Generation of a 3D Liver Model Comprising Human Extracellular Matrix in an Alginate/Gelatin-Based Bioink by Extrusion Bioprinting for Infection and Transduction Studies.

Bioprinting is a novel technology that may help to overcome limitations associated with two-dimensional (2D) cell cultures and animal experiments, as it allows the production of three-dimensional (3D) tissue models composed of human cells. The present study describes the optimization of a bioink composed of alginate, gelatin and human extracellular matrix (hECM) to print human HepaRG liver cells with a pneumatic extrusion printer. The resulting tissue model was tested for its suitability for the study of transduction by an adeno-associated virus (AAV) vector and infection with human adenovirus 5 (hAdV5). We found supplementation of the basic alginate/gelatin bioink with 0.5 and 1 mg/mL hECM provides desirable properties for the printing process, the stability of the printed constructs, and the viability and metabolic functions of the printed HepaRG cells. The tissue models were efficiently transduced by AAV vectors of serotype 6, which successfully silenced an endogenous target (cyclophilin B) by means of RNA interference. Furthermore, the printed 3D model supported efficient adenoviral replication making it suitable to study virus biology and develop new antiviral compounds. We consider the approach described here paradigmatic for the development of 3D tissue models for studies including viral vectors and infectious viruses.

Virotherapy Research in Germany: From Engineering to Translation.

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.

A novel artificial microRNA expressing AAV vector for phospholamban silencing in cardiomyocytes improves Ca2+ uptake into the sarcoplasmic reticulum.

In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.

Molecular genetic and expression analysis of alpha-tocopherol transfer protein mRNA in German shepherd dogs with degenerative myelopathy.

Degenerative Myelopathy (DM) is a progressive neurological disorder of the spinal cord preferentially occurring in German shepherd dogs. The pathogenesis of the disease is unknown. However, there are indications that vitamin E deficiency may be involved in the pathogenesis of DM. Therefore, we analyzed the expression and the nucleotide sequence of the canine alpha-tocopherol transfer protein (alpha Ttp) of German shepherd dogs with DM in order to determine whether a deficiency or a defect of the alpha Ttp could be a primary factor in the pathogenesis of DM, as found in human patients with Ataxia with vitamin E deficiency (AVED). The cDNA of the coding region of the canine alpha Ttp-mRNA was generated from total liver RNA using RT-PCR and 5' RACE technique. We determined the sequence of 707 out of 834 base pairs or 84.8% of the canine alpha Ttp coding region. Sequence comparison of canine alpha Ttp between affected and control dogs revealed no differences in either nucleotide or predicted amino acid sequence. Using Northern blot analysis alpha Ttp-mRNA expression was solely found in the liver of the dogs, rats and humans, while various other organs showed no alpha Ttp-mRNA expression. No significant differences in expression levels of canine alpha Ttp mRNA were found between DM and control dogs. Our data suggest that the canine alpha Ttp gene is unlikely to be involved in the pathogenesis of DM in German shepherd dogs.