The functional characteristics of optogenetic gene therapy for vision restoration

Optogenetic strategies to restore vision in patients blind from end-stage retinal degenerations aim to render remaining retinal neurons light-sensitive. We present an innovative combination of multi-electrode array recordings together with a complex pattern-generating light source as a toolset to determine the extent to which neural retinal responses to complex light stimuli can be restored following viral delivery of red-shifted channelrhodopsin in the retinally degenerated mouse. Our data indicate that retinal output level spatiotemporal response characteristics achieved by optogenetic gene therapy closely parallel those observed for normal mice but equally reveal important limitations, some of which could be mitigated using bipolar-cell targeted gene-delivery approaches. As clinical trials are commencing, these data provide important new information on the capacity and limitations of channelrhodopsin-based gene therapies. The toolset we established enables comparing optogenetic constructs and stem-cell-based techniques, thereby providing an efficient and sensitive starting point to identify future approaches for vision restoration

A systematic comparison of optogenetic approaches to visual restoration

During inherited retinal degenerations (IRDs), vision is lost due to photoreceptor cell death; however, a range of optogenetic tools have been shown to restore light responses in animal models. Restored response characteristics vary between tools and the neuronal cell population to which they are delivered: the interplay between these is complex, but targeting upstream neurons (such as retinal bipolar cells) may provide functional benefit by retaining intraretinal signal processing. In this study, our aim was to compare two optogenetic tools: mammalian melanopsin (hOPN4) and microbial red-shifted channelrhodopsin (ReaChR) expressed within two subpopulations of surviving cells in a degenerate retina. Intravitreal adeno-associated viral vectors and mouse models utilising the Cre/lox system restricted expression to populations dominated by bipolar cells or retinal ganglion cells and was compared with non-targeted delivery using the chicken beta actin (CBA) promoter. In summary, we found bipolar-targeted optogenetic tools produced faster kinetics and flatter intensity-response relationships compared with non-targeted or retinal-ganglion-cell-targeted hOPN4. Hence, optogenetic tools of both mammalian and microbial origins show advantages when targeted to bipolar cells. This demonstrates the advantage of bipolar-cell-targeted optogenetics for vision restoration in IRDs. We therefore developed a bipolar-cell-specific gene delivery system employing a compressed promoter with the potential for clinical translation

ON-bipolar cell gene expression during retinal degeneration: Implications for optogenetic visual restoration.

PURPOSE: Retinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter. METHODS: Fluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6brd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wildtype retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration. RESULTS: A total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes (Srm2, Slf2, Anxa7 & Cntn1), implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration. CONCLUSION: Our findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies

A lack of functional NK1 receptors explains most, but not all, abnormal behaviours of NK1R-/- mice

Mice lacking functional neurokinin-1 receptors (NK1R-/-) display abnormal behaviours seen in Attention Deficit Hyperactivity Disorder (hyperactivity, impulsivity and inattentiveness). These abnormalities were evident when comparing the behaviour of separate (inbred: 'Hom') wildtype and NK1R-/- mouse strains. Here, we investigated whether the inbreeding protocol could influence their phenotype by comparing the behaviour of these mice with that of wildtype (NK1R+/+) and NK1R-/- progeny of heterozygous parents ('Het', derived from the same inbred strains). First, we recorded the spontaneous motor activity of the two colonies/genotypes, over 7 days. This continuous monitoring also enabled us to investigate whether the diurnal rhythm in motor activity differs in the two colonies/genotypes. NK1R-/- mice from both colonies were hyperactive compared with their wildtypes and their diurnal rhythm was also disrupted. Next, we evaluated the performance of the four groups of mice in the 5-Choice Serial Reaction-Time Task (5-CSRTT). During training, NK1R-/- mice from both colonies expressed more impulsive and perseverative behaviour than their wildtypes. During testing, only NK1R-/- mice from the Hom colony were more impulsive than their wildtypes, but NK1R-/- mice from both colonies were more perseverative. There were no colony differences in inattentiveness. Moreover, a genotype difference in this measure depended on time of day. We conclude that the hyperactivity, perseveration and, possibly, inattentiveness of NK1R-/- mice is a direct consequence of a lack of functional NK1R. However, the greater impulsivity of NK1R-/- mice depended on an interaction between a functional deficit of NK1R and other (possibly environmental and/or epigenetic) factors. Comparison of the homozygous progeny of homozygous and heterozygous breeding-pairs reveals that certain behavioural abnormalities in NK1R-/- mice are explained by their genotype, alone, but others rest on an interaction between genotype and epigenetic/environmental factors

New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR

We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead

Model based analysis of real-time PCR data from DNA binding dye protocols

BACKGROUND: Reverse transcription followed by real-time PCR is widely used for quantification of specific mRNA, and with the use of double-stranded DNA binding dyes it is becoming a standard for microarray data validation. Despite the kinetic information generated by real-time PCR, most popular analysis methods assume constant amplification efficiency among samples, introducing strong biases when amplification efficiencies are not the same. RESULTS: We present here a new mathematical model based on the classic exponential description of the PCR, but modeling amplification efficiency as a sigmoidal function of the product yield. The model was validated with experimental results and used for the development of a new method for real-time PCR data analysis. This model based method for real-time PCR data analysis showed the best accuracy and precision compared with previous methods when used for quantification of in-silico generated and experimental real-time PCR results. Moreover, the method is suitable for the analyses of samples with similar or dissimilar amplification efficiency. CONCLUSION: The presented method showed the best accuracy and precision. Moreover, it does not depend on calibration curves, making it ideal for fully automated high-throughput applications

Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions

The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition and to introduce/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems this invention solves are universal to all qPCR reactions, and it performs all necessary qPCR set-up calculations in about 52 seconds (using a pentium 4 processor) for up to seven qPCR targets and seventy-two samples at a time – calculations that commonly take capable investigators days to finish. We have named this custom Excel-based file system "FocusField2-6GallupqPCRSet-upTool-001" (FF2-6-001 qPCR set-up tool), and are in the process of transforming it into professional qPCR set-up software to be made available in 2007. The current prototype is already fully functional

Errors in CGAP xProfiler and cDNA DGED: the importance of library parsing and gene selection algorithms

<p>Abstract</p> <p>Background</p> <p>The Cancer Genome Anatomy Project (CGAP) xProfiler and cDNA Digital Gene Expression Displayer (DGED) have been made available to the scientific community over a decade ago and since then were used widely to find genes which are differentially expressed between cancer and normal tissues. The tissue types are usually chosen according to the ontology hierarchy developed by NCBI. The xProfiler uses an internally available flat file database to determine the presence or absence of genes in the chosen libraries, while cDNA DGED uses the publicly available UniGene Expression and Gene relational databases to count the sequences found for each gene in the presented libraries.</p> <p>Results</p> <p>We discovered that the CGAP approach often includes libraries from dependent or irrelevant tissues (one third of libraries were incorrect on average, with some tissue searches no correct libraries being selected at all). We also discovered that the CGAP approach reported genes from outside the selected libraries and may omit genes found within the libraries. Other errors include the incorrect estimation of the significance values and inaccurate settings for the library size cut-off values. We advocated a revised approach to finding libraries associated with tissues. In doing so, libraries from dependent or irrelevant tissues do not get included in the final library pool. We also revised the method for determining the presence or absence of a gene by searching the UniGene relational database, revised calculation of statistical significance and sorted the library cut-off filter.</p> <p>Conclusion</p> <p>Our results justify re-evaluation of all previously reported results where NCBI CGAP expression data and tools were used.</p

Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP

The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST–XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at −80°C for at least 60 days. M30-Apoptosense™ plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at −80°C, while at 37°C it had a half-life of 80–100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited

Neuronal pentraxin II is highly upregulated in Parkinson’s disease and a novel component of Lewy bodies

Neuronal pentraxin II (NPTX2) is the most highly upregulated gene in the Parkinsonian substantia nigra based on our whole genome expression profiling results. We show here that it is a novel component of Lewy bodies and Lewy neurites in sporadic Parkinson’s disease (PD). NPTX2 is also known as the neuronal activity-regulated protein (Narp), which is secreted and involved in long-term neuronal plasticity. Narp further regulates AMPA receptors which have been found to mediate highly selective non-apoptotic cell death of dopaminergic neurons. NPTX2/Narp is found in close association with alpha-synuclein aggregates in both substantia nigra and cerebral cortex in PD but unlike alpha-synuclein gene expression, which is down-regulated in the Parkinsonian nigra, NPTX2 could represent a driver of the disease process. In view of its profound (>800%) upregulation and its established role in synaptic plasticity as well as dopaminergic nerve cell death, NPTX2 is a very interesting novel player which is likely to be involved in the pathway dysregulation which underlies PD