Alphaviral Vectors for Gene Transfer into Neurons

Alphaviruses are small, enveloped positive-strand RNA viruses that have been successfully transformed into expression vectors in the case of Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus. Compared to other viral vectors, their advantages are easy and fast generation of recombinant viral particles, rapid onset, and high-level transgene expression. When applied to neuronal tissue, SFV and SIN vectors possess the additional advantage of efficiently and preferentially transducing neurons rather than non-neuronal cells. This article gives an overview of the biology of SFV and SIN, their generation into expression vectors, and their application in neurobiology, with particular emphasis on the transduction of hippocampal neurons. In addition, it describes the more recent development of alphaviral vectors with decreased or absent cytotoxicity and lowered transgene expression, temperature-controllable gene expression, and altered host-cell specificity in the central nervous system (CNS). Finally, the review evaluates the use of SFV and SIN vectors in hippocampal tissue cultures vs recombinant lentivirus, adenovirus type 5, adeno-associated virus type 2, and measles viru

Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature sensitivity for long-term enhancement of transgene expression.

Alphaviral vectors inhibit host cell protein synthesis and are cytotoxic. To overcome these limitations, we modified the nonstructural protein-2 (nsP2) gene in the Semliki Forest virus (SFV) vector, pSFV1. Packaging of SFV replicons with two point mutations in nsP2 resulted in high-titer recombinant SFV(PD) particles. SFV(PD) led to more efficient host cell protein synthesis, exhibited reduced cytotoxicity and improved cell survival, and allowed greater and prolonged transgene expression than the original vector, SFV. In dissociated hippocampal neurons and organotypic rat hippocampal slices, SFV(PD) infection preserved neuronal morphology and synaptic function more efficiently than SFV. Combination of the two point mutations with a replication-persistent mutation in nsP2 resulted in a highly temperature-sensitive vector, SFV(PD713P), which efficiently transduced neurons in hippocampal slice cultures. At 31 °C, SFV(PD713P) allowed continuous transgene expression in BHK cells, at amounts comparable to SFV(PD). These new SFV mutants are expected to substantially broaden the application of alphaviral vectors in neurons and other mammalian cells

Advanced modular self‐inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction

In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult‐to‐transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit‐like self‐inactivating lentiviral expression vector family which is compatible to state‐of‐the‐art packaging and pseudotyping technologies and contains, besides essential cis‐acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate‐early promoter (PhCMV) or the human elongation factor 1α (PhEF1α), (iii) PhCMV- or PPGK- (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein‐based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size‐optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5′U3, R‐U5‐ψ+‐SD, cPPT‐RRE‐SA and 3′LTRΔU3 modules or placed within the 5′U3 (-78 bp) and 3′LTRΔU3 (8666 bp). pLentiModule enables straightforward cassette‐type module swapping between lentiviral expression vector family members and facilitates the design of Tat‐independent (replacement of 5′LTR by heterologous promoter elements), regulated and self‐excisable proviruses (insertion of responsive operators or LoxP in the 3′LTRΔU3 element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi‐purpose construction kit‐like vector series described here is compatible with itself as well as many other (non‐viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human disease

Alphaviral cytotoxicity and its implication in vector development

A great variety of viruses have been engineered to serve as expression vectors. Among them, the alphaviruses Semliki Forest virus and Sindbis virus represent promising tools for heterologous gene expression in a wide variety of host cells. Several applications have already been described in neurobiological studies, in gene therapy, for vaccine development and in cancer therapy. Both viruses trigger stress pathways in the cells they infect, sometimes culminating in the death of the host. This inherent property is either an advantage or a drawback, depending on the type of application. This review covers the development and applications of alphavirus vectors and, as our work has been mainly with Semliki Forest virus, we have focused on this virus with special emphasis on how the understanding of Semliki Forest virus cytotoxicity enables it to be manipulated and used

Gesangsvariabilität der Rohrammer Emberiza schoeniclus in der Schweiz

The song of the Reed Bunt­ing is characterized by a large variability between individuals. Within Switzerland, the subspecies schoeniclus is breeding north and south of the Alps in wetlands, which are often small and unconnected. The local isolation might favour the development of local song characteristics, i.e. dialects. We therefore analysed 260 songs of 30 different Reed Buntings from five separate locations (Greifensee, Chatzensee, Fanel, Gletterens, and Bolle di Magadino). For quantification, we determined seven song variables and compared them within and between the local populations. Our results show considerable song variability within local populations. In addition, we found significant differences between various local populations for the variables song diversity and maximal frequency. Song diversity further differed between songs from populations north and the one south of the Alps. Our findings thus support the existence of local song dialects of the Reed Bunting in Switzerland; however, our results also confirm that song variability within local populations is extensive

Homer/Vesl Proteins and Their Roles in CNS Neurons

Since their initial discovery in 1997, Homer/Vesl proteins have become increasingly investigated as putative regulators of receptor and ion-channel function in the central nervous system. Within a relatively brief period, numerous research reports have described manifold effects of Homer proteins, including the modulation of the trafficking of type I metabotropic glutamate receptors (mGluRs), axonal pathfinding, mGluR coupling to calcium and potassium channels, agonist-independent mGluR activity, ryanodine receptor regulation, locomotor activity, and behavioral plasticity. This review summarizes our current knowledge on the induction, expression, and structure of the various forms of Homer proteins, as well as their roles in neuronal function. In addition, we provide an outlook on novel developments with regard to the involvement of Homer-1a in hippocampal synaptic functio