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Generation of an Isogenic Hereditary Hemorrhagic Telangiectasia Model via Prime Editing in Human Induced Pluripotent Stem Cells
Abstract
Prime editing (PE) is a recently developed genome-editing technique that enables versatile editing. Despite its flexibility and potential, applying PE in human induced pluripotent stem cells (hiPSCs) has not been extensively addressed. Genetic disease models using patient-derived hiPSCs have been used to study mechanisms and drug efficacy. However, genetic differences between patient and control cells have been attributed to the inaccuracy of the disease model, highlighting the significance of isogenic hiPSC models. Hereditary hemorrhagic telangiectasia 1 (HHT1) is a genetic disorder caused by an autosomal dominant mutation in endoglin (ENG). Although previous HHT models using mice and HUVEC have been used, these models did not sufficiently elucidate the relationship between the genotype and disease phenotype in HHT, demanding more clinically relevant models that reflect human genetics. Therefore, in this study, we used PE to propose a method for establishing an isogenic hiPSC line. Clinically reported target mutation in ENG was selected, and a strategy for PE was designed. After cloning the ENGineered PE guide RNA, hiPSCs were nucleofected along with PEmax and hMLH1dn plasmids. As a result, hiPSC clones with the intended mutation were obtained, which showed no changes in pluripotency or genetic integrity. Furthermore, introducing the ENG mutation increased the expression of proangiogenic markers during endothelial organoid differentiation. Consequently, our results suggest the potential of PE as a toolkit for establishing isogenic lines, enabling disease modeling based on hiPSC-derived disease-related cells or organoids. This approach is expected to stimulate mechanistic and therapeutic studies on genetic diseases.a.
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Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3′ ends of CRISPR–Cas guide RNAs¹. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3′ ends of RNA polymerase III transcripts². We found that La functionally interacts with the 3′ ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.
Owing to the advances in genome editing technologies, research on human pluripotent stem cells (hPSCs) have recently undergone breakthroughs that enable precise alteration of desired nucleotide bases in hPSCs for the creation of isogenic disease models or for autologous ex vivo cell therapy. As pathogenic variants largely consist of point mutations, precise substitution of mutated bases in hPSCs allows researchers study disease mechanisms with “disease-in-a-dish” and provide functionally repaired cells to patients for cell therapy. To this end, in addition to utilizing the conventional homologous directed repair system in the knock-in strategy based on endonuclease activity of Cas9 (i.e., ‘scissors’ like gene editing), diverse toolkits for editing the desirable bases (i.e., ‘pencils’ like gene editing) that avoid the accidental insertion and deletion (indel) mutations as well as large harmful deletions have been developed. In this review, we summarize the recent progress in genome editing methodologies and employment of hPSCs for future translational applications.
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. Previously we showed that adenine base editing (ABE) can efficiently correct a nonsense point mutation in a DMD mouse model. Here we explored the feasibility of base editing-mediated exon skipping as a therapeutic strategy for DMD using cardiomyocyte derived from human induced pluripotent stem cells (hiPSCs). We first generated a DMD hiPSC line with a large deletion spanning exon 48 through 54 (ΔE48-54) using CRISPR/Cas9 gene editing. Dystrophin expression was disrupted in DMD hiPSC-derived cardiomyocytes (iCMs) as examined by RT-PCR, Western blot and immunofluorescence staining. Transfection of ABE and a guide RNA (gRNA) targeting the splice acceptor led to efficient conversion of AG to GG (35.9 ± 5.7%) and enabled exon 55 skipping. Complete AG to GG conversion in a single clone restored dystrophin expression (42.5 ± 11.0% of WT) in DMD iCMs. Moreover, we designed gRNAs to target the splice sites of exons 6, 7, 8, 43, 44, 46 and 53 in the mutational hotspots, and demonstrated their efficiency to induce exon skipping in iCMs. These results highlight the great promise of ABE-mediated exon skipping as a promising therapeutic approach for DMD.
Prime editing (PE), as a “search-and-replace” genome editing technology, has shown the attractive potential of versatile genome editing ability, which is, in principle, currently superior to other well-established genome-editing technologies in the all-in-one operation scope. However, essential technological solutions of PE technology, such as the improvement of genome editing efficiency, the inhibition of potential off-targets and intended edits accounting for unexpected side-effects, and the development of effective delivery systems, are necessary to broaden its application. Since the advent of PE, many optimizations have been performed on PE systems to improve their performance, resulting in bright prospects for application in many fields. This review briefly discusses the development of PE technology, including its functional principle, noteworthy barriers restraining its application, current efforts in technical optimization, and its application directions and potential risks. This review may provide a concise and informative insight into the burgeoning field of PE, highlight the exciting prospects for this powerful tool, and provide clues for questions that may propel the field forward.
Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus. However, the efficiency of PE varies widely and depends not only on the genomic region targeted, but also on the genetic background of the edited cell. Here, to determine which cellular factors affect PE efficiency, we carry out a focused genetic screen targeting 32 DNA repair factors, spanning all reported repair pathways. We show that, depending on cell line and type of edit, ablation of mismatch repair (MMR) affords a 2–17 fold increase in PE efficiency, across several human cell lines, types of edits and genomic loci. The accumulation of the key MMR factors MLH1 and MSH2 at PE sites argues for direct involvement of MMR in PE control. Our results shed new light on the mechanism of PE and suggest how its efficiency might be optimised. Prime Editing is a versatile genome engineering tool. Here, the authors identify the DNA repair pathway known as mismatch repair as inhibitory for Prime Editing, thus, loss of mismatch repair enhances the efficiency of Prime Editing.
The possibility of reprogramming human somatic cells to pluripotency has opened unprecedented opportunities for creating genuinely human experimental models of disease. Inborn errors of metabolism (IEMs) constitute a greatly heterogeneous class of diseases that appear, in principle, especially suited to be modeled by iPSC-based technology. Indeed, dozens of IEMs have already been modeled to some extent using patient-specific iPSCs. Here, we review the advantages and disadvantages of iPSC-based disease modeling in the context of IEMs, as well as particular challenges associated to this approach, together with solutions researchers have proposed to tackle them. We have structured this review around six lessons that we have learnt from those previous modeling efforts, and that we believe should be carefully considered by researchers wishing to embark in future iPSC-based models of IEMs.
Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme.
Therapeutic advances for neurological disorders are challenging due to limited accessibility of the human central nervous system and incomplete understanding of disease mechanisms. Many neurological diseases lack precision treatments, leading to significant disease burden and poor outcome for affected patients. Induced pluripotent stem cell (iPSC) technology provides human neuronal cells that facilitate disease modeling and development of therapies. The use of genome editing, in particular CRISPR-Cas9 technology, has extended the potential of iPSCs, generating new models for a number of disorders, including Alzheimers and Parkinson Disease. Editing of iPSCs, in particular with CRISPR-Cas9, allows generation of isogenic pairs, which differ only in the disease-causing mutation and share the same genetic background, for assessment of phenotypic differences and downstream effects. Moreover, genome-wide CRISPR screens allow high-throughput interrogation for genetic modifiers in neuronal phenotypes, leading to discovery of novel pathways, and identification of new therapeutic targets. CRISPR-Cas9 has now evolved beyond altering gene expression. Indeed, fusion of a defective Cas9 (dCas9) nuclease with transcriptional repressors or activation domains allows down-regulation or activation of gene expression (CRISPR interference, CRISPRi; CRISPR activation, CRISPRa). These new tools will improve disease modeling and facilitate CRISPR and cell-based therapies, as seen for epilepsy and Duchenne muscular dystrophy. Genome engineering holds huge promise for the future understanding and treatment of neurological disorders, but there are numerous barriers to overcome. The synergy of iPSC-based model systems and gene editing will play a vital role in the route to precision medicine and the clinical translation of genome editing-based therapies.
Objectives:
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by recurrent epistaxis, telangiectasias, and visceral arteriovenous malformations (AVMs). Activin A receptor-like type 1 (ACVRL1/ALK1) and Endoglin (ENG) are the principal genes whose mutations cause HHT. A multicenter study to investigate the correlation between genetic variations and clinical outcomes in Korean HHT patients has been lacking.
Methods:
Seventy-two members from 40 families suspected of HHT based on symptoms were genetically screened for pathogenic variants in ACVRL1 and ENG. Patients with genetically diagnosed HHT were also evaluated.
Results:
In the HHT genetic screening, 42 patients from 24 of the 40 families had genetic variants that met the pathogenic criteria (pathogenic very strong, pathogenic strong, pathogenic moderate, or pathogenic supporting) based on ACMG Standards and Guidelines in either ENG or ACVRL1; 26 from 12 families (50%) in ENG, and 16 from 12 families (50%) in ACVRL1. The diagnostic screening of 42 genetically positive HHT patients based on the Curaçao criteria revealed that 24 patients (57%) were in the definite group, 17 patients (41%) were in the probable group, and 1 patient (2%) was in the unlikely group. Epistaxis was the most common clinical presentation (38/42, 90%), followed by visceral AVMs (24/42, 57%), and telangiectasia (21/42, 50%). Five patients (12%) did not have a family history of HHT clinical symptoms.
Conclusion:
Among patients having ACVRL1 or ENG genetic variants, only about half of them could be clinically diagnosed as definite HHT, suggesting that genetic screening is important to confirm the diagnosis.
Human pluripotent stem cells (hPSCs) are a powerful platform for disease modeling and drug discovery. However, the introduction of known pathogenic mutations into hPSCs is a time-consuming and labor-intensive process. Base editing is a newly developed technology that enables facile introduction of point mutations into specific loci within the genome of living cells. Here, we design an all-in-one episomal vector that expresses a single guide RNA (sgRNA) with an adenine base editor (ABE) or a cytosine base editor (CBE). Both ABE and CBE can efficiently introduce mutations into cells, A-to-G and C-to-T, respectively. We introduce disease-specific mutations of long QT syndrome into hPSCs to model LQT1, LQT2, and LQT3. Electrophysiological analysis of hPSC-derived cardiomyocytes (hPSC-CMs) using multi-electrode arrays (MEAs) reveals that edited hPSC-CMs display significant increases in duration of the action potential. Finally, we introduce the novel Brugada syndrome-associated mutation into hPSCs, demonstrating that this mutation can cause abnormal electrophysiology. Our study demonstrates that episomal encoded base editors (epi-BEs) can efficiently generate mutation-specific disease hPSC models.
Prime editing enables the introduction of virtually any small-sized genetic change without requiring donor DNA or double-strand breaks. However, evaluation of prime editing efficiency requires time-consuming experiments, and the factors that affect efficiency have not been extensively investigated. In this study, we performed high-throughput evaluation of prime editor 2 (PE2) activities in human cells using 54,836 pairs of prime editing guide RNAs (pegRNAs) and their target sequences. The resulting data sets allowed us to identify factors affecting PE2 efficiency and to develop three computational models to predict pegRNA efficiency. For a given target sequence, the computational models predict efficiencies of pegRNAs with different lengths of primer binding sites and reverse transcriptase templates for edits of various types and positions. Testing the accuracy of the predictions using test data sets that were not used for training, we found Spearman's correlations between 0.47 and 0.81. Our computational models and information about factors affecting PE2 efficiency will facilitate practical application of prime editing.
Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.
CRISPR-mediated DNA base editors, which include cytosine base editors (CBEs) and adenine base editors (ABEs), are promising tools that can induce point mutations at desired sites in a targeted manner to correct or disrupt gene expression. Their high editing efficiency, coupled with their ability to generate a targeted mutation without generating a DNA double strand break (DSB) or requiring a donor DNA template, suggests that DNA base editors will be useful for treating genetic diseases, among other applications. However, this hope has recently been challenged by the discovery of DNA base editor shortcomings, including off-target DNA editing, the generation of bystander mutations, and promiscuous deamination effects in both DNA and RNA, which arise from the main DNA base editor constituents, a Cas nuclease variant and a deaminase. In this review, we summarize information about the DNA base editors that have been developed to date, introduce their associated potential challenges, and describe current efforts to minimize or mitigate those issues of DNA base editors.
Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by the expansion of CAG repeats in exon 1 of the huntingtin gene (HTT). Despite its monogenic nature, HD pathogenesis is still not fully understood, and no effective therapy is available to patients. The development of new techniques such as genome engineering has generated new opportunities in the field of disease modeling and enabled the generation of isogenic models with the same genetic background. These models are very valuable for studying the pathogenesis of a disease and for drug screening. Here, we report the generation of a series of homozygous HEK 293T cell lines with different numbers of CAG repeats at the HTT locus and demonstrate their usefulness for testing therapeutic reagents. In addition, using the CRISPR-Cas9 system, we corrected the mutation in HD human induced pluripotent stem cells and generated a knock-out of the HTT gene, thus providing a comprehensive set of isogenic cell lines for HD investigation.
Hereditary Hemorrhagic Telangiectasia (HHT), also known as Rendu-Osler syndrome, is a genetic vascular disorder affecting 1 in 5000-8000 individuals worldwide. This rare disease is characterized by various vascular defects including epistaxis, blood vessel dilations (telangiectasia) and arteriovenous malformations (AVM) in several organs. About 90% of the cases are associated with heterozygous mutations of ACVRL1 or ENG genes, that respectively encode a bone morphogenetic protein receptor (activin receptor-like kinase 1, ALK1) and a co-receptor named endoglin. Less frequent mutations found in the remaining 10% of patients also affect the gene SMAD4 which is part of the transcriptional complex directly activated by this pathway. Presently, the therapeutic treatments for HHT are intended to reduce the symptoms of the disease. However, recent progress has been made using drugs that target VEGF (vascular endothelial growth factor) and the angiogenic pathway with the use of bevacizumab (anti-VEGF antibody). Furthermore, several exciting high-throughput screenings and preclinical studies have identified new molecular targets directly related to the signaling pathways affected in the disease. These include FKBP12, PI3-kinase and angiopoietin-2. This review aims at reporting these recent developments that should soon allow a better care of HHT patients.
Most genetic variants that contribute to disease¹ are challenging to correct efficiently and without excess byproducts2–5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay–Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.
Purpose:
Hereditary hemorrhagic telangiectasia (HHT), a rare genetic vascular disorder, has been rarely reported in South Korea. We investigated the current prevalence and presenting patterns of genetically confirmed HHT in South Korea.
Materials and methods:
We defined HHT patients as those with proven mutations on known HHT-related genes (ENG, ACVRL1, SMAD4, and GDF2) or those fulfilling 3 or 4 of the Curaçao criteria. A computerized systematic search was performed in PubMed and KoreaMed using the following search term: ("hereditary hemorrhagic telangiectasia" AND "Korea") OR ("Osler-Weber-Rendu" AND "Korea"). We also collected government health insurance data. HHT genetic testing results were collected from three tertiary hospitals in which the genetic tests were performed. We integrated patient data by analyzing each case to obtain the prevalence and presenting pattern of HHT in South Korea.
Results:
We extracted 90 cases from 52 relevant articles from PubMed and KoreaMed. An additional 22 cases were identified from the three Korean tertiary hospitals after excluding seven cases that overlapped with those in the published articles. Finally, 112 HHT patients were identified (41 males and 71 females, aged 4-82 years [mean±standard deviation, 45.3±20.6 years]). The prevalence of HHT in South Korea is about 1 in 500,000, with an almost equal prevalence among men and women. Forty-nine patients underwent genetic testing, of whom 28 had HHT1 (ENG mutation) and 19 had HHT2 (ACVRL1 mutation); the other two patients were negative for ENG, ACVRL1, and SMAD4 mutations.
Conclusion:
The prevalence of HHT is underestimated in Korea. The rate of phenotypic presentation seems to be similar to that found worldwide. Korean health insurance coverage is limited to representative genetic analysis to detect ENG and ACVRL1 mutations. Further genetic analyses to detect HHT3, HHT4, and other forms of HHT should be implemented.
Targeted mutagenesis via genome editing technologies holds great promise in developing improved crop varieties to meet future demands. Point mutations or single nucleotide polymorphisms (SNPs) often determine important agronomic traits of crops. Genome editing‐based single‐base changes could generate elite trait variants in crop plants which help in accelerating crop improvement. Among the genome editing technologies, base editing has emerged as a novel and efficient genome editing approach which enables direct and irreversible conversion of one target base into another in a programmable manner. A base editor is a fusion of catalytically inactive CRISPR–Cas9 domain (Cas9 variants) and cytosine or adenosine deaminase domain that introduces desired point mutations in the target region enabling precise editing of genomes. In the present review, we have summarized the development of different base editing platforms. Then, we have focussed on the current advances and the potential applications of this precise technology in crop improvement. The review also sheds light on the limitations associated with this technology. Finally, the future perspectives of this emerging technology towards crop improvement have been highlighted. This article is protected by copyright. All rights reserved.
Hereditary hemorrhagic telangiectasia (HHT), also known as Osler-Weber-Rendu syndrome, is an autosomal dominant disorder that causes abnormal blood vessel formation. The diagnosis of hereditary hemorrhagic telangiectasia is clinical, based on the Curaçao criteria. Genetic mutations that have been identified include ENG, ACVRL1/ALK1, and MADH4/SMAD4, among others. Patients with HHT may have telangiectasias and arteriovenous malformations in various organs and suffer from many complications including bleeding, anemia, iron deficiency, and high-output heart failure. Families with the same mutation exhibit considerable phenotypic variation. Optimal treatment is best delivered via a multidisciplinary approach with appropriate diagnosis, screening and local and/or systemic management of lesions. Anti-angiogenic agents such as bevacizumab have emerged as a promising systemic therapy in reducing bleeding complications but are not curative. Other pharmacological agents include iron supplementation, antifibrinolytics and hormonal treatment. This review discusses the biology of HHT, management issues that face the practising hematologist, and considerations of future directions in HHT treatment.
The advent of human induced pluripotent stem cells (iPSCs) presents unprecedented opportunities to model human diseases. Differentiated cells derived from iPSCs in two-dimensional (2D) monolayers have proven to be a relatively simple tool for exploring disease pathogenesis and underlying mechanisms. In this Spotlight article, we discuss the progress and limitations of the current 2D iPSC disease-modeling platform, as well as recent advancements in the development of human iPSC models that mimicin vivotissues and organs at the three-dimensional (3D) level. Recent bioengineering approaches have begun to combine different 3D organoid types into a single '4D multi-organ system'. We summarize the advantages of this approach and speculate on the future role of 4D multi-organ systems in human disease modeling.
The advent of human-induced pluripotent stem cell (hiPSC) technology has provided a unique opportunity to establish cellular models of disease from individual patients, and to study the effects of the underlying genetic aberrations upon multiple different cell types, many of which would not normally be accessible. Combining this with recent advances in genome editing techniques such as the clustered regularly interspaced short palindromic repeat (CRISPR) system has provided an ability to repair putative causative alleles in patient lines, or introduce disease alleles into a healthy “WT” cell line. This has enabled analysis of isogenic cell pairs that differ in a single genetic change, which allows a thorough assessment of the molecular and cellular phenotypes that result from this abnormality. Importantly, this establishes the true causative lesion, which is often impossible to ascertain from human genetic studies alone. These isogenic cell lines can be used not only to understand the cellular consequences of disease mutations, but also to perform high throughput genetic and pharmacological screens to both understand the underlying pathological mechanisms and to develop novel therapeutic agents to prevent or treat such diseases. In the future, optimising and developing such genetic manipulation technologies may facilitate the provision of cellular or molecular gene therapies, to intervene and ultimately cure many debilitating genetic disorders.
Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder characterized by a multi-systemic vascular dysplasia and hemorrhage. The precise factors leading to these vascular malformations are not yet understood and robust animal models of HHT are essential to gain a detailed understanding of the molecular and cellular events that lead to clinical symptoms, as well as to test new therapeutic modalities. Most cases of HHT are caused by mutations in either endoglin (ENG) or activin receptor-like kinase 1 (ACVRL1, also known as ALK1). Both genes are associated with TGFβ/BMP signaling, and loss of function mutations in the co-receptor ENG are causal in HHT1, while HHT2 is associated with mutations in the signaling receptor ACVRL1. Significant advances in mouse genetics have provided powerful ways to study the function of Eng and Acvrl1 in vivo, and to generate mouse models of HHT disease. Mice that are null for either Acvrl1 or Eng genes show embryonic lethality due to major defects in angiogenesis and heart development. However mice that are heterozygous for mutations in either of these genes develop to adulthood with no effect on survival. Although these heterozygous mice exhibit selected vascular phenotypes relevant to the clinical pathology of HHT, the phenotypes are variable and generally quite mild. An alternative approach using conditional knockout mice allows us to study the effects of specific inactivation of either Eng or Acvrl1 at different times in development and in different cell types. These conditional knockout mice provide robust and reproducible models of arteriovenous malformations, and they are currently being used to unravel the causal factors in HHT pathologies. In this review, we will summarize the strengths and limitations of current mouse models of HHT, discuss how knowledge obtained from these studies has already informed clinical care and explore the potential of these models for developing improved treatments for HHT patients in the future.
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.
The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual's gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care.
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder affecting 1 in 5000–8000 individuals. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is the most common HHT and manifests as diverse vascular malformations ranging from mild symptoms such as epistaxis and mucosal and cutaneous telangiectases to severe arteriovenous malformations (AVMs) in the lungs, brain or liver. HHT1 is caused by heterozygous mutations in the ENG gene, which encodes endoglin, the TGFβ homodimeric co‐receptor. It was previously shown that some endoglin HHT1‐causing variants failed to traffic to the plasma membrane due to their retention in the endoplasmic reticulum (ER) and consequent degradation by ER‐associated degradation (ERAD). Endoglin is a homodimer formed in the ER, and we therefore hypothesized that mixed heterodimers might form between ER‐retained variants and WT protein, thus hampering its maturation and trafficking to the plasma membrane causing dominant negative effects. Indeed, HA‐tagged ER‐retained mutants formed heterodimers with Myc‐tagged WT endoglin. Moreover, variants L32R, V105D, P165L, I271N and C363Y adversely affected the trafficking of WT endoglin by reducing its maturation and plasma membrane localization. These results strongly suggest dominant negative effects exerted by these ER‐retained variants aggravating endoglin loss of function in patients expressing them in the heterozygous state with the WT allele. Moreover, this study may help explain some of the variability observed among HHT1 patients due to the additional loss of function exerted by the dominant negative effects in addition to that due to haploinsufficiency. These findings might also have implications for some of the many conditions impacted by ERAD.
Biomedical research is undergoing a paradigm shift towards approaches centred on human disease models owing to the notoriously high failure rates of the current drug development process. Major drivers for this transition are the limitations of animal models, which, despite remaining the gold standard in basic and preclinical research, suffer from interspecies differences and poor prediction of human physiological and pathological conditions. To bridge this translational gap, bioengineered human disease models with high clinical mimicry are being developed. In this Review, we discuss preclinical and clinical studies that benefited from these models, focusing on organoids, bioengineered tissue models and organs-on-chips. Furthermore, we provide a high-level design framework to facilitate clinical translation and accelerate drug development using bioengineered human disease models.
Prime editing (PE) is a precision gene editing technology that enables the programmable installation of substitutions, insertions and deletions in cells and animals without requiring double-strand DNA breaks (DSBs). The mechanism of PE makes it less dependent on cellular replication and endogenous DNA repair than homology-directed repair-based approaches, and its ability to precisely install edits without creating DSBs minimizes indels and other undesired outcomes. The capabilities of PE have also expanded since its original publication. Enhanced PE systems, PE4 and PE5, manipulate DNA repair pathways to increase PE efficiency and reduce indels. Other advances that improve PE efficiency include engineered pegRNAs (epegRNAs), which include a structured RNA motif to stabilize and protect pegRNA 3′ ends, and the PEmax architecture, which improves editor expression and nuclear localization. New applications such as twin PE (twinPE) can precisely insert or delete hundreds of base pairs of DNA and can be used in tandem with recombinases to achieve gene-sized (>5 kb) insertions and inversions. Achieving optimal PE requires careful experimental design, and the large number of parameters that influence PE outcomes can be daunting. This protocol describes current best practices for conducting PE and twinPE experiments and describes the design and optimization of pegRNAs. We also offer guidelines for how to select the proper PE system (PE1 to PE5 and twinPE) for a given application. Finally, we provide detailed instructions on how to perform PE in mammalian cells. Compared with other procedures for editing human cells, PE offers greater precision and versatility, and can be completed within 2–4 weeks. This protocol describes prime editing (PE) and twinPE experiments as well as the design and optimization of pegRNAs. The authors provide guidelines for selecting the proper PE system for a given application and how to perform PE in mammalian cells.
Mitochondrial dysfunction plays a key role in the pathogenesis and progression of Alzheimer’s Disease. Our previous studies showed that over expression of AD‐associated mutant APP led to abnormalities of mitochondrial biogenesis and mitophagy, leading to mitochondrial dysfunction. However the mechanism remains unclear. In this study, we investigated the effect of orexin‐A on mitochondrial biogenesis, mitophagy and mitochondrial structure in over expression of AD‐associated mutant APP cells. We used 20E2 cells as the AD cell model. 20E2 cells were treated with orexin‐A (50, 100 nM). The effect of different concentrations of orexin‐A on cell activity was detected by MTT. As compared with the non‐treated 20E2 cells, orexin‐A‐treated 20E2 cells showed increased expression of APP, decreased cell viability and decreased adenosine triphosphate (ATP) level, decreased levels of regulatory proteins of mitochondrial biogenesis (peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha [PGC‐1α], nuclear respiratory factor 1/2 [NRF1/2], mitochondrial transcription factor A [TFAM]), increased levels of regulatory proteins of mitophagy (Parkin, PTEN‐induced putative kinase 1 [PINK1], microtubule‐associated protein light chain 3 II/I [LC3‐II/LC3‐I]) and decreased p62 level, with damaged mitochondrial structure. Orexin‐A may reduce mitochondrial biogenesis, enhance mitophagy and damage mitochondrial structure in AD.
Base editing — the introduction of single-nucleotide variants (SNVs) into DNA or RNA in living cells — is one of the most recent advances in the field of genome editing. As around half of known pathogenic genetic variants are due to SNVs, base editing holds great potential for the treatment of numerous genetic diseases, through either temporary RNA or permanent DNA base alterations. Recent advances in the specificity, efficiency, precision and delivery of DNA and RNA base editors are revealing exciting therapeutic opportunities for these technologies. We expect the correction of single point mutations will be a major focus of future precision medicine.
The development of new CRISPR–Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR–Cas-derived genome editing agents—nucleases, base editors, transposases/recombinases and prime editors—are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics. A growing arsenal of CRISPR-based tools enables increasingly sophisticated genome editing applications.
Full text-
https://authors.elsevier.com/c/1Zj4fc9XEVOUw
Abstract:
Genome editing with CRISPR/Cas has rapidly gained popularity. Base editing, a new CRISPR/Cas-based approach, can precisely convert one nucleotide to another in DNA or RNA without inducing a double-strand DNA break (DSB). A combination of catalytically impaired nuclease variants with different deaminases has yielded diverse base-editing platforms that aim to address the key limitations such as specificity, protospacer adjacent motif (PAM) compatibility, editing window length, bystander editing, and sequence context preference. Because new base editors significantly reduce unintended editing in the genome, they hold great promise for treating genetic diseases and for developing superior agricultural crops. We review here the development of various base editors, assess their technical advantages and limitations, and discuss their broad applications in basic research, medicine, and agriculture.
The derivation of human embryonic stem cells (hESCs) and the stunning discovery that somatic cells can be reprogrammed into human induced pluripotent stem cells (hiPSCs) holds the promise to revolutionize biomedical research and regenerative medicine. In this Review, we focus on disorders of the central nervous system and explore how advances in human pluripotent stem cells (hPSCs) coincide with evolutions in genome engineering and genomic technologies to provide realistic opportunities to tackle some of the most devastating complex disorders. Advances in stem cell biology are paving new paths toward their use in the clinic, especially toward understanding and treating neurological and neurodegenerative disease.
Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
Hereditary hemorrhagic telangiectasia (HHT) is a dominantly inherited vascular disorder that is heterogeneous in terms of age of onset and clinical manifestations, Endoglin is the gene mutated in HHT1, which is associated with a higher prevalence of pulmonary arteriovenous malformations than HHT2, where ALK-1 is the mutated gene. Endoglin is constitutively expressed on endothelial cells and inducible on peripheral blood activated monocytes so that protein levels can be measured by metabolic labeling and immunoprecipitation. We report the analysis of umbilical vein endothelial cells in 28 newborns from 24 families with a clinical diagnosis of HHT. Reduced levels of endoglin were observed in umbilical vein endothelial cells in 15/28 subjects and in activated monocytes of all clinically affected relatives tested, suggesting that these individuals had HHT1. No mutant protein was expressed at the cell surface in any of these cases, and a transient intracellular species was seen in samples of only two families, supporting a haploinsufficiency model. Quantitative multiplex PCR fragment analysis was established for the endoglin gene and revealed six mutations that were confirmed by automated DNA sequencing. An additional 10 mutations were identified in newborns by sequencing all exons. Of the 16 mutations, 10 were novel, three had been independently identified in related families, and three were previously known. Our data confirm that endoglin levels correlate with the presence or absence of mutation in HHT1 families, allowing the early identification of affected newborns that should be screened clinically to avoid serious complications of this disorder, such as cerebral arteriovenous malformations.
DNA double-strand breaks (DSBs) are highly toxic lesions that can drive genetic instability. To preserve genome integrity, organisms have evolved several DSB repair mechanisms, of which nonhomologous end-joining (NHEJ) and homologous recombination (HR) represent the two most prominent. It has recently become apparent that multiple layers of regulation exist to ensure these repair pathways are accurate and restricted to the appropriate cellular contexts. Such regulation is crucial, as failure to properly execute DSB repair is known to accelerate tumorigenesis and is associated with several human genetic syndromes. Here, we review recent insights into the mechanisms that influence the choice between competing DSB repair pathways, how this is regulated during the cell cycle, and how imbalances in this equilibrium result in genome instability.
The classical genetic approach for exploring biological pathways typically begins by identifying mutations that cause a phenotype of interest. Overexpression or misexpression of a wild-type gene product, however, can also cause mutant phenotypes, providing geneticists with an alternative yet powerful tool to identify pathway components that might remain undetected using traditional loss-of-function analysis. This review describes the history of overexpression, the mechanisms that are responsible for overexpression phenotypes, tests that begin to distinguish between those mechanisms, the varied ways in which overexpression is used, the methods and reagents available in several organisms, and the relevance of overexpression to human disease.
Hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu syndrome) is a disorder of development of the vasculature characterized by telangiectases and arteriovenous malformations in specific locations. It is one of most common monogenic disorders, but affected individuals are frequently not diagnosed. The most common features of the disorder, nosebleeds, and telangiectases on the lips, hands, and oral mucosa are often quite subtle. Optimal management requires an understanding of the specific presentations of these vascular malformations, especially their locations and timing during life. Telangiectases in the nasal and gastrointestinal mucosa and brain arteriovenous malformations generally present with hemorrhage. However, complications of arteriovenous malformations in the lungs and liver are generally the consequence of blood shunting through these abnormal blood vessels, which lack a capillary bed and thus result in a direct artery-to-vein connection. Mutations in at least five genes are thought to result in hereditary hemorrhagic telangiectasia, but mutations in two genes (ENG and ACVRL1/ALK1) cause approximately 85% of cases. The frequency of arteriovenous malformations in particular organs and the occurrence of certain rare symptoms are dependent on the gene involved. Molecular genetic testing is used to establish the genetic subtype of hereditary hemorrhagic telangiectasia in a clinically affected individual and family, and for early diagnosis to allow for appropriate screening and preventive treatment.
Reprogramming of human somatic cells uses readily accessible tissue, such as skin or blood, to generate embryonic-like induced pluripotent stem cells (iPSCs). This procedure has been applied to somatic cells from patients who are classified into a disease group, thus creating "disease-specific" iPSCs. Here, we examine the challenges and assumptions in creating a disease model from a single cell of the patient. Both the kinetics of disease onset and progression as well as the spatial localization of disease in the patient's body are challenges to disease modeling. New tools in genetic modification, reprogramming, biomaterials, and animal models can be used for addressing these challenges.
Medicine. iPSC disease modeling
- F Soldner
- R Jaenisch
Soldner F, Jaenisch R. Medicine. iPSC disease modeling.
Science 2012;338:1155-1156