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Epithelial cells may play an important role in the pathologic process of chronic rhinosinusitis with nasal polyps. Therefore, providing epithelial cells from a biobank could greatly contribute to further research. In the present work, the isolation of epithelial cells from long-term cryopreserved tissue is demonstrated. Polyp tissues were cryopreserved in a commercially available freezing medium with dimethyl sulfoxide and stored in liquid nitrogen. The outgrowth and proliferation of epithelial cells from cryopreserved tissue were evaluated and compared to that of fresh tissue. Flow cytometric analysis with anti-cytokeratin, anti-p63, and anti-Ki-67 was performed to identify epithelial cells and determine differentiation and proliferation. A functionality test was performed by determining type 2–relevant proteins, representatively thymic stromal lymphopoietin (TSLP) and periostin, using ELISA. Primary epithelial cells could be isolated from cryopreserved tissues. Cells from cryopreserved tissues showed comparable outgrowth and proliferation to that of fresh tissue. Isolated epithelial cells showed high cytokeratin, p63, and Ki-67 expression and secreted TSLP and periostin. In the present study, a method for long-term cryopreservation of polyp tissue was established, thereby enabling the isolation and cell culture of primary cell culture at a later time. Epithelial cell availability should be greatly improved by including this method in a biobank.

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ObjectivesEpithelial cells are an important part of the pathomechanism in chronic rhinosinusitis with nasal polyps. It is therefore essential to establish a robust method for the isolation and culture of epithelial cells from nasal polyps to enable further research. In this study, the feasibility of the outgrowth technique for the isolation of the epithelial cells from the nasal polyps was evaluated.MethodsThe outgrowth technique was performed to isolate the epithelial cells. Proliferation was evaluated up to the 3rd passage. Epithelial cells were identified and differentiation and proliferation were evaluated using flow cytometry with anti-cytokeratin, anti-p63, and anti-Ki-67. A functionality test was assessed by determining type 2–relevant proteins using ELISA, representatively, interleukin-33 and periostin.ResultsUsing the outgrowth technique, epithelial cells could be isolated from all tissue samples. Isolated epithelial cells showed a proliferation rate of approximately 7- to 23-fold every 6 days up to the 3rd passage. Over 97% of isolated cells were shown to be cytokeratin- and p63-positive, and over 86% of them were Ki-67–positive in flow cytometry. Interleukin-33 and periostin were detectable in the supernatant.ConclusionsWe introduce a simple, low-cost, and well-performing method for isolating epithelial cells from nasal polyps with the outgrowth technique.

Background: Human breast milk has a high microRNA (miRNA) content. It remains unknown whether and how milk miRNAs might affect intestinal gene regulation and homeostasis of the developing microbiome after initiating enteral nutrition. However, this requires that relevant milk miRNA amounts survive the gastrointestinal (GI) passage, are taken up by cells, and become available to the RNA interference machinery. It seems important to dissect the fate of these miRNAs after oral ingestion and GI passage. Objectives: Our goal was to analyze the potential transmissibility of milk miRNAs via the gastrointestinal system in neonate humans and a porcine model in vivo to contribute to the discussion of whether milk miRNAs could influence gene regulation in neonates and thus might vertically transmit developmental relevant signals. Methods: We performed cross-species profiling of miRNAs via deep sequencing and utilized dietary xenobiotic taxon-specific milk miRNA (xenomiRs) as tracers in human and porcine neonates, followed by functional studies in primary human fetal intestinal epithelial cells using adenovirus-type 5-mediated miRNA gene transfer. Results: Mammals share many milk miRNAs yet exhibit taxon-specific miRNA fingerprints. We traced bovine-specific miRNAs from formula nutrition in human preterm stool and 9 d after the onset of enteral feeding in intestinal cells (ICs) of preterm piglets. Thereafter, several xenomiRs accumulated in the ICs. Moreover, a few hours after introducing enteral feeding in preterm piglets with supplemented reporter miRNAs (cel-miR-39-5p/-3p), we observed their enrichment in blood serum and in argonaute RISC catalytic component 2 (AGO2)-immunocomplexes from intestinal biopsies. Conclusions: Milk-derived miRNAs survived GI passage in human and porcine neonates. Bovine-specific miRNAs accumulated in ICs of preterm piglets after enteral feeding with bovine colostrum/formula. In piglets, colostrum supplementation with cel-miR-39-5p/-3p resulted in increased blood concentrations of cel-miR-39-3p and argonaute RISC catalytic component 2 (AGO2) loading in ICs. This suggests the possibility of vertical transmission of miRNA signaling from milk through the neonatal digestive tract. Am J Clin Nutr 2023;xx:xx–xx.

The adenoviral vector based AstraZeneca and Janssen COVID vaccines have been associated with rare cases of thrombosis, a condition which depends on adenovirus binding to the blood protein Platelet Factor 4 (PF4).In order to identify adenoviruses with low or absent affinity for PF4, we screened dozens of types from various adenovirus species, and Adenovirus type 5 (Ad5) derived vectors carrying genetic or chemical modifications of different hexon hyper-variable regions (HVR). For this purpose, we established an armamentarium of techniques including ELISA-qPCR and Aggregate Pull-Down (APD), which enabled fast and sensitive assessments of virus-protein interactions.Unlike most tested serotypes, Ad34 did not bind to PF4. Likewise, the deletion or shielding of the HVR1 loop of Ad5 seemingly ablated its PF4 binding. Therefore, we showed that PF4 binds to adenovirus hexon through interactions dependent on HVR1, and identified vectors that may avoid or decrease the risk of thrombosis and represent safer candidates for vaccine or gene therapy vector development.

The efficient delivery and stable transgene expression are critical for applications in gene therapy. While carefully selected and engineered viral vectors allowed for remarkable clinical successes, they still bear significant safety risks. Thus, nonviral vectors are a sound alternative and avoid genotoxicity and adverse immunological reactions. Nonviral vector systems have been extensively studied and refined during the last decades. Emerging knowledge of the epigenetic regulation of replication and spatial chromatin organisation, as well as new technologies, such as Crispr/Cas, were employed to enhance the performance of different nonviral vector systems. Thus, nonviral vectors are in focus and hold some promising perspectives for future applications in gene therapy. This review addresses three prominent nonviral vector systems: the Sleeping Beauty transposase, S/MAR-based episomes, and viral plasmid replicon-based EBV vectors. Exemplarily, we review different utilities, modifications, and new concepts that were pursued to overcome limitations regarding stable transgene expression and mitotic stability. New insights into the nuclear localisation of nonviral vector molecules and the potential consequences thereof are highlighted. Finally, we discuss the remaining limitations and provide an outlook on possible future developments in nonviral vector technology.

Abstract Arming of oncolytic viruses with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown as a viable approach to increase the antitumor efficacy in melanoma. However, melanoma cells may be partially or completely resistant to TRAIL or develop TRAIL resistance, thus counteracting the antitumor efficiency of TRAIL-armed oncolytic viruses. Recently, we found that TRAIL resistance in melanoma cells can be overcome by inhibition of antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1). Here, we investigated whether the cytotoxicity of AdV-TRAIL, an oncolytic adenovirus, which expresses TRAIL after induction by doxycycline (Dox), can be improved in melanoma cells by silencing of Mcl-1. Two melanoma cell lines, the TRAIL-resistant MeWo and the TRAIL-sensitive Mel-HO were investigated. Treatment of both cell lines with AdV-TRAIL resulted in a decrease of cell viability, which was caused by an increase of apoptosis and necrosis. The proapoptotic effects were dependent on induction of TRAIL by Dox and were more pronounced in Mel-HO than in MeWo cells. SiRNA-mediated silencing of Mcl-1 resulted in a further significant decrease of cell viability and a further increase of apoptosis and necrosis in AdV-TRAIL-infected MeWo and Mel-HO cells. However, while in absolute terms, the effects were more pronounced in Mel-HO cells, in relative terms, they were stronger in MeWo cells. These results show that silencing of Mcl-1 represents a suitable approach to increase the cytotoxicity of a TRAIL-armed oncolytic adenovirus in melanoma cells. Key messages • Cytotoxicity of TRAIL-expressing adenovirus can be enhanced by silencing of Mcl-1. • The effect occurs in TRAIL-sensitive and TRAIL-resistant melanoma cells. • Increase of apoptosis is the main mechanism induced by Mcl-1 silencing.

There is high interest in investigating the daily dynamics of gene expression in mammalian organs, for example, in liver. Such studies help to elucidate how and with what kinetics peripheral clocks integrate circadian signals from the suprachiasmatic nucleus, which harbors the circadian master pacemaker, with other systemic and environmental cues, such as those associated with feeding and hormones. Organ sampling around the clock, followed by the analysis of RNA and/or proteins, is the most commonly used procedure in assessing rhythmic gene expression. However, this method requires large cohorts of animals and is only applicable to behaviorally rhythmic animals whose phases are known. Real-time recording of gene expression rhythms using luciferase reporters has emerged as a powerful method to acquire continuous, high-resolution datasets from freely moving individual mice. Here, we share our experience and protocols with this technique, using the RT-Biolumicorder setup.

BackgroundHuman breast milk has a high microRNA (miRNA) content. It remains unknown whether and how milk miRNAs might affect intestinal gene regulation and homeostasis of the developing microbiome after initiation of enteral nutrition. However, this requires that relevant milk miRNA amounts survive gastrointestinal passage, are taken up by cells, and become available to the RNA interference (RNAi) machinery. It seems important to dissect the fate of these miRNAs after oral ingestion and gastrointestinal passage.ObjectiveOur goal was to analyze the potential transmissibility of milk miRNAs via the gastrointestinal system in neonate humans and a porcine modelin vivoto contribute to the discussion whether milk miRNAs could influence gene regulation in neonates and thus might vertically transmit developmental relevant signals.DesignWe performed cross-species profiling of miRNAs via deep-sequencing and utilized dietary xenobiotic taxon-specific milk miRNA (xenomiRs) as tracers in human and porcine neonates, followed by functional studies in primary human fetal intestinal epithelial cells (HIEC-6) using Ad5-mediated miRNA-gene transfer.ResultsMammals share many milk miRNAs yet exhibit taxon-specific miRNA fingerprints. We traced bovine-specific miRNAs from formula-nutrition in human preterm stool and 9 days after onset of enteral feeding in intestinal cells of preterm piglets. Thereafter, several xenomiRs accumulated in the intestinal cells. Moreover, few hours after introducing enteral feeding in preterm piglets with supplemented reporter miRNAs (cel-miR-39-5p/-3p), we observed their enrichment in blood serum and in AGO2-immunocomplexes from intestinal biopsies.ConclusionsMilk-derived miRNAs survived gastrointestinal passage in human and porcine neonates. Bovine-specific miRNAs accumulated in intestinal cells of preterm piglets after enteral feeding with bovine colostrum/formula. In piglets, colostrum supplementation with cel-miR-39-5p/-3p resulted in increased blood levels of cel-miR-39-3p and argonaute RISC catalytic component 2 (AGO2) loading in intestinal cells. This suggests the possibility of vertical transmission of miRNA signaling from milk through the neonatal digestive tract.

The surface of proteins is heterogeneous with sophisticated but precise hydrophobic and hydrophilic patches, which is essential for their diverse biological functions. To emulate such distinct surface patterns on macromolecules, we used rigid spherical synthetic dendrimers (polyphenylene dendrimers) to provide controlled amphiphilic surface patches with molecular precision. We identified an,. I optimal spatial arrangement of these patches on certain dendrimers that enabled their interaction with human adenovirus 5 (Ads). Patchy dendrimers bound to the surface of Ads formed a synthetic polymer corona that greatly altered various host interactions of Ads as well as in vivo distribution. The dendrimer corona (1) improved the ability of Ad5-derived gene transfer vectors to transduce cells deficient for the primary Ad5 cell membrane receptor and (2) modulated the binding of Ads to blood coagulation factor X, one of the most critical virus host interactions in the bloodstream. It significantly enhanced the transduction efficiency of Ad5 while also protecting it from neutralization by natural antibodies and the complement system in human whole blood. Ads with a synthetic dendrimer corona revealed profoundly altered in vivo distribution, improved transduction of heart, and dampened vector sequestration by liver and spleen. We propose the design of bioactive polymers that bind protein surfaces solely based on their amphiphilic surface patches and protect against a naturally occurring protein corona, which is highly attractive to improve Ad5-based in vivo gene therapy applications.

It has been assumed that the suprachiasmatic nucleus (SCN) synchronizes peripheral circadian oscillators. However, this has never been convincingly shown, since biochemical time series experiments are not feasible in behaviorally arrhythmic animals. By using long-term bioluminescence recording in freely moving mice, we show that the SCN is indeed required for maintaining synchrony between organs. Surprisingly, however, circadian oscillations persist in the livers of mice devoid of an SCN or oscillators in cells other than hepatocytes. Hence, similar to SCN neurons, hepatocytes can maintain phase coherence in the absence of Zeitgeber signals produced by other organs or environmental cycles.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the “phosphate tweezer” CLR01, a “carboxylate tweezer” CLR05, and a “phosphate clip” PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.

Adenovirus-based vectors comprise the most frequently used vector type in clinical studies to date. Both intense lab research and insights from the clinical trials reveal the importance of a comprehensive understanding of vector-host interactions. Especially for systemic intravenous adenovirus vector delivery, it is paramount to develop safe and efficacious vectors. Very early vector-host interactions that take place in blood long before the first cell is being transduced are phenomena triggered by the surface, shape, and size of the adenovirus vector particles. Not surprisingly, a multitude of different technologies ranging from genetics to chemistry has been developed to alter the adenovirus vector surface. In this review, we discuss the most important technologies and evaluate them for their suitability to overcome hurdles imposed by early vector-host interactions.

Immune defense against hepatotropic viruses such as hepatitis B (HBV) and hepatitis C (HCV) poses a major challenge for therapeutic approaches. Intrahepatic cytotoxic CD8 T cells that are crucial for an immune response against these viruses often become exhausted resulting in chronic infection. We elucidated the T cell response upon therapeutic vaccination in inducible transgenic mouse models in which variable percentages of antigen-expressing hepatocytes can be adjusted, providing mosaic antigen distribution and reflecting the varying viral antigen loads observed in patients. Vaccination induced endogenous CD8 T cells could eliminate low antigen loads in liver but were functionally impaired if confronted with elevated antigen loads. Strikingly, only by conditioning the liver environment with TLR9 ligand prior and early after peripheral vaccination, successful immunization against high intrahepatic antigen density with its elimination was achieved. Moreover, TLR9 immunomodulation was also indispensable for functional memory recall after high frequency antigen challenge. Together, the results indicate that TLR9-mediated conditioning of liver environment during therapeutic vaccination or antigen reoccurrence is crucial for an efficacious intrahepatic T cell response.

Currently, virus-based vectors, namely derivatives of the adenovirus, are frequently used in a wide variety of ex vivo or local gene therapeutic applications. However, the efficacy of virus-based vectors in systemic applications is presently still extremely limited. Complex interactions of the various vector types with the patient's organism hinder successful vector deployment. Exemplary, here we summarize barriers to systemic application of Adenovirus-based vectors leading either to acute toxic effects or rapid vector neutralization and discuss strategies to overcome these barriers aiming to develop more efficient vector types.

Adenovirus-based gene transfer vectors are the most frequently used vector type in gene therapy clinical trials to date, and they play an important role as genetic vaccine candidates during the ongoing SARS-CoV-2 pandemic. Immediately upon delivery, adenovirus-based vectors exhibit multiple complex vector-host interactions and induce innate and adaptive immune responses. This can severely limit their safety and efficacy, particularly after delivery through the blood stream. In this review article we summarize two strategies to modulate Ad vector-induced immune responses: extensive genomic and chemical capsid modifications. Both strategies have shown beneficial effects in a number of preclinical studies while potential synergistic effects warrant further investigations.

The surface of proteins is heterogeneous with sophisticated but precise hydrophobic and hydrophilic patches, which is essential for their diverse biological functions. To emulate such distinct surface patterns on macromolecules, we used rigid spherical synthetic dendrimers (polyphenylene dendrimers) to provide controlled amphiphilic surface patches with molecular precision. We identified an optimal spatial arrangement of these patches on certain dendrimers that enabled their interaction with human adenovirus 5 (Ad5). Patchy dendrimers bound to the surface of Ad5 formed a synthetic polymer corona that greatly altered various host interactions of Ad5 as well as

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.

Viral pathogens continue to constitute a heavy burden on healthcare and socioeconomic systems. Efforts to create antiviral drugs repeatedly lag behind the advent of pathogens and growing understanding is that broad‐spectrum antiviral agents will make strongest impact in future antiviral efforts. This work performs selection of synthetic polymers as novel broadly active agents and demonstrates activity of these polymers against Zika, Ebola, Lassa, Lyssa, Rabies, Marburg, Ebola, influenza, herpes simplex, and human immunodeficiency viruses. Results presented herein offer structure–activity relationships for these pathogens in terms of their susceptibility to inhibition by polymers, and for polymers in terms of their anionic charge and hydrophobicity that make up broad‐spectrum antiviral agents. The identified leads cannot be predicted based on prior data on polymer‐based antivirals and represent promising candidates for further development as preventive microbicides., Broad spectrum antiviral agents are designed through systematic variation of the polymer composition, specifically the nature of the anionic charge of the polymer and hydrophobicity of the backbone. Structure–function relationship reveals unexpected lead candidates with inhibitory antiviral activity against all tested enveloped viruses.

Vectors based on human adenovirus are highly efficient tools for transient genetic modifications of cells or tissues in vitro and in vivo. They can be utilized for gene addition strategies, knockdown strategies and as transfer vectors for designer nucleases and CRISPR/Cas. They are characterized by high genomic stability and can be produced to high titers. This chapter describes the method how to produce, purify and titrate adenovirus vectors based on human adenovirus type 5.

Background Genetic treatments of chronic arthritic conditions are essentially dependent on safe and efficient vector systems. To combine features of the efficient transduction of adenovirus vectors with the advantage of stable integration into the host cell genome of apathogenic prototype foamy virus vectors, hybrid vectors (FAD) have been established. In the present study, we have generated and investigated the use of safe FAD vectors for direct gene delivery to joints. Methods We generated recombinant FAD encoding enhanced green fluorescent protein (EGFP) or human interleukin 1 receptor antagonist protein (IL1RA) cDNA, and explored their transgene expression profile, as well as the bioactivity of the IL1RA transgene in vitro. The feasibility of IL1RA gene delivery to articular tissues was investigated in a pilot study employing direct FAD injections to the knee joints of Wistar rats. Results FAD vectors efficiently transduced human or rat fibroblasts with EGFP or IL1RA transgene in vitro. Levels of IL1RA transgene expression were high, stable and functional in vitro. Transduced synovial fibroblasts and high levels of IL1RA protein (10–35 ng/ml) could be detected in vivo in the synovium of Wistar rats 3–5 days after injection of FAD vectors to the knee joints. Conclusions Our results indicate that FAD vectors are capable of efficient in vivo gene transfer to synovium and merit further investigation as a means of providing efficient and long-term intra-articular transgene expression for treatment of the arthritides. Copyright © 2013 John Wiley & Sons, Ltd.

Viral infections lead to alarmin release and elicit potent cytotoxic effector T lymphocyte (CTLeff) responses. Conversely, the induction of protective tumour-specific CTLeff and their recruitment into the tumour remain challenging tasks. Here we show that lymphocytic choriomeningitis virus (LCMV) can be engineered to serve as a replication competent, stably-attenuated immunotherapy vector (artLCMV). artLCMV delivers tumour-associated antigens to dendritic cells for efficient CTL priming. Unlike replication-deficient vectors, artLCMV targets also lymphoid tissue stroma cells expressing the alarmin interleukin-33. By triggering interleukin-33 signals, artLCMV elicits CTLeff responses of higher magnitude and functionality than those induced by replication-deficient vectors. Superior anti-tumour efficacy of artLCMV immunotherapy depends on interleukin-33 signalling, and a massive CTLeff influx triggers an inflammatory conversion of the tumour microenvironment. Our observations suggest that replicating viral delivery systems can release alarmins for improved anti-tumour efficacy. These mechanistic insights may outweigh safety concerns around replicating viral vectors in cancer immunotherapy., Viruses trigger potent cytotoxic T cell responses, whereas anti-tumour immunity has been difficult to establish. Here the authors engineer a replicating viral delivery system for tumour-associated antigens, which induces alarmin release, innate activation and protective anti-tumour immunity in mice.

Virotherapy is a unique modality for the treatment of cancer with oncolytic viruses (OVs) that selectively infect and lyse tumor cells, spread within tumors, and activate anti-tumor immunity. Various viruses are being developed as OVs preclinically and clinically, several of them engineered to encode therapeutic proteins for tumor-targeted gene therapy. Scientists and clinicians in German academia have made significant contributions to OV research and development, which are highlighted in this review paper. Innovative strategies for "shielding," entry or postentry targeting, and "arming" of OVs have been established, focusing on adenovirus, measles virus, parvovirus, and vaccinia virus platforms. Thereby, new-generation virotherapeutics have been derived. Moreover, immunotherapeutic properties of OVs and combination therapies with pharmacotherapy, radiotherapy, and especially immunotherapy have been investigated and optimized. German investigators are increasingly assessing their OV innovations in investigator-initiated and sponsored clinical trials. As a prototype, parvovirus has been tested as an OV from preclinical proof-of-concept up to first-in-human clinical studies. The approval of the first OV in the Western world, T-VEC (Imlygic), has further spurred the involvement of investigators in Germany in international multicenter studies. With the encouraging developments in funding, commercialization, and regulatory procedures, more German engineering will be translated into OV clinical trials in the near future.

Adenovirus type 5 (Ad) is an efficient gene vector with high gene transduction potential, but its efficiency depends on its native cell receptors coxsackie- and adenovirus receptor (CAR) for cell attachment and α(v)β(3/5) integrins for internalization. To enable transduction of CAR negative cancer cell lines, we have coated the negatively charged Ad by noncovalent charge interaction with cationic PAMAM (polyamidoamine) dendrimers. The specificity for tumor cell infection was increased by targeting the coated Ad to the epidermal growth factor receptor using the peptide ligand GE11, which was coupled to the PAMAM dendrimer via a 2 kDa PEG spacer. Particles were examined by measuring surface charge and size, the degree of coating was determined by transmission electron microscopy. The net positive charge of PAMAM coated Ad enhanced cellular binding and uptake leading to increased transduction efficiency, especially in low to medium CAR expressing cancer cell lines using enhanced green fluorescent protein or luciferase as transgene. While PAMAM coated Ad allowed for efficient internalization, coating with linear polyethylenimine induced excessive particle aggregation, elevated cellular toxicity and lowered transduction efficiency. PAMAM coating of Ad enabled successful transduction of cells in vitro even in the presence of neutralizing antibodies. Taken together, this study clearly proves noncovalent, charge-based coating of Ad vectors with ligand-equipped dendrimers as a viable strategy for efficient transduction of cells otherwise refractory to Ad infection.

Adenovirus vectors belong to the most frequently used vector type in gene therapy approaches. In addition, adenovirus vector particles and adenovirus subviral particles offer a great potential for the direct delivery of proteins into cells. In this review we discuss this potential and the technology of adenovirus as a protein delivery platform for applications ranging from vaccination to gene therapy.

Background The ability to induce cytotoxic T lymphocyte (CTL) responses that are multispecific is considered to comprise an essential feature for an efficacious genetic vaccine against many pathogens including HIV and hepatitis C virus. ΔE1Ad vectors are promising vectored vaccines but have been shown to induce antigen-specific CTLs with only limited multispecificity. In the present study, we investigated the applicability of gene-deleted high-capacity adenovirus (HC-Ad) vectors and focused on the induction of multispecific CTL responses. Methods We generated ΔE1 and HC-Ad vectors expressing hepatitis B virus small surface antigen (HBsAg). We comparatively analyzed the CTL profiles against various transgene product- and vector-derived epitopes in several mouse strains and HBsAg- and vector-directed antibody responses. Results HC-Ad vectors efficiently induced multispecific HBsAg-directed CTLs. By contrast, ΔE1Ad vectors mainly primed CTLs against one immunodominant epitope of HBsAg. This absence of multispecific CTL responses correlated with the induction of CTLs against viral epitopes generated by de novo expression of Ad genes from the ΔE1Ad vector. However, Ad-specific CTLs induced in trans did not impair HC-AdS-induced multispecific CTL responses against HBsAg. Finally, HC-Ad vectors also induced higher HBsAg antibody titers compared to ΔE1Ad vectors. Conclusions De novo expression of viral genes from ΔE1Ad vector genomes restricts the multispecificity of transgene product-specific CTLs by immunodominance effects. HC-Ad vectors devoid of Ad genes are favorable for the induction of both multispecific CD8 T-cell responses and high antibody responses. Our results suggest the deletion of Ad genes as an important means for developing potent Ad-based vectored vaccines. Copyright © 2011 John Wiley & Sons, Ltd.

Background The merozoite surface protein (MSP)-1 of Plasmodium falciparum, the causative agent of malaria tropica, is considered to be a promising vaccine candidate. Although its stable cloning and expression has been difficult in the past, adenoviral vectors expressing the complex protein are described in the present study. Methods Codon-optimized msp-1 was used to construct a set of first generation (ΔE1Ad) and high-capacity adenovirus (HC-Ad) vectors, and cellular and humoral immune responses induced by the vectors were characterized in detail in mice. Results Generation of stable ΔE1Ad and HC-Ad vectors expressing full-length MSP-1 and their production to high vector titers was found to be feasible. Epitope identification and analysis of frequencies of specific CD8 T-cells revealed that MSP-1 expressing HC-Ad vectors induced higher frequencies of interferon-γ + CD8 T-cells than ΔE1 vectors. Irrespective of the vector format, higher titers of MSP-1 specific antibodies were generated by Ad vectors expressing MSP-1 from a chicken β-actin (CAG) promoter comprising the cytomegalovirus early enhancer element and the chicken β-actin promoter. Conclusions The findings of the present study suggest that Ad vectors expressing full-length codon-optimized MSP-1 are promising candidate vaccines against P. falciparum infections. Use of the HC-Ad vector type for delivery, as well as the CAG promoter to control MSP-1 expression, may further increase the efficacy of this vaccine candidate. Copyright © 2011 John Wiley & Sons, Ltd.

Adenovirus vectors based on human serotype 5 can induce potent CD8 T cell responses to vector-encoded transgenic antigens. However, the individual contribution of different cell types expressing antigen upon adenovirus vector injection to the generation of antigen-directed adaptive immune responses is poorly understood so far. We investigated the role of hepatocytes, skeletal muscle, and hematopoietic cells for the induction of cellular and humoral immune responses by miRNA-mediated tissue-specific silencing of antigen expression. Using hepatitis B small surface antigen (HBsAg) as the vector-encoded transgene we show that adenovirus vector dissemination from an intramuscular (i.m.) injection site into the liver followed by HBsAg expression in hepatocytes can limit early priming of CD8 T cells and the generation of anti-HBsAg antibody responses. However, hepatocyte-specific miRNA122a-mediated silencing of HBsAg expression overcame these limitations. Early clonal expansion of K(b)/S(190-197)-specific CD8 T cells was significantly enhanced and improved polyfunctionality of CD8 T cells was found. Furthermore, miRNA122a-mediated antigen silencing induced significantly higher anti-HBsAg antibody titers allowing an up to 100-fold vector dose reduction. These results indicate that miRNA-mediated regulation of antigen expression in the context of adenovirus vectors can significantly improve transgene product-directed immune responses. This finding could be of interest for future adenovirus vaccine vector development.

Essentiell fur die Realisierung gentherapeutischer Behandlungsstrategien ist die Verfugbarkeit von geeigneten “Genfahren” (Vektoren). Mit der Optimierung der Vektoren befasst sich ein eigenes Forschungsgebiet, die Vektorologie. Ein Teilbereich der Vektorologie fokussiert auf die Verbesserung der Gentransfereffizienz und -spezifitat. Hierzu wurde unter anderem die Cell-surface-targeting-Technologie entwickelt, die in diesem Beitrag vorgestellt werden wird.

The unique region of the VP1 capsid protein of adeno-associated viruses (AAV) in common with autonomously replicating parvoviruses comprises a secreted phospholipase A2 (sPLA2) homology domain. While the sPLA2 domain of Minute Virus of Mice has recently been shown to mediate endosomal escape by lipolytic pore formation, experimental evidence for a similar function in AAV infection is still lacking. Here, we explored the function of the sPLA2 domain of AAV by making use of the serotype 2 mutant 76HD/AN. The sPLA2 defect in 76HD/AN, which severely impairs AAV's infectivity, could be complemented in trans by co-infection with wild-type AAV2. Furthermore, co-infection with endosomolytically active, but not with inactive adenoviral variants partially rescued 76HD/AN, providing the first evidence for a function of this domain in endosomal escape of incoming AAV particles.

In vivo gene transfer with adenovirus vectors would significantly benefit from a tight control of the adenovirus-inherent liver tropism. For efficient hepatocyte transduction, adenovirus vectors need to evade from Kupffer cell scavenging while delivery to peripheral tissues or tumors could be improved if both scavenging by Kupffer cells and uptake by hepatocytes were blocked. Here, we provide evidence that a single point mutation in the hexon capsomere designed to enable defined chemical capsid modifications may permit both detargeting from and targeting to hepatocytes with evasion from Kupffer cell scavenging. Vector particles modified with small polyethylene glycol (PEG) moieties specifically on hexon exhibited decreased transduction of hepatocytes by shielding from blood coagulation factor binding. Vector particles modified with transferrin or, surprisingly, 5,000 Da PEG or dextran increased hepatocyte transduction up to 18-fold independent of the presence of Kupffer cells. We further show that our strategy can be used to target high-capacity adenovirus vectors to hepatocytes emphasizing the potential for therapeutic liver-directed gene transfer. Our approach may lead to a detailed understanding of the interactions between adenovirus vectors and Kupffer cells, one of the most important barriers for adenovirus-mediated gene delivery.

Summary The free radical theory of aging postulates that the production of mitochondrial reactive oxygen species is the major determinant of aging and lifespan. Its role in aging of the connective tissue has not yet been established, even though the incidence of aging-related disorders in connective tissue-rich organs is high, causing major disability in the elderly. We have now addressed this question experimentally by creating mice with conditional deficiency of the mitochondrial manganese superoxide dismutase in fibroblasts and other mesenchyme-derived cells of connective tissues in all organs. Here, we have shown for the first time that the connective tissue-specific lack of superoxide anion detoxification in the mitochondria results in reduced lifespan and premature onset of aging-related phenotypes such as weight loss, skin atrophy, kyphosis (curvature of the spine), osteoporosis and muscle degeneration in mutant mice. Increase in p16INK4a, a robust in vivo marker for fibroblast aging, may contribute to the observed phenotype. This novel model is particularly suited to decipher the underlying mechanisms and to develop hopefully novel connective tissue-specific anti-aging strategies.

Adenoviral (Ad) vectors are widely used for gene therapy approaches. Because of the high abundance of the natural adenoviral receptors (coxsackievirus-adenovirus receptor and integrins) on a wide variety of cells, numerous methods have been developed to redirect the virions to specific receptors on target cell surfaces. Importantly, an increasing number of publications have shown that the success of targeting not only depends on receptor binding and cellular uptake, but also on intracellular trafficking processes. Therefore, improved knowledge about the intracellular fate of targeted Ad vector particles is mandatory for a rational design of targeted Ad vectors. However, the technologies currently available for fluorescent labeling of Ad vectors have significant limitations: (1) at present capsids are labeled all over the particle surface, and this imposes the risk of interference with particle infectivity; (2) capsomer-specific labeling requires extensive genetic modifications and has been demonstrated only at protein IX; and (3) two-color labeling approaches are not available. Here we present a novel, robust, and straightforward labeling procedure that overcomes these limitations. It allows for specific labeling of the capsomer's fiber, protein IX, or hexon and permits two-color labeling. We demonstrate the potential of this labeling technology by analyzing two different bioresponsive bonds that can be used for the attachment of shielding or targeting moieties to the capsids: disulfide and hydrazone bonds. We demonstrate that in contrast to disulfide bonds, hydrazone bonds are quickly hydrolyzed after uptake of the virions and are thus favorable for the generation of bioresponsive vectors.

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8(+) T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.