Connie Cepko

Potent Delivery of Functional Proteins into Mammalian Cells in Vitro and in Vivo Using a Supercharged Protein

The inability of proteins to potently penetrate mammalian cells limits their usefulness as tools and therapeutics. When fused to superpositively charged GFP, proteins rapidly (within minutes) entered five different types of mammalian cells with potency up to ∼100-fold greater than that of corresponding fusions with known protein transduction domains (PTDs) including Tat, oligoarginine, and penetratin. Ubiquitin-fused supercharged GFP when incubated with human cells was partially deubiquitinated, suggesting that proteins delivered with supercharged GFP can access the cytosol. Likewise, supercharged GFP delivered functional, nonendosomal recombinase enzyme with greater efficiencies than PTDs in vitro and also delivered functional recombinase enzyme to the retinae of mice when injected in vivo.

Clustering analysis of SAGE data using a Poisson approach.

Serial analysis of gene expression (SAGE) data have been poorly exploited by clustering analysis owing to the lack of appropriate statistical methods that consider their specific properties. We modeled SAGE data by Poisson statistics and developed two Poisson-based distances. Their application to simulated and experimental mouse retina data show that the Poisson-based distances are more appropriate and reliable for analyzing SAGE data compared to other commonly used distances or similarity measures such as Pearson correlation or Euclidean distance.

Comparison of hybridization-based and sequencing-based gene expression technologies on biological replicates

BackgroundHigh-throughput systems for gene expression profiling have been developed and have matured rapidly through the past decade. Broadly, these can be divided into two categories: hybridization-based and sequencing-based approaches. With data from different technologies being accumulated, concerns and challenges are raised about the level of agreement across technologies. As part of an ongoing large-scale cross-platform data comparison framework, we report here a comparison based on identical samples between one-dye DNA microarray platforms and MPSS (Massively Parallel Signature Sequencing).ResultsThe DNA microarray platforms generally provided highly correlated data, while moderate correlations between microarrays and MPSS were obtained. Disagreements between the two types of technologies can be attributed to limitations inherent to both technologies. The variation found between pooled biological replicates underlines the importance of exercising caution in identification of differential expression, especially for the purposes of biomarker discovery.ConclusionBased on different principles, hybridization-based and sequencing-based technologies should be considered complementary to each other, rather than competitive alternatives for measuring gene expression, and currently, both are important tools for transcriptome profiling.

Vesicular stomatitis virus with the rabies virus glycoprotein directs retrograde transsynaptic transport among neurons in vivo

Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of recombinant VSV (rVSV) with the rabies virus glycoprotein (RABV-G), and provide examples of its activity relative to the anterograde transsynaptic tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based anterograde transsynaptic tracer.

Neurobiology: Developing order

Different types of nerve cells are made in a particular sequence during development. But how? Studies of fruitflies reveal a temporal order in the expression of the genes that regulate these decisions.

Giving in to the blues

The molecular players that direct the development of mammalian photoreceptors are slowly coming into focus. A study that elucidates the cause of enhanced S-cone syndrome, a disorder that may be caused by a distortion of retinal cell fate, reveals one such factor—although questions about its effects remain.

Lassa-Vesicular Stomatitis Chimeric Virus Safely Destroys Brain Tumors

ABSTRACT High-grade tumors in the brain are among the deadliest of cancers. Here, we took a promising oncolytic virus, vesicular stomatitis virus (VSV), and tested the hypothesis that the neurotoxicity associated with the virus could be eliminated without blocking its oncolytic potential in the brain by replacing the neurotropic VSV glycoprotein with the glycoprotein from one of five different viruses, including Ebola virus, Marburg virus, lymphocytic choriomeningitis virus (LCMV), rabies virus, and Lassa virus. Based on in vitro infections of normal and tumor cells, we selected two viruses to test in vivo. Wild-type VSV was lethal when injected directly into the brain. In contrast, a novel chimeric virus (VSV-LASV-GPC) containing genes from both the Lassa virus glycoprotein precursor (GPC) and VSV showed no adverse actions within or outside the brain and targeted and completely destroyed brain cancer, including high-grade glioblastoma and melanoma, even in metastatic cancer models. When mice had two brain tumors, intratumoral VSV-LASV-GPC injection in one tumor (glioma or melanoma) led to complete tumor destruction; importantly, the virus moved contralaterally within the brain to selectively infect the second noninjected tumor. A chimeric virus combining VSV genes with the gene coding for the Ebola virus glycoprotein was safe in the brain and also selectively targeted brain tumors but was substantially less effective in destroying brain tumors and prolonging survival of tumor-bearing mice. A tropism for multiple cancer types combined with an exquisite tumor specificity opens a new door to widespread application of VSV-LASV-GPC as a safe and efficacious oncolytic chimeric virus within the brain. IMPORTANCE Many viruses have been tested for their ability to target and kill cancer cells. Vesicular stomatitis virus (VSV) has shown substantial promise, but a key problem is that if it enters the brain, it can generate adverse neurologic consequences, including death. We tested a series of chimeric viruses containing genes coding for VSV, together with a gene coding for the glycoprotein from other viruses, including Ebola virus, Lassa virus, LCMV, rabies virus, and Marburg virus, which was substituted for the VSV glycoprotein gene. Ebola and Lassa chimeric viruses were safe in the brain and targeted brain tumors. Lassa-VSV was particularly effective, showed no adverse side effects even when injected directly into the brain, and targeted and destroyed two different types of deadly brain cancer, including glioblastoma and melanoma.

Vision research special issue: Sight restoration: Prosthetics, optogenetics and gene therapy.

As a species that cherishes the sense of vision, we fear blindness more than any infirmity, other than cancer (National Alliance For Eye and Vision Research & Alliance For Eye and Vision Research, 2014). Although blind individuals now have more tools and opportunities than ever before (see Fig. 1), learning the skills required to be a functional blind individual, especially late in life, remains a daunting task. This special issue is inspired by the wide variety of innovative approaches that are currently being developed to prolong or partially restore vision. Our primary goal was to provide an integrated discussion of these various approaches in a single volume. Current sight restoration research involves a wide range of technologies that vary greatly both in their underlying scientific methodologies and in the challenges that they face. As a result, different approaches have rarely been discussed within an integrated literature, making it difficult to compare their relative opportunities and challenges in this rapidly developing field. This is a particularly timely moment for such a special issue. An extraordinary variety of potential therapies have begun, or are about to begin clinical trials, and one variant of retinal prosthesis is on the market in both Europe and America. This editorial review is heavily inspired by the many contributors to the US National Eye Institute’s Audacious Goals meeting of 2013, in which leaders in the field were given the opportunity to discuss the opportunities and challenges offered by current sight restoration technologies. Probably all of the interesting ideas of this editorial should be attributed to one of the many attendees who contributed to the discussion. Any incorrect or foolish ideas are of course entirely our own. A wide variety of approaches towards sight restoration are currently being actively researched (e.g. Mellough et al., 2014; NagelWolfrum et al., 2014; Pearson et al., 2014). This special issue focusses on three main approaches that primarily focus on alleviating the effects of photoreceptor diseases and are currently in, or are approaching, clinical trial status. Gene therapies use the delivery of genes to directly compensate for the loss of function of a disease gene, or, alternatively, use genes such as growth factors to generically prolong the life and functioning of photoreceptors. Optogenetics and small molecule photoswitches endow retinal cells with the ability to sense light by creating novel (or modulating existing) light-sensitive ion channels or pumps. Finally retinal and cortical prostheses use electrical stimulation to directly elicit neural responses. 2. Photoreceptor disease

Cell metabolism: Sugar for sight

Retinitis pigmentosa causes the death of cone cells, leading to blindness. A factor secreted from rod cells, RdCVF, promotes cone survival in a mouse model of the disease. It now emerges that RdCVF works by increasing glucose uptake in cones.

Validation of oligoarrays for quantitative exploration of the transcriptome

BackgroundOligoarrays have become an accessible technique for exploring the transcriptome, but it is presently unclear how absolute transcript data from this technique compare to the data achieved with tag-based quantitative techniques, such as massively parallel signature sequencing (MPSS) and serial analysis of gene expression (SAGE). By use of the TransCount method we calculated absolute transcript concentrations from spotted oligoarray intensities, enabling direct comparisons with tag counts obtained with MPSS and SAGE. The tag counts were converted to number of transcripts per cell by assuming that the sum of all transcripts in a single cell was 5·105. Our aim was to investigate whether the less resource demanding and more widespread oligoarray technique could provide data that were correlated to and had the same absolute scale as those obtained with MPSS and SAGE.ResultsA number of 1,777 unique transcripts were detected in common for the three technologies and served as the basis for our analyses. The correlations involving the oligoarray data were not weaker than, but, similar to the correlation between the MPSS and SAGE data, both when the entire concentration range was considered and at high concentrations. The data sets were more strongly correlated at high transcript concentrations than at low concentrations. On an absolute scale, the number of transcripts per cell and gene was generally higher based on oligoarrays than on MPSS and SAGE, and ranged from 1.6 to 9,705 for the 1,777 overlapping genes. The MPSS data were on same scale as the SAGE data, ranging from 0.5 to 3,180 (MPSS) and 9 to1,268 (SAGE) transcripts per cell and gene. The sum of all transcripts per cell for these genes was 3.8·105 (oligoarrays), 1.1·105 (MPSS) and 7.6·104 (SAGE), whereas the corresponding sum for all detected transcripts was 1.1·106 (oligoarrays), 2.8·105 (MPSS) and 3.8·105 (SAGE).ConclusionThe oligoarrays and TransCount provide quantitative transcript concentrations that are correlated to MPSS and SAGE data, but, the absolute scale of the measurements differs across the technologies. The discrepancy questions whether the sum of all transcripts within a single cell might be higher than the number of 5·105 suggested in the literature and used to convert tag counts to transcripts per cell. If so, this may explain the apparent higher transcript detection efficiency of the oligoarrays, and has to be clarified before absolute transcript concentrations can be interchanged across the technologies. The ability to obtain transcript concentrations from oligoarrays opens up the possibility of efficient generation of universal transcript databases with low resource demands.