BIOMED PhD Thesis Defense

Title:
Interaction of Lipid Nanoparticles with Pulmonary Innate Immune Cells in the Context of Acute Respiratory Distress Syndrome (ARDS)

Speaker:
Marco Zamora, PhD Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University
 
Advisors:
Jacob S. Brenner, MD, PhD
Assistant Professor
Perelman School of Medicine
Associate Director, Penn Health-Tech
University of Pennsylvania

Kara L. Spiller, PhD
URBN Professor of Biomedical Innovation
School of Biomedical Engineering, Science and Health Systems
Drexel University

Details:
The field of nanomedicine aims to achieve targeted delivery of therapeutic agents to specific organs and cell types. However, achieving this goal has been challenging for most targets. In this work we discuss two methods to achieve targeting of lipid nanoparticles (LNPs) to the lung. The first of these methods is antibody-mediated targeting to the pulmonary endothelium. Lung targeting using this method directs binding utilizing antibodies against endothelial surface markers like platelet endothelial cell adhesion molecule (PECAM), intercellular cell adhesion molecule (ICAM), or plasmalemma vesicle associated protein (PLVAP). The second method is physicochemical targeting which utilizes intrinsic physical properties (size, shape, charge) or chemical features (binding to specific endogenous proteins because of the nanoparticles’ chemical makeup) to effect organ tropism. For lung tropism, the use of a permanently charged cationic lipid introduced into the formulation allows for this targeting.
 
For both methods, it has not been fully understood what cell types in the lung take up these nanoparticles and what effects this may have on therapeutic translation of these modalities and if it changes in the context of acute lung injury (ALI). Here, we answer both questions and show that for both antibody-mediated and physicochemical targeting, the marginated neutrophils of the lung are avid players in the uptake of these nanoparticles. We further show that these marginated neutrophils actively take up these nanoparticles, effectively achieving co-equal uptake, underscoring their role as part of the reticuloendothelial system (RES). In models of acute lung injury, we go on to show that our lung-targeted nanoparticles are cleared faster likely due to activated marginated neutrophils.
 
Exploration of these mechanisms show that for antibody-mediated targeting, complement activation as well as non-specific recognition of the fragment crystallizable region (Fc) region on the antibodies covalently conjugated on the LNPs drove this uptake. However, for physicochemical targeted LNPs, thrombosis and coagulation was the key driving force in lung localization. This work further proposes methods to help reduce uptake by marginated neutrophils and prevent undesired side effects in these nano particles. This work provides insights into these mechanisms and their implications for therapeutic translation of LNPs.

BIOMED Seminar

Title:
Circuit Mechanisms and Molecular Determinants of a Visuomotor Transformation (VMT)

Speaker:
Mark Dombrovski, PhD
Postdoctoral Associate, Zipursky Lab
University of California, Los Angeles; Howard Hughes Medical Institute

Details:
Using interdisciplinary approaches (EM-connectomics, single-cell transcriptomics, genetics and physiology), Dr. Dombrovski and colleagues investigate how vision is transformed into action in the fruit fly brain.

Visuomotor transformation (VMT), a vital process by which the brain translates vision into action, requires precise synaptic connectivity between sensory and motor neural circuits. However, the developmental and molecular underpinnings of VMT remain poorly understood. We address this gap by examining the visuomotor interface in Drosophila, leveraging single-cell transcriptomics, EM-connectomics, genetics, and functional approaches to causally link genes and molecules with circuit function.

In our earlier work, we uncovered a novel wiring mechanism governing VMT known as synaptic gradients. This mechanism, operating between feature-detecting Visual Projection Neurons (VPN) and premotor Descending Neurons (DN), transforms object locations in the eye coordinates into directional body movements. Notably, synaptic gradients are independent of axonal and dendritic topography: individual neurons of the same VPN cell type exhibit gradients of synaptic weights with specific DNs reflecting visual field region they sample. This within-cell-type synaptic specificity results in the conversion of a visual space map into a gradient of synapses, representing a fundamentally new mechanism of neuronal connectivity.

But how do synaptic gradients emerge amid lack of spatial cues? Through single-cell transcriptomic analysis of VPNs during development, we discovered significant transcriptomic heterogeneity across individual neuronal types. Spatial gradients of Cell Adhesion Molecules (CAMs) regulating synaptic specificity, topographically aligned with synaptic gradients. We show that within-cell-type molecular gradients of DIP/Dpr and Beat/Side families of CAMs regulate axonal and dendritic synaptic gradients in looming escape and motion detection circuits, respectively. Thus, we propose a model where CAM gradients determine within-cell-type synaptic specificity independently of axonal and dendritic spatial organization. Ongoing functional experiments aim to validate this model.

This work expands our understanding of VMT origins, raises questions about cell type definition, and underscores the importance of multiomic approaches in deciphering the logic of brain wiring.

Biosketch:
Mark Dombrovski, PhD, received his BS degree in Biochemistry from Moscow State University in Russia in 2012, following which he dedicated two years to research at the Russian Center for Pediatric Oncology and Immunology, focusing on innovating anti-cancer drug delivery methods.

In 2014, Dr. Dombrovski started a PhD program in Neurobiology at the University of Virginia, supported by a Jefferson Fellowship under the mentorship of Barry Condron. Over five years, he investigated neural mechanisms underlying social behavior and introduced a new experimental model system featuring cooperative foraging behavior in fruit flies, which shed light on how socially relevant traits emerge during critical developmental windows.

After graduating in 2019, Dr. Dombrovski started a postdoc at the laboratory of Larry Zipursky, focusing on circuit wiring mechanisms in the fly visual system. His proposal to establish a new model system investigating synaptic connectivity patterns in the fly visuomotor interface received recognition through the Helen Hay Whitney postdoctoral fellowship. Collaborating with Dr. Gwyneth Card, Dr. Dombrovski uncovered a novel wiring strategy governing visuomotor transformations, prompting further investigation into its molecular determinants. He is now looking to establish his own Research Laboratory that will focus on further understanding the molecular origins of neuronal connectivity underlying complex behaviors.

BIOMED PhD Thesis Defense

Title:
Single Molecule Telomere Length Measurement and Its Application in Investigation of ALT

Speaker:
Heba Z. Abid, PhD Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University

Advisor:
Ming Xiao, PhD
Professor
School of Biomedical Engineering, Science and Health Systems
Drexel University

Details:
Telomere is a repeated sequence (TTAGGG)N that caps the end of the chromosomes to protect them from damage during DNA replication prior to cell division. Telomere length is essential to the cell's genomic stability and biological function. Each division shortened the telomere by approximately 40 to 200bp. After multiple rounds of cell division, telomere shortening leads to genomic instability, which triggers DNA Damage Response (DDR), leading to senescence or apoptosis. In rare cases, 1 out of 10 million cells activates the telomere maintenance mechanisms (TMMs) to maintain cell immortality and lead to cancer.  Approximately 85 to 90% of cancers maintain telomere length by activating telomerase expression, while the remaining 10-15% of cancers use Alternative Lengthening of Telomeres (ALT). Recent studies indicate that Homology-Dependent Repair (HDR) plays a role in the ALT pathway. Unlike Telomerase, HDR doesn't use a standard template; instead, HDR searches for a DNA template complement to the broken DNA strand. As a result, ALT telomeres are characterized by highly heterogeneous telomeres, including very long telomeres and telomere-free chromosome end, and unique telomere structures such as extrachromosomal telomeric DNA.

ALT cancers include some of the deadliest and most difficult-to-treat cancers. Understanding telomere repair mechanisms in ALT cancer presents a potential for finding biomarkers for cancer diagnosis and helps explore targets for anticancer therapy. Here, single-molecule telomere assay via Optical Mapping (SMTA-OM), an innovative technology that enables single telomere characterization and measurement, was utilized to study telomere repair and replication in ALT cancer cells. SMTA-OM allowed the identification of ALT-unique telomere structures, such as internal telomere-like sequences (ITS), which other telomere measurement methods couldn’t identify. Furthermore, SMTA-OM  is utilized to study the effects of inhibiting the replication stress response by depleting DNA2 and FANCM proteins in ALT cancer cells. The result shows that most chromosome arms exhibited longer telomeres increase in fusion/ITS+ frequency in comparison to the control cells, suggesting that the break-induced replication (BIR) pathway may be activated by the damaged telomeres. Additionally, the role of POLD3 and PIF1 in the BIR telomere Repair Pathway in ALT Cancer cells was investigated using SMTA-OM. The results provide insight into telomere maintenance and repair mechanisms in ALT cells and help identify ALT biomarkers and potential anticancer treatment targets.

BIOMED PhD Thesis Defense

Title:
Development of a Platform Technology To Impart Immunomodulatory Activity to Complex Biomaterials

Speaker:
Victoria Nash, PhD Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University

Advisor:
Kara Spiller, PhD
URBN Professor of Biomedical Innovation
School of Biomedical Engineering, Science and Health Systems
Drexel University

Details:
Tissue regeneration is a complex series of events, driven by highly plastic immune cells: macrophages. Typically, “pro-inflammatory” macrophages act early to support angiogenesis, while later acting “pro-reparative” macrophages support newly sprouted vasculature, assisting in tissue repair. Approaches used in biomaterials engineering to temporally influence macrophage phenotype are surface coating or encapsulation of cytokines, however these are not amenable to a variety of biomaterials. Affinity interactions, such as heparin or albumin have been leveraged for drug delivery and rely on weak interactions, like hydrogen bonding, to retain and deliver the drug. However, these systems require specific biomaterial formulations for drug delivery. While these systems work for small molecules and some amino acids, they are limited for cytokine delivery because weak interactions are not stable enough for effective delivery.

Biotin-avidin affinity becomes a favorable option because biotin, avidin (or its variants), can be directly conjugated to proteins, biomaterials, and even cells, without altering its bioactivity. Historically, avidin was first used as a model adjuvant, then explored as a protein carrier for adjuvants in vaccines, but modern uses of the affinity pair, biotin-avidin, range from analytical assays to targeted radioimmunotherapy. Biotin-avidin interactions are rarely used for drug release due to biotin’s low dissociation rate from avidin. However, release can be triggered by introducing free biotin to the system, promoting the release of biotinylated molecules from avidin. Yet it is not known how bioconjugation parameters can lead to spontaneous release between biotin and avidin. Or even how biotin or avidin influence macrophage phenotype. Therefore, the goal of this thesis is to determine how bioconjugation parameters can control biotin-avidin interactions to release an immunomodulatory cytokine from a model biomaterial to influence macrophage phenotype.

BIOMED PhD Thesis Defense

Title:
Characterizing Pharmacokinetic-Pharmacodynamic Relationships of VSV-GP

Speaker:
Richard M. Dambra, PhD Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University
Scientist
Boehringer Ingelheim

Advisors:
Joseph Ashour, PhD
Senior Principal Scientist
Boehringer Ingelheim

Lin Han, PhD
Professor
School of Biomedical Engineering, Science and Health Systems
Drexel University

Details:
Oncolytic viruses (OVs) are a re-emerging treatment modality that have the potential to destroy tumors, drive adaptive antitumor immune responses, and break barriers of immune tolerance to other immunotherapies. This premise of using replicating viruses as cancer therapeutics has led to numerous clinical trials and an FDA-approved product. However, OVs pose unique challenges for pharmacology-based drug development compared to traditional drugs. Pharmacokinetic (PK) and pharmacodynamic (PD) assessments – the study of drug fate and drug effect, respectively – are complicated by the viruses’ ability to replicate and their multifaceted mechanisms of action. Consequently, key PK-PD characteristics for OVs remain abstruse.

This thesis provides an in-depth characterization of the PK-PD relationships for a model OV, VSV-GP, after systemic administration in mice. First, methods were developed to quantify and differentiate the exposure contribution from the input virus and its replication using an inactivated tool virus, which provided a valuable PK characterization of viral replication that was applied throughout subsequent studies. Biodistribution (BD) was also assessed to quantify disposition and replication throughout tissues, revealing tissue-specific differences in distribution and permissivity to viral replication which was dependent on tissue resident macrophages. Based on this finding, experiments were then conducted to further evaluate the impacts of innate immune responses on VSV-GP PK/BD. These experiments highlighted the role of the type-I interferon response as a determinant of PK and BD. Finally, dose-exposure-response relationships for VSV-GP were thoroughly evaluated in tumor-bearing mice by modulating dose level and infusion rate and measuring several PK and PD markers. In these experiments, VSV-GP exhibited dose-proportional PK/BD which was impacted by the presence of a permissive tumor, but not by infusion rate. Furthermore, VSV-GP exhibited dose dependent PK-PD relationships which were predominantly driven by the overall systemic exposure.

Altogether, these efforts refined the PK/BD/PD profile for a replicating OV, elucidated PK-PD relationships, and developed a framework which could be applied to other replicating vectors. These advances may be helpful to inform safety and efficacy considerations, develop predictive PK-PD models, and optimize dose regimens to help realize the full potential of oncolytic virotherapy.

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