Academic literature on the topic 'CRISPR, Gene editing, Parkinson, Gene therapy'
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Journal articles on the topic "CRISPR, Gene editing, Parkinson, Gene therapy"
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Chung, Sun-Ku, and Seo-Young Lee. "Advances in Gene Therapy Techniques to Treat LRRK2 Gene Mutation." Biomolecules 12, no. 12 (December 5, 2022): 1814. http://dx.doi.org/10.3390/biom12121814.
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Leucine-rich repeat kinase 2 (LRRK2) gene mutation is an autosomal dominant mutation associated with Parkinson’s disease (PD). Among LRRK2 gene mutations, the LRRK2 G2019S mutation is frequently involved in PD onset. Currently, diverse gene correction tools such as zinc finger nucleases (ZFNs), helper-dependent adenoviral vector (HDAdV), the bacterial artificial chromosome-based homologous recombination (BAC-based HR) system, and CRISPR/Cas9-homology-directed repair (HDR) or adenine base editor (ABE) are used in genome editing. Gene correction of the LRRK2 G2019S mutation has been applied whenever new gene therapy tools emerge, being mainly applied to induced pluripotent stem cells (LRRK2 G2019S-mutant iPSCs). Here, we comprehensively introduce the principles and methods of each programmable nuclease such as ZFN, CRISPR/Cas9-HDR or ABE applied to LRRK2 G2019S, as well as those of HDAdV or BAC-based HR systems used as nonprogrammable nuclease systems.
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Rahman, Mujeeb ur, Muhammad Bilal, Junaid Ali Shah, Ajeet Kaushik, Pierre-Louis Teissedre, and Małgorzata Kujawska. "CRISPR-Cas9-Based Technology and Its Relevance to Gene Editing in Parkinson’s Disease." Pharmaceutics 14, no. 6 (June 13, 2022): 1252. http://dx.doi.org/10.3390/pharmaceutics14061252.
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Parkinson’s disease (PD) and other chronic and debilitating neurodegenerative diseases (NDs) impose a substantial medical, emotional, and financial burden on individuals and society. The origin of PD is unknown due to a complex combination of hereditary and environmental risk factors. However, over the last several decades, a significant amount of available data from clinical and experimental studies has implicated neuroinflammation, oxidative stress, dysregulated protein degradation, and mitochondrial dysfunction as the primary causes of PD neurodegeneration. The new gene-editing techniques hold great promise for research and therapy of NDs, such as PD, for which there are currently no effective disease-modifying treatments. As a result, gene therapy may offer new treatment options, transforming our ability to treat this disease. We present a detailed overview of novel gene-editing delivery vehicles, which is essential for their successful implementation in both cutting-edge research and prospective therapeutics. Moreover, we review the most recent advancements in CRISPR-based applications and gene therapies for a better understanding of treating PD. We explore the benefits and drawbacks of using them for a range of gene-editing applications in the brain, emphasizing some fascinating possibilities.
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De Plano, Laura M., Giovanna Calabrese, Sabrina Conoci, Salvatore P. P. Guglielmino, Salvatore Oddo, and Antonella Caccamo. "Applications of CRISPR-Cas9 in Alzheimer’s Disease and Related Disorders." International Journal of Molecular Sciences 23, no. 15 (August 5, 2022): 8714. http://dx.doi.org/10.3390/ijms23158714.
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Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease represent some of the most prevalent neurodegenerative disorders afflicting millions of people worldwide. Unfortunately, there is a lack of efficacious treatments to cure or stop the progression of these disorders. While the causes of such a lack of therapies can be attributed to various reasons, the disappointing results of recent clinical trials suggest the need for novel and innovative approaches. Since its discovery, there has been a growing excitement around the potential for CRISPR-Cas9 mediated gene editing to identify novel mechanistic insights into disease pathogenesis and to mediate accurate gene therapy. To this end, the literature is rich with experiments aimed at generating novel models of these disorders and offering proof-of-concept studies in preclinical animal models validating the great potential and versatility of this gene-editing system. In this review, we provide an overview of how the CRISPR-Cas9 systems have been used in these neurodegenerative disorders.
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Katzmann, Julius L., Arjen J. Cupido, and Ulrich Laufs. "Gene Therapy Targeting PCSK9." Metabolites 12, no. 1 (January 12, 2022): 70. http://dx.doi.org/10.3390/metabo12010070.
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The last decades of research in cardiovascular prevention have been characterized by successful bench-to-bedside developments for the treatment of low-density lipoprotein (LDL) hypercholesterolemia. Recent examples include the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) with monoclonal antibodies, small interfering RNA and antisense RNA drugs. The cumulative effects of LDL cholesterol on atherosclerosis make early, potent, and long-term reductions in LDL cholesterol desirable—ideally without the need of regular intake or application of medication and importantly, without side effects. Current reports show durable LDL cholesterol reductions in primates following one single treatment with PCSK9 gene or base editors. Use of the CRISPR/Cas system enables precise genome editing down to single-nucleotide changes. Provided safety and documentation of a reduction in cardiovascular events, this novel technique has the potential to fundamentally change our current concepts of cardiovascular prevention. In this review, the application of the CRISPR/Cas system is explained and the current state of in vivo approaches of PCSK9 editing is presented.
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Tang, Xuanting. "CRISPR/Cas9-based genome engineering in HIV gene therapy." E3S Web of Conferences 233 (2021): 02004. http://dx.doi.org/10.1051/e3sconf/202123302004.
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In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) technology has become the most heated genome editing technique. Comparing to earlier genetic engineering methods, the CRISPR/Cas system is more advantageous due to its simple convenient design, high efficiency, cost-effectiveness, and the ability to perform multi-sites editing simultaneously. As the most effective gene editing tool, it utilizes a simple short RNA-guided mechanism to direct Cas-mediated DNA cleavage at the target genome locus and exploits the endogenous DNA repair pathways to achieve site-specific gene modifications. Initially discovered as a part of the bacterial adaptive immune system, the CRISPR/Cas system has now been widely used to study a broad range of biological genomes. Besides its contribution to our understanding of the basic genetic science, the application of the CRISPR/Cas system also expands rapidly into the medical fields, showing great potentials in the research of genetic diseases, viral infectious diseases, and cancers. In this review, the latest research progress of CRISPR/Cas technology is summarized based on its development, mechanism, and application in HIV/AIDS intervention. This review also examines the existing weaknesses and the future prospects of this promising technology.
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S., Manasa M. "CRISPR-Cas9 gene editing technology in human gene therapy: the new realm of medicine." International Journal of Advances in Medicine 9, no. 4 (March 24, 2022): 513. http://dx.doi.org/10.18203/2349-3933.ijam20220796.
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Gene therapy has a huge clinical relevance in the present therapeutic world and is one of the many research fields of biology which received many benefits from the recent advancements of modern clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing technology. Researchers are on the way to make significant changes in the ways of treating genetic abnormalities. An increase in the number of approved clinical trials of CRISPR based gene therapy shows we are not too far from eliminating deadly diseases such as acquired immunodeficiency syndrome (AIDS), cancer and many inherited genetic conditions from the society. However, there are some challenges associated with the development of CRISPR technology in medical field most of which revolves around its safety, efficiency and ethics. Lack of an optimized method by which the CRISPR-Cas9 expression cassette can be delivered to cells is one of the main challenges when it comes to its application in human gene therapy. Although viral vectors are the most common delivery systems used in gene therapy, recent researches show promising results on using lipid- based l delivery systems such as liposome-templated hydrogel nanoparticles (LHNPs). As these could eliminate the safety concerns of using viral vectors, it is expected to have potential therapeutic applications in future. Nevertheless, the efficiency of non-viral systems is still not fully comparable with that of viral vectors. Hence, CRISPR based therapies might take longer than expected to be prevalent in the medical field. In this short review, the recent advances of CRISPR technology in gene therapy is discussed along with its challenges and limitations.
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Preece, Roland, and Christos Georgiadis. "Emerging CRISPR/Cas9 applications for T-cell gene editing." Emerging Topics in Life Sciences 3, no. 3 (April 2, 2019): 261–75. http://dx.doi.org/10.1042/etls20180144.
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Abstract Gene editing tools are being rapidly developed, accelerating many areas of cell and gene therapy research. Each successive gene editing technology promises increased efficacy, improved specificity, reduced manufacturing cost and design complexity; all of which are currently epitomised by the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas9) platform. Since its conceptualisation, CRISPR-based gene editing has been applied to existing methodologies and has further allowed the exploration of novel avenues of research. Implementation of CRISPR/Cas9 has been instrumental to recent progress in the treatment of cancer, primary immunodeficiency, and infectious diseases. To this end, T-cell therapies have attempted to harness and redirect antigen recognition function, and through gene editing, broaden T-cell targeting capabilities and enhance their potency. The purpose of this review is to provide insights into emerging applications of CRISPR/Cas9 in T-cell therapies, to briefly address concerns surrounding CRISPR-mediated indel formation, and to introduce CRISPR/Cas9 base editing technologies that hold vast potential for future research and clinical translation.
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Liu, Wenlou, Chunsheng Yang, Yanqun Liu, and Guan Jiang. "CRISPR/Cas9 System and its Research Progress in Gene Therapy." Anti-Cancer Agents in Medicinal Chemistry 19, no. 16 (January 23, 2020): 1912–19. http://dx.doi.org/10.2174/1871520619666191014103711.
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Genome editing refers to changing the genome sequence of an organism by knockout, insertion, and site mutation, resulting in changes in the genetic information of the organism. The clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein-9 nuclease (Cas9) system is a genome editing technique developed by the acquired immune system in the microbes, such as bacteria and archaebacteria, which targets and edits genome sequences according to the principle of complementary base pairing. This technique can be used to edit endogenous genomic DNA sequences in organisms accurately and has been widely used in fields, such as biotechnology, cancer gene therapy, and dermatology. In this review, we summarize the history, structure, mechanism, and application of CRISPR/Cas9 in gene therapy and dermatological diseases.
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Salsman, Jayme, and Graham Dellaire. "Precision genome editing in the CRISPR era." Biochemistry and Cell Biology 95, no. 2 (April 2017): 187–201. http://dx.doi.org/10.1139/bcb-2016-0137.
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With the introduction of precision genome editing using CRISPR–Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it is possible to engineer DNA double strand breaks (DSB) at specific genomic loci. DSB repair by the error-prone non-homologous end-joining (NHEJ) pathway can disrupt a target gene by generating insertions and deletions. Alternatively, Cas9-mediated DSBs can be repaired by homology-directed repair (HDR) using an homologous DNA repair template, thus allowing precise gene editing by incorporating genetic changes into the repair template. HDR can introduce gene sequences for protein epitope tags, delete genes, make point mutations, or alter enhancer and promoter activities. In anticipation of adapting this technology for gene therapy in human somatic cells, much focus has been placed on increasing the fidelity of CRISPR–Cas9 and increasing HDR efficiency to improve precision genome editing. In this review, we will discuss applications of CRISPR technology for gene inactivation and genome editing with a focus on approaches to enhancing CRISPR–Cas9-mediated HDR for the generation of cell and animal models, and conclude with a discussion of recent advances and challenges towards the application of this technology for gene therapy in humans.
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Kanu, Gayathri A., Javad B. M. Parambath, Raed O. Abu Odeh, and Ahmed A. Mohamed. "Gold Nanoparticle-Mediated Gene Therapy." Cancers 14, no. 21 (October 31, 2022): 5366. http://dx.doi.org/10.3390/cancers14215366.
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Gold nanoparticles (AuNPs) have gained increasing attention as novel drug-delivery nanostructures for the treatment of cancers, infections, inflammations, and other diseases and disorders. They are versatile in design, synthesis, modification, and functionalization. This has many advantages in terms of gene editing and gene silencing, and their application in genetic illnesses. The development of several techniques such as CRISPR/Cas9, TALEN, and ZFNs has raised hopes for the treatment of genetic abnormalities, although more focused experimentation is still needed. AuNPs, however, have been much more effective in trending research on this subject. In this review, we highlight recently well-developed advancements that are relevant to cutting-edge gene therapies, namely gene editing and gene silencing in diseases caused by a single gene in humans by taking an edge of the unique properties of the AuNPs, which will be an important outlook for future research.
Dissertations / Theses on the topic "CRISPR, Gene editing, Parkinson, Gene therapy"
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Giada, Beligni. "Application of the CRISPR-Cas9 genome editing approach for the correction of the p.Gly2019Ser (c.6055G>A) LRRK2 variant in Parkinson Disease." Doctoral thesis, Università di Siena, 2022. https://hdl.handle.net/11365/1220257.
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Parkinson’s disease (PD) is one of the most common long-term degenerative disorders that affect the nervous system. Clinical symptoms are bradykinesia, resting tremor and postural imbalance due to the loss of dopaminergic neurons in the substantia nigra pars compacta. Heterozygous mutations in the Leucine Rich Repeat Kinase 2 gene (LRRK2) have been identified both in familial and sporadic cases of PD. The most common variant is the p.Gly2019Ser substitution (c.6055G>A). To date there is no effective treatment available.
The genome editing tool CRISPR/Cas9 has recently transformed the field of biotechnology and biomedical discovery, posing the basis for the development of innovative treatments. Using CRISPR/Cas9 technology and Homology Directed Repair, our project aims to validate gene editing as an alternative therapeutic approach for PD through the genetic correction of the pathogenic p.Gly2019Ser LRRK2 mutation restoring the wild-type sequence both in human and mouse models. Specifically, we tested various strategies, based on the CRISPR/Cas9-based genome editing technique, for the correction of LRRK2 p.Gly2019Ser (c.6055G>A) variant in primary mouse and human fibroblasts with promising results. If the correction experiments in in vitro models will confirm the good efficiency of the approach, these experiments will represent a fundamental step for the subsequent evaluation of the potential of gene therapy for the treatment of PD as well as other brain disorders for which no therapy is currently available.
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Pigini, Paolo <1991>. "Neuroblastoma targeted therapy: employment of CRISPR gene-editing to explore relevant markers and potential targets in aggressive tumours." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2019. http://amsdottorato.unibo.it/8752/1/Pigini_Paolo_tesi.pdf.
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Neuroblastoma is a tumour originating from the sympathetic nervous system, and represents the most common extracranial solid cancer in childhood. Despite the malignancy is extremely heterogeneous, about 25% of all cases is characterized by MYCN-gene amplification, aggressive tumour and poor survival. The network of genes that are deregulated in this group of patients represents a focal point for targeted-therapy discovery. Along this research line, the first objective of the present project was to investigate the prognostic significance of a single nucleotide polymorphism (SNP) located in the promoter of ODC1, a neuroblastoma prognostic marker involved in polyamine biosynthesis. The SNP genotype was first associated with survival of a large cohort of patients with aggressive neuroblastoma. Then, CRISPR-editing revealed that the SNP genotype affects ODC1 expression and proliferation of neuroblastoma cells. At last, the SNP was found to influence cell sensibility to DFMO, an ODC1 inhibitor that is currently under trial for treatment of aggressive neuroblastoma. The second objective was to investigate the role in neuroblastoma development and progression of RUNX1T1, a poorly studied transcription repressor involved in distinct development events and cancers. Survival analysis of a cohort of neuroblastoma patients revealed that RUNX1T1 is a potential oncosuppressor. In apparent contrast, RUNX1T1 knockout by CRISPR-editing demonstrated that the gene promotes aggressiveness of neuroblastoma cells. Transcriptome analysis of the mutant cells then evidenced deregulation of a significant number of genes and pathways that are prognostic markers in neuroblastoma, therefore depicting a multifunctional regulation network that could be exploited for new therapies. The third and last objective was to test a novel therapeutic approach based on MYCN-amplification targeting via CRISPR-cleavage. In vitro experiments demonstrated that the system efficiently and specifically impairs the survival of aggressive neuroblastoma cells, thus providing a proof of principle for the development of an innovative therapy.
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Yang, Luhan. "Development of Human Genome Editing Tools for the Study of Genetic Variations and Gene Therapies." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11117.
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The human genome encodes information that instructs human development, physiology, medicine, and evolution. Massive amount of genomic data has generated an ever-growing pool of hypothesis. Genome editing, broadly defined as targeted changes to the genome, posits to deliver the promise of genomic revolution to transform basic science and personalized medicine. This thesis aims to contribute to this scientific endeavor with a particular focus on the development of effective human genome engineering tools.
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Fine, Eli Jacob. "A toolkit for analysis of gene editing and off-target effects of engineered nucleases." Diss., Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/54875.
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Several tools were developed to help researchers facilitate clinical translation of the use of engineered nucleases towards their disease gene of interest. Two major issues addressed were the inability to accurately predict nuclease off-target sites by user-friendly \textit{in silico} methods and the lack of a high-throughput, sensitive measurement of gene editing activity at endogenous loci. These objectives were accomplished by the completion of the following specific aims. An online search interface to allow exhaustive searching of a genome for potential nuclease off-target sites was implemented. Previously discovered off-target sites were collated and ranking algorithms developed that preferentially score validated off-target sites higher than other predictions. HEK-293T cells transfected with newly developed TALENs and ZFNs targeting the beta-globin gene were analyzed at the off-target sites predicted by the tool. Many samples of genomic DNA from cells treated with different ZFNs and TALENs were analyzed for off-target effects to generate a greatly expanded training set of bona fide off-target sites. Modifications to the off-target prediction algorithm parameters were evaluated for improvement through Precision-Recall analysis and several other metrics. An analysis pipeline was developed to process SMRT reads to simultaneously measure the rates of different DNA repair mechanisms by directly examining the DNA sequences. K562 cells were transfected with different types of nucleases and donor repair templates in order to optimize conditions for repairing the beta-globin gene. This work will have significant impact on future studies as the methods developed herein allow other laboratories to optimize nuclease-based therapies for single gene disorders.
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Amaya, Colina Anais Karime. "Towards the Treatment of Human Genetic Liver Disease by AAV-Mediated Genome Editing and Selective Expansion of Repaired Hepatocytes." Thesis, The University of Sydney, 2019. https://hdl.handle.net/2123/21893.
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Gene repair involves the correction of the genetic mutation directly at the defective locus with retention of physiological expression. The biggest challenge of this approach, however, is that gene repair by homologous recombination occurs at levels that are unlikely to be sufficient to confer therapeutic benefit in the majority of cell-autonomous liver disease phenotypes, such as OTC deficiency, the most common urea cycle disorder. To overcome this challenge, gene correction can be complemented by selective expansion strategies designed to expand repaired hepatocytes to frequencies required for therapeutic benefit. In vivo expansion can be achieved, for instance, by conferring a selective advantage to gene-corrected cells. In this study, human-specific genetic inhibitors were designed to exploit a selective expansion strategy based on the modulation of the tyrosine catabolism pathway and were successfully validated in humanised (Fah-/-, Rag2-/-, IL2rg-/-) FRG mice. Another way to increase the frequency of gene repair is to use nucleases to create DNA breaks at the target site to promote homology-directed repair (HDR). Recombinant AAV vectors carrying human-specific reagents for CRISPR/Cas9-mediated genome editing were developed in order to correct a single nucleotide mutation in exon 9 of the OTC gene. Initially, the editing reagents were evaluated in OTC-deficient mice with a transposed engineered “minigene” version of the OTC gene. Editing reagents functionally validated in this model were then evaluated in vivo on the native OTC locus in primary human hepatocytes, including patient-derived hepatocytes, xenografted into FRG mice. Availability of novel synthetic AAV capsids, such as NP59, facilitated high targeting efficiency of human hepatocytes which in turn resulted in up to 29% OTC alleles being corrected by HDR. The studies described in this thesis show for the first time precise gene repair of a disease-causing mutation in primary human hepatocytes in vivo.
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Foster, Robert Graham. "Development of a modular in vivo reporter system for CRISPR-mediated genome editing and its therapeutic applications for rare genetic respiratory diseases." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33040.
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Rare diseases, when considered as a whole, affect up to 7% of the population, which would represent 3.5 million individuals in the United Kingdom alone. However, while 'personalised medicine' is now yielding remarkable results using recent sequencing technologies in terms of diagnosing genetic conditions, we have made much less headway in translating this patient information into therapies and effective treatments. Even with recent calls for greater research into personalised treatments for those affected by a rare disease, progress in this area is still severely lacking, in part due to the astronomical cost and time involved in bringing treatments to the clinic. Gene correction using the recently-described genome editing technology CRISPR/Cas9, which allows precise editing of DNA, offers an exciting new avenue of treatment, if not cure, for rare diseases; up to 80% of which have a genetic component. This system allows the researcher to target any locus in the genome for cleavage with a short guide-RNA, as long as it precedes a highly ubiquitous NGG sequence motif. If a repair sequence is then also provided, such as a wild-type copy of the mutated gene, it can be incorporated by homology-directed repair (HDR), leading to gene correction. As both guide-RNA and repair template are easily generated, whilst the machinery for editing and delivery remain the same, this system could usher in the era of 'personalised medicine' and offer hope to those with rare genetic diseases. However, currently it is difficult to test the efficacy of CRISPR/Cas9 for gene correction, especially in vivo. Therefore, in my PhD I have developed a novel fluorescent reporter system which provides a rapid, visual read-out of both non-homologous end joining (NHEJ) and homology-directed repair (HDR) driven by CRISPR/Cas9. This system is built upon a cassette which is stably and heterozygously integrated into a ubiquitously expressed locus in the mouse genome. This cassette contains a strong hybrid promoter driving expression of membrane-tagged tdTomato, followed by a strong stop sequence, and then membrane-tagged EGFP. Unedited, this system drives strong expression of membrane-tdTomato in all cell types in the embryo and adult mouse. However, following the addition of CRISPR/Cas9 components, and upon cleavage, the tdTomato is rapidly excised, resulting via NHEJ either in cells without fluorescence (due to imperfect deletions) or with membrane-EGFP. If a repair template containing nuclear tagged-EGFP is also supplied, the editing machinery may then use the precise HDR pathway, which results in a rapid transition from membrane-tdTomato to nuclear- EGFP. Thereby this system allows the kinetics of editing to be visualised in real time and allows simple scoring of the proportion of cells which have been edited by NHEJ or corrected by HDR. It therefore provides a simple, fast and scalable manner to optimise reagents and protocols for gene correction by CRISPR/Cas9, especially compared to sequencing approaches, and will prove broadly useful to many researchers in the field. Further to this, I have shown that methods which lead to gene correction in our reporter system are also able to partially repair mutations found in the disease-causing gene, Zmynd10; which is implicated in the respiratory disorder primary ciliary dyskinesia (PCD), for which there is no effective treatment. PCD is an autosomal-recessive rare disorder affecting motile cilia (MIM:244400), which results in impaired mucociliary clearance leading to neonatal respiratory distress and recurrent airway infections, often progressing to lung failure. Clinically, PCD is a chronic airway disease, similar to CF, with progressive deterioration of lung function and lower airway bacterial colonization. However, unlike CF which is monogenic, over 40 genes are known to cause PCD. The high genetic heterogeneity of this rare disease makes it well suited to such a genome editing strategy, which can be tailored for the correction of any mutated locus.
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Hsu, Patrick David. "Development of the CRISPR nuclease Cas9 for high precision mammalian genome engineering." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13068392.
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Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. To facilitate successful and specific Cas9 targeting, we first optimize the guide RNAs (sgRNA) to significantly enhance gene editing efficiency and consistency. We also systematically characterize Cas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target mutagenesis. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that Cas9-mediated cleavage is unaffected by DNA methylation and that the dosage of Cas9 and sgRNA can be titrated to minimize off-target modification. Additionally, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses. We next demonstrate that Cas9 nickase mutants can be used with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs can reduce off-target activity by over 1,500-fold in human cells. In collaboration with researchers at the University of Tokyo, we further identified a PAM-interacting domain of the Cas9 nuclease that dictates Cas9 target recognition specificity. Finally, we present protocols that provide experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks. Taken together, this work enables a variety of genome engineering applications from basic biology to biotechnology and medicine.
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MINGOIA, MAURA. "Terapia genica della β Talassemia mediante editing del DNA." Doctoral thesis, Università degli Studi di Cagliari, 2016. http://hdl.handle.net/11584/266632.
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β (HBB) gene, resulting in absence (β0) or deficiency (β+) of β globin chain synthesis. This genetic disorder occurs most frequently in people from Mediterranean countries, such as Italy. In particular, the data indicates that about 12.6% of the Sardinian subjects are carriers of β thalassemia and these are among the highest frequencies of thalassemia genes found in a Caucasian population. In Sardinia, the disease is generally determined by a nonsense mutation at codon 39 (E39X) of exon 2 causing the interruption of β globin synthesis. Patients homozygous with E39X mutation have a severe anemia and require frequent transfusions and iron chelation. The only definitive cure today possible for β chain hemoglobinopathies is the hematopoietic stem cells transplantation, but it is limited by availability of HLA matched donors. However, in the last few years new therapeutic approaches for this genetic disease are taking place. The correction of disease-causing mutation through the technique of Genome-Editing in patient-specific stem cells and subsequent autologous transplantation would be the ideal approach for the treatment of monogenic diseases such as β thalassemia. However, due to difficulties in obtaining sufficient homologous recombination percentages for therapeutic purposes, the aim of my PhD project is to reproduce artificially the HPFH mutations identified in non-coding regions of the β globin cluster, using the system CRISPR/Cas9 associated with NHEJ pathway. In this way, we hope to restore at therapeutic levels the expression of HBG genes and consequently the synthesis of a functional HbF in order to ameliorate the phenotype of β thalassemia.
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Kennedy, Zachary C. "Optimizing CRISPR/Cas9 for Gene Silencing of SOD1 in Mouse Models of ALS." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1047.
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Mutations in the SOD1 gene are the best characterized genetic cause of amyotrophic lateral sclerosis (ALS) and account for ~20% of inherited cases and 1-3% of sporadic cases. The gene-editing tool Cas9 can silence mutant genes that cause disease, but effective delivery of CRISPR-Cas9 to the central nervous system (CNS) remains challenging. Here, I developed strategies using canonical Streptococcus pyogenes Cas9 to silence SOD1. In the first strategy, I demonstrate effectiveness of systemic delivery of guide RNA targeting SOD1 to the CNS in a transgenic mouse model expressing human mutant SOD1 and Cas9. Silencing was observed in both the brain and the spinal cord. In the second strategy, I demonstrate the effectiveness of delivering both guide RNA and Cas9 via two AAVs into the ventricles of the brain of SOD1G93A mice. Silencing was observed in the brain and in motor neurons within the spinal cord. For both strategies, treated mice had prolonged survival when compared to controls. Treated mice also had improvements in grip strength and rotarod function. For ICV treated mice, we detected a benefit of SOD1 silencing using net axonal transport assays, a novel method to detect motor neuron function in mice before onset of motor symptoms. These studies demonstrate that Cas9-mediated genome editing can mediate disease gene silencing in motor neurons and warrants further development for use as a therapeutic intervention for SOD1-linked ALS patients.
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Schneider, Sara Jane. "Delivery of CRISPR/Cas9 RNAs into Blood Cells of Zebrafish: Potential for Genome Editing in Somatic Cells." Thesis, University of North Texas, 2017. https://digital.library.unt.edu/ark:/67531/metadc1011754/.
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Factor VIII is a clotting factor found on the intrinsic side of the coagulation cascade. A mutation in the factor VIII gene causes the disease Hemophilia A, for which there is no cure. The most common treatment is administration of recombinant factor VIII. However, this can cause an immune response that renders the treatment ineffective in certain hemophilia patients. For this reason a new treatment, or cure, needs to be developed. Gene editing is one solution to correcting the factor VIII mutation. CRISPR/Cas9 mediated gene editing introduces a double stranded break in the genomic DNA. Where this break occurs repair mechanisms cause insertions and deletions, or if a template oligonucleotide can be provided point mutations could be introduced or corrected. However, to accomplish this goal for editing factor VIII mutations, a way to deliver the components of CRISPR/Cas9 into somatic cells is needed. In this study, I confirmed that the CRISPR/Cas9 system was able to create a mutation in the factor VIII gene in zebrafish. I also showed that the components of CRISPR/Cas9 could be piggybacked by vivo morpholino into a variety of blood cells. This study also confirmed that the vivo morpholino did not interfere with the gRNA binding to the DNA, or Cas9 protein inducing the double stranded break.
Book chapters on the topic "CRISPR, Gene editing, Parkinson, Gene therapy"
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Hung, Sandy S., Fan Li, Jiang-Hui Wang, Anna E. King, Bang V. Bui, Guei-Sheung Liu, and Alex W. Hewitt. "Methods for In Vivo CRISPR/Cas Editing of the Adult Murine Retina." In Retinal Gene Therapy, 113–33. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7522-8_9.
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María Vaschetto, Luis. "CRISPR/Cas and Gene Therapy: An Overview." In CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases, 85–89. New York: CRC Press, 2021. http://dx.doi.org/10.1201/9781003088516-5.
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Gouw, Arvin M. "Challenging the Therapy/Enhancement Distinction in CRISPR Gene Editing." In The Palgrave Handbook of Philosophy and Public Policy, 493–508. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-319-93907-0_38.
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López-Márquez, Arístides, Ainhoa Martínez-Pizarro, Belén Pérez, Eva Richard, and Lourdes R. Desviat. "Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells." In Methods in Molecular Biology, 167–84. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2010-6_10.
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AbstractThe field of splice modulating RNA therapy has gained new momentum with FDA approved antisense-based drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate splicing. However, minigenes do not include the full genomic context of the exons under study and patients’ samples are not always available, especially if the gene is expressed solely in certain tissues (e.g. liver or brain). This is the case for specific inherited metabolic diseases such as phenylketonuria (PKU) caused by mutations in the liver-expressed PAH gene.Herein we describe the generation of mutation-specific hepatic cellular models of PKU using CRISPR/Cas9 system, which is a versatile and easy-to-use gene editing tool. We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant, which should result in the accurate recapitulation of the splicing defect.
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Mandal, Shubhjeet, and Piyush Kumar Tiwari. "Gene Therapy and Gene Editing for Cancer Therapeutics." In Handbook of Research on Advancements in Cancer Therapeutics, 116–204. IGI Global, 2021. http://dx.doi.org/10.4018/978-1-7998-6530-8.ch004.
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Over the past two decades, developments in human genomics have shown that cancer in the host genome is caused by somatic aberration. This discovery has inspired interest among cancer researchers; many are now using genetic engineering therapeutic methods to improve the cancer regression and seeking a possible cure for the disease. The large gene therapy sector offers a variety of therapies which are likely to become effective in preventing cancer deaths. The latest clinical trials of third generation vaccines for a wide variety of cancers have produced promising results. Cancer virotherapy, which uses viral particles replicating within the cancer cell, is an emerging method of treatment which shows great promise. The latest developments in gene editing techniques, such as CRISPR, Cas9, TALENs, and ZFNs, are being used to help to make cancer a manageable condition. Gene therapy is expected to play a significant role in potential cancer therapy as a part of a multi-modality procedure.
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Scherman, Daniel, and Jean-Louis Mandel. "GENOME ENGINEERING AND GENOME EDITING USING CRISPR/CAS9–RNA-GUIDED NUCLEASE." In Advanced Textbook on Gene Transfer, Gene Therapy and Genetic Pharmacology, 115–30. WORLD SCIENTIFIC (EUROPE), 2019. http://dx.doi.org/10.1142/9781786346889_0008.
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Ramirez, Juan C. "Gene Editing and CRISPR Therapeutics: Strategies Taught by Cell and Gene Therapy." In Progress in Molecular Biology and Translational Science, 115–30. Elsevier, 2017. http://dx.doi.org/10.1016/bs.pmbts.2017.08.003.
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Becerra, Edgardo, Valeria Soto-Ontiveros, and Guadalupe García Alcocer. "CRISPR-Cas9-based Strategies for Acute Lymphoblastic Leukemia Therapy." In Leukemia - From Biology to Diagnosis and Treatment [Working Title]. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.106702.
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Defeating cancer as leukemia has been an up and down challenge. However, leukemia must be treated from the roots. Nowadays, the CRISPR-Cas9 system provided scientists the ability to manipulate the genetic information to correct mutations, rewrite genetic code, or edit immune cells for immunotherapy purposes. Additionally, such system is used for basic and clinical approaches in leukemia therapy. Lymphoid cancers including acute lymphoblastic leukemia (ALL) can be treated by performing gene editing or enhancing immune system through CART cells. Here, we present and detail therapeutic applications of the CRISPR/Cas9 system for immune cell therapy, and knock-out or knock-in of main genes promoting leukemogenesis or ALL progression. We also described current and future challenges, and optimization for the application of CRISPR/Cas9 system to treat lymphoid malignancies.
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Rao, J. Venkateshwara, R. Ravindar Naik, S. Venkanna, and N. Ramesh Kumar. "Application of 21st Century Genetic Engineering Tools and CRISPR-Cas9 Technologies to Treat Most Advanced Cardiovascular Diseases of Humans." In Advancements in Cardiovascular Research and Therapeutics: Molecular and Nutraceutical Perspectives, 79–103. BENTHAM SCIENCE PUBLISHERS, 2022. http://dx.doi.org/10.2174/9789815050837122010008.
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21st Century Genome-editing technologies have been rapidly emerging as the most powerful tool capable of creating genetically altered cells or organisms for explicit gene functions and mechanisms for causing several human ailments. While clinical gene therapy celebrates its first taste of success, with several products approved for clinical usage and several thousands of them awaiting stages in pipelines, unfortunately, there are no gene therapy treatment methods available for many cardiovascular diseases (CVD). Despite sustained medical advances over the last 50 years in CVD, the main cause of death is still uncertain in the developed world. The management of genetic expression by using small molecule RNA therapeutics and the development of accurate gene corrections may lead to several applications, such as cardiac revitalization after myocardial infarctions and gene corrections for the inherited cardiomyopathies but certainly with some limitations. CRISPR/Cas9 technology can be utilized to realign DNA modifications ranging from a single base pair to multiplepairs of mutations in both in vitro and in vivo models. This book chapter emphasizes various types of applications by CRISPR technologies in cardio-vascular research, and genome-editing novel therapies for future medicines.
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Ejaz, Mahnoor, Areena Suhail Khan, Faiza Naseer, and Alvina Gul. "Metabolic Syndromes." In Omics Technologies for Clinical Diagnosis and Gene Therapy: Medical Applications in Human Genetics, 242–68. BENTHAM SCIENCE PUBLISHERS, 2022. http://dx.doi.org/10.2174/9789815079517122010018.
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Metabolic Syndromes (MetS) are recognized as a cluster of risk factors which are known to increase the likelihood of obesity, type 2 diabetes (T2D) and cardiovascular disorders (CVDs). It is significant to understand disease pathology in order to discover a pathological mechanism leading to the development of MetS. Elevated triglycerides, increased blood pressure, hyperglycemia (increased blood glucose levels), low levels of High-density lipoprotein (HDL) cholesterol and elevated waist circumference are key parameters in diagnosing MetS. Various therapeutic interventions have been developed for treating metabolic diseases like polypills which are commonly known as combination pills, along with the fixed dose combinations. In addition to pharmacological handling, surgical treatment is also showing success in treating MetS such as Bariatric treatment. With the emerging experimental techniques, gene therapy allows the replacement of a defective gene with a healthy one, which may eventually reverse the disease. Leptin Gene Therapy, ZFN Gene Editing, CRISPR/ Cas9 genome editing are different platforms of gene therapy which are showing promising results in treating the metabolic disease. Novel experimental approaches and pharmacological treatments can provide a better insight into metabolic syndrome and its related complications, thereby reducing its global burden.
Conference papers on the topic "CRISPR, Gene editing, Parkinson, Gene therapy"
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Divita, Gilles, Elodie Czuba, Melanie Guidetti, Audrey Gunenberger, Leslie Tempremant, Veronique Josserand, and Neil Desai. "Abstract 1826: CRISPR mediated KRAS mutant gene editing in pancreatic and colorectal cancer therapy." In Proceedings: AACR Annual Meeting 2020; April 27-28, 2020 and June 22-24, 2020; Philadelphia, PA. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1538-7445.am2020-1826.
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