Ananth Annapragada - US grants

Abstract: We seek to use a novel cell-internalization SELEX (Systemic Evolution of Ligands by Exponential enrichment) to isolate thioaptamer-conjugated liposomal nanoparticles (TA-NP) that internalize and deliver its siRNA into the cytosol of tumor initiating cells (TIC) of oral squamous cell carcinoma, minimizing off-target delivery. In vivo delivery and release of siRNA from the endosomes to cytosol remain the two biggest obstacles for translating anticancer siRNA drugs to the clinic. Oral squamous cell carcinoma commonly known as oral cancer (OC) is the most common malignancy of head and neck with high global public health impact. Fanconi anemia (FA) is a hereditary cancer syndrome that predisposes one to OC. Evidence points to TIC as the key driver of field cancerization, resistance to therapy and disease relapse. A therapeutic vehicle that selectively targets and delivers anticancer drugs to TIC offers great promise for OC treatment. Our data and published studies show: (1).TIC is more enriched in FA-OC than sporadic OC; (2) Chk1 and CD147 promote TIC survival and chemoresistance and its overexpression contribute to poor prognosis in OC. The main goal of this proposal is to use FA- OC-TIC as target cells to develop TA- NP for the cytosolic delivery of siRNA. We hypothesize that isolated TA-NP will facilitate targeted delivery of siRNA to OC-TIC cytosol, and silence CD-147/Chk1 in OC-TIC's, disrupting their niche and rendering them chemosensitive and susceptible to elimination by chemotherapy with gemcitabine. Aims:(1): Elucidate the effect of RNAi mediated silencing of CD147/Chk1 in FA-OC-TIC; (2A): Select a list of TA-Liposomal NP (TA-NP) specific for TIC fraction of FA-OC cells, while avoiding hepatocyte uptake, using a positive and negative cell-uptake based SELEX; (2B): Determine the selected TA-NP's targeting specificity and therapeutic efficacy in in vitro and in murine OC xenografts. We will isolate ALDH+ TIC in FA-OC cell lines and examine the effects of siRNA-mediated silencing of CD147 and Chk1. (2) We will use a modified conjugate SELEX with positive and negative selections to identify OC-TIC-specific internalizing TA-NP. (3) We will validate TIC-targeting specificity and silencing efficacy of isolated TA-NP-sRNA in vitro and in murine orthotopic FA-OC xenografts. RNAi mediated inhibition of CD147 and Chk1 in OC-TIC will yield valuable insight into their potential role in the tumor propagating and chemoresistant properties of OC-TIC. The proposed TA-NP will have important clinical potentials for in vivo delivery of therapeutic and imaging agents targeting the OC-TIC.

This Small Business Technology Transfer (STTR) Phase I project?s primary objective is to develop a glucose-responsive platform for 24-hour delivery of insulin for type 1 and type 2 diabetes, which will cover the patient?s entire daily dose in a single administration. The company?s drug delivery technology platform is designed to passively deliver the right amount of insulin at the right time. Successful development of this technology is expected to 1) make basal and prandial insulin obsolete (a projected $32 B market in 2018), 2) reduce the need for constant monitoring and multiple injections per day, 3) improve glucose control, via a self-regulating system that requires no patient intervention post-dose, 4) better protect patients against long-term diabetic complications, and most importantly, 5) give those with diabetes a chance at a normal life.

The broader impact/commercial potential of this project is substantial. If successfully, the proposed platform could enable hundreds, if not thousands, of other stimulus-responsive medicines. The proposed delivery system can encapsulate many other types of drugs within the core particle system, and we have identified stimulus responsive elements that respond to hypoxia, inflammation, etc. The possibilities are limited only by the imagination of scientists. If medicines are released on a controlled basis, upon other types of stimulus, this technology may open up a world of continually personalized medicine that will substantially improve the health and quality of life for patients worldwide.

We  propose  a  novel  platform  technology  based  on  liposomal  MRI  imaging  agents  that  provide  methodology  for  safe,  facile  vascular  and  molecular  imaging  of  the  placenta.  We  focus  in  this  application  on  Morbidly  Adherent  Placenta  (MAP:  classified  as  placenta  accreta,  increta  or  percreta),  and  will  demonstrate  the  power  of  this  technology  for  the  study  of  this  condition,  noting  that  the  technology  once  developed  will  be  applicable  to  numerous  other  placental  conditions.  Diagnosis  of  MAP  remains  challenging,  even  with  ultrasound  followed  by  MRI  in  indeterminate  cases:  only  about  half  of  the  cases  of  MAP  are  suspected  prior  to  childbirth15.  MAP  results  in  massive  blood  loss  (25%  of  cases),  hysterectomy (70% of cases) and ICU admission (30% of cases), at rates far higher than the non-­?MAP population.   The  detection  of  a  ?retroplacental  clear  space?  is  a  marker  of  normal  placentation.  Further,  the  level  of  adrenomedullin and its receptor (the CRCLR/RAMP2 complex) is thought to be an early marker of MAP. The specific aims  of this project are therefore  1. Quantify placental margin delineation with the liposomal Gd contrast agent, in rodent models.  a. Test  the  visualization  of  the  ?retroplacental  clear  space?,  a  poorly  vascularized  layer  between  the  placenta and the myometrial wall, as a measure of margin delineation, as a function of gestational age.  b. Compare detection of the retroplacental clear space and placental margins with non-­?contrast MRI and  conventional Gd chelates, through the course of gestation.  2. Test whether the spatial expression of the Adrenomedullin receptor (CRCLR/RAMP2) in the placenta and uterine  wall correlate with placental invasion, across a range of gestational ages.  a. Visualize  and  quantify  CRCLR/RAMP2  with  MRI  using  an  adrenomedullin  targeted  Gd  liposome,  and  validate using immunohistochemistry  b. Correlate CRCLR/RAMP2 levels with imaging based margin delineation and histologically determined  placental invasion throughout gestation.   

PROJECT SUMMARY AND ABSTRACT: Radiation therapy (RT) is a mainstay of cancer treatment. However many tumors are resistant to RT, making it possible for microscopic tumor cells to remain or travel to other parts of the body and cause cancer recurrence at a later date. Immunotherapies can train the patient?s immune system to seek out and identify hidden tumor cells. The combination of RT with immunotherapy is a very exciting approach, but RT can have both immune- stimulating and immune suppressive effects, and further study is needed to understand how best to combine RT with immunotherapy. Many scientists now believe that understanding the tumor microenvironment ? the types of cells which make up a tumor, and their interactions ? is required to maximize the immune-stimulating effects of RT. We have discovered a strategy to alter the balance of cells in the tumor microenvironment of oral cancer and other solid tumor types, by simultaneously targeting immunosuppressive myeloid derived suppressor cells (MDSC) and regulatory T cells (Treg), so that immune-stimulating effects of RT predominate. Studies in mouse tumors show that this strategy is particularly effective when combined with an immunotherapy approach called ?checkpoint inhibition? that targets molecules that limit effectiveness of anti- tumor T cells. This leads to our scientific hypothesis that tumor-infiltrating MDSC and Treg render the tumor microenvironment resistant to immune activation by RT and/or checkpoint inhibition, and limit the induction of tumor-specific CD8+ T cells and other immune effector immune cells. The goals of this proposal are to 1) determine whether modulating the tumor immune microenvironment enhances responsiveness of oral cancer to RT and/or checkpoint inhibition, leading to long-lasting and powerful anti-tumor effects; 2) determine the immunological mechanisms which make these combination therapies effective; and 3) develop a novel drug formulation which will make this approach more effective and suitable for testing in clinical trials. We will accomplish these goals by carrying out the following specific aims: In Aim 1 we will functionally dissect the immune mechanisms by which MDSC contribute to radioresistance in mouse oral cancer models. In Aim 2 we will assess the ability of dual targeting of MDSC and Treg to sensitize oral cancer to treatment with RT + anti-PD-1 and induce durable protective memory responses. In Aim 3 we will develop a novel drug delivery system that can enhance delivery of inhibitors of MDSC function directly to the tumor and tumor-infiltrating MDSC.

This work is directed at characterizing pediatric COVID-19 and stratifying incoming patients by projected (future) disease severity. Such stratification has several implications: immediately improving treatment planning, and as disease mechanistic pathways are uncovered, directing treatment. Predicting future severity will inform the risks of outpatient treatment; to the patients themselves, their family, other caregivers/cohabitants, and to schools and employers. As varying levels of ?reopening? are adopted across the country (and the world), such prognostication will inform policy on the handling of pediatric carriers in the community. Based on our preliminary analysis we assert that a combination of novel assays including quantitative serology inflammatory markers (cytokine/chemokine profiles, immune profiles), transcriptomics, epigenomics, longitudinal physiological monitoring, time series analysis, imaging, radiomics and clinical observation including social determinants of health, contains adequate information even at early stages of infection to stratify the disease and predict disease severity. We propose an artificial intelligence/machine learning approach to integrate this rich and heterogeneous dataset, characterize the spectrum of disease and identify biosignatures that predict severity in progressive disease. To facilitate translation of the approaches developed in this work to a wide user community, we incorporate a Translational Development function, to oversee the design-control process and ensure readiness of our methods for regulatory review. Incorporated into our timelines are appropriate regulatory milestones intended to conform with the Emergency Use Authorization (EUA) programs in effect for SARS- CoV-2 diagnostics.