A nebulized gelatin nanoparticle-based CpG formulation is effective in immunotherapy of allergic horses.

PURPOSE: In the recent years, nanotechnology has boosted the development of potential drug delivery systems and material engineering on nanoscale basis in order to increase drug specificity and reduce side effects. A potential delivery system for immunostimulating agents such as cytosine-phosphate-guanine-oligodeoxynucleotides (CpG-ODN) needs to be developed to maximize the efficacy of immunotherapy against hypersensitivity. In this study, an aerosol formulation of biodegradable, biocompatible and nontoxic gelatin nanoparticle-bound CpG-ODN 2216 was used to treat equine recurrent airway obstruction in a clinical study. METHODS: Bronchoalveolar lavage fluid was obtained from healthy and allergic horses to quantify Th1/Th2 cytokine levels before and after inhalation regimen. Full clinical examinations were performed to evaluate the therapeutic potential of this nebulized gelatin nanoparticle-based CpG formulation. RESULTS: Most remarkable was that regulatory anti-inflammatory and anti-allergic cytokine IL-10 expression was significantly triggered by five consecutive inhalations. Thorough assessment of clinical parameters following nanoparticle treatment indicated a partial remission of the allergic condition. CONCLUSION: Thus this study, for the first time, showed effectiveness of colloidal nanocarrier-mediated immunotherapy in food-producing animals with potential future applicability to other species including humans.

Reduced Acquisition Time [18F]GE-180 PET Scanning Protocol Replaces Gold-Standard Dynamic Acquisition in a Mouse Ischemic Stroke Model.

AIM: Understanding neuroinflammation after acute ischemic stroke is a crucial step on the way to an individualized post-stroke treatment. Microglia activation, an essential part of neuroinflammation, can be assessed using [18F]GE-180 18 kDa translocator protein positron emission tomography (TSPO-PET). However, the commonly used 60-90 min post-injection (p.i.) time window was not yet proven to be suitable for post-stroke neuroinflammation assessment. In this study, we compare semi-quantitative estimates derived from late time frames to quantitative estimates calculated using a full 0-90 min dynamic scan in a mouse photothrombotic stroke (PT) model. MATERIALS AND METHODS: Six mice after PT and six sham mice were included in the study. For a half of the mice, we acquired four serial 0-90 min scans per mouse (analysis cohort) and calculated standardized uptake value ratios (SUVRs; cerebellar reference) for the PT volume of interest (VOI) in five late 10 min time frames as well as distribution volume ratios (DVRs) for the same VOI. We compared late static 10 min SUVRs and the 60-90 min time frame of the analysis cohort to the corresponding DVRs by linear fitting. The other half of the animals received a static 60-90 min scan and was used as a validation cohort. We extrapolated DVRs by using the static 60-90 min p.i. time window, which were compared to the DVRs of the analysis cohort. RESULTS: We found high linear correlations between SUVRs and DVRs in the analysis cohort for all studied 10 min time frames, while the fits of the 60-70, 70-80, and 80-90 min p.i. time frames were the ones closest to the line of identity. For the 60-90 min time window, we observed an excellent linear correlation between SUVR and DVR regardless of the phenotype (PT vs. sham). The extrapolated DVRs of the validation cohort were not significantly different from the DVRs of the analysis group. CONCLUSION: Simplified quantification by a reference tissue ratio of the late 60-90 min p.i. [18F]GE-180 PET image can replace full quantification of a dynamic scan for assessment of microglial activation in the mouse PT model.

Targeting the Retinoblastoma/E2F repressive complex by CDK4/6 inhibitors amplifies oncolytic potency of an oncolytic adenovirus.

CDK4/6 inhibitors (CDK4/6i) and oncolytic viruses are promising therapeutic agents for the treatment of various cancers. As single agents, CDK4/6 inhibitors that are approved for the treatment of breast cancer in combination with endocrine therapy cause G1 cell cycle arrest, whereas adenoviruses induce progression into S-phase in infected cells as an integral part of the their life cycle. Both CDK4/6 inhibitors and adenovirus replication target the Retinoblastoma protein albeit for different purposes. Here we show that in combination CDK4/6 inhibitors potentiate the anti-tumor effect of the oncolytic adenovirus XVir-N-31 in bladder cancer and murine Ewing sarcoma xenograft models. This increase in oncolytic potency correlates with an increase in virus-producing cancer cells, enhanced viral genome replication, particle formation and consequently cancer cell killing. The molecular mechanism that regulates this response is fundamentally based on the reduction of Retinoblastoma protein expression levels by CDK4/6 inhibitors.

Neoadjuvant gene delivery of feline granulocyte-macrophage colony-stimulating factor using magnetofection for the treatment of feline fibrosarcomas: a phase I trial.

Despite aggressive pre- or postoperative treatment, feline fibrosarcomas have high recurrence rates. Immunostimulatory gene therapy is a promising approach in veterinary oncology. This phase I dose-escalation study was performed to determine toxicity and feasibility of gene therapy with feline granulocyte-macrophage colony-stimulating factor (feGM-CSF) in cats with fibrosarcomas. Twenty cats were treated with plasmid coding for feGM-CSF attached to magnetic nanoparticles in doses of 50, 250, 750 and 1250 microg. Two preoperative intratumoral injections followed by magnetofection were given. Four control cats received only surgical treatment. Adverse events were recorded and correlated according to the veterinary co-operative oncology group toxicity scale. An enzyme-linked immunosorbent assay was performed to detect plasma feGM-CSF concentrations. No significant treatment related toxicity was observed. Preliminary recurrence results were encouraging as, on day 360, ten of 20 treated cats were recurrence-free. In conclusion, 1250 microg of feGM-CSF plasmid DNA applied by magnetofection is safe and feasible for phase II testing.

Aerosol gene delivery to the murine lung is mouse strain dependent.

The cationic polymer polyethylenimine (PEI) has been previously demonstrated to efficiently deliver genes to the lungs of mice in vivo via nebulization. Although within these studies various mouse strains were used in individual experiments, no direct comparison of gene delivery to different mouse strains via aerosol application has been published to date. With respect to the widespread use of mice as animal models of inherited and acquired diseases, such data could be of relevance to select the most appropriate mouse genetic background for preclinical mouse models. We investigated PEI-based aerosol gene delivery in two commonly used mouse strains, BALB/c and NMRI, and mixed 129/Sv x C57BL/6 mice. Gene expression in BALB/c mice was significantly 3.2- and 3.8-fold higher than in NMRI and 129/Sv x C57BL/6 mice, respectively. Lung deposition rates of radioactively labeled plasmid DNA (I(123)) complexed with PEI were not significantly different between each of the mouse strains. The kinetics of pDNA clearance from the lungs of BALB/c mice was slightly faster than from NMRI mice. Whereas gene expression increased until day 3 after treatment, the levels of pDNA decreased over the same period of time. Repeated aerosol application in a 3-day time interval could maintain gene expression at high levels compared with a single application. Furthermore, PEI-pDNA aerosol application led to reproducible gene expression in independent experiments. These data suggest that the genetic background of mice could be important for nonviral aerosol gene delivery which should be considered in transgenic animal mouse models of inherited and acquired diseases for aerosol gene delivery studies.

Thyroid hormone (T3)-modification of polyethyleneglycol (PEG)-polyethyleneimine (PEI) graft copolymers for improved gene delivery to hepatocytes.

Targeting of gene vectors to liver hepatocytes could offer the opportunity to cure various acquired and inherited diseases. Efficient gene delivery to the liver parenchyma has been obscured from efficient targeting of hepatocytes. Here we show that the thyroid hormone, triiodothyronine (T3), can be used to improve the gene transfer efficiency of nonviral gene vectors to hepatocytes in vitro and to the liver of mice in vivo. T3 conjugated to the distal ends of fluorescent labeled PEG-g-dextran resulted in T3-specific cellular endosomal uptake into the hepatocellular cell line HepG2. PEG-g-PEI graft copolymers with increasing molar PEG-ratios were synthesized, complexed with plasmid DNA, and transfected into HepG2 or HeLa cells. Gene transfer efficiency decreased as the number of PEG blocks increased. T3 conjugation to PEI and the distal ends of PEG blocks resulted in T3 specific gene transfer in HepG2 cells as evidenced by reduction of gene transfer efficiency after pre-incubation of cells with excess of T3. In vivo application of T3-PEG-g-PEI based gene vectors in mice after tail vein injection resulted in a significantly 7-fold increase of gene expression in the liver compared with PEG-g-PEI based gene vectors.

In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects.

To examine a retroviral gene transfer to chondrocytes in vitro and in vivo in tissue-engineered cell-collagen constructs articular chondrocytes from rabbits and humans were isolated and transduced with VSV.G pseudotyped murine leukemia virus-derived retroviral vectors. Viral supernatants were generated by transient transfection of 293T cells using the pBullet retroviral vector carrying the nlslacZ gene, a Moloney murine leukemia virus gag/pol plasmid and a VSV.G coding plasmid. Transduction efficiency was analyzed by fluorescence-activated-cell-sorter analysis and transduced autologous chondrocytes from rabbits were seeded on collagen-scaffolds and implanted into osteochondral defects in the patellar groove of the rabbit's femur (n=10). LacZ-expression was analyzed by X-gal staining on total knee explants and histological sections. Retroviral transduction efficiency exceeded 92.3% (SEM+/-3.5%) in rabbit articular chondrocytes, 74.7% (SEM+/-1.8%) in human articular chondrocytes and 52.7% (SEM+/-5.8%) in osteoarthritic human chondrocytes. Reporter gene expression remained high after 15 weeks in 75.7% (SEM+/-8.2%) of transduced rabbit articular chondrocytes. In vivo, intraarticular beta-galactosidase activity could be determined in the majority of implanted chondrocytes in the osteochondral defects after 4 weeks.

Adjuvant immunotherapy of feline fibrosarcoma with recombinant feline interferon-omega.

BACKGROUND: Recombinant feline interferon-omega (rFeIFN-omega) was tested as a treatment option for cats with fibrosarcoma to assess safety and feasibility. HYPOTHESIS: Treatment with rFeIFN-omega in cats with fibrosarcoma is safe and feasible. ANIMALS: Twenty domestic cats. METHODS: In an open-labeled uncontrolled clinical trial 12 injections of 1 x 10(6) U/kg rFeIFN-omega were administered over a 5-week period: the 1st through 4th injections were given intratumorally, and the 5th through 12th injections were administered subcutaneously at the tumor excision site. Wide surgical excision of the tumors was carried out after the 4th injection and before the 5th injection of rFeIFN-omega. A Common Terminology Criteria for Adverse Events (CTCAE) analysis was conducted. Flow cytometry of fibrosarcoma cells after incubation with rFeIFN-omega and recombinant feline interferon-gamma was performed to assess the biological effect of rFeIFN-omega. RESULTS: Changes in blood cell count, increases in serum aspartate-amino-transferase activity, serum bilirubin concentration, serum creatinine and serum electrolyte concentrations, weight loss, anorexia, increased body temperature, and reduced general condition were observed but were mostly minor (grade 1 and 2) and self limiting. Eosinophilia (P = .025), neutropenia (P = .021), and weight loss (P < .001) were statistically correlated with rFeIFN-omega-treatment (analysis of parameters before treatment and after 3 injections of rFeIFN-omega). Flow cytometry of 5 unrelated feline fibrosarcoma cell lines showed increased expression of major histocompatibility complex (MHC) class I molecules (P = .026) in response to in vitro incubation with rFeIFN-omega, whereas expression of MHC class II molecules was not affected significantly. CONCLUSIONS AND CLINICAL IMPORTANCE: RFeIFN-omega for the treatment of feline fibrosarcoma is safe, well tolerated, and can be easily performed in practice. To assess the efficacy of the treatment, it should be tested in a placebo-controlled trial.

Functional analysis of bioactivated and antiinfective PDLLA - coated surfaces.

Common scaffold surfaces such as titanium can have side effects; for example, infections, cytotoxicity, impaired osseointegration, or low regeneration rates for bone tissue. These effects lead to poor implant integration or even implant loss. Therefore, bioactive implants are promising instruments in tissue regeneration. Osteoinductive elements-such as growth factors and anti-infectives-support wound healing and bone growth and thereby enable faster osseointegration, even in elderly patients. In this study, titanium surfaces were coated with a poly-(d,l-lactide) (PDLLA) layer containing different concentrations of copolymer-protected gene vectors (COPROGs) to locally provide bone morphogenetic protein-2 (BMP-2) or activated anti-infective agents, such as chlorhexidine gluconate, triclosan, and metronidazole, to prevent peri-implantitis. The coated titanium implants were then loaded with osteoblasts, NIH 3T3 fibroblasts, and human mesenchymal stem cells in 96-well plates. When shielded by COPROGs as a protective layer and resuspended in PDLLA, BMP-2-encoding pDNA at relatively low doses (5.63 µg/implant) induced the local expression of BMP-2. A linear dose dependence, which is common for recombinant growth factors, was not found, probably due to the retention property of the PDLLA surface. PDLLA, in general, successfully retains additional elements, such as osteoconductive growth factors (BMP-2) and anti-infective agents, which was demonstrated using metronidazole, and thus prevents the systemic application of excessive doses. These bioactive implant surfaces that provide the local release of therapeutic gene vectors or anti-infective agents allow the controlled stimulation of the implant and scaffold osseointegration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1672-1683, 2017.

Effects of nanoparticle coatings on the activity of oncolytic adenovirus-magnetic nanoparticle complexes.

Limitations to adenovirus infectivity can be overcome by association with magnetic nanoparticles and enforced infection by magnetic field influence. Here we examined three core-shell-type iron oxide magnetic nanoparticles differing in their surface coatings, particle sizes and magnetic properties for their ability to enhance the oncolytic potency of adenovirus Ad520 and to stabilize it against the inhibitory effects of serum or a neutralizing antibody. It was found that the physicochemical properties of magnetic nanoparticles are critical determinants of the properties which govern the oncolytic productivities of their complexes with Ad520. Although high serum concentration during infection or a neutralizing antibody had strong inhibitory influence on the uptake or oncolytic productivity of the naked virus, one particle type was identified which conferred high protection against both inhibitory factors while enhancing the oncolytic productivity of the internalized virus. This particle type equipped with a silica coating and adsorbed polyethylenimine, displaying a high magnetic moment and high saturation magnetization, mediated a 50% reduction of tumor growth rate versus control upon intratumoral injection of its complex with Ad520 and magnetic field influence, whereas Ad520 alone was inefficient. The correlations between physical properties of the magnetic particles or virus complexes and oncolytic potency are described herein.

MagnetofectionTM platform: from magnetic nanoparticles to novel nucleic acid therapeutics.

Nucleic acid delivery to cells to make them produce a desired protein or to shut down the expression of endogenous genes opens unique possibilities for research and therapy. During the last decade, to realize the potential of this approach, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as Magnetofection. Our research group at the Institute of Experimental Oncology and Therapy Research, located at the University Hospital Klinikum rechts der Isar in the center of Munich, Germany, develops new magnetic nanomaterials and, their formulations with gene-delivery vectors and technologies to allow localized and efficient gene delivery in vitro and in vivo for a variety of research, diagnostic and therapeutic applications.

Immunostimulation of bronchoalveolar lavage cells from recurrent airway obstruction-affected horses by different CpG-classes bound to gelatin nanoparticles.

Recurrent airway obstruction (RAO) in horses has become a common problem in stabled horses in industrialized countries and deserves new therapeutic strategies. CpG-oligodeoxynucleotides (CpG-ODNs) were developed as effective immunostimulating agents to induce a Th2/Th1 shift. These agents showed a beneficial therapeutic effect in allergic diseases with predominant Th2 immunoresponse. CpG-ODN delivery by gelatin nanoparticles (GNPs) resulted in enhanced cellular uptake in murine and human in vitro studies and was a starting point for the present trial. The aim of this study was to identify an optimal stimulating CpG motif in horses with regard to species specificity on equine bronchoalveolar lavage (BAL) cells, in terms of a possible specific immunomodulation effect (Th2/Th1 shift) by used CpG-ODN. Accordingly, GNPs were evaluated as a delivery system to improve CpG-ODN immunostimulation in equine BAL cells. BAL fluid (BALF) was obtained from seven horses with moderate RAO and from four healthy horses and was subsequently incubated with five different CpG-ODN sequences (from A-, B- and C-class) and one ODN without any CpG motif. Release of three key cytokines (IL-4, IL-10 and IFN-γ) was quantified by ELISA to detect an allergy mediated Th2 immunoresponse (IL-4) as well as a proinflammatory Th1 response (IFN-γ). Due to its specific anti-inflammatory and anti-allergic effects, IL-10 was considered as a beneficial agent in pathophysiology of RAO. Results showed a significant upregulation of IL-10 and IFN-γ on the one hand and a downregulation of IL-4 on the other hand in RAO affected horses. Cell cultures from healthy horses had a significantly stronger response in cytokine release to all the applied stimuli in contrast to RAO derived cells. Comparing all five CpG sequences, A-class 2216 significantly showed the highest immunomodulatory effects on equine BALF cells and, hence, was chosen for follow-up preliminary clinical studies.

Successful anatomic repair of fetoscopic access sites in the mid-gestational rabbit model using amnion cell engineering.

BACKGROUND/AIM: Our aim was to evaluate the impact of in vitro cultured amnion cells, injected and/or seeded in different scaffolds, on in vivo fetal membrane repair. MATERIALS AND METHODS: Amnion cells, isolated from allogeneic fetal membranes, were cultured on three different scaffolds for 14 to 21 days. In 33 mid-gestational rabbits, fetoscopic access sites were randomly allocated to four closure study groups: conventional collagen plug, as well as collagen plug, collagen foil, and fibrin glue as scaffolds for the cultured amnion cells. All membrane access sites were sealed with fibrin glue, and the myometrium closed with sutures. Fetal survival, amnion membrane integrity, and the presence of amniotic fluid were evaluated one week later. RESULTS: Cultures showed good survival in the collagen scaffolds. The use of collagen plug as a scaffold for the in vitro cultured amnion cells improved the integrity of fetal membranes to 80%, better than that of any other study group. CONCLUSION: Despite the need for additional studies, the present data suggest that amnion cells can be a practical and important source of cells for the engineering of constructs for sealing of the fetal membrane.

The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects.

Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n=32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.

A fibrin glue composition as carrier for nucleic acid vectors.

PURPOSE: Gene delivery from biomaterials has become an important tool in tissue engineering. The purpose of this study was to generate a gene vector-doted fibrin glue as a versatile injectable implant to be used in gene therapy supported tissue regeneration. METHODS: Copolymer-protected polyethylenimine(PEI)-DNA vectors (COPROGs), naked DNA and PEI-DNA were formulated with the fibrinogen component of the fibrin glue TISSUCOL and lyophilized. Clotting parameters upon rehydration and thrombin addition were measured, vector release from fibrin clots was determined. Structural characterizations were carried out by electron microscopy. Reporter and growth factor gene delivery to primary keratinocytes and chondrocytes in vitro was examined. Finally,chondrocyte colonized clots were tested for their potency in cartilage regeneration in a osteochondral defect model. RESULTS: The optimized glue is based on the fibrinogen component of TISSUCOL, a fibrin glue widely used in the clinics, co-lyophilized with copolymer-protected polyethylenimine(PEI)- DNA vectors (COPROGs). This material, when rehydrated, forms vector-containing clots in situ upon thrombin addition and is suitable to mediate growth factor gene delivery to primary keratinocytes and primary chondrocytes admixed before clotting. Unprotected PEI-DNA in the same setup was comparatively unsuitable for clot formation while naked DNA was ineffective in transfection. Naked DNA was released rapidly from fibrin clots (>70% within the first seven days) in contrast to COPROGs which remained tightly immobilized over extended periods of time (0.29% release per day). Electron microscopy of chondrocytecolonized COPROG-clots revealed avid endocytotic vector uptake. In situ BMP-2 gene transfection and subsequent expression in chondrocytes grown in COPROG clots resulted in the upregulation of alkaline phosphatase expression and increased extracellular matrix formation in vitro. COPROG-fibrinogen preparations with admixed autologous chondrocytes when clotted in situ in osteochondral defects in the patellar grooves of rabbit femura gave rise to luciferase reporter gene expression detectable for two weeks (n=3 animals per group). However, no significant improvement in cartilage formation in osteochondral defects filled with autologous chondrocytes in BMP-2-COPROG clots was achieved in comparison to controls (n=8 animals per group). CONCLUSIONS: COPROGs co-lyophilized with fibrinogen are a simple basis for an injectable fibrin gluebased gene-activated matrix. The preparation can be used is complete analogy to fibrin glue preparations that are used in the clinics. However, further improvements in transgene expression levels and persistence are required to yield cartilage regeneration in the osteochondral defect model chosen in this study.

Amnion cells engineering: a new perspective in fetal membrane healing after intrauterine surgery?

In this study we aimed to set up an in vitro culture of the rabbit amnion in order to support in vivo fetal membrane healing capacity following fetoscopy. Fetal membranes were collected from a mid-gestational rabbit, and cultured on collagen support material for 14 days. 34 rabbits at 22-23 days gestational age (GA) underwent fetoscopy. The entry site was randomly allocated to 4 closure technique study groups: group I, human amnion membrane (n = 23); group II, collagen foil (n = 16); group III, collagen plug (n = 19), and group IV, collagen plug with cultured amnion cells (n = 19). In all groups membrane access sites were additionally sealed with fibrin sealant, and the myometrium was closed with sutures. Fetal survival, amnion membrane integrity, and the presence of amniotic fluid were evaluated at 30 days GA. Cultures showed good survival in the collagen support material. Increased cellularity, survival and proliferations were observed. The amnion at the access site resealed in 58-64% of cases in groups II-IV, but none of the tested techniques was significantly better than the other. Histological examination indirectly revealed the anatomic repair of the membranes, since no entrapment of the membranes could be demonstrated in the myometrial wound.

Aerosolized nanogram quantities of plasmid DNA mediate highly efficient gene delivery to mouse airway epithelium.

The lung is an important target of gene therapeutic interventions. In contrast to intratracheal instillation, inhalation would be the most practical route of administration in clinical applications. Here we show that aerosolized nanogram quantities of pDNA complexed to PEI (350 ng) yielded transfection levels 15-fold higher than a 140-fold higher dose (50 microg) of the same vector applied directly to the lungs of mice via intratracheal intubation. An important efficacy parameter is the osmolarity of the aerosol and not biophysical properties of the nebulized vector. Vectors formulated and nebulized in hypoosmotic distilled water yielded 57- and 185-fold higher expression levels than those in isotonic 5% glucose or Hepes-buffered saline, respectively. Pretreatment of mice with nebulized indomethacin, which prevents water-induced airway alteration, resulted in lower gene expression, whereas pretreatment with EGTA or polidocanol, which modulate tight-junction activity, had no effect. These results, together with histological analysis of regional lung deposition and gene expression, suggest that a temporary water-induced hypoosmotic shock permeabilizes the epithelium sufficiently to allow vector uptake. The so far observed inefficiency of nonviral gene delivery to the airways may be the result of an inappropriate method of vector administration.

Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells.

We constructed multimers of the TAT-(47-57) peptide. This polycationic peptide is known to be a protein and particle transduction domain and at the same time to comprise a nuclear localization function. Here we show that oligomers of the TAT-(47-57) peptide compact plasmid DNA to nanometric particles and stabilize DNA toward nuclease degradation. At optimized vector compositions, these peptides mediated gene delivery to cells in culture 6-8-fold more efficiently than poly-L-arginine or the mutant TAT(2)-M1. When DNA was precompacted with TAT peptides and polyethyleneimine (PEI), Superfect, or LipofectAMINE was added, transfection efficiency was enhanced up to 390-fold compared with the standard vectors. As early as after 4 h of transfection, reporter gene expression mediated by TAT-containing complexes was higher than the 24-h transfection level achieved with a standard PEI transfection. When cells were cell cycle-arrested by serum starvation or aphidicolin, TAT-mediated transfection was 3-fold more efficient than a standard PEI transfection in proliferating cells. In primary nasal epithelial cells and upon intratracheal instillation in vivo, TAT-containing complexes were superior to standard PEI vectors. These data together with confocal imaging of TAT-DNA complexes in cells support the hypothesis that the TAT nuclear localization sequence function is involved in enhancing gene transfer.

Nonviral vector loaded collagen sponges for sustained gene delivery in vitro and in vivo.

BACKGROUND: Naked DNA and standard vectors have previously been used for gene delivery from implantable carrier matrices with great potential for gene therapeutic assistance of wound healing or tissue engineering. We have previously developed copolymer-protected gene vectors which are inert towards opsonization. Here we examine their potency in carrier-mediated gene delivery in comparison to standard vectors using a vector-loaded collagen sponge model. METHODS: Equine collagen type I sponges were loaded by a lyophilization method with naked DNA, polyethylenimine (PEI)-DNA, DOTAP/cholesterol-DNA and copolymer-protected PEI-DNA. These preparations were characterized in terms of vector-release, cell growth on the matrices and reporter gene expression by cells colonizing the sponges in vitro and in vivo. Subcutaneous implantation of sponges in rats served as an in vivo model. RESULTS: At the chosen low vector dose, the loading efficiency was at least 86%. Naked DNA-loaded collagen matrices lost 77% of the DNA dose in an initial burst in aqueous buffer in vitro. The other preparations examined displayed a sustained vector release. There was no difference in cell growth and invasion of the sponges between vector-loaded and untreated collagen grafts. Reporter gene expression from cells colonizing the sponges in vitro was observed for not more than 7 days with naked DNA, whereas the lipoplex and polyplex preparations yielded long-term expression throughout the experimental period of up to 56 days. The highest expression levels were achieved with the PEI-DNA-PROCOP (protective copolymer) formulation. Upon subcutaneous implantation in rats, no luciferase expression was detected with naked DNA preparations. DOTAP/cholesterol-DNA and PEI-DNA-loaded implants lead to reporter gene expression for at least 3 days, but with poor reproducibility. PEI-DNA-PROCOP collagen matrices yielded consistently the highest reporter gene expression levels for at least 7 days with good reproducibility. CONCLUSIONS: With the preparation method chosen, lipoplex- and polyplex-loaded collagen sponges are superior in mediating sustained gene delivery in vitro and local transfection in vivo as compared to naked DNA-loaded sponges. Protective copolymers are particularly advantageous in promoting the tranfection capacity of polyplex-loaded sponges upon subcutaneous implantation, likely due to their stabilizing and opsonization-inhibiting properties.

Application of novel solid lipid nanoparticle (SLN)-gene vector formulations based on a dimeric HIV-1 TAT-peptide in vitro and in vivo.

PURPOSE: To optimize gene delivery of SLN-based gene vectors by incorporation of a dimeric HIV-1 TAT peptide (TAT2) into SLN gene vectors. METHODS: Plasmid DNA was complexed with two SLN preparations either with or without pre-compaction of DNA by TAT2, poly-L-arginine, or the mutant TAT2-M1. DNA complexed with polyethylenimine (PEI) served as a standard. Gene expression was analyzed upon transfection of bronchial epithelial cells in vitro and after intratracheal instillation or aerosol application to the lungs of mice in vivo. Stability of DNA was analyzed by agarose gel electrophoresis. RESULTS: Incorporation of TAT2 into SLN gene vectors induced an up to 100-fold sequence-dependent increase of gene expression as compared with the mutant TAT2-M1 and was 4- to 8-times higher as compared with PEI in vitro. In vivo application of TAT2-SLN gene vectors via jet nebulization increased SLN-based gene expression but was accompanied with DNA degradation. DNA degradation was not observed when an innovative device operating on the principle of a perforated vibrating membrane was used. CONCLUSIONS: Incorporation of TAT2 into SLN gene vectors is suitable to optimize gene transfer in vitro. The use of a mild nebulization technology avoids DNA degradation and offers the opportunity for further studies in large animal models.