CRISPR technologies for the treatment of Duchenne muscular dystrophy.

The emerging clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing technologies have progressed remarkably in recent years, opening up the potential of precise genome editing as a therapeutic approach to treat various diseases. The CRISPR-CRISPR-associated (Cas) system is an attractive platform for the treatment of Duchenne muscular dystrophy (DMD), which is a neuromuscular disease caused by mutations in the DMD gene. CRISPR-Cas can be used to permanently repair the mutated DMD gene, leading to the expression of the encoded protein, dystrophin, in systems ranging from cells derived from DMD patients to animal models of DMD. However, the development of more efficient therapeutic approaches and delivery methods remains a great challenge for DMD. Here, we review various therapeutic strategies that use CRISPR-Cas to correct or bypass DMD mutations and discuss their therapeutic potential, as well as obstacles that lie ahead.

CRISPR RNAs trigger innate immune responses in human cells.

Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5'-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4+ T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5'-hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.

CRISPR-Cas12a with an oAd Induces Precise and Cancer-Specific Genomic Reprogramming of EGFR and Efficient Tumor Regression.

CRISPR-Cas12a represents a class 2/type V CRISPR RNA-guided endonuclease, holding promise as a precise genome-editing tool in vitro and in vivo. For efficient delivery of the CRISPR-Cas system into cancer, oncolytic adenovirus (oAd) has been recognized as a promising alternative vehicle to conventional cancer therapy, owing to its cancer specificity; however, to our knowledge, it has not been used for genome editing. In this study, we show that CRISPR-Cas12a mediated by oAd disrupts the oncogenic signaling pathway with excellent cancer specificity. The intratumoral delivery of a single oAd co-expressing a Cas12a and a CRISPR RNA (crRNA) targeting the epidermal growth factor receptor (EGFR) gene (oAd/Cas12a/crEGFR) induces efficient and precise editing of the targeted EGFR gene in a cancer-specific manner, without detectable off-target nuclease activity. Importantly, oAd/Cas12a/crEGFR elicits a potent antitumor effect via robust induction of apoptosis and inhibition of tumor cell proliferation, ultimately leading to complete tumor regression in a subset of treated mice. Collectively, in this study we show precise genomic reprogramming via a single oAd vector-mediated CRISPR-Cas system and the feasibility of such system as an alternative cancer therapy.

Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration.

RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene-specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody- and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.

Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus.

Genome editing with CRISPR systems provides an unprecedented opportunity to modulate cellular responses in pathological conditions by inactivating undruggable targets, such as transcription factors. Previously, we demonstrated that the smallest Cas9 ortholog characterized to date, from Campylobacter jejuni (CjCas9) targeted to Hif1a and delivered in an adeno-associated virus (AAV) vector, effectively suppressed pathological choroidal neovascularization in the mouse retina. Before implementation of CjCas9 as an in vivo therapeutic modality, it is essential to investigate the long-term effects of target gene disruption via AAV-mediated delivery of CjCas9 in vivo. In this study, histologic and electroretinographic analyses demonstrated that CjCas9 targeted to Hif1a did not induce any definite toxicity in the retina, although the target gene was mutated with a frequency ranging from 45% to 79% in retinal or retinal pigment epithelial cells. Importantly, at 14 months after injection, no indels were detected at potential off-target sites identified using Digenome-seq and Cas-OFFinder, suggesting that long-term expression of CjCas9 does not aggravate off-target effects. Taken together, our results show that intravitreal injection of AAV encoding CjCas9 targeted to Hif1a effectively induced and maintained mutations in retinal tissues for more than 1 year and did not affect retinal histologic integrity or functions.

Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9.

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle-wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the EGFP gene in the tibialis anterior muscle of the Dmd knockout mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out of frame to in frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9, has great potential for the treatment of DMD and other neuromuscular diseases.

Selective disruption of an oncogenic mutant allele by CRISPR/Cas9 induces efficient tumor regression.

Approximately 15% of non-small cell lung cancer cases are associated with a mutation in the epidermal growth factor receptor (EGFR) gene, which plays a critical role in tumor progression. With the goal of treating mutated EGFR-mediated lung cancer, we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system to discriminate between the oncogenic mutant and wild-type EGFR alleles and eliminate the carcinogenic mutant EGFR allele with high accuracy. We targeted an EGFR oncogene harboring a single-nucleotide missense mutation (CTG > CGG) that generates a protospacer-adjacent motif sequence recognized by the CRISPR/Cas9 derived from Streptococcus pyogenes. Co-delivery of Cas9 and an EGFR mutation-specific single-guide RNA via adenovirus resulted in precise disruption at the oncogenic mutation site with high specificity. Furthermore, this CRISPR/Cas9-mediated mutant allele disruption led to significantly enhanced cancer cell killing and reduced tumor size in a xenograft mouse model of human lung cancer. Taken together, these results indicate that targeting an oncogenic mutation using CRISPR/Cas9 offers a powerful surgical strategy to disrupt oncogenic mutations to treat cancers; similar strategies could be used to treat other mutation-associated diseases.

Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides.

Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2'Omethyl phosphorothioate (2'OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2'OMe as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2'OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.

Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases.

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

Survivin enhances hippocampal neurogenesis and cognitive function in Alzheimer's disease mouse model.

AIMS: Cognitive impairment is associated with reduced hippocampal neurogenesis; however, the causes of decreased hippocampal neurogenesis remain highly controversial. Here, we investigated the role of survivin in the modulation of hippocampal neurogenesis in AD. METHODS: To investigate the effect of survivin on neurogenesis in neural stem cells (NSCs), we treated mouse embryonic NSCs with a survivin inhibitor (YM155) and adeno-associated viral survivin (AAV-Survivin). To explore the potential role of survivin expression in AD, AAV9-Survivin or AAV9-GFP were injected into the dentate gyrus (DG) of hippocampus of 7-month-old wild-type and 5XFAD mice. Cognitive function was measured by the Y maze and Morris water maze. Neurogenesis was investigated by BrdU staining, immature, and mature neuron markers. RESULTS: Our results indicate that suppression of survivin expression resulted in decreased neurogenesis. Conversely, overexpression of survivin using AAV-Survivin restored neurogenesis in NSCs that had been suppressed by YM155 treatment. Furthermore, the expression level of survivin decreased in the 9-month-old 5XFAD compared with that in wild-type mice. AAV-Survivin-mediated overexpression of survivin in the DG in 5XFAD mice enhanced neurogenesis and cognitive function. CONCLUSION: Hippocampal neurogenesis can be enhanced by survivin overexpression, suggesting that survivin could serve as a promising therapeutic target for the treatment of AD.

Targeted dual base editing with Campylobacter jejuni Cas9 by single AAV-mediated delivery.

Various CRISPR‒Cas9 orthologs are used in genome engineering. One of the smallest Cas9 orthologs is cjCas9 derived from Campylobacter jejuni, which is a highly specific genome editing tool. Here, we developed cjCas9-based base editors including a cytosine base editor (cjCBEmax) and an adenine base editor (cjABE8e) that can successfully induce endogenous base substitutions by up to 91.2% at the HPD gene in HEK293T cells. Analysis of the base editing efficiency of 13 endogenous target sites showed that the active windows of cjCBEmax and cjABE8e are wider than those of spCas9-based base editors and that their specificities are slightly lower than that of cjCas9. Importantly, engineered cjCas9 and gRNA scaffolds can improve the base editing efficiency of cjABE8e by up to 6.4-fold at the HIF1A gene in HEK293T cells. Due to its small size, cjABE8e can be packaged in a single adeno-associated virus vector with two tandem arrays of gRNAs, and the delivery of the resulting AAV could introduce base substitutions at endogenous ANGPT2 and HPD target sites. Overall, our findings have expanded the potential of the use of base editors for in vivo or ex vivo therapeutic approaches.

Expanded targeting scope of LbCas12a variants allows editing of multiple oncogenic mutations.

RNA-guided CRISPR-Cas12a endonucleases are promising tools for genome engineering. Here we demonstrate that LbCas12a variants derived from Lachnospiraceae bacterium show a broad PAM preference, recognizing certain non-canonical PAMs with high efficiency. Furthermore, we engineered LbABE8e to carry G532R and/or K595R mutations, altering its original PAM specificities; these variants exhibited superior base editing activity in human cells compared with wild-type LbABE8e at sites with non-canonical PAMs. Based on this finding, we utilized the most effective LbCas12a and LbABE8e variants to demonstrate multiplexed and mutant-allele-specific gene editing in oncogenes, made possible by the variant's recognition of non-canonical PAMs. Importantly, LbCas12a-G532R/K595R and LbABE8e-G532R/K595R with optimized crRNA arrays targeted to triple oncogenic mutations inhibited colon cancer cell proliferation. Taken together, these results demonstrate the potential of engineered LbCas12a and LbABE8e as tools for targeting sites with alternative PAMs for genome engineering and therapeutic editing in cancer cells.

Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus.

Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5' terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA-crRNA complementary region, a penta(uridinylate) sequence, the 3' terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.

Long-term functional adeno-associated virus-microdystrophin expression in the dystrophic CXMDj dog.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe, inherited, muscle-wasting disorder caused by mutations in the dystrophin gene. Preclinical studies of adeno-associated virus gene therapy for DMD have been described in mouse and dog models of this disease. However, low and transient expression of microdystrophin in dystrophic dogs and a lack of long-term microdystrophin expression associated with a CD8(+) T-cell response in DMD patients suggests that the development of improved microdystrophin genes and delivery strategies is essential for successful clinical trials in DMD patients. METHODS: We have previously shown the efficiency of mRNA sequence optimization of mouse microdystrophin in ameliorating the pathology of dystrophic mdx mice. In the present study, we generated adeno-associated virus (AAV)2/8 vectors expressing an mRNA sequence-optimized canine microdystrophin under the control of a muscle-specific promoter and injected intramuscularly into a single canine X-linked muscular dystrophy (CXMDj) dog. RESULTS: Expression of stable and high levels of microdystrophin was observed along with an association of the dystrophin-associated protein complex in intramuscularly injected muscles of a CXMDj dog for at least 8 weeks without immune responses. Treated muscles were highly protected from dystrophic damage, with reduced levels of myofiber permeability and central nucleation. CONCLUSIONS: The data obtained in the present study suggest that the use of canine-specific and mRNA sequence-optimized microdystrophin genes in conjunction with a muscle-specific promoter results in high and stable levels of microdystrophin expression in a canine model of DMD. This approach will potentially allow the reduction of dosage and contribute towards the development of a safe and effective AAV gene therapy clinical trial protocol for DMD.

Recent advances in CRISPR technologies for genome editing.

The discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, and its development into a set of powerful tools for manipulating the genome, has revolutionized genome editing. Precise, targeted CRISPR/Cas-based genome editing has become the most widely used platform in organisms ranging from plants to animals. The CRISPR/Cas system has been extensively modified to increase its efficiency and fidelity. In addition, the fusion of various protein motifs to Cas effector proteins has facilitated diverse set of genetic manipulations, such as base editing, transposition, recombination, and epigenetic regulation. The CRISPR/Cas system is undergoing continuous development to overcome current limitations, including off-target effects, narrow targeting scope, and issues associated with the delivery of CRISPR components for genome engineering and therapeutic approaches. Here, we review recent progress in a diverse array of CRISPR/Cas-based tools. We also describe limitations and concerns related to the use of CRISPR/Cas technologies.

Template Engineering of Metal-to-Insulator Transitions in Epitaxial Bilayer Nickelate Thin Films.

Understanding metal-to-insulator phase transitions in solids has been a keystone not only for discovering novel physical phenomena in condensed matter physics but also for achieving scientific breakthroughs in materials science. In this work, we demonstrate that the transport properties (i.e., resistivity and transition temperature) in the metal-to-insulator transitions of perovskite nickelates are tunable via the epitaxial heterojunctions of LaNiO3 and NdNiO3 thin films. A mismatch in the oxygen coordination environment and interfacial octahedral coupling at the oxide heterointerface allows us to realize an exotic phase that is unattainable in the parent compound. With oxygen vacancy formation for strain accommodation, the topmost LaNiO3 layer in LaNiO3/NdNiO3 bilayer thin films is structurally engineered and it electrically undergoes a metal-to-insulator transition that does not appear in metallic LaNiO3. Modification of the NdNiO3 template layer thickness provides an additional knob for tailoring the tilting angles of corner-connected NiO6 octahedra and the linked transport characteristics further. Our approaches can be harnessed to tune physical properties in complex oxides and to realize exotic physical phenomena through oxide thin-film heterostructuring.

Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy.

Adenine base editors (ABEs) composed of an engineered adenine deaminase and the Streptococcus pyogenes Cas9 nickase enable adenine-to-guanine (A-to-G) single-nucleotide substitutions in a guide RNA (gRNA)-dependent manner. Here we demonstrate application of this technology in mouse embryos and adult mice. We also show that long gRNAs enable adenine editing at positions one or two bases upstream of the window that is accessible with standard single guide RNAs (sgRNAs). We introduced the Himalayan point mutation in the Tyr gene by microinjecting ABE mRNA and an extended gRNA into mouse embryos, obtaining Tyr mutant mice with an albino phenotype. Furthermore, we delivered the split ABE gene, using trans-splicing adeno-associated viral vectors, to muscle cells in a mouse model of Duchenne muscular dystrophy to correct a nonsense mutation in the Dmd gene, demonstrating the therapeutic potential of base editing in adult animals.

CRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration.

LbCpf1, derived from Lachnospiraceae bacterium ND2006, is a CRISPR RNA-guided endonuclease and holds promise for therapeutic applications. Here we show that LbCpf1 can be used for therapeutic gene editing in a mouse model of age-related macular degeneration (AMD). The intravitreal delivery of LbCpf1, targeted to two angiogenesis-associated genes encoding vascular endothelial growth factor A (Vegfa) and hypoxia inducing factor 1a (Hif1a), using adeno-associated virus, led to efficient gene disruption with no apparent off-target effects in the retina and retinal pigment epithelium (RPE) cells. Importantly, LbCpf1 targeted to Vegfa or Hif1a in RPE cells reduced the area of laser-induced choroidal neovascularization as efficiently as aflibercept, an anti-VEGF drug currently used in the clinic, without inducing cone dysfunction. Unlike aflibercept, LbCpf1 targeted to Vegfa or Hif1a achieved a long-term therapeutic effect on CNV, potentially avoiding repetitive injections. Taken together, these results indicate that LbCpf1-mediated in vivo genome editing to ablate pathologic angiogenesis provides an effective strategy for the treatment of AMD and other neovascularization-associated diseases.

Therapeutic applications of CRISPR RNA-guided genome editing.

The rapid development of programmable nuclease-based genome editing technologies has enabled targeted gene disruption and correction both in vitro and in vivo This revolution opens up the possibility of precise genome editing at target genomic sites to modulate gene function in animals and plants. Among several programmable nucleases, the type II clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) system has progressed remarkably in recent years, leading to its widespread use in research, medicine and biotechnology. In particular, CRISPR-Cas9 shows highly efficient gene editing activity for therapeutic purposes in systems ranging from patient stem cells to animal models. However, the development of therapeutic approaches and delivery methods remains a great challenge for biomedical applications. Herein, we review therapeutic applications that use the CRISPR-Cas9 system and discuss the possibilities and challenges ahead.