Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

Prior activation state shapes the microglia response to antihuman TREM2 in a mouse model of Alzheimer's disease.

Triggering receptor expressed on myeloid cells 2 (TREM2) sustains microglia response to brain injury stimuli including apoptotic cells, myelin damage, and amyloid ß (Aß). Alzheimer's disease (AD) risk is associated with the TREM2R47H variant, which impairs ligand binding and consequently microglia responses to Aß pathology. Here, we show that TREM2 engagement by the mAb hT2AB as surrogate ligand activates microglia in 5XFAD transgenic mice that accumulate Aß and express either the common TREM2 variant (TREM2CV) or TREM2R47H scRNA-seq of microglia from TREM2CV-5XFAD mice treated once with control hIgG1 exposed four distinct trajectories of microglia activation leading to disease-associated (DAM), interferon-responsive (IFN-R), cycling (Cyc-M), and MHC-II expressing (MHC-II) microglia types. All of these were underrepresented in TREM2R47H-5XFAD mice, suggesting that TREM2 ligand engagement is required for microglia activation trajectories. Moreover, Cyc-M and IFN-R microglia were more abundant in female than male TREM2CV-5XFAD mice, likely due to greater Aß load in female 5XFAD mice. A single systemic injection of hT2AB replenished Cyc-M, IFN-R, and MHC-II pools in TREM2R47H-5XFAD mice. In TREM2CV-5XFAD mice, however, hT2AB brought the representation of male Cyc-M and IFN-R microglia closer to that of females, in which these trajectories had already reached maximum capacity. Moreover, hT2AB induced shifts in gene expression patterns in all microglial pools without affecting representation. Repeated treatment with a murinized hT2AB version over 10 d increased chemokines brain content in TREM2R47H-5XFAD mice, consistent with microglia expansion. Thus, the impact of hT2AB on microglia is shaped by the extent of TREM2 endogenous ligand engagement and basal microglia activation.

miR-212 and miR-132 Are Downregulated in Neurally Derived Plasma Exosomes of Alzheimer's Patients.

It was recently discovered that brain cells release extracellular vesicles (EV) which can pass from brain into blood. These findings raise the possibility that brain-derived EV's present in blood can be used to monitor disease processes occurring in the cerebrum. Since the levels of certain micro-RNAs (miRNAs) have been reported to be altered in Alzheimer's disease (AD) brain, we sought to assess miRNA dysregulation in AD brain tissue and to determine if these changes were reflected in neural EVs isolated from blood of subjects with AD. To this end, we employed high-content miRNA arrays to search for differences in miRNAs in RNA pools from brain tissue of AD (n = 5), high pathological control (HPC) (n = 5), or cognitively intact pathology-free controls (n = 5). Twelve miRNAs were altered by >1.5-fold in AD compared to controls, and six of these were also changed compared to HPCs. Analysis of hits in brain extracts from 11 AD, 7 HPCs and 9 controls revealed a similar fold difference in these six miRNAs, with three showing statistically significant group differences and one with a strong trend toward group differences. Thereafter, we focused on the four miRNAs that showed group differences and measured their content in neurally derived blood EVs isolated from 63 subjects: 16 patients with early stage dementia and a CSF Aß42+ tau profile consistent with AD, 16 individuals with mild cognitive impairment (MCI) and an AD CSF profile, and 31 cognitively intact controls with normal CSF Aß42+ tau levels. ROC analysis indicated that measurement of miR-132-3p in neurally-derived plasma EVs showed good sensitivity and specificity to diagnose AD, but did not effectively separate individuals with AD-MCI from controls. Moreover, when we measured the levels of a related miRNA, miR-212, we found that this miRNA was also decreased in neural EVs from AD patients compared to controls. Our results suggest that measurement of miR-132 and miR-212 in neural EVs should be further investigated as a diagnostic aid for AD and as a potential theragnostic.

Diverse Long RNAs Are Differentially Sorted into Extracellular Vesicles Secreted by Colorectal Cancer Cells.

The regulation and functional roles of secreted coding and long noncoding RNAs (lncRNAs; >200 nt) are largely unknown. We previously showed that mutant KRAS colorectal cancer (CRC) cells release extracellular vesicles (EVs) containing distinct proteomes, microRNAs (miRNAs), and circular RNAs. Here, we comprehensively identify diverse classes of CRC extracellular long RNAs secreted in EVs and demonstrate differential export of specific RNAs. Distinct noncoding RNAs, including antisense transcripts and transcripts derived from pseudogenes, are enriched in EVs compared to cellular profiles. We detected strong enrichment of Rab13 in mutant KRAS EVs and demonstrate functional delivery of Rab13 mRNA to recipient cells. To assay functional transfer of lncRNAs, we implemented a CRISPR/Cas9-based RNA-tracking system to monitor delivery to recipient cells. We show that gRNAs containing export signals from secreted RNAs can be transferred from donor to recipient cells. Our data support the existence of cellular mechanisms to selectively export diverse classes of RNA.

Detection of Aggregation-Competent Tau in Neuron-Derived Extracellular Vesicles.

Progressive cerebral accumulation of tau aggregates is a defining feature of Alzheimer's disease (AD). A popular theory that seeks to explain the apparent spread of neurofibrillary tangle pathology proposes that aggregated tau is passed from neuron to neuron. Such a templated seeding process requires that the transferred tau contains the microtubule binding repeat domains that are necessary for aggregation. While it is not clear how a protein such as tau can move from cell to cell, previous reports have suggested that this may involve extracellular vesicles (EVs). Thus, measurement of tau in EVs may both provide insights on the molecular pathology of AD and facilitate biomarker development. Here, we report the use of sensitive immunoassays specific for full-length (FL) tau and mid-region tau, which we applied to analyze EVs from human induced pluripotent stem cell (iPSC)-derived neuron (iN) conditioned media, cerebrospinal fluid (CSF), and plasma. In each case, most tau was free-floating with a small component inside EVs. The majority of free-floating tau detected by the mid-region assay was not detected by our FL assays, indicating that most free-floating tau is truncated. Inside EVs, the mid-region assay also detected more tau than the FL assay, but the ratio of FL-positive to mid-region-positive tau was higher inside exosomes than in free solution. These studies demonstrate the presence of minute amounts of free-floating and exosome-contained FL tau in human biofluids. Given the potential for FL tau to aggregate, we conclude that further investigation of these pools of extracellular tau and how they change during disease is merited.

Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes.

Recent studies have shown that circular RNAs (circRNAs) are abundant, widely expressed in mammals, and can display cell-type specific expression. However, how production of circRNAs is regulated and their precise biological function remains largely unknown. To study how circRNAs might be regulated during colorectal cancer progression, we used three isogenic colon cancer cell lines that differ only in KRAS mutation status. Cellular RNAs from the parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically-matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only) were analyzed using RNA-Seq. We developed a bioinformatics pipeline to identify and evaluate circRNA candidates from RNA-Seq data. Hundreds of high-quality circRNA candidates were identified in each cell line. Remarkably, circRNAs were significantly down-regulated at a global level in DLD-1 and DKO-1 cells compared to DKs-8 cells, indicating a widespread effect of mutant KRAS on circRNA abundance. This finding was confirmed in two independent colon cancer cell lines HCT116 (KRAS mutant) and HKe3 (KRAS WT). In all three cell lines, circRNAs were also found in secreted extracellular-vesicles, and circRNAs were more abundant in exosomes than cells. Our results suggest that circRNAs may serve as promising cancer biomarkers.

KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes.

Secretion of RNAs in extracellular vesicles is a newly recognized form of intercellular communication. A potential regulatory protein for microRNA (miRNA) secretion is the critical RNA-induced silencing complex (RISC) component Argonaute 2 (Ago2). Here, we use isogenic colon cancer cell lines to show that overactivity of KRAS due to mutation inhibits localization of Ago2 to multivesicular endosomes (MVEs) and decreases Ago2 secretion in exosomes. Mechanistically, inhibition of mitogen-activated protein kinase kinases (MEKs) I and II, but not Akt, reverses the effect of the activating KRAS mutation and leads to increased Ago2-MVE association and increased exosomal secretion of Ago2. Analysis of cells expressing mutant Ago2 constructs revealed that phosphorylation of Ago2 on serine 387 prevents Ago2-MVE interactions and reduces Ago2 secretion into exosomes. Furthermore, regulation of Ago2 exosomal sorting controls the levels of three candidate miRNAs in exosomes. These data identify a key regulatory signaling event that controls Ago2 secretion in exosomes.

KRAS-dependent sorting of miRNA to exosomes.

Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the composition of secreted microRNAs (miRNAs), we compared small RNAs of cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status. We show that exosomal profiles are distinct from cellular profiles, and mutant exosomes cluster separately from wild-type KRAS exosomes. miR-10b was selectively increased in wild-type exosomes, while miR-100 was increased in mutant exosomes. Neutral sphingomyelinase inhibition caused accumulation of miR-100 only in mutant cells, suggesting KRAS-dependent miRNA export. In Transwell co-culture experiments, mutant donor cells conferred miR-100-mediated target repression in wild-type-recipient cells. These findings suggest that extracellular miRNAs can function in target cells and uncover a potential new mode of action for mutant KRAS in CRC.

RNAi-mediated gene silencing in zebrafish triggered by convergent transcription.

RNAi based strategies to induce gene silencing are commonly employed in numerous model organisms but have not been extensively used in zebrafish. We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes. Convergent transcription enabled detection of both sense and antisense transcripts and silencing was suppressed upon Dicer knockdown, indicating processing of double stranded RNA. By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame. Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

miR-153 regulates SNAP-25, synaptic transmission, and neuronal development.

SNAP-25 is a core component of the trimeric SNARE complex mediating vesicle exocytosis during membrane addition for neuronal growth, neuropeptide/growth factor secretion, and neurotransmitter release during synaptic transmission. Here, we report a novel microRNA mechanism of SNAP-25 regulation controlling motor neuron development, neurosecretion, synaptic activity, and movement in zebrafish. Loss of miR-153 causes overexpression of SNAP-25 and consequent hyperactive movement in early zebrafish embryos. Conversely, overexpression of miR-153 causes SNAP-25 down regulation resulting in near complete paralysis, mimicking the effects of treatment with Botulinum neurotoxin. miR-153-dependent changes in synaptic activity at the neuromuscular junction are consistent with the observed movement defects. Underlying the movement defects, perturbation of miR-153 function causes dramatic developmental changes in motor neuron patterning and branching. Together, our results indicate that precise control of SNAP-25 expression by miR-153 is critically important for proper neuronal patterning as well as neurotransmission.

Melanoma vulvo vaginal / Vulvar and vaginal melanoma

Los melanomas de mucosas son raros de observar. El melanoma de vulva representa entre un 8-11 por ciento de los cánceres con esa localización. Presentamos una mujer de 72 años con una lesión pigmentada de bordes irregulares, con proyección a vagina, midiendo en su diámetro mayor 3-4 cm. Se realiza una biopsia que permite el diagnóstico de melanoma maligno. Se realiza tratamiento quirúrgico: vulvectomía total y colpectomía parcial. El informe histopatológico refiere: clítoris: melanoma lentiginoso nivel IV de Clark con espesor de 1 cm. En cara anterior de vagina, el informe es similar. El diagnóstico de melanoma genital suele realizarse tardíamente por la frecuente imposibilidad de observación de la lesión por parte del paciente. El pronóstico del melanoma vulvo vaginal es malo. La sobrevida a los 5 años varía entre el 20 por ciento y 50 por ciento

Tumor fibroepitelial de Pinkus: forma botriomicoide, localización infrecuente / Fibroepithelial tumor of Pinkus: botriomycoid type, infrequent localization

El tumor fibroepitelial (T.FE) descrito por Pinkus en 1953, constituye una variedad poco frecuente de carcinoma basocelular (CA.B.). Presenta diversas formas clínicas, y aunque su histopatología es muy característica, es de difícil diagnóstico clínico. Presentamos un caso con localización (brazo) y forma clínica (botriomicoide) infrecuentes. Desarrollamos una clasificación de las variantes clínicas que puede tener este tumor. Las formas clínicas son: 1-tumoral: verrugosa (tipo queratosis seborreica), polipoide (tipo fibroma blando), botriomicoide, cuerno cutáneo y noduloide. 2-en placa: (simulando un CA.B. superficial o pagetoide y una enfermedad de Bowen. 3-papuloide. 4-micropapuloide o liquenoide)

Granuloma facial: Hallazgos de inmunofluorescencia directa en cuatro casos / Facial Granuloma

El granuloma facial es una entidad clínica e histológica bien definida, cuya etiología permanece desconocida. Los hallazgos por inmunofluorescencia directa de depósitos de inmunoglobulinas y complemento en las lesiones, inician un nuevo camino de investigación que aún no ha sido bien aclarado, ya que se alternan estudios positivos con otros megativos. Estudiamos 4 pacientes, 3 hombres y una mujer, con un rango de edad de 33 a 55 años; el tiempo de evolución de la enfermedad osciló entre 1 y 14 años. Todos los pacientes tenían compromiso facial. Uno presentó lesiones múltiples y otro se acompañaba de lesiones extrafaciales. La inmunofluorescencia directa fue positiva en dos casos, hallando depósitos granulares de IgA en zona de membrana basal. La IgG, IgM, IgE y complemento fueron negativos en los 4 casos. Las distintas características de las inmunoglobulinas más un factor ambiental (rayos ultravioletas) u otro, llevaría a la IgA de la circulación a los tejidos. Posteriormente lo haría la IgG en la respuesta secundaria

Tumor de células granulosas doble localización en lengua / Granular cell tumor: double localization on the tongue

El tumor de células granulosas es una neoformación de curso habitualmente benigno. Entidad clínica infrecuente y de histología definida. Su localización principal es en mucosa bucal, sobre todo en lengua, piel y tejido celular subcutáneo; menos frecuente en distintos órganos. Se presenta como un tumor único, noduloide, asintomático,duro y pequeño. Presentamos un caso de sexo masculino, caracterizado por tener dos tumores aislados, localizados en ambos bordes de la lengua; uno de aspecto noduloide ulcerado, y el otro tipo placa indurada, de dos años y medio de evolución, doloroso al roce y a la palpación. Ambos fueron extirpados quirúrgicamente, sin recaídas

Arsenicismo cronico medicamentoso / Chronic medicinal arsenic

Se presentan cuatro casos de arsenicismo medicamentoso. Tres de ellos fueron tratados con Neosalvarsán antes de la era penicilínica por presentar sífilis y un casos por ingesta de Licor de Fowler durante 15 años por padecer psoriasis. En los 4 pacientes se desarrollaron epiteliomas basocelulares múltiples, predominando en sitios cubiertos, siendo la forma clínica más frecuente la superficial. El caso 3 desarrolló un adenocarcinoma infiltrante de glándula de Bartolino y una enfermedad de Bowen de clítoris, asociado a un Hodgkin. Ninguno de nuestros casos desarrolló melanodermia, queratodermia palmo-plantar ni cáncer visceral. La motivación de este trabajo es de recordar a los médicos la existencia de epiteliomas baso-celulares múltiples por terapéuticas arsenicales

Epitelioma espinocelular de cavidad bucal / Squamous cell carcinoma of the oral cavity

Se describen 4 pacientes con carcinoma espinocelular de boca, mayores de 40 años, con antecedentes de deficiente higiene bucal y/o estado dentario. Tres de ellos fumadores crónicos y en dos se comprobó sinergismo entre tabaco y alcohol. El tamaño del tumor fue mayor de 4 cm en todos los casos, forma clínica úlcerovegetante, extendidos a piso de boca y lengua y compromiso ganglionar vervical. La primera consulta fue tardía, entre uno y dos años. A los ocho y once meses de tratamiento radiante y quimioterapia fallecen dos pacientes, sólo con metástasis ganglionares cervicales y submaxilares, comprobado por autopsia. Dividimos a la población en dos grupos, de bajo y alto riesgo, aconsejando en el primero exámenes periódicos cada año y, en el otro grupo, control semestral para posibilitar un diagnóstico precoz y el tratamiento con mejor pronóstico