Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

The Xist long noncoding RNA orchestrates X chromosome inactivation, a process that entails chromosome-wide silencing and remodeling of the three-dimensional (3D) structure of the X chromosome. Yet, it remains unclear whether these changes in nuclear structure are mediated by Xist and whether they are required for silencing. Here, we show that Xist directly interacts with the Lamin B receptor, an integral component of the nuclear lamina, and that this interaction is required for Xist-mediated silencing by recruiting the inactive X to the nuclear lamina and by doing so enables Xist to spread to actively transcribed genes across the X. Our results demonstrate that lamina recruitment changes the 3D structure of DNA, enabling Xist and its silencing proteins to spread across the X to silence transcription

Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

The Xist long noncoding RNA orchestrates X chromosome inactivation, a process that entails chromosome-wide silencing and remodeling of the three-dimensional (3D) structure of the X chromosome. Yet, it remains unclear whether these changes in nuclear structure are mediated by Xist and whether they are required for silencing. Here, we show that Xist directly interacts with the Lamin B receptor, an integral component of the nuclear lamina, and that this interaction is required for Xist-mediated silencing by recruiting the inactive X to the nuclear lamina and by doing so enables Xist to spread to actively transcribed genes across the X. Our results demonstrate that lamina recruitment changes the 3D structure of DNA, enabling Xist and its silencing proteins to spread across the X to silence transcription

Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus

Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus

Identification and validation of novel human genomic safe harbor sites

Numerous gene addition methods are gaining increasing popularity in the field of gene therapy, where replacement of the mutated copy of the gene is required, as well as in cell engineering, in which synthetic receptors can be introduced into a cell or a group of cells to create artificial gene circuits capable of eliciting therapeutic or tissue enhancing functions. Existing gene addition tools suffer from heterogeneity of transgene expression levels and may cause aberration to normal transcriptomic profile due to up- or down-regulation of both protein coding and non-protein coding genes. With the advent of targeted gene integration methods, the necessity for the identification of genomic loci, which would support durable and safe transgene expression – Genomic Safe Harbor (GSH) sites – became ever more urgent. In this dissertation I describe a pipeline for computational prediction and experimental validation of novel human GSH sites using existing as well as newly introduced genomic safety criteria. In chapter 1 I explain the use of a rational approach to verify computationally predicted genomic sites by targeted integration of reporter as well as therapeutic genes into select computationally predicted locations. This approach yielded the identification of two candidate GSHs, which showed robust and durable expression in investigated cell lines and were later confirmed in primary human T cells and primary human dermal fibroblasts. The safety of transgene expression upon integration into these two sites was subsequently verified using bulk and single-cell transcriptomic analyses, which showed minimal changes in global RNA expression levels following transgene integration. Overall, these two newly identified GSH sites create a broad platform for safer and more reliable gene addition-based gene and cell therapies, facilitating their transition into clinical practice. In chapter 2 I describe an attempt to implement a multiplexed experimental search of novel GSHs using high-throughput library-based approach. Specifically, described method would allow for a rapid screen of thousands GSH sites exploiting a library of guide RNAs targeting various computationally predicted GSH locations and a non-homologous end joining pathway to drive targeted insertion of a reporter transgene into a genomic locus determined by a guide RNA library member. Such pooled approach would allow to reveal a set of highly transcribed loci, allowing for their subsequent validation by individual transgene integration and transcriptomics assessment. This study, however, was associated with numerous experimental hurdles and was eventually discontinued with suggestions on further optimizations in the future. To date, only three empirically validated sites in the human genome have been reported for durable expression in different cellular contexts. However, all three of them are located in gene dense regions surrounded by proven oncogenes, significantly increasing the risk of integration-induced tumorigenesis. Furthermore, they do not support the rapid pace of innovation in synthetic biology that enables multiple transgene integration and genetic circuits to rewire and reprogram cellular function. Two novel, computationally and experimentally validated GSH sites described in this thesis open new opportunities for safer and more predictable genome engineering of human cells, expanding the toolkit for diverse cell therapy and synthetic biology applications, from the treatment of inherited disorders by replacing mutated genes with their functional copies, to creating synthetic networks in immune cells to drive multi-input response, to augmenting properties of cells and tissues by safe addition of enhancing transgenes. Finally, thanks to long-term high levels of transgene expression, identified GSH sites can be used for large-scale therapeutic protein manufacturing in human hosts

Восстановление сенсорной фузии у детей методом попеременного разобщения полей зрения

Введение. Реабилитация зрительных функций в детском возрасте является актуальной проблемой детской офтальмологии. Поиски новых реабилитационных мероприятий при таких заболеваниях, как миопия и ретинопатия недоношенных, постоянно ведутся исследователями. Но не менее важным является своевременное эффективное проведение реабилитационных мероприятий для восстановления бинокулярных функций при косоглазии. Цель. Оценка эффективности восстановления сенсорной фузии и бинокулярного зрения жидкокристаллическими (ЖК) очками в сравнении с ортоптическим лечением на синоптофоре. Материал и методы. Проведено лечение 99 пациентам с оперированным содружественным сходящимся косоглазием, остаточным углом косоглазия после оперативного вмешательства до 10° и отсутствием сенсорной фузии. все пациенты были с гиперметропической рефракцией: по сферическому компоненту рефракция была не более 4,75 диоптрий, по цилиндрическому – не более 1,5 диоптрий. При этом допускалось наличие амблиопии слабой степени. Исключали пациентов с вертикальным косоглазием и циклотропией, выявленных при обследовании на синоптофоре и исследовании циклотропии посредством палочек Maddox, а также пациентов с сопутствующей офтальмологической патологией. всем пациентам проводили стандартное офтальмологическое исследование, а также определение характера зрения 4-х точечным тестом, исследование на синоптофоре с определением объективного и субъективного углов косоглазия. в основную группу были включены 46 пациентов, которые проходили лечение попеременным разобщением полей зрения ЖК-очками постоянно в течение 4 часов в день, контрольная группа включала 53 пациента, получавших ортоптическое лечение на синоптофоре курсами по 10 дней (3-4 курса). Период наблюдения составил 12 месяцев. Результаты. Эффективность восстановления сенсорной фузии и бинокулярного зрения была существенно выше при лечении ЖК-очками по сравнению с синоптофором. Так, устойчивая сенсорная фузия сформировалась у 32 (69,5%) и 11 (21%) детей, неустойчивая – у 9 (19,6%) и 2 (4%) детей; лечение было неэффективным у 5 (10,9%) и 40 (75%) детей соответственно. Бинокулярное зрение непосредственно после лечения восстановилось у 18 (39,1%) и 11 (21%) детей, а после проведения диплоптических процедур еще у 12 (66,7%) детей основной группы (все различия между группами достоверны (p<0,005). Заключение. После проведенной хирургии косоглазия в состоянии ортотропии лечение методом попеременного разобщения полей зрения ЖК-очками является более эффективным методом восстановления сенсорной фузии и реабилитации бинокулярных функций по сравнению с лечением на синоптофоре

Discovery and validation of human genomic safe harbor sites for gene and cell therapies

Existing approaches to therapeutic gene transfer are marred by the transient nature of gene expression following non-integrative gene delivery and by safety concerns due to the random mechanism of viral-mediated genomic insertions. The disadvantages of these methods encourage future research in identifying human genomic sites that allow for durable and safe expression of genes of interest. We conducted a bioinformatic search followed by the experimental characterization of human genomic sites, identifying two that demonstrated the stable expression of integrated reporter and therapeutic genes without malignant changes to the cellular transcriptome. The cell-type agnostic criteria used in our bioinformatic search suggest widescale applicability of identified sites for engineering of a diverse range of tissues for clinical and research purposes, including modified T cells for cancer therapy and engineered skin to ameliorate inherited diseases and aging. In addition, the stable and robust levels of gene expression from identified sites allow for the industry-scale biomanufacturing of proteins in human cells

High-throughput T cell receptor engineering by functional screening identifies candidates with enhanced potency and specificity

A major challenge in adoptive T cell immunotherapy is the discovery of natural T cell receptors (TCRs) with high activity and specificity to tumor antigens. Engineering synthetic TCRs for increased tumor antigen recognition is complicated by the risk of introducing cross-reactivity and by the poor correlation that can exist between binding affinity and activity of TCRs in response to antigen (peptide-MHC). Here, we developed TCR-Engine, a method combining genome editing, computational design, and deep sequencing to engineer the functional activity and specificity of TCRs on the surface of a human T cell line at high throughput. We applied TCR-Engine to successfully engineer synthetic TCRs for increased potency and specificity to a clinically relevant tumor-associated antigen (MAGE-A3) and validated their translational potential through multiple in vitro and in vivo assessments of safety and efficacy. Thus, TCR-Engine represents a valuable technology for engineering of safe and potent synthetic TCRs for immunotherapy applications

LRPPRC mutations cause early-onset multisystem mitochondrial disease outside of the French-Canadian population

Mitochondrial Complex IV [cytochrome c oxidase (COX)] deficiency is one of the most common respiratory chain defects in humans. The clinical phenotypes associated with COX deficiency include liver disease, cardiomyopathy and Leigh syndrome, a neurodegenerative disorder characterized by bilateral high signal lesions in the brainstem and basal ganglia. COX deficiency can result from mutations affecting many different mitochondrial proteins. The French-Canadian variant of COX-deficient Leigh syndrome is unique to the Saguenay-Lac-Saint-Jean region of Québec and is caused by a founder mutation in the LRPPRC gene. This encodes the leucine-rich pentatricopeptide repeat domain protein (LRPPRC), which is involved in post-transcriptional regulation of mitochondrial gene expression. Here, we present the clinical and molecular characterization of novel, recessive LRPPRC gene mutations, identified using whole exome and candidate gene sequencing. The 10 patients come from seven unrelated families of UK-Caucasian, UK-Pakistani, UK-Indian, Turkish and Iraqi origin. They resemble the French-Canadian Leigh syndrome patients in having intermittent severe lactic acidosis and early-onset neurodevelopmental problems with episodes of deterioration. In addition, many of our patients have had neonatal cardiomyopathy or congenital malformations, most commonly affecting the heart and the brain. All patients who were tested had isolated COX deficiency in skeletal muscle. Functional characterization of patients’ fibroblasts and skeletal muscle homogenates showed decreased levels of mutant LRPPRC protein and impaired Complex IV enzyme activity, associated with abnormal COX assembly and reduced steady-state levels of numerous oxidative phosphorylation subunits. We also identified a Complex I assembly defect in skeletal muscle, indicating different roles for LRPPRC in post-transcriptional regulation of mitochondrial mRNAs between tissues. Patient fibroblasts showed decreased steady-state levels of mitochondrial mRNAs, although the length of poly(A) tails of mitochondrial transcripts were unaffected. Our study identifies LRPPRC as an important disease-causing gene in an early-onset, multisystem and neurological mitochondrial disease, which should be considered as a cause of COX deficiency even in patients originating outside of the French-Canadian population