AAV capsid bioengineering in primary human retina models

Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells

Signalling molecules involved in the maintenance and differentiation of human pluripotent stem cells and retinal pigment epithelial cells

© 2017 Dr. Grace Ellie LidgerwoodTo study diseases affecting the retina, we rely on appropriate models to investigate causes of degenerative onset and progression. Human pluripotent stem cells (hPSCs), and in particular patient-specific induced pluripotent stem cells (iPSCs), provide an excellent source of material for generating retinal cells that can be used as disease models to explore disease-causing mutations, genotype-phenotype relationships, therapeutic drug screening and potentially cell replacement therapy. I developed a rapid, one-step differentiation protocol to differentiate hPSCs to the retinal pigment epithelium (RPE), the outermost monolayer of cells in the retina that are damaged in age-related macular degeneration (AMD). Using a simple growth factor treatment regime, functional RPE could be differentiated from hPSCs in 60 days for use in disease modelling and basic fundamental research. Given the protocol’s simplicity, it was easily adapted to an automated platform, enabling the generation of hundreds of patient-specific RPE lines simultaneously for large-scale disease modelling purposes. Retinal degeneration may be mediated by the loss off the outer blood retinal barrier (BRB) integrity, caused by injury of the retinal pigment epithelium. Lysophosphatidic acid (LPA) and its producing enzyme autotaxin (ATX) have been implicated in inflammation, angiogenesis and apoptosis. Given that ATX is a signature marker of RPE, I hypothesized that it plays an important role in maintaining the microenvironment of the outer retina, including the BRB. Using the differentiation protocol I developed, hPSCs were differentiated into RPE and were shown to express high levels of ATX and LPA receptors (LPARs) by quantitative real time polymerase chain reaction (qRT-PCR). Measurements of endogenous LPA and ATX were determined using liquid chromatography mass spectrometry (LC-MS) and western blot respectively, and indicate that hPSC-RPE cells secrete low basal amounts of LPA (0.1-0.4 nM), but secrete large amounts of functional ATX at the apical surface, towards the photoreceptor layer. It is possible that the low detectible amounts of LPA were a result of the short half-life of LPA in culture or the potential lack of the lipid substrate for LPA synthesis in RPE monocultures. It is more plausible that in vivo, given the high expression of ATX, the amount of local LPA produced is much higher. Addition of exogenous LPA to hPSC-RPE cultures resulted in increased expression of tight junction proteins ZO-1 and Occludin at the cell junction, which was validated at the functional level by increased transepithelial resistance (TER), indicative of increased barrier function and reduced paracellular permeability. This data suggests that RPE-derived LPA acts in an autocrine manner to maintain the tight junction barrier, one of the key function of RPE in vivo. To assess the potential paracrine actions of RPE-derived ATX and LPA, the mouse 661W photoreceptor line and optic cup-derived CD73 photoreceptors were treated with different doses of LPA. At high doses (>10 µM), cytoskeletal changes consistent with cellular retraction were observed, as indicated by myosin light chain (MLC) and its phosphorylated form (p-MLC). These results indicate the RPE-derived ATX and LPA act in a paracrine manner, and dysregulation of this pathway could play a role in photoreceptor dysfunction. Endothelial cells that line the outside of the BRB may also potentially contribute to its destruction. RPE-ATX was also secreted basally, although at much lower levels, and therefore it’s highly plausible that the amount of local LPA produced in this microenvironment have an effect on BRB maintenance. Treatment of CD31+ iPSC-endothelial cells with LPA indicated that high doses of LPA caused reduced tubule formation. This indicates that in the retina, LPA may function to suppress angiogenesis. Furthermore, I showed that treatment of mouse-derived CD4+ and CD8+ T cell with LPA resulted in reduced activation, again suggesting RPE-derived LPA may protect against loss of BRB by supressing inflammatory mediators. In conclusion, this Thesis has highlighted a potential novel role for RPE-derived ATX and LPA in the maintenance of the BRB, and that possible dysregulation of this signalling axis may be involved in retinal disease onset and progression where the BRB is compromised

A novel role for the Pol I transcription factor TBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes

Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity)

L'analyse des mesures conjointes

Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity)

Kinase Inhibitor Screening Identifies Cyclin-Dependent Kinases and Glycogen Synthase Kinase 3 as Potential Modulators of TDP-43 Cytosolic Accumulation during Cell Stress

<div><p>Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an <i>in vitro</i> model of TDP-43-positve stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219–414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities for kinase modulation-based therapeutic intervention in ALS and FTLD.</p></div

Effect of selected kinase inhibitors on TDP-43 expression.

<p>SH-SY5Y cells were treated with paraquat overnight in the presence or absence of 10 µM LY294002 (#7, PI3K); olomoucine (#12, CDKs); ZM 449829 (#15, JAK3); GW 5074 (#17, Raf); SB 203580 (#19, p38); SB 415286 (#29, GSK3); arctigenin (#30, MEK); SB 239063 (#32, p38); (1 µM) aminopurvalanol A (#35, CDKs); TBB (#40, CK2); HA 1100 (#42, ROCK); BIBX 1382 (#43, EGFR); CGP 53353 (#44, PKC); arcyriaflavin A (#45, CDKs). Western blot analysis of TDP-43 expression was determined and represented as densitometric analysis of expression compared to untreated control. *P<0.05 compared to untreated control.</p

Phosphorylation of hnRNP K by cyclin-dependent kinase 2 controls cytosolic accumulation of TDP-43

Cytosolic accumulation of TAR DNA binding protein 43 (TDP-43) is a major neuropathological feature of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the mechanisms involved in TDP-43 accumulation remain largely unknown. Previously, we reported that inhibitors of cyclin-dependent kinases (CDKs) prevented cytosolic stress granule accumulation of TDP-43, correlating with depletion of heterogeneous ribonucleoprotein (hnRNP) K from stress granules. In the present study, we further investigated the relationship between TDP-43 and hnRNP K and their control by CDKs. Inhibition of CDK2 abrogated the accumulation of TDP-43 into stress granules. Phosphorylated CDK2 co-localized with accumulated TDP-43 and phosphorylated hnRNP K in stress granules. Inhibition of CDK2 phosphorylation blocked phosphorylation of hnRNP K, preventing its incorporation into stress granules. Due to interaction between hnRNP K with TDP-43, the loss of hnRNP K from stress granules prevented accumulation of TDP-43. Mutation of Ser216 and Ser284 phosphorylation sites on hnRNP K inhibited hnRNP K- and TDP-43-positive stress granule formation in transfected cells. The interaction between hnRNP K and TDP-43 was further confirmed by the loss of TDP-43 accumulation following siRNA-mediated inhibition of hnRNP K expression. A substantial decrease of CDK2 and hnRNP K expression in spinal cord motor neurons in ALS patients demonstrates a potential key role for these proteins in ALS and TDP-43 accumulation, indicating that further investigation of the association between hnRNP K and TDP-43 is warranted. Understanding how kinase activity modulates TDP-43 accumulation may provide new pharmacological targets for disease intervention.15 page(s