Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma.

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

Quantitative MRI Evaluation of Ferritin Overexpression in Non-Small-Cell Lung Cancer.

Cancer cells frequently present elevated intracellular iron levels, which are thought to facilitate an enhanced proliferative capacity. Targeting iron metabolism within cancer cells presents an avenue to enhance therapeutic responses, necessitating the use of non-invasive models to modulate iron manipulation to predict responses. Moreover, the ubiquitous nature of iron necessitates the development of unique, non-invasive markers of metabolic disruptions to develop more personalized approaches and enhance the clinical utility of these approaches. Ferritin, an iron storage enzyme that is often upregulated as a response to iron accumulation, plays a central role in iron metabolism and has been frequently associated with unfavorable clinical outcomes in cancer. Herein, we demonstrate the successful utility, validation, and functionality of a doxycycline-inducible ferritin heavy chain (FtH) overexpression model in H1299T non-small-cell lung cancer (NSCLC) cells. Treatment with doxycycline increased the protein expression of FtH with a corresponding decrease in labile iron in vitro and in vivo, as determined by calcein-AM staining and EPR, respectively. Moreover, a subsequent increase in TfR expression was observed. Furthermore, T2* MR mapping effectively detected FtH expression in our in vivo model. These results demonstrate that T2* relaxation times can be used to monitor changes in FtH expression in tumors with bidirectional correlations depending on the model system. Overall, this study describes the development of an FtH overexpression NSCLC model and its correlation with T2* mapping for potential use in patients to interrogate iron metabolic alterations and predict clinical outcomes.

Depletion of Labile Iron Induces Replication Stress and Enhances Responses to Chemoradiation in Non-Small-Cell Lung Cancer.

The intracellular redox-active labile iron pool (LIP) is weakly chelated and available for integration into the iron metalloproteins that are involved in diverse cellular processes, including cancer cell-specific metabolic oxidative stress. Abnormal iron metabolism and elevated LIP levels are linked to the poor survival of lung cancer patients, yet the underlying mechanisms remain unclear. Depletion of the LIP in non-small-cell lung cancer cell lines using the doxycycline-inducible overexpression of the ferritin heavy chain (Ft-H) (H1299 and H292), or treatment with deferoxamine (DFO) (H1299 and A549), inhibited cell growth and decreased clonogenic survival. The Ft-H overexpression-induced inhibition of H1299 and H292 cell growth was also accompanied by a significant delay in transit through the S-phase. In addition, both Ft-H overexpression and DFO in H1299 resulted in increased single- and double-strand DNA breaks, supporting the involvement of replication stress in the response to LIP depletion. The Ft-H and DFO treatment also sensitized H1299 to VE-821, an inhibitor of ataxia telangiectasis and Rad2-related (ATR) kinase, highlighting the potential of LIP depletion, combined with DNA damage response modifiers, to alter lung cancer cell responses. In contrast, only DFO treatment effectively reduced the LIP, clonogenic survival, cell growth, and sensitivity to VE-821 in A549 non-small-cell lung cancer cells. Importantly, the Ft-H and DFO sensitized both H1299 and A549 to chemoradiation in vitro, and Ft-H overexpression increased the efficacy of chemoradiation in vivo in H1299. These results support the hypothesis that the depletion of the LIP can induce genomic instability, cell death, and potentiate therapeutic responses to chemoradiation in NSCLC.

Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma.

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

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

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.

Disulfiram causes selective hypoxic cancer cell toxicity and radio-chemo-sensitization via redox cycling of copper.

Therapies for lung cancer patients initially elicit desirable responses, but the presence of hypoxia and drug resistant cells within tumors ultimately lead to treatment failure. Disulfiram (DSF) is an FDA approved, copper chelating agent that can target oxidative metabolic frailties in cancer vs. normal cells and be repurposed as an adjuvant to cancer therapy. Clonogenic survival assays showed that DSF (50-150 nM) combined with physiological levels of Cu (15 µM CuSO4) was selectively toxic to H292 NSCLC cells vs. normal human bronchial epithelial cells (HBEC). Furthermore, cancer cell toxicity was exacerbated at 1% O2, relative to 4 or 21% O2. This selective toxicity of DSF/Cu was associated with differential Cu ionophore capabilities. DSF/Cu treatment caused a >20-fold increase in cellular Cu in NSCLCs, with nearly two-fold higher Cu present in NSCLCs vs. HBECs and in cancer cells at 1% O2vs. 21% O2. DSF toxicity was shown to be dependent on the retention of Cu as well as oxidative stress mechanisms, including the production of superoxide, peroxide, lipid peroxidation, and mitochondrial damage. DSF was also shown to selectively (relative to HBECs) enhance radiation and chemotherapy-induced NSCLC killing and reduce radiation and chemotherapy resistance in hypoxia. Finally, DSF decreased xenograft tumor growth in vivo when combined with radiation and carboplatin. These results support the hypothesis that DSF could be a promising adjuvant to enhance cancer therapy based on its apparent ability to selectively target fundamental differences in cancer cell oxidative metabolism.

Ocular-specific ER stress reduction rescues glaucoma in murine glucocorticoid-induced glaucoma.

Administration of glucocorticoids induces ocular hypertension in some patients. If untreated, these patients can develop a secondary glaucoma that resembles primary open-angle glaucoma (POAG). The underlying pathology of glucocorticoid-induced glaucoma is not fully understood, due in part to lack of an appropriate animal model. Here, we developed a murine model of glucocorticoid-induced glaucoma that exhibits glaucoma features that are observed in patients. Treatment of WT mice with topical ocular 0.1% dexamethasone led to elevation of intraocular pressure (IOP), functional and structural loss of retinal ganglion cells, and axonal degeneration, resembling glucocorticoid-induced glaucoma in human patients. Furthermore, dexamethasone-induced ocular hypertension was associated with chronic ER stress of the trabecular meshwork (TM). Similar to patients, withdrawal of dexamethasone treatment reduced elevated IOP and ER stress in this animal model. Dexamethasone induced the transcriptional factor CHOP, a marker for chronic ER stress, in the anterior segment tissues, and Chop deletion reduced ER stress in these tissues and prevented dexamethasone-induced ocular hypertension. Furthermore, reduction of ER stress in the TM with sodium 4-phenylbutyrate prevented dexamethasone-induced ocular hypertension in WT mice. Our data indicate that ER stress contributes to glucocorticoid-induced ocular hypertension and suggest that reducing ER stress has potential as a therapeutic strategy for treating glucocorticoid-induced glaucoma.

BBS mutations modify phenotypic expression of CEP290-related ciliopathies.

Ciliopathies are a group of heterogeneous disorders associated with ciliary dysfunction. Diseases in this group display considerable phenotypic variation within individual syndromes and overlapping phenotypes among clinically distinct disorders. Particularly, mutations in CEP290 cause phenotypically diverse ciliopathies ranging from isolated retinal degeneration, nephronophthisis and Joubert syndrome, to the neonatal lethal Meckel-Gruber syndrome. However, the underlying mechanisms of the variable expressivity in ciliopathies are not well understood. Here, we show that components of the BBSome, a protein complex composed of seven Bardet-Biedl syndrome (BBS) proteins, physically and genetically interact with CEP290 and modulate the expression of disease phenotypes caused by CEP290 mutations. The BBSome binds to the N-terminal region of CEP290 through BBS4 and co-localizes with CEP290 to the transition zone (TZ) of primary cilia and centriolar satellites in ciliated cells, as well as to the connecting cilium in photoreceptor cells. Although CEP290 still localizes to the TZ and connecting cilium in BBSome-depleted cells, its localization to centriolar satellites is disrupted and CEP290 appears to disperse throughout the cytoplasm in BBSome-depleted cells. Genetic interactions were tested using Cep290(rd16)- and Bbs4-null mutant mouse lines. Additional loss of Bbs4 alleles in Cep290(rd16/rd16) mice results in increased body weight and accelerated photoreceptor degeneration compared with mice without Bbs4 mutations. Furthermore, double-heterozygous mice (Cep290(+/rd16);Bbs4(+/-)) have increased body weight compared with single-heterozygous animals. Our data indicate that genetic interactions between BBSome components and CEP290 could underlie the variable expression and overlapping phenotypes of ciliopathies caused by CEP290 mutations.

ARL13B, PDE6D, and CEP164 form a functional network for INPP5E ciliary targeting.

Mutations affecting ciliary components cause a series of related genetic disorders in humans, including nephronophthisis (NPHP), Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), and Bardet-Biedl syndrome (BBS), which are collectively termed "ciliopathies." Recent protein-protein interaction studies combined with genetic analyses revealed that ciliopathy-related proteins form several functional networks/modules that build and maintain the primary cilium. However, the precise function of many ciliopathy-related proteins and the mechanisms by which these proteins are targeted to primary cilia are still not well understood. Here, we describe a protein-protein interaction network of inositol polyphosphate-5-phosphatase E (INPP5E), a prenylated protein associated with JBTS, and its ciliary targeting mechanisms. INPP5E is targeted to the primary cilium through a motif near the C terminus and prenyl-binding protein phosphodiesterase 6D (PDE6D)-dependent mechanisms. Ciliary targeting of INPP5E is facilitated by another JBTS protein, ADP-ribosylation factor-like 13B (ARL13B), but not by ARL2 or ARL3. ARL13B missense mutations that cause JBTS in humans disrupt the ARL13B-INPP5E interaction. We further demonstrate interactions of INPP5E with several ciliary and centrosomal proteins, including a recently identified ciliopathy protein centrosomal protein 164 (CEP164). These findings indicate that ARL13B, INPP5E, PDE6D, and CEP164 form a distinct functional network that is involved in JBTS and NPHP but independent of the ones previously defined by NPHP and MKS proteins.

A novel protein LZTFL1 regulates ciliary trafficking of the BBSome and Smoothened.

Many signaling proteins including G protein-coupled receptors localize to primary cilia, regulating cellular processes including differentiation, proliferation, organogenesis, and tumorigenesis. Bardet-Biedl Syndrome (BBS) proteins are involved in maintaining ciliary function by mediating protein trafficking to the cilia. However, the mechanisms governing ciliary trafficking by BBS proteins are not well understood. Here, we show that a novel protein, Leucine-zipper transcription factor-like 1 (LZTFL1), interacts with a BBS protein complex known as the BBSome and regulates ciliary trafficking of this complex. We also show that all BBSome subunits and BBS3 (also known as ARL6) are required for BBSome ciliary entry and that reduction of LZTFL1 restores BBSome trafficking to cilia in BBS3 and BBS5 depleted cells. Finally, we found that BBS proteins and LZTFL1 regulate ciliary trafficking of hedgehog signal transducer, Smoothened. Our findings suggest that LZTFL1 is an important regulator of BBSome ciliary trafficking and hedgehog signaling.

Mice defective in Trpm6 show embryonic mortality and neural tube defects.

The syndrome of hypomagnesemia with secondary hypocalcemia is caused by defective TRPM6. This protein is an ion channel that also contains a kinase in its C-terminus. It is usually diagnosed in childhood and, without treatment with supplemental Mg, affected children suffer from mental retardation, seizures and retarded development. We developed a mouse lacking Trpm6 in order to understand in greater detail the function of this protein. In contrast to our expectations, Trpm6(-/-) mice almost never survived to weaning. Many mice died by embryonic day 12.5. Most that survived to term had neural tube defects consisting of both exencephaly and spina bifida occulta, an unusual combination. Feeding dams a high Mg diet marginally improved offspring survival to weaning. The few Trpm6(-/-) mice that survived were fertile but matings between Trpm6(-/-) mice produced no viable pregnancies. Trpm6(+/-) mice had normal electrolytes except for modestly low plasma [Mg]. In addition, some Trpm6(+/-) mice died prematurely. Absence of Trpm6 produces an apparently different phenotype in mice than in humans. The presence of neural tube defects identifies a previously unsuspected role of Trpm6 in effecting neural tube closure. This genetic defect produces one of very few mouse models of spina bifida occulta. These results point to a critical role of Trpm6 in development and suggest an important role in neural tube closure.

Comparative genomics and gene expression analysis identifies BBS9, a new Bardet-Biedl syndrome gene.

Bardet-Biedl syndrome (BBS) is an autosomal recessive, genetically heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Eight BBS genes representing all known mapped loci have been identified. Mutation analysis of the known BBS genes in BBS patients indicate that additional BBS genes exist and/or that unidentified mutations exist in the known genes. To identify new BBS genes, we performed homozygosity mapping of small, consanguineous BBS pedigrees, using moderately dense SNP arrays. A bioinformatics approach combining comparative genomic analysis and gene expression studies of a BBS-knockout mouse model was used to prioritize BBS candidate genes within the newly identified loci for mutation screening. By use of this strategy, parathyroid hormone-responsive gene B1 (B1) was found to be a novel BBS gene (BBS9), supported by the identification of homozygous mutations in BBS patients. The identification of BBS9 illustrates the power of using a combination of comparative genomic analysis, gene expression studies, and homozygosity mapping with SNP arrays in small, consanguineous families for the identification of rare autosomal recessive disorders. We also demonstrate that small, consanguineous families are useful in identifying intragenic deletions. This type of mutation is likely to be underreported because of the difficulty of deletion detection in the heterozygous state by the mutation screening methods that are used in many studies.