Models Of Human Genetic Diseases Research Articles

45 Genome Editing of Livestock for Production Agriculture and Biomedical Applications

Abstract Continued population growth and clear evidence of climate change are placing unprecedented strain on food production and health care systems worldwide. Agricultural production goals that highlight environmental sustainability are at the forefront of social dialog, with animal agriculture routinely targeted for inefficiencies, welfare concerns and impact on climate. Genomic selection has transformed animal production and the rate of genetic gain in each generation continues to increase. Nonetheless, it is apparent that the rate of production gains will fall short of the necessary goals of increased efficiency and adaptation to the changing environment. Genetic engineering may have an important role in enhancing livestock production as well as providing new models for research in human health. The CRISPR/Cas and related systems for targeted gene editing has revolutionized livestock genetic engineering by increasing the rate of targeted mutation, thus drastically reducing the cost of producing founder animals with the desired trait. Intentional insertion of well-known mutations into genes such as myostatin have been shown to impart the expected increase in meat production, but have also resulted in undesirable associated traits affecting fertility, marbling and the skeleton. Thus, the approach to increasing meat production may require nuances such as promotor targeting and multiple gene targeting to obtain the desired production characteristics. Efforts to increase disease resistance could have dramatic impacts on animal welfare and production rates, potentially saving millions of animals from morbidity and mortality. However, confronting a single disease with each genetic alteration will be costly and inefficient. Considerations on how broad scale disease resistance could be achieved through genetic engineering will be instrumental in developing livestock with advantages to avoid infection and/or transmission of multiple pathogens. Gene editing also allows correction of mutations that arise in livestock through natural mechanisms while under intense genomic selection for increased production. Removing disease causing mutations in otherwise high value livestock will allow continued production from these lines without concern for inheriting the causative mutation. The same process can be utilized to insert genetic mutations known to phenocopy human conditions providing valuable large animal models of human genetic disease. Increased milk, meat, or eggs per animal unit will sustainably improve high quality, nutrient dense food production while large animal models of human disease will provide a more translational biomedical model for accelerated development of drugs and treatments. These goals are achievable, but require continued investment in innovation, clear regulatory framework for commercialization, education, and public engagement to ensure success.

Generating a Heat-Tolerance Mouse Model.

Creating mouse models of human genetic disease (Gurumurthy and Lloyd, Dis Models Mech 12(1):dmm029462, 2019) and livestock trait (Schering et al. Arch Physiol Biochem 121(5):194-205, 2015; Habiela et al. J Gen Virol 95 (Pt 11):2329-2345, 2014) have been proven to be a useful tool for understanding the mechanism behind the phenotypes and fundamental and applied research in livestock. A single base pair deletion of prolactin receptor (PRLR) has an impact on hair morphology phenotypes beyond its classical roles in lactation in cattle, the so-called slick cattle (Littlejohn et al. Nat Commun 5:5861, 2014). Here, we generate a knock-in mouse model by targeting the specific locus of PRLR gene using Cas9-mediated genome editing via homology-directed repair (HDR) in mouse zygotes. The mouse model carrying the identical PRLR mutation in slick cattle may provide a useful animal model to study the pathway of thermoregulation and the mechanism of heat-tolerance in the livestock.

Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases.

The use of prime editing-a gene-editing technique that induces small genetic changes without the need for donor DNA and without causing double strand breaks-to correct pathogenic mutations and phenotypes needs to be tested in animal models of human genetic diseases. Here we report the use of prime editors 2 and 3, delivered by hydrodynamic injection, in mice with the genetic liver disease hereditary tyrosinemia, and of prime editor 2, delivered by an adeno-associated virus vector, in mice with the genetic eye disease Leber congenital amaurosis. For each pathogenic mutation, we identified an optimal prime-editing guide RNA by using cells transduced with lentiviral libraries of guide-RNA-encoding sequences paired with the corresponding target sequences. The prime editors precisely corrected the disease-causing mutations and led to the amelioration of the disease phenotypes in the mice, without detectable off-target edits. Prime editing should be tested further in more animal models of genetic diseases.

Genome engineering technologies in rabbits.

The rabbit has been recognized as a valuable model in various biomedical and biological research fields because of its intermediate size and phylogenetic proximity to primates. However, the technology for precise genome manipulations in rabbit has been stalled for decades, severely limiting its applications in biomedical research. Novel genome editing technologies, especially CRISPR/Cas9, have remarkably enhanced precise genome manipulation in rabbits, and shown their superiority and promise for generating rabbit models of human genetic diseases. In this review, we summarize the brief history of transgenic rabbit technology and the development of novel genome editing technologies in rabbits.

Mouse Models and Online Resources for Functional Analysis of Osteoporosis Genome-Wide Association Studies.

Osteoporosis is a complex genetic disease in which the number of loci associated with the bone mineral density, a clinical risk factor for fracture, has increased at an exponential rate in the last decade. The identification of the causative variants and candidate genes underlying these loci has not been able to keep pace with the rate of locus discovery. A large number of tools and data resources have been built around the use of the mouse as model of human genetic disease. Herein, we describe resources available for functional validation of human Genome Wide Association Study (GWAS) loci using mouse models. We specifically focus on large-scale phenotyping efforts focused on bone relevant phenotypes and repositories of genotype-phenotype data that exist for transgenic and mutant mice, which can be readily mined as a first step toward more targeted efforts designed to deeply characterize the role of a gene in bone biology.

Base Editing: Chemistry on a Target Nucleotide in the Genome of Living Cells

Point mutations represent the majority of known human genetic variants associated with disease but are difficult to correct cleanly and efficiently using standard nuclease‐based genome editing methods. In this lecture I will describe the development, application, and evolution of base editing, a new approach to genome editing that directly converts a target base pair to another base pair in living cells without requiring DNA backbone cleavage or donor DNA templates. Through a combination of protein engineering and protein evolution, we recently developed two classes of base editors (BE4 and ABE) that together enable all four types of transition mutations (C to T, T to C, A to G, and G to A) to be efficiently and cleanly installed or corrected at target positions in genomic DNA. The four transition mutations collectively account for most known human pathogenic point mutations. Base editing has been widely used by many laboratories around the world in a wide range of organisms including bacteria, fungi, plants, fish, frogs, insects, mammals, and even human embryos. We have recently expanded the scope of base editing by enhancing its efficiency, product purity, targeting scope, and DNA specificity. By optimizing base editor expression, we developed “max” versions of cytosine and adenine base editors with greatly increase editing efficiency in mammalian cells. We also show that base editing can function in vivo in post‐mitotic somatic cells that do not support homology‐directed repair. To improve the targeting scope of base editing, we used our phage‐assisted continuous evolution (PACE) system to rapidly evolve Cas9 and base editor variants with both broadened PAM compatibility and higher DNA specificity. Finally, we integrated several of these developments to address cell and animal models of human genetic disease. Base editing can be used to correct pathogenic point mutations, introduce disease‐suppressing mutations, and create new models of genetic diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Myotonia congenita in a Labrador Retriever with truncated CLCN1

An eight week old Labrador Retriever puppy presented with stiff-legged robotic gait. Abnormal gait was most evident after rest and improved with prolonged activity. On occasions, initiation of sudden movements would result in collapse with rigidity of the trunk and stiff extended limbs for several seconds. Other clinical signs were excitement-induced upper airway stridor and oropharyngeal dysphagia. Myotonia congenita was diagnosed based on clinical signs, abundant myotonic discharges on electromyography and exclusion of structural myopathies on histology. Whole exome sequencing revealed a case-specific homozygous variant in CLCN1, c.2275A > T resulting in a premature stop codon, p.R759X. The CLCN1 variant was absent from the genomes of 127 Labrador Retriever controls and 474 control dogs from other breeds. This study expands the spectrum of identified canine CLCN1 mutations and the list of affected breeds in myotonia congenita and highlights the potential value of dogs as translational large animal models of human genetic diseases.

Genome-editing applications of CRISPR-Cas9 to promote in vitro studies of Alzheimer's disease.

Genetic variations play an important role in the clinical presentation and progression of Alzheimer’s disease (AD), especially early-onset Alzheimer’s disease. Hundreds of mutations have been reported with the majority resulting from alterations in β-amyloid precursor protein (APP), presenilin 1 (PSEN1), or presenilin 2 (PSEN2) genes. The roles of these mutations in the pathogenesis of AD have been classically confirmed or refuted through functional studies, where the mutations are cloned, inserted into cell lines, and monitored for changes in various properties including cell survival, amyloid production, or Aβ42/40 ratio. However, these verification studies tend to be expensive, time consuming, and inconsistent. Recently, the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR–Cas9) system was developed, which improves sequence-specific gene editing in cell lines, organs, and animals. CRISPR–Cas9 is a promising tool for the generation of models of human genetic diseases and could facilitate the establishment of new animal AD models and the observation of dynamic bioprocesses in AD. Here, we recapitulated the history of CRISPR technology, recent progress, and, especially, its potential applications in AD-related genetic, animal modeling, and functional studies.

Zebrafish: A Premier Vertebrate Model for Biomedical Research in Indian Scenario.

The zebrafish (Danio rerio) is a versatile model organism that has been used in biomedical research for several decades to study a wide range of biological phenomena. There are many technical advantages of using zebrafish over other vertebrate models. They are readily available, hardy, easy, and inexpensive to maintain in the laboratory, have a short life cycle, and have excellent fecundity. Due to its optical clarity and reproducible capabilities, it has become one of the predominant models of human genetic diseases. Zebrafish research has made rapid strides in the United States and Europe, but in India the field is at an early stage and many researchers still remain unaware of the full research potential of this tiny fish. The zebrafish model system was introduced into India in the early 2000s. Up to now, more than 200 scientific referred articles have been published by Indian researchers. This review gives an overview of the current state of knowledge for zebrafish research in India, with the aim of promoting wider utilization of zebrafish for high level biological studies.

Editing mammalian genomes: ethical considerations.

Genome editing is facilitating the manipulation of genomes on an unprecedented scale. It promises to revolutionize our ability to study gene function and generate models of human genetic disease in a range of organisms, most notably in mammals such as the mouse. Is this new technology likely to be disruptive to our research practices in any way? Will it alter the ways in which we implement the ethical imperatives of the 3Rs? In short, what ethical questions are raised by genome editing of mammals in a biomedical research context?

An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase.

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.

Exome screening to identify loss-of-function mutations in the rhesus macaque for development of preclinical models of human disease.

BackgroundExome sequencing has been utilized to identify genetic variants associated with disease in humans. Identification of loss-of-function mutations with exome sequencing in rhesus macaques (Macaca mulatta) could lead to valuable animal models of genetic disease. Attempts have been made to identify variants in rhesus macaques by aligning exome data against the rheMac2 draft genome. However, such efforts have been impaired due to the incompleteness and annotation errors associated with rheMac2. We wished to determine whether aligning exome reads against our new, improved rhesus genome, MacaM, could be used to identify high impact, loss-of-function mutations in rhesus macaques that would be relevant to human disease.ResultsWe compared alignments of exome reads from four rhesus macaques, the reference animal and three unrelated animals, against rheMac2 and MacaM. Substantially more reads aligned against MacaM than rheMac2. We followed the Broad Institute’s Best Practice guidelines for variant discovery which utilizes the Genome Analysis Toolkit to identify high impact mutations. When rheMac2 was used as the reference genome, a large number of apparent false positives were identified. When MacaM was used as the reference genome, the number of false positives was greatly reduced. After examining the variant analyses conducted with MacaM as reference genome, we identified two putative loss-of-function mutations, in the heterozygous state, in genes related to human health. Sanger sequencing confirmed the presence of these mutations. We followed the transmission of one of these mutations (in the butyrylthiocholine gene) through three generations of rhesus macaques. Further, we demonstrated a functional decrease in butyrylthiocholinesterase activity similar to that observed in human heterozygotes with loss-of-function mutations in the same gene.ConclusionsThe new MacaM genome can be effectively utilized to identify loss-of-function mutations in rhesus macaques without generating a high level of false positives. In some cases, heterozygotes may be immediately useful as models of human disease. For diseases where homozygous mutants are needed, directed breeding of loss-of-function heterozygous animals could be used to create rhesus macaque models of human genetic disease. The approach we describe here could be applied to other mammals, but only if their genomes have been improved beyond draft status.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2509-5) contains supplementary material, which is available to authorized users.

CRISPR-Cas9 for in vivo Gene Therapy: Promise and Hurdles.

Owing to its easy-to-use and multiplexing nature, the genome editing tool CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease 9) is revolutionizing many areas of medical research and one of the most amazing areas is its gene therapy potentials. Previous explorations into the therapeutic potentials of CRISPR-Cas9 were mainly conducted in vitro or in animal germlines, the translatability of which, however, is either limited (to tissues with adult stem cells amenable to culture and manipulation) or currently impermissible (due to ethic concerns). Recently, important progresses have been made on this regard. Several studies have demonstrated the ability of CRISPR-Cas9 for in vivo gene therapy in adult rodent models of human genetic diseases delivered by methods that are potentially translatable to human use. Although these recent advances represent a significant step forward to the eventual application of CRISPR-Cas9 to the clinic, there are still many hurdles to overcome, such as the off-target effects of CRISPR-Cas9, efficacy of homology-directed repair, fitness of edited cells, immunogenicity of therapeutic CRISPR-Cas9 components, as well as efficiency, specificity, and translatability of in vivo delivery methods. In this article, we introduce the mechanisms and merits of CRISPR-Cas9 in genome editing, briefly retrospect the applications of CRISPR-Cas9 in gene therapy explorations and highlight recent advances, later we discuss in detail the challenges lying ahead in the way of its translatability, propose possible solutions, and future research directions.

Large animal models of rare genetic disorders: sheep as phenotypically relevant models of human genetic disease.

Animals that accurately model human disease are invaluable in medical research, allowing a critical understanding of disease mechanisms, and the opportunity to evaluate the effect of therapeutic compounds in pre-clinical studies. Many types of animal models are used world-wide, with the most common being small laboratory animals, such as mice. However, rodents often do not faithfully replicate human disease, despite their predominant use in research. This discordancy is due in part to physiological differences, such as body size and longevity. In contrast, large animal models, including sheep, provide an alternative to mice for biomedical research due to their greater physiological parallels with humans. Completion of the full genome sequences of many species, and the advent of Next Generation Sequencing (NGS) technologies, means it is now feasible to screen large populations of domesticated animals for genetic variants that resemble human genetic diseases, and generate models that more accurately model rare human pathologies. In this review, we discuss the notion of using sheep as large animal models, and their advantages in modelling human genetic disease. We exemplify several existing naturally occurring ovine variants in genes that are orthologous to human disease genes, such as the Cln6 sheep model for Batten disease. These, and other sheep models, have contributed significantly to our understanding of the relevant human disease process, in addition to providing opportunities to trial new therapies in animals with similar body and organ size to humans. Therefore sheep are a significant species with respect to the modelling of rare genetic human disease, which we summarize in this review.

Effect of Genetic Variation in a Drosophila Model of Diabetes-Associated Misfolded Human Proinsulin

The identification and validation of gene–gene interactions is a major challenge in human studies. Here, we explore an approach for studying epistasis in humans using a Drosophila melanogaster model of neonatal diabetes mellitus. Expression of the mutant preproinsulin (hINSC96Y) in the eye imaginal disc mimics the human disease: it activates conserved stress-response pathways and leads to cell death (reduction in eye area). Dominant-acting variants in wild-derived inbred lines from the Drosophila Genetics Reference Panel produce a continuous, highly heritable distribution of eye-degeneration phenotypes in a hINSC96Y background. A genome-wide association study (GWAS) in 154 sequenced lines identified a sharp peak on chromosome 3L, which mapped to a 400-bp linkage block within an intron of the gene sulfateless (sfl). RNAi knockdown of sfl enhanced the eye-degeneration phenotype in a mutant-hINS-dependent manner. RNAi against two additional genes in the heparan sulfate (HS) biosynthetic pathway (ttv and botv), in which sfl acts, also modified the eye phenotype in a hINSC96Y-dependent manner, strongly suggesting a novel link between HS-modified proteins and cellular responses to misfolded proteins. Finally, we evaluated allele-specific expression difference between the two major sfl-intronic haplotypes in heterozygtes. The results showed significant heterogeneity in marker-associated gene expression, thereby leaving the causal mutation(s) and its mechanism unidentified. In conclusion, the ability to create a model of human genetic disease, map a QTL by GWAS to a specific gene, and validate its contribution to disease with available genetic resources and the potential to experimentally link the variant to a molecular mechanism demonstrate the many advantages Drosophila holds in determining the genetic underpinnings of human disease.

Methemoglobinemia In Rhesus Monkeys Due To M-Hemoglobinopathy

M-hemoglobinopathies in humans are usually due to amino acid changes, which tend to stabilize the heme iron in ferric state and inaccessible to methemoglobin reductase. Consequently, blood methemoglobin concentrations are elevated despite normal methemoglobin reductase activity. No naturally-occurring animal models for M-hemoglobinopathies have been described. In contrast to humans where thousands of hereditary disorders and genetic predispositions to disease have been described, inborn errors of metabolism in non-human primates have been very rarely documented (e.g. globoid cell leukodystrophy). As part of an NIH-supported National Referral Center for Animal Models of Human Genetic Disease, which focuses on homologous hereditary diseases in dogs and cats, we present here the discovery and characterization of an M-hemogobinopathy in rhesus monkeys (Macaca mulatta). Adult rhesus monkeys of Chinese origin with persistent cyanotic mucous membranes and healthy controls were studied from two institutions. Despite the apparent cyanosis, these primates did clinically not show any other overt signs. Routine complete blood cell counts, chemistry screens and urinalyses as well as cardiopulmonary examinations did not reveal any abnormalities. Fresh EDTA blood samples were analyzed to determine the degree and cause of methemoglobinemia and results were compared to healthy rhesus monkeys from the same institutions. Blood methemoglobin concentrations were persistently increased to 15-30% in affected monkeys, while control animals had <1% methemoglobin levels. Methemoglobin (cytochrome b5) reductase activities in erythrocytes were repeatedly measured in vitro and found to be normal compared to controls. Based upon the hemoglobin-oxygen dissociation curves and p50 values (14.8 mmHg; compared to 14.5 and 14.6 mmHg for controls) the oxygen transport function was normal. Both cation-exchange as well as reversed-phase and high performance liquid chromatography analyses revealed normal elution patterns of hemoglobin isotype and globin chains. DNA studies identified a disease-associated polymorphism in the alpha globin gene (CAC → CAA) in the affected animals. This change in the alpha globin protein was confirmed by mass spectrometry (α78 His → Gln). Our findings indicate an electrophoretically silent α-globin polymorphism is associated with methemoglobinemia in these rhesus monkeys. This is the first description of methemoglobinemia in a non-human primate and represents the first animal model of an M-hemoglobinopathy. While clinical signs were relatively mild in these rhesus monkeys, our findings represent one of the first inborn errors of metabolism described in non-human primates, thus emphasizing how unusual disorders caused by “experiments of nature” provide rare opportunities to gain a better understanding of both medicine and biology. Rhesus monkeys represent unique, valuable animal models for developing and testing new treatment options for hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.

2012 William Allan Award Introduction: Uta Francke

2012 William Allan Award Introduction: Uta Francke

An ENU-Induced Mutation of Nrg1 Causes Dilated Pupils and a Reduction in Muscarinic Receptors in the Sphincter Pupillae

BackgroundN-ethyl-N-nitrosourea (ENU)-induced mutagenesis is a powerful tool for the study of gene function and the generation of human disease models. A large number of mouse mutants obtained by ENU-induced mutagenesis with a variety of phenotypes have been recovered. However, after genetic confirmation testing, only approximately 50% of the abnormal phenotypes were found to be heritable.Methodology/Principal FindingsA mouse mutant, Dp1, with a dilated pupil phenotype was induced with an N-ethyl-N-nitrosourea (ENU) mutagenesis strategy. Sequence analysis for Nrg1 reveals a G>A base substitution that flanks exon E59, encoding for an EGFβ domain, in the 5′ splice donor site. The mutation affects but does not abolish the splicing of EGFβ-type Nrg1 mRNA in Dp1 mice and produces several different transcripts by activating other, cryptic splice sites. These types of protein isoforms are expected, and the result shows that, in the mutant, the effect is a decrease in but not an elimination of the high affinity EGFβ-type Nrg1 isoforms. This is partially compensated for by an increase in expression of the low affinity alpha forms or inactive proteins, suggesting that the mutation results in a hypomorphic allele. Interestingly, genetic model testing shows that Dp1 is a mutation that results in a dilated pupil phenotype that is inherited with very low penetrance when heterozygous and with complete penetrance when homozygous. Pharmacological and immunohistochemical tests show a reduction of muscarinic (M) receptors in the sphincter pupillae of Dp1 mice, which is a major cause of dilated pupils.Conclusions/SignificanceThis study is the first report of an Nrg1 mutation being associated with a dilated pupil phenotype and the reduction of M receptors. This report may help in establishing more mutant mouse lines and models of human genetic disease and can be applied to other organisms. Dp1 mice are a valuable resource for the further clarification of Nrg1 biological function.

Animal models of human genetic diseases: do they need to be faithful to be useful?

With the advances in molecular genetics, animal models of human diseases are becoming more numerous and more refined every year. Despite this, one must recognize that they generally do not faithfully and comprehensively mimic the homologous human disease. Faced with these imperfections, some geneticists believe that these models are of little value, while for others, on the contrary, they are important tools. We agree with this second statement, and in this review, we examine the reasons that may explain the observed differences and suggest means to circumvent or even exploit them. Our opinion is that animal models should be regarded more as tools capable of answering specific questions rather than mere replicas, at a smaller scale, of a given human disease. Far from disappointing they are probably called for a promising future.

Generation and clinical application of human T cell-derived induced pluripotent stem cells

Pluripotent stem (iPS) cells are a very promising cell source for models of human genetic diseases and revolutionary new therapies. Successful reprogramming of human blood cells has been reported and is likely to advance the clinical application of iPS cells. In terms of a patient’s own somatic cells, generating iPS cells from peripheral blood cells has advantages for clinical applications because these cells are an easily accessible cell source. Of the human peripheral blood cells, T cells can be readily cultured in vitro and proliferate rapidly. Furthermore, only a small amount of peripheral blood is needed to generate iPS cells from T cells, thus increasing the number of patients in whom the technique can be used. iPS cells that contain T-cell receptor (TCR) rearrangements in their genome also have the potential to be traceable markers when establishing novel transplantation therapies. The present review summarizes recent progress in the methods used to generate iPS cells and the future potential of human T cell-derived iPS cells. Rec./Acc.10/11/2011, pp393-398