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Principal Investigator: Kenan Christopher GARCIA
Organization: STANFORD UNIVERSITY
Fiscal Year: 2020
Award: $553,964
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
ABSTRACT
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
has threatened global health. The severity of disease and rising number of deaths from SARS-CoV-2
have raised an urgent need for effective therapies. Besides respiratory symptoms, 20-50% of patients
exhibit gastrointestinal symptoms such as diarrhea and emesis. In addition, clinical evidence shows
that viral RNA can be found in rectal swabs, indicating that the intestine may be a critical target of
SARS-CoV-2 infection. In this proposal, we engineer novel high-affinity blocking agents for known entry
receptors of SARS-CoV-2 to prevent infection of human intestinal cells and pursue a longer-term goal
of structure-based discovery of novel receptor targets.
Aim 1 designs blocking agents that target the known interaction of SARS-CoV-2 S protein with
its primary entry receptor ACE2 (angiotensin-converting enzyme 2), as well as with a novel co-receptor,
CD147 (accessory protein for monocarboxylate transporters), both of which are expressed in human
small intestinal and colon epithelial cells. In Aim 1 we will engineer an ACE2/CD147 bi-specific agent
that can simultaneously target both SARS-CoV-2 S protein receptors to improve the efficiency and
specificity of viral blockade. We utilize in vitro protein evolution by yeast cell surface display to generate
high-affinity ACE2 and CD147 ECDs with improved affinity for SARS-CoV-2 S protein versus the wild-
type ECDs These will be combined into a single bispecific agent containing both ACE2 and CD147
affinity-matured ECDs and assayed in human intestinal organoids. In particular, we deploy intestinal
organoids with a “flipped polarity” where the apical ACE2-expressing aspect faces outwards towards
the surrounding ECM/media instead of towards the interior lumen to better model physiologic viral
infection. In Aim 2, we will screen a CRISPRa activating library for additional human SARS-CoV-2
secretome targets. The SARS-CoV-2 secretome, i.e. virus-encoded secreted or surface-exposed
transmembrane proteins, also facilitates infection of host cells and provides novel targets for SARS-
CoV-2 therapeutics. This proposal leverages expertise of Chris Garcia (Multi-PI of the parental R01)
in protein engineering, immunotherapeutics, and structural biology with Calvin Kuo (Multi-PI of the
parental R01) expertise in organoid generation and disease modelling to design targeted therapeutics
for SARS-CoV-2. We also utilize collaboration from the Manuel Amieva and Catherine Blish groups in
organoid apical-basal polarity inversion and BSL3 SARS-CoV-2 infection, respectively.
Terms: <2019 novel coronavirus><2019-nCoV><Affinity><Angiotensin Converting Enzyme><Angiotensin I-Converting Enzyme><Apical><Assay><Bioassay><Biologic Assays><Biological Assay><Biomedical Engineering><CD143 Antigens><COVID-19><COVID-19 epidemic><COVID-19 pandemic><COVID19><COVID19 epidemic><COVID19 pandemic><Carboxycathepsin><Cell Body><Cell Therapy><Cell surface><Cell-Extracellular Matrix><Cells><Cessation of life><Clinical><Collaborations><Colon><Death><Diarrhea><Dipeptidyl Peptidase A><Disease><Disease model><Disorder><ECM><Emesis><Engineering><Epithelial Cells><Evolution><Exhibits><Extracellular Matrix><Face><Generations><Goals><Human><Immunotherapeutic agent><In Vitro><Infection><Infection prevention><Integral Membrane Protein><Intestinal><Intestines><Intrinsic Membrane Protein><Kininase A><Kininase II><Libraries><Modern Man><Organoids><Patients><Peptidyl-Dipeptidase A><Prevent infection><Protein Engineering><Proteins><Receptor Protein><Respiratory Signs and Symptoms><SARS Virus><SARS corona virus><SARS coronavirus><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV-2><SARS-CoV2><SARS-Related Coronavirus><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related coronavirus 2><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe acute respiratory syndrome coronavirus 2><Severity of illness><Small Intestines><Specificity><Structure><Surface><Swab><Therapeutic><Transmembrane Protein><Transmembrane Protein Gene><Viral><Viral Diseases><Virus><Virus Diseases><Vomiting><Wuhan coronavirus><Yeasts><airway symptom><base><bio-engineered><bio-engineers><bioengineering><bowel><cell engineering><cell mediated therapies><cell-based therapy><cellular engineering><cellular therapy><corona virus disease 2019><corona virus disease 2019 epidemic><corona virus disease 2019 pandemic><coronavirus disease 2019><coronavirus disease 2019 epidemic><coronavirus disease 2019 pandemic><design><designing><disease severity><disorder model><effective therapy><effective treatment><faces><facial><gastrointestinal symptom><genetic protein engineering><global health><immune drugs><immune-based therapeutics><immunologic preparation><immunologic therapeutics><immunotherapeutics><immunotherapy agent><improved><novel><pandemic><pandemic disease><physiologic model><prevent><preventing><protein design><pulmonary symptom><receptor><rectal><respiratory><respiratory symptom><severe acute respiratory syndrome-CoV><small bowel><stem cell biology><structural biology><targeted agent><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><viral RNA><viral infection><virus RNA><virus infection><virus-induced disease>