(4i) Genome Engineering and Systems Biology Tools for Probing Endo-Lysosomal Pathophysiology to Develop Novel Therapeutics

I am a trained physician with PhD in Chemical Engineering and have received multidisciplinary training in diverse fields. The overarching goal of my research is to leverage systems biology and genome engineering tools to transform how we understand endo-lysosomal and related organelle biogenesis and exploit them to engineer novel therapies.

Research Interests

Endosomes and lysosomes organelles(ELOs) are membrane-bound organelles. They are centers of hydrolytic activity required for the maintenance of cellular homeostasis. In addition, specific cell types such as cytotoxic T lymphocytes, mast cells, osteoclasts, melanocytes and endothelial cells contain lysosome-related organelles (LROs). They share a common origin with the endo-lysosomes and serve as specialized secretory organelles for the host cell. ELOs act as nutrient-sensing centers by integrating intra and extracellular metabolic inputs. ELOs also regulate cellular metabolic flux by recycling biomolecules from plasma membrane and cytosol, and controlling their degradation in the lysosomes. ELOs’ biochemical status is, therefore, a good indicator of overall cell health. ELOs dysfunction is a hallmark of several metabolic and degenerative diseases, cancers and aging. Moreover, many human viruses such as coronaviruses (SARS-CoV-2) hijack endolysosomal pathways to enter/exit host cells highlighting ELOs’ prominent role in viral pathogenesis.

Current metabolomics and proteomics approaches measure metabolites and proteins at whole cell level. ELOs typically occupy ~2-3% of a cell’s volume. Therefore, bulk whole-cell biochemical (proteomic and metabolomic) assays may not be able to capture subtle organelle-specific changes in diseased states underscoring the need to develop technologies that enable organelle-level resolution.

To address this, I have engineered cells in which organelles are tagged with short peptide sequences. Using antibodies against tags, I have purified intact ELOs/LROs from cell lysates and performed quantitative biochemical assays including proteomics and metabolomics.

Using unique organelle probing capabilities, I aim to investigate biochemical changes occurring in ELOs/LROs during health and disease. Furthermore, organelle measurements will be complemented with whole cell level quantitative transcriptomic and epigenomic studies in order to identify genes and chromatin regulatory landscapes mediating organelle level biochemical changes. These studies will identify potential pathways that will serve as target for development of novel therapies.

I will establish a leading research program focusing on the following three focus areas.

1. Uncovering Biochemical Pathways in Melanoma ELOs

Cancer cells display increased density as well as volume of acidic ELOs making them metabolically more fit than host cells thus enhancing their survival capacity. Understanding these biochemical differences and the genes driving these differences is key to find novel targets against cancers. My lab will perform quantitative biochemical (proteomic and metabolomic) profiling of purified ELOs, and whole cell transcriptomic and epigenomic studies on human melanoma. These studies will identify pathways that are hypo or hyperactive in melanoma cells and molecular machinery that leads to the expansion of cell’s acid compartment. These pathways will be targeted for novel drug development.

2. Improving Functionality of Cardiovascular Tissue Engineered Grafts by ELOs Characterization

Patient specific tissue engineered cardiovascular grafts require successful differentiation of patient derived adult stem cells/ induced pluripotent stem cells (iPSC) into cardiovascular lineages. However, metabolic heterogeneity among differentiated cells populations leads to reduced performance of engineered tissues. My lab will perform biochemical characterization of differentiated cell’s ELOs and compare them against gold-standard native human cardiovascular tissues. This similarity metric will be used to ascertain robustness of stem cell differentiation protocols, which we will test in a high-throughput manner employing design of experiments multivariate analysis (DOE). These strategies will enable manufacturing of metabolically stable clinical-grade homogenous cell populations for tissue engineering applications. Minimizing metabolic heterogeneity of cells as ascertained by quantitative biochemical (metabolomic/proteomic) studies on ELOs, will enable engineering of functionally robust and lasting tissue/organs and cell therapies.

3. Re-engineer the Genetic Circuitry of Aged ELOs to Reverse Cellular Aging

Aging leads to ELOs dysfunction likely due to structural and organelle acidity changes and contributes to pathophysiology of neurodegenerative disorders such as Alzheimer’s and Parkinson diseases. In order to decipher genetic circuitry and biochemical pathways involved in ELOs aging process, my lab will use progeroid-iPSC derived cells, aged patient cells and mouse models of aging to first document aging induced structural and biochemical changes in ELOs by quantitative metabolomics and proteomics as well as quantitative confocal and transmission electron microscopy studies. These studies complemented with RNA-seq, chromatin accessibility analyses will uncover molecular mechanisms behind ELOs aging. Using the CRISPR-Cas9 toolkit, we will re-engineer the defective genetic circuitry in the aged cell in order to mitigate effects of aging and restore ELOs functionality.

Impact of proposed studies

My proposed research program will address an enormous unmet need across spectrum of multiple human diseases and enable development of novel anti-cancer and cell and tissue engineering therapies.

Relevant Training

1. Postdoctoral Fellowship (Chemical and Systems Biology, Stanford University)

In the lab of Prof. Joanna Wysocka, I have led a Department of Defense and Stanford Institute for Stem Cell Biology and Regenerative Medicine funded study on melanosomes (ELOs/LROs) biogenesis/maturation and epigenomic reprogramming in human melanoma development. I have conducted a genome-wide CRISPR screen in melanocytes and have discovered several novel genes which regulate melanosome maturation and consequently melanogenesis in humans. The newly discovered genes have been validated by deleting them in-vitro in human melanocytes and in-vivo in animals and assaying their effect on biochemical composition immune-selected LROs/ ELOs by proteomic and metabolomic studies, electron/confocal microscopy and quantitative changes in pigmentation. Furthermore, I have deciphered the biochemical function of a novel candidate gene, which exerts its influence in melanosome maturation by affecting acidity (pH) of LROs/ELOs.

Reference: A genome-wide genetic screen uncovers novel determinants of human pigmentation (in revision)

In another study, I set out to identify earliest biomolecular events that reprogram normal melanocytes into melanoma cell upon acquiring cancerous mutations. I conducted epigenomic, transcriptomic, tumor xenograft and in-vivo imaging studies on primary human melanocytes and iPSC derived melanocytes after genetically modifying them to express oncogenes. This study identified a transcriptional network that activated upon oncogenic initiation and have discovered over 700 active enhancers (DNA elements) that likely drive melanoma initiation specific transcriptional program.

Reference: Transcriptional and epigenomic reprogramming landscape of BRAFv600e induced human melanomagenesis (in preparation)

2. Doctoral training (Chemical and Biological Engineering, SUNY-Buffalo)

I pursued my PhD in the lab of Prof. Stelios Andreadis, where I studied human stem cell reprogramming for cardiovascular tissue engineering applications. I developed novel method of differentiating smooth muscle cells (SMC) from patient iPSC in a stepwise manner, where I first induced epithelial-to-mesenchymal transition in iPSC, followed by differentiation into mesenchymal stem cells (MSC) and then mature SMC as evidenced by contractility of tissue engineered vascular grafts made of SMC. I further demonstrated that iPSC derived MSC were rejuvenated in terms of their mesenchymal differentiation capacity compared to old parental MSC from which they were derived.

In another study, I reprogrammed human skin keratinocytes into functional neural crest (NC) stem cells using chemical treatment without any genetic modification. I demonstrated that KC derived NC (KC-NC) could differentiate both in-vitro and in-vivo into all NC derivative cell types such as melanocytes, peripheral neurons, fat, bone, cartilage, Schwann cells. Each of these lineages were functionally mature highlighting KC-NC application in tissue engineering and regenerative medicine. (I co-wrote a grant proposal for this work which received a $1.7 million R01 grant from NIBIB, 1R01EB023114)

In another study, I collaboratively demonstrated that human hair follicle derived MSCs were clonally multipotent and not a collection of progenitor cells. I also contributed in a study where we functionally reversed the effects of aging in senescent and progeria patient muscle cells by introducing a pluripotency factor NANOG to the cells.

Funding

$235,500 PI Bajpai Dept. of the Army – USAMRAA 07/01/2017 – 06/30/2020

Teaching Interests

As I have been mentored/taught by many great people during my training over the years, I strongly believe in forwarding the gained knowledge. I have been involved in training students during my career. I believe in learning-by-doing principle and apply in teaching students. I taught medical physiology PGY451&PGY452 (summer2014, fall2014, spring2015) to a class of 25 students in Post-Baccalaureate Program, Associated Medical Schools of New York. Students in this program were from underrepresented minority students from a diversity of backgrounds. The teaching that I imparted to these students helped them learn basic physiology concepts and relate them with pathophysiological conditions. I keep receiving emails from them and it gives me immense happiness to see them succeed in their MD and post-MD careers across different US Medical Schools. In PhD, I was invited to take guest lectures for Tissue Engineering (CE 564, Fall 2015) and have tutored for Biomechanics & Biomaterials course (BSE 457, Fall 2007) course. Given my PhD in Chemical engineering, I am comfortable in teaching core chemical engineering courses (e.g. transport, reaction engineering, thermodynamics). I mentored five graduate students in PhD and have trained a California Institute of Regenerative Medicine scholar and graduate student in my postdoc. I am interested in developing a course that introduces engineering students to human pathophysiological principles and newer bioengineering technologies.

Publications (Published 13, 5 first authored, average impact factor, 11.7)

Full publications list link ⇐ Click here

https://scholar.google.com/citations?user=OWaeryMAAAAJ&hl=en&oi=ao