Team:DNHS SanDiego CA/Poster

Poster: DNHS_SanDiego_CA

Team DNHS_SanDiego_CA: Hannah Chen, Aashi Mittal, Saurav Nagpal, Kaavya Raamkumar, Anya Sabhnani, Anusri Thokachichu, Aishani Vora, Yajat Yadav, Iris Yang, Flora Yuan

Advisors: Dia Koupantsis, Dr. Sumeet Salaniwal, Dr. Jeff Chen, Dr. Misha Golynskiy, Dr. Ranjeet Sinha, Dr. Shilpa Mathew, Dr. Chun Yang

Sponsors: Del Norte High School, Qualcomm
Preeclampsia is a dangerous condition, characterized by high blood pressure during pregnancy. It affects many pregnancies globally, and although most women survive preeclampsia, untreated it can lead to severe complications, and even death. Despite extensive research, there is, unfortunately, no reliable treatment for preeclampsia. The goal of this project is to produce an effective theoretical siRNA treatment using recombinant lentiviral vectors to carry and deliver shRNA to the trophoblast to inhibit the translation of soluble fms-like tyrosine kinase (sFlt1-14), an antiangiogenic pseudo-receptor that captures placental growth factor (PlGF) and prevents it from binding the proper receptor, which would signal for angiogenesis. We would test several different shRNA sequences to see which causes the most effective reduction of sFlt1-14. By degrading the mRNA of sFlt1-14 using siRNA, we aim to reduce the placental levels of this molecule (which is overproduced in preeclamptic patients, leading to hypertension) and thus alleviate symptoms.

Through our project, we hope to:
Preeclampsia is a dangerous condition, characterized by high blood pressure during pregnancy. It affects 3%-7% of all pregnancies globally [2], and although most women survive preeclampsia, untreated it can lead to severe complications, and even death. Despite extensive research, there is unfortunately no reliable treatment for preeclampsia besides premature delivery to alleviate the symptoms, which is risky. Knowing this, we attempted to theorize and model a lasting treatment using synthetic biology.

Problem and Solution
Human Practices
How RNAi Works

Once in the cell, the lentivirus genome is transcribed to produce the shRNA.The shRNA is processed and one strand associates with RISC. The RNA component guides RISC to a complementary sequence on the mRNAs present in the cell, leading to the mRNA’s degradation [1].

Generating shRNA Sequences

First,we retrieved the mRNA sequence of s-FLT1-14 [2]. Then, using several design softwares online, such as the Invivogen siRNA wizard [3] and the Stanford Design Center, we corroborated a list of several siRNA sequences. Next, we used BLAST to eliminate siRNAs that showed high off-target binding. Finally, we converted our siRNA sequences into shRNA by adding a 9 nucleotide loop proven to be effective [6].

Generation of Lentiviruses

After designing the shRNA, in the lab, we plan to use our narrowed list of 4 shRNA sequences and anneal each one into the plasmids. We plan on transforming these plasmids into 293T cells, along with other necessary plasmids like the packaging plasmids, so that the cells can synthesize viral particles containing our desired shRNA.

Measuring sFLT-1 Expression Changes

In order to measure our shRNA’s efficiency, we plan on using qRT-PCR. After placental tissue has been transduced with shRNA-containing lentiviruses in vitro, we plan on using qRT-PCR, with a few potential housekeeping genes in mind, in order to measure sFLT-1 mRNA levels for each shRNA treatment.
We submitted the cDNA sequences for following 4 shRNA sequences to the Parts Registry, designed using the approach described in Methodology section

We additionally submitted a regulatory sequence (Part BBa_K3652006) to precede these shRNA sequences in the lentiviral plasmid Tet-pLKO-neo for optimal expression. Our shRNA sequences were inserted in the transfer plasmid [12], which was under a H1 promoter. Using this lentiviral plasmid also ensured that shRNA would only be produced if an additional inducer was present (Tet-On system).
The following diagram illustrates the operation of the tet-on system:

This genetic circuit shows the theoretical pathway of gene expression using our designed constructs:

The next diagram shows a theoretical and designed Tet-pLKO-neo packaging plasmid using our constructs:

sFLT-1(green) binds with VEGF(blue): Proteins obtained from Protein Data Bank and input into PYMOL to create the model. Shows sFLT-1 binding to VEGF, preventing VEGF from starting the angiogenic signaling process. This binding interaction causes symptoms of preeclampsia.

sFLT-1(green) binds with PLGF(blue): Proteins obtained from Protein Data Bank and input into PYMOL to create the model. Shows sFLT-1 binding to PLGF, preventing PLGF from taking part in the angiogenic signaling process. The binding occurs as a result of the overexpression of sFLT-1, causing more sFLT-1 molecules to exist in the placenta than PLGF molecules. This interaction causes symptoms of preeclampsia.
Safety and Future Directions
Experimental Validation
  • After plasmid is subcloned into a lentiviral vector to transfect target cells, leads to further analysis
  • Chemical testing: Western blotting, qRT-PCR assays
  • Animal models: biodistribution of our vectors, any off-target binding, efficacy of the treatment, safe dosage window

  • Local injection to deliver lentiviral vector with the siRNA sequence targeting the sFlt-1 gene
  • Orally consumed doxycycline induces the sequence, which would otherwise be inactive, to carry out RNA knockdown
  • Decide optimal dosage, treatment length, and injection frequency empirically with an emphasis on the relationship between dosages of RNAi therapy, doxycycline, and sFLT-1 levels, allowing for control of sFLT-1 knockdown through different dosages of doxycycline
  • Goals:
    • Monitor the typical ratio of sFLT-1 and PIGF levels
    • Proper angiogenesis and placental perfusion
    • Symptoms of preeclampsia, including hypertension, subside
    • Prevent underexpression of antiangiogenic factors and potential hemorrhage
    • Prevent excess administration of doxycycline to pregnant patients
    • Accessibility for all preeclamptic patients, regardless of severity

Safety Measures
  • Generation 3 plasmid with a lentiviral vector that reaches biosafety level 2 due to its potential to integrate into the human genome
    • Deletions at the 5’ and 3’ LTR regions inactivates it upon integration
    • Proper angiogenesis and placental perfusion
    • Packaging system reduces the number of lentiviral genes expressed as a chimeric 5’ LTR made it so that Tat didn’t need to be expressed for transactivation, thus reducing the chance of producing replicable viruses.
  • Tet-On promoter allows orally administered doxycycline to induce our locally administered shRNA sequence, preventing undue activation
  • shRNA designed to have high binding specificity for target and low chances for off-target binding, but must be tested in chemical and human models
    • However, shRNA target is generally only expressed in placental cells and the sequence will not be passed down through generations
References and Acknowledgements
[1]Murphy, A. T. (2018, June 08). Pre-eclampsia, Uterus Didelphys, and Fibroids! Retrieved from
[2]Preeclampsia: MedlinePlus Medical Encyclopedia. (n.d.). Retrieved from

Human Practices
[1]Created with

[1] ShRNA Process and shRNA Diagram. (n.d.). Retrieved November 07, 2020, from
[2] Sela, S. (2007, December 30). Homo sapiens soluble VEGF receptor 1-14 (FLT1) mRNA, complete cds - Nucleotide - NCBI. Retrieved October 23, 2020, from
[3] Find siRNA sequences - Standard search. (n.d.). Retrieved October 23, 2020, from
[4] Kay Lab siRNA/shRNA/Oligo Optimal Design. (n.d.). Retrieved October 23, 2020, from
[5] Li, L., Lin, X., Khvorova, A., Fesik, S., & Shen, Y. (2013, October). Defining the optimal parameters for hairpin-based knockdown constructs. Retrieved October 23, 2020, from

[2] Bank, R. P. (n.d.). 2XV7: Crystal structure of vascular endothelial growth factor D. Retrieved from
[3] PyMOL is a user-sponsored molecular visualization system on an open-source foundation, maintained and distributed by Schrödinger. We are happy to introducePyMOL 2.4! (n.d.). Retrieved from