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Achilles tendinopathy is a painful overuse injury that is extremely common in athletes, especially those who participate in running and jumping sports. In addition to pain, Achilles tendinopathy is accompanied by alterations in the tendon's structure and mechanical properties, altered lower extremity function, and fear of movement. Cumulatively, these impairments limit sport participation and performance. A thorough evaluation and comprehensive treatment plan, centered on progressive tendon loading, is required to ensure full recovery of tendon health and to minimize the risk of reinjury. In this review, we will provide an update on the evidence-based evaluation, outcome assessment, treatment, and return-to-sport planning for Achilles tendinopathy. Furthermore, we will provide the strength of evidence for these recommendations using the Strength of Recommendation Taxonomy system.

Complement-mediated damage to the neuromuscular junction (NMJ) is a key mechanism of pathology in myasthenia gravis (MG), and therapeutics inhibiting complement have shown evidence of efficacy in the treatment of MG. In this study, we describe the development of a subcutaneously administered N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C5 component of complement that silences C5 expression in the liver (ALN-CC5). Treatment of wild-type rodents with ALN-CC5 resulted in robust and durable suppression of liver C5 expression. Dose-dependent serum C5 suppression was observed in non-human primates, with a lowering of serum C5 of up to 97.5% and the concomitant inhibition of serum complement activity. C5 silencing was efficacious in ameliorating disease symptoms in two standard rat models of MG, demonstrating the key role of circulating C5 in pathology at the NMJ. Improvement in disease activity scores and NMJ pathology was observed at intermediate levels of complement activity inhibition, suggesting that complete ablation of complement activity may not be required for efficacy in MG. The pre-clinical studies of ALN-CC5 and efficacy of C5 silencing in rat models of MG support further clinical development of ALN-CC5 as a potential therapeutic for the treatment of MG and other complement-mediated disorders. Keywords: myasthenia gravis, small-interfering RNA, complement pathway, siRNA therapeutic

Tendon mechanical properties have been proposed as a biomarker of tendon health to track response to injury and treatment. Prior to utilizing these properties in an injured population, it is critical to understand how these are influenced by age and sex in an uninjured population. A retrospective analysis was conducted of 118 uninjured Achilles tendons to evaluate the relationship between tendon mechanical properties, age and sex. Mechanical properties (shear modulus and viscosity) were assessed using continuous shear wave elastography. A moderator regression analysis was completed to examine the relationship between tendon mechanical properties, age and sex, after adjusting for body mass index and physical activity level. There was an interaction between age and sex for shear modulus (p=0.049, R(2) change=0.034). Females had a negative relationship between age and shear modulus (p=0.030, β=−0.350) but no relationship was observed for males (p=0.78, β=0.031). A positive relationship was found between age and viscosity (p=0.034, β=0.214). Increased viscosity was related to increased age with no difference between sexes. The effect of aging on shear modulus differed between men and women and may help explain sex specific injury risks and their differing response to mechanical load.

ObjectiveTo perform a systematic review and meta-analysis identifying (1) potential modifiable risk factors and (2) associated modifiable factors for patellar tendinopathy in athletes.DesignA systematic review and meta-analysis was conducted. Risk of bias was assessed using the Newcastle-Ottawa Scale and grouped based on study design. Meta-analytic statistics were performed for items reported by five or more studies. A strength of evidence rating is provided for items not appropriate for meta-analysis.Data sourcesPubMed, Web of Science, Scopus and Cinahl were searched on 14 November 2017.Eligibility criteriaQuantitative, original research reporting potential modifiable risk factors or associated factors, comparing athletes with patellar tendinopathy with a group without the injury.Results862 records were screened and 31 articles were included (6 prospective, 25 cross-sectional). There was a lack of strong evidence for any potential modifiable risk factor or associated factors. There was limited or conflicting evidence that decreased ankle dorsiflexion range of motion, decreased posterior thigh and quadriceps flexibility, greater volume of jump training, more volleyball sets played per week, greater countermovement jump (CMJ) height and greater activity volume are potential modifiable risk factors. Meta-analysis supported greater activity volume (Cohen’s d=0.22, 95% CI 0.06 to 0.39, p=0.008), higher body weight (0.36, 0.17 to 0.55, pConclusionsThere is a lack of strong evidence for any potential modifiable risk factors or associated factors. Factors with lower levels of support may be of interest in designing prevention programmes but require further research in high-quality, prospective studies.

Tendinopathy is one of the most common overuse injuries in basketball players, with patellar and Achilles tendinopathy occurring most frequently. In athletic populations, these injuries typically result from excessive load (acute or chronic) placed upon the tendon with inadequate time for recovery. A variety of non-invasive treatment options are available for tendinopathy, but controlled tendon loading is supported by the highest level of evidence. In this chapter, we will describe key tendinopathy rehabilitation principles. Furthermore, we will describe how these principles are applied to patellar and Achilles tendinopathy.

Jumper's knee is not synonymous with patellar tendinopathy. The term includes patellar tendinopathy and quadriceps tendinopathy. Although the patellar and quadriceps tendons work in tandem as part of the extensor mechanism of the knee, they have distinct anatomy and functional roles. As a result, there are probable differences in risk factors, etiology, and response to treatment. It is time to clinically separate patellar tendinopathy and quadriceps tendinopathy and design more specific rehabilitation programs. In this Viewpoint, the authors will (1) provide a rationale for distinguishing the 2 clinical entities-patellar tendinopathy and quadriceps tendinopathy-for treatment decision making, and (2) identify areas of research priority in quadriceps tendinopathy. J Orthop Sports Phys Ther 2019;49(9):627-630. doi:10.2519/jospt.2019.0611.

This article explores Horatian versification, specifically the variable rhythms of the Latin word accent within the flow of a fixed meter. After outlining the prominence of asclepiadean forms in Horatian lyric, ancient and modern scansions of the common 12-syllable asclepiad (a.k.a., lesser asclepiadean), and the distribution of word accents therein, this article considers Horace’s lively exploitation of accentual pattern and variation in the four asclepiadean poems of the “parade odes,” culminating in some rare and rhetorically expressive accents in 1.6. A consequence is a generous sense of rhythm in Horace’s versification, with an occasional concatenation of sound and sense.

Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

Meter, as taught in ancient Rome, entailed not just the sounds of the words, but also the expectation of verse beat (ictus). Even Latin lyrics can include significant coincidence and counterpoint between the performed accent and a latent, learned ictus. This essay surveys evidence for ictus and accent in Sapphics, then considers counterpoint and coincidence in Horace's Odes , a poem of Ausonius, and Horace's Carmen Saeculare , where a regular caesura falling one syllable later than expected reflects a different relationship of accent to beat. This relationship explains Horace's directions to his youthful chorus ( Carm 4.6.35-36) and helps us notice an acoustic feature of Latin lyric.

RNA interference (RNAi) has generated significant interest as a strategy to suppress viral infection, but in some cases antiviral activity of unmodified short-interfering RNA (siRNA) has been attributed to activation of innate immune responses. We hypothesized that immunostimulation by unmodified siRNA could mediate both RNAi as well as innate immune stimulation depending on the mode of drug delivery. We investigated the potential of immunostimulatory RNAs (isRNAs) to suppress influenza A virus in vivo in the mouse lung. Lipidoid 98N12-5(1) formulated with unmodified siRNA targeting the influenza nucleoprotein gene exhibited antiviral activity. Formulations were optimized to increase antiviral activity, but the antiviral activity of lipidoid-delivered siRNA did not depend on sequence homology to the influenza genome as siRNA directed against unrelated targets also suppressed influenza replication in vivo. This activity was primarily attributed to enhancement of innate immune stimulation by lipidoid-mediated delivery, which indicates increased toll-like receptor (TLR) activation by siRNA. Certain chemical modifications to the siRNA backbone, which block TLR7/8 activation but retain in vitro RNAi activity, prevented siRNA-mediated antiviral activity despite enhanced lipidoid-mediated delivery. Here, we demonstrate that innate immune activation caused by unmodified siRNA can have therapeutically relevant effects, and that these non-RNAi effects can be controlled through chemical modifications and drug delivery.

Modulating T cell function by down-regulating specific genes using RNA interference (RNAi) holds tremendous potential in advancing targeted therapies in many immune-related disorders including cancer, inflammation, autoimmunity, and viral infections. Hematopoietic cells, in general, and primary T lymphocytes, in particular, are notoriously hard to transfect with small interfering RNAs (siRNAs). Herein, we describe a novel strategy to specifically deliver siRNAs to murine CD4(+) T cells using targeted lipid nanoparticles (tLNPs). To increase the efficacy of siRNA delivery, these tLNPs have been formulated with several lipids designed to improve the stability and efficacy of siRNA delivery. The tLNPs were surface-functionalized with anti-CD4 monoclonal antibody to permit delivery of the siRNAs specifically to CD4(+) T lymphocytes. Ex vivo, tLNPs demonstrated specificity by targeting only primary CD4(+) T lymphocytes and no other cell types. Systemic intravenous administration of these particles led to efficient binding and uptake into CD4(+) T lymphocytes in several anatomical sites including the spleen, inguinal lymph nodes, blood, and the bone marrow. Silencing by tLNPs occurs in a subset of circulating and resting CD4(+) T lymphocytes. Interestingly, we show that tLNP internalization and not endosome escape is a fundamental event that takes place as early as 1 h after systemic administration and determines tLNPs' efficacy. Taken together, these results suggest that tLNPs may open new avenues for the manipulation of T cell functionality and may help to establish RNAi as a therapeutic modality in leukocyte-associated diseases.

Objective— Adhesive interactions between cells and the extracellular matrix play an important role in inflammatory diseases like atherosclerosis. We investigated the role of the collagen-binding integrin α1β1 in atherosclerosis. Methods and Results— ApoE−/− mice were α1-deficient or received early or delayed anti-α1 antibody treatment. Deficiency in α1 integrin reduced the area of atherosclerotic plaques and altered plaque composition by reducing inflammation and increasing extracellular matrix. In advanced plaques, α1-deficient mice had a reduced macrophage and CD3+ cell content, collagen and smooth muscle cell content increased, lipid core sizes decreased, and cartilaginous metaplasia occurred. Anti-α1 antibody treatment reduced the macrophage content in initial plaques after early and delayed treatment, decreased the CD3+ cell content in advanced plaques after delayed treatment, and increased the collagen content in initial and advanced plaques after delayed treatment. Migration assays performed on α1-deficient macrophages on collagen I and IV substrata revealed that α1-deficient cells can migrate on collagen I, but not IV. Anti-α1 antibody treatment of ApoE−/− macrophages also inhibited migration of cells on collagen IV. Conclusions— Our results suggest that α1β1 integrin is involved in atherosclerosis by mediating the migration of leukocytes to lesions by adhesion to collagen IV. Blocking this integrin reduces atherosclerosis and induces a stable plaque phenotype.

Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1)beta(1) parallels that of viral-specific effector CD8(+) T cells (defined by tetramer and IFN-gamma staining). In an adoptive transfer model, mAb-mediated blockade of these integrins on activated effector and memory T cells inhibited Ag-specific delayed-type hypersensitivity responses; similar decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect of alpha(1)beta(1) blockade on the delayed-type hypersensitivity response could be bypassed by direct injection of Ag-specific T cells to inflammatory sites, demonstrating for the first time in vivo that collagen-binding integrins are involved in leukocyte migration into tissues.

Inflammation occurs in the context of integrin-mediated adhesive interactions of cells with their extracellular matrix environment. We investigated the role of the collagen binding integrin alpha1beta1 in a model of colitis. alpha1beta1 was expressed on lamina propria T cells and monocytes during disease. Both alpha1 deficiency and anti-alpha1 mAb treatment (prophylactic and therapeutic) protected against colitis. In vivo alpha1beta1 blockade improved macroscopic and histologic scores, decreased inflammatory cytokine production, and profoundly affected the ability of lamina propria mononuclear cells to proliferate and produce IFN-gamma in vitro. Development and alpha1-mediated inhibition of colitis can be lymphocyte independent, suggesting that activated monocytes also represent a key alpha1beta1-expressing cell type involved in colitis. These results underscore the importance of innate immunity and, specifically, of leukocyte/matrix interactions in regulating local inflammatory responses.

Introduction: Cardiovascular disease remains the top cause of mortality in the United States. We have developed, and validated in human trials, a platform for reducing the synthesis of genes expressed in the liver. The platform utilizes a GalNAc sugar ligand attached to the 3’end of the sense strand of an RNAi molecule to enable delivery specifically to the liver. Here we present data from this platform on multiple targets of interest in cardiovascular disease, including PCSK9, ANGPLT3 and ApoC3. Hypothesis: RNAi therapeutics targeting liver genes of interest (such as PCSK9, ApoC3 and Angptl3) will allow for control of lipid levels and reduce the risk of cardiovascular disease. Methods: Chemically modified siRNAs were designed using bioinformatic algorithms and were screened for potency in vitro . pM active siRNA molecules were developed targeting PCSK9, ANGPLT3 and ApoC3. Results: ALN-PCSsc: In NHPs a single dose of ALN-PCSsc at ≥ 6 mg/kg reduced circulating PCSK9 levels up to 97% and LDL-C up to 67%. Moreover the nadir effect ( where LDL-C is clamped) lasted ~90 days suggesting that once monthly to quarterly dosing could be supported. Multidose studies in NHP at 2 mg/kg reduced circulating PCSK9 levels up to 94% with a subsequent lowering of LDL-C up to 67%. Safety studies in rat and NHP at doses up to 300 mg/kg (multi-dose) showed that ALN-PCSsc was safe demonstrating potential for a very wide therapeutic index. ALN-PCSsc was selected as a development candidate and is being advanced towards an IND for the treatment of hypercholesterolemia. ALN-ANGsc: Was tested in two models of hyperlipidemia, the OB/OB mouse and the hCETP-ApoB mouse. In the Ob/Ob model, treatment with ALN-ANGsc at 3mg/kg resulted in a significant lowering ANGPLT3 protein (>95%), cholesterol (>60%) and triglycerides (>85%). ALN-ApoC3: Was tested in the db/db mouse model of hyperlipidemia. A robust lowering of plasma ApoC3 (>90%) lead to a >50% lowering of triglycerides. Screening for both programs is underway for a development candidates. Conclusions: We have developed a modular, robust and reliable platform for the delivery of RNAi therapeutics to the liver with a large therapeutic index. This small volume, subcutaneous, platform has a remarkable duration of effect.

Aim: There remains high unmet medical need for therapies to treat cardio/metabolic diseases. We validated in human trials, a platform for reducing the synthesis of genes expressed in the liver. The platform utilizes a GalNAc ligand attached to the 3’ end of the sense strand of an RNAi molecule to enable delivery specifically to the liver. Here we extend the platform to targets of interest in cardiovascular disease, including PCSK9, ANGPLT3 and ApoC3. METHODS: Chemically modified siRNAs were designed and were screened for potency in vitro . pM active siRNA molecules were developed targeting PCSK9, ANGPLT3 and ApoC3. The siRNAs were tested in either rodents or in non-human primates (NHPs) for activity. RESULTS: In NHPs a single dose of ALN-PCSsc at 6 mg/kg reduced PCSK9 levels up to 97% and LDL-C up to 67%. Moreover the nadir effect (without any rebound of LDL-C) lasted >30 days indicating that once a month or longer dosing frequency in clinic should be supported. Multidose studies in NHP at 2mg/kg reduced circulating PCSK9 levels up to 94% with a subsequent lowering of LDL-C up to 67%. The effects on both PCSK9 and LDL-C was also very durable with levels of LDL-C returning to baseline > 140 days post the last dose. Safety studies in rat at doses up to 225mg/kg (multi-dose) indicate that ALN-PCSsc is safe demonstrating a very wide therapeutic index. ALN-PCSsc was selected as a development candidate and is being advanced towards an IND ALN-ANGsc (an siRNA targeting ANGPTL3) was tested in two models of hyperlipidemia, the OB/OB mouse and the hCETP-ApoB mouse. In the Ob/Ob model, treatment with ALN-ANGsc at 3mg/kg resulted in a significant lowering ANGPLT3 protein (>95%), total cholesterol(>60%), and triglycerides (>85%). Finally, we have developed a prototype molecule targeting Apoc3 with an ED90 for ApoC3 protein of CONCLUSION: We have developed a modular, robust and durable platform for the delivery of RNAi therapeutics to the liver. This platform is administered as a small volume subcutaneous dose and has a remarkable duration of effect in rodent NHP models. We have extended this platform to several targets of high interest includingPCSK9,ANGPLT3 and ApoC3.

Mantle cell lymphoma is characterized by a genetic translocation results in aberrant overexpression of the CCND1 gene, which encodes cyclin D1. This protein functions as a regulator of the cell cycle progression, hence is considered to play an important role in the pathogenesis of the disease. In this study, we used RNA interference strategies to examine whether cyclin D1 might serve as a therapeutic target for mantle cell lymphoma. Knocking down cyclin D1 resulted in significant growth retardation, cell cycle arrest, and most importantly, induction of apoptosis. These results mark cyclin D1 as a target for mantle cell lymphoma and emphasize the therapeutic potential hidden in its silencing.

u Abstract. This essay explores the metrical as well as rhythmical aspects of the acoustic contour of the Latin Alcaic, focusing on patterns of natural, audible, performed word accents in coincidence and syncopation with the fixed pattern of the meter, in both the ancient and modern scansions of the stanza. The meter was measured in antiquity with a learned, latent expectation or undercurrent of regular verse beats to scan aloud, to measure for the ear, the pattern of long and short syllables. Within the fixed framework of the meter, variable patterns of accent provide a rhythm, and that rhythm is the focus of this essay. Very little attention falls on sound and sense: the coda argues that sound need not be sub- ordinate to meaning, need not be sound effect, nor explicitly rhetorical, to be worth our attention. By accentuating weight and pause, Horace points up a principal beauty of the Alcaic, its shifts in rhythm in midcourse, its calculated imbalance as the iambic and choriambic first two lines yield to iambs in the third and resolve in racy dactyls in the fourth. —Rosanna Warren 2008, 29 ThiS eSSAy exploreS The meTricAl And rhyThmicAl ASpecTS of the acoustic contour of the Latin Alcaic stanza. The particular focus is on the patterns of natural, audible, performed word accents in coincidence and syncopation with the fixed pattern of the meter, in both the ancient and modern understanding of the stanza. The meter was measured in antiquity with a learned, latent expectation or undercurrent of regular verse-beats, used to scan aloud—to measure for the ear—the pattern of long and short syllables. Within the framework of the meter, patterns of accent provide a rhythm, and that rhythm is the focus here. 1 Very little

A lymphotoxin-β (LTβ) receptor-Ig fusion protein (LTβR-Ig) was used to evaluate the importance of the lymphotoxin/LIGHT axis in the development and perpetuation of arthritis. Prophylactic treatment with the inhibitor protein LTβR-Ig blocked the induction of collagen-induced arthritis in mice and adjuvant arthritis in Lewis rats. Treatment of mice with established collagen-induced arthritis reduced the severity of arthritic symptoms and joint tissue damage. However, in a passive model of anti-collagen Ab-triggered arthritis, joint inflammation was not affected by LTβR-Ig treatment precluding LT/LIGHT involvement in the very terminal immune complex/complement/FcR-mediated effector phase. Collagen-II and Mycobacterium-specific T cell responses were not impaired, yet there was evidence that the overall response to the mycobacterium was blunted. Serum titers of anti-collagen-II Abs were reduced especially during the late phase of disease. Treatment with LTβR-Ig ablated follicular dendritic cell networks in the draining lymph nodes, suggesting that impaired class switching and affinity maturation may have led to a decreased level of pathological autoantibodies. These data are consistent with a model in which the LT/LIGHT axis controls microenvironments in the draining lymph nodes. These environments are critical in shaping the adjuvant-driven initiating events that impact the subsequent quality of the anti-collagen response in the later phases. Consequently, blockade of the LT/LIGHT axis may represent a novel approach to the treatment of autoimmune diseases such as rheumatoid arthritis that involve both T cell and Ab components.

Excessive complement activation plays a pivotal role in a variety of disorders. Complement component C5 is a clinically validated therapeutic target for treatment of both paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic-uremic syndrome. We developed a robust RNAi therapeutics platform for the delivery of siRNAs to the liver using trivalent GalNAc conjugates, enabling specific silencing of hepatocyte-expressed genes following subcutaneous (SC) injection. The liver produces essentially the entirety of C5 and other complement pathway proteins. We are developing ALN-CC5, an investigational RNAi therapeutic targeting human, primate and rodent C5. C5 silencing and complement activity inhibition were examined in rodents and primates. Multi-dose SC ALN-CC5 treatment resulted in sustained lowering of cyno serum C5 with ≤3% residual protein remaining. C5 reduction was associated with >90% and >95% inhibition of classical and alternative complement pathways as measured by ELISA-based assays. Additionally, >80% lowering of complement serum hemolytic activity was observed. ALN-CC5 was safe and well tolerated in both rat and non-human primate toxicology studies. In addition to wild type animals, ALN-CC5 was tested in several animal models of disease in which complement activation plays a prominent role. Silencing of murine C5 was highly efficacious in a model of anti-collagen antibody-induced arthritis with a disease modifying activity equivalent to that of an anti-C5 antibody. Furthermore, C5 silencing was effective at reducing proteinuria in a rat model of membranous nephropathy. Up-regulation of C5 expression, observed in both models, had no effect on the extent of C5 silencing, suggesting that ALN-CC5 could be efficacious in the context of inflammation. These data demonstrate a prominent role for circulating, liver-derived C5 in mediating pathology at extrahepatic sites and the potential utility of an RNAi therapeutic targeting C5. In summary, RNAi-mediated silencing of liver-derived C5 is a promising novel therapeutic approach for inhibiting systemic complement activity, with the potential to enable, low volume, subcutaneous treatment for patients with PNH and other disorders where complement activation plays a role in disease progression. Disclosures Borodovsky: Alnylam: Employment. Yucius:Alnylam: Employment. Sprague:Alnylam: Employment. Banda:Alnylam: Research Funding. Holers:Alnylam: Research Funding. Vaishnaw:Alnylam Pharmaceuticals: Employment, Equity Ownership. Maier:Alnylam: Employment. Kallanthottathil:Alnylam: Employment. Charisse:Alnylam: Employment. Kuchimanchi:Alnylam: Employment. Manoharan:Alnylam: Employment. Salant:Alnylam: Honoraria. Fitzgerald:Alnylam: Employment. Meyers:Alnylam: Employment. Sorensen:Alnylam Pharmaceuticals: Employment, Equity Ownership.

Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAb's against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.

The complement system is a pivotal player in multiple hematological conditions. Antibody blockade of the C5 component of complement has been approved as a treatment for both paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic-uremic syndrome (aHUS), validating C5 as an important therapeutic target. Recently, we developed a robust RNAi therapeutics platform for the delivery of siRNAs to the liver using trivalent GalNAc conjugates, enabling silencing of hepatocyte-expressed genes following subcutaneous (SC) injection. The liver is a major source of C5 and other complement pathway components. The GalNAc conjugate technology allows rapid development of siRNAs targeting multiple members of the complement cascade and evaluation of their silencing in pre-clinical models. To examine the utility of the siRNA approach for targeting complement pathway components we designed and synthesized GalNAc conjugated siRNAs targeting rodent, primate and human C5. Potent siRNA duplexes, showing greater than 95% silencing of C5 mRNA were selected using in vitro screening in human cell lines and mouse primary hepatocytes. C5 silencing and serum hemolytic activity inhibition were evaluated in rodents using single and multi-dose SC treatment regimens. A C5-targeting siRNA conjugate demonstrated a single dose ED50 of 0.625 mg/kg in the mouse with greater than 90% silencing of serum C5 achievable at higher doses. Serum C5 silencing was durable, with recovery starting two weeks after a single SC injection We went on to examine the efficacy of C5 silencing in the rat and observed robust lowering of serum C5 with 2.5 and 5 mg/kg multi-dose regimens, resulting in up to ∼90% inhibition of complement classical pathway hemolytic activity. Evaluation of the translation of this approach to higher species is in progress. Since PNH erythrocyte lysis is thought to be mediated by the activation of the alternative pathway of complement we initiated work on the development of siRNA conjugates targeting Factor B, an essential component of the alternative pathway C3 convertase. siRNAs targeting rodent, primate and human Factor B were identified by in vitro screening and demonstrate >90% silencing of Factor B mRNA in human cell lines and primary mouse hepatocytes. Evaluation of Factor B silencing in rodent models is ongoing. siRNA-mediated silencing of liver-derived complement components is a promising novel therapeutic approach for inhibiting the activity of C5 and other complement pathway targets, with the potential to enable subcutaneous treatment for patients with PNH and related disorders. Disclosures: Borodovsky: Alnylam: Employment. Yucius:Alnylam: Employment. Sprague:Alnylam: Employment. Butler:Alnylam: Employment. Fishman:Alnylam: Employment. Nguyen:Alnylam: Employment. Vaishnaw:Alnylam: Employment. Maier:Alnylam: Employment. Kallanthottathil:Alnylam: Employment. Kuchimanchi:Alnylam: Employment. Manoharan:Alnylam: Employment. Meyers:Alnylam: Employment. Fitzgerald:Alnylam: Employment.

A common feature of many infections is that many pathogen-specific memory T cells become established in diverse nonlymphoid tissues. A mechanism that promotes the retention and survival of the memory T cells in diverse tissues has not been described. Our studies show that the collagen binding α1β1 integrin, VLA-1, is expressed by the majority of influenza-specific CD8 T cells recovered from nonlymphoid tissues during both the acute and memory phases of the response. Antibody treatment or genetic deficiency of VLA-1 decreased virus-specific CTL in the lung and other nonlymphoid tissues, and increased them in the spleen. In spite of the increase in the spleen, secondary heterosubtypic immunity against flu was compromised. This suggests that VLA-1 is responsible for retaining protective memory CD8 T cells in the lung and other tissues via attachment to the extracellular matrix.