Harnessing the Potential of Human Pluripotent Stem Cells and Gene Editing for the Treatment of Retinal Degeneration

PURPOSE OF REVIEW: A major cause of visual disorders is dysfunction and/or loss of the light-sensitive cells of the retina, the photoreceptors. To develop better treatments for patients, we need to understand how inherited retinal disease mutations result in the dysfunction of photoreceptors. New advances in the field of stem cell and gene editing research offer novel ways to model retinal dystrophies in vitro and present opportunities to translate basic biological insights into therapies. This brief review will discuss some of the issues that should be taken into account when carrying out disease modelling and gene editing of retinal cells. We will discuss (i) the use of human induced pluripotent stem cells (iPSCs) for disease modelling and cell therapy; (ii) the importance of using isogenic iPSC lines as controls; (iii) CRISPR/Cas9 gene editing of iPSCs; and (iv) in vivo gene editing using AAV vectors. RECENT FINDINGS: Ground-breaking advances in differentiation of iPSCs into retinal organoids and methods to derive mature light sensitive photoreceptors from iPSCs. Furthermore, single AAV systems for in vivo gene editing have been developed which makes retinal in vivo gene editing therapy a real prospect. SUMMARY: Genome editing is becoming a valuable tool for disease modelling and in vivo gene editing in the retina

TRF2-Mediated Stabilization of hREST4 Is Critical for the Differentiation and Maintenance of Neural Progenitors

Abstract Telomere repeat binding factor 2 (TRF2) is a component of the shelterin complex that is known to bind and protect telomeric DNA, yet the detection of TRF2 in extra-telomeric regions of chromosomes suggests other roles for TRF2 besides telomere protection. Here, we demonstrate that TRF2 plays a critical role in antagonizing the repressive function of neuron-restrictive silencer factor, also known as repressor element-1 silencing transcription factor (REST), during the neural differentiation of human embryonic stem cells (hESCs) by enhancing the expression of a truncated REST splice isoform we term human REST4 (hREST4) due to its similarity to rodent REST4. We show that TRF2 is specifically upregulated during hESC neural differentiation concordantly with an increase in the expression of hREST4 and that both proteins are highly expressed in NPCs. Overexpression of TRF2 in hESCs increases hREST4 levels and induces their neural differentiation, whereas TRF2 knockdown in hESCs and NPCs reduces hREST4 expression, hindering their ability to differentiate to the neural lineage. Concurrently, we show that TRF2 directly interacts with the C-terminal of hREST4 through its TRF2 core binding motif [F/Y]xL, protecting hREST4 from ubiquitin-mediated proteasomal degradation and consequently furthering neural induction. Thus, the TRF2-mediated counterbalance between hREST4 and REST is vital for both the generation and maintenance of NPCs, suggesting an important role for TRF2 in both neurogenesis and function of the central nervous system. Stem Cells  2014;32:2111–2122</jats:p

Antioxidant and lipid supplementation improve the development of photoreceptor outer segments in pluripotent stem cell-derived retinal organoids

The generation of retinal organoids from human pluripotent stem cells (hPSC) is now a well-established process that in part recapitulates retinal development. However, hPSC-derived photoreceptors that exhibit well-organized outer segment structures have yet to be observed. To facilitate improved inherited retinal disease modeling, we determined conditions that would support outer segment development in maturing hPSC-derived photoreceptors. We established that the use of antioxidants and BSA-bound fatty acids promotes the formation of membranous outer segment-like structures. Using new protocols for hPSC-derived retinal organoid culture, we demonstrated improved outer segment formation for both rod and cone photoreceptors, including organized stacked discs. Using these enhanced conditions to generate iPSC-derived retinal organoids from patients with X-linked retinitis pigmentosa, we established robust cellular phenotypes that could be ameliorated following adeno-associated viral vector-mediated gene augmentation. These findings should aid both disease modeling and the development of therapeutic approaches for the treatment of photoreceptor disorders

Assessment of AAV vector tropisms for mouse and human pluripotent stem cell-derived RPE and photoreceptor cells

Adeno-associated viral vectors are showing great promise as gene therapy vectors for a wide range of retinal disorders. To date, evaluation of therapeutic approaches has depended almost exclusively on the use of animal models. With recent advances in human stem cell technology, stem-cell derived retina now offers the possibility to assess efficacy in human organoids in vitro. Here we test 6 AAV serotypes (AAV2/2, AAV2/9, AAV2/8, AAV2/8T(Y733F), AAV2/5 and ShH10) to determine their efficiency in transducing mouse and human pluripotent stem cell (PSC)-derived RPE and photoreceptor cells in vitro. All the serotypes tested were capable of transducing RPE and photoreceptor cells in vitro. AAV ShH10 and AAV2/5 are the most efficient vectors at transducing both mouse and human RPE, while AAV2/8 and ShH10 achieved similarly robust transduction of human ESC-derived cone photoreceptors. Furthermore, we show that hESC-derived photoreceptors can be used to establish promoter specificity in human cells in vitro. The results of this study will aid capsid selection and vector design for pre-clinical evaluation of gene therapy approaches, such as gene editing, that require the use of human cells and tissues

Role of telomere binding protein TRF2 in neural differentiation of human embryonic stem cells

Telomere repeat binding factor 2 (TRF2) is reported to be a key component of shelterin, a multi-protein complex that binds telomeric deoxyribonucleic acid (DNA) to protect chromosome ends and maintain genome stability. However, in recent years, TRF2 has been found to also bind non-telomeric regions and to act as a protein hub, interacting with a wide range of non-telomeric proteins and thus raising the possibility that it may serve functions independent of telomere maintenance. Despite the importance of TRF2, there is little information about how TRF2 is expressed during development and whether it could have an extratelomeric role in this process. Human embryonic stem cells (hESCs) derived from pre-implantation embryos are able to differentiate into most, if not all, tissues of the adult body, thereby provide a good cell model to tackle the problem. Given the abundance of TRF2 in the human brain and its potential for extratelomeric roles, this study focused on neural differentiation. TRF2 protein levels were found dramatically increased upon differentiation of hESCs to neural progenitor cells (NPCs) and these high levels, similarly to what is observed in vivo, were specific to the neural lineage. Gain and loss of function approaches revealed that exogenous expression of TRF2 in hESCs induced neural differentiation while, in contrast, TRF2 knockdown in NPCs drastically hindered their ability to terminally differentiate into neurons and glia. This enhancing neural function of TRF2 is achieved through the ability of TRF2 to inhibit the proteasomal degradation of REST4, an alternative splice variant of RE1-Silencing Transcription factor (REST), which alleviates REST repression over neural genes, hence consolidating neural progenitor identity and potency. This study identifies TRF2 as a novel component of neural differentiation, suggesting its importance in central nervous system development as well as in neurological disorders.Open Acces

SOP002 Human pluripotent stem cell HDR-mediated gene editing using CRISPR/Cas9 and PiggyBac technology v1

The piggyBac transposon system can be combined with CRISPR/Cas9 to efficiently perform genetic manipulations in animal models or cells lines, including stem cells, without leaving behind any accessory DNA sequences like for example it happens in the Cre system. Simply put, the piggyBac transposon (e.g. our in-house pMV vector or Transposagen commercially available vectors) containing Puro positive and Thymidine kinase negative selection markers is included in a standard homology directed repair template to facilitate the selection of cells containing your desired edit. The selection cassette is then seamlessly removed using a piggyBac excision only transposase (e.g. pBX). To achieve this type of gene editing a CRISPR/Cas9 + specific gRNA plasmid is transfected into your cells of interest along the piggyBac transposon containing your repair template (e.g. mutation you want to insert or correct) and selection cassette. After the Cas9 nuclease + gRNA generates a site-specific DNA cleavage, the piggyBac donor is used by the cell’s host machinery for DNA repair by homologous recombination, which results in the incorporation of your specific edit into the host genome together with the selection cassette (Figure 1). You can now select your gene edited cells with puromycin so you don’t have to screen hundreds of clones (like it occurs with other methods). Once you isolate gene edited stem cell clones that carry out your edit of interest you can remove the selection cassette by transfecting your cells with an excision-only piggyBac transposase (e.g. PBx). The removal of the cassette by pBX is not 100% efficient. It is likely some of your cells will still carry the cassette. To remove all traces of cells containing the cassette you can take advantage of the thymidine kinase negative selection marker. Add Ganciclovir to your cells post pBX transfection and recovery to kill any remaining cells that still carry the cassette. The complete and detailed protocol below leads to Footprint-Free precise genome editing of PSCs. </p

Identification of Factors in Regulating a Protein Ubiquitination by Immunoprecipitation: a Case Study of TRF2 on Human REST4 Ubiquitination.

Ubiquitination is the first step of the ubiquitin-proteasome pathway that regulates cells for their homeostatic functions and is an enzymatic, protein post-translational modification process in which ubiquitin is transferred to a target protein substrate by a set of three ubiquitin enzymes (Weissman et al., 2011; Bhattacharyya et al., 2014; Ristic et al., 2014). Given the importance of this process, it is plausible that ubiquitination is under strict control by many factors and that the regulatory machineries are protein-specific. An assay for the detection of a specific protein ubiquitination will enable us to examine whether a factor has a function to regulate the ubiquitination of this protein. Here we describe a protocol that detects the ubiquitination status of the human REST4 protein in cultured cells, a neural alternative splicing isoform of REST (RE-1 silencing transcription factor), that antagonizes the repressive function of REST on neural differentiation and neuron formation. Using this protocol, we show that the telomere binding protein TRF2 stabilizes the expression of the human REST4 by inhibiting its ubiquitination. This indicates that TRF2 plays a positive role in neural differentiation (Ovando-Roche et al., 2014). This protocol is also useful for the detection of ubiquitination of other proteins of interest

Specific Localization of the Drosophila Telomere Transposon Proteins and RNAs, Give Insight in Their Behavior, Control and Telomere Biology in This Organism

Drosophila telomeres constitute a remarkable exception to the telomerase mechanism. Although maintaining the same cytological and functional properties as telomerase maintain telomeres, Drosophila telomeres embed the telomere retrotransposons whose specific and highly regulated terminal transposition maintains the appropriate telomere length in this organism. Nevertheless, our current understanding of how the mechanism of the retrotransposon telomere works and which features are shared with the telomerase system is very limited. We report for the first time a detailed study of the localization of the main components that constitute the telomeres in Drosophila, HeT-A and TART RNAs and proteins. Our results in wild type and mutant strains reveal localizations of HeT-A Gag and TART Pol that give insight in the behavior of the telomere retrotransposons and their control. We find that TART Pol and HeT-A Gag only co-localize at the telomeres during the interphase of cells undergoing mitotic cycles. In addition, unexpected protein and RNA localizations with a well-defined pattern in cells such as the ovarian border cells and nurse cells, suggest possible strategies for the telomere transposons to reach the oocyte, and/or additional functions that might be important for the correct development of the organism. Finally, we have been able to visualize the telomere RNAs at different ovarian stages of development in wild type and mutant lines, demonstrating their presence in spite of being tightly regulated by the piRNA mechanism.This work was supported by a grant from the Spanish Ministry of Science and Innovation BFU2009-08318/BMC to EC. A National Institutes of Health grant R01GM067758 to ERG. EL-P acknowledges the following short term Fellowships: Ruth Lee Kennedy (Fulbright), EMBO short term, Journal Cell Science traveling Fellowship, and the Spanish Society of Genetics traveling fellowship.Peer reviewe