Binding and neutralizing anti-AAV antibodies: Detection and implications for rAAV-mediated gene therapy.

Assessment of anti-adeno-associated virus (AAV) antibodies in patients prior to systemic gene therapy administration is an important consideration regarding efficacy and safety of the therapy. Approximately 30%-60% of individuals have pre-existing anti-AAV antibodies. Seroprevalence is impacted by multiple factors, including geography, age, capsid serotype, and assay type. Anti-AAV antibody assays typically measure (1) transduction inhibition by detecting the neutralizing capacity of antibodies and non-antibody neutralizing factors, or (2) total anti-capsid binding antibodies, regardless of neutralizing activity. Presently, there is a paucity of head-to-head data and standardized approaches associating assay results with clinical outcomes. In addition, establishing clinically relevant screening titer cutoffs is complex. Thus, meaningful comparisons across assays are nearly impossible. Although complex, establishing screening assays in routine clinical practice to identify patients with antibody levels that may impact favorable treatment outcomes is achievable for both transduction inhibition and total antibody assays. Formal regulatory approval of such assays as companion diagnostic tests will confirm their suitability for specific recombinant AAV gene therapies. This review covers current approaches to measure anti-AAV antibodies in patient plasma or serum, their potential impact on therapeutic safety and efficacy, and investigative strategies to mitigate the effects of pre-existing anti-AAV antibodies in patients.

AAV-mediated expression of galactose-1-phosphate uridyltransferase corrects defects of galactose metabolism in classic galactosemia patient fibroblasts.

Classic galactosemia (CG) is a rare disorder of autosomal recessive inheritance. It is caused predominantly by point mutations as well as deletions in the gene encoding the enzyme galactose-1-phosphate uridyltransferase (GALT). The majority of the more than 350 mutations identified in the GALT gene cause a significant reduction in GALT enzyme activity resulting in the toxic buildup of galactose metabolites that in turn is associated with cellular stress and injury. Consequently, developing a therapeutic strategy that reverses both the oxidative and ER stress in CG cells may be helpful in combating this disease. Recombinant adeno-associated virus (AAV)-mediated gene therapy to restore GALT activity offers the potential to address the unmet medical needs of galactosemia patients. Here, utilizing fibroblasts derived from CG patients we demonstrated that AAV-mediated augmentation of GALT protein and activity resulted in the prevention of ER and oxidative stress. We also demonstrate that these CG patient fibroblasts exhibit reduced CD109 and TGFßRII protein levels and that these effectors of cellular homeostasis could be restored following AAV-mediated expression of GALT. Finally, we show initial in vivo proof-of-concept restoration of galactose metabolism in a GALT knockout mouse model following treatment with AAV-GALT.

Substrate reduction therapy using Genz-667161 reduces levels of pathogenic components in a mouse model of neuronopathic forms of Gaucher disease.

Most lysosomal storage diseases (LSDs) have a significant neurological component, including types 2 and 3 Gaucher disease (neuronal forms of Gaucher disease; nGD). No therapies are currently available for nGD since the recombinant enzymes used in the systemic form of Gaucher disease do not cross the blood-brain barrier (BBB). However, a number of promising approaches are currently being tested, including substrate reduction therapy (SRT), in which partial inhibition of the synthesis of the glycosphingolipids (GSLs) that accumulate in nGD lowers their accumulation. We now induce nGD in mice by injection with conduritol B-epoxide (CBE), an irreversible inhibitor of acid beta-glucosidase (GCase), the enzyme defective in nGD, with or without co-injection with Genz-667161, a prototype for SRT which crosses the BBB. Significant neuropathology, and a reduction in lifespan, was observed upon CBE injection, and this was largely reversed by co-injection with Genz-667161, along with a reduction in glucosylceramide and glucosylsphingosine levels. Analysis of gene expression by RNAseq revealed that Genz-667161 largely reversed the changes in genes and pathways that were differentially expressed upon CBE injection, specifically pathways of GSL metabolism, lipoproteins and other lipid metabolic pathways, lipid droplets, astrocyte activation, neuronal function, and to some extent, neuroinflammation. Together, this demonstrates the efficacy of SRT to reverse the effects of substrate accumulation on pathological components and pathways in nGD brain.

HENMT1 is involved in the maintenance of normal female fertility in the mouse.

PIWI-interacting small RNAs (piRNAs) maintain genome stability in animal germ cells, with a predominant role in silencing transposable elements. Mutations in the piRNA pathway in the mouse uniformly lead to failed spermatogenesis and male sterility. By contrast, mutant females are fertile. In keeping with this paradigm, we previously reported male sterility and female fertility associated with loss of the enzyme HENMT1, which is responsible for stabilising piRNAs through the catalysation of 3'-terminal 2'-O-methylation. However, the Henmt1 mutant females were poor breeders, suggesting they could be subfertile. Therefore, we investigated oogenesis and female fertility in these mice in greater detail. Here, we show that mutant females indeed have a 3- to 4-fold reduction in follicle number and reduced litter sizes. In addition, meiosis-II mutant oocytes display various spindle abnormalities and have a dramatically altered transcriptome which includes a down-regulation of transcripts required for microtubule function. This down-regulation could explain the spindle defects observed with consequent reductions in litter size. We suggest these various effects on oogenesis could be exacerbated by asynapsis, an apparently universal feature of piRNA mutants of both sexes. Our findings reveal that loss of the piRNA pathway in females has significant functional consequences.

biAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

NMN does not protect the ovarian reserve from cancer treatments.

Primordial follicle oocytes are extremely vulnerable to DNA damage caused by exogenous agents, such as those commonly used to treat cancer. Consequently, female cancer patients often have diminished ovarian reserve, which if severe enough, can cause premature ovarian failure and early menopause. Advances in cancer therapies have resulted in significantly improved cancer survival rates; therefore, it is becoming increasingly important to devise strategies to protect the ovarian reserve from cancer treatments, to avoid loss of fertility and endocrine dysfunction. In this study, we aimed to determine whether supplementation with nicotinamide mononucleotide (NMN) could preserve the ovarian reserve following exposure to DNA-damaging cancer treatments. Adult female mice (n = 5-6/group) received saline or NMN (500 mg/kg/day) for 8 days. Mice were left untreated or exposed to γ-irradiation (0.1 Gy) or cyclophosphamide (150 mg/kg) on day 7 and ovaries and serum collected for analysis on day 12. We report that γ-irradiation treatment significantly reduced the number of primordial follicles, but supplementation with NMN did not prevent the observed follicle loss. Similarly, cyclophosphamide treatment significantly reduced primordial follicle numbers, but these losses were not prevented by NMN supplementation. In conclusion, depletion of the ovarian reserve following γ-irradiation or cyclophosphamide was not protected by NMN supplementation under the conditions employed in this study.

Moderate episodic prenatal alcohol does not impact female offspring fertility in rats.

Prenatal alcohol exposure (PAE) has been associated with reproductive dysfunction in offspring. However, studies in females, particularly examining long-term infertility or impacts on ovarian reserve, are lacking. The current study utilised a moderate, episodic exposure model in rats to mimic 'special occasion' drinking, which is reported to be common during pregnancy. Our objective was to examine the consequences of this prenatal alcohol exposure on reproductive parameters in female offspring. Pregnant Sprague-Dawley rats were treated with either an ethanol gavage (1 g EtOH/kg body weight), or an equivalent volume of saline, on embryonic days 13.5 and 14.5 of pregnancy, resulting in a peak blood alcohol concentration of ~0.04%. Neonatal female offspring were examined for molecular markers regulating early follicle numbers in the ovary, and unbiased stereology was used to quantify primordial and early growing follicle numbers. Puberty onset (age at vaginal opening and first estrous) was measured post-weaning, and estrous cycles, reproductive hormones (progesterone and estradiol) and pregnancy success was measured in adults (5-6 months of age). We found no evidence that any of these reproductive parameters were significantly altered by PAE in this model. This animal study provides some reassurance for women who may have consumed a small amount of alcohol during their pregnancy. However, previously published effects on offspring metabolism using this model reinforce avoidance of alcohol during pregnancy.

Substrate Reduction Therapy for Sandhoff Disease through Inhibition of Glucosylceramide Synthase Activity.

Neuronopathic glycosphingolipidoses are a sub-group of lysosomal storage disorders for which there are presently no effective therapies. Here, we evaluated the potential of substrate reduction therapy (SRT) using an inhibitor of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide (GL1) and related glycosphingolipids. The substrates that accumulate in Sandhoff disease (e.g., ganglioside GM2 and its nonacylated derivative, lyso-GM2) are distal to the drug target, GCS. Treatment of Sandhoff mice with a GCS inhibitor that has demonstrated CNS access (Genz-682452) reduced the accumulation of GL1 and GM2, as well as a variety of disease-associated substrates in the liver and brain. Concomitant with these effects was a significant decrease in the expression of CD68 and glycoprotein non-metastatic melanoma B protein (Gpnmb) in the brain, indicating a reduction in microgliosis in the treated mice. Moreover, using in vivo imaging, we showed that the monocytic biomarker translocator protein (TSPO), which was elevated in Sandhoff mice, was normalized following Genz-682452 treatment. These positive effects translated in turn into a delay (∼28 days) in loss of motor function and coordination, as measured by rotarod latency, and a significant increase in longevity (∼17.5%). Together, these results support the development of SRT for the treatment of gangliosidoses, particularly in patients with residual enzyme activity.

DNA damage-induced primordial follicle oocyte apoptosis and loss of fertility require TAp63-mediated induction of Puma and Noxa.

Trp63, a transcription factor related to the tumor suppressor p53, is activated by diverse stimuli and can initiate a range of cellular responses. TAp63 is the predominant Trp53 family member in primordial follicle oocyte nuclei and is essential for their apoptosis triggered by DNA damage in vivo. After γ-irradiation, induction of the proapoptotic BH3-only members Puma and Noxa was observed in primordial follicle oocytes from WT and Trp53(-/-) mice but not in those from TAp63-deficient mice. Primordial follicle oocytes from mice lacking Puma or both Puma and Noxa were protected from γ-irradiation-induced apoptosis and, remarkably, could produce healthy offspring. Hence, PUMA and NOXA are critical for DNA damage-induced, TAp63-mediated primordial follicle oocyte apoptosis. Thus, blockade of PUMA may protect fertility during cancer therapy and prevent premature menopause, improving women's health.

Glucosylceramide synthase inhibition alleviates aberrations in synucleinopathy models.

Mutations in the glucocerebrosidase gene (GBA) confer a heightened risk of developing Parkinson's disease (PD) and other synucleinopathies, resulting in a lower age of onset and exacerbating disease progression. However, the precise mechanisms by which mutations in GBA increase PD risk and accelerate its progression remain unclear. Here, we investigated the merits of glucosylceramide synthase (GCS) inhibition as a potential treatment for synucleinopathies. Two murine models of synucleinopathy (a Gaucher-related synucleinopathy model, GbaD409V/D409V and a A53T-α-synuclein overexpressing model harboring wild-type alleles of GBA, A53T-SNCA mouse model) were exposed to a brain-penetrant GCS inhibitor, GZ667161. Treatment of GbaD409V/D409V mice with the GCS inhibitor reduced levels of glucosylceramide and glucosylsphingosine in the central nervous system (CNS), demonstrating target engagement. Remarkably, treatment with GZ667161 slowed the accumulation of hippocampal aggregates of α-synuclein, ubiquitin, and tau, and improved the associated memory deficits. Similarly, prolonged treatment of A53T-SNCA mice with GZ667161 reduced membrane-associated α-synuclein in the CNS and ameliorated cognitive deficits. The data support the contention that prolonged antagonism of GCS in the CNS can affect α-synuclein processing and improve behavioral outcomes. Hence, inhibition of GCS represents a disease-modifying therapeutic strategy for GBA-related synucleinopathies and conceivably for certain forms of sporadic disease.

Tetrahydrobiopterin deficiency in the pathogenesis of Fabry disease.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent accumulation of glycosphingolipids (mainly globotriaosylceramide, Gb3), leading to multisystem organ dysfunction. Oxidative stress and nitric oxide synthase (NOS) uncoupling are thought to contribute to Fabry cardiovascular diseases. We hypothesized that decreased tetrahydrobiopterin (BH4) plays a role in the pathogenesis of Fabry disease. We found that BH4 was decreased in the heart and kidney but not in the liver and aorta of Fabry mice. BH4 was also decreased in the plasma of female Fabry patients, which was not corrected by enzyme replacement therapy (ERT). Gb3 levels were inversely correlated with BH4 levels in animal tissues and cultured patient cells. To investigate the role of BH4 deficiency in disease phenotypes, 12-month-old Fabry mice were treated with gene transfer-mediated ERT or substrate reduction therapy (SRT) for 6 months. In the Fabry mice receiving SRT but not ERT, BH4 deficiency was restored, concomitant with ameliorated cardiac and renal hypertrophy. Additionally, glutathione levels were decreased in Fabry mouse tissues in a sex-dependent manner. Renal BH4 levels were closely correlated with glutathione levels and inversely correlated with cardiac and kidney weight. In conclusion, this study showed that BH4 deficiency occurs in Fabry disease and may contribute to the pathogenesis of the disease through oxidative stress associated with a reduced antioxidant capacity of cells and NOS uncoupling. This study also suggested dissimilar efficacy of ERT and SRT in correcting pre-existing pathologies in Fabry disease.

The capacity of oocytes for DNA repair.

Female fertility and offspring health are critically dependent on the maintenance of an adequate supply of high-quality oocytes. Like somatic cells, oocytes are subject to a variety of different types of DNA damage arising from endogenous cellular processes and exposure to exogenous genotoxic stressors. While the repair of intentionally induced DNA double strand breaks in gametes during meiotic recombination is well characterised, less is known about the ability of oocytes to repair pathological DNA damage and the relative contribution of DNA repair to oocyte quality is not well defined. This review will discuss emerging data suggesting that oocytes are in fact capable of efficient DNA repair and that DNA repair may be an important mechanism for ensuring female fertility, as well as the transmission of high-quality genetic material to subsequent generations.

Rationally designed AAV2 and AAVrh8R capsids provide improved transduction in the retina and brain.

The successful application of adeno-associated virus (AAV) gene delivery vectors as a therapeutic paradigm will require efficient gene delivery to the appropriate cells in affected organs. In this study, we utilized a rational design approach to introduce modifications to the AAV2 and AAVrh8R capsids and the resulting variants were evaluated for transduction activity in the retina and brain. The modifications disrupted either capsid/receptor binding or altered capsid surface charge. Specifically, we mutated AAV2 amino acids R585A and R588A, which are required for binding to its receptor, heparan sulfate proteoglycans, to generate a variant referred to as AAV2-HBKO. In contrast to parental AAV2, the AAV2-HBKO vector displayed low-transduction activity following intravitreal delivery to the mouse eye; however, following its subretinal delivery, AAV2-HBKO resulted in significantly greater photoreceptor transduction. Intrastriatal delivery of AAV2-HBKO to mice facilitated widespread striatal and cortical expression, in contrast to the restricted transduction pattern of the parental AAV2 vector. Furthermore, we found that altering the surface charge on the AAVrh8R capsid by modifying the number of arginine residues on the capsid surface had a profound impact on subretinal transduction. The data further validate the potential of capsid engineering to improve AAV gene therapy vectors for clinical applications.

Glucocerebrosidase modulates cognitive and motor activities in murine models of Parkinson's disease.

Mutations in GBA1, the gene encoding glucocerebrosidase, are associated with an enhanced risk of developing synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies. A higher prevalence and increased severity of motor and non-motor symptoms is observed in PD patients harboring mutant GBA1 alleles, suggesting a link between the gene or gene product and disease development. Interestingly, PD patients without mutations in GBA1 also exhibit lower levels of glucocerebrosidase activity in the central nervous system (CNS), implicating this lysosomal enzyme in disease pathogenesis. Here, we investigated whether modulation of glucocerebrosidase activity in murine models of synucleinopathy (expressing wild type Gba1) affected α-synuclein accumulation and behavioral phenotypes. Partial inhibition of glucocerebrosidase activity in PrP-A53T-SNCA mice using the covalent inhibitor conduritol-B-epoxide induced a profound increase in soluble α-synuclein in the CNS and exacerbated cognitive and motor deficits. Conversely, augmenting glucocerebrosidase activity in the Thy1-SNCA mouse model of PD delayed the progression of synucleinopathy. Adeno-associated virus-mediated expression of glucocerebrosidase in the Thy1-SNCA mouse striatum led to decrease in the levels of the proteinase K-resistant fraction of α-synuclein, amelioration of behavioral aberrations and protection from loss of striatal dopaminergic markers. These data indicate that increasing glucocerebrosidase activity can influence α-synuclein homeostasis, thereby reducing the progression of synucleinopathies. This study provides robust in vivo evidence that augmentation of CNS glucocerebrosidase activity is a potential therapeutic strategy for PD, regardless of the mutation status of GBA1.

Intravitreous injection of AAV2-sFLT01 in patients with advanced neovascular age-related macular degeneration: a phase 1, open-label trial.

BACKGROUND: Long-term intraocular injections of vascular endothelial growth factor (VEGF)-neutralising proteins can preserve central vision in many patients with neovascular age-related macular degeneration. We tested the safety and tolerability of a single intravitreous injection of an AAV2 vector expressing the VEGF-neutralising protein sFLT01 in patients with advanced neovascular age-related macular degeneration. METHODS: This was a phase 1, open-label, dose-escalating study done at four outpatient retina clinics in the USA. Patients were assigned to each cohort in order of enrolment, with the first three patients being assigned to and completing the first cohort before filling positions in the following treatment groups. Patients aged 50 years or older with neovascular age-related macular degeneration and a baseline best-corrected visual acuity score of 20/100 or less in the study eye were enrolled in four dose-ranging cohorts (cohort 1, 2 × 108 vector genomes (vg); cohort 2, 2 × 109 vg; cohort 3, 6 × 109 vg; and cohort 4, 2 × 1010 vg, n=3 per cohort) and one maximum tolerated dose cohort (cohort 5, 2 × 1010 vg, n=7) and followed up for 52 weeks. The primary objective of the study was to assess the safety and tolerability of a single intravitreous injection of AAV2-sFLT01, through the measurement of eye-related adverse events. This trial is registered with ClinicalTrials.gov, number NCT01024998. FINDINGS: 19 patients with advanced neovascular age-related macular degeneration were enrolled in the study between May 18, 2010, and July 14, 2014. All patients completed the 52-week trial period. Two patients in cohort 4 (2 × 1010 vg) experienced adverse events that were possibly study-drug related: pyrexia and intraocular inflammation that resolved with a topical steroid. Five of ten patients who received 2 × 1010 vg had aqueous humour concentrations of sFLT01 that peaked at 32·7-112·0 ng/mL (mean 73·7 ng/mL, SD 30·5) by week 26 with a slight decrease to a mean of 53·2 ng/mL at week 52 (SD 17·1). At baseline, four of these five patients were negative for anti-AAV2 serum antibodies and the fifth had a very low titre (1:100) of anti-AAV2 antibodies, whereas four of the five non-expressers of sFLT01 had titres of 1:400 or greater. In 11 of 19 patients with intraretinal or subretinal fluid at baseline judged to be reversible, six showed substantial fluid reduction and improvement in vision, whereas five showed no fluid reduction. One patient in cohort 5 showed a large decrease in vision between weeks 26 and 52 that was not thought to be vector-related. INTERPRETATION: Intravitreous injection of AAV2-sFLT01 seemed to be safe and well tolerated at all doses. Additional studies are needed to identify sources of variability in expression and anti-permeability activity, including the potential effect of baseline anti-AAV2 serum antibodies. FUNDING: Sanofi Genzyme, Framingham, MA, USA.

Priapism in a Fabry disease mouse model is associated with upregulated penile nNOS and eNOS expression.

Fabry disease is a glycosphingolipidosis caused by deficient activity of α-galactosidase A; it is one of a few diseases that are associated with priapism, an abnormal prolonged erection of the penis. The goal of this study was to investigate the pathogenesis of Fabry disease-associated priapism in a mouse model of the disease. We found that Fabry mice develop late-onset priapism. Neuronal nitric oxide synthase (nNOS), which was predominantly present as the 120-kDa N-terminus-truncated form, was significantly upregulated in the penis of 18-month-old Fabry mice compared to wild type controls (~fivefold). Endothelial NOS (eNOS) was also upregulated (~twofold). NO level in penile tissues of Fabry mice was significantly higher than wild type controls at 18 months. Gene transfer-mediated enzyme replacement therapy reversed abnormal nNOS expression in the Fabry mouse penis. The penile nNOS level was restored by antiandrogen treatment, suggesting that hyperactive androgen receptor signaling in Fabry mice may contribute to nNOS upregulation. However, the phosphodiesterase-5A expression level and the adenosine content in the penis, which are known to play roles in the development of priapism in other etiologies, were unchanged in Fabry mice. In conclusion, these data suggested that increased nNOS (and probably eNOS) content and the consequential elevated NO production and high arterial blood flow in the penis may be the underlying mechanism of priapism in Fabry mice. Furthermore, in combination with previous findings, this study suggested that regulation of NOS expression is susceptible to α-galactosidase A deficiency, and this may represent a general pathogenic mechanism of Fabry vasculopathy.

Targeting nuclear RNA for in vivo correction of myotonic dystrophy.

Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat and are retained in the nucleus. The mutant RNA exerts a toxic gain-of-function effect, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target messenger RNAs. Here we show that nuclear-retained transcripts containing expanded CUG (CUG(exp)) repeats are unusually sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUG(exp) RNA in skeletal muscle, correcting the physiological, histopathologic and transcriptomic features of the disease. The effect was sustained for up to 1 year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat1, a long non-coding RNA (lncRNA) that is retained in the nucleus. These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence.

CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10.

As the most common subtype of Leber congenital amaurosis (LCA), LCA10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. The most frequent mutation found in patients with LCA10 is a deep intronic mutation in CEP290 that generates a cryptic splice donor site. The large size of the CEP290 gene prevents its use in adeno-associated virus (AAV)-mediated gene augmentation therapy. Here, we show that targeted genomic deletion using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system represents a promising therapeutic approach for the treatment of patients with LCA10 bearing the CEP290 splice mutation. We generated a cellular model of LCA10 by introducing the CEP290 splice mutation into 293FT cells and we showed that guide RNA pairs coupled with SpCas9 were highly efficient at removing the intronic splice mutation and restoring the expression of wild-type CEP290. In addition, we demonstrated that a dual AAV system could effectively delete an intronic fragment of the Cep290 gene in the mouse retina. To minimize the immune response to prolonged expression of SpCas9, we developed a self-limiting CRISPR/Cas9 system that minimizes the duration of SpCas9 expression. These results support further studies to determine the therapeutic potential of CRISPR/Cas9-based strategies for the treatment of patients with LCA10.

Glycosphingolipids are modulators of disease pathogenesis in amyotrophic lateral sclerosis.

Recent genetic evidence suggests that aberrant glycosphingolipid metabolism plays an important role in several neuromuscular diseases including hereditary spastic paraplegia, hereditary sensory neuropathy type 1, and non-5q spinal muscular atrophy. Here, we investigated whether altered glycosphingolipid metabolism is a modulator of disease course in amyotrophic lateral sclerosis (ALS). Levels of ceramide, glucosylceramide, galactocerebroside, lactosylceramide, globotriaosylceramide, and the gangliosides GM3 and GM1 were significantly elevated in spinal cords of ALS patients. Moreover, enzyme activities (glucocerebrosidase-1, glucocerebrosidase-2, hexosaminidase, galactosylceramidase, α-galactosidase, and ß-galactosidase) mediating glycosphingolipid hydrolysis were also elevated up to threefold. Increased ceramide, glucosylceramide, GM3, and hexosaminidase activity were also found in SOD1(G93A) mice, a familial model of ALS. Inhibition of glucosylceramide synthesis accelerated disease course in SOD1(G93A) mice, whereas infusion of exogenous GM3 significantly slowed the onset of paralysis and increased survival. Our results suggest that glycosphingolipids are likely important participants in pathogenesis of ALS and merit further analysis as potential drug targets.

Dual roles for Id4 in the regulation of estrogen signaling in the mammary gland and ovary.

The HLH transcriptional regulator Id4 exerts important roles in different organs, including the neural compartment, where Id4 loss usually results in early lethality. To explore the role of this basally restricted transcription factor in the mammary gland, we generated a cre-inducible mouse model. MMTV- or K14-cre-mediated deletion of Id4 led to a delay in ductal morphogenesis, consistent with previous findings using a germ-line knockout mouse model. A striking increase in the expression of ERα (Esr1), PR and FoxA1 was observed in both the basal and luminal cellular subsets of Id4-deficient mammary glands. Together with chromatin immunoprecipitation of Id4 on the Esr1 and Foxa1 promoter regions, these data imply that Id4 is a negative regulator of the ERα signaling axis. Unexpectedly, examination of the ovaries of targeted mice revealed significantly increased numbers of secondary and antral follicles, and reduced Id4 expression in the granulosa cells. Moreover, expression of the cascade of enzymes that are crucial for estrogen biosynthesis in the ovary was decreased in Id4-deficient females and uterine weights were considerably lower, indicating impaired estrogen production. Thus, compromised ovarian function and decreased circulating estrogen likely contribute to the mammary ductal defects evident in Id4-deficient mice. Collectively, these data identify Id4 as a novel regulator of estrogen signaling, where Id4 restrains ERα expression in the basal and luminal cellular compartments of the mammary gland and regulates estrogen biosynthesis in the ovary.