Active Projects

Antibody-based therapeutics against Nipah virus

Haley Varnum, Vesna Brusic, and Jonathan Abraham

The spillover of emerging RNA viruses from their natural reservoirs into human populations continuously threatens global health. Antiviral monoclonal antibodies are an attractive approach to combating infection by emerging viruses, and FDA-approved drugs are now available to treat SARS-CoV-2 and Ebola virus infection. Nipah virus causes respiratory illness and encephalitis in humans with high fatality rates (50–100%) and can also be transmitted from person to person. However, there are no vaccines or drugs against

Nipah virus infection, highlighting a critical gap in pandemic preparedness. Our laboratory has extensive experience isolating monoclonal antibodies from the peripheral blood mononuclear cells (PBMCs) of human convalescent donors. We will use antigen-specific B-cell sorting to isolate monoclonal antibodies from the PBMCs of survivors of Nipah virus infection. We will test antibodies for in vitro activity against Nipah virus and a related virus (Hendra virus). We will use structural studies to determine the basis for the antiviral activity of the most potent neutralizing antibodies and use in vitro assays to map potential

resistance pathways. Our work will thus identify antiviral monoclonal antibodies that can be combined as cocktails to resist antibody neutralization escape and help meet an urgent need for medical countermeasures against Nipah virus.

Brain-Penetrant Kinase Inhibitors Targeting Alzheimer's and Related Dementias

Caitlin Mills, Mirra Chung, Paul Charifson, Peter Sorger, and Mark Albers (MGH, LSP)

Alzheimer’s dementia (AD) affects nearly 6 million US patients and costs > $250B/year. No therapies slow its devastating course. We have discovered a root cause of neuronal cell death in disease-relevant regions of autopsied AD brains: inflammation spatially coincident with genomically-encoded cytoplasmic double stranded RNA (cdsRNA). In mouse models, genomically encoded cdsRNA triggers neuroinflammation and neuronal death in a non-cell autonomous mechanism. We recapitulated this cascade of cdsRNA, neuroinflammation, and neuronal death in cultured differentiated human neural cells. This proposal focuses on small molecule kinases inhibitors that we have shown to block this cascade of cdsRNA-mediated neuronal death. Some of the active molecules are annotated as JAK inhibitors, but this class of FDA approved compounds is known to have complex poly-pharmacology and safety concerns. Moreover, a panel of JAK inhibitors heterogeneously rescued human neural cell death in culture. In our genome-wide CRISPR/Cas9 screen one protein kinase was the only JAK family member that conferred rescue when knocked out. In our pilot QFASTR award, we identified another protein kinase as an “off-target” kinase that was preferentially bound by neuroprotective JAK inhibitors relative to inactive JAK inhibitors. Moreover, we demonstrated that inhibitors of the two kinases rescue the neuronal cell death in two human neural cell lines. Here, we propose to focus on one of these protein kinases, validate it as a target by generating knockout human neural cell line using CRISPR/Cas9, and to proceed with SAR and in silico docking to generate leads that target the kinase with brain penetrant properties. Candidate leads that rescue in our phenotypic assay will be tested for brain penetration in our in vivo mouse model system. Successful execution will generate leads for further optimization.

Agents modulating B cell activation through the CD81-CD19 co-receptor complex

Katherine Susa, Anthony Banks, Andrew Kruse, and Steve Blacklow

B cell activation and antibody production are essential in the immune response to infection and for the development of lasting protective immunity. Inappropriate B cell activation, however, contributes to a wide variety of autoimmune diseases, including rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). Moreover, aberrant, sustained B cell receptor activation plays an important pathogenic role in chronic lymphocytic leukemia (CLL). Current therapies for these diseases have undesirable toxicities because they target all B cells (e.g. CAR-T cells for B cell leukemias), leading to immunosuppression. The B cell co-receptor, which includes the tetraspanin protein CD81, the signaling subunit CD19, and the complement-binding component CD21, lowers the threshold for B cell activation by approximately 1000-fold. Thus, agents blocking B cell co-receptor function will be powerful modulators of B cell activation. Here, we will develop agents targeting the B cell co-receptor subcomplex consisting of CD19 and CD81and assess their ability to modulate the activation threshold in primary human B cells, and their effect on the B cell signaling response, with the goal of identifying therapeutic candidates for treatment of B cell-driven autoimmune diseases and BCR-driven leukemias. These agents should be preferable to therapies targeting all B cells because they will suppress chronic signaling activity without depleting normal B cells.

A kinase inhibitor for malignant brain tumors

Dolen Emma, Tae Yeon Yoo, and Tim Mitchison

Malignant brain tumors, mostly glioblastoma, account for ~25,000 diagnoses and 16,000 deaths per year in the USA. Median survival from diagnosis is less than a year. Targeted- and immuno-therapies have made little impact. An effective small molecule drug would be transformative. We identified a protein kinase as a potential glioma/glioblastoma target using DepMap data as one of very few proteins that are much more essential in cell lines derived from CNS tumors than other cancers. The kinase helps re-build the nuclear envelope after mitosis and may have other functions. Recent preprints showed that it is selectively essential in CNS tumor lines because they tend to lack expression of its paralog. The aim of this grant is to screen for specific inhibitors of this kinase.

Identification of druggable targets against Vibrio pathogens

Raghuvir Viswanatha, William Robins, John Mekalanos, and Norbert Perrimon

Toxigenic Vibrio species cause disease in farmed shrimp, costing tens of billions of dollars of damage to the global shrimp industry. Building on previous work that contributed to understanding the causative agent, we now propose to develop neutralizing toxin-binders for use in rearing and farming food shrimp. Peptide based inhibitors have been successfully tested on pathogenic shrimp viruses, but not yet implemented to target bacterial toxins. Peptides that are efficacious in preventing shrimp disease will be of significant global economic value as they are cheap to produce and can be conveniently deployed in yeast, which are already a component of shrimp feed. The project objectives are to determine whether peptides identified by a combination of large-scale peptide panning and genome-wide CRISPR screening are protective against two Vibrio-borne toxins that cause disease, vPirAB and nigritoxin. Overlaying peptide panning on genetic screening reveals candidate peptides and a rationale for their inhibition, which could mimic binding between the toxin and the host receptor. For vPirAB, we have already conducted both screens, revealing partial overlap between the hits as predicted. Here, we propose to follow up on this finding by validating the genetic screens, optimizing the peptides using our cell culture model, testing the peptides on shrimp with a toxin challenge, and producing the expressed peptides cheaply in yeast. To our knowledge, this is the first merged application of these two approaches to identify host factors required for toxin activity and inhibitors, and could pave the way for expanded use of peptide therapeutics in aquaculture.

Evaluating antisense oligos for therapeutic impact in liver fibrosis and hepatocellular carcinoma

Benjamin Martin, Allen Mullen (UMASS Chan medical school) and Karen Adelman

Liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) are devastating diseases affecting millions of

people, with poor prognosis and few treatment options. Liver fibrosis develops from chronic liver injury, and its progression leads to cirrhosis, end stage liver disease, and liver failure. Advanced fibrosis is also a major risk factor for HCC. Liver fibrosis is reversible, so therapies that target fibrosis have the promise of preventing the progression of disease. There are currently no FDA-approved treatments that target the process of fibrosis and very limited options for medical management of HCC.

Several epigenetic factors are implicated in liver fibrosis and subsequent HCC, including chromatin remodeling and histone modifying complexes. In particular, amplification or over-expression of several epigenetic factors has been associated with dysregulated gene expression and poor prognosis in HCC. Accordingly, interventions that decrease expression levels of these proteins have been shown to reduce fibrosis and tumorigenicity in animal models of HCC. Here we propose to develop antisense oligos (ASOs) to specifically deplete selected epigenetic regulators. ASOs can be readily targeted to the liver, thus enabling a promising novel approach to deplete proteins selectively in this tissue, for targeted treatment or prevention of fibrosis, cirrhosis and HCC.

Developing Novel Agonists of Cytokine Receptors by Targeting Receptor Pre-Clusters

Anissa Belfetmi and James Chou

Members of the tumor necrosis factor receptor superfamily (TNFRSF) regulate proliferation of immune cells or induce programmed cell death. Developing antibodies to selectively activate or inhibit these receptors is of tremendous implication in the treatment of cancer or autoimmune diseases. In earlier study, we made an unexpected finding that, for several members of the TNFRSF, there exists a defined autoinhibitory, pre-clustered state formed by the receptor ectodomain, and that proteolytic removal of the ectodomain led to full receptor activation. Recently, we have obtained direct structural evidence of such preligand cluster, raising the prospect of developing new agonistic antibodies that act by disrupting the receptor preligand association. In this pilot research, we propose to develop agonistic single-domain antibodies (sdAbs) that specifically activate TNF receptors by disrupting the preligand association for enhancing antitumor activity of T cells, using a combination of Yeast Display and the SPLANDID antigen presentation technology.

Methods and applications of semi-permeable capsules with current application to high-throughput single cell multi-omics

Ignas Mazelis and Allon Klein

The focus of this proposal is the development of capsules, a technological platform with applications to isolation, growth, sorting, dialysis and genomic analysis of single cells or molecules. The platform addresses multiple needs ranging from measurement and diagnostics, screening, and potentially cell-based therapy. We recently developed a method to produce capsules robustly, with a novel composition that satisfies design requirements for these applications. A report-of-invention has been filed with HMS OTD (filing number HU8902). In this pilot grant, we have two specific aims: first, to extend the diversity of capsule compositions, and to characterize their biophysical properties and their biocompatibility. Second, to develop applications in single cell genomics. These efforts will characterize capsules as a novel and versatile reagent.

Manipulating mitobiogenesis by targeting ME2

Juan Menendz-Gonzalez, Azeem Sharda and David Scadden

Acute myeloid leukemia (AML) remains a highly lethal cancer and is increasing in incidence. Targeted therapies are modestly improving patient outcomes but only in small subsets of patients. We focused on the long-noted features of AML of increased mitochondrial number and increased oxidative phosphorylation dependence across genotypes to discover a distinct vulnerability to malic enzyme2 (ME2). We demonstrated that ME2 connects TCA activity with mitobiogenesis by serving as a fumarate sensor and regulating mitochondrial gene translation: mitochondrial genes encode proteins of the electron transport pathway. When ME2 is inhibited, metabolic crisis ensues and AML cells undergo apoptosis. This is due to the fumarate binding and not the catalytic activity of ME2. Animals

bearing human AML cells have markedly improved survival with ME2-fumarate binding inhibition. Notably, normal human and mouse hematopoietic cells express lower levels of ME2 and normal mouse hematopoietic cells were unaffected by ME2 inhibition in vivo. We have encouraging early data for primary and secondary screening assays for ME2 inhibition based on the dimerization of ME2 induced by fumarate affecting its catalytic activity. We seek to optimize and apply these assays in a high throughput manner to define small molecule inhibitors of ME2 dimerization with the goal of advancing genotype agnostic therapy for AML.

Evaluating splicing modulators for therapeutic impact in cohesin-mutant AML and MDS cells

Benjamin Martin, Zuzana Tothova (DFCI), and Karen Adelman

Cohesin is a multi-subunit protein complex that forms a ring-like structure around DNA. Cohesin is essential for sister chromatid cohesion, chromosome organization into looped domains, DNA damage repair and transcription regulation. Germline loss-of-function mutations in cohesin subunits cause a family of developmental disorders termed cohesinopathies. In addition, cohesin is one of the most frequently mutated protein complexes in cancer, including myeloid malignancies, with recurrent somatic loss-of-function mutations in core components of the cohesin ring and its modulators. Importantly, cancer-associated mutations in cohesin rarely affect chromosome integrity, but instead selectively impair gene-regulatory functions. However, how cohesin affects gene activity remains enigmatic, offering no clues towards intervention. Consequently, there are no targeted therapeutic approaches available to treat disease involving cohesin mutations. Recently, we discovered that cohesin mutations common in myeloid malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) disrupt RNA splicing. We propose to define the aberrant RNA species generated in cohesin-mutant AML cells, using state-of-the-art molecular and computational approaches to capture nascent transcripts and characterize RNA processing. Through detailed investigation of gene activity and splicing profiles in cohesin-mutant AML cells, we hope to discover genes with alternative splicing events that may present opportunities for therapeutic targeting. Our goal is to leverage this knowledge to develop targeted approaches that will selectively kill cohesin-mediated disease. The findings from these studies have the potential to lead directly to one or more clinical trials within the next several years.

Application of the SPLANDID Antigen Presentation Technology to HIV-1 Vaccine Development

Alessandro Piai, Yongfei Cai, Bing Chen, and James J. Chou.

The HIV-1 envelope glycoprotein (Env) is a transmembrane protein sitting in the viral membrane and it is also a major target for B-cell based vaccine development. Previous large clinical trials of vaccine candidates involving recombinant Env preparations mostly focused on various soluble forms of its ectodomain. These efforts have been disappointing, however, probably because we do not fully understand how effective antibodies are generated in some infected patients and what is the best form of the Env that can induce such an antibody response by vaccination. To facilitate Env immunogen design, we have recently developed an antigen presentation technology known as SPLANDID that allows presentation of transmembrane immunogens in a membrane environment on nanoparticles suitable for in vivo immunogenicity studies. This HMS patented technology now affords the opportunity to test novel HIV-1 Env immunogens that include the membrane-related components in a membrane environment and better mimic the native Env on the virion surface. We plan to use the new technology to display membrane-bound Env immunogens and evaluate their potential as vaccine candidates by defining their antigenicity in vitro and testing their immunogenicity in animals.

A novel biologic and therapeutic target for the treatment of diabetes

José Rivera-Feliciano, Timothy R. Kunz, Elias S. Peterson and Douglas A. Melton

It is estimated that more than 422 million people in the world have diabetes; by 2021 the cost burden of diabetes in the United States is on track to be $512 billion. Diabetes, characterized by elevated blood glucose (sugar) levels, results from either beta-cell malfunction (Type 2) or beta-cell demise after an autoimmune attack (Type 1). The hormone insulin, produced by pancreatic beta-cells, reduces circulating levels of glucose after a meal primarily by shuttling glucose to muscle and fat. Current strategies to treat diabetics include insulin injection, augmenting endogenous insulin secretion, increasing glucose absorption, or increasing glucose excretion. Hypoglycemic (low blood glucose) episodes after insulin injection are life threatening and this risk usually results in patients underdosing. Here we describe the discovery of a novel peptide hormone, we call ERSEQ08, present in human beta-cells, that can reduce blood glucose levels in mice. Importantly, this effect is independent of insulin action. Surprisingly, ERSEQ08 lowers blood glucose, in a glucose dependent manner, without causing hypoglycemia—thus overcoming one of the major setbacks with current diabetic treatments. While it is well known that pancreatic beta cells make Insulin, this new finding may represent a second system by which beta cells regulate glucose metabolism.

The potential to bypass insulin resistance presents a novel therapeutic paradigm to treat diabetes. As a biologic, this hormone may complement or replace some of the therapies used as the current standard of care in diabetes treatment. As an inroad into a novel signaling system controlling glucose homeostasis, this discovery has the potential to lead to a new kind of drug for diabetics.

Our main objectives using the Q-FASTR funds are: to test the activity of recombinant human ERSEQ08 protein to lower blood glucose levels in mice; to develop a cell-based assay to identify ERSEQ08’s mechanism of action; and, to find the minimal active fragment required for ERSEQ08’s function.

Development of an inexpensive at-home influenza kit and detection technology

Sarah Boswell, Jason Qian, Zhixiang Lu, Mary Pettit, and Michael Springer

Influenza is a major drain on the US economy. Antiviral drugs are most effective against influenza when taken early, often before patients seek medical help. Tests exist for influenza but are only effective when influenza titer is high and/or require sophisticated medical equipment. Here we will build off of methods we are combining and developing for the specific detection of barcoded microorganisms (a DARPA funded project) to develop and optimize a quantitative, cheap, rapid, sensitive, selective, and field-deployable method for detecting influenza. We aim to develop this influenza detection system into an affordable ‘at-home’ system allowing individuals to detect influenza early, thereby increasing the efficacy of antiviral drugs and eliminating unnecessary trips to the ER.

Protein therapeutics for treatment of chronic and inflammatory pain

Bruce Bean, Clifford Woolf, Jeff Way, and Peter Sorger

Chronic and inflammatory pain affect millions and new treatments are urgently

needed. Despite significant investment in opioid safety and formulation, there is little exploration of drugs with fundamentally novel mechanisms of action. Our goal is to develop such medicines based on novel protein therapeutics that block pain sensation by silencing multiple NaV sodium channels in nociceptors. The failure of NaV1.7 inhibitors as analgesics1 suggests that blocking more than one channel will be necessary. We will therefore take an approach involving a multivalent scaffold and small proteins that inhibit several NaV channels with tunable selectivity and potency. Activity will be measured by differential screening against human iPSC-derived sensory, cortical, and motor neurons. Over a two year period we expect to achieve hit/lead generation for 10-30 distinct molecules and optimization of the most active for testing in animals.

Structure-guided discovery of novel antibiotics inhibiting bacterial cell wall formation

Francois Thelot and Maofu Liao

The emergence of drug-resistant Gram-negative bacteria causes a significant global health problem, because the available antibiotics are limited and the discovery of new compounds cannot keep pace with the emergence of drug-resistant strains. The difficulty to combat these pathogens is largely due to their unique dual-membrane cell wall which efficiently blocks the entry of antibiotics. Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria plays key roles in cell wall formation and antibiotic resistance. Thus, LPS biosynthesis is a particularly attractive target for developing new classes of antibiotics. LPS is synthesized in the inner membrane and subsequently transported to the outer membrane, a process critically dependent on two ATP-binding cassette transporters (MsbA and LptB₂FGC). My laboratory has made important contributions in characterizing the structure and function of these LPS transporters. Here we propose to leverage our experience and mechanistic insights of MsbA to develop novel antibiotics. This will be achieved by establishing a targeted screening pipeline that comprises chemical screen, cryo-electron microscopy (cryo-EM), computational docking, and activity assays. Once established, this pipeline can be applied to target many other bacterial membrane transporters.

Developing novel therapeutic approaches for CNS drug delivery by targeting a newly identified key regulator of the blood brain barrier

Urs Langen and Chenghua Gu

A major obstacle in treating neurological diseases and brain tumors is to deliver drugs or antibodies across the ‘blood brain barrier’ (BBB). My lab’s recent discoveries have changed our understanding of how the BBB restricts blood-brain communication. The BBB is formed by a single layer of endothelial cells that lines the blood vessel walls and act as a gatekeeper for the brain. Historically, the restricted permeability of brain vasculature has been attributed to tight junctions. However, substances can also cross endothelial cells by transcytosis, and we discovered that transcytosis is actively inhibited in brain endothelial cells. We identified a

novel multi-transmembrane protein Mfsd2a as a key regulator for BBB function, and demonstrated that interfering with Mfsd2a and its downstream pathway upregulates transcytosis and causes the BBB to become permeable. We propose to develop therapeutic agents that specifically target Mfsd2a function as a strategy to facilitate drug delivery across the BBB.