LipidSIM: Inferring mechanistic lipid biosynthesis perturbations from lipidomics with a flexible, low-parameter, Markov modeling framework.

Lipid metabolism is a complex and dynamic system involving numerous enzymes at the junction of multiple metabolic pathways. Disruption of these pathways leads to systematic dyslipidemia, a hallmark of many pathological developments, such as nonalcoholic steatohepatitis and diabetes. Recent advances in computational tools can provide insights into the dysregulation of lipid biosynthesis, but limitations remain due to the complexity of lipidomic data, limited knowledge of interactions among involved enzymes, and technical challenges in standardizing across different lipid types. Here, we present a low-parameter, biologically interpretable framework named Lipid Synthesis Investigative Markov model (LipidSIM), which models and predicts the source of perturbations in lipid biosynthesis from lipidomic data. LipidSIM achieves this by accounting for the interdependency between the lipid species via the lipid biosynthesis network and generates testable hypotheses regarding changes in lipid biosynthetic reactions. This feature allows the integration of lipidomics with other omics types, such as transcriptomics, to elucidate the direct driving mechanisms of altered lipidomes due to treatments or disease progression. To demonstrate the value of LipidSIM, we first applied it to hepatic lipidomics following Keap1 knockdown and found that changes in mRNA expression of the lipid pathways were consistent with the LipidSIM-predicted fluxes. Second, we used it to study lipidomic changes following intraperitoneal injection of CCl4 to induce fast NAFLD/NASH development and the progression of fibrosis and hepatic cancer. Finally, to show the power of LipidSIM for classifying samples with dyslipidemia, we used a Dgat2-knockdown study dataset. Thus, we show that as it demands no a priori knowledge of enzyme kinetics, LipidSIM is a valuable and intuitive framework for extracting biological insights from complex lipidomic data.

The Mediterranean diet is not associated with neuroimaging or cognition in middle-aged adults: a cross-sectional analysis of the PREVENT dementia programme.

BACKGROUND AND PURPOSE: The Mediterranean diet (MedDiet) has been associated with reduced dementia incidence in several studies. It is important to understand if diet is associated with brain health in midlife, when Alzheimer's disease and related dementias are known to begin. METHODS: This study used data from the PREVENT dementia programme. Three MedDiet scores were created (the Pyramid, Mediterranean Diet Adherence Screener [MEDAS] and MEDAS continuous) from a self-reported food frequency questionnaire. Primary outcomes were hippocampal volume and cube-transformed white matter hyperintensity volume. Secondary outcomes included cornu ammonis 1 and subiculum hippocampal subfield volumes, cortical thickness and measures of cognition. Sex-stratified analyses were run to explore differential associations between diet and brain health by sex. An exploratory path analysis was conducted to study if any associations between diet and brain health were mediated by cardiovascular risk factors for dementia. RESULTS: In all, 504 participants were included in this analysis, with a mean Pyramid score of 8.10 (SD 1.56). There were no significant associations between any MedDiet scoring method and any of the primary or secondary outcomes. There were no differences by sex in any analyses and no significant mediation between the Pyramid score and global cognition by cardiovascular risk factors. CONCLUSIONS: Overall, this study did not find evidence for an association between the MedDiet and either neuroimaging or cognition in a midlife population study. Future work should investigate associations between the MedDiet and Alzheimer's disease and related dementias biomarkers as well as functional neuroimaging in a midlife population.

Site-specific incorporation of 5'-methyl DNA enhances the therapeutic profile of gapmer ASOs.

We recently showed that site-specific incorporation of 2'-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5'-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5'-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2'-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5'-Me and R-5'-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5'-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs.

Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides.

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.

Artificial intelligence for diagnostic and prognostic neuroimaging in dementia: A systematic review.

INTRODUCTION: Artificial intelligence (AI) and neuroimaging offer new opportunities for diagnosis and prognosis of dementia. METHODS: We systematically reviewed studies reporting AI for neuroimaging in diagnosis and/or prognosis of cognitive neurodegenerative diseases. RESULTS: A total of 255 studies were identified. Most studies relied on the Alzheimer's Disease Neuroimaging Initiative dataset. Algorithmic classifiers were the most commonly used AI method (48%) and discriminative models performed best for differentiating Alzheimer's disease from controls. The accuracy of algorithms varied with the patient cohort, imaging modalities, and stratifiers used. Few studies performed validation in an independent cohort. DISCUSSION: The literature has several methodological limitations including lack of sufficient algorithm development descriptions and standard definitions. We make recommendations to improve model validation including addressing key clinical questions, providing sufficient description of AI methods and validating findings in independent datasets. Collaborative approaches between experts in AI and medicine will help achieve the promising potential of AI tools in practice. HIGHLIGHTS: There has been a rapid expansion in the use of machine learning for diagnosis and prognosis in neurodegenerative disease Most studies (71%) relied on the Alzheimer's Disease Neuroimaging Initiative (ADNI) dataset with no other individual dataset used more than five times There has been a recent rise in the use of more complex discriminative models (e.g., neural networks) that performed better than other classifiers for classification of AD vs healthy controls We make recommendations to address methodological considerations, addressing key clinical questions, and validation We also make recommendations for the field more broadly to standardize outcome measures, address gaps in the literature, and monitor sources of bias.

CAIDE dementia risk score relates to severity and progression of cerebral small vessel disease in healthy midlife adults: the PREVENT-Dementia study.

BACKGROUND: Markers of cerebrovascular disease are common in dementia, and may be present before dementia onset. However, their clinical relevance in midlife adults at risk of future dementia remains unclear. We investigated whether the Cardiovascular Risk Factors, Ageing and Dementia (CAIDE) risk score was associated with markers of cerebral small vessel disease (SVD), and if it predicted future progression of SVD. We also determined its relationship to systemic inflammation, which has been additionally implicated in dementia and SVD. METHODS: Cognitively healthy midlife participants were assessed at baseline (n=185) and 2-year follow-up (n=158). To assess SVD, we quantified white matter hyperintensities (WMH), enlarged perivascular spaces (EPVS), microbleeds and lacunes. We derived composite scores of SVD burden, and subtypes of hypertensive arteriopathy and cerebral amyloid angiopathy. Inflammation was quantified using serum C-reactive protein (CRP) and fibrinogen. RESULTS: At baseline, higher CAIDE scores were associated with all markers of SVD and inflammation. Longitudinally, CAIDE scores predicted greater total (p<0.001), periventricular (p<0.001) and deep (p=0.012) WMH progression, and increased CRP (p=0.017). Assessment of individual CAIDE components suggested that markers were driven by different risk factors (WMH/EPVS: age/hypertension, lacunes/deep microbleeds: hypertension/obesity). Interaction analyses demonstrated that higher CAIDE scores amplified the effect of age on SVD, and the effect of WMH on poorer memory. CONCLUSION: Higher CAIDE scores, indicating greater risk of dementia, predicts future progression of both WMH and systemic inflammation. Findings highlight the CAIDE score's potential as both a prognostic and predictive marker in the context of cerebrovascular disease, identifying at-risk individuals who might benefit most from managing modifiable risk.

Understanding the effect of controlling phosphorothioate chirality in the DNA gap on the potency and safety of gapmer antisense oligonucleotides.

Therapeutic oligonucleotides are often modified using the phosphorothioate (PS) backbone modification which enhances stability from nuclease mediated degradation. However, substituting oxygen in the phosphodiester backbone with sulfur introduce chirality into the backbone such that a full PS 16-mer oligonucleotide is comprised of 215 distinct stereoisomers. As a result, the role of PS chirality on the performance of antisense oligonucleotides (ASOs) has been a subject of debate for over two decades. We carried out a systematic analysis to determine if controlling PS chirality in the DNA gap region can enhance the potency and safety of gapmer ASOs modified with high-affinity constrained Ethyl (cEt) nucleotides in the flanks. As part of this effort, we examined the effect of systematically controlling PS chirality on RNase H1 cleavage patterns, protein mislocalization phenotypes, activity and toxicity in cells and in mice. We found that while controlling PS chirality can dramatically modulate interactions with RNase H1 as evidenced by changes in RNA cleavage patterns, these were insufficient to improve the overall therapeutic profile. We also found that controlling PS chirality of only two PS linkages in the DNA gap was sufficient to modulate RNase H1 cleavage patterns and combining these designs with simple modifications such as 2'-OMe to the DNA gap resulted in dramatic improvements in therapeutic index. However, we were unable to demonstrate improved potency relative to the stereorandom parent ASO or improved safety over the 2'-OMe gap-modified stereorandom parent ASO. Overall, our work shows that while controlling PS chirality can modulate RNase H1 cleavage patterns, ASO sequence and design are the primary drivers which determine the pharmacological and toxicological properties of gapmer ASOs.

Proximity to dementia onset and multi-modal neuroimaging changes: The prevent-dementia study.

BACKGROUND: First-degree relatives of people with dementia (FH+) are at increased risk of developing Alzheimer's disease (AD). Here, we investigate "estimated years to onset of dementia" (EYO) as a surrogate marker of preclinical disease progression and assess its associations with multi-modal neuroimaging biomarkers. METHODS: 89 FH+ participants in the PREVENT-Dementia study underwent longitudinal MR imaging over 2 years. EYO was calculated as the difference between the parental age of dementia diagnosis and the current age of the participant (mean EYO = 23.9 years). MPRAGE, ASL and DWI data were processed using Freesurfer, FSL-BASIL and DTI-TK. White matter lesion maps were segmented from FLAIR scans. The SPM Sandwich Estimator Toolbox was used to test for the main effects of EYO and interactions between EYO, Time, and APOE-ε4+. Threshold free cluster enhancement and family wise error rate correction (TFCE FWER) was performed on voxelwise statistical maps. RESULTS: There were no significant effects of EYO on regional grey matter atrophy or white matter hyperintensities. However, a shorter EYO was associated with lower white matter Fractional Anisotropy and elevated Mean/Radial Diffusivity, particularly in the corpus callosum (TFCEFWERp < 0.05). The influence of EYO on white matter deficits were significantly stronger compared to that of normal ageing. APOE-ε4 carriers exhibited hyperperfusion with nearer proximity to estimated onset in temporo-parietal regions. There were no interactions between EYO and time, suggesting that EYO was not associated with accelerated imaging changes in this sample. CONCLUSIONS: Amongst cognitively normal midlife adults with a family history of dementia, a shorter hypothetical proximity to dementia onset may be associated with incipient brain abnormalities, characterised by white matter disruptions and perfusion abnormalities, particularly amongst APOE-ε4 carriers. Our findings also confer biological validity to the construct of EYO as a potential stage marker of preclinical progression in the context of sporadic dementia. Further clinical follow-up of our longitudinal sample would provide critical validation of these findings.

Rate of, and risk factors for, white matter hyperintensity growth: a systematic review and meta-analysis with implications for clinical trial design.

BACKGROUND: White matter hyperintensities (WMHs) are a highly prevalent MRI marker of cerebral small vessel disease (SVD), which predict stroke and dementia risk, and are being increasingly used as a surrogate marker in clinical trials. However, the influence of study population selection on WMH progression rate has not been studied and the effect of individual patient factors for WMH growth are not fully understood. METHODS: We performed a systematic review and meta-analysis of the literature on progression of WMHs in longitudinal studies to determine rates of WMH growth, and how these varied according to population characteristics and cardiovascular risk factors. We used these data to calculate necessary sample sizes for clinical trials using WMH as an endpoint. RESULTS: WMH growth rate was highest in SVD (2.50cc/year), intermediate in unselected stroke patients (1.29cc/year) and lower in patients with non-stroke cardiovascular disease, and with cognitive impairment. Age was significantly associated with progression (correlation coefficient 0.15cc/year, 95% CI 0.02 to 0.28cc/year) as was baseline lesion volume (0.6cc/year, 95% CI 0.13 to 1.06 cc/year). Both hypertension (OR 1.72, 95% CI 1.19 to 2.46) and current smoking (OR 1.48, 95% CI 1.02 to 2.16) were associated with WMH growth. Sample sizes for a clinical trial varied greatly with patient population selection and baseline lesion volume; estimates are provided. CONCLUSIONS: WMH progression varies markedly according to the characteristics of the population being studied and this will have a major impact on sample sizes required in a clinical trial. Our sample size estimates provide data for planning clinical trials using WMH as an outcome measure. PROSPERO REGISTRATION NUMBER: CRD42020191781.

Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins.

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.

Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle.

Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.

Conjugation of hydrophobic moieties enhances potency of antisense oligonucleotides in the muscle of rodents and non-human primates.

We determined the effect of attaching palmitate, tocopherol or cholesterol to PS ASOs and their effects on plasma protein binding and on enhancing ASO potency in the muscle of rodents and monkeys. We found that cholesterol ASO conjugates showed 5-fold potency enhancement in the muscle of rodents relative to unconjugated ASOs. However, they were toxic in mice and as a result were not evaluated in the monkey. In contrast, palmitate and tocopherol-conjugated ASOs showed enhanced potency in the skeletal muscle of rodents and modest enhancements in potency in the monkey. Analysis of the plasma-protein binding profiles of the ASO-conjugates by size-exclusion chromatography revealed distinct and species-specific differences in their association with plasma proteins which likely rationalizes their behavior in animals. Overall, our data suggest that modulating binding to plasma proteins can influence ASO activity and distribution to extra-hepatic tissues in a species-dependent manner and sets the stage to identify other strategies to enhance ASO potency in muscle tissues.

In vivo neuroinflammation and cerebral small vessel disease in mild cognitive impairment and Alzheimer's disease.

INTRODUCTION: Associations between cerebral small vessel disease (SVD) and inflammation have been largely examined using peripheral blood markers of inflammation, with few studies measuring inflammation within the brain. We investigated the cross-sectional relationship between SVD and in vivo neuroinflammation using [11C]PK11195 positron emission tomography (PET) imaging. METHODS: Forty-two participants were recruited (according to NIA-AA guidelines, 14 healthy controls, 14 mild Alzheimer's disease, 14 amyloid-positive mild cognitive impairment). Neuroinflammation was assessed using [11C]PK11195 PET imaging, a marker of microglial activation. To quantify SVD, we assessed white matter hyperintensities (WMH), enlarged perivascular spaces, cerebral microbleeds and lacunes. Composite scores were calculated for global SVD burden, and SVD subtypes of hypertensive arteriopathy and cerebral amyloid angiopathy (CAA). General linear models examined associations between SVD and [11C]PK11195, adjusting for sex, age, education, cognition, scan interval, and corrected for multiple comparisons via false discovery rate (FDR). Dominance analysis directly compared the relative importance of hypertensive arteriopathy and CAA scores as predictors of [11C]PK11195. RESULTS: Global [11C]PK11195 binding was associated with SVD markers, particularly in regions typical of hypertensive arteriopathy: deep microbleeds (ß=0.63, F(1,35)=35.24, p<0.001), deep WMH (ß=0.59, t=4.91, p<0.001). In dominance analysis, hypertensive arteriopathy score outperformed CAA in predicting [11C]PK11195 binding globally and in 28 out of 37 regions of interest, especially the medial temporal lobe (ß=0.66-0.76, t=3.90-5.58, FDR-corrected p (pFDR)=<0.001-0.002) and orbitofrontal cortex (ß=0.51-0.57, t=3.53-4.30, pFDR=0.001-0.004). CONCLUSION: Microglial activation is associated with SVD, particularly with the hypertensive arteriopathy subtype of SVD. Although further research is needed to determine causality, our study suggests that targeting neuroinflammation might represent a novel therapeutic strategy for SVD.

Construct validity of the Visual Cognitive Assessment Test (VCAT)-a cross-cultural language-neutral cognitive screening tool.

BACKGROUND: The Visual Cognitive Assessment Test (VCAT) is a language-neutral cognitive screening tool designed for use in culturally diverse populations without the need for translations or adaptations. While it has been established to be language-neutral, the VCAT's construct validity has not been investigated. METHODS: 471 participants were recruited, comprising 233 healthy comparisons, 117 mild cognitive impairment (MCI), and 121 mild Alzheimer's disease (AD) patients. VCAT and domain-specific neuropsychological tests were administered in the same sitting. Construct validity was assessed by analyzing domain-specific associations between the VCAT and well-established cognitive assessments. Reliability (internal consistency) was measured by Cronbach's alpha. Diagnostic ability (area under the curve) and recommended cutoffs were determined by receiver operating characteristic (ROC) analysis. RESULTS: The VCAT and its subdomains demonstrated good construct validity in terms of both convergent and divergent validity and good internal consistency (α = .74). ROC analysis found that the VCAT was on par with the Mini Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) at distinguishing between healthy comparisons, MCI, and mild AD. Consistent with previous studies, VCAT scores were not affected by language of administration or ethnicity in our cohort. Findings suggest the following cutoffs: Dementia 0-19, MCI 20-24, Normal 25-30. CONCLUSION: This study established the construct validity of the VCAT, which is vital to ensure its subdomains effectively measure the cognitive processes they were designed to. The VCAT is capable of detecting early cognitive impairments and allows for meaningful cross-cultural comparisons, especially useful for international collaborations and clinical trials, and for clinical use in diverse multiethnic populations.

Hippocampal subfield atrophy of CA1 and subicular structures predict progression to dementia in idiopathic Parkinson's disease.

BACKGROUND: Global hippocampal atrophy is a hallmark of Alzheimer's dementia and has been similarly reported in Parkinson's disease dementia (PDD). However, there is limited literature on the differential involvement of hippocampal subfields in predicting conversion to PDD. This study is an extension of previous findings on progression to mild cognitive impairment in Parkinson's disease (PD). METHODS: This cohort study recruited 73 non-demented participants with idiopathic PD (age 65.80±8.17, 75.3% male) from an outpatient neurology clinic. All participants underwent clinical assessment, neuropsychological testing and 3T MRI scans at baseline and 18 months while on prescribed dopaminergic medication. Hippocampal subfield volumes were obtained using automatic segmentation in FreeSurfer V.6.0. Participants who progressed to PDD and those who did not were compared on hippocampal subfield atrophy and cognitive change (episodic memory, attention, executive functions, language, visuospatial abilities). Subfields were further examined for their abilities to predict PDD conversion and distinguish PDD from non-demented PD using receiver operating characteristic analysis. RESULTS: Smaller baseline global hippocampal volume, cornu ammonis (CA) subfield CA1, subiculum and presubiculum volumes were observed in participants who went on to develop dementia, and predicted PDD conversion. Those who progressed to PDD saw greater decline in global hippocampal volume, granule cell layer of the dentate gyrus, presubiculum, parasubiculum and fimbria. Decline in subiculum and fimbria volume corresponded to cognitive decline in attention and executive functions, respectively. CONCLUSIONS: Early atrophy of CA1, subiculum and presubiculum preceded, and predicted, PDD conversion. Differential patterns of subfield atrophy were also observed among those who progressed to PDD and were associated with impaired executive functions.

Characterizing the effect of GalNAc and phosphorothioate backbone on binding of antisense oligonucleotides to the asialoglycoprotein receptor.

Targeted delivery of antisense oligonucleotides (ASO) to hepatocytes via the asialoglycoprotein receptor (ASGR) has improved the potency of ASO drugs ∼30-fold in the clinic (1). In order to fully characterize the effect of GalNAc valency, oligonucleotide length, flexibility and chemical composition on ASGR binding, we tested and validated a fluorescence polarization competition binding assay. The ASGR binding, and in vitro and in vivo activities of 1, 2 and 3 GalNAc conjugated single stranded and duplexed ASOs were studied. Two and three GalNAc conjugated single stranded ASOs bind the ASGR with the strongest affinity and display optimal in vitro and in vivo activities. 1 GalNAc conjugated ASOs showed 10-fold reduced ASGR binding affinity relative to three GalNAc ASOs but only 2-fold reduced activity in mice. An unexpected observation was that the ASGR also appears to play a role in the uptake of unconjugated phosphorothioate modified ASOs in the liver as evidenced by the loss of activity of GalNAc conjugated and unconjugated ASOs in ASGR knockout mice. Our results provide insights into how backbone charge and chemical composition assist in the binding and internalization of highly polar anionic single stranded oligonucleotides into cells and tissues.

Serious infection risk after 1 year between patients with rheumatoid arthritis treated with rituximab or with a second TNFi after initial TNFi failure: results from The British Society for Rheumatology Biologics Register for Rheumatoid Arthritis.

Objectives: Both TNF inhibitors (TNFi) and rituximab (RTX), a B-cell depleting biologic, can disrupt the immune system in RA. RTX is licensed in Europe for use following TNFi failure. However, safety data on serious infections (SIs) are scarce for RTX in daily practice. This analysis aims to compare the risk of SIs in the first year after a switch to either TNFi or RTX in patients who have failed a first TNFi. Methods: This study included patients with RA registered with the British Society for Rheumatology Biologics Register (BSRBR-RA) who switched to either a second TNFi or RTX after failing a first TNFi. Patients were followed until first SI, treatment discontinuation, last recorded follow-up or the end of the first year after the switch, whichever came first. SI was defined as requiring hospitalization, intravenous antibiotics or resulting in death. The risk of first SI was compared between TNFi and RTX using Cox proportional hazard models adjusted using propensity scores using inverse probability of treatment weighting. Results: This analysis included 3419 TNFi and 1396 RTX patients contributing 2765 and 1224 person-years (pyrs), respectively. SI occurred in 164 (4.8%) TNFi and 81 (5.8%) RTX patients giving a crude rate of 59 and 66 SI/1000 pyrs, respectively. The adjusted hazard ratio for SI was 1.0 (95% CI: 0.7, 1.4). Conclusion: The risk of SIs was comparable over the first year of treatment between TNFi and RTX treatment in patients who had failed a single prior TNFi.

Evaluation of the effect of 2'-O-methyl, fluoro hexitol, bicyclo and Morpholino nucleic acid modifications on potency of GalNAc conjugated antisense oligonucleotides in mice.

The potency of antisense oligonucleotide (ASO) drugs has significantly improved in the clinic after exploiting asialoglycoprotein receptor (ASGR) mediated delivery to hepatocytes. To further this technology, we evaluated the structure-activity relationships of oligonucleotide chemistry on in vivo potency of GalNAc-conjugated Gapmer ASOs. GalNAc conjugation improved potency of ASOs containing 2'-O-methyl (2'-O-Me), 3'-fluoro hexitol nucleic acid (FHNA), locked nucleic acid (LNA), and constrained ethyl bicyclo nucleic acid (cEt BNA) 10-20-fold compared to unconjugated ASOs. We further demonstrate that GalNAc conjugation improves activity of 2'-O-(2-methoxyethyl) (2'-O-MOE) and Morpholino ASOs designed to correct splicing of survival motor neuron (SMN2) pre-mRNA in liver after subcutaneous administration. GalNAc modification thus represents a viable strategy for enhancing potency of ASO with diverse nucleic acid modifications and mechanisms of action for targets expressed in hepatocytes.

Relationship between exposure to tumour necrosis factor inhibitor therapy and incidence and severity of myocardial infarction in patients with rheumatoid arthritis.

OBJECTIVES: Patients with rheumatoid arthritis (RA) are at increased risk of myocardial infarction (MI) compared with subjects without RA, with the increased risk driven potentially by inflammation. Tumour necrosis factor inhibitors (TNFi) may modulate the risk and severity of MI. We compared the risk and severity of MI in patients treated with TNFi with that in those receiving synthetic disease-modifying antirheumatic drugs (sDMARDs). METHODS: This analysis included patients with RA recruited from 2001 to 2009 to the British Society for Rheumatology Biologics Register for Rheumatoid Arthritis starting TNFi (etanercept/infliximab/adalimumab) and a biologic-naïve comparator cohort receiving sDMARD. All patients were followed via physician and patient questionnaires and national death register linkage. Additionally, all patients were linked to the Myocardial Ischaemia National Audit Project, a national registry of hospitalisations for MI. Patients were censored at first verified MI, death, 90 days following TNFi discontinuation, last physician follow-up or 20 April 2010, whichever came first. The risk of first MI was compared between cohorts using COX regression, adjusted with propensity score deciles (PD). MI phenotype and severity were compared using descriptive statistics. 6-month mortality post MI was compared using logistic regression. RESULTS: 252 verified first MIs were analysed: 58 in 3058 patients receiving sDMARD and 194 in 11 200 patients receiving TNFi (median follow-up per person 3.5 years and 5.3 years, respectively). The PD-adjusted HR of MI in TNFi referent to sDMARD was 0.61 (95% CI 0.41 to 0.89). No statistically significant differences in MI severity or mortality were observed between treatment groups. CONCLUSIONS: Patients with RA receiving TNFi had a decreased risk of MI compared with patients with RA receiving sDMARD therapy over the medium term. This might be attributed to a direct action of TNFi on the atherosclerotic process or better overall disease control.

Risk of lymphoma in patients exposed to antitumour necrosis factor therapy: results from the British Society for Rheumatology Biologics Register for Rheumatoid Arthritis.

OBJECTIVES: Patients with rheumatoid arthritis (RA) are at increased risk of lymphoma compared with the general population. There are concerns that tumour necrosis factor inhibitors (TNFi) may exacerbate this risk. However, since the excess risk of lymphoma in RA is related to the cumulative burden of inflammation, TNFi may conversely reduce the risk of lymphoma by decreasing the burden of inflammation. The aim of this study was to compare the risk of lymphoma in subjects with RA treated with TNFi with those treated with non-biological therapy. METHODS: Subjects diagnosed by a rheumatologist with RA enrolled in the British Society for Rheumatology Rheumatoid Arthritis Register (BSRBR-RA), a prospective cohort study, were followed until first lymphoma, death or until 30 November 2013. Rates of lymphoma in the TNFi and non-biological-treated cohorts were compared using Cox regression. RESULTS: 11 931 TNFi-treated patients were compared with 3367 biological-naive patients. 84 lymphomas (88 (95% CI 70 to 109) per 100 000 person-years) were reported in the TNFi cohort and 30 lymphomas (154 (95% CI 104 to 220)) in the biological-naive cohort. After adjusting for differences in baseline characteristics, there was no difference in the risk of lymphoma for the TNFi versus the biological-naive group: HR 1.00 (95% CI 0.56 to 1.80). No risk differences were observed for individual TNFi. CONCLUSIONS: In medium-term follow-up, there is no evidence that tumour necrosis factor inhibition influences the risk of lymphoma over the background risk in subjects with RA.