Quantifying the activity profile of ASO and siRNA conjugates in glioblastoma xenograft tumors in vivo.

Glioblastoma multiforme is a universally lethal brain tumor that largely resists current surgical and drug interventions. Despite important advancements in understanding GBM biology, the invasiveness and heterogeneity of these tumors has made it challenging to develop effective therapies. Therapeutic oligonucleotides-antisense oligonucleotides and small-interfering RNAs-are chemically modified nucleic acids that can silence gene expression in the brain. However, activity of these oligonucleotides in brain tumors remains inadequately characterized. In this study, we developed a quantitative method to differentiate oligonucleotide-induced gene silencing in orthotopic GBM xenografts from gene silencing in normal brain tissue, and used this method to test the differential silencing activity of a chemically diverse panel of oligonucleotides. We show that oligonucleotides chemically optimized for pharmacological activity in normal brain tissue do not show consistent activity in GBM xenografts. We then survey multiple advanced oligonucleotide chemistries for their activity in GBM xenografts. Attaching lipid conjugates to oligonucleotides improves silencing in GBM cells across several different lipid classes. Highly hydrophobic lipid conjugates cholesterol and docosanoic acid enhance silencing but at the cost of higher neurotoxicity. Moderately hydrophobic, unsaturated fatty acid and amphiphilic lipid conjugates still improve activity without compromising safety. These oligonucleotide conjugates show promise for treating glioblastoma.

A combinatorial approach for achieving CNS-selective RNAi.

RNA interference (RNAi) is an endogenous process that can be harnessed using chemically modified small interfering RNAs (siRNAs) to potently modulate gene expression in many tissues. The route of administration and chemical architecture are the primary drivers of oligonucleotide tissue distribution, including siRNAs. Independently of the nature and type, oligonucleotides are eliminated from the body through clearance tissues, where their unintended accumulation may result in undesired gene modulation. Divalent siRNAs (di-siRNAs) administered into the CSF induce robust gene silencing throughout the central nervous system (CNS). Upon clearance from the CSF, they are mainly filtered by the kidneys and liver, with the most functionally significant accumulation occurring in the liver. siRNA- and miRNA-induced silencing can be blocked through substrate inhibition using single-stranded, stabilized oligonucleotides called antagomirs or anti-siRNAs. Using APOE as a model target, we show that undesired di-siRNA-induced silencing in the liver can be mitigated through administration of liver targeting GalNAc-conjugated anti-siRNAs, without impacting CNS activity. Blocking unwanted hepatic APOE silencing achieves fully CNS-selective silencing, essential for potential clinical translation. While we focus on CNS/liver selectivity, coadministration of differentially targeting siRNA and anti-siRNAs can be adapted as a strategy to achieve tissue selectivity in different organ combinations.

Silencing Apoe with divalent-siRNAs improves amyloid burden and activates immune response pathways in Alzheimer's disease.

INTRODUCTION: The most significant genetic risk factor for late-onset Alzheimer's disease (AD) is APOE4, with evidence for gain- and loss-of-function mechanisms. A clinical need remains for therapeutically relevant tools that potently modulate APOE expression. METHODS: We optimized small interfering RNAs (di-siRNA, GalNAc) to potently silence brain or liver Apoe and evaluated the impact of each pool of Apoe on pathology. RESULTS: In adult 5xFAD mice, siRNAs targeting CNS Apoe efficiently silenced Apoe expression and reduced amyloid burden without affecting systemic cholesterol, confirming that potent silencing of brain Apoe is sufficient to slow disease progression. Mechanistically, silencing Apoe reduced APOE-rich amyloid cores and activated immune system responses. DISCUSSION: These results establish siRNA-based modulation of Apoe as a viable therapeutic approach, highlight immune activation as a key pathway affected by Apoe modulation, and provide the technology to further evaluate the impact of APOE silencing on neurodegeneration.

Mutant huntingtin messenger RNA forms neuronal nuclear clusters in rodent and human brains.

Mutant messenger RNA (mRNA) and protein contribute to the clinical manifestation of many repeat-associated neurological disorders, with the presence of nuclear RNA clusters being a common pathological feature. Yet, investigations into Huntington's disease-caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene-have primarily focused on toxic protein gain-of-function as the primary disease-causing feature. To date, mutant HTT mRNA has not been identified as an in vivo hallmark of Huntington's disease. Here, we report that, in two Huntington's disease mouse models (YAC128 and BACHD-97Q-ΔN17), mutant HTT mRNA is retained in the nucleus. Widespread formation of large mRNA clusters (∼0.6-5 µm3) occurred in 50-75% of striatal and cortical neurons. Cluster formation was independent of age and driven by expanded repeats. Clusters associate with chromosomal transcriptional sites and quantitatively co-localize with the aberrantly processed N-terminal exon 1-intron 1 mRNA isoform, HTT1a. HTT1a mRNA clusters are observed in a subset of neurons from human Huntington's disease post-mortem brain and are likely caused by somatic expansion of repeats. In YAC128 mice, clusters, but not individual HTT mRNA, are resistant to antisense oligonucleotide treatment. Our findings identify mutant HTT/HTT1a mRNA clustering as an early, robust molecular signature of Huntington's disease, providing in vivo evidence that Huntington's disease is a repeat expansion disease with mRNA involvement.

PK-modifying anchors significantly alter clearance kinetics, tissue distribution, and efficacy of therapeutics siRNAs.

Effective systemic delivery of small interfering RNAs (siRNAs) to tissues other than liver remains a challenge. siRNAs are small (∼15 kDa) and therefore rapidly cleared by the kidneys, resulting in limited blood residence times and tissue exposure. Current strategies to improve the unfavorable pharmacokinetic (PK) properties of siRNAs rely on enhancing binding to serum proteins through extensive phosphorothioate modifications or by conjugation of targeting ligands. Here, we describe an alternative strategy for enhancing blood and tissue PK based on dynamic modulation of the overall size of the siRNA. We engineered a high-affinity universal oligonucleotide anchor conjugated to a high-molecular-weight moiety, which binds to the 3' end of the guide strand of an asymmetric siRNA. Data showed a strong correlation between the size of the PK-modifying anchor and clearance kinetics. Large 40-kDa PK-modifying anchors reduced renal clearance by ∼23-fold and improved tissue exposure area under the curve (AUC) by ∼26-fold, resulting in increased extrahepatic tissue retention (∼3- to 5-fold). Furthermore, PK-modifying oligonucleotide anchors allowed for straightforward and versatile modulation of blood residence times and biodistribution of a panel of chemically distinct ligands. The effects were more pronounced for conjugates with low lipophilicity (e.g., N-Acetylgalactosamine [GalNAc]), where significant improvement in uptake by hepatocytes and dose-dependent silencing in the liver was observed.

Delivering siRNA Compounds During HOPE to Modulate Organ Function: A Proof-of-concept Study in a Rat Liver Transplant Model.

BACKGROUND: Apoptosis contributes to the severity of ischemia-reperfusion injury (IRI), limiting the use of extended criteria donors in liver transplantation (LT). Machine perfusion has been proposed as a platform to administer specific therapies to improve graft function. Alternatively, the inhibition of genes associated with apoptosis during machine perfusion could alleviate IRI post-LT. The aim of the study was to investigate whether inhibition of an apoptosis-associated gene (FAS) using a small interfering RNA (siRNA) approach could alleviate IRI in a rat LT model. METHODS: In 2 different experimental protocols, FASsiRNA (500 µg) was administered to rat donors 2 h before organ procurement, followed by 22 h of static cold storage, (SCS) or was added to the perfusate during 1 h of ex situ hypothermic oxygenated perfusion (HOPE) to livers previously preserved for 4 h in SCS. RESULTS: Transaminase levels were significantly lower in the SCS-FASsiRNA group at 24 h post-LT. Proinflammatory cytokines (interleukin-2, C-X-C motif chemokine 10, tumor necrosis factor alpha, and interferon gamma) were significantly decreased in the SCS-FASsiRNA group, whereas the interleukin-10 anti-inflammatory cytokine was significantly increased in the HOPE-FASsiRNA group. Liver absorption of FASsiRNA after HOPE session was demonstrated by confocal microscopy; however, no statistically significant differences on the apoptotic index, necrosis levels, and FAS protein transcription between treated and untreated groups were observed. CONCLUSIONS: FAS inhibition through siRNA therapy decreases the severity of IRI after LT in a SCS protocol; however the association of siRNA therapy with a HOPE perfusion model is very challenging. Future studies using better designed siRNA compounds and appropriate doses are required to prove the siRNA therapy effectiveness during liver HOPE liver perfusion.

Comparative route of administration studies using therapeutic siRNAs show widespread gene modulation in Dorset sheep.

siRNAs comprise a class of drugs that can be programmed to silence any target gene. Chemical engineering efforts resulted in development of divalent siRNAs (di-siRNAs), which support robust and long-term efficacy in rodent and nonhuman primate brains upon direct cerebrospinal fluid (CSF) administration. Oligonucleotide distribution in the CNS is nonuniform, limiting clinical applications. The contribution of CSF infusion placement and dosing regimen on relative accumulation, specifically in the context of large animals, is not well characterized. To our knowledge, we report the first systemic, comparative study investigating the effects of 3 routes of administration - intrastriatal (i.s.), i.c.v., and intrathecal catheter to the cisterna magna (ITC) - and 2 dosing regimens - single and repetitive via an implanted reservoir device - on di-siRNA distribution and accumulation in the CNS of Dorset sheep. CSF injections (i.c.v. and ITC) resulted in similar distribution and accumulation across brain regions. Repeated dosing increased homogeneity, with greater relative deep brain accumulation. Conversely, i.s. administration supported region-specific delivery. These results suggest that dosing regimen, not CSF infusion placement, may equalize siRNA accumulation and efficacy throughout the brain. These findings inform the planning and execution of preclinical and clinical studies using siRNA therapeutics in the CNS.

Cell Type Impacts Accessibility of mRNA to Silencing by RNA Interference.

RNA interference (RNAi) is a potent mechanism that silences mRNA and protein expression in all cells and tissue types. RNAi is known to exert many of its functional effects in the cytoplasm, and thus, the cellular localization of target mRNA may impact observed potency. Here, we demonstrate that cell identity has a profound impact on accessibility of apolipoprotein E (ApoE) mRNA to RNAi. We show that, whereas both neuronal and glial cell lines express detectable ApoE mRNA, in neuronal cells, ApoE mRNA is not targetable by RNAi. Screening of a panel of thirty-five chemically modified small interfering RNAs (siRNAs) did not produce a single hit in a neuronal cell line, whereas up to fifteen compounds showed strong efficacy in glial cells. Further investigation of the cellular localization of ApoE mRNA demonstrates that ApoE mRNA is partially spliced and preferentially localized to the nucleus (∼80%) in neuronal cells, whereas more than 90% of ApoE mRNA is cytoplasmic in glial cells. Such an inconsistency in intracellular localization and splicing might provide an explanation for functional differences in RNAi compounds. Thus, cellular origin might have an impact on accessibility of mRNA to RNAi and should be taken into account during the screening process.

A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system.

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington's disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin.

Huntington's disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ∼50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA subcellular localization might play a role in important neuronal regulatory mechanisms.

Immediate Implant Reconstruction Is Associated With a Reduced Risk of Lymphedema Compared to Mastectomy Alone: A Prospective Cohort Study.

OBJECTIVE: We sought to determine the risk of lymphedema associated with immediate breast reconstruction compared to mastectomy alone. BACKGROUND: Immediate breast reconstruction is increasingly performed at the time of mastectomy. Few studies have examined whether breast reconstruction impacts development of lymphedema. METHODS: A total of 616 patients with breast cancer who underwent 891 mastectomies between 2005 and 2013 were prospectively screened for lymphedema at our institution, with 22.2 months' median follow-up. Mastectomies were categorized as immediate implant, immediate autologous, or no reconstruction. Arm measurements were performed preoperatively and during postoperative follow-up using a Perometer. Lymphedema was defined as 10% or more arm volume increase compared to preoperative. Kaplan-Meier and Cox regression analyses were performed to determine lymphedema rates and risk factors. RESULTS: Of 891 mastectomies, 65% (580/891) had immediate implant, 11% (101/891) immediate autologous, and 24% (210/891) no reconstruction. The two-year cumulative incidence of lymphedema was as follows: 4.08% [95% confidence interval (CI): 2.59-6.41%] implant, 9.89% (95% CI: 4.98-19.1%) autologous, and 26.7% (95% CI: 20.4-34.4%) no reconstruction. By multivariate analysis, immediate implant [hazards ratio (HR): 0.352, P < 0.0001] but not autologous (HR: 0.706, P = 0.2151) reconstruction was associated with a significantly reduced risk of lymphedema compared to no reconstruction. Axillary lymph node dissection (P < 0.0001), higher body mass index (P < 0.0001), and greater number of nodes dissected (P = 0.0324) were associated with increased lymphedema risk. CONCLUSIONS: This prospective study suggests that in patients for whom implant-based reconstruction is available, immediate implant reconstruction does not increase the risk of lymphedema compared to mastectomy alone.

Impact of Ipsilateral Blood Draws, Injections, Blood Pressure Measurements, and Air Travel on the Risk of Lymphedema for Patients Treated for Breast Cancer.

PURPOSE: The goal of this study was to investigate the association between blood draws, injections, blood pressure readings, trauma, cellulitis in the at-risk arm, and air travel and increases in arm volume in a cohort of patients treated for breast cancer and screened for lymphedema. PATIENTS AND METHODS: Between 2005 and 2014, patients undergoing treatment of breast cancer at our institution were screened prospectively for lymphedema. Bilateral arm volume measurements were performed preoperatively and postoperatively using a Perometer. At each measurement, patients reported the number of blood draws, injections, blood pressure measurements, trauma to the at-risk arm(s), and number of flights taken since their last measurement. Arm volume was quantified using the relative volume change and weight-adjusted change formulas. Linear random effects models were used to assess the association between relative arm volume (as a continuous variable) and nontreatment risk factors, as well as clinical characteristics. RESULTS: In 3,041 measurements, there was no significant association between relative volume change or weight-adjusted change increase and undergoing one or more blood draws (P = .62), injections (P = .77), number of flights (one or two [P = .77] and three or more [P = .91] v none), or duration of flights (1 to 12 hours [P = .43] and 12 hours or more [P = .54] v none). By multivariate analysis, factors significantly associated with increases in arm volume included body mass index ≥ 25 (P = .0236), axillary lymph node dissection (P < .001), regional lymph node irradiation (P = .0364), and cellulitis (P < .001). CONCLUSION: This study suggests that although cellulitis increases risk of lymphedema, ipsilateral blood draws, injections, blood pressure readings, and air travel may not be associated with arm volume increases. The results may help to educate clinicians and patients on posttreatment risk, prevention, and management of lymphedema.

Impact of adjuvant taxane-based chemotherapy on development of breast cancer-related lymphedema: results from a large prospective cohort.

Taxane-based chemotherapy for the treatment of breast cancer is associated with fluid retention in the extremities; however, its association with development of breast cancer-related lymphedema is unclear. We sought to determine if adjuvant taxane-based chemotherapy increased risk of lymphedema or mild swelling of the upper extremity. 1121 patients with unilateral breast cancer were prospectively screened for lymphedema with perometer measurements. Lymphedema was defined as a relative volume change (RVC) of ≥10 % from preoperative baseline. Mild swelling was defined as RVC 5- <10 %. Clinicopathologic characteristics were obtained via medical record review. Kaplan-Meier and Cox proportional hazard analyses were performed to determine lymphedema rates and risk factors. 29 % (324/1121) of patients were treated with adjuvant taxane-based chemotherapy. The 2-year cumulative incidence of lymphedema in the overall cohort was 5.27 %. By multivariate analysis, axillary lymph node dissection (ALND) (p < 0.0001), higher body mass index (p = 0.0007), and older age at surgery (p = 0.04) were significantly associated with increased risk of lymphedema; however, taxane chemotherapy was not significant when compared to no chemotherapy and non-taxane chemotherapy (HR 1.14, p = 0.62; HR 1.56, p = 0.40, respectively). Chemotherapy with docetaxel was significantly associated with mild swelling on multivariate analysis in comparison to both no chemotherapy and non-taxane chemotherapy groups (HR 1.63, p = 0.0098; HR 2.15, p = 0.02, respectively). Patients who receive taxane-based chemotherapy are not at an increased risk of lymphedema compared to patients receiving no chemotherapy or non-taxane adjuvant chemotherapy. Those treated with docetaxel may experience mild swelling, but this does not translate into subsequent lymphedema.

Tbx6, Mesp-b and Ripply1 regulate the onset of skeletal myogenesis in zebrafish.

During embryonic development, the paraxial mesoderm becomes segmented into somites, within which proliferative muscle progenitors and muscle fibers establish the skeletal musculature. Here, we demonstrate that a gene network previously implicated in somite boundary formation, involving the transcriptional regulators Tbx6, Mesp-b and Ripply1, also confers spatial and temporal regulation to skeletal myogenesis in zebrafish. We show that Tbx6 directly regulates mesp-b and ripply1 expression in vivo, and that the interactions within the regulatory network are largely conserved among vertebrates. Mesp-b is necessary and sufficient for the specification of a subpopulation of muscle progenitors, the central proportion of the Pax3(+)/Pax7(+) dermomyotome. Conditional ubiquitous expression indicates that Mesp-b acts by inhibiting myogenic differentiation and by inducing the dermomyotome marker meox1. By contrast, Ripply1 induces a negative-feedback loop by promoting Tbx6 protein degradation. Persistent Tbx6 expression in Ripply1 knockdown embryos correlates with a deficit in dermomyotome and myotome marker gene expression, suggesting that Ripply1 promotes myogenesis by terminating Tbx6-dependent inhibition of myogenic maturation. Together, our data suggest that Mesp-b is an intrinsic upstream regulator of skeletal muscle progenitors and that, in zebrafish, the genes regulating somite boundary formation also regulate the development of the dermomyotome in the anterior somite compartment.

The impact of breast cancer-related lymphedema on the ability to perform upper extremity activities of daily living.

We sought to assess the association of breast cancer-related lymphedema (BCRL) with the ability to perform upper extremity activities of daily living (ADL) in our patient population. 324 breast cancer patients who had received treatment for unilateral breast cancer at our institution between 2005 and 2014 were prospectively screened for lymphedema. Bilateral arm measurements were performed pre-operatively and during post-operative follow-up using a Perometer. Patients completed an extensive quality of life (QOL) questionnaire at the time of each study assessment. Lymphedema was defined as a relative volume change (RVC) of ≥10% from the patient's pre-operative baseline measurement. Linear regression models were used to evaluate the relationship between post-operative arm function score (as a continuous variable) and RVC, demographic, clinical, and QOL factors. By multivariate analysis, greater fear of lymphedema (p < 0.0001), more pain (p < 0.0001), body mass index >25 (p = 0.0015), mastectomy (p = 0.0001), and having an axillary node dissection (p = 0.0045) were all associated with lower functional scores. Higher emotional well-being score (p < 0.0001) and adjuvant chemotherapy (p = 0.0005) were associated with higher post-operative functional score. Neither low-level volume changes (5-10 % RVC) nor BCRL (RVC ≥10 %) were associated with ability to perform upper extremity ADL as measured by self-report (p = 0.99, p = 0.79). This prospective study demonstrates that low-level changes in arm volume (RVC 5-10 %) as well as clinically significant BCRL (RVC ≥10 %) did not impact the self-reported ability to use the affected extremity for ADL. These findings may help to inform clinicians and patients on the importance of prospective screening for lymphedema and QOL which enables early detection and intervention.