USF Health researchers applied CRISPR technology to study the very large human non-protein coding gene expressed only in placenta, stem cells and certain cancers

TAMPA, Fla (March 16, 2020) — The placenta, an organ which attaches to the lining of the uterus during pregnancy, supplies maternal oxygen and nutrients to the growing fetus. Abnormal formation and growth of the placenta is considered an underlying cause of various pregnancy complications such as miscarriages, stillbirth, preeclampsia and fetal growth restriction. Yet, much remains to be learned about molecular mechanisms regulating development of this blood-vessel rich organ so vital to the health of a pregnant woman and her developing fetus.

Hana Totary-Jain, PhD, an associate professor of molecular pharmacology and physiology in the USF Health Morsani College of Medicine, was senior author of the study published in Scientific Reports.

University of South Florida Health (USF Health) Morsani College of Medicine researchers recently discovered how a very large human non-protein coding gene regulates epithelial-to-mesenchymal transition (EMT) – a process that contributes to placental development during early pregnancy, but can also promote cancer progression.

During the first trimester, fetal-derived placental cells known as trophoblasts invade the maternal uterine lining and modify its blood vessels to allow oxygenated blood to flow from the mother to fetus. However, trophoblast invasion requires tight regulation of EMT. If inadequate, trophoblast invasion is too shallow to adequately remodel the maternal blood vessels, and adverse pregnancy outcomes can occur. Conversely, excess EMT can cause exaggerated trophoblast invasion through the uterine wall leading to placenta accreta, a condition that can cause hemorrhage and often requires hysterectomy at delivery.

The USF Health researchers used a powerful genome editing technology called CRISPR (shorthand for “CRISPR-dCas9) to activate all of the chromosome 19 microRNA cluster (known as C19MC), so they could study the gene’s function in early pregnancy. C19MC — one of the largest microRNA gene clusters in the human genome — is normally turned off but becomes expressed only in the placenta, embryonic stem cells and certain cancers.

Dr. Totary-Jain discusses the molecular aspects of placenta development and pregnancy complications with research collaborator Umit Kayisli, PhD, a professor of obstetrics and gynecology at USF Health.

In their cell model study, published Feb. 20 in Scientific Reports, a Nature research journal, the USF Health team showed that robust activation of C19MC inhibited EMT gene expression, which has been shown to reduce trophoblast invasion.

But when trophoblast-like cells were exposed to hypoxia – a lack of oxygen similar to that occurring in early placental development — C19MC expression was significantly reduced, the researchers found. The loss of C19MC function causes differentiation of trophoblasts from stem-like epithelial cells into mesenchymal-like cells that can migrate and invade much like metastatic tumors. This EMT process helps explain trophoblast invasion and early placental formation.

“We were the first to use CRISPR to efficiently activate the entire gene, not just a few regions of this huge gene, in human cell lines,” said the paper’s senior author Hana Totary-Jain, PhD, an associate professor in the Department of Molecular Pharmacology and Physiology, USF Health Morsani College of Medicine. “Our study indicates C19MC plays a key role in regulating many genes important in early implantation and placental development and function. The regulation of these genes is critical for proper fetal growth.”

Above: Chromosome 19 microRNA cluster (stained purple) expressed in first-trimester placenta.  Below: In preparation for pregnancy, fetal trophoblast cells (brown) from which the placenta arises invade maternal decidual cells (pink) in the uterus lining. | Images courtesy of Hana Totary-Jain, originally published in Scientific Reports: doi.org/10.1038/s41598-020-59812-8

“You need EMT, but at some point the process needs to cease to prevent adverse pregnancy outcomes,” Dr. Totary-Jain said. “You really need a balance between not enough invasion and too much invasion, and C19MC is important in maintaining that balance.”

Dr. Totary-Jain and others in her department collaborated with colleagues in the medical college’s Department of Obstetrics and Gynecology on the project.

“The USF Health study offers new insight into how trophoblasts interact with the maternal uterine environment to become more invasive or less invasive in the formation of the placenta,” said coauthor Umit Kayisli, PhD, a USF Health professor of Obstetrics and Gynecology. “More research on microRNA expression and how it inhibits EMT may help us better understand the causes and potential prevention of preeclampsia and fetal growth restriction, which account for 5-to-10 percent of all pregnancy complications as well as spontaneous preterm births.”

Investigating the effects of altered C19MC expression on cell differentiation and trophoblast invasion has implications not only for a better understanding of normal and abnormal placental development, but also for cancer and stem cell research, Dr. Totary-Jain added.

Dr. Totary-Jain and Dr. Kayisli

Photos by Freddie Coleman, USF Health Communications and Marketing

The peptide-siRNA nanoparticle technology applied in the preclinical study was developed by USF Health Heart Institute researchers

Advanced ovarian and uterine cancers are deadly diseases. Ovarian cancers, in particular, present with vague symptoms common to other diseases, and often are not diagnosed until a late stage when cancer has spread throughout the abdomen.  More options are needed to effectively treat these metastasized gynecological cancers and improve patient survival rates.

A preclinical study published recently in Scientific Reports demonstrates that nanoparticle-delivered small interfering RNA (siRNA) targeting production of the protein AXL (AXL siRNA) inhibits metastasis of ovarian and uterine cancer cells.  The study was conducted by researchers at Washington University School of Medicine, St. Louis, Mo., and the USF Health Heart Institute, University of South Florida Morsani College of Medicine, Tampa, Fla.

3D illustration of ovarian cancer

The research team used a new nanoparticle system developed by USF Health co-investigators Samuel Wickline, MD, and Hua Pan, MBA, PhD, to test the experimental nanotherapy in human uterine and ovarian tumor cells and in immunodeficient mice implanted with these cancer cells.

“We’ve figured how to package in a simple peptide all the critical steps needed to efficiently get this particular small interfering RNA (also known as silencing RNA) into tumor cells and then release the siRNA so it can do its job,” said Dr. Wickline, a professor of cardiovascular sciences who direct the USF Health Heart Institute. “The nanoparticle basically hijacks the tumor cells’ biological machinery to get the siRNA where it needs to go – without being destroyed along the way, or creating harmful side effects.”

The nanoparticle combines two components in one delivery package 100 times smaller than a red blood cell: AXL siRNA and the peptide p5RHH.  AXL siRNA is designed to target and silence the expression of AXL, a key molecule that drives uterine and ovarian cancers. The p5RHH nanoparticles are derived from a major substance of bee venom called melittin, detoxified and selectively modified to facilitate timely escape of AXL siRNA from the nanostructure once the silencing RNA is delivered inside the targeted tumor cells.

Among the findings of the Washington University-USF Health study:

• In cell culture, treatment with p5RHH-siAXL nanoparticles decreased the ability of uterine and ovarian cancer cells to migrate and invade neighboring normal tissues.
• Mice with established uterine and ovarian tumors were intravenously and abdominally (intraperitoneally) injected with nanoparticles containing p5RHH and fluorescent control siRNA. The peptide nanoparticles localized to and released their contents into both tumor cell types regardless of the injection route, but fluorescent imaging showed that intraperitoneal administration was more effective than IV administration.
• In the mouse models, p5RHH-siALX treatment significantly reduced metastasis of both uterine and ovarian cancer without toxic effects.

USF Health Heart Institute Director Samuel Wickline, MD, and biomedical engineer Hua Pan, PhD, build nanoparticles to safely and efficiently deliver drugs or other therapeutic agents to specific cell types.

Overall, the study demonstrates this nanoparticle approach shows promise for treating patients with ovarian or uterine cancers, the authors conclude.

Challenges in translating preclinical successes into patient care remain, but Dr. Wickline believes nanoparticle-mediated delivery of siRNA has applications beyond just suppressing one target (AXL) implicated in other cancers in addition to uterine and ovarian.

The power of harnessing tiny nanotechnology for gene therapies lies in its flexibility, he said.

“As we identify new disease-modifying targets, it offers the potential to attack multiple different targets at the same time.  So, one nanoparticle could deliver a whole host of genetic materials – a combination of RNA interference drugs, or other types of synthetic RNA or DNA-based drugs —  to hit any specific cell types where treatment is needed.”

A new University of South Florida preclinical study finds that the regenerative cell therapy boosts motor nerve cell survival by repairing the blood-spinal cord barrier

TAMPA, Fla. (April 2, 2019) — Transplantation of human bone marrow-derived endothelial progenitor cells (EPCs) into mice mimicking symptoms of amyotrophic lateral sclerosis (ALS) helped more motor neurons survive and slowed disease progression by repairing damage to the blood-spinal cord barrier (BSCB), University of South Florida researchers report.

The study was published March 27 in Scientific Reports, one of the Nature journals. The findings contribute to a growing body of work exploring cell therapy approaches to barrier repair in ALS and other neurodegenerative diseases.

Human bone marrow-derived endothelial progenitor cells in vitro

The progressive degeneration of nerve cells that control muscle movement (motor neurons) eventually leads to total paralysis and death from ALS. Each day, an average of 15 Americans are diagnosed with the disease, according to the ALS Association.

Damage to the barrier between the blood circulatory system and the central nervous system has been recognized as a key factor in the development of ALS. A breach in this protective wall opens the brain and spinal cord to immune/inflammatory cells and other potentially harmful substances circulating in peripheral blood. The cascade of biochemical events leading to ALS includes alterations of endothelial cells lining the inner surface of tiny blood vessels near damaged spinal cord motor neurons.

This latest study by lead author Svitlana Garbuzova-Davis, PhD, and colleagues at the USF Health Morsani College of Medicine’s Center of Excellence for Aging & Brain Repair, builds upon a previous study showing that human bone marrow-derived stem cells improved motor functions and nervous system conditions in symptomatic ALS mice by advancing barrier repair. However, in that earlier USF study the beneficial effect was delayed until several weeks after cell transplant and some severely damaged capillaries were detected even after a high-dose treatment. So in this study, the researchers tested whether human EPCs – cells harvested from bone marrow but more genetically similar to vascular endothelial cells than undifferentiated stem cells – would provide even better BSCB restoration.

Svitlana Garbuzova-Davis, PhD

ALS mice were intravenously administered a dose of human bone-marrow derived EPCs.  Four weeks after transplant, the results of the active cell treatment was compared against findings from two other groups of mice:  ALS mice receiving a media (saline) treatment and untreated healthy mice.

The symptomatic ALS mice receiving EPC treatments demonstrated significantly improved motor function, increased motor neuron survival and slower disease progression than their symptomatic counterparts injected with media. The researchers suggest that these benefits leading to BSCB repair may have been promoted by widespread attachment of EPCs to capillaries in the spinal cord. To support this proposal, they point to evidence of substantially restored capillaries, less capillary leakage, and re-establishment of structural support cells (perivascular astrocytes) that play a role in helping form a protective barrier in the spinal cord and brain.

Further research is needed to clearly define the mechanisms of EPC barrier repair.  But, the study authors conclude: “From a translational viewpoint, the initiation of cell treatment at the symptomatic disease stage offered robust restoration of BSCB integrity and shows promise as a future clinical therapy for ALS.”

The USF study was supported by a grant from the National Institute of Neurological Disorders and Stroke.

Article citation:
Svitlana Garbuzova-Davis, Crupa Kurien, Edward Haller, David J. Eve, Stephanie Navarro, George Steiner, Ajay Mahendrasah, Surafuale Hailu, Mohammed Khatib, Kayla J. Boccio, Cesario V. Borlongan, Harry R. Van Loveren, Stanley H. Appel and Paul R. Sanberg. Human Bone Marrow Endothelial Progenitor Cell Transplantation into Symptomatic ALS Mice Delays Disease Progression and Increases Motor Neuron Survival by Repairing Blood-Spinal Cord Barrier, Scientific Reports, March 27, 2019. https://doi.org/10.1038/s41598-019-41747-4.

Tampa, FL (Nov. 10, 2015) — A University of South Florida (USF) Center for Global Health & Infectious Diseases Research team has demonstrated a new screening model to classify antimalarial drugs and to identify drug targets for the most lethal strain of malaria, Plasmodium falciparum.

The National Institutes of Health-funded study appeared online Nov. 6 in the journal Scientific Reports.

Half the world’s population is at risk for malaria, a mosquito-borne disease becoming increasingly resistant to the drug artemisinin.

The malaria parasite is becoming increasingly resistant to the drug artemisinin as the front-line treatment to combat the mosquito-borne disease, even though artemisinin is given as a combination therapy with another antimalarial drug.

The USF research provides a better understanding how antimalarial drugs work, thus adding ammunition in the race to overcome the spread of multidrug-resistant malaria – a public health threat that could  potentially undermine the success of global malaria control efforts.

The global health researchers used a collection of malaria parasite mutants that each had altered metabolism linked to defect in a single P. falciparum gene. They then screened 53 drugs and compounds against 71 of these P. falciparum piggyBac single insertion mutant parasites. Computational analysis of the response patterns linked the different antimalarial drug candidates and metabolic inhibitors to the specific gene defect.

This novel chemogenomic profiling revealed new insights into the drugs’ mechanisms of action and most importantly identified six new genes critically involved P. falciparum’s response to artemisinin, but with increased susceptibility to the drugs tested.

“That represents six new targets potentially as effective as artemisinin for killing the malaria parasite,” said the study’s co-senior author Dennis Kyle, PhD, a Distinguished USF Health Professor in the Department of Global Health, USF College of Public Health.  “There is definitely a sense of urgency for discovering new antimalarial drugs that may replace artemisinin, or work better with artemisinin, to prevent or delay drug resistance.”

From left, Dr. John Adams, Dr. Rays Jiang and Dr. Dennis Kyle are members of USF’s Center for Global Health & Infectious Diseases Research team.

The multi-faceted team of USF scientists worked with researchers from the University of Notre Dame’s Eck Institute for Global Health to undertake the chemogenomic profiling of P. falciparum for the first time.

“The methodology used in the study highlights the importance of team-based interdisciplinary research for cutting-edge scientific innovation by combining the tools of drug discovery methods with functional genomics and computational biology analysis. We are very happy to have such an important result published in the first year of a five-year NIH grant,” said co-senior author John Adams, PhD, Distinguished University Health Professor in the Department of Global Health, USF College of Public Health. “Equally important are the enormous efforts by the cadre of talented postdoctoral researchers and graduate students who were critical for making this type of challenging scientific study a success.”

“That interdisciplinary collaboration is where the power of this work comes to light,” Dr. Kyle said. “It helps us develop the tools, the molecular techniques we need to rapidly mine huge amounts of data and to discover new drug targets in ways not previously feasible.”

P. falciparum causes three-quarters of all malaria cases in Africa, and 95 percent of malaria deaths worldwide. It is transmitted to humans by the bite of an infected mosquito, which injects the one-celled malaria parasites from its salivary glands into the person’s bloodstream.

Half the world’s population is at risk of contracting malaria, so any decrease in artemisinin’s effectiveness could result in more deaths.

The USF study was supported by grants from the NIH, National Institute of Allergy and Infectious Diseases (NAID).

Microscopic image of malaria parasite P. falciparum

Article citation:
Anupam Pradhan, Geoffrey H. Siwo, Naresh Singh, Brian Martens, Bharath Balu, Katrina Button-Simons, Asako Tan, Min Zhang, Kenneth O. Udenze, Rays H.Y. Jiang, Michael T. Ferdig, John H. Adams & Dennis E. Kyle. “Chemogenomic profiling of plasmodium falciparum as a tool to aid antimalarial drug discovery.” Scientific Reports, 5, Article number 15930 (2015). doi: 10.1038/srep15930.

About USF Health
USF Health’s mission is to envision and implement the future of health. It is the partnership of the USF Health Morsani College of Medicine, the College of Nursing, the College of Public Health, the College of Pharmacy, the School of Physical Therapy and Rehabilitation Sciences, and the USF Physician’s Group. The University of South Florida is a Top 50 research university in total research expenditures among both public and private institutions nationwide, according to the National Science Foundation.  For more information, visit www.health.usf.edu

Media contact:
Anne DeLotto Baier, USF Health Communications
(813) 974-3303 or abaier@health.usf.edu