# Active Projects

## 2021 Development Grants

### Small molecule modulators of bile acid metabolism to treat NAFLD

Small molecule modulators of bile acid metabolism to treat NAFLD/NASH

Snehal Chaudhari, Yoojin Lee (MGH), Raymond T. Chung (MGH), and A. Sloan Devlin

Liver diseases are growing worldwide health concerns and place significant burdens on healthcare systems. The prevalence of non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and liver cirrhosis is rising. There is a large, unmet need for effective treatments for these conditions. There are currently no approved medications for NAFLD/NASH, and while off-label usage of type 2 diabetes medications has become common, evidence for benefits has been limited. Drugs in the pipeline target later stages of fibrosis and display modest effects. Our data indicate that conjugated bile acids, a large class of host-produced endogenous metabolites, protect against pathogenic intestinal permeability, an early feature of NAFLD/NASH that contributes to the development of liver inflammation and injury. Our goal is to use small molecule inhibitors of gut bacterial enzymes that chemically modify bile acids to modulate the in vivo pool of these metabolites. We will test whether these inhibitors shift the bile acid pool toward beneficial conjugated bile acids and away from damaging unconjugated bile acids and thereby present a novel treatment strategy for NAFLD/NASH.

### A natural beta interferon-inducing therapeutic glycolipid

A natural beta interferon-inducing therapeutic glycolipid from the microbiome that prevents and treats severe viral infection

Tiandi Yang, Sungwhan Oh (BWH), Seung Bum Park (Seoul National University), and Dennis Kasper

One important clinical aspect of the current COVID-19 pandemic, as well as in influenza infection, is the wide variety of clinical presentations. Infections with either virus can lead to a clinical course that is anywhere on a spectrum from asymptomatic nasopharyngeal carriage to rapidly progressive fatal systemic disease. It has been shown that an important regulator of viral disease severity is host interferon beta (IFNβ)which is a crucial mediator of antiviral immunity and homeostatic immunoregulation. Our work has found that host natural IFNβ is regulated by the microbiome and specifically by glycolipids present on the surface of microbes from the phylum Bacteroides. We discovered that the critical Bacteroides molecule responsible for IFNβ induction is the outer membrane-associated lipooligosaccharide(LOS). LOS molecules regulate the IFNβ response both locally and systemically through induction in colonic dendritic cells and we have identified the molecular mechanism by which this occurs. The LOS of Bacteroides exists naturally in multiple structural variations. Our goal in this proposal is to identify a specific structural analog of the numerous glycolipid structures belonging to the lipooligosaccharides(LOS) of the Bacteroides phylum. Once we purify a specific IFNβ inducing structure, we will test it for prophylaxis and therapy in models of influenza and COVID-19 infection. Since these structures can be synthesized, our approach presents an exciting opportunity to develop a low-cost approach to minimizing severity of viral diseases as well as other diseases for which type 1 interferons can be used therapeutically.

### Gene therapy for the treatment of Wolfram Syndrome II

Gene therapy for the treatment of Wolfram Syndrome II

Michael Florea, David Anderson, Luk Vandenberghe (MEEI), and Amy Wagers

Wolfram Syndrome II is a progeria (early aging disease) characterized by accelerated senescence and death by mid-adulthood. There is no existing treatment. The disease affects multiple organ systems, and is caused by loss of function of the Cisd2 gene, which normally is expressed in many different cell types. As a result, it is thought that effective treatment of Wolfram Syndrome II will require multi-organ rescue of Cisd2 expression. Unfortunately, standard gene transfer technologies have not achieved sufficiently broad and uniform gene delivery to meet this therapeutic goal. We therefore created a new system (DAEUS) that overcomes the limitations of standard vector systems, and applied it successfully to ameliorate disease pathology in Cisd2 knockout mice. With Q-FASTR, we aim to perform dose-ranging studies and optimize component ratios of the DAEUS-Cisd2 system, ultimately deriving a dose-guiding model for achieving optimal therapeutic effects. This effort will de-risk our approach and lay a foundation for further development of multi-organ gene therapies.

## 2021 Pilot Grants

### Novel biologics for cancer therapy

Novel biologics for cancer therapy

Chun-Ting Wu, Raphael Ferreira, and George Church

Despite recent successful CAR-T outcomes in liquid tumors, its effect on solid tumors has not been achieved. This is mostly because the immunosuppressive tumor microenvironment inhibits the functions of T cells. Here, we have designed a novel biologic, which interacts with tumors in a different manner, that overcomes the CAR-T therapies’ limitations. This novel cancer-elimination mechanism is independent of inherent immune functions, such that it would not be inhibited by the tumor microenvironment. Our technology opens up brand new approaches for engineered cells to eliminate cancer.

### Studies on Immunogenic Lipids from the Gut Microbiome

Studies on Immunogenic Lipids from the Gut Microbiome

Munhyung Bae, David Szamosvari, Sunghee Bang, and Jon Clardy

The gut microbiome’s composition influences human health both positively and negatively, and most of these influences involve regulating inflammatory immune responses. (1) For example, multiple studies, including in vivo human studies, have shown that Muciniphila akkermansia has positive effects on metabolism and response to cancer immunotherapy, but the underlying molecular mechanism for these responses remains unknown. Finding the answer to this and many similar questions is complicated because it requires deconvoluting the interaction of two complex systems: the gut microbiome and our immune systems. In collaboration with Ramnik Xavier’s laboratory (MGH, Broad, and Department of Genetics) we believe that we have found the answer: a small phospholipid making up part of the M. akkermansia’s cell membrane. Synthetic lipid recapitulates the effects of isolated lipid and of the intact bacteria. We plan to explore the structure-activity space to develop therapeutic agents that could be used as vaccine adjuvants, metabolic disease modifiers, and cancer chemotherapy enhancers.

### CNS drug delivery by identifying small molecule inhibitors

CNS drug delivery by identifying small molecule inhibitors of a newly identified key regulator of the blood brain barrier

Joseph Amick 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 discoveries have changed our understanding of how the BBB restricts blood-brain communication1-4. 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 junctions5. However, substances can also cross endothelial cells by transcytosis, and we discovered that transcytosis is actively inhibitedin 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 permeable1-3. We propose to develop small molecule inhibitors as therapeutic agents that specifically target Mfsd2a function as a strategy to facilitate drug delivery across the BBB

### A novel monoclonal antibody that targets SARS-COV-2 3a

Development of a novel monoclonal antibody that targets SARS-COV-2 3a

William Robins and John Mekalanos

The damage to the lung and other organs during SARS-CoV-2 infection is driven by inflammation induced by the virus. Independent of vaccine-based strategies, distinct efforts to curtail severe acute and chronic lung disease by targeting inflammation following infection have not yet been proven completely safe and efficacious. The complex interactions between SARS-CoV-2 virus and the immune system during virus replication appear to be distinct from that of other respiratory viruses and are not yet entirely understood. There are SARS-CoV/SARS-CoV-2 gene products demonstrated to induce and modulate inflammation. This includes viroporin 3a, the virus-encoded K+ efflux membrane protein on the cell surface that activates the NLRP3 inflammasome. The exposed N-terminal 42 amino acid 3a ectodomain extends out from the channel pore and appears to be under strong immune selection based on our pan-genome covariance analysis of lineage B b-coronaviruses. We propose screening and identifying human mAbs with high affinity to exposed extracellular regions of 3a including ectodomain. Using cloned and purified mAbs as candidates, we will use established screens to find those that inhibit 3a activities including viroporin ion channel activity, and may then reduce both inflammation and pathogenesis.

### Small molecule drugs for advanced cancer with ecDNA amplicons

Small molecule drugs for advanced cancer with ecDNA amplicons

Tae Yoo, Patrick Flynn, and Timothy Mitchison

Impact: There is a huge need for drugs to treat advanced and metastatic solid cancers. Cells in such cancers usually have aberrant genomes with highly amplified oncogenes. Drugs have been developed to target certain amplified oncogenes as proteins, but this has been difficult in many cases. Our idea is to target the amplified DNA itself by exploiting fundamental differences in its chromosomal organization compared to normal DNA. Recent analysis showed that 20-60% of common solid tumors contain amplified DNA in the form of small, circular chromosomes called extrachromosomal DNA (ecDNA). These autonomous genetic elements, previously called “double minutes”, are typically 0.2-2MB in length, present at 2-100copies per cancer cell and absent from normal cells. They lack centromeres and segregate inefficiently at mitosis, resulting in copy number fluctuations that allow rapid evolution of cancer genotype. ecDNA is a highly abnormal state of chromosome organization that is specific to cancer cells. We propose it constitutes a druggable vulnerability and have identified a candidate target based on its DepMap dependency scores and cell biology.
Research Objectives: Develop an enzyme assay suited for inhibitor screening on the candidate target and test inhibitor scaffolds from in silico prediction or/and HTS screens. In parallel, develop cell-based assays for genetic depletion of the candidate target and test if it is selectively required for mitosis in cancer cells with high ecDNA copy number.

### A platform for transcriptome time-series

A platform for transcriptome time-series in single cells with applications to hematopoietic stem cells

Charlotte Strandkvist, Allon Klein, and Johan Paulsson

We will integrate live time-lapse imaging with genome-wide sequencing of RNA and chromatin, where one sister cell is harvested at every division in low-stress environments with exceptional uniformity in time and space. This provides vertical transcriptome time-series along lineages of single cells, while also providing information about growth, morphology and spatiotemporal reporter dynamics. We established feasibility of all key steps. As a proof-of-principle application we will use the platform to establish long-term maintenance, expansion and monitoring of primary tissue cells -- a ‘holy grail’ in regenerative biology and cell-based therapies. Specifically, we will expand hematopoeitic stem cells in culture for transplantation, while continuously tracking cell states along the lineages. If successful, this pilot will set the stage for optimizing additional stem cell and immunotherapy protocols, and produce (1) a commercializable research platform, (2) derivative methods to expand cells for therapy, and (3) cellular products that result from them.

### Enzyme prospecting for better diagnostics

Enzyme prospecting for better diagnostics

Seungwoo Chang, Joseph Loparo, and Michael Springer

The SARS-CoV-2 pandemic has highlighted the limitations in diagnostic testing approaches. Cheap, robust, and field-deployable tests that did not require instrumentation would change the nature of how we could respond and contain disease spread. A major limitation in the generation of point-of-need (PON) assays is enzyme performance. Many enzymes have been optimized to work well with high end machines, but similar effort has not been put into enzyme engineering for enzyme that will make field-deployable assay work well. Specifically, we need enzymes that perform rapidly and robustly, in vitro, at ambient temperature as opposed to elevated temperatures. Here we will use a combination of enzyme prospecting and enzyme engineering together with high throughput screening approaches to identify enzymes that have promissing characteristic for PON test development.

## 2020 Development Grants

### Evaluating novel transcription modulators for therapeutic impact

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.

### HIV-1 Vaccine using SPLANDID Antigen Presentation Technology

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 diabetes

A novel biologic and therapeutic target for the treatment of diabetes

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

### Novel therapeutic targets for treating pain/chronic itch

Identifying novel therapeutic targets for treating pain and chronic itch

David Ginty, Nikhil Sharma, Jing Peng

The perception of painful stimuli begins with detection of noxious stimuli by somatosensory neurons called nociceptors. Itch-inducing compounds, on the other hand, are detected by neurons called pruriceptors. We have recently identified the genes expressed in all somatosensory neuron subtypes, including nociceptors and pruriceptors, throughout development and into adulthood. This analysis revealed six transcriptionally distinct subtypes of nociceptors and two subtypes of pruriceptors, each with strikingly distinct gene expression profiles. Importantly, our findings reveal new candidate therapeutic targets to block nociceptors and pruriceptors. We found that select members of the GPCR superfamily, which account for nearly 1/3 of known drug targets, are expressed in nociceptors and pruriceptors, but not other sensory neuron subtypes. We will to leverage our knowledge of somatosensory neurons and the newly identified GPCRs they express to develop new drugs for treating pain and chronic itch while avoiding undesirable side effects. We will focus on drugs that activate Gi/o-coupled GPCRs expressed in nociceptors and pruriceptors because activation of these GPCR subtypes silence neuronal activity.

### Structure-guided discovery of novel antibiotics

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.

## 2019 Development Grants

### Monoclonal antibody therapeutics for Argentine hemorrhagic fever

Monoclonal antibody-based therapeutics for Argentine hemorrhagic fever

Lars Clark, Brian Gowen (USU), and Jonathan Abraham

Viral hemorrhagic fevers (VHFs) pose continuing threats to public health. Most have limited treatment options or vaccines, which underscores a point of vulnerability in public health. Passive immunization is an attractive treatment strategy; for example, transfusion of the monoclonal antibody (mAb) cocktail ZMappTM was a lead approach during the 2014-2016 Ebola virus outbreak. Results of testing in humans with ZMappTM did not reach statistical significance, suggesting that further studies would be required to fine-tune the approach. A mAb derived from the blood of an Ebola survivor, mAb114, was also recently deployed for testing in humans in the ongoing Ebola virus outbreak in the Democratic Republic of Congo.

While these investigational therapies are promising, there remains a large, unmet need for therapies against all agents that cause VHFs. We chose to focus on Argentine hemorrhagic fever (AHF) caused by the arenavirus Junin because antibody transfusions have a well-established track record of successfully treating this infection. We will isolate mAbs from the blood of AHF survivors, characterize the molecular basis for their antiviral activity, and test these for therapeutic effect in small animal models. We will also determine if the antibodies we isolate cross-react with Machupo, Guanarito, Sabia, and Chapare viruses, which are related arenaviruses that cause VHFs in South America and lack effective therapies. Once completed, the work would position candidate mAbs for pre-clinical testing in non-human primates, thus facilitating their translation into human use.

### Targeting apoptotic pathways for ALS, AD and PD

Targeting apoptotic pathways for ALS, Alzheimer’s Disease and Parkinson’s Disease

Sooyeon Jo, Laurel Heckman (BCH), Clifford Woolf (BCH), and Bruce Bean

The proposed work follows our recent discovery that targeting a class of voltage-dependent potassium channels inhibits death of motor neurons derived from induced pluripotent stem cells (iPSCs) from patients with ALS. These experiments were motivated by single cell RNA-expression data showing altered expression of a channel regulatory subunit in ALS motor neurons compared to their isogenic controls. This discovery serendipitously converged with an ongoing project developing small molecule modulators of these channels as investigational tools. We have now found that one of these compounds mitigates cell death of ALS patient iPSC-derived motor neurons. We therefore propose to test its ability to delay disease progression in a mouse model of ALS, to develop more potent inhibitors with drug-like properties, and to test the efficacy of these modulators in animal models of Parkinson’s disease and Alzheimer’s disease as a new treatment paradigm for preventing neurodegeneration by inhibiting apoptosis.

### Developing novel therapeutic approaches for CNS drug delivery

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.

## 2018 Development Grants

### Development of a new precision therapeutic for an important cancer target

Development of a new precision therapeutic for an important cancer target
Vidyasiri Vemulapalli, Jonathan LaRochelle, and Stephen Blacklow

The focus of this proposal is to develop a new class of potent and selective small molecule inhibitors of a genetically validated cancer target for further therapeutic development by enhancing the potency of our initial hit. Our inhibitors will occupy a unique niche in the landscape of small molecule precision therapeutics in cancer.