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.

Herein, we review advances in gene delivery vectors targeting the liver and more specifically hepatocytes, covering strategies based on gene addition and gene editing, as well as the exciting results obtained with the use of RNA as a therapeutic molecule. Ex vivo gene therapy starts with an extraction of cells that are transduced with the vector carrying the therapeutic gene and then reintroduced into the body. In vivo gene therapy is based on the direct administration of the gene delivery vector or genetic material to the organism and can utilise different types of vectors, including non-viral and viral vectors. 64 Additionally, LNPs are now being used for the delivery of gene editing molecules such as ZFNs and Cas9 mRNA together with a single guideRNA. mRNA encoding ZFNs formulated into LNP have enabled >90% knockout of gene expression in mice by targeting the TTR or PCSK9 gene for the treatment of TTR amyloidosis and FH, respectively.

The rapid advance of CRISPR gene editing has evoked a predictable debate regarding "Reprogramming DNA," similar to the concerns regarding genetic manipulation that have been raised since the earliest days of gene cloning. Ethical support for providing gene editing therapies in a somatic cell is much like any other therapy directed at and for the patient, grounded in the principles of autonomy, beneficence, and nonmaleficence. In the United States the FDA Center for Biologics Evaluation and Research regulates cellular and gene therapy products on the basis of Section 351 of the Public Health Service Act on Cellular, Tissue and Gene Therapies. Given the ease and facility with which CRISPR gene editing has been able to correct many different disease causing gene defects in the laboratory, we need to now move forward with developing new CRISPR gene editing therapies to treat, or even cure, the many patients currently suffering from a wide range of devastating diseases.

Advances in nanotechnology are helping overcome systemic delivery hurdles in the development of gene editing therapies to cure diseases, according to a review of the sector in the journal Biomaterials. Gene editing tools Zinc Finger Nucleases, Transcription Activator-Like Effector Nucleases and the Clustered Regularly Interspaced Short Palindromic Repeat system offer the potential to tackle illnesses caused by faulty genes, but require safe and efficient delivery to the cell nucleus. Gene editors can be delivered using nanoparticulate lipid-like and polymeric materials, which can bypass delivery obstacles and move the emerging field closer to clinical use.

Current clinical gene therapy trials of gene supplementation in the eye involve the delivery of exogenous genetic material into cells with inherited genetic defects, while genome surgery focuses on the precise modification of endogenous genomes to correct mutant alleles. For dominant-negative conditions, scientists have focused on genetic tools to modulate gene expression, such as RNAi, or tools that modify the patient's genome to mutate the pathogenic allele, such as site-specific nucleases like CRISPR-Cas. While adenoviruses are not frequently used for the transduction of eye cells, efficient transduction, episomal nature, and large genome size make them attractive for use in gene therapy of the eye. While groups have observed larger gene delivery using AAVs, such as the case of a 8.9-kb ABCA4 expression cassette delivery in a mouse model of Stargardt disease, this is now believed to be the result of recombination of viral packaged gene fragments.

Inherited retinal diseases (IRDs) can be treated using gene-editing technology based on clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) and an RNA that guides the Cas protein to a predetermined region of the genome, according to researchers from the University of Modena and Reggio Emilia. The genome editing technology can permanently and precisely replace or remove genetic mutations causative of disease, without the need for supplemental therapies that are often inefficient and expensive. The researchers highlighted the potential use of CRISPR/Cas genome editing in retinal and patient-derived induced pluripotent stem cells in the retina as several emerging treatment options.

The possibility of curing inherited retinal diseases through gene therapy has gained momentum with the approval of Luxturna by the US Food and Drug Administration for treating Leber congenital amaurosis caused by RPE65 mutations. However, the success of gene therapy depends on several factors specific to the disease being treated. While gene replacement, silencing, and editing are being tested with different delivery methods and injection sites, ongoing clinical trials are expected to continue advancing the field of gene therapy. For now, gene therapy is a relatively safe approach that holds great promise for treating retinal diseases.