In contrast to RNAi , which lowers but never fully abrogates gene expression, NHEJ-based genome editing can completely disrupt gene expression in cells that have INDELs that inactivate all of the alleles of a gene. While NHEJ-based gene editing and RNAi have both largely proved efficient in the study of gene function, there are also Cas9-sgRNA: Making It Work in Primary Cells In the immediate years after the first demonstration of the use of the Cas9-sgRNA system for gene editing purposes, hundreds of papers were published using it but the majority employed the system in easily manipulated cell lines using plasmid delivery. Delivery of ZFN-encoding plasmids to primary cell types like T cells and human hematopoietic stem and progenitor cells has yielded relatively low Ex Vivo Gene Editing in Hematopoietic Stem Cells Ex vivo gene editing offers the benefit of confining the process of genetic manipulation to a defined subset of cells that is extracted from a patient and transplanted back after gene editing. Can gene editing be applied to treat multigenic diseases? How do we overcome current limitations of delivery efficiency and tissue specificity in in vivo gene editing while balancing nuclease exposure and activity? What impact will off-target nuclease activity have on gene editing therapies and which genotoxicity assays will be required to assess Glossary Adeno-associated virus a single-stranded, nonpathogenic virus that has been used extensively as a gene vector for gene transfer into a variety of cell types.

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To help readers to grasp the essence and to better organize the diverse applications, we categorize them under four gene therapy strategies: gene replacement therapy for monogenic diseases, gene addition for complex disorders and infectious diseases, gene expression alteration targeting RNA, and gene editing to introduce targeted changes in host genome. Integrating viral vectors such as gamma-retroviral vectors and lentiviral vectors have been used to deliver genes into isolated HSCs, and to replace the adenosine deaminase gene and the interleukin-2 receptor subunit gamma gene for ADA-SCID and X-linked SCID, respectively. Gene addition for complex disorders and infectious diseases In contrast to a single gene defect underlying monogenic diseases, the combination effects of multiple genes and environmental factors cause complex disorders such as cancer and heart diseases, rendering gene replacement not feasible for these disorders. In Duchene muscular dystrophy, many mutations in the DMD gene disrupt reading frame and generate a premature stop codon in mature mRNA. Since the DMD gene is too big to be packaged in the most efficient in vivo gene delivery vector AAV for gene replacement, DNA or vector-derived RNA AONs are designed to induce exon skipping, and to restore the correct reading frame, rather than replacing the entire DMD gene.

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Cargo Genes Encoding Therapeutic Proteins The question that comes together with the development of suitable vectors for gene transfer in the CNS is which genes to transfer. Apart from genes encoding therapeutic proteins there are other cargo options for CNS gene therapy vectors, like gene editing, chemogenetic, and optogenetic tools. Gene Editing Tools A step forward was made in gene therapy with the development of gene editing tools that can correct genetic defects directly in the host DNA. These tools generate a double strand break at a precisely desired location, and the break allows to take advantage of the fine strategies that cells have evolved to detect and repair DNA damage. Post-Transcriptional Gene Regulation The term "Post-transcriptional gene regulation" refers to approaches designed to enhance degradation or block translation of a target mRNA. The prototypical example is RNA interference, a physiological and evolutionarily conserved gene silencing mechanism normally present in eukaryotic cells, which is mediated by a group of noncoding small RNAs.

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Classical gene therapy approaches Proof-of-concept studies in cell cultures and animal models have demonstrated the effectiveness of gene therapy in treating many neurological disorders , usually by either overexpression of a therapeutic gene, by either boosting expression of the non-mutated wild-type gene or by expressing a gene unrelated to the channelopathy that permits improvement of pathology. The direct modification of the single mutation in an ion channel gene, the disruption of a faulty gene as well as the regulation of endogenous gene expression have advantages compared to the current strategies such as drug administration, optogenetics , chemogenetics, channel overexpression or RNA interference. Lentivirus Lentiviral vectors are a popular vector for CNS gene therapy as they result in a long-lasting gene expression within neurons without inducing a significant host immune response. Distinct approaches can be used to treat a channelopathy using a CRISPR-Cas strategy: disruption of gene expression, thereby reducing mutant protein levels; regulation of endogenous gene expression and epigenetic modifications ; and finally, the repair of point mutations in a faulty gene, restoring normal function to the translated protein.

Therapeutic genome editing was born out of the idea that the ideal therapy for monogenic diseases would be to develop a method that can correct the disease-causing mutations directly; but as genome editing has developed in concert with continuing improvements in our understanding of the genetic contribution to non-monogenic diseases, the principle of genome editing is being developed not only to cure monogenic diseases but also to cure more common diseases that have multifactorial origins. Table 1 Contrasting characteristics of the four standard nuclease platforms Full size table The only tool needed for NHEJ-mediated genome editing is an engineered nuclease, but HR-mediated genome editing also requires an engineered donor vector. Studies of the globin switch have demonstrated that Bcl11-A is a transcriptional repressor of HBG and that repression of Bcl11A results in the de-repression of HBG [56 ]. Moreover, when used as a research tool, genome editing demonstrated that deletion of a specific regulatory element in the Bcl11A gene, the erythroid enhancer, could repress Bcl11A in the erythroid lineage but not in the B-cell lineage, thus validating the inactivation of this element by NHEJ-mediated genome editing in HSPCs as a therapeutic strategy [53 ]. A different strategy using NHEJ-mediated genome editing is being developed to treat Duchenne's muscular dystrophy, a monogenic disease cause by mutations in the Dystrophin gene. In vivo nuclease-mediated genome editing has been used to mutate the PCSK9 gene in livers, with a resultant drop in cholesterol levels [69 , 70 ]. Although there are multiple caveats to these experiments, they do show, in principle, how in vivo editing might be used to treat multifactorial diseases whose course could be modified by using genome editing to create a clinically useful genotype.

Breakdown of Responses for Survey Items Used to Gauge Participant's Agreement for Various Applications of Human Gene Editing Putative predictors for support or opposition to these applications were explored using multiple regression. We did not find an association between self-reported wealth and support or resistance to gene editing in humans, but participants from countries with high Gross Domestic Product per capita were generally more supportive of all health-related applications of gene editing as well as its use in GM food. While the scientific community appears unified regarding the merits of somatic cell editing, the issue of germline editing is more divisive, demanding greater public discussion. Some have voiced concern that public backlash regarding germline editing will hinder somatic cell research, and the recent international summit on human gene editing concluded, "It would be irresponsible to proceed with any clinical use of germline editing unless there is broad societal consensus about the appropriateness of the proposed application." Interestingly, our study participants generally supported the application of embryonic editing for life-threatening or debilitating diseases.