Objective: DNL343 is being investigated as a potential therapeutic agent for Amyotrophic Lateral Sclerosis (ALS). Background: ALS is a fatal neurodegenerative disease with TDP-43 inclusion pathology in 95% of patients. Chronic activation of the integrated stress response (ISR) may contribute to ALS by blocking translation, altering RNA and endosomal trafficking, and increasing formation of TDP-43-containing stress granules. DNL343 is a small molecule that activates a key ISR regulator, eIF2B, which inhibits ISR stress granule formation in cellular models and promotes neuroprotection in animal models. Design/Methods: The safety, pharmacokinetics (PK) and pharmacodynamics (PD) of DNL343 were evaluated in a Phase 1 randomized, placebo-controlled trial (RCT) in healthy volunteers (NCT04268784) and a 28-day Phase 1b RCT in ALS participants (NCT05006352), with an ongoing 18-month open label extension (OLE). ISR inhibition was evaluated by measuring CHAC1 gene expression and ATF4 protein in stimulated peripheral blood mononuclear cells (PBMCs). Results: In the Phase 1 study, ninety-five healthy participants were randomized (n=48 SAD, n=47 MAD). DNL343 was generally safe and well-tolerated with no serious adverse events (SAEs) or discontinuations related to study drug. DNL343 plasma concentrations were dose-dependent, with a plasma half-life of 38–46 hours and CSF-to-unbound plasma concentration ratio of 0.66–0.92. DNL343 attenuated two ISR biomarkers across the dosing period and at trough 24-hours after the last dose (CHAC1 [66–94%] and ATF4 [50–73%]) in all MAD cohorts. Safety, pharmacokinetics and ISR pharmacodynamics from the 28-day Phase 1b study in ALS participants will be presented. Conclusions: DNL343 is generally safe and well-tolerated at doses that demonstrate robust inhibition of ISR through CHAC1 and ATF4 inhibition. The pharmacokinetic profile supports once daily oral dosing and there is extensive CSF distribution. Data from these early-stage studies in HV and ALS patients support further development of DNL343 as a potential therapeutic for the treatment of ALS. Disclosure: Dr. Sun has received personal compensation for serving as an employee of Denali Therapeutics. Dr. Sun has stock in Denali Therapeutics. Dr. Tsai has received personal compensation for serving as an employee of Denali Therapeutics. Dr. Tsai has stock in Denali Therapeutics. Dr. Yulyaningsih has received personal compensation for serving as an employee of Denali Therapeutics. Dr. Yulyaningsih has stock in Denali Therapeutics. Dr. Yulyaningsih has stock in 23 & ME. Dr. Yulyaningsih has stock in Ardelyx. Dr. Yulyaningsih has stock in Amylyx. Dr. Fanok has received personal compensation for serving as an employee of Denali Therapeutics . An immediate family member of Dr. Fanok has received personal compensation in the range of $0-$499 for serving as an officer or member of the Board of Directors for Cincor Pharma. Dr. Fanok has stock in Denali Therapeutics. Mr. Vissers has received personal compensation for serving as an employee of Centre for Human Drug Research. Dr. Heuberger has received personal compensation for serving as an employee of Centre for Human Drug Research. Dr. Flores has nothing to disclose. Fen Huang has nothing to disclose. Dr. Kane has received personal compensation for serving as an employee of Denali Therapeutics. Dr. Kane has stock in Denali Therapeutics. Dr. Cohen has received personal compensation for serving as an employee of Denali. Dr. Cohen has stock in Denali. Dr. Dhuria has received personal compensation for serving as an employee of Denali Therapeutics. Dr. Dhuria has stock in Denali Thereapeutics. Dr. Fang has received personal compensation for serving as an employee of Denali Therapeutics. Dr. Fang has stock in Denali therapeutics. Dr. Estrada has stock in Denali Therapeutics . Dr. Osipov has received personal compensation for serving as an employee of Denali Therapeutics. Dr. Osipov has stock in Denali Therapeutics. Dr. Osipov has received intellectual property interests from a discovery or technology relating to health care. Mr. Willman-Yoswa has received personal compensation for serving as an employee of Denali Therapeutics. Mr. Maciuca has received personal compensation for serving as an employee of Denali Therapeutics. Mr. Maciuca has received stock or an ownership interest from Denali Therapeutics. Ms. Dobbins has received personal compensation for serving as an employee of Denali Therapeutics. Ms. Dobbins has stock in Denali Therapeutics. Miss Chau has received personal compensation for serving as an employee of Denali Therapeutics. Miss Chau has stock in Denali Therapeutics. Timothy Earr has received personal compensation for serving as an employee of Denali Therapeutics. Timothy Earr has stock in Denali Therapeutics. Ms. Nguyen has received personal compensation for serving as an employee of DENALI THERAPEUTICS. Ms. Nguyen has stock in Denali Therapeutics. Mrs. Lopez has nothing to disclose. Kimberly Scearce-Levie has received personal compensation for serving as an employee of Denali Therapeutics. Kimberly Scearce-Levie has stock in Denali Therapeutics. Mr. Bunte has nothing to disclose. Dr. Van den Berg has nothing to disclose. Carole Ho has nothing to disclose. Geert-Jan Groeneveld has nothing to disclose. Dr. Troyer has received personal compensation for serving as an employee of Denali Therapeutics Inc. Dr. Troyer has stock in Denali Therapeutics Inc. Dr. Troyer has stock in Merck & Co., Inc. Dr. Troyer has stock in Eli Lilly.

ABSTRACT The macrophage checkpoint receptor SIRPα signals against phagocytosis by binding CD47 expressed on all cells – including macrophages. Here, we found that inhibiting cis interactions between SIRPα and CD47 on the same macrophage increased engulfment (‘eating’) by approximately the same level as inhibiting trans interactions. Antibody blockade of CD47, as pursued in clinical trials against cancer, was applied separately to human-derived macrophages and to red blood cell (RBC) targets for phagocytosis, and both scenarios produced surprisingly similar increases in RBC engulfment. Blockade of both macrophages and targets resulted in hyper-phagocytosis, and knockdown of macrophage-CD47 likewise increased engulfment of ‘foreign’ cells and particles, decreased the baseline inhibitory signaling of SIRPα, and linearly increased binding of soluble CD47 in trans, consistent with cis-trans competition. Many cell types express both SIRPα and CD47, including mouse melanoma B16 cells, and CRISPR-mediated deletions modulate B16 phagocytosis, consistent with cis-trans competition. Additionally, soluble SIRPα binding to human CD47 displayed on Chinese hamster ovary (CHO) cells was suppressed by SIRPα co-display, and atomistic computations confirm SIRPα bends and binds CD47 in cis. Safety and efficacy profiles for CD47–SIRPα blockade might therefore reflect a disruption of both cis and trans interactions.

Lentiviruses infect many cell types and are now widely used for gene delivery in vitro, but in vivo uptake of these foreign vectors by macrophages is a limitation. Lentivectors are produced here from packaging cells that overexpress "Marker of Self" CD47, which inhibits macrophage uptake of cells when prophagocytic factors are also displayed. Single particle analyses show"hCD47-Lenti" display properly oriented human-CD47 for interactions with the macrophage's inhibitory receptor SIRPA. Macrophages derived from human and NOD/ SCID/Il2rg(-/-) (NSG) mice show a SIRPA-dependent decrease in transduction, i.e., transgene expression, by hCD47-Lenti compared to control Lenti. Consistent with known "Self" signaling pathways, macrophage transduction by control Lenti is decreased by drug inhibition of Myosin-II to the same levels as hCD47-Lenti. In contrast, human lung carcinoma cells express SIRPA and use it to enhance transduction by hCD47-Lenti- as illustrated by more efficient gene deletion using CRISPR/Cas9. Intravenous injection of hCD47-Lenti into NSG mice shows hCD47 prolongs circulation, unless a blocking anti-SIRPA is preinjected. in vivo transduction of spleen and liver macrophages also decreases for hCD47-Lenti while transduction of lung carcinoma xenografts increases. hCD47 could be useful when macrophage uptake is limiting on other viral vectors that are emerging in cancer treatments (e.g., Measles glycoprotein-pseudotyped lentivectors) and also in targeting various SIRPA-expressing tumors such as glioblastomas.

Macrophages and dendritic cells take up foreign microbes from the circulation and other tissues and often present microbial components to the adaptive immune system. Uptake of viruses including Lentiviral Vectors (LVs) by macrophages not only makes viral delivery inefficient, but also contributes to an unwanted immune response to vector components as well as delivered gene products. Macrophage uptake of micron-size particles and cells is inhibited by CD47 display on the surface of target particles by an interaction with macrophage receptor SIRPa. A novel LV was engineered to present an oriented human CD47-GFP fusion protein on the vector envelope with the aim of reducing uptake by macrophages without affecting transduction of other cells – which is indeed demonstrated. While fluorescent microscopy, flow cytometry, and western blotting confirm that virus as well as the producing cells indeed express CD47-GFP, the key physical question is whether virus displays CD47-GFP in the proper orientation. AFM-coupled nano-fluorescence imaging of viral vectors displaying envelope CD47 demonstrated colocalization of the GFP tagged protein with acridine orange stained RNA, and AFM imaging also showed viral vectors were rigid and quasi-spherical, ranging in diameter from 100 to 300 nanometers as expected. Kinetics of lentiviral vector binding specifically to anti-CD47 coverslips established the proper orientation of CD47-GFP on the viral surface. This result confirms the development of a novel lentiviral vector that properly displays CD47 to specifically minimize macrophage uptake and subsequent immune activation to virus.

Foreign particles and cells are cleared from the body by phagocytes that must also recognize and avoid clearance of "self" cells. The membrane protein CD47 is reportedly a "marker of self" in mice that impedes phagocytosis of self by signaling through the phagocyte receptor CD172a. Minimal "Self" peptides were computationally designed from human CD47 and then synthesized and attached to virus-size particles for intravenous injection into mice that express a CD172a variant compatible with hCD47. Self peptides delay macrophage-mediated clearance of nanoparticles, which promotes persistent circulation that enhances dye and drug delivery to tumors. Self-peptide affinity for CD172a is near the optimum measured for human CD172a variants, and Self peptide also potently inhibits nanoparticle uptake mediated by the contractile cytoskeleton. The reductionist approach reveals the importance of human Self peptides and their utility in enhancing drug delivery and imaging.

Immune response to viral gene therapy vectors and their transgene products is a significant problem in the field of gene therapy. Viral vectors, because they are derived from viruses, can induce an immune response. This makes gene delivery inefficient and can pose a significant danger to patients. Macrophages act as immunological gatekeepers at the interface of tissue and lymph. They take up antigens from the extracellular environment and then present them to the immune system. Macrophage uptake has been shown to be inhibited by CD47 interaction with SIRP alpha. Viral vectors presenting CD47 on their surface should show reduced levels of phagocytosis by macrophages, and thus reduced presentation and clearance by the immune system. In this work, HEK 293T cells were transduced, using a lentiviral vector, to over-express CD47 with green fluorescent protein (GFP) at the C-terminus. These transduced cells were then transfected to produce a second set of lentiviral vectors. Since the lentivirus takes a piece of the cell membrane to make its envelope when it buds from the cell, these vectors express CD47 on their envelope. The main goal of this work is to qualitatively and quantitatively characterize the presentation of CD47 by these lentiviral vectors. Fluorescent microscopy of equilibrium density gradient fractions indicates that these lentiviral vectors present CD47-GFP. The fluorescence intensity of individual and aggregated viral vectors was quantified. This will be the first step in using CD47 expression as a method to reduce immune response to lentiviral vectors in order to increase the efficacy and safety of lentiviral vector mediated gene therapy.

CD47 is a transmembrane protein that is a marker of “self”. CD47 binding to its cognate receptor in leukocytes and macrophages, signal-regulatory protein alpha (SIRPα), causes inhibition of inflammatory cell attachment. We hypothesized that immobilization of recombinant CD47 on polymeric surfaces would reduce inflammation. Recombinant CD47 was appended to polyvinyl chloride (PVC) or polyurethane (PU) surfaces via photoactivation chemistry. Cell culture studies showed that CD47 immobilization significantly reduced human neutrophil (HL-60) and human monocyte derived macrophage (MDM) (THP-1) attachment to PVC and PU respectively. A neutralizing antibody, directed against SIRPα, inhibited THP-1 and HL-60 binding to PU and PVC surfaces respectively. This antibody also increased the level of SIRPα tyrosine phosphorylation, thereby indicating a direct role for SIRPα mediated signaling in preventing inflammatory cell attachment. Studies using human blood in an ex vivo flow-loop showed that CD47 modified PVC tubing significantly reduced cell binding and neutrophil activation compared to unmodified tubing or poly-2-methoxy-ethylacrylate (PMEA) coated tubing. In ten-week rat subdermal implants, CD47 functionalized PU films showed a significant reduction in markers of MDM mediated oxidative degradation compared to unmodified PU. In conclusion, CD47 functionalized surfaces can resist inflammatory cell interactions both in vitro and in vivo.

Cells make a number of key decisions by actively applying forces to the objects that they 'touch'. How a macrophage decides to adhere to and 'eat'a foreign object (microbes to drug carriers) while leaving 'self'cells alone is a central decision in macrophage function. The molecularly specific adhesion systems that activate (or inhibit) will be shown to function from nanometer to micron length scales and to be made efficient by signaling (or not) to force-generating myosin motors in macrophages. Additional effects of particle shape - relevant to filamentous viruses perhaps - will be discussed. Collectively, the results suggest new means of achieving compatibility and the CD47 findings even appear relevant to engraftment of stem cells.

Phagocytes engulf foreign cells but not 'self' in part because self cells express CD47 as a ligand for signal regulatory protein SIRPalpha, which inhibits phagocytosis. Motivated by reports of upregulation of CD47 on both normal and cancerous stem cells [1: Jaiswal et al., 2009] and also by polymorphisms in SIRPalpha [2: Takenaka et al., 2007], we show here that inhibition of engulfment correlates with affinity of CD47 for SIRPalpha - but only at low levels of CD47. One common human polymorph of SIRPalpha is studied and binds more strongly to human-CD47 than to mouse-CD47 (K(d) approximately 0.12 microM and 6.9 microM, respectively) and does not bind sheep red blood cells (RBCs) - which are well-established targets of human macrophages; in comparison, a common mouse polymorph of SIRPalpha binds with similar affinity to human and mouse CD47 (K(d) approximately 0.22 microM). Using immunoglobulin (IgG)-opsonized particles with varying levels of either human- or mouse-CD47, the effective inhibition constants K(i) for blocking phagocytosis are then determined with both human- and mouse-derived macrophages. Only human phagocytes show significant differences in man versus mouse K(i)'s and only at CD47 levels below normal densities for RBCs. While phospho-signaling through human-SIRPalpha shows similar trends, consistent again with the affinity differences, saturating levels of CD47 (>K(i)) can signal and inhibit phagocytosis regardless of man versus mouse. Quantitative analyses here prompt more complete characterizations of both CD47 levels and SIRPalpha polymorphisms when attempting to study in vivo effects of these key proteins in innate immunity.

Phagocytosis of foreign cells or particles by macrophages is a rapid process that is inefficient when faced with "self" cells that display CD47-although signaling mechanisms in self-recognition have remained largely unknown. With human macrophages, we show the phagocytic synapse at cell contacts involves a basal level of actin-driven phagocytosis that, in the absence of species-specific CD47 signaling, is made more efficient by phospho-activated myosin. We use "foreign" sheep red blood cells (RBCs) together with CD47-blocked, antibody-opsonized human RBCs in order to visualize synaptic accumulation of phosphotyrosine, paxillin, F-actin, and the major motor isoform, nonmuscle myosin-IIA. When CD47 is functional, the macrophage counter-receptor and phosphatase-activator SIRPalpha localizes to the synapse, suppressing accumulation of phosphotyrosine and myosin without affecting F-actin. On both RBCs and microbeads, human CD47 potently inhibits phagocytosis as does direct inhibition of myosin. CD47-SIRPalpha interaction initiates a dephosphorylation cascade directed in part at phosphotyrosine in myosin. A point mutation turns off this motor's contribution to phagocytosis, suggesting that self-recognition inhibits contractile engulfment.

Interaction of spherical particles with cells and within animals has been studied extensively, but the effects of shape have received little attention. Here we use highly stable, polymer micelle assemblies known as filomicelles to compare the transport and trafficking of flexible filaments with spheres of similar chemistry. In rodents, filomicelles persisted in the circulation up to one week after intravenous injection. This is about ten times longer than their spherical counterparts and is more persistent than any known synthetic nanoparticle. Under fluid flow conditions, spheres and short filomicelles are taken up by cells more readily than longer filaments because the latter are extended by the flow. Preliminary results further demonstrate that filomicelles can effectively deliver the anticancer drug paclitaxel and shrink human-derived tumours in mice. Although these findings show that long-circulating vehicles need not be nanospheres, they also lend insight into possible shape effects of natural filamentous viruses.