Evaluating the oral delivery of GalNAc-conjugated siRNAs in rodents and non-human primates.

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.

Implementation of a Clinical Vessel Wall MR Imaging Program at an Academic Medical Center.

BACKGROUND AND PURPOSE: The slow adoption of new advanced imaging techniques into clinical practice has been a long-standing challenge. Principles of implementation science and the reach, effectiveness, adoption, implementation, maintenance (RE-AIM) framework were used to build a clinical vessel wall imaging program at an academic medical center. MATERIALS AND METHODS: Six phases for implementing a clinical vessel wall MR imaging program were contextualized to the RE-AIM framework. Surveys were designed and distributed to MR imaging technologists and clinicians. Effectiveness was measured by surveying the perceived diagnostic value of vessel wall imaging among MR imaging technologists and clinicians, trends in case volumes in the clinical vessel wall imaging examination, and the number of coauthored vessel wall imaging-focused publications and abstracts. Adoption and implementation were measured by surveying stakeholders about workflow. Maintenance was measured by surveying MR imaging technologists on the value of teaching materials and online tip sheets. The Integration dimension was measured by the number of submitted research grants incorporating vessel wall imaging protocols. Feedback during the implementation phases and solicited through the survey is qualitatively summarized. Quantitative results are reported using descriptive statistics. RESULTS: Six phases of the RE-AIM framework focused on the following: 1) determining patient and disease representation, 2) matching resource availability and patient access, 3) establishing vessel MR wall imaging (VWI) expertise, 4) forming interdisciplinary teams, 5) iteratively refining workflow, and 6) integrating for maintenance and scale. Survey response rates were 48.3% (MR imaging technologists) and 71.4% (clinicians). Survey results showed that 90% of the MR imaging technologists agreed that they understood how vessel wall MR imaging adds diagnostic value to patient care. Most clinicians (91.3%) reported that vessel wall MR imaging results changed their diagnostic confidence or patient management. Case volumes of clinical vessel wall MR imaging performed from 2019 to 2022 rose from 22 to 205 examinations. Workflow challenges reported by MR imaging technologists included protocoling examinations and scan length. Feedback from ordering clinicians included the need for education about VWI indications, limitations, and availability. During the 3-year implementation period of the program, the interdisciplinary teams coauthored 27 publications and abstracts and submitted 13 research grants. CONCLUSIONS: Implementation of a clinical imaging program can be successful using the principles of the RE-AIM framework. Through iterative processes and the support of interdisciplinary teams, a vessel wall MR imaging program can be integrated through a dedicated clinical pipeline, add diagnostic value, support educational and research missions at an academic medical center, and become a center for excellence.

RNA interference in the era of nucleic acid therapeutics.

Two decades of research on RNA interference (RNAi) have transformed a breakthrough discovery in biology into a robust platform for a new class of medicines that modulate mRNA expression. Here we provide an overview of the trajectory of small-interfering RNA (siRNA) drug development, including the first approval in 2018 of a liver-targeted siRNA interference (RNAi) therapeutic in lipid nanoparticles and subsequent approvals of five more RNAi drugs, which used metabolically stable siRNAs combined with N-acetylgalactosamine ligands for conjugate-based liver delivery. We also consider the remaining challenges in the field, such as delivery to muscle, brain and other extrahepatic organs. Today's RNAi therapeutics exhibit high specificity, potency and durability, and are transitioning from applications in rare diseases to widespread, chronic conditions.

Stream hydrology and a pulse subsidy shape patterns of fish foraging.

Pulsed subsidy events create ephemeral fluxes of hyper-abundant resources that can shape annual patterns of consumption and growth for recipient consumers. However, environmental conditions strongly affect local resource availability for much of the year, and can heavily impact consumer foraging and growth patterns prior to pulsed subsidy events. Thus, a consumer's capacity to exploit pulse subsidy resources may be influenced by antecedent environmental conditions, but this has rarely been shown in nature and is unknown in aquatic ecosystems. Here, we sought to understand the importance of hydrologic variation and a salmon pulse subsidy on the foraging and growth patterns of two stream salmonids in a coastal southeast Alaska drainage. To do this, we sampled fish stomach contents at a high temporal frequency (daily-weekly measurements) and analyzed fish consumption rates in relation to streamflow and pulse subsidy resource availability. We then explored the influence of interannual hydrologic variation on access to pulse subsidy resources (i.e. whether fish exceeded an egg consumption gape limit) in a bioenergetic simulation. Prior to Pink Salmon spawning, Dolly Varden and Coho Salmon displayed distinct and nonlinear flow-foraging relationships, where forage for both species consisted primarily of macroinvertebrates. During this time period, consumption maxima coincided with baseflow and the highest observed flow conditions, and consumption minima were observed at severe low-water and intermediate flow values. After salmon spawning began, forage was not significantly related to flow and consisted primarily of salmon eggs. Further, consumption rates increased overall, and foraging patterns did not appear to be affected by flow in either species. Bioenergetic simulations revealed that patterns of interannual hydrologic variation may shift Coho Salmon growth trajectories among years. Together, our results suggest that access to marine pulse subsidy resources may depend on whether antecedent hydrologic conditions are suitable for juvenile salmonids to grow large enough to consume salmon eggs by the onset of spawning.

New Approaches for Targeting PCSK9: Small-Interfering Ribonucleic Acid and Genome Editing.

There is overwhelming clinical and genetic evidence supporting the concept that low-density-lipoprotein cholesterol should be as low as possible for as long as possible in patients at very high cardiovascular risk. Despite the wide availability of effective lipid-lowering therapies, the majority of patients still fail to reach guideline-based lipid goals. Advances in novel approaches targeting PCSK9 (proprotein convertase subtilisin/kexin type 9) through small-interfering RNA and genome editing hold the potential to bridge this gap, by offering long-acting alternatives, which may overcome adherence and other challenges in the current chronic care model. In this review, we discuss the history of targeting PCSK9 with the use of mRNA and small-interfering ribonucleic acid. We also shed light on targeting PCSK9 with genome editing, including discussion of the VERVE-101 clustered regularly interspaced short palindromic repeats-base editing medicine currently being evaluated in a clinical trial and others in development.

Fructose induced KHK-C can increase ER stress independent of its effect on lipogenesis to drive liver disease in diet-induced and genetic models of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform leads to unresolved endoplasmic reticulum (ER) stress when coupled with a HFD intake. Conversely, a liver-specific knockdown of KHK in mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in mice with genetically induced obesity or metabolic dysfunction, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes.

Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein hepatocyte-directed AAV transgene expression is silenced using the clinically validated modality of chemically modified small interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.

Social Isolation Activates Dormant Mammary Tumors, and Modifies Inflammatory and Mitochondrial Metabolic Pathways in the Rat Mammary Gland.

Although multifactorial in origin, one of the most impactful consequences of social isolation is an increase in breast cancer mortality. How this happens is unknown, but many studies have shown that social isolation increases circulating inflammatory cytokines and impairs mitochondrial metabolism. Using a preclinical Sprague Dawley rat model of estrogen receptor-positive breast cancer, we investigated whether social isolation impairs the response to tamoxifen therapy and increases the risk of tumors emerging from dormancy, and thus their recurrence. We also studied which signaling pathways in the mammary glands may be affected by social isolation in tamoxifen treated rats, and whether an anti-inflammatory herbal mixture blocks the effects of social isolation. Social isolation increased the risk of dormant mammary tumor recurrence after tamoxifen therapy. The elevated recurrence risk was associated with changes in multiple signaling pathways including an upregulation of IL6/JAK/STAT3 signaling in the mammary glands and tumors and suppression of the mitochondrial oxidative phosphorylation (OXPHOS) pathway. In addition, social isolation increased the expression of receptor for advanced glycation end-products (RAGE), consistent with impaired insulin sensitivity and weight gain linked to social isolation. In socially isolated animals, the herbal product inhibited IL6/JAK/STAT3 signaling, upregulated OXPHOS signaling, suppressed the expression of RAGE ligands S100a8 and S100a9, and prevented the increase in recurrence of dormant mammary tumors. Increased breast cancer mortality among socially isolated survivors may be most effectively prevented by focusing on the period following the completion of hormone therapy using interventions that simultaneously target several different pathways including inflammatory and mitochondrial metabolism pathways.

Fructose Induced KHK-C Increases ER Stress and Modulates Hepatic Transcriptome to Drive Liver Disease in Diet-Induced and Genetic Models of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform increases endoplasmic reticulum (ER) stress in a dose dependent fashion, so when fructose is coupled with a HFD intake it leads to unresolved ER stress. Conversely, a liver-specific knockdown of KHK in C57BL/6J male mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in genetically obesity ob/ob, db/db and lipodystrophic FIRKO male mice, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

Liver-specific FGFR4 knockdown in mice on an HFD increases bile acid synthesis and improves hepatic steatosis.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease with increased risk in patients with metabolic syndrome. There are no FDA-approved treatments, but FXR agonists have shown promising results in clinical studies for NAFLD management. In addition to FXR, fibroblast growth factor receptor FGFR4 is a key mediator of hepatic bile acid synthesis. Using N-acetylgalactosamine-conjugated siRNA, we knocked down FGFR4 specifically in the liver of mice on chow or high-fat diet and in mouse primary hepatocytes to determine the role of FGFR4 in metabolic processes and hepatic steatosis. Liver-specific FGFR4 silencing increased bile acid production and lowered serum cholesterol. Additionally, we found that high-fat diet-induced liver steatosis and insulin resistance improved following FGFR4 knockdown. These improvements were associated with activation of the FXR-FGF15 axis in intestinal cells, but not in hepatocytes. We conclude that targeting FGFR4 in the liver to activate the intestinal FXR-FGF15 axis may be a promising strategy for the treatment of NAFLD and metabolic dysfunction.

Folate receptor alpha in ovarian cancer tissue and patient serum is associated with disease burden and treatment outcomes.

BACKGROUND: Survival rates for ovarian cancer remain poor, and monitoring and prediction of therapeutic response may benefit from additional markers. Ovarian cancers frequently overexpress Folate Receptor alpha (FRα) and the soluble receptor (sFRα) is measurable in blood. Here we investigated sFRα as a potential biomarker. METHODS: We evaluated sFRα longitudinally, before and during neo-adjuvant, adjuvant and palliative therapies, and tumour FRα expression status by immunohistrochemistry. The impact of free FRα on the efficacy of anti-FRα treatments was evaluated by an antibody-dependent cellular cytotoxicity assay. RESULTS: Membrane and/or cytoplasmic FRα staining were observed in 52.7% tumours from 316 ovarian cancer patients with diverse histotypes. Circulating sFRα levels were significantly higher in patients, compared to healthy volunteers, specifically in patients sampled prior to neoadjuvant and palliative treatments. sFRα was associated with FRα cell membrane expression in the tumour. sFRα levels decreased alongside concurrent tumour burden in patients receiving standard therapies. High concentrations of sFRα partly reduced anti-FRα antibody tumour cell killing, an effect overcome by increased antibody doses. CONCLUSIONS: sFRα may present a non-invasive marker for tumour FRα expression, with the potential for monitoring patient response to treatment. Larger, prospective studies should evaluate FRα for assessing disease burden and response to systemic treatments.

Rare loss of function variants in the hepatokine gene INHBE protect from abdominal obesity.

Identifying genetic variants associated with lower waist-to-hip ratio can reveal new therapeutic targets for abdominal obesity. We use exome sequences from 362,679 individuals to identify genes associated with waist-to-hip ratio adjusted for BMI (WHRadjBMI), a surrogate for abdominal fat that is causally linked to type 2 diabetes and coronary heart disease. Predicted loss of function (pLOF) variants in INHBE associate with lower WHRadjBMI and this association replicates in data from AMP-T2D-GENES. INHBE encodes a secreted protein, the hepatokine activin E. In vitro characterization of the most common INHBE pLOF variant in our study, indicates an in-frame deletion resulting in a 90% reduction in secreted protein levels. We detect associations with lower WHRadjBMI for variants in ACVR1C, encoding an activin receptor, further highlighting the involvement of activins in regulating fat distribution. These findings highlight activin E as a potential therapeutic target for abdominal obesity, a phenotype linked to cardiometabolic disease.

From bench to bedside: Improving the clinical safety of GalNAc-siRNA conjugates using seed-pairing destabilization.

Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2'-5'-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.

The Essentials of Multiomics.

Within the last decade, the science of molecular testing has evolved from single gene and single protein analysis to broad molecular profiling as a standard of care, quickly transitioning from research to practice. Terms such as genomics, transcriptomics, proteomics, circulating omics, and artificial intelligence are now commonplace, and this rapid evolution has left us with a significant knowledge gap within the medical community. In this paper, we attempt to bridge that gap and prepare the physician in oncology for multiomics, a group of technologies that have gone from looming on the horizon to become a clinical reality. The era of multiomics is here, and we must prepare ourselves for this exciting new age of cancer medicine.

Repurposing the Homing Endonuclease I-SceI for Positive Selection and Development of Gene-Editing Technologies.

Prokaryote genomes encode diverse programmable DNA endonucleases with significant potential for biotechnology and gene editing. However, these endonucleases differ significantly in their properties, which must be screened and measured. While positive selection screens based on ccdB and barnase have been developed to evaluate such proteins, their high levels of toxicity make them challenging to use. Here, we develop and validate a more robust positive selection screen based on the homing endonuclease I-SceI. Candidate endonucleases target and cure the I-SceI expression plasmid preventing induction of I-SceI-mediated double strand DNA breaks that lead to cell death in E. coli. We validated this screen to measure the relative activity of SpCas9, xCas9, and eSpCas9 and demonstrated an ability to enrich for more active endonuclease variants from a mixed population. This system may be applied in high throughput to rapidly characterize novel programmable endonucleases and be adapted for directed evolution of endonuclease function.

The Nonclinical Disposition and Pharmacokinetic/Pharmacodynamic Properties of N-Acetylgalactosamine-Conjugated Small Interfering RNA Are Highly Predictable and Build Confidence in Translation to Human.

Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N-acetylgalactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterolemia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor-mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. SIGNIFICANCE STATEMENT: Several nonclinical ADME studies have been conducted in order to provide a comprehensive overview of the disposition and elimination of GalNAc-conjugated siRNAs and the pharmacokinetic/pharmacodynamic translation between species. These studies demonstrate that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species, building confidence in the ability to extrapolate to human.

NgAgo possesses guided DNA nicking activity.

Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-editing as they do not require sequence motifs adjacent to their targets for function, unlike popular CRISPR/Cas systems. One promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), has been the subject of debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with most of the protein being insoluble and inactive even after refolding. However, we report that the soluble fraction does indeed act as a nicking DNA endonuclease. NgAgo shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single-stranded DNA binding domain (repA). Both repA and the canonical PIWI domains participate in DNA cleavage activities of NgAgo. NgAgo can be programmed with guides to nick targeted DNA in Escherichia coli and in vitro 1 nt outside the 3' end of the guide sequence. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results demonstrate the potential of NgAgo for gene-editing and provide new insight into seemingly contradictory reports.

Association of the transthyretin variant V122I with polyneuropathy among individuals of African ancestry.

Hereditary transthyretin-mediated (hATTR) amyloidosis is an underdiagnosed, progressively debilitating disease caused by mutations in the transthyretin (TTR) gene. V122I, a common pathogenic TTR mutation, is found in 3-4% of individuals of African ancestry in the United States and has been associated with cardiomyopathy and heart failure. To better understand the phenotypic consequences of carrying V122I, we conducted a phenome-wide association study scanning 427 ICD diagnosis codes in UK Biobank participants of African ancestry (n = 6062). Significant associations were tested for replication in the Penn Medicine Biobank (n = 5737) and the Million Veteran Program (n = 82,382). V122I was significantly associated with polyneuropathy in the UK Biobank (odds ratio [OR] = 6.4, 95% confidence interval [CI] 2.6-15.6, p = 4.2 × 10-5), which was replicated in the Penn Medicine Biobank (OR = 1.6, 95% CI 1.2-2.4, p = 6.0 × 10-3) and Million Veteran Program (OR = 1.5, 95% CI 1.2-1.8, p = 1.8 × 10-4). Polyneuropathy prevalence among V122I carriers was 2.1%, 9.0%, and 4.8% in the UK Biobank, Penn Medicine Biobank, and Million Veteran Program, respectively. The cumulative incidence of common hATTR amyloidosis manifestations (carpal tunnel syndrome, polyneuropathy, cardiomyopathy, heart failure) was significantly enriched in V122I carriers compared with non-carriers (HR = 2.8, 95% CI 1.7-4.5, p = 2.6 × 10-5) in the UK Biobank, with 37.4% of V122I carriers having at least one of these manifestations by age 75. Our findings show that V122I carriers are at increased risk of polyneuropathy. These results also emphasize the underdiagnosis of disease in V122I carriers with a significant proportion of subjects showing phenotypic changes consistent with hATTR amyloidosis. Greater understanding of the manifestations associated with V122I is critical for earlier diagnosis and treatment.

Neurofilament Light Chain as a Biomarker of Hereditary Transthyretin-Mediated Amyloidosis.

OBJECTIVE: To identify changes in the proteome associated with onset and progression of hereditary transthyretin-mediated (hATTR) amyloidosis, also known as ATTRv amyloidosis, we performed an observational, case-controlled study that compared proteomes of patients with ATTRv amyloidosis and healthy controls. METHODS: Plasma levels of >1,000 proteins were measured in patients with ATTRv amyloidosis with polyneuropathy who received either placebo or patisiran in a Phase 3 study of patisiran (APOLLO), and in healthy controls. The effect of patisiran on the time profile of each protein was determined by linear mixed model at 0, 9, and 18 months. Neurofilament light chain (NfL) was further assessed with an orthogonal quantitative approach. RESULTS: Levels of 66 proteins were significantly changed with patisiran vs placebo, with NfL change most significant (p < 10-20). Analysis of changes in protein levels demonstrated that the proteome of patients treated with patisiran trended toward that of healthy controls at 18 months. Healthy controls' NfL levels were 4-fold lower than in patients with ATTRv amyloidosis with polyneuropathy (16.3 pg/mL vs 69.4 pg/mL, effect -53.1 pg/mL [95% confidence interval -60.5 to -45.9]). NfL levels at 18 months increased with placebo (99.5 pg/mL vs 63.2 pg/mL, effect 36.3 pg/mL [16.5-56.1]) and decreased with patisiran treatment (48.8 pg/mL vs 72.1 pg/mL, effect -23.3 pg/mL [-33.4 to -13.1]) from baseline. At 18 months, improvement in modified Neuropathy Impairment Score +7 score after patisiran treatment significantly correlated with reduced NfL (R = 0.43 [0.29-0.55]). CONCLUSIONS: Findings suggest that NfL may serve as a biomarker of nerve damage and polyneuropathy in ATTRv amyloidosis, enable earlier diagnosis of patients with ATTRv amyloidosis, and facilitate monitoring of disease progression. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that NfL levels may enable earlier diagnosis of polyneuropathy in patients with ATTRv amyloidosis and facilitate monitoring of disease progression.

Global loss of Tfr2 with concomitant induced iron deficiency greatly ameliorates the phenotype of a murine thalassemia intermedia model.

ß-thalassemias result from mutations in ß-globin, causing ineffective erythropoiesis and secondary iron overload due to inappropriately low levels of the iron regulatory hormone hepcidin. Mutations in transferrin receptor 2 (TFR2) lead to hereditary hemochromatosis (HH) as a result of inappropriately increased iron uptake from the diet, also due to improperly regulated hepcidin. TFR2 is also thought to be required for efficient erythropoiesis through its interaction with the erythropoietin receptor in erythroid progenitors. Transmembrane serine protease 6 (TMPRSS6), a membrane serine protease expressed selectively in the liver, participates in regulating hepcidin production in response to iron stores by cleaving hemojuvelin (HJV). We have previously demonstrated that inhibiting TMPRSS6 expression with a hepatocyte-specific siRNA formulation, induces hepcidin, mitigates anemia, and reduces iron overload in murine models of ß-thalassemia intermedia and HH. Here, we demonstrate that Tmprss6 siRNA treatment of double mutant Tfr2Y245X/Y245X HH Hbbth3/+ thalassemic mice induces hepcidin and diminishes tissue and serum iron levels. Importantly, treated double mutant animals produce more mature red blood cells and have a nearly 50% increase in hemoglobin compared to untreated ß-thalassemic mice. Furthermore, we also show that treatment of Tfr2Y245X/Y245X HH mice leads to increased hepcidin expression and reduced total body iron burden. These data indicate that siRNA suppression of Tmprss6, in conjunction with the targeting of TFR2, may be superior to inhibiting Tmprss6 alone in the treatment of the anemia and secondary iron loading in ß-thalassemia intermedia and may be useful as a method of suppressing the primary iron overload in TFR2-related (type 3) hereditary hemochromatosis.