The Nissim Lab
Synthetic biology is a multidisciplinary field that aims at rational and predictable assembly of artificial genetic building blocks, such as artificial promoters and engineered transcription factors, to generate gene circuits with well-defined cellular functions. This approach provides powerful tools for basic research and biomedicine. Our lab combines biology, medicine and engineering principles to develop synthetic biology platforms as effective, practical solutions for human therapeutics and to decipher human biology.
Synthetic gene circuits for cancer immunotherapy
We develop synthetic biology platforms to overcome major challenges in cancer immunotherapy, including the rarity of targetable tumor-specific antigens, tumor-mediated immune suppression and the cytotoxicity caused by systemic immunomodulators administration. For example, we design and optimize synthetic gene circuits that precisely identifies tumor-specific gene regulation patterns and generates the co-expression of multiple synthetic and native immunostimulatory outputs only from within cancer cells. Thus, the circuit selectively converts cancer cells into ‘Trojan horses’ that initiate potent anti-tumor immune response, capable of significantly reducing tumor size in vivo and prolong mouse survival. This approach has the potential to enable powerful new immunotherapies and to study the tumor immunology.
Synthetic promoters engineering
Cell-state specific promoters are useful for both basic and applicative research, but are challenging to find. We developed a high-throughput synthetic promoters engineering and screening approach that could potentially provide compact synthetic promoters with enhanced specificity to virtually any cell-state without prior gene regulation data. This platform was applied to discover promoters specific to sub-tissues of induced pluripotent stem cells (iPSCs) derived organoids, breast cancer and glioma cancer stem-cells.
(A) Panel 1: Immunomodulatory synthetic gene circuit designed to integrate the activity of two tumor-specific synthetic promoters (P1 and P2) with an RNA-based AND gate mechanism, and generate combinatorial immunomodulators output only when the input promoters are mutually active. When activated, the circuit drives the co-expression of Surface T-cell Engager (STE; See figure 1B), CCL21, IL12, and an anti-PD1 antibody (namely, SCIP). Panel 2: The circuits drive the expression of SCIP in cancer cells (red), but not normal cells (blue). Panel 3: Cancer-specific SCIP expression triggers effective T-cell mediated killing of the cancer cells. (B) Schematic drawing of the STE. This design is based on membrane-anchored anti-CD3 scFv that binds the non-variable regions of the T-cell receptor. Consequently, STE-expressing cells are designated for T-cell mediated killing regardless of T-cell receptor antigen specificity. (C) Top: Design of synthetic tumor-specific promoters, S(TF)p, regulating mKate2 expression. Each promoter comprises multiple binding sites for a cancer-specific transcription factor (TF-BS) cloned in tandem upstream of a minimal promoter (lateADEp). Bottom: Synthetic tumor-specific promoters regulating mKate2 exhibited enhanced tumor specificity compared to representative native tumor-specific promoters, SSX1p and H2A1p. Parentheses indicate the transcription factor binding sites used to construct each synthetic tumor-specific promoter. aHDF (adult human dermal fibroblast) and HOV (human ovarian epithelium) are normal primary cells. OV8 (OVCAR8) is a human ovarian cancer cell line. Error bars represent S.E.M. (D) In vivo lentiviral delivery of the SCIP-expressing circuit significantly reduced ovarian cancer burden. NSG mice were injected intraperitoneally (IP) with OVCAR8 cells at day 0. Lentiviruses encoding a control circuit generating a non-immunogenic output protein (Control), or combination immunomodulators (SCIP) were then injected IP on day 7. These mice were also periodically injected IP with human T cells (day 9, 16 and 23). Error bars represent S.E.M.; p<0.005. (E) Lentiviral delivery of the SCIP-expressing circuit significantly increased mice survival, as shown by Kaplan-Meier survival curves of SCIP and control circuits groups. A log-rank (Mantel-Cox) test was performed to compare survival between groups (p<0.005).
The RNA-Only Multi-Output AND Gate Design: Module 1 of the AND gate is designed as an auto-inhibitory loop such that it represses its own transcript, which encodes the synthetic transcription factor GAD. Module 2 is designed to inhibit the auto-inhibition of module 1. Module 1 and module 2 are regulated by the cancer-specific synthetic promoters S(cMYC)p and S(E2F1)p, respectively. The synthetic transcription factor (GAD) that activate downstream gene expression as the output of module 3 (mKate2 ·· Output N) is expressed at a high level only when both S(cMYC)p and S(E2F1)p are active, which enhances the tumor specificity of the circuit.
All four possible input states and their respective output states for the RNA-only AND gate (input states are defined within the square brackets by whether module 1 and module 2 are active, where 0 means inactive and 1 means active):
- In state [1,0], S(cMYC)p is active and the GAD transcript is expressed (GAD-Ex1 and GAD-Ex2 denote GAD exon1 and GAD exon2, respectively). However, this transcript encodes miR1 within the GAD gene that inhibits the GAD transcript by targeting BS(Pe)s in the 30 UTR. Thus, the GAD levels are minimal.
- In state [0,1], S(cMYC)p is inactive, so the output protein GAD is not expressed.
- In state [0,0], neither S(cMYC)p nor S(cMYC)p are active, and thus, the output protein GAD is not expressed.
- In state [1,1], the S(cMYC)p promoter expresses a sponge for miR1 that is based on BS(B)s. This enables sequestration of miR1 away from inhibiting the GAD transcript expressed by S(cMYC)p, thus allowing GAD to activate mKate2 and additional outputs expression from module 3.
Nissim, L.*, Wu, M.R.*, Pery, E., Binder-Nissim, A., Suzuki, H.I., Stupp, D., Wehrspaun, C., Tabach, Y., Sharp, P.A., and Lu, T.K. (2017). Synthetic RNA-based immunomodulatory gene circuits for cancer immunotherapy. Cell 171, 1138-1150
Lee, G., Atia, L., Lan, B., Sharma, Y., Nissim, L., Wu, M.R., Pery, E., Lu, T.K., Park, C.Y., Butler, J.P., et al. (2017). Contact guidance and collective migration in the advancing epithelial monolayer. Connect Tissue Res.
Morel, M., Shtrahman, R., Rotter, V., Nissim, L.**, and Bar-Ziv, R.H**. (2016). Cellular heterogeneity mediates inherent sensitivity-specificity tradeoff in cancer targeting by synthetic circuits. Proc Natl Acad Sci U S A 113, 8133-8138.
Nissim, L.*, Perli, S.D.*, Fridkin, A., Perez-Pinera, P., and Lu, T.K. (2014). Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Molecular Cell 54, 698-710.
Jusiak, B., Daniel, R., Farzadfard, F., Nissim, L., Purcell, O., Rubens , J., and Lu, T.K. (2014). Synthetic Gene Circuits. Encyclopedia of Molecular Cell Biology and Molecular Medicine.
Nissim, L., and Bar-Ziv, R.H. (2010). A tunable dual-promoter integrator for targeting of cancer cells. Molecular systems biology 6, 444.
Nissim, L., Beatus, T., and Bar-Ziv, R. (2007). An autonomous system for identifying and governing a cell's state in yeast. Physical biology 4, 154-163.
Matas, D., Milyavsky, M., Shats, I., Nissim, L., Goldfinger, N., and Rotter, V. (2004). p53 is a regulator of macrophage differentiation. Cell death and differentiation 11, 458-467.
Peller, S., Frenkel, J., Lapidot, T., Kahn, J., Rahimi-Levene, N., Yona, R., Nissim, L., Goldfinger, N., Sherman, D.J., and Rotter, V. (2003). The onset of p53-dependent apoptosis plays a role in terminal differentiation of human normoblasts. Oncogene 22, 4648-4655.
Media & News
HIGH-THROUGHPUT PROMOTER ENGINEERING AND SCREENING
In preparation for submission by the Massachusetts Institute of Technology
Inventors: Timothy K. Lu, Lior Nissim, Ming-Ru Wu
U.S. 62/181906, M0656.70356
Inventors: Timothy K. Lu, Lior Nissim, Ming-Ru Wu
METHODS AND COMPOSITIONS FOR THE PRODUCTION OF GUIDE RNA
Inventors: Timothy K. Lu, Lior Nissim, Samuel Perli
NUCLEIC ACID CONSTRUCT SYSTEMS CAPABLE OF DIAGNOSING OR TREATING A CELL STATE
WO 2009/007980 (15.01.2009 Gazette 2009/03)
Inventors: Lior Nissim, Roy H. Bar-Ziv
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