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A Minimally Invasive Method for Generating a Syngeneic Orthotopic Mouse Model of Lung Cancer

Non-small cell lung cancer (NSCLC) continues to be the number one cause of cancer-related death for both women and men worldwide. More information needs ...

University of Iowa Health Care

The genetic mapping of disease loci involves the use of patient phenotype and genotype data in the search for genetic markers that segregate, or are associated with, a trait or disorder. Genetically isolated populations offer many advantages for such studies. The high degree of inbreeding and/or founder effects in some small population isolates result in an increased incidence of recessive disorders. Monogenic disorders are less likely to show non-allelic heterogeneity in isolated populations than in more diverse populations. The use of isolated populations also reduces the complexity of polygenic disorders by reducing the number of loci probably involved in the disorder. Finally, a variety of strategies can be used with particular efficacy for the mapping of disease genes in isolated populations.

Use of isolated inbred human populations for identification of disease genes.

Bardet-Biedl syndrome (BBS) has been shown to be a genetically heterogeneous disorder involving genes mapping to at least six known loci. One BBS gene (MKKS) has been identified and the form of the disorder caused by this gene is allelic to McKusick-Kaufman syndrome. MKKS codes for a putative chaperonin, suggesting that other BBS genes may also code for components of chaperone complexes or be substrates of chaperone function.

The molecular genetics of Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a pleiotropic genetic disorder with the cardinal features of obesity, photoreceptor degeneration, polydactyly, hypogenitalism, renal abnormalities, and developmental delay. Other associated clinical findings in BBS patients include diabetes, hypertension, and congenital heart defects. The clinical diagnosis is based on the presence of at least four of the cardinal symptoms. BBS is recognized to be a genetically heterogeneous autosomal recessive disorder mapping to eight known loci. Positional cloning and candidate gene evaluation have resulted in the identification of six BBS genes. Mutation of one of these genes, BBS6, also causes McKusick-Kaufman syndrome. The BBS6 gene is predicted to code for a protein with sequence similarity to the chaperonin family of proteins. The predicted BBS1, BBS2, BBS4, BBS7, and BBS8 gene products do not seem to be molecular chaperones, on the basis of a lack of sequence similarity to the chaperonin family of proteins. The identification of BBS8 suggests a possible role in cilia function for BBS gene products. It remains to be determined whether the multiple BBS proteins are part of a multisubunit complex or do not directly interact with each other but are part of a common pathway. The study of BBS illustrates the value of using isolated inbred populations for the study of human genetic diseases and suggests strategies for facilitating the study of complex diseases and traits.

Use of isolated populations in the study of a human obesity syndrome, the Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder affecting multiple organ systems and resulting in blindness, obesity, cognitive impairment, and congenital defects. Interest in the etiology of this disorder stems, in part, from the fact that patients with BBS develop common clinical problems, including obesity, diabetes and hypertension. Twelve genes independently causing BBS have been identified. The heterogeneity is explained by the existence of two BBS complexes, the BBSome consisting of seven known BBS proteins, and the BBS chaperone complex consisting of three known BBS proteins. The formation of the BBSome requires the function of the BBS chaperone complex. Both mouse and zebrafish data support a role for BBS genes in cilia function, and in intracellular and intraflagellar trafficking. From the work described here, a common primary function of BBS proteins has emerged, specifically the mediation and regulation of microtubule-based intracellular transport.

The blind leading the obese: the molecular pathophysiology of a human obesity syndrome.

Chromosomal microarrays (CMAs) are routinely used in both research and clinical laboratories; yet, little attention has been given to the estimation of genome-wide true and false negatives during the assessment of these assays and how such information could be used to calibrate various algorithmic metrics to improve performance. Low-throughput, locus-specific methods such as fluorescence in situ hybridization (FISH), quantitative PCR (qPCR), or multiplex ligation-dependent probe amplification (MLPA) preclude rigorous calibration of various metrics used by copy number variant (CNV) detection algorithms. To aid this task, we have established a comparative methodology, CNV-ROC, which is capable of performing a high throughput, low cost, analysis of CMAs that takes into consideration genome-wide true and false negatives. CNV-ROC uses a higher resolution microarray to confirm calls from a lower resolution microarray and provides for a true measure of genome-wide performance metrics at the resolution offered by microarray testing. CNV-ROC also provides for a very precise comparison of CNV calls between two microarray platforms without the need to establish an arbitrary degree of overlap. Comparison of CNVs across microarrays is done on a per-probe basis and receiver operator characteristic (ROC) analysis is used to calibrate algorithmic metrics, such as log2 ratio threshold, to enhance CNV calling performance. CNV-ROC addresses a critical and consistently overlooked aspect of analytical assessments of genome-wide techniques like CMAs which is the measurement and use of genome-wide true and false negative data for the calculation of performance metrics and comparison of CNV profiles between different microarray experiments.

CNV-ROC: A cost effective, computer-aided analytical performance evaluator of chromosomal microarrays.

Primary open-angle glaucoma (POAG) is a leading cause of irreversible vision loss worldwide, with elevated intraocular pressure (IOP) a major risk factor. Myocilin () dominant gain-of-function mutations have been reported in ∼4% of POAG cases.  mutations result in protein misfolding, leading to endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that regulates IOP. We use CRISPR-Cas9-mediated genome editing in cultured human TM cells and in a MYOC mouse model of POAG to knock down expression of mutant MYOC, resulting in relief of ER stress. In vivo genome editing results in lower IOP and prevents further glaucomatous damage. Importantly, using an ex vivo human organ culture system, we demonstrate the feasibility of human genome editing in the eye for this important disease.

CRISPR-Cas9-based treatment of myocilin-associated glaucoma.

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease with severe vision impairment leading to blindness. About 10-15% of RP cases are caused by mutations in the  gene, with RPGR mutations accounting for 70% of X-linked RP cases. The mechanism by which RPGR mutations cause photoreceptor cell dysfunction is not well understood. In this study, we show that the two isoforms of RPGR (RPGR and RPGR) interact with endogenous PDE6D, INPP5E, and RPGRIP1L. The RPGR isoform contains two PDE6D binding sites with the C-terminal prenylation site being the predominant PDE6D binding site. The C terminus of RPGR that contains the prenylation site regulates its interaction with PDE6D, INPP5E, and RPGRIP1L. Only the RPGR isoform localizes to cilia in cultured RPE1 cells. Missense variations found in RPGR patients disrupt the interaction between RPGR isoforms and their endogenous interactors INPP5E, PDE6D, and RPGRIP1L. We evaluated a RPGR missense variation (M58K) found in a family with X-linked retinitis pigmentosa (XLRP) and show that this missense variation disrupts the interaction of RPGR isoforms with their endogenous interactors. The M58K variation also disrupts the ciliary localization of the RPGR isoform. Using this assay, we also show that some of the RPGR missense variants reported in the literature might not actually be disease causing. Our data establishes an in vitro assay that can be used to validate the potential pathogenicity of RPGR missense variants.

Disruption of RPGR protein interaction network is the common feature of RPGR missense variations that cause XLRP.

In humans, dysfunctional primary cilia result in Bardet-Biedl syndrome (BBS), which presents with clinical features including intellectual disabilities, obesity, and retinal degeneration, and, in mouse models, the added feature of hydrocephalus. We observed increased Glial Fibrillary Acidic Protein (GFAP) immunoreactivity in BBS mouse brains. Increased GFAP expression is a hallmark of astrocyte reactivity that is associated with microglia activation and neuro-inflammation. To gain a better understanding of reactive astrocytes observed in BBS mice, we used two mouse models of BBS8, a BBSome protein, to characterize the reactive astrocyte phenotype. The finding of reactive astrocytes in young BBS mouse brains led us to hypothesize that loss of BBSome function leads to reactive astrocytes prior to hydrocephalus and obesity. By using two mouse models of BBS8, a congenital BBS8 knockout with hydrocephalus, and a tamoxifen-inducible BBS8 knockout without hydrocephalus, we were able to molecularly phenotype the reactive astrocytes. Molecular phenotype of reactive astrocytes shows differential regulation of inducers of Pan, A1 neurotoxic, and A2 neuroprotective astrocytes that are significantly altered in brains of both congenital and induced knockouts of BBS8, but without microglia activation. We find evidence for neuroinflammation in the brains of congenital knockout mice, but not in induced knockout mice. Protein levels of GFAP, SERPINA3N and post-synaptic density 95 (PSD95) are significantly increased in congenital knockout mice, but remain unchanged in induced knockout mice. Thus, despite the reactive astrocyte phenotype being present in both models, the molecular signature of reactive astrocytes in BBS8 mice models are distinct. Together, these findings suggest that BBS8, and by extension the BBSome, plays a role in neuro-astrocyte functions independent of hydrocephalus, and its dysregulation is associated with astrocyte reactivity without microglia activation. (Total word count 278).

Absence of BBSome function leads to astrocyte reactivity in the brain.

Photoreceptors possess ribbon synapses distinct from the conventional synapses in the brain. Little is known about the function of the BBSome, a complex integral in ciliary and intracellular trafficking, in ribbon synaptic formation. We performed immunohistochemistry using retinas from Bardet-Biedl Syndrome (BBS) mouse models and found that BBS mutant animals have significantly fewer ribbon synapses in the outer plexiform layer and increased ectopic synapses in the outer nuclear layer compared to controls. Many ectopic synapses in BBS mutant retinas are associated with horizontal cell axonal processes that aberrantly intrude into the outer nuclear layer. To determine whether this horizontal cell phenotype is a consequence of retinal degeneration, we examined this phenotype in mice with photoreceptor-specific inactivation of the BBSome induced by Cre recombinase driven by the rhodopsin promoter. At three months of age, despite retinal degeneration, Bbs8; Rho-Cre mice lack the aberrant intrusion of horizontal cell processes. At 6 months, some horizontal cell processes intrude into the outer nuclear layer in Bbs8; Rho-Cre mice, but the phenotype does not recapitulate the phenotypic severity observed in young congenital BBS mutant mice. Therefore, the lack of BBSome function negatively impacts retinal synaptogenesis, and causes horizontal cell defects in a potentially cell-autonomous fashion.

The absence of BBSome function decreases synaptogenesis and causes ectopic synapse formation in the retina.

Aberrant redox signaling underlies the pathophysiology of many chronic metabolic diseases, including type 2 diabetes (T2D). Methodologies aimed at rebalancing systemic redox homeostasis have had limited success. A noninvasive, sustained approach would enable the long-term control of redox signaling for the treatment of T2D. We report that static magnetic and electric fields (sBE) noninvasively modulate the systemic GSH-to-GSSG redox couple to promote a healthier systemic redox environment that is reducing. Strikingly, when applied to mouse models of T2D, sBE rapidly ameliorates insulin resistance and glucose intolerance in as few as 3 days with no observed adverse effects. Scavenging paramagnetic byproducts of oxygen metabolism with SOD2 in hepatic mitochondria fully abolishes these insulin sensitizing effects, demonstrating that mitochondrial superoxide mediates induction of these therapeutic changes. Our findings introduce a remarkable redox-modulating phenomenon that exploits endogenous electromagneto-receptive mechanisms for the noninvasive treatment of T2D, and potentially other redox-related diseases.

Exposure to Static Magnetic and Electric Fields Treats Type 2 Diabetes.

The field of gene therapy has made significant strides over the last several decades toward the treatment of previously untreatable genetic disease. Gene therapy techniques have been aimed at mitigating disease features of recessive and dominant disorders, as well as several cancers and other diseases. While there have been numerous disease targets of gene therapy trials, only four therapies have reached FDA and/or EMA approval for clinical use. Gene correction using CRISPR-Cas9 is an extension of gene therapy that has received considerable attention in recent years and boasts many possible uses beyond classical gene therapy approaches. While there is significant therapeutic potential using gene therapy and gene correction strategies, a number of hurdles remain to be overcome before they become more common in clinical use, particularly with regards to safety and efficacy. As research progresses in this exciting field, it is likely that these therapies will become first-line treatments and will have tremendous positive impacts on the lives of patients with genetic disorders.

Gene therapy and gene correction: targets, progress, and challenges for treating human diseases.

Bardet-Biedl syndrome (BBS) is a rare ciliopathy for which there are no current effective treatments. BBS is a genetically heterogeneous disease, though the M390R mutation in BBS1 is involved in ~25% of all genetic diagnoses of BBS. The principle features of BBS include retinal degeneration, obesity, male infertility, polydactyly, intellectual disability, and renal abnormalities. Patients with mutations in BBS genes often present with night blindness within the first decade of life, which progresses to complete blindness. This is due to progressive loss of photoreceptor cells. Male infertility is caused by a lack of spermatozoa flagella, rendering them immobile. In this study, we have crossed the wild-type human BBS1 gene, driven by the CAG promoter, onto the Bbs1 mouse model to determine if ectopic expression of BBS1 rescues male infertility and retinal degeneration. qRT-PCR indicates that the BBS1 transgene is expressed in multiple tissues throughout the mouse, with the highest expression seen in the testes, and much lower expression in the eye and hypothalamus. Immunohistochemistry of the transgene in the eye showed little if any expression in the photoreceptor outer nuclear layer. When male Bbs1;BBS1 mice are housed with WT females, they are able to sire offspring, indicating that the male infertility phenotype of BBS is rescued by the transgene. Using electroretinography (ERGs) to measure retinal function and optical coherence tomography to measure retinal thickness, we show that the transgene does not confer protection against retinal degeneration in Bbs1;BBS1 mice. The results of this study indicate that the male infertility aspect of BBS is an attractive target for gene therapy.

Ectopic expression of BBS1 rescues male infertility, but not retinal degeneration, in a BBS1 mouse model.

The primary cilium is a highly specialized and evolutionary conserved organelle in eukaryotes that plays a significant role in cell signaling and trafficking. Over the past few decades tremendous progress has been made in understanding the physiology of cilia and the underlying pathomechanisms of various ciliopathies. Syndromic ciliopathies consist of a group of disorders caused by ciliary dysfunction or abnormal ciliogenesis. These disorders have multiorgan involvement in addition to retinal degeneration underscoring the ubiquitous distribution of primary cilia in different cell types. Genotype-phenotype correlation is often challenging due to the allelic heterogeneity and pleiotropy of these disorders. In this review, we discuss the clinical and genetic features of syndromic ciliopathies with a focus on Bardet-Biedl syndrome (BBS) as a representative disorder. We discuss the structure and function of primary cilia and their role in retinal photoreceptors. We describe the progress made thus far in understanding the functional and genetic characterization including expression quantitative trait locus (eQTL) analysis of BBS genes. In the future directions section, we discuss the emerging technologies, such as gene therapy, as well as anticipated challenges and their implications in therapeutic development for ciliopathies.

Article	Year
Use of isolated inbred human populations for identification of disease genes. Trends in genetics : TIG\| PubMed ID: 9820027	1998
The molecular genetics of Bardet-Biedl syndrome. Current opinion in genetics & development\| PubMed ID: 11377969	2001
Use of isolated populations in the study of a human obesity syndrome, the Bardet-Biedl syndrome. Pediatric research\| PubMed ID: 15155861	2004
The blind leading the obese: the molecular pathophysiology of a human obesity syndrome. Transactions of the American Clinical and Climatological Association\| PubMed ID: 20697559	2010
CNV-ROC: A cost effective, computer-aided analytical performance evaluator of chromosomal microarrays. Journal of biomedical informatics\| PubMed ID: 25595567	2015
CRISPR-Cas9-based treatment of myocilin-associated glaucoma. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 28973933	2017
Disruption of RPGR protein interaction network is the common feature of RPGR missense variations that cause XLRP. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30622176	2019
Absence of BBSome function leads to astrocyte reactivity in the brain. Molecular brain\| PubMed ID: 31072410	2019
The absence of BBSome function decreases synaptogenesis and causes ectopic synapse formation in the retina. Scientific reports\| PubMed ID: 32433491	2020
Exposure to Static Magnetic and Electric Fields Treats Type 2 Diabetes. Cell metabolism\| PubMed ID: 33027675	2020
Gene therapy and gene correction: targets, progress, and challenges for treating human diseases. Gene therapy\| PubMed ID: 33037407	2022
Ectopic expression of BBS1 rescues male infertility, but not retinal degeneration, in a BBS1 mouse model. Gene therapy\| PubMed ID: 33664503	2022
Retinal ciliopathies through the lens of Bardet-Biedl Syndrome: Past, present and future. Progress in retinal and eye research\| PubMed ID: 34929400	2022

Val C. Sheffield

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