Exploring the therapeutic potential of CRISPR/Cas9 for the treatment of PelizaeusMerzbacher Disease

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By IMPC

Published 17th October 2018

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 Bum-Jun Kim (ASHG talk: Wednesday, 4:30pm – 4:45pm in Room 6D)

1p36 deletion syndrome is a genetic disorder caused by deletion of specific regions in p arm of chromosome 1 (1p36). Deletion of chromosome 1p36 is one of the most common terminal deletions in humans and found in children with incidence of 1 in 5,000 newborns. People carrying terminal or interstitial deletions have problems in the heart, the brain, growth, vision, and intelligence. A few genes are suggested as a causative gene for defects seen in 1p36 deletion syndrome due to wide range of deletion length. We have unveiled that RERE is one of the causative genes for symptoms seen in these individuals by using the mouse models (RERE-deficient mice) in which function of RERE is limited. RERE-deficient mice show similar defects developed in 1p36 deletion syndrome. We also described that individuals with single mutations in RERE gene present many of defects seen in 1p36 deletion syndrome.

Congenital heart defect is seen in approximately 70% of individuals with 1p36 deletions, with 23% having ventricular septal defects (VSDs), and 28% having atrial septal defects (ASDs). We have described that RERE-deficient mice also show VSDs with other congenital heart defects. In order to understand how RERE deficiency develops VSDs, we investigated RERE-deficient mice and heart specific RERE-deficient mice in which Rere is ablated from the heart by Tie2 Cre. In wild type mice, the atrioventricular cushions (AVCs) are filled with mesenchymal cells produced by epithelial-to-mesenchymal transition (EMT) and contribute to complete development of ventricular septum. However, RERE-deficient mice show decreased number of mesenchymal cells and abnormal EMT in the AVCs, which leads VSDs. Heart specific RERE deficiency also results in same defects seen in the heart of RERE-deficient mice. A gene called, Gata4 is known to be important for development of the heart and ablation of GATA4 in the heart causes similar phenotypes developed in the heart of RERE-deficient mice. Expression of Gata4 is reduced in the heart of RERE-deficient mice. In addition, we demonstrated that RERE regulates transcriptional activity of Gata4 promoter in vitro systems. The main finding of this study is that RERE-deficiency leads to decrease of GATA4 expression and development of VSDs with abnormal EMT.

Our research is currently published in Disease Models & Mechanisms 2018

ASHG 2018 Annual Meeting – research using mouse models

Guest post by Basel Al-Barghouthi (ASHG talk: Wed Oct 17, 9:15am – 9:30am in Room 6D)

The sequencing of the human genome and other scientific advances have led to approaches that have revolutionized our ability to understand genetic contributions to disease. One example, genome-wide association studies (GWASs), have successfully identified thousands of associations for hundreds of complex diseases. In the case of osteoporosis, a complex disease characterized by reduced bone strength and increased incidence of fracture, human GWASs have focused on dissecting the genetic basis of bone mineral density (BMD). BMD, however, does not explain all of the phenotypic variance in bone strength and there are many other aspects of bone that influence its strength, such as bone size. Unfortunately, bone strength cannot be measured directly in humans. Therefore, we are performing GWAS for over 60 traits related to bone strength in mice, in order to bridge existing gaps in our knowledge.

In this project, we used the Diversity Outbred (DO), an outbred mouse population derived from eight genetically diverse mouse strains to perform a GWAS for size measurements of mouse femurs. The DO is particularly well-suited for high-resolution GWAS. In the DO, we measured bone size and identified a strong association influencing the width of femurs on Chromosome (Chr.) 1. To identify the gene responsible, we queried the Chr. 1 locus for associated genetic variants that potentially impacted protein activity; however, none were found, suggesting the locus was due to a genetic variant influencing gene expression. We then scanned the locus for variants affecting gene expression. Of all genes within the locus, Quiescin Sulfhydryl Oxidase 1 (Qsox1) was the only one whose expression in bone was regulated by the same variants associated with femoral width in a manner consistent with it being causal. The genetic data suggested that decreased Qsox1 levels would lead to wider bones.

Using CRISPR/Cas9 genome-editing, we tested this prediction by generating Qsox1 mutant mice that completely lacked active QSOX1 protein. Consistent with the genetic data, we observed significantly wider femurs in the absence of QSOX1. We also determined that the bones were wider specifically due to increased formation of bone along the left and right sides of the femur.

These data identify Qsox1 as a genetic determinant of bone size and highlight the power of the DO for the genetic analysis of complex traits, which can be particularly useful for traits that are difficult to measure in humans.

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

Damian Smedley will give a talk on Wednesday from 9:30am to 9:45am in Ballroom 20A

The 100,000 Genomes Project is applying whole genome sequencing in a diagnostic setting to rare disease and cancer patients from the National Health Service (NHS) of the UK. Damian Smedley will describe how the clinical phenotype data collected on each rare disease patient is used in automated variant prioritisation software (Exomiser) to identify 68%, 78% or 81% of diagnoses in the top 1, 3 and 5 matches respectively. This software takes advantage of a number of reference disease and model organism genotype to phenotype databases including the International Mouse Phenotyping Consortium (IMPC).

ASHG 2018 Annual Meeting – research using mouse models

Guest post by John Morris (ASHG talk: Wed Oct 17, 9:00am – 9:15am in Room 6D)

Osteoporosis is a common, aging-related disease characterized by decreased bone strength and, consequently, increased fracture risk. Bone mineral density (BMD), a non-invasive measurement, is the most clinically relevant risk factor for diagnosing osteoporosis and is highly heritable (i.e. determined by genetics). To understand the genetic determinants of osteoporosis, we performed a genome-wide association study (GWAS) in 426,824 UK Biobank participants to identify regions of the genome associated with BMD estimated from quantitative heel ultrasound (eBMD). This approach is unbiased in that it systematically tests millions of single nucleotide polymorphisms (SNPs) in the human genome—sites of common, uncommon, or rare genetic variation in the general population—for association with eBMD measurements. BMD-associated SNPs can then be used to identify novel bone genes, but such genes would require further study in human cells or animal models to understand their function. Therefore, we collaborated with the Origins of Bone and Cartilage Disease (OBCD, www.boneandcartilage.com) to examine genes in knockout mice. Such genes, when validated by knockout mouse skeletal phenotyping, represent strong candidates for developing new therapies to prevent and treat osteoporosis.

Our eBMD GWAS identified 518 significant regions of the genome, 301 of which were novel findings. Next, to identify target genes, we performed statistical fine-mapping and integrative bone cell functional genomics data analyses. First, by leveraging SNP association summary statistics and SNP-by-SNP correlations, we can identify a subset of plausibly causal SNPs. Then, by intersecting this list of plausibly causal SNPs with genomic characteristics that indicate function (e.g. coding SNPs, osteoblast open chromatin, osteoblast 3D contacts with gene promoters), we can identify a list of target genes likely to be functional in bone cells. These orthogonal approaches resulted in a list of 515 target genes, identified by plausibly causal and putatively functional SNPs, that we found were strongly enriched for known bone genes and osteoporosis drug targets. We sought to examine the effects of as many of these genes as possible in knockout mice and found the OBCD had skeletal phenotyping data on 126. Importantly, the OBCD receives all knockout mouse lines for skeletal phenotyping at random from the International Mouse Phenotyping Consortium (IMPC), therefore it is not known beforehand if a given knockout mouse has a skeletal phenotype. These 126 genes were found to be enriched for outlier skeletal phenotypes, providing strong evidence that our target genes are disease-relevant, and we focused on one such gene in further detail: disheveled-associated activator of morphogenesis 2 (DAAM2).

Mice with hypomorphic Daam2 alleles were found to have increased cortical porosity and markedly reduced bone strength, even though all other cortical bone parameters, including BMD, were normal. We performed further analyses on DAAM2, such as CRISPR-Cas9 mediated knockouts in human osteoblast cell lines, revealing a decreased ability of this crucial bone-forming cell to mineralize. We concluded that DAAM2 is a novel risk gene for osteoporosis meriting further study and highlighted five other strong candidates for follow-up: CBX1, WAC, DSCC1, RGCC, and YWHAE. In summary, we have generated an atlas of genetic influences on osteoporosis in humans and mice, more fully describing its genetic architecture. Human and mouse genetics identified DAAM2 and other genes previously unknown to function in bone biology. We expect the genes identified here to include new drug targets for the treatment of osteoporosis, where novel therapeutic options are a health priority.

Our work is currently available on bioRxiv at www.biorxiv.org/content/early/2018/07/27/338863

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.

We are excited to be attending the the ASHG 2018 Annual Meeting in San Diego next week. We are looking forward to engaging with researchers and to raise awareness of the IMPC as a resource. If you are attending the conference and want to learn more about the IMPC please visit stand 225 in the non-profit section of the exhibition hall. We will be tweeting relevant research and news during the conference so please follow us on Twitter for updates. We are also adding guest blog posts from attendees presenting research that utilize mouse models, click here to read them.

As well as documentation and information about the latest research , we will also be giving out coasters, pens, notebooks and toy mice. Please don’t hesitate to come to our stand if you are interested in hearing more about the IMPC or if you want to pick up some handouts. Pilar Cacheiro and Damian Smedley will also be presenting research that mentions IMPC resources, both on Wednesday the 17th, and we will have copies of the poster on the stand. More information on the aims and uses of IMPC can be found below.

The International Mouse Phenotyping Consortium (IMPC) is an international effort to identify the function of every gene in the mouse genome. The entire genome of many species has now been published and whole genome sequencing is becoming relatively quick and cheap to complete. Despite these advancements the function of the majority of genes remains unknown.

This is where the IMPC comes in, with the goal of phenotyping all 20,000 or so protein coding mouse genes. To achieve this, genes in the mouse genome are switched off 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.

IMPC data can be used in a variety of ways, such as to investigate basic biology mechanisms that can lead to new therapeutic targets or to narrow down a suspected list of genes in patients. In the last few years the IMPC have made major discoveries in parts of the genome that were hitherto unexplored, with new genes discovered relating to areas such as deafness, diabetes, and rare diseases. Summaries of five recent research articles that highlight the diversity of how IMPC data can be used are listed below. These include inferring mammalian gene function, studies on specific human conditions, sex differences in medical research, and even using IMPC data to help in wildlife conservation.

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By IMPC

Published 10th October 2018

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