Correction Of Mutation Research Articles

CRISPR-Cas9: A cornerstone for the evolution of precision medicine.

Modern genetic therapy incorporates genomic testing and genome editing. It is the finest approach for precision medicine. Genome editing is a state-of-the-art technology to manipulate gene expression thus generating a particular genotype. It encompasses multiple programmable nuclease-based approaches leading to genetic changes. Not surprisingly, this method triggered internationally a wide array of controversies in the scientific community and in the public since it transforms the human genome. Given its importance, the pace of this technology is exceptionally fast. In this report, we introduce one aspect of genome editing, the CRISPR/Cas9 system, highlight its potential to correct genetic mutations and explore its utility in clinical setting. Our goal is to enlighten health care providers about genome editing and incite them to take part of this vital debate.

Use and application of 3D-organoid technology.

The capacity of the 3D-organoid cultures to resemble a near-physiological tissue organization and to mimic - to a certain degree - organ functionality, make organoids an excellent model for applications spanning from basic developmental/stem cell research to personalized medicine. Here, we review key findings achieved through organoid technology, and we discuss applications such as disease - and tumour modelling, correction of genetic mutations and understanding gene - and cell functions. Finally, we discuss future developments in the field.

Recent Advances in Therapeutic Genome Editing in China.

Editing of the genome to correct disease-causing mutations is a promising approach for the treatment of human diseases. Recent advances in the development of programmable nuclease-based genome editing tools have substantially improved the ability to make precise changes in the human genome. Genome editing technologies are already being used to correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional gene therapies. Chinese scientists have made remarkable breakthroughs in the field of therapeutic genome editing, particularly with the first clinical trial involving the clustered regularly interspaced short palindromic repeats-caspase 9 system that began in China. Herein, current progress toward developing programmable nuclease-based gene therapies is introduced, as well as future prospects and challenges in China.

CRISPR: Established Editor of Human Embryos?

Off-target effects and mosaicism are major concerns for applying CRISPR-Cas9 to correct genetic mutations. A recent article in Nature by Ma etal. (2017) uses an elegant CRISPR-Cas9 approach that repairs a genetic mutation in human embryos with negligible mosaicism and no off-target effects, bringing this editing tool closer to clinical application.

Cellular Reprogramming, Genome Editing, and Alternative CRISPR Cas9 Technologies for Precise Gene Therapy of Duchenne Muscular Dystrophy.

In the past decade, the development of two innovative technologies, namely, induced pluripotent stem cells (iPSCs) and the CRISPR Cas9 system, has enabled researchers to model diseases derived from patient cells and precisely edit DNA sequences of interest, respectively. In particular, Duchenne muscular dystrophy (DMD) has been an exemplary monogenic disease model for combining these technologies to demonstrate that genome editing can correct genetic mutations in DMD patient-derived iPSCs. DMD is an X-linked genetic disorder caused by mutations that disrupt the open reading frame of the dystrophin gene, which plays a critical role in stabilizing muscle cells during contraction and relaxation. The CRISPR Cas9 system has been shown to be capable of targeting the dystrophin gene and rescuing its expression in in vitro patient-derived iPSCs and in vivo DMD mouse models. In this review, we highlight recent advances made using the CRISPR Cas9 system to correct genetic mutations and discuss how emerging CRISPR technologies and iPSCs in a combined platform can play a role in bringing a therapy for DMD closer to the clinic.

CRISPR-Cas9 gene editing: Delivery aspects and therapeutic potential

The CRISPR-Cas9 gene editing system has taken the biomedical science field by storm, initiating rumors about future Nobel Prizes and heating up a fierce patent war, but also making significant scientific impact. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with CRISPR-associated proteins (Cas) are a part of the prokaryotic adaptive immune system and have successfully been repurposed for genome editing in mammalian cells. The CRISPR-Cas9 system has been used to correct genetic mutations and for replacing entire genes, opening up a world of possibilities for the treatment of genetic diseases. In addition, recently some new CRISPR-Cas systems have been discovered with interesting mechanistic variations. Despite these promising developments, many challenges have to be overcome before the system can be applied therapeutically in human patients and enabling delivery technology is one of the key challenges. Furthermore, the relatively high off-target effect of the system in its current form prevents it from being safely applied directly in the human body. In this review, the transformation of the CRISPR-Cas gene editing systems into a therapeutic modality will be discussed and the currently most realistic in vivo applications will be highlighted.

Neural Crest Stem Cells/Progenitors and Their Potential Applications in Disease Therapies

The neural crest is a remarkable embryonic population generated transiently during early vertebrate development. Because of their multipotency and extensive migratory capacity, neural crest progenitors harbor stem cell characteristics with self-renewal capacity and contribute to a variety of differentiated cell types from cranio-facial skeletal tissues to peripheral nervous system in the trunk. Multiple molecules including signaling factors, transcription factors and components of migratory machinery are expressed at different stages of neural crest development. Gain- and loss-of -function studies in several vertebrate species have revealed the functional relationship of these molecules and assembled them into a gene regulatory network that define the process of neural crest induction, specification, migration and differentiation. These studies form the fundamental criteria for the subsequent establishment and molecular validation of neural crest stem cells/progenitors derived by various strategies. We present here in vivo and in vitro characterization of neural crest stem cells isolated from embryonic, fetal and adult tissues as well as the latest experimental approaches for their derivation from embryonic stem cells, induced pluripotent stem cells and skin fibroblasts. We further provide an overview of the recent development in applying neural crest stem/progenitor cells for the treatment of neural crest-associated diseases. Future work is required to explore the possibility in directing neural crest -derived specific lineages from fibroblasts using transcription factor-mediated reprogramming strategy, characterize the differentiation potential of adult-derived neural crest stem cell from different tissue origin, and use genomic editing approach to correct genetic mutations in patient-derived NCSCs for transplantation therapy. These endeavors should further unravel and enhance the therapeutic potential of neural crest stem cells/progenitors in clinical setting.

The necessity of cell banks

Current progress in cell biology is connected with the development of somatic cell reprogramming technology. As a result of this technology, it is possible to produce induced pluripotent stem cells (iPSCs) from human somatic cells, for instance, from skin cells. As well as embryonic stem cells, these iPSCs possess pluripotency. Production of iPSCs opened new horizons for patient-specific cell therapy. Many researchers consider iPSCs a real basis for future regenerative medicine. Production of a patient’s iPSCs, their differentiation into somatic cells, and subsequent transplantation to a patient would allow them to avoid immunological rejection. In addition, a recently developed technology of directed genome modification, CRISPR/Cas, allows correction of genetic mutations in iPSCs. Thus, genetic mutations could be corrected in vitro, and after differentiation into a desired cell type, these cells could be transplanted to a patient. In addition, CRISPR/Cas could be used to introduce practically any mutations into iPSCs for the creation of disease-specific model cell lines that would facilitate disease mechanism studies and pharmaceutical drug testing. It is possible to turn off any gene or genes as well as to insert a genetic construct into a selected genomic region to temporarily turn on and off genes and remove chromosomal regions. Cell banks that are open to general use are necessary for efficient usage of iPSCs in biomedical research. Currently, there are no pluripotent stem cell lines in Russian Federation cell banks. Moreover, it is essential to develop standardized practice of culture and storage of that cell type. This mini-review focuses on the necessity of the creation of a pluripotent stem cell bank in the Russian Federation, a detailed description, and a recommended protocol for cell line deposition and usage.

British Society for Gene and Cell Therapy Annual Conference Glasgow9–11th June 2015Conference Abstracts

British Society for Gene and Cell Therapy Annual Conference Glasgow9–11th June 2015Conference Abstracts

Curing genetic mutations for the next generation: CRISPR/Cas9-mediated gene correction in mouse spermatogonial stem cells

The CRISPR/Cas system is a very powerful and versatile tool for genomic manipulation. A recent study published in Cell Research by Wu et al. [1] reported a Cas9-mediated strategy to correct a disease-causing mutation in the mouse gammaC-crystallin(Crygc) locus in spermatogonial stem cells (SSCs) to cure the resulting cataracts disease in the offspring with 100 % efficiency. Importantly, no off-target mutations were identified through whole-genome sequencing. This study is a perfect combination of three different high biological technologies, SSCs manipulation, CRISPR/ Cas gene editing and round spermatid injection (ROSI), shedding light on clinical applications for repair of genetic mutations in the progeny. Many genetic disorders are caused by DNA mutation(s) which is usually inherited from a parental genome and potentially passes to the next generation. Although some symptoms of certain genetic diseases are alleviated with medical or surgical treatments, the disease is hard to cure due to limitations of available approaches to manipulate the human genome efficiently. In recent years, with the emergence of programmable nucleases, scientists are able to edit specific genomic sites by creating DNA doublestrand breaks (DSBs) which stimulate DNA repair either through nonhomologous end joining (NHEJ) or homologydirected repair (HDR) pathways depending on the presence of the repair template [2]. Compared with zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system is more popular due to its simplicity, efficiency and flexibility [3]. The CRISPR/Cas system is derived from the adaptive immune system in bacteria and archaea [4]. It consists of the endonuclease Cas9 (from S. pyogenes) and a customized single-guide RNA (sgRNA) that leads the Cas9 to the DNA target following base-paring rules [4]. With the CRISPR/ Cas system, researchers have demonstrated quick generation of gene-edited model organisms [5, 6] or correction of genetic mutations in animals [7, 8]. Although both somatic and one-cell embryo injection of CRISPR/Cas-mediated gene repair alleviates disease symptoms, these studies neither could cure the disease nor avoid off-target digestion of Cas9 endonuclease. More importantly, the mutations were still transmissable to the next generation. Spermatogonial stem cells (SSCs) are self-renewing male germ line stem cells that are responsible for producing numerous spermatozoa and transmitting genetic information through fertilization. The techniques of in vitro culture and proliferation of SSCs from different species including humans have been well developed [9, 10]. Recently, by taking advantages of SSC manipulation, the CRISPR/Cas system and ROSI, Wu et al. took the lead in gene editing in mouse SSCs and the generation of genetically modified animal models. Moreover, they also used the strategy to correct a genetic disease in the new born pups with 100 % efficiency [1]. To test the feasibility, Wu et al. first disrupted an eGFP transgene in the SSCs in vitro by electroporation of plasmids expressing Cas9 and sgRNA targeting eGFP. The SSCs derived from actin-eGFP transgenic male mice were stably transduced with an mRFP marker to facilitate tracing. Two weeks after transfection of Cas9–sgRNA plasmids, the SSCs expressing mRFP alone were sorted. The sorted SSCs were transplanted into the seminiferous tubules of busulfan-treated males. Round spermatids from the recipient males were isolated and injected into wildtype oocytes to produce mice. After sequencing, half of the D. Li (&) Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China e-mail: dlli@bio.ecnu.edu.cn

Therapeutic genome editing: prospects and challenges.

Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease-based therapies as well as future prospects and challenges.

Gene therapy for inherited muscle diseases: where genetics meets rehabilitation medicine.

The development of clinical vectors to correct genetic mutations that cause inherited myopathies and related disorders of skeletal muscle is advancing at an impressive rate. Adeno-associated virus vectors are attractive for clinical use because (1) adeno-associated viruses do not cause human disease and (2) these vectors are able to persist for years. New vectors are now becoming available as gene therapy delivery tools, and recent preclinical experiments have demonstrated the feasibility, safety, and efficacy of gene therapy with adeno-associated virus for long-term correction of muscle pathology and weakness in myotubularin-deficient canine and murine disease models. In this review, recent advances in the application of gene therapies to treat inherited muscle disorders are presented, including Duchenne muscular dystrophy and x-linked myotubular myopathy. Potential areas for therapeutic synergies between rehabilitation medicine and genetics are also discussed.

Variable correction of Artemis deficiency by I-Sce1-meganuclease-assisted homologous recombination in murine hematopoietic stem cells

The correction of genetic mutations by homologous recombination is an attractive approach to gene therapy. We used the DNA double-strand breaks introduced by the site-specific endonuclease I-Sce1 as a means of increasing homologous recombination of an exogenous DNA template in murine hematopoietic stem cells (mHSCs). To develop this approach, we chose an Artemis knockout (Art(-/-)) mouse in which exon 12 of the Artemis gene had been replaced by an I-Sce1 recognition site. The I-Sce1 enzyme and the Artemis correction template were each delivered by a self-inactivating (SIN)-integrase-defective lentiviral vector (SIN-IDLV-CMV-ISce1 and SIN-IDLV-Art, respectively). Transduction of Art(-/-) mHSCs with the two vectors successfully reverted the Art(-/-) phenotype in 2 of our 10 experiments. Even though the potential for genotoxicity has yet to be evaluated, this new approach to gene editing appears to be promising. Improving the efficacy of this approach will require further technical work.

Forthcoming Issues

Targeted gene-editing strategies have emerged as promising therapeutic approaches for the permanent treatment of inherited genetic diseases. However, precise gene correction and insertion approaches using homology-directed repair are still limited by low efficiencies. Consequently, many gene-editing strategies have focused on removal or disruption, rather than repair, of genomic DNA. In contrast, homology-independent targeted integration (HITI) has been reported to effectively insert DNA sequences at targeted genomic loci. This approach could be particularly useful for restoring full-length sequences of genes affected by a spectrum of mutations that are also too large to deliver by conventional adeno-associated virus (AAV) vectors. Here, we utilize an AAV-based, HITI-mediated approach for correction of full-length dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy (DMD). We co-deliver CRISPR-Cas9 and a donor DNA sequence to insert the missing human exon 52 into its corresponding position within the DMD gene and achieve full-length dystrophin correction in skeletal and cardiac muscle. Additionally, as a proof-of-concept strategy to correct genetic mutations characterized by diverse patient mutations, we deliver a superexon donor encoding the last 28 exons of the DMD gene as a therapeutic strategy to restore full-length dystrophin in >20% of the DMD patient population. This work highlights the potential of HITI-mediated gene correction for diverse DMD mutations and advances genome editing toward realizing the promise of full-length gene restoration to treat genetic disease.

Reading Frame Correction by Targeted Genome Editing Restores Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients

Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template. However, many genetic diseases, such as Duchenne muscular dystrophy (DMD), can be treated simply by correcting a disrupted reading frame. We show that genome editing with transcription activator-like effector nucleases (TALENs), without a repair template, can efficiently correct the reading frame and restore the expression of a functional dystrophin protein that is mutated in DMD. TALENs were engineered to mediate highly efficient gene editing at exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from Duchenne patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. Finally, exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein-coding regions of the genome, as predicted by in silico target site analysis. This strategy integrates the rapid and robust assembly of active TALENs with an efficient gene-editing method for the correction of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts or premature stop codons.

Gene therapy for deafness.

Hearing loss is the most common sensory deficit in humans and can result from genetic, environmental or combined etiologies that prevent normal function of the cochlea, the peripheral sensory organ. Recent advances in understanding the genetic pathways that are critical for the development and maintenance of cochlear function, as well as the molecular mechanisms that underlie cell trauma and death, have provided exciting opportunities for modulating these pathways to correct genetic mutations, to enhance the endogenous protective pathways for hearing preservation and to regenerate lost sensory cells with the possibility of ameliorating hearing loss. A number of recent animal studies have used gene-based therapies in innovative ways toward realizing these goals. With further refinement, some of the protective and regenerative approaches reviewed here may become clinically applicable.

371. Optimizing Custom Zinc-Finger Nucleases for Use in Human Cells

Genome engineering is a powerful instrument to study basic biological questions or to create treatment options for genetic diseases. A safe way to correct genetic mutations is through homologous recombination (HR), during which a mutant sequence is replaced with the corresponding wild-type sequence. While HR is predominant in yeast, it is a rare event in mammalian cells where non-homologous recombination is much more frequent. The creation of a targeted DNA double-strand break (DSB) has been shown to locally stimulate HR several hundred-fold by activating the cellular machinery for double-strand break repair, but it presupposes the availability of a site-specific endonuclease to create the DSB. Some initial experiments with artificial nucleases in human cells revealed that cytotoxicity of such enzymes is a major issue, most likely through cleavage at non-specific sites. Here, we present a rapid two-step method to evaluate and optimize novel, rationally designed endonucleases. In a first step, the DNA-binding domain was synthesized by assembling predefined C2H2-zinc-finger modules and fused to a transcriptional activation domain. Each zinc-finger module is designed to recognize 3 bp of DNA and the modules can be assembled in any order necessary to recognize any given sequence in a target locus. DNA-binding specificity of these artificial transcription factors was assessed in a reporter assay. In a second step, the DNA-binding domain was attached to the catalytic domain of the FokI restriction endonuclease to generate the custom nucleases. Upon co-transfection with a repair matrix, they were assessed for their ability to stimulate HR in an episomal gene repair assay. We have engineered several kinds of custom nucleases and demonstrate that high specificity, fine-tuned expression kinetics and protein design are absolutely critical to promote HR. Importantly, the same parameters are also crucial for a low level of cytotoxicity. After optimization, expression of the custom nucleases stimulated both episomal and chromosomal gene repair and HR was achieved in 35% or 2.5% of transfected cells, respectively. Thus, custom nucleases will be a valuable tool to stimulate gene repair for therapeutic purposes. Moreover, they can be easily combined with viral vector systems for efficient delivery.

A dispersion model for viral transport in tissues

Gene therapy of cancer is the transfer of genetic material to correct genetic mutations in malignant cells or destroy cancer cells through cytolysis or other mechanisms. Intratumoural injection is often used for direct delivery of viral particles (VP) into tumours. The temporal and spatial distributions of VP in the tissue of interest (TOI) following the injection may closely correlate with the therapeutic results. Thus, a theoretical model as a dispersion process is introduced to describe VP transport processes in the TOI. Which include mechanical flow, molecular diffusion, blood uptake, cellular absorption, and viral death and replication.

Screening for antisense modulation of dystrophin pre-mRNA splicing

Most gene therapy approaches to genetic disorders aim to compensate loss-of-function by introducing recombinant cDNA-based minigenes into diseased tissues. The current report represents an ongoing series of studies designed to correct genetic mutations at the post-transcriptional level. This strategy modifies the binding of components of the spliceosome by high affinity hybridisation of small complementary (antisense) RNA oligonucleotides to specific pre-mRNA sequences. These, so-called ‘splicomer’ reagents are chemically modified to impart bio-stability, and are designed to cause skipping of mutant frame-shifting exon sequences leading to restoration of the reading frame and an internally deleted but partially functional gene product. For instance, Duchenne muscular dystrophy is generally caused by frame-shift mutations in the dystrophin gene, whereas in-frame deletions of up to 50% of the central portion of the gene cause Becker muscular dystrophy, a much milder myopathy, which in some cases can remain asymptomatic to old age. In the mdx mouse model of Duchenne muscular dystrophy, a mutation in exon 23 of the dystrophin gene creates a stop codon and leads to a dystrophin-deficient myopathy in striated muscle. In previous studies, we have demonstrated that forced skipping of this mutant exon by treatment of mdx muscle cells with splicomer oligonucleotides can generate in-frame dystrophin transcripts and restore dystrophin expression. Here, we report the results of an optimisation of splicomer sequence design by the use of both high-throughput arrays and biological screens. This has resulted in specific and, importantly, exclusive skipping of the targeted exon in greater than 60% of dystrophin mRNA, leading to the de novo synthesis and localisation of dystrophin protein in cultured mdx muscle cells.