Special Issue "Lentiviral Vectors"

Lentiviral vectors (LV) have been developed upon knowledge accumulated in the virology field, in particular intensive research on HIV biology since its discovery in 1983 [...]

Systemic remodeling of the redox regulatory network due to RNAi perturbations of glutaredoxin 1, thioredoxin 1, and glucose-6-phosphate dehydrogenase

<p>Abstract</p> <p>Background</p> <p>Cellular clearance of reactive oxygen species is dependent on a network of tightly coupled redox enzymes; this network rapidly adapts to oxidative conditions such as aging, viral entry, or inflammation. Current widespread use of shRNA as a means to perturb specific redox couples may be misinterpreted if the targeted effects are not monitored in the context of potential global remodeling of the redox enzyme network.</p> <p>Results</p> <p>Stable cell lines containing shRNA targets for glutaredoxin 1, thioredoxin 1, or glucose-6-phosphate dehydrogenase were generated in order to examine the changes in expression associated with altering cytosolic redox couples. A qRT PCR array revealed systemic off-target effects of altered antioxidant capacity and reactive oxygen species formation. Empty lentiviral particles generated numerous enzyme expression changes in comparison to uninfected cells, indicating an alteration in antioxidant capacity irrespective of a shRNA target. Of the three redox couples perturbed, glutaredoxin 1, attenuation produced the most numerous off-target effects with 10/28 genes assayed showing statistically significant changes. A multivariate analysis extracted strong co-variance between glutaredoxin 1 and peroxiredoxin 2 which was subsequently experimentally verified. Computational modeling of the peroxide clearance dynamics associated with the remodeling of the redox network indicated that the compromised antioxidant capacity compared across the knockdown cell lines was unequally affected by the changes in expression of off-target proteins.</p> <p>Conclusions</p> <p>Our results suggest that targeted reduction of redox enzyme expression leads to widespread changes in off-target protein expression, changes that are well-insulated between sub-cellular compartments, but compensatory in both the production of and protection against intracellular reactive oxygen species. Our observations suggest that the use of lentivirus can in itself have off-target effects on dynamic responses to oxidative stress due to the changes in species concentrations.</p

Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP

X-linked lymphoproliferative disease is a rare inherited immune disorder, caused by mutations or deletions in the SH2D1A gene that encodes an intracellular adapter protein SAP (Slam-associated protein). SAP is essential for mediating several key immune processes and the immune system - T cells in particular - are dysregulated in its absence. Patients present with a spectrum of clinical manifestations, including haemophagocytic lymphohistiocytosis (HLH), dysgammaglobulinemia, lymphoma and autoimmunity. Treatment options are limited, and patients rarely survive to adulthood without an allogeneic haematopoietic stem cell transplant (HSCT). However, this procedure can have poor outcomes in the mismatched donor setting or in the presence of active HLH, leaving an unmet clinical need. Autologous haematopoeitic stem cell or T cell therapy may offer alternative treatment options, removing the need to find a suitable donor for HSCT and any risk of alloreactivity. SAP has a tightly controlled expression profile that a conventional lentiviral gene delivery platform may not be able to fully replicate. A gene editing approach could preserve more of the endogenous regulatory elements that govern SAP expression, potentially providing a more optimum therapy. Here, we assessed the ability of TALEN, CRISPR-Cas9 and CRISPR-Cas12a nucleases to drive targeted insertion of SAP cDNA at the first exon of the SH2D1A locus using an adeno-associated virus serotype 6 (AAV6)-based vector containing the donor template. All nuclease platforms were capable of high efficiency gene editing, which was optimised using a serum-free AAV6 transduction protocol. We show that T cells from XLP patients corrected by gene editing tools have restored physiological levels of SAP gene expression and restore SAP-dependent immune functions, indicating a new therapeutic opportunity for XLP patients

Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 30 long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivatorfree, all-one-one system capable of generating Dox-inducible ATMPs.Spanish ISCIII Health Research FundEuropean Union (EU) PI18/00337 PI21/00298 RD21/0017/0004 PI18/00330 PI17/00672Red TerAvJunta de Andalucia FEDER/European Cohesion Fund (FSE) for AndalusiaSpanish Government PI18/00337 PI21/00298European Union-NextGenerationEU - Maria Zambrano Senior Program RD21/0017/0004 PI18/00330 PI17/00672Ministry of Health 2016000073332-TRA PI-57069 CARTPI-0001-201 PE-CART-0031-2020 PI-0014-2016 PECART-0027-2020 ProyExcel_00875 PEER-0286-2019European Cooperation in Science and Technology (COST) 00123009/SNEO-20191072MINECO - European Regional Development Fund PLEC2021-008094Spanish Government 0006/2018FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades CA21113Spanish Government SAF2015-71589-PMCI RYC-2016-21395German Research Foundation (DFG) PY20_00619 y A-CTS-28_UGR20Biomedicine Program of the University of Granada (Spain) FPU16/05467 FPU17/02268 FPU17/04327 DIN2018-010180 DIN2020-011550 PEJ-2018-001760-

Methods to Record Transcription Factor Binding and Enhancer Activity throughout Cellular Differentiation

The ability to create distinct cell types is fundamental for the development of multicellular organisms. Since all cells in an organism contain the same genes, cellular diversity is achieved through the transcriptional network where transcription factors (TFs) interacts with cis-regulatory elements, leading to the selective transcription of different sets of genes. To better understand the functions of TFs and regulatory elements underlying cell fate decisions, we developed methods that are able to record their activities throughout cellular differentiation. In Chapter 2, we developed a degradation domain based induction system for ҃alling CardsӠmethod which maps the binding sites of TFs using piggybac transposons. The induction ҃alling CardsӠmethod offers an alternative to chromatin immunoprecipitation (ChIP) methods and furthermore has the ability to record TF binding at different time periods of the development. In Chapter 3, we applied the ҃alling CardӠmethod to study the role of master regulatory Brd4-bound enhancers for sex differences in glioblastoma. We revealed a set of sex-specific regulatory genes and networks, which are indicative of sex-specific transcriptional programs regulated by Brd4-bound enhancers. Finally, to record the activity of regulatory elements or enhancers, in Chapter 4, we developed a CRE recombinase-mediated method for high-throughput functional identification of active enhancers at different time periods of the development, named as Developmental Enhancer Sequencing (DevEn-seq). We demonstrated that DevEn-seq is able to detect enhancers more efficiently than regular reporter methods and trace enhancer activities throughout cellular differentiation without being disturbed by the gene silencing effect caused by lentiviral sequences. With an in vitro neural differentiation protocol, we identified two neural progenitor-specific enhancers near HB9 and Olig2 genes respectively. In summary, this dissertation contributes to the field of developmental biology by providing useful methods for recording TF binding events and enhancer activities throughout development

Viral vector-mediated RNA interference in the retina

RNA interference (RNAi) is a highly conserved post-transcriptional gene silencing process triggered by double-stranded RNA (dsRNA) in eukaryotic cells. Elucidation of the RNAi regulatory pathway and its components has led to the identification of endogenous dsRNA molecules, termed microRNAs (miRNAs), which are transcribed as a single hairpin molecule prior to their maturation into a cytoplasmic dsRNA. The efficient gene silencing achieved by these short hairpin RNA (shRNA) molecules and the cumulative understanding of the RNAi pathway has prompted the development of hairpin expression vectors capable of mediating stable gene silencing in vitro and in vivo. The aim of this thesis is to evaluate the efficacy of viral vector-mediated RNAi in the retina using recombinant adeno-associated viruses (AAV) and lentiviruses that contain silencing hairpin cassettes to target four genes in murine photoreceptors and the retinal pigment epithelium (RPE). A detailed assessment of the utility and extend of RNAi in the retina using different viral vectors and hairpin designs is presented in this thesis. Lentiviral and AAV vectors were firstly used to silence GFP in vitro and in vivo as a proof of concept for vector mediated RNAi in the retina. Subsequently, we used lentivirally-mediated RNAi to study disease processes in the retina concentrating on tight junction (TJ) modulators ZO-1 and ZONAB and their role in RPE homeostasis, cell-cycle progression and epithelial-mesenchymal transition (EMT). Here we demonstrated how TJ misregulation can lead to RPE loss, proliferation or dedifferentiation; processes involved in pathological conditions such as atrophic age-related macular degeneration (AMD) and proliferative vitroretinopathy (PVR). Whilst lentivirally-mediated RNAi was used to elucidate aspects of retinal function and disease, AAV-mediated RNAi was used to probe the therapeutic potential of shRNAs by silencing Peripherin-2 (Prph2), the second most abundant retinal protein, using a miRNA-based hairpin. AAV2/8 particles were used to target endogenous Prph2 and evasion of silencing was demonstrated using an engineered Prph2 cDNA that could be used in a suppression and replacement approach for the treatment of dominant retinal disorders

Neuropathic MORC2 mutations perturb GHKL ATPase dimerization dynamics and epigenetic silencing by multiple structural mechanisms

Missense mutations in MORC2 cause neuropathies including spinal muscular atrophy and Charcot-Marie-Tooth disease. We recently identified MORC2 as an effector of epigenetic silencing by the human silencing hub (HUSH). Here we report the biochemical and cellular activities of MORC2 variants, alongside crystal structures of wild-type and neuropathic forms of a human MORC2 fragment comprising the GHKL-type ATPase module and CW-type zinc finger. This fragment dimerizes upon binding ATP and contains a hinged, functionally critical coiled coil insertion absent in other GHKL ATPases. We find that dimerization and DNA binding of the MORC2 ATPase module transduce HUSH-dependent silencing. Disease mutations change the dynamics of dimerization by distinct structural mechanisms: destabilizing the ATPase-CW module, trapping the ATP lid or perturbing the dimer interface. These defects lead to modulation of HUSH function, thus providing a molecular basis for understanding MORC2-associated neuropathies

Investigating the Phenotypic Effects of RING1- and YY1-Binding Protein (RYBP) in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer that has horrendous survival outcomes with the use of current therapies. Further study into its molecular mechanisms will inform development of new, more effective treatments. The Polycomb protein RING1- and YY1- Binding Protein (RYBP) has emerged as an important gene in multiple cancers. In complex with other Polycomb proteins, RYBP acts to repress regions of chromatin, though it also performs other functions independent of these complexes. RYBP has a tumor suppressive role in various cancers, but may act as an oncogene in others, demonstrating its context-specific effects. The role of RYBP in GBM has not yet been elucidated. In GBM, RYBP expression is frequently downregulated compared to normal brain tissue, suggesting it may act as a tumor suppressor in GBM. Thus, we hypothesized that forced expression of RYBP in GBM cell lines would activate apoptosis while decreasing cell invasion, migration, and proliferation. We transduced U-118 or T98G GBM cell lines with lentivirus expressing RYBP or a GFP control and established stable cell lines. RYBP-expressing U-118 and T98G cells showed decreased migration in wound- healing assays and invasion in Matrigel-coated Boyden chamber assays when compared to control cells. SDS-PAGE and Western blots were performed to measure changes in epithelial-to-mesenchymal transition (EMT) and apoptotic protein markers among transduced cells, and WST-1 assays were conducted to study the changes in proliferation. Overall, our findings suggest RYBP exerts anti-tumor effects in GBM and acts as a tumor suppressor gene. Future work should investigate the mechanism of RYBP’s phenotypic effects in GBM

Sox2/PARylated Parp1 complexes regulate pluripotency

Embryonic stem cells (ESCs) were first derived from the inner cell mass (ICM) of blastocyst-stage embryos. ESCs are distinguished from other cell types by their pluripotency and unlimited self-replication. ESCs are able to differentiate into multiple cell types that represent all three primary germ layers (ectoderm, mesoderm and endoderm) and can form an entire adult organism. Under defined conditions, ESCs are self-renewable and have an extended life-span without compromising pluripotency. The plasticity of ESCs provides opportunities for regenerative medicine. The recent production of induced pluripotent stem cells (iPSC) offers an ideal personalized substitute for ESCs. Overexpression of four transcription factors, Oct4, Sox2 , Klf4 and c-Myc can reprogram mouse somatic fibroblasts to iPSCs which are similar to ESCs in many biological properties including gene expression patterns, DNA methylation status and the differentiation ability. I demonstrate an advanced protocol for iPSC reprogramming. By using single lentiviral-based vector combined with Cre-loxP system, I established transgene- free iPSC lines from adult mouse fibroblasts. Dissecting the mechanisms that control transcription of pluripotency core factors is critical for understanding the mechanisms that induce and maintain pluripotency. I propose a novel mechanism in which FGF signaling fine-tunes Sox2 activity through post-translational modification of a critical interacting protein, Parp1, and balances the maintenance of ESC pluripotency and differentiation. In addition, I demonstrated that regulation of Sox2 activity by Parp1 is critical for efficient generation of induced pluripotent stem cells (iPSCs)