general metabolism.

A hollow Co3-xCuxS4 with glutathione depleting and photothermal properties for synergistic dual-enhanced chemodynamic/photothermal cancer therapy.

Chemodynamic therapy has become an emerging cancer treatment strategy, in which tumor cells are killed through toxic reactive oxygen species (ROS), especially hydroxyl radicals (˙OH) produced by the Fenton reaction. Nevertheless, low ROS generation efficiency and ROS depletion by cellular antioxidant systems are still the main obstacles in chemodynamic therapy. In the present work, we propose a dually enhanced chemodynamic therapy obtained by inhibiting ˙OH consumption and promoting ˙OH production based on the administration of bimetallic sulfide Co3-xCuxS4 nanoparticles functionalized by polyethylene glycol. These bimetallic nanoparticles display glutathione depleting and photothermal properties. The nanoparticles are gradually degraded in a tumor microenvironment, resulting in Co2+ and Cu2+ release. The released Co2+ triggers a Fenton-like reaction that turns endogenous hydrogen peroxide into highly toxic ˙OH. In the cellular environment, Cu2+ ions are reduced to Cu+ by endogenous GSH, which decreases the intracellular antioxidant capacity and additionally up-regulates ˙OH production via the Cu+-induced Fenton-like reaction. Moreover, under near-infrared light irradiation, the bimetallic nanoparticles display a photothermal conversion efficacy of 46.7%, which not only improves chemodynamic therapy via boosting a Fenton-like reaction but results in photothermal therapy through hyperthermia. Both in vitro cancer cell killing and in vivo tumor ablation experiments show that the bimetallic nanoparticles display outstanding therapeutic efficacy and negligible systemic toxicity, indicating their anticancer potential.

Combination of thermal ablation by focused ultrasound, pFAR4-IL-12 transfection and lipidic adjuvant provide a distal immune response.

Aim: Gene-based immunotherapy against cancer is limited by low gene transfer efficiency. In the literature, interleukin-12 (IL-12) encoding plasmid associated with sonoporation has been shown to enhance antitumoral activity. Moreover, non-viral carriers and high-frequency ultrasound have both been shown to promote immune response activation. Here, IL-12 encoding plasmid, non-viral carrier stimulating the immune response and focused ultrasound were combined in order to improve the antitumoral efficiency. Methods: In order to enhance a gene-based antitumoral immune response, home-made lipids Toll-like receptor 2 (TLR2) agonists and plasmid free of antibiotic resistance version 4 (pFAR4), a mini-plasmid, encoding the IL-12 cytokine were combined with high-intensity focused ultrasound (HIFU). The lipid composition and the combination conditions were selected following in vitro and in vivo preliminary studies. The expression of IL-12 from our plasmid construct was measured in vitro and in vivo. The combination strategy was evaluated in mice bearing colon carcinoma cells (CT26) tumors following their weight, tumor volume, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) levels in the serum and produced by splenocytes exposed to CT26 tumor cells. Results: Lipid-mediated cell transfection and intratumoral injection into CT26 tumor mice using pFAR4-IL-12 led to the secretion of the IL-12 cytokine into cell supernatant and mice sera, respectively. Conditions of thermal deposition using HIFU were optimized. The plasmid encoding pFAR4-IL-12 or TLR2 agonist alone had no impact on tumor growth compared with control mice, whereas the complete treatment consisting of pFAR4-IL-12, TLR2 lipid agonist, and HIFU limited tumor growth. Moreover, only the complete treatment increased significantly mice survival and provided an abscopal effect on a metastatic CT26 model. Conclusions: The HIFU condition was highly efficient to stop tumor growth. The combined therapy was the most efficient in terms of IL-12 and IFN-γ production and mice survival. The study showed the feasibility and the limits of this combined therapy which has the potential to be improved.

Persistent luminescence nanoparticles functionalized by polymers bearing phosphonic acid anchors: synthesis, characterization, and in vivo behaviour.

Optical in vivo imaging has become a widely used technique and is still under development for clinical diagnostics and treatment applications. For further development of the field, researchers have put much effort into the development of inorganic nanoparticles (NPs) as imaging probes. In this trend, our laboratory developed ZnGa1.995O4Cr0.005 (ZGO) nanoparticles, which can emit a bright persistent luminescence signal through the tissue transparency window for dozens of minutes and can be activated in vivo with visible irradiation. These properties endow them with unique features, allowing us to recover information over a long-time study with in vivo imaging without any background. To target tissues of interest, ZGO must circulate long enough in the blood stream, a phenomenon which is limited by the mononuclear phagocyte system (MPS). Depending on their size, charge and coating, the NPs are sooner or later opsonized and stored into the main organs of the MPS (liver, spleen, and lungs). The NPs therefore have to be coated with a hydrophilic polymer to avoid this limitation. To this end, a new functionalization method using two different polyethylene glycol phosphonic acid polymers (a linear one, later named lpPEG and a branched one, later named pPEG) has been studied in this article. The coating has been optimized and characterized in various aqueous media. The behaviour of the newly functionalized NPs has been investigated in the presence of plasmatic proteins, and an in vivo biodistribution study has been performed. Among them ZGOpPEG exhibits a long circulation time, corresponding to low protein adsorption, while presenting an effective one-step process in aqueous medium with a low hydrodynamic diameter increase. This new method is much more advantageous than another strategy we reported previously that used a two-step PEG silane coating performed in an organic solvent (dimethylformamide) for which the final hydrodynamic diameter was twice the initial diameter.

A tale of two robots: Operating times and learning curves in robot-assisted lumbar fusion.

Robotic assistance technologies are being incorporated into minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) to minimize radiation exposure to the patient and operating staff. However, they introduce new issues including increased operating time and difficult incorporation into surgical workflow. This study, conducted with 42 patients under the care of one neurosurgeon in Sydney, Australia, investigates the operating time increase with three different robotic modalities, and the learning curves they pose to the surgeon. Between the comparable modalities of freehand MIS-TLIF and Mazor Renaissance® CT to Fluoro, there was a significant increase in time from patient draping to insertion of the final K-wire (p = 0.0019), and a non-significant increase in time per K-wire (p = 0.55) using Mazor Renaissance®. Comparing the ROSA® and Mazor Renaissance® Scan and Plan, there were significant increases in drape to final K-wire time and time per K-wire using ROSA® assistance (p = 0.000068 and p = 0.011). ROSA® also had a steeper learning curve compared to both Mazor Renaissance® modalities, which were similar. Our study shows that Mazor Renaissance® modalities are superior to ROSA® in minimizing extra operating time, and also have easier learning curves; however, both modalities increase operating time compared with freehand MIS-TLIF. This study, to our knowledge, is the first to compare multiple robotic techniques in MIS-TLIF. Though these results highlight important differences between robotic modalities that are crucial for spinal surgeons to understand, the low sample size and variability in data reveal the need for larger, multi-centre studies in this field.

Cationic lipid nanoparticle production by microfluidization for siRNA delivery.

Microfluidization has been investigated as a new, scalable, and basic component saving method to produce cationic lipid nanoparticles, in particular for the delivery of short interfering RNAs (siRNAs). The design of experiment (DoE) allowed to reach optimized characteristics in terms of nanocarrier size reduction and low polydispersity. The structure of cationic liposomes and siRNA-lipoplexes was characterized. The optimized preparation parameters were identified as three microfluidization passages at a pressure of 10,000 psi, with a thin film hydration volume of 4 ml. Microfluidized liposomes mean size was 160 nm, with a polydispersity index of 0.2-0.3 and a zeta potential of +40 mV to +60 mV. Positive versus negative charge ratio between the charges of the cationic lipid and the phosphate charges of the siRNAs is a key factor determining the structure and silencing efficacy of siRNA lipoplexes. At a (+/-) charge ratio of 8, a proportion of 88% of the siRNA was associated to microfluidized lipoplexes, which remained stable for one month. These lipoplexes exhibited moderate cytotoxicity and gene silencing efficacy, which should be further optimized.

Viscous Core Liposomes Increase siRNA Encapsulation and Provides Gene Inhibition When Slightly Positively Charged.

Since its discovery, evidence that siRNA was able to act as an RNA interference effector, led to its acceptation as a novel medicine. The siRNA approach is very effective, due to its catalytic mechanism, but still the limitations of its cellular delivery should be addressed. One promising form of non-viral gene delivery system is liposomes. The variable and versatile nature of the lipids keeps the possibility to upgrade the liposomal structure, which makes them suitable for encapsulation and delivery of drugs. However, to avoid the limitation of fast release for the hydrophilic drug, we previously designed viscous core liposomes. We aimed in this work to evaluate if these viscous core liposomes (NvcLs) could be of interest for siRNA encapsulation. Then, we sought to add a limited amount of positive charges to provide cell interaction and transfection. Cationic lipid dimyristoylaminopropylaminopropyl or the polymer poly(ethylenimine) were incorporated in NvcL to produce positively charged viscous core liposomes (PvcL) by a customized microfluidic device. We found that NvcLs increased the encapsulation efficiency and loading content with regards to the neutral liposome. Both PvcLPEI and PvcLDMAPAP exhibited transfection and GFP knock-down (≈40%) in both 2D and 3D cell cultures. Finally, the addition of slight positive charges did not induce cell toxicity.

PLGA Based Nanospheres as a Potent Macrophage-Specific Drug Delivery System.

Macrophages possess an innate ability to scavenge heterogenous objects from the systemic circulation and to regulate inflammatory diseases in various organs via cytokine production. That makes them attractive targets for nanomedicine-based therapeutic approaches to inflammatory diseases. In the present study, we have prepared several different poly(lactic-co-glycolic acid) (PLGA) polymer nanospheres for macrophage-targeted drug delivery using both nanoprecipitation and emulsification solvent evaporation methods. Two experimental linear PLGA polymers with relatively low molar weight, one experimental branched PLGA with unique star-like molecular architecture, and a commercially available PLGA, were used for nanosphere formulation and compared to their macrophage uptake capacity. The nanosphere formulations labelled with loaded fluorescent dye Rhodamine B were further tested in mouse bone marrow-derived macrophages and in hepatocyte cell lines AML-12, HepG2. We found that nanospheres larger than 100 nm prepared using nanoprecipitation significantly enhanced distribution of fluorescent dye selectively into macrophages. No effects of nanospheres on cellular viability were observed. Additionally, no significant proinflammatory effect after macrophage exposure to nanospheres was detected as assessed by a determination of proinflammatory cytokines Il-1ß and Tnfα mRNA. All experimental PLGA nanoformulations surpassed the nanospheres obtained with the commercially available polymer taken as a control in their capacity as macrophage-specific carriers.

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells using the Sleeping Beauty Transposon System.

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 µL of resuspension buffer and combined with 2 µL of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

Coating Persistent Luminescence Nanoparticles With Hydrophilic Polymers for in vivo Imaging.

Persistent luminescence nanoparticles (PLNPs) are innovative nanomaterials highly useful for bioimaging applications. Indeed, due to their particular optical properties, i.e., the ability to store the excitation energy before slowly releasing it for a prolonged period of time, they allow in vivo imaging without auto-fluorescence and with a high target to background ratio. However, as for most nanoparticles (NPs), without any special surface coating, they are rapidly opsonized and captured by the liver after systemic injection into small animals. To overcome this issue and prolong nanoparticle circulation in the bloodstream, a new stealth strategy was developed by covering their surface with poly(N-2-hydroxypropyl)methacrylamide (pHPMA), a highly hydrophilic polymer widely used in nanomedicine. Preliminary in vivo imaging results demonstrated the possibility of pHPMA as an alternative strategy to cover ZnGa2O4:Cr NPs to delay their capture by the liver, thereby providing a new perspective for the formulation of stealth NPs.

Polymer Nanomedicines with Ph-Sensitive Release of Dexamethasone for the Localized Treatment of Inflammation.

Polymer-drug conjugates have several advantages in controlled drug delivery to inflammation as they can accumulate and release the drug in inflamed tissues or cells, which could circumvent the shortcomings of current therapy. To improve the therapeutic potential of polymer-drug conjugates in joint inflammation, we synthesized polymer conjugates based on N-(2-hydroxypropyl) methacrylamide) copolymers labeled with a near-infrared fluorescent dye and covalently linked to the anti-inflammatory drug dexamethasone (DEX). The drug was bound to the polymer via a spacer enabling pH-sensitive drug release in conditions mimicking the environment inside inflammation-related cells. An in vivo murine model of adjuvant-induced arthritis was used to confirm the accumulation of polymer conjugates in arthritic joints, which occurred rapidly after conjugate application and remained until the end of the experiment. Several tested dosage schemes of polymer DEX-OPB conjugate showed superior anti-inflammatory efficacy. The highest therapeutic effect was obtained by repeated i.p. application of polymer conjugate (3 × 1 mg/kg of DEX eq.), which led to a reduction in the severity of inflammation in the ankle by more than 90%, compared to 40% in mice treated with free DEX.

Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver.

Non-viral gene delivery into the liver generally mediates a transient transgene expression. A comparative analysis was performed using two gene vectors, pFAR4 and pKAR4, which differ by the absence or presence of an antibiotic resistance marker, respectively. Both plasmids carried the same eukaryotic expression cassette composed of a sulfamidase (Sgsh) cDNA expressed from the human alpha antitrypsin liver-specific promoter. Hydrodynamic injection of the pFAR4 construct resulted in prolonged sulfamidase secretion from the liver, whereas delivery of the pKAR4 construct led to a sharp decrease in circulating enzyme. After induction of hepatocyte division, a rapid decline of sulfamidase expression occurred, indicating that the pFAR4 derivative was mostly episomal. Quantification analyses revealed that both plasmids were present at similar copy numbers, whereas Sgsh transcript levels remained high only in mice infused with the pFAR4 construct. Using a chromatin immunoprecipitation assay, it was established that the 5' end of the expression cassette carried by pKAR4 exhibited a 7.9-fold higher heterochromatin-to-euchromatin ratio than the pFAR4 construct, whereas a bisulfite treatment did not highlight any obvious differences in the methylation status of the two plasmids. Thus, by preventing transgene expression silencing, the pFAR4 gene vector allows a sustained transgene product secretion from the liver.

Degenerate-disc infection study with contaminant control (DISC): a multicenter prospective case-control trial.

BACKGROUND: A bacterial cause of disc degeneration has evoked several controversies and, if true, would lead to a major shift in treatment paradigm. Earlier studies analyzing the relationship of bacterial disc infection within a degenerative cohort featured prolonged cultures susceptible to contamination. The degenerate-disc infection study with contaminant control (DISC) trial aims to investigate this theory further by examining infection rates using a non-degenerative control cohort in comparison to a degenerative internal control cohort and a sham cohort (sampling only sterile paraspinal tissue). To our knowledge, the current study is the largest evaluating the growth of organisms (or possible contamination rate) in paraspinal tissue if prolonged cultures are performed. Protocols on methodology have been previously published. PURPOSE: (1) To investigate the infection rates across cohorts (degenerative vs. nondegenerative control; paraspinal and/or disc controls vs. combined sampling cohorts) using stringent standardized aseptic surgical technique and laboratory processing. (2) To compare our findings to that of the literature and make a statement in support and/or against a possible contamination theory to positive cultures. STUDY DESIGN: Multicenter, multisurgeon case-control trial. PATIENT SAMPLE: In all, 812 surgical samples were retrieved across a 3.5-year period (2013-2016) including 25 trauma controls (nondegenerative), 550 "disc and paraspinal" samples (degenerative cases with internal control), 190 disc-only samples (degenerative cases without internal control), and 46 paraspinal only controls (sham group). OUTCOME MEASURES: Growth and/or Contamination rate (%) per cohort. Chi-square of growth in disc versus paraspinal samples as a means of examining the distribution of false positive and contaminant growth. The impact of previous injections and/or surgery on positive disc or paraspinal growth. Correlation of Modic changes with positive growth rates analyzed with the Kruskal-Wallis Test. The distribution of species in positive samples were also analyzed. METHODS: The DISC trial is registered under Australian and New Zealand clinical trials registry-ACTRN12616000541404. Institutional ethics review was obtained (HREC northern sector 13/218) at the primary center and further centers (n=6) were recruited. Patients undergoing spinal surgery with discectomy were eligible for trial entry with tissue specimens obtained using strict aseptic technique for microbiological examination. All specimens were handled with sterile instruments only and by a fresh instrument to a sterile pot that was closed immediately. Separate pots were used for the disc and paraspinal tissue respectively with similar stringent processing during microbiological assessment. A cohort of the degenerative cases at one single institution also underwent an additional histopathological examination. RESULTS: There was an expected significant difference in gender and age associated with the non-degenerative control group (due to trauma patients) compared with other cohorts. There was a higher percentage of positive-growth in the control group in comparison to the disc and paraspinal and disc only groups across positive disc growth (48% vs. 27% vs. 17%, p<.001). A similar infection rate was observed in the paraspinal samples across the equivalent controls (44% vs. 36% vs. 37%, p=.739). There was a significant difference in the proportions of positive growth with a large proportion of false positives (growth in both disc and paraspinal samples; p<.001). There was no difference in true positive growth between the case and control groups (16.0 vs. 7.7%, p=.112). These trends were preserved across all cohorts and when stratifying by spinal segment (cervical or lumbar). There was no correlation between Modic changes and positive disc culture growth (p=.398, n=144 samples). Cutibacterium (formerly Propionibacterium) acnes was the most dominant pathogen isolated, representing between 50% and 70% of positive disc and paraspinal specimens, followed by staphylococcal species. CONCLUSIONS: Our study failed to find a difference in true infection rates between the nondegenerative and degenerative disc populations. These findings are suggestive of a contamination theory and against a common infective etiology in the setting of discogenic back and neck pain. We believe the rationale for antibiotic therapy in the management of discogenic back pain warrants further evidence to establish efficacy.

Drug repurposing in rare diseases: Myths and reality.

While nearly 8000 rare diseases have been identified, only 5 percent have licensed treatments. As most of these diseases are life threatening, it underscores the urgent need for new drugs. Drug repurposing (also called drug repositioning) consists in identifying new uses for approved or investigational drugs that are outside the scope of the original medical indication. It represents an opportunity for rare diseases and patients with unmet needs. It is an alternative option in drug development and is often presented as being a viable, risk-managed strategy for pharmaceutical companies developing orphan drugs. Drug repurposing is presented as offering various advantages over developing an entirely new drug for a given indication: fewer risks, lower costs and shorter timelines. However, matters are not as simple as this. There are notable successes for drug repurposing. Nevertheless, repurposing does not always succeed. The repurposed drug may fail to demonstrate a benefits-harms balance in clinical trials. Moreover, there are legal and regulatory issues which are specific barriers to drug repurposing and which have to be carefully analyzed before any development of repurposed drugs. The objective of this article is to identify major challenges and opportunities of drug repurposing in rare diseases and to separate fact from fiction.