CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors.
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ABSTRACT:
SUBMITTER: Wang D
PROVIDER: S-EPMC7236621 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Degenerative retinal diseases such as retinitis pigmentosa (RP) and Leber's congenital amaurosis (LCA) may lead to blindness without effective treatment. With the rapid advancement of the CRISPR/Cas9 genome editing technology, in vivo application of CRISPR/Cas9 holds immense potential for treatment of these diseases. Adeno-associated virus (AAV) vectors are an ideal gene transfer tool for delivery of CRISPR components to the retina. Here, we describe a protocol for utilizing an AAV-based CRISPR/Cas9 system for in vivo genome editing in the retina.
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Project description:Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans.
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Project description:Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a "protecting oligo"), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.
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Project description:Since its mechanism discovery in 2012 and the first application for mammalian genome editing in 2013, CRISPR-Cas9 has revolutionized the genome engineering field and created countless opportunities in both basic science and translational medicine. The first clinical trial of CRISPR therapeutics was initiated in 2016, which employed ex vivo CRISPR-Cas9 edited PD-1 knockout T cells for the treatment of non-small cell lung cancer. So far there have been dozens of clinical trials registered on ClinicalTrials.gov in regard to using the CRISPR-Cas9 genome editing as the main intervention for therapeutic applications; however, most of these studies use ex vivo genome editing approach, and only a few apply the in vivo editing strategy. Compared to ex vivo editing, in vivo genome editing bypasses tedious procedures related to cell isolation, maintenance, selection, and transplantation. It is also applicable to a wide range of diseases and disorders. The main obstacles to the successful translation of in vivo therapeutic genome editing include the lack of safe and efficient delivery system and safety concerns resulting from the off-target effects. In this review, we highlight the therapeutic applications of in vivo genome editing mediated by the CRISPR-Cas9 system. Following a brief introduction of the history, biology, and functionality of CRISPR-Cas9, we showcase a series of exemplary studies in regard to the design and implementation of in vivo genome editing systems that target the brain, inner ear, eye, heart, liver, lung, muscle, skin, immune system, and tumor. Current challenges and opportunities in the field of CRISPR-enabled therapeutic in vivo genome editing are also discussed.
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Project description:The recently developed CRISPR/Cas9 technology has revolutionized the genome engineering field. Since 2016, increasing number of studies regarding CRISPR therapeutics have entered clinical trials, most of which are focusing on the ex vivo genome editing. In this review, we highlight the ex vivo cell-based CRISPR/Cas9 genome editing for therapeutic applications. In these studies, CRISPR/Cas9 tools were used to edit cells in vitro and the successfully edited cells were considered as therapeutics, which can be introduced into patients to treat diseases. Considering a large number of previous reviews have been focused on the CRISPR/Cas9 delivery methods and materials, this review provides a different perspective, by mainly introducing the targeted conditions and design strategies for ex vivo CRISPR/Cas9 therapeutics. Brief descriptions of the history, functionality, and applications of CRISPR/Cas9 systems will be introduced first, followed by the design strategies and most significant results from previous research that used ex vivo CRISPR/Cas9 genome editing for the treatment of conditions or diseases. The last part of this review includes general information about the status of CRISPR/Cas9 therapeutics in clinical trials. We also discuss some of the challenges as well as the opportunities in this research area.
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Project description:Therapeutic genome editing technology has been widely used as a powerful tool for directly correcting genetic mutations in target pathological tissues and cells to cure of diseases. The modification of specific genomic sequences can be achieved by utilizing programmable nucleases, such as Meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly-interspaced short palindromic repeat-associated nuclease Cas9 (CRISPR/Cas9). However, given the properties, such as large size, negative charge, low membrane penetrating ability, as well as weak tolerance for serum, and low endosomal escape, of these nucleases genome editing cannot be successfully applied unless in vivo delivery of related programmable nucleases into target organisms or cells is achieved. Here, we look back at delivery strategies having been used in the in vivo delivery of three main genome editing nucleases, followed by methodologies currently undergoing testing in clinical trials, and potential delivery strategies provided by analyzing characteristics of nucleases and commonly used vectors.
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Project description:The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle-mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.
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Project description:The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) systems have emerged as a robust and versatile genome editing platform for gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. However, the insufficient transfection and off-target risks have seriously hampered the potential biomedical applications of CRISPR/Cas9 technology. Herein, we review the recent progress towards CRISPR/Cas9 system delivery based on viral and non-viral vectors. We summarize the CRISPR/Cas9-inspired clinical trials and analyze the CRISPR/Cas9 delivery technology applied in the trials. The rational-designed non-viral vectors for delivering three typical forms of CRISPR/Cas9 system, including plasmid DNA (pDNA), mRNA, and ribonucleoprotein (RNP, Cas9 protein complexed with gRNA) were highlighted in this review. The vector-derived strategies to tackle the off-target concerns were further discussed. Moreover, we consider the challenges and prospects to realize the clinical potential of CRISPR/Cas9-based genome editing.
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Project description:CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy has been hindered by efficient delivery of CRISPR/Cas9 materials into target cells. Direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single guide RNA (sgRNA) has emerged as a powerful and widespread method for genome editing due to its advantages of transient genome editing and reduced off-target effects. In this review, we summarized the current Cas9 RNP delivery systems including physical approaches and synthetic carriers. The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed. Examples in the development of stimuli-responsive and targeted carriers for RNP delivery are highlighted. Finally, the challenges of current Cas9 RNP delivery systems and perspectives in rational design of next generation materials for this promising field will be discussed.
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Project description:The recent progress in harnessing the efficient and precise method of DNA editing provided by CRISPR/Cas9 is one of the most promising major advances in the field of gene therapy. However, the development of safe and optimally efficient delivery systems for CRISPR/Cas9 elements capable of achieving specific targeting of gene therapy to the location of interest without off-target effects is a primary challenge for clinical therapeutics. Nanoparticles (NPs) provide a promising means to meet such challenges. In this review, we present the most recent advances in developing innovative NP-based delivery systems that efficiently deliver CRISPR/Cas9 constructs and maximize their effectiveness.
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