Therapeutic efficacy of antisense oligonucleotides in mouse models of CLN3 Batten disease.

CLN3 Batten disease is an autosomal recessive, neurodegenerative, lysosomal storage disease caused by mutations in CLN3, which encodes a lysosomal membrane protein1-3. There are no disease-modifying treatments for this disease that affects up to 1 in 25,000 births, has an onset of symptoms in early childhood and typically is fatal by 20-30 years of life4-7. Most patients with CLN3 Batten have a deletion encompassing exons 7 and 8 (CLN3∆ex7/8), creating a reading frameshift7,8. Here we demonstrate that mice with this deletion can be effectively treated using an antisense oligonucleotide (ASO) that induces exon skipping to restore the open reading frame. A single treatment of neonatal mice with an exon 5-targeted ASO-induced robust exon skipping for more than a year, improved motor coordination, reduced histopathology in Cln3∆ex7/8 mice and increased survival in a new mouse model of the disease. ASOs also induced exon skipping in cell lines derived from patients with CLN3 Batten disease. Our findings demonstrate the utility of ASO-based reading-frame correction as an approach to treat CLN3 Batten disease and broaden the therapeutic landscape for ASOs in the treatment of other diseases using a similar strategy.

An Improved Breast Epithelial Sampling Method for Molecular Profiling and Biomarker Analysis in Women at Risk for Breast Cancer.

BACKGROUND: There is a strong need to define the molecular changes in normal at-risk breast epithelium to identify biomarkers and new targets for breast cancer prevention and to develop a molecular signature for risk assessment. Improved methods of breast epithelial sampling are needed to promote whole-genome molecular profiling, increase ductal epithelial cell yield, and reduce sample cell heterogeneity. METHODS: We developed an improved method of breast ductal sampling with ductal lavage through a 22-gauge catheter and collection of ductal samples with a microaspirator. Women at normal risk or increased risk for breast cancer were studied. Ductal epithelial samples were analyzed for cytopathologic changes, cellular yield, epithelial cell purity, quality and quantity of DNA and RNA, and use in multiple downstream molecular applications. RESULTS: We studied 50 subjects, including 40 subjects at normal risk for breast cancer and 37 subjects with non-nipple aspirate fluid-yielding ducts. This method provided multiple 1.0 mL samples of high ductal epithelial cell content (median ≥8 samples per subject of ≥5,000 cells per sample) with 80%-100% epithelial cell purity. Extraction of a single intact ductal sample (fluid and cells) or the separate frozen cellular component provided DNA and RNA for multiple downstream studies, including quantitative reverse transcription- polymerase chain reaction (PCR) for microRNA, quantitative PCR for the human telomerase reverse transcriptase gene, whole-genome DNA amplification, and array comparative genomic hybridization analysis. CONCLUSION: An improved breast epithelial sampling method has been developed, which should significantly expand the acquisition and biomarker analysis of breast ductal epithelium in women at risk for breast cancer.

Targeting RNA splicing for disease therapy.

Splicing of pre-messenger RNA into mature messenger RNA is an essential step for the expression of most genes in higher eukaryotes. Defects in this process typically affect cellular function and can have pathological consequences. Many human genetic diseases are caused by mutations that cause splicing defects. Furthermore, a number of diseases are associated with splicing defects that are not attributed to overt mutations. Targeting splicing directly to correct disease-associated aberrant splicing is a logical approach to therapy. Splicing is a favorable intervention point for disease therapeutics, because it is an early step in gene expression and does not alter the genome. Significant advances have been made in the development of approaches to manipulate splicing for therapy. Splicing can be manipulated with a number of tools including antisense oligonucleotides, modified small nuclear RNAs (snRNAs), trans-splicing, and small molecule compounds, all of which have been used to increase specific alternatively spliced isoforms or to correct aberrant gene expression resulting from gene mutations that alter splicing. Here we describe clinically relevant splicing defects in disease states, the current tools used to target and alter splicing, specific mutations and diseases that are being targeted using splice-modulating approaches, and emerging therapeutics.

Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness.

Hearing impairment is the most common sensory disorder, with congenital hearing impairment present in approximately 1 in 1,000 newborns. Hereditary deafness is often mediated by the improper development or degeneration of cochlear hair cells. Until now, it was not known whether such congenital failures could be mitigated by therapeutic intervention. Here we show that hearing and vestibular function can be rescued in a mouse model of human hereditary deafness. An antisense oligonucleotide (ASO) was used to correct defective pre-mRNA splicing of transcripts from the USH1C gene with the c.216G>A mutation, which causes human Usher syndrome, the leading genetic cause of combined deafness and blindness. Treatment of neonatal mice with a single systemic dose of ASO partially corrects Ush1c c.216G>A splicing, increases protein expression, improves stereocilia organization in the cochlea, and rescues cochlear hair cells, vestibular function and low-frequency hearing in mice. These effects were sustained for several months, providing evidence that congenital deafness can be effectively overcome by treatment early in development to correct gene expression and demonstrating the therapeutic potential of ASOs in the treatment of deafness.

Sensitive PCR-based quantitation of cell-free circulating microRNAs.

Cell-free microRNAs (miRNAs) that circulate in the blood are promising surrogate biomarkers of disease and physiological processes. The ease of quantifying specific miRNA species using made-to-order approaches based on Taq-polymerase has led to numerous studies that have identified changes in the abundance of circulating cell-free miRNA species that correlate with pathology or other events. The growing interest in developing miRNAs as blood biomarkers necessitates the careful consideration of the unique properties of such body fluids that can make the reproducible and quantitative assessment of RNA abundance challenging. For example, enzymes involved in the amplification and analysis of RNA can be affected by blood components that copurify with miRNA. Thus, if miRNAs are to be effectively utilized as biomarkers, it is important to establish standardized protocols for blood collection and miRNA analysis to ensure accurate quantitation. Here we outline several considerations, including the type of collection tube used in sampling, the influence of added anticoagulants and stabilizers, sample processing, enrichment of vesicular and other miRNA species, RNA extraction approaches and enzyme selection, that affect quantitation of miRNA isolated from plasma and should be considered in order to achieve reproducible, sensitive and accurate quantitation.

MicroRNAs are exported from malignant cells in customized particles.

MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body.

Biogenesis of mammalian microRNAs by a non-canonical processing pathway.

Canonical microRNA biogenesis requires the Microprocessor components, Drosha and DGCR8, to generate precursor-miRNA, and Dicer to form mature miRNA. The Microprocessor is not required for processing of some miRNAs, including mirtrons, in which spliceosome-excised introns are direct Dicer substrates. In this study, we examine the processing of putative human mirtrons and demonstrate that although some are splicing-dependent, as expected, the predicted mirtrons, miR-1225 and miR-1228, are produced in the absence of splicing. Remarkably, knockout cell lines and knockdown experiments demonstrated that biogenesis of these splicing-independent mirtron-like miRNAs, termed 'simtrons', does not require the canonical miRNA biogenesis components, DGCR8, Dicer, Exportin-5 or Argonaute 2. However, simtron biogenesis was reduced by expression of a dominant negative form of Drosha. Simtrons are bound by Drosha and processed in vitro in a Drosha-dependent manner. Both simtrons and mirtrons function in silencing of target transcripts and are found in the RISC complex as demonstrated by their interaction with Argonaute proteins. These findings reveal a non-canonical miRNA biogenesis pathway that can produce functional regulatory RNAs.

Plasma components affect accuracy of circulating cancer-related microRNA quantitation.

Circulating microRNAs (miRNAs) have emerged as candidate biomarkers of various diseases and conditions including malignancy and pregnancy. This approach requires sensitive and accurate quantitation of miRNA concentrations in body fluids. Herein we report that enzyme-based miRNA quantitation, which is currently the mainstream approach for identifying differences in miRNA abundance among samples, is skewed by endogenous serum factors that co-purify with miRNAs and anticoagulant agents used during collection. Of importance, different miRNAs were affected to varying extent among patient samples. By developing measures to overcome these interfering activities, we increased the accuracy, and improved the sensitivity of miRNA detection up to 30-fold. Overall, the present study outlines key factors that prevent accurate miRNA quantitation in body fluids and provides approaches that enable faithful quantitation of miRNA abundance in body fluids.

A feedback loop regulates splicing of the spinal muscular atrophy-modifying gene, SMN2.

Spinal muscular atrophy (SMA) is a neurological disorder characterized by motor neuron degeneration and progressive muscle paralysis. The disease is caused by a reduction in survival of motor neuron (SMN) protein resulting from homozygous deletion of the SMN1 gene. SMN protein is also encoded by SMN2. However, splicing of SMN2 exon 7 is defective, and consequently, the majority of the transcripts produce a truncated, unstable protein. SMN protein itself has a role in splicing. The protein is required for the biogenesis of spliceosomal snRNPs, which are essential components of the splicing reaction. We now show that SMN protein abundance affects the splicing of SMN2 exon 7, revealing a feedback loop inSMN expression. The reduced SMN protein concentration observed in SMA samples and in cells depleted of SMN correlates with a decrease in cellular snRNA levels and a decrease in SMN2 exon 7 splicing. Furthermore, altering the relative abundance or activity of individual snRNPs has distinct effects on exon 7 splicing, demonstrating that core spliceosomal snRNPs influence SMN2 alternative splicing. Our results identify a feedback loop in SMN expression by which low SMN protein levels exacerbate SMN exon 7 skipping, leading to a further reduction in SMN protein. These results imply that a modest increase in SMN protein abundance may cause a disproportionately large increase in SMN expression, a finding that is important for assessing the therapeutic potential of SMA treatments and understanding disease pathogenesis.

Selective release of microRNA species from normal and malignant mammary epithelial cells.

MicroRNAs (miRNAs) in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.

Cell-to-cell fusion as a link between viruses and cancer.

The ability to fuse cells is shared by many viruses, including common human pathogens and several endogenous viruses. Here we will discuss how cell fusion can link viruses to cancer, what types of cancers it can affect, how the existence of this link can be tested and how the hypotheses that we propose might affect the search for human oncogenic viruses. In particular, we will focus on the ability of cell fusion that is caused by viruses to induce chromosomal instability, a common affliction of cancer cells that has been thought to underlie the malignant properties of cancerous tumours.

Control of pre-mRNA splicing by the general splicing factors PUF60 and U2AF(65).

Pre-mRNA splicing is a crucial step in gene expression, and accurate recognition of splice sites is an essential part of this process. Splice sites with weak matches to the consensus sequences are common, though it is not clear how such sites are efficiently utilized. Using an in vitro splicing-complementation approach, we identified PUF60 as a factor that promotes splicing of an intron with a weak 3' splice-site. PUF60 has homology to U2AF(65), a general splicing factor that facilitates 3' splice-site recognition at the early stages of spliceosome assembly. We demonstrate that PUF60 can functionally substitute for U2AF(65)in vitro, but splicing is strongly stimulated by the presence of both proteins. Reduction of either PUF60 or U2AF(65) in cells alters the splicing pattern of endogenous transcripts, consistent with the idea that regulation of PUF60 and U2AF(65) levels can dictate alternative splicing patterns. Our results indicate that recognition of 3' splice sites involves different U2AF-like molecules, and that modulation of these general splicing factors can have profound effects on splicing.

A virus causes cancer by inducing massive chromosomal instability through cell fusion.

Chromosomal instability (CIN) underlies malignant properties of many solid cancers and their ability to escape therapy, and it might itself cause cancer [1, 2]. CIN is sustained by deficiencies in proteins, such as the tumor suppressor p53 [3-5], that police genome integrity, but the primary cause of CIN in sporadic cancers remains uncertain [6, 7]. The primary suspects are mutations that deregulate telomere maintenance, or mitosis, yet such mutations have not been identified in the majority of sporadic cancers [6]. Alternatively, CIN could be caused by a transient event that destabilizes the genome without permanently affecting mechanisms of mitosis or proliferation [5, 8]. Here, we show that an otherwise harmless virus rapidly causes massive chromosomal instability by fusing cells whose cell cycle is deregulated by oncogenes. This synergy between fusion and oncogenes "randomizes" normal diploid human fibroblasts so extensively that each analyzed cell has a unique karyotype, and some produce aggressive, highly aneuploid, heterogeneous, and transplantable epithelial cancers in mice. Because many viruses are fusogenic, this study suggests that viruses, including those that have not been linked to carcinogenesis, can cause chromosomal instability and, consequently, cancer by fusing cells.

A primate virus generates transformed human cells by fusion.

Amodel that explains both the origin and sporadic nature of cancer argues that cancer cells are a chance result of events that cause genomic and epigenetic variability. The prevailing view is that these events are mutations that affect chromosome segregation or stability. However, genomic and epigenetic variability is also triggered by cell fusion, which is often caused by viruses. Yet, cells fused by viruses are considered harmless because they die. We provide evidence that a primate virus uses both viral and exosomal proteins involved in cell fusion to produce transformed proliferating human cells. Although normal cells indeed fail to proliferate after fusion, expression of an oncogene or a mutated tumor suppressor p53 in just one of the fusion partners is sufficient to produce heterogeneous progeny. We also show that this virus can produce viable oncogenically transformed cells by fusing cells that are otherwise destined to die. Therefore, we argue that viruses can contribute to carcinogenesis by fusing cells.