The inhibition of alkaptonuria by the chemical nitisinone in a mouse model led to a full-scale clinical trial funded by the European Commission – the results of which are expected to produce medicine for a condition for which there was previously no treatment available.

Alkaptonuria (AKU) is caused by a deficiency of homogentisate 1,2 dioxygenase, an enzyme that is required for breaking down the two amino acids tyrosine and phenylalanine. The lack of a functional copy of this enzyme results in a vast increase in circulating concentration of homogentisic acid (HGA), leading to a darkening of the urine upon exposure to air.

High levels of homogentisic acid within the human body eventually leads to ochronosis – a progressive pigmentation of connective tissues and eventually severe joint disease, which can become fatal later in life.

Whilst AKU is a serious condition, for many years it was not seen as prevalent enough among the population to be lucrative for drug developers. However, physicians had long suspected that nitisinone, a chemical that was first developed for use as a weed-killer, could be used to treat AKU – nitisinone was being used effectively to treat tyrosinemia (a condition that results from disruption to the same metabolic pathway as AKU).

There was a small-scale clinical trial run in the US between 2005 and 2008, but too few patients were recruited and the clinical endpoint (the success of the treatment) was defined very narrowly. No positive results were obtained.

In order for a clinical trial to succeed, a more reliable endpoint would have to be suggested, and this required more knowledge of how the disease developed. With this in mind, funding obtained from Britain’s Big Lottery Fund went to a research team at the University of Liverpool to develop a mouse model of AKU.

A colony of Hgd-/- (the genetic equivalent of human AKU) mice were bred by the research team in Liverpool and the mice were established as a model of the plasma biochemistry of AKU and its associated arthropathy. The concentration of plasma HGA was found to be 0.149 ± 0.019 mM, whilst HGA levels in WT mice were below the level of UV detection. The researchers were also able to determine that pigmentation of connective tissue increased linearly with increasing age. Perhaps most importantly though, a significant difference was found between HGA concentrations before and after treatment with nitisinone. Levels of plasma HGA dropped by 88% – a figure that was maintained over the mouse lifetime, with joint tissue from nitisinone-treated AKU mice showing no pigmented tissue.

The results of this mouse model study led to a full-scale clinical trial, funded by the European Commission, with use of nitisinone as a treatment for AKU expected to be approved across Europe once the trial comes to an end.

The aim of the IMPC is to generate similar knock out lines – on a much broader scale, with the goal of producing knock out mutations in embryonic stem cells for 20,000 known and predicted mouse genes, and determining the function of each of these genes. The mouse’s genetic similarity to humans (95% at the gene level) means that data generated by the IMPC could be a powerful tool as we seek to understand the genetic basis of human disease. As our knowledge of rare genetic diseases increases in line with rapid technological advancement, the desire for treatments targeted to small groups of sufferers with these diseases will also likely increase – the case involving alkaptonuria is likely to be just one instance of a mouse model proving to be a driver behind new treatments.

Research article: Ochronotic osteoarthropathy in a mouse model of alkaptonuria, and its inhibition by nitisinone

For the first time scientists have identified how to halt kidney disease in a life-limiting genetic condition, which may pave the way for personalised treatment in the future. Researchers at Newcastle University, UK, have shown in a cell model and in a mouse model that gene editing could be used for Joubert syndrome to stop kidney damage in patients who have the CEP290 faulty gene.

Joubert syndrome is a brain disorder, causing varying degrees of physical, mental and sometimes visual impairments. The condition affects approximately one in 80,000 newborns, and one third also get kidney failure. Not all patients with Joubert syndrome carry the CEP290 gene, but those who do will develop kidney disease during their lifetime and may require a transplant or dialysis.

The study, which was funded by Kidney Research UK, has found it is possible to use a strand of engineered DNA to trick the cells’ own editing machinery to bypass the CEP290 mutation that causes kidney damage – a technique known as ‘exon-skipping’.

Professor John Sayer, from the Institute of Genetic Medicine, Newcastle University, led the research that is published in the Proceedings of the National Academy of Sciences (PNAS). He said: “This is the first time that gene editing within the kidney has been performed, even in a mouse model, as the design and delivery of the gene editing to the kidney has previously been thought to be too difficult.

“Our research is a major step forwards as we now know how we may be able to offer a therapy that corrects the gene mistake within kidney cells and prevent the development of genetic kidney disease.

“This work paves the way towards personalised genetic therapies in patients with the inherited kidney disease.”

The European study used kidney cells from patients with Joubert syndrome and a mouse model to progress the research. Experts used urine samples to grow kidney cells in the laboratory to see how the cells responded to gene editing. They also performed gene editing to halt kidney disease in a mouse that had Joubert syndrome and rodents suffering from kidney cysts and kidney failure.

Scientists are now looking to work with a drug manufacturing company to bring the exon-skipping technology into patients’ clinics.

Research article: Targeted exon skipping of a CEP290 mutation rescues Joubert syndrome phenotypes in vitro and in a murine model

ASHG 2018 Annual Meeting – research using mouse models

Guest post by Eleonora Maino (ASHG poster: Wednesday 2-3pm, poster number 1107)

Pelizaeus Merzbacher Disorder (PMD) is a rare X-linked pediatric leukodystrophy, that affects approximately 1:100,000 children at birth. The disease is associated with severe motor and cognitive impairment and a limited life expectancy. PMD is caused by mutations in the PLP1 gene, encoding proteolipid protein 1, one of the main components of myelin. In healthy individuals, myelin forms an insulating layer around the nerve fibers allowing fast and efficient signal conduction in the nervous system. Mutations in the PLP1 gene interrupt this process, leading to the hallmark symptoms of PMD. While a variety of mutations can lead to PMD, the vast majority of cases are the result of duplications of the X chromosome region containing the PLP1 gene. Currently, there is no cure and treatment options are limited to symptom management, which fail to have any considerable impact on the quality of life or lifespan of PMD patients. Accordingly, there is an urgent need for the development of effective therapies for PMD patients.

Since the discovery of the novel genome editing technology CRISPR/Cas9, a variety of innovative strategies have been developed to correct genetic defects, including genome rearrangements such as duplication mutations. Here, we are implementing a CRISPR/Cas9-based approach to remove the Plp1 duplication and ameliorate disease manifestation in a PMD mouse model, with the eventual goal of providing a new treatment strategy not only for PMD, but for all genetic disorders caused by genomic duplications. To date, we have characterized a PMD mouse model containing a Plp1 duplication generated in the laboratory of Dr. Grace Hobson. This mouse model is an excellent model to test CRISPR/Cas9 strategies in vivo since it recapitulates both PMD human mutations and disease phenotypes.

To test the genome editing approach in vivo, we administered the CRISPR/Cas9 components via intracerebroventricular injection in the brain of newborn PMD pups, utilizing adeno-associated viral vectors 9 (AAV9) as a delivery vector. Preliminary analyses suggest that, 12 days after the injection, Plp1 expression both at the mRNA and protein level is reduced in PMD mice treated with CRISPR/Cas9 compared to GFP injected control mice. These data suggest that CRISPR/Cas9 promoted the removal of the Plp1 duplication in the treated mice. Next, we will optimize the treatment and assay for a potential attenuation of disease phenotypes.

Once completed, this project will provide the essential in vivo proof of concept to further develop the CRISPR/Cas9 system as a therapeutic option for PMD patients, opening a novel potential avenue for the treatment of genetic disorders caused by genomic duplications.

ASHG 2018 Annual Meeting – research using mouse models

Guest post by Dwi Kemaladewi (ASHG talk: Wednesday, 6:15pm – 6:30pm in Room 6F)

Identification of protective and/or pathogenic genetic modifiers provides important insight into the heterogeneity of disease presentations in individuals affected by neuromuscular disorders (NMDs), despite having well-defined pathogenic variants. Targeting modifier genes to improve disease phenotypes could be especially beneficial in cases where the causative genes are large, structurally complex and the mutations are heterogeneous.

At the American Society of Human Genetics conference, I will be presenting our work on a mutation-independent strategy to upregulate expression of a compensatory disease-modifying gene in Congenital Muscular Dystrophy type 1A (MDC1A) using a CRISPR/dCas9-based transcriptional activation system.

MDC1A is caused by nonfunctional Laminin α2, which compromises muscle fibers stability and axon myelination in peripheral nerves. Transgenic overexpression of Lama1, encoding a structurally similar protein Laminin α1, ameliorates muscle wasting and paralysis in the MDC1A mouse models, demonstrating its role as a protective disease modifier. Yet, upregulation of Lama1 as a postnatal gene therapy is hampered by its large size, which exceeds the current genome packaging capacity of clinically relevant delivery vehicles such as adeno-associated viral vectors (AAVs).

In this study, we sought to upregulate Lama1 using CRISPR/dCas9-based transcriptional activation system, comprised of catalytically inactive S. aureus Cas9 (dCas9) fused to VP64 transactivation domains and sgRNAs targeting the Lama1 promoter. We packaged these CRISPR/dCas9 components into AAV-9, which has high serotype in skeletal muscles and nerves, injected into dy2j/dy2j mouse model of MDC1A and assessed whether systemic upregulation of Lama1 would yield therapeutic benefits.

Indeed, when the intervention was started early in pre-symptomatic dy2j/dy2j mice, Lama1 upregulation prevented muscle fibrosis and hindlimb paralysis.

An important question for future therapeutic approaches for a variety of disorders concerns the therapeutic window and phenotypic reversibility. This is particularly true for muscular dystrophies as it has long been hypothesized that fibrotic changes in skeletal muscle represent an irreversible disease state that would impair any therapeutic intervention at advanced stages of the disease. In this work, we also demonstrate that dystrophic features and disease progression were significantly improved and partially reversed when the treatment was initiated in symptomatic 3-week old dy2j/dy2j mice with already-apparent hind limb paralysis and significant muscle fibrosis.

Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR/dCas9-mediated modulation of a disease modifier gene, which opens up an entirely new and mutation-independent treatment approach for all MDC1A patients. Moreover, this treatment strategy provides evidence that muscle fibrosis can be reversible, thus extending the therapeutic window for this disorder. Our data provide a proof-of-concept strategy that can be applied to a variety of disease modifier genes and a powerful therapeutic approach for various inherited and acquired diseases.

ASHG 2018 Annual Meeting – research using mouse models

Guest post by Jennifer Zieba

Osteogenesis Imperfecta (OI) is the most common genetic bone dysplasia that is phenotypically and genetically complex. It is characterized by bone deformities and fractures caused by low bone mass and impaired bone quality. Roughly 85-90% of cases are dominantly inherited and result from mutations in genes encoding type I collagen (COL1A1 and COL1A2), the major protein of the bone matrix. 10-15% of OI cases are recessively inherited and the majority of those result from mutations in members of the prolyl-3-hydroxylation complex including Cartilage Associated Protein (CRTAP) involved in collagen posttranslational modification.

OI patients are at an increased risk of fracture throughout their lifetimes and anecdotal evidence suggests successful fracture recovery. However, non-union has been reported in 24% of fractures and 52% of osteotomies and many stabilization techniques result in additional surgery due to re-fracture. Re-fractures typically go unreported making the frequency of re-fractures in OI patients unknown. Thus, there is an unmet need to better understand the mechanisms by which OI affects fracture healing. Assessing fracture healing in human patients is a difficult task as neither X-Ray nor CT analysis provide accurate information concerning fracture callus composition, remodeling rate, or the final bone composition. Mouse models for OI have been proven to accurately reflect OI pathogenesis and phenotype. Furthermore, using mice as models for fracture healing allow us to observe in greater detail the lengthy process of fracture healing in a smaller time frame with more informative in vivo techniques such as histochemistry, uCT analysis and biomechanical testing of the fracture tissue at several timepoints. It is our hypothesis that OI fractures undergo suboptimal healing and that this process results in ultimately weaker bone leading to the increased possibility of re-fracture and we are using two murine models to assess this hypothesis.

Using an open tibial fracture model, we show a decrease in callus size in both Col1a1G610c/+ and Crtap–/– OI mouse models post-fracture indicating delayed healing and decreased cartilage content indicating decreased callus cell proliferation. Additionally, fracture calluses in both models exhibited a significant decrease in polar moment of inertia (pMOI) indicating a decrease in resistance to torsional stress supporting a potential functional deficit in newly healed bone. This data provides valuable insight into the effect of the ECM on fracture healing, a poorly understood mechanism. Most importantly, we performed biomechanical testing via three-point-bending of fully healed Crtap–/– tibia to determine the mechanical strength of the fracture site. In wild type bone, the healed fracture site resulted in stronger bone when compared to the unfractured tibia. However, Crtap–/– healed fractured tibia are mechanically weaker than the contralateral unfractured bone. This implies the possibility that OI fractures do not heal properly and may be a prime location for re-fracture. These data may support aggressive prevention of primary fractures as well as a need for therapies during fracture healing to decrease incidence of refracture and deformity in OI patients.

ASHG 2018 Annual Meeting – research using mouse models

Guest post by Robert Erickson (ASHG poster: Wed Oct 17th, 2:00pm – 3:00pm)

Human dominant, gain-of-function mutations in connexin 47 can cause lymphedema but are not always penetrant.  We sought to better understand the causes of variable penetrance and expressivity of one such mutant, R260C, by using CRISPR technology to create this mutant in mice of different genetic backgrounds.  Only mice homozygous for the mutation on the C57BL/6J genetic background showed a lymphatic phenotype.  As with other mouse models for human lymphedema-causing mutations, overt limb swelling was not seen.  Instead, there were increased numbers and size of lymph nodes, with increased lobulation.  In addition, there was abnormal chylous reflux and increased lymphatic branching in the ears.  Mice on genetic backgrounds which were 75% 129/J or A/J did not show abnormalities even when homozygous for the mutation.  These results suggest that modifying genes are important for the e expression of human CX47 gain-of-function mutations.

New research finds that scientists are still studying the same 10% of human genes and ignoring the rest, highlighting the importance of international projects such as the IMPC.

Historical bias is a key reason why biomedical researchers continue to study the same 10 percent of all human genes while ignoring many genes known to play roles in disease, according to a study publishing in PLOS Biology. The researchers involved in this study suggest this bias is bolstered by research funding mechanisms and social forces. The International Mouse Phenotyping Consortium (IMPC) aims to address this issue by creating a platform that characterizes all 20,000 protein-coding mouse genes, which can then give valuable information into homologous genes in the human genome.

Recent studies have reported that researchers actively study only about 2,000 human protein-coding genes, so the researchers set out to find why. They compiled 36 distinct resources describing various aspects of biomedical research and analyzed the large database for answers. The team found that well-meaning policy interventions to promote exploratory or innovative research actually result primarily in additional work on the most established research topics — those genes first characterized in the 1980s and 1990s, before completion of the Human Genome Project. The researchers also discovered that postdoctoral fellows and Ph.D. students who focus on poorly characterized genes have a 50 percent lower chance of becoming an independent researcher.

The researchers applied a systems approach to the data — which included chemical, physical, biological, historical and experimental data — to uncover underlying patterns. In addition to explaining why some genes are not studied, they also explain the extent to which an individual gene is studied. And they can do that for approximately 15,000 genes.

The Human Genome Project — the identification and mapping of all human genes, completed in 2003 — promised to expand the scope of scientific study beyond the small group of genes scientists had studied since the 1980s. But the Northwestern researchers found that 30 percent of all genes have never been the focus of a scientific study and less than 10 percent of genes are the subject of more than 90 percent of published papers.

“The bias to study the exact same human genes is very high,” said Amaral, the Erastus Otis Haven Professor of Chemical and Biological Engineering and a co-author of the study. “The entire system is fighting the very purpose of the agencies and scientific knowledge which is to broaden the set of things we study and understand. We need to make a concerted effort to incentivize the study of other genes important to human health.”

The International Mouse Phenotyping Consortium

This new research highlights the need for projects such as the IMPC, in which the aim is to characterize approximately all 20,000 or so protein coding genes. To achieve this, genes in the mouse genome are switched off, or ‘knocked out’, then standardised physiological tests undertaken across a range of biological systems. This data is then made freely available to the research community. As well as completing large scale comparative studies, the overall aim of the project is to create a platform for this data where researchers/clinicians can search for genes or diseases of interest to help them understand human health and disease. Data for over 6,000 genes is now available on the IMPC website and the project should therefore help address some of the main issues outlined above.

“The IMPC is leveling the playing field by freely providing robust phenotype data for poorly characterized genes. Nothing pleases me more than helping researchers overcome these barriers and discover new research areas none of us have dreamed of. This will be lasting legacy of the IMPC and is why our global partners continue to generate and phenotype mutant mice every day.” said Terry Meehan, a member of IMPC, and the Mouse Informatics Coordinator at the European Bioinformatics Institute.

Lluis Montoliu, researcher at the National Center for Biotechnology (CNB-CSIC) and member of the IMPC said “In the scientific community we all know of ‘famous’ genes and genes that are not so. Fashions also prevail in the scientific community and, indeed, there are many very relevant genes that, due to their difficulty and scarce literature, remain largely unknown. For example, huntingtin, whose gene causes Huntington’s deadly neurodegenerative disease. The IMPC aims to generate and phenotype mutant mice for each and every one of the 20,000 genes, whether or not they are famous, in order to correlate the data obtained with their homologous genes in the human genome.”

PLOS Biology research article: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2006643

More information on the IMPC: https://www.mousephenotypetest.org/what-is-the-impc/

In novel study, mice engineered to lack the same gene run stronger, longer and with less fatigue

Two to three million years ago, the functional loss of a single gene triggered a series of significant changes in what would eventually become the modern human species, altering everything from fertility rates to increasing cancer risk from eating red meat.

In a new paper, published in the Proceedings of the Royal Society B, researchers at University of California San Diego School of Medicine report on studies of mice engineered to lack the same gene, called CMAH, and resulting data that suggest the lost gene may also have contributed to humanity’s well-documented claim to be among the best long-distance runners in the animal kingdom.

At roughly the same time as the CMAH mutation took hold, human ancestors were transitioning from forest dwellers to life primarily upon the arid savannahs of Africa. While they were already walking upright, the bodies and abilities of these early hominids were evolving dramatically, in particular major changes in skeletal biomechanics and physiology that resulted in long, springy legs, big feet, powerful gluteal muscles and an expansive system of sweat glands able to dissipate heat much more effectively than other larger mammals.

Such changes may have helped fuel the emergence of the human ability to run long distances relatively tirelessly, allowing ancestors to hunt in the heat of the day when other carnivores were resting and to pursue prey to their point of exhaustion, a technique called persistence hunting.

“We discovered this first clear genetic difference between humans and our closest living evolutionary relatives, the chimpanzees, more than 20 years ago,” said senior author Ajit Varki, MD, Distinguished Professor of Medicine and Cellular and Molecular Medicine at UC San Diego School of Medicine and co-director of the UC San Diego/Salk Center for Academic Research and Training in Anthropogeny.

Given the approximate timing of the mutation and its documented impact on fertility in a mouse model with the same mutation, Varki and Pascal Gagneux, PhD, professor of anthropology and pathology, began investigating how the genetic difference might have contributed to the origin of Homo, the genus that includes modern Homo sapiens and extinct species like Homo habilis and Homo erectus.

“Since the mice were also more prone to muscle dystrophy, I had a hunch that there was a connection to the increased long distance running and endurance of Homo,” said Varki, “but I had no expertise on the issue and could not convince anyone in my lab to organize this long-shot experiment.”

Ultimately, a graduate student named Jon Okerblom took up the task, building mouse running wheels and borrowing a mouse treadmill. “We evaluated the exercise capacity (of mice lacking the CMAH gene), and noted an increased performance during treadmill testing and after 15 days of voluntary wheel running,” said Okerblom, the study’s first author. The researchers then consulted Ellen Breen, PhD, a research scientist in the division of physiology, part of the Department of Medicine in the UC San Diego School of Medicine, who added observations that the mice displayed greater resistance to fatigue, increased mitochondrial respiration and hind-limb muscle, with more capillaries to increase blood and oxygen supply.

Taken together, Varki said the data suggest CMAH loss contributed to improved skeletal muscle capacity for oxygen utilization. “And if the findings translate to humans, they may have provided early hominids with a selective advantage in their move from trees to becoming permanent hunter-gatherers on the open range.”

When the CMAH gene mutated in the genus Homo two to three million years ago, perhaps in response to evolutionary pressures caused by an ancient pathogen, it altered how subsequent hominids and modern humans used sialic acids — a family of sugar molecules that coat the surfaces of all animal cells, where they serve as vital contact points for interaction with other cells and with the surrounding environment.

The human mutation causes loss of a sialic acid called N-glycolylneuraminic acid (Neu5Gc), and accumulation of its precursor, called N-acetylneuraminic acid or Neu5Ac, which differs by only a single oxygen atom.

This seemingly minor difference affects almost every cell type in the human body — and has proved to be a mixed blessing. Varki and others have linked the loss of the CMAH gene and sialic acids to not just improved long-distance running ability, but also enhanced innate immunity in early hominids. Sialic acids may also be a biomarker for cancer risk.

Conversely, they have also reported that certain sialic acids are associated with increased risk of type 2 diabetes; may contribute to elevated cancer risk associated with red meat consumption; and trigger inflammation.

“They are a double-edged sword,” said Varki. “The consequence of a single lost gene and a small molecular change that appears to have profoundly altered human biology and abilities going back to our origins.”

Research article: Human-like Cmah inactivation in mice increases running endurance and decreases muscle fatigability: implications for human evolution

Obesity and its related ailments like type 2 diabetes and fatty liver disease pose a major global health burden, but researchers report in Nature Communications that blocking an RNA-silencing protein in the livers of mice keeps the animals from getting fat and diabetic conditions.

Takahisa Nakamura, and colleagues at Cincinnati Children’s Hospital Medical Center genetically deleted a protein called Argonaute 2 (Ago2) from the livers of mice. Ago2 controls the silencing of RNA in cells, affecting energy metabolism in the body, according to the study. When Ago2 silences RNA in the liver, it slows metabolism and liver’s ability to process a high-fat diet, the scientists report.

When they deleted Ago2 from the livers of mice, it was not toxic to the animals but it did stabilize energy metabolism. This helped stave off obesity and prevented the mice from developing diabetes and fatty liver disease, which can severely damage the vital organ–which helps rid the body of toxic substances.

“Although this is still basic science, we propose that there may be important translational implications for our findings for chronic metabolic disorders like diabetes, fatty liver diseases, and other obesity associated illnesses,” said Nakamura, senior investigator and a member of the Division of Endocrinology. “This allows us to explore the potential of finding a novel therapeutic approach that alters energy balance in obesity and modulates the associated diseases.”

The scientists caution the research is early stage. Their findings still need additional study and verification in laboratory models and the development of a practical therapeutic to inhibit Ago2 in a clinical setting for patients. But the current paper provides a solid basis for subsequent work.

Ago2 was identified after the researchers conducted a thorough screen and analysis of the activity of genes and their molecular targets in the liver, such as critical proteins. They analyzed wild type and genetically modified mice with high-fat diets by deleting certain proteins that are critical to liver metabolism–such as one called AMPK (AMP-activated protein kinase).

Nakamura said that identifying Ago2’s role in the process “connects the dots” between how proteins are translated in the liver, how energy is produced and consumed and the activity of AMPK in these processes. He pointed out that disruption of these events is already a common feature in obesity and its related illnesses.

Journal article: Hepatic Ago2-mediated RNA silencing controls energy metabolism linked to AMPK activation and obesity-associated pathophysiology