Nano medicine therapeutics for targeted Gene Silencing in Ovarian cancer

Dr. Manu Smriti Singh’s interview with Bio Patrika hosting “Vigyaan Patrika”, a series of author interviews. Dr. Singh completed her Doctorate from University of Bonn. She completed her Post-Doctoral Research from Tel Aviv University and Hebrew University of Jerusalem, Israel in 2020. At present, she is a Faculty at Department of Biotechnology, Bennett University with focus on tumor microenvironment and Cancer Nano medicine. Here, Manu Smriti talks about her work titled “Therapeutic Gene Silencing Using Targeted Lipid Nanoparticles in Metastatic Ovarian Cancer” published in Small journal.

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How would you explain your paper’s key results to the non-scientific community?

Nucleic Acids are prone to degradation when administered in vivo. Also, negative charge makes the delivery into cells challenging due to the barrier posed by the anionic cell membrane. Covid19 vaccines manufactured by Moderna and Pfizer/ BioNTech are based on messenger RNA (mRNA) – a transient genetic material that carries information from DNA (in nucleus) to form protein (in cytoplasm). In my work, we have used the same delivery vehicle as Moderna’s vaccine but to deliver small interfering RNA (siRNA) instead of mRNA. The common RNA delivery vehicle we have used are termed Lipid Nanoparticles (LNPs).

Clinically, chemotherapy is the conventional treatment for cancer but is accompanied by unwanted toxicity to healthy tissues. Gene therapy is increasingly gaining clearance from FDA following successful clinical trials and would become more mainstream in cancer treatment as well. For example- Tisagenlecleucel and Patisiran for the treatment of B-cell Acute Lymphoblastic Leukemia and Transthyretin-mediated amyloidosis respectively. Here, we used siRNA as the therapeutic modality which works on the principle of RNA interference for which Craig Mello and Andrew Fire shared the Nobel Prize just 15 years ago (2006). Delivery of siRNA to cancer cells can lead to downregulation of target genes and reduce the production of desired protein, crucial for proliferation of cancer cell.

For this work we chose two targets- eIF3c and PLK1. eIF3c (Eukaryotic Initiation Factor Subunit 3c) which plays a role in starting protein translation process and drives tumorigenesis. PLK1 (Polo Like Kinase 1) plays a role in malignant transformation by triggering cell division. LNPs encapsulating siRNA against PLK1 (TKM-080301) has shown promising leads so far and has already cleared Phase I/II trial for hepatocellular carcinoma. The siRNA against both targets- (si-eIF3c and si-PLK1) were co-encapsulated in LNPs facilitated by a microfluidic mixing device. These LNPs were further coated with Hyaluronan (HA) which specially binds to cell surface receptor called CD44.

CD44 is highly expressed in ovarian cancer cells (also observed in patient’s ovary tumor and ascites samples). The HA-coating was to enable active targeting of cancer cells by LNPs (tNP) as opposed to uncoated LNPs (uNP). Our LNP were ~60nm in size.

Key results:

  • We confirmed the functional activity of both siRNA’s individually on cancer cells growing in 2D and 3D cell culture. si-eIF3c-LNP inhibited global protein synthesis and si-PLK1-LNP arrested cells at G2/M-phase of cell cycle; both the process leading to death of cancer cells.
  • The above results were due to robust gene silencing of both genes in vitro and in vivo (in mice cancer tissues)
  • Another important observation was the selective and enhanced targeting to CD44 expressing cells in mice tumor tissue with tNP (in comparison to uNP)
  • After 4 injections, we found a durable response in mice in the combination treatment group which improved the median survival and overall survival. 60% mice survived in combination treatment as compared to none of the mice surviving in the control group or with empty nanoparticles (only carrier).

What are the possible consequences of these findings for your research area?

Clinically, ovarian cancer is diagnosed in advanced stages (III/IV) at which the patient’s 5-year survival is only 30%. Most studies on ovarian cancer start an early treatment on day 5 onwards when the tumor is not even established in vivo. We deliberately injected mice with siRNA-encapsulated-LNPs on day 15 onwards to mimic clinical settings of late stage patients. In the combination siRNA treatment group, we observed 60% survival at a low dose of 1mg/kg mice body weight.

The fact that the two siRNA’s we encapsulated, can be replaced with other siRNA targets for a more personalized approach for an individual’s treatment makes LNPs a promising candidate in future therapies. This implies that depending on the signature profile of the patient – chemotherapy resistant/ recurring/ unique mutation leading to altered metabolism/ quiescent gene in the hypoxic core etc. can be identified, and tailor-made LNPs encapsulating siRNA1 + siRNA2 or more can be formulated and administered to the patient.

Gene therapy is highly specific in terms of targeting specific genes, which are up regulated in cancer, thereby reducing chances of undue toxicity to healthy tissue. In addition, HA-coating can provide cancer-tissue specific delivery as we observed in our mice studies.

What was the exciting moment (eureka moment) during your research?

While we knew that there is a robust gene silencing and in vitro anti-tumor activity, I was not sure if the gene therapy will be strong enough for an advanced stage metastasized tumor. Almost 80% of the ovarian cancer patients come to the gynaecologist in advanced stages with swollen abdomen filled with a fluid called ascites. The fluid comprises of tumor-cell clusters which shedfrom the growing tumor in the ovary. So, we were assuming to observe silencing of the target genes in ascites of the mice because it was closer to the site of injection (intraperitoneal). But the most exciting part was to observe a robust and reproducible silencing in the key tissues associated with ovarian cancer pathophysiology- ovary, omentum and distant from site of injection. This was a direct evidence of functional activity of siRNA activity in vivo.

What do you hope to do next?

In a prior work, I developed an in vivo tumor model based on 3D spheroids (ovarian cancer) and observed the role of tumor microenvironment in delivery of chemotherapy, biological therapy and nanomedicine. As I set up my lab, my focus will be in understanding non-cancer supportive cells of the tumor microenvironment specifically carcinoma-associated fibroblasts and endothelial cells along with developing nanomedicine to target them besides cancer cells.

Another area I am interested in exploring is early cancer diagnosis, which I think should be given more attention than cancer therapeutics as we move ahead.

Where do you seek scientific inspiration?

As much as I would like to give a philosophical answer to that, the reality is – review of literature. I try to do a thorough reading before starting a project and also as it continues. I believe that the scientists are a very curious lot and most of the ideas that come to the mind have already been worked upon. I try to look for the questions that have remained unanswered and try to work on those gaps to find solutions.

Of course, both my Post-doctoral mentors Prof. Dan Peer (Tel Aviv University) and Professor Emeritus Yechezkel Barenholz (Hebrew University of Jerusalem) have been and will continue to be a constant source of inspiration.

How do you intend to help Indian science improve?

My research experience at Israeli Universities has exposed me to the entrepreneurship-driven academic learning. In India, the greatest asset is talented students and researchers. On one hand, I aim to focus on predominantly cancer-related diagnostic and therapeutic research, while on the other hand to translate potential projects into commercial products. In this direction, I am teaching Intellectual Property Right (IPR) at Bennett University to the students to understand IP issues related to research products commercialization.

I would also like to work at the interface of Industry and Hospitals because I believe that academicians cannot work in isolation and must try to understand the problems at hand (in Indian context here). Academia-Industry-Hospital collaborations can address problems in a more concerted and timely manner in terms of translational output.

Reference

Singh, M. S., Ramishetti, S., Landesman-Milo, D., Goldsmith, M., Chatterjee, S., Palakuri, R., Peer, D., Therapeutic Gene Silencing Using Targeted Lipid Nanoparticles in Metastatic Ovarian Cancer. Small 2021, 17, 2100287. https://doi.org/10.1002/smll.202100287

Website: https://www.bennett.edu.in/faculties/dr-manu-smriti-singh/

Edited by: Pratibha Siwach

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