New application

It should be noted that this RNAi strategy against HCC, if works, is a proof of concept. Once the AND-gated system is proven to properly function as proposed here, it is nearly as flexible as the technology of RNA interference itself. Such disease-specific approach would be highly suitable in particular for diseases which are related to inflammation, which encompasses a long list of major diseases which increase in prevalence as we age (atherosclerosis, dementia, arthritis and so on)^[24]. Here we give three examples: other types of cancer, Parkinson's disease and atherosclerosis.

Specifically, the promoters (“keys” to the AND gate) here can be replaced with other disease-specific promoters, in gene therapy for the corresponding diseases. And the target can be any messenger RNA. Indeed, such significance has prompted us to sign for the New Application Track in this year’s competition, rather than the Therapeutics Track we originally thought would apply.

1.Other types of cancer

AND-gated systems targeting cancer have been seen in the previous iGEM seasons, such as Bladder Cancer Sniper (SZU_China, 2015) and The Boomerang system (BGU_Israel, 2015). They all took advantage of cancer-specific promoters, including hTERT promoter discussed also in our work. While hTERT is indeed very popular among cancer researchers^[1][2][3], we do have other options.

Survivin is an inhibitor of the apoptosis protein that is expressed in many human cancer cells but not in most normal adult tissues^[4]. In cancer cells, the main role of Survivin is the inhibition of apoptosis. The human survivin promoter has been investigated in extensive detail. Its specificity in cancer can be explained by many features of its sequence, including a canonical CpG island, numerous Sp1 sites, CDEs (Cell-cycle Dependent Elements) and a C/G polymorphism in tumour cells^[5]. Till date several researches verified the ability of the human survivin promoter to specifically target different type of cancers from tumor cells to small animal models^[6][7].

Our project highlights the feasibility of utilizing promoters of certain long-non-coding RNA (lncRNA) genes as switches of cancer gene therapy.Recently there have been explosive discoveries of new lncRNAs, obtained by progress in second-generation sequencing. These transcripts have been found to be major participants in various physiologic processes and diseases. In human cancers, lncRNAs have been found to function as novel types of oncogenes and tumor suppressors through various mechanisms, which endow them with the potential of serving as reliable biomarkers and novel therapeutic targets for cancers. Few efforts have been put into utilizing these in genetic engineering. Our AND-system should be one starting example.

Similar to HULC in HCC, H19 has been found to be specific for gastric cancer^[8], PTCSC3 for papillary thyroid carcinoma^[9] and PCAT-1 for prostate cancer^[10]. We recommend further research into these tissue- and/or tumor-specific lncRNAs as applicable in cancer gene therapy. If proven to be up/down-regulated in the corresponding cancer cell lines at transcription level, each of them could likewise serve as a “key” (or in combination as “keys”) to various logic gates for delivery of suicide genes.

For cancer therapy targets, there are even more options. We can develop strategies to inhibit overexpressed mRNAs (oncogenic mRNAs) or, to induce the biogenesis of under-expressed miRNAs (tumor suppressor miRNAs) in tumor cells. For relevancy, we discuss the anti‐apoptotic protein B‐cell lymphoma 2 (Bcl‐2). Inhibition of the activity of Bcl-2 restores apoptotic processes in tumor cells and can trigger cell death. Zhang et al. successfully designed a similar RNAi-regulatory device with pri-miRNA analogue targeting Bcl-2 in MCF-7 cells but with an embedded theophylline aptamer as a sensor domain^[11]. However, we have to point out the fact that there is inconsistency between the amount of pri-miRNA and the mature miRNA in certain cases^[12][13]. You may want to look at specific literature to find out whether your engineered pri-miRNA analogue would indeed not be hampered somewhere in the post-transcriptional gene silencing pathway.

2.Parkinson's disease

Parkinson disease (PD) is a common neurodegenerative disorder caused by environmental and genetic factors. The human synapsin-1 (hSYN) minimal promoter has previously been demonstrated to direct neuronal-specific expression of eGFP when incorporated into an AAV-vector and delivered into the rat brain^[14]. Wettergren et al. evaluated several disease-relevant promoter candidates using lentiviral vectors in rat striatum. DNAJC3, MAP1a and RNF25 all had a neuronal specificity of 91–100%, and efficiencies comparable to the widely used ubiquitous promoters CMV, EF1α and CAG (7–18%)^[15]. Specificity could be further improved using miRNA detargeting. Wettergren et al. designed an astrocytic-specific vector combining fibrillary acidic promoter (GFAP) and miRNA-based regulation using miRNA 124^[16]. Transgene expression could potentially be silenced in unwanted cell types highly expressing this miRNA (mature neurons), thus the tropism of the vectors could be shifted from a neuronal one to an astrocytic one.

To our surprise, no efforts towards RNAi therapeutics for PD has ever been paid in the iGEM community. Perhaps it’s because that RNAi is always about de-regulating certain gene products but the canonical idea behind current PD therapies is about L-DOPA production^[17][18]. But there has been some progress outside demonstrating RNAi therapeutic potential in cell and animal models for PD^[21]. Two of the known genes associated with PD are the aggregate-forming alpha-synuclein (α-syn) seen in Lewy Bodies, and leucine-rich repeat kinase-2 (LRRK2). Many such studies have been focusing on targeting single-nucleotide polymorphisms (SNPs) in α-syn or LRRK2.

Instead of deciding on a specific target, we can just express known good miRNAs in the pathogenic cells. Interestingly, over-expression of miR-124 mentioned above could effectively attenuate LPS-induced expression of pro-inflammatory cytokines and promote the secretion of neuroprotective factors^[19]. For patients with known alpha synuclein gene multiplications, delivery of miR-7 and miR-153 may represent an appealing therapeutic strategy to promote neuroprotection^[20]. Other relevant miRNAs include miR-133b and miR-433^[21].

3.Atherosclerosis

Atherosclerosis can be described as an inflammation of arteries. Efforts within the iGEM community around this disease mainly focused on engineering bacteria or external enzymes for the direct degradation of atherosclerotic plaques^[23][24]. No team has ever exploited disease-specific promoters or RNAi technology for treating this disease.

LOX1 encodes for a scavenger receptor which binds oxidized low-density lipoprotein and is expressed by activated endothelial cells (EC), smooth muscle cells, and macrophages, which are all major cell types involved in atherosclerosis. Its promoter (LOX1pr) indeed proved to be a useful disease-specific promoter for expression of therapeutic genes to counteract atherogenesis^[25]. Levin et al. found that the exclusive introduction of the 150-bp proximal part of the CD68 promoter improves the efficiency and specificity of this well-known macrophage-specific promoter^[26]. Kang et al. successfully developed an artificial promoter SP146-C1, combined with a p47^phox promoter element to also target macrophage^[27].

MAP4K4 is also a key signalling node that promotes immune cell recruitment in atherosclerosis^[28][29]. Reduction or loss of endothelial MAP4K4 expression profoundly ameliorates atherosclerotic lesion development in mice. Ath29 is a major atherosclerosis susceptibility locus affecting both early and advanced lesion formation in mice^[30]. Knockdown of Rcn2 on this locus appeared to be an appropriate approach for pharmacological interventions of atherosclerosis.
Atherosclerosis

© 2018 Shuyao Zhou, CPU_CHINA

[1]https://2016.igem.org/Team:SCUT-China_B/Protumor

[2]Schepelmann, S., Hallenbeck, P., Ogilvie, L. M., Hedley, D., Friedlos, F., Martin, J., ... & Springer, C. J. (2005). Systemic gene-directed enzyme prodrug therapy of hepatocellular carcinoma using a targeted adenovirus armed with carboxypeptidase G2. Cancer research, 65(12), 5003-5008.

[3]https://2016.igem.org/Team:Tongji_Shanghai/Results

[4]Chen, J. S., Liu, J. C., Shen, L., Rau, K. M., Kuo, H. P., Li, Y. M., ... & Hung, M. C. (2004). Cancer-specific activation of the survivin promoter and its potential use in gene therapy. Cancer gene therapy, 11(11), 740.

[5]Boidot, R., Végran, F., & Lizard-Nacol, S. (2014). Transcriptional regulation of the survivin gene. Molecular biology reports, 41(1), 233-240.

[6]Van Houdt, W. J., Haviv, Y. S., Lu, B., Wang, M., Rivera, A. A., Ulasov, I. V., ... & Dirven, C. M. (2006). The human survivin promoter: a novel transcriptional targeting strategy for treatment of glioma. Journal of neurosurgery, 104(4), 583-592.

[7]Li, B., Liu, X., Fan, J., Qi, R., Bo, L., Gu, J., ... & Liu, X. (2006). A survivin‐mediated oncolytic adenovirus induces non‐apoptotic cell death in lung cancer cells and shows antitumoral potential in vivo. The Journal of Gene Medicine: A cross‐disciplinary journal for research on the science of gene transfer and its clinical applications, 8(10), 1232-1242.

[8]Zhang, E. B., Han, L., Yin, D. D., Kong, R., De, W., & Chen, J. (2014). c-Myc-induced, long, noncoding H19 affects cell proliferation and predicts a poor prognosis in patients with gastric cancer. Medical oncology, 31(5), 914.

[9]Jendrzejewski, J., Thomas, A., Liyanarachchi, S., Eiterman, A., Tomsic, J., He, H., ... & de la Chapelle, A. (2015). PTCSC3 is involved in papillary thyroid carcinoma development by modulating S100A4 gene expression. The Journal of Clinical Endocrinology & Metabolism, 100(10), E1370-E1377.

[10]Prensner, J. R., Iyer, M. K., Balbin, O. A., Dhanasekaran, S. M., Cao, Q., Brenner, J. C., ... & Cao, X. (2011). Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression. Nature biotechnology, 29(8), 742.

[11]Zhang, Y., Wang, J., Cheng, H., Sun, N., Liu, M., Wu, Z., & Pei, R. (2017). Inducible Bcl-2 gene RNA interference mediated by aptamer-integrated HDV ribozyme switch. Integrative Biology, 9(7), 619-626.

[12]Wang, X., Cao, L. E. I., Wang, Y., Wang, X., Liu, N., & You, Y. (2012). Regulation of let-7 and its target oncogenes. Oncology letters, 3(5), 955-960.

[13]Lin, S., & Gregory, R. I. (2015). MicroRNA biogenesis pathways in cancer. Nature reviews cancer, 15(6), 321.

[14]Kügler, S., Kilic, E., & Bähr, M. (2003). Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene therapy, 10(4), 337.

[15]Wettergren, E. E., Gussing, F., Quintino, L., & Lundberg, C. (2012). Novel disease-specific promoters for use in gene therapy for Parkinson's disease. Neuroscience letters, 530(1), 29-34.

[16]Wettergren, E. E., Quintino, L., Manfré, G., & Lundberg, C. (2014). Gene Therapy for Parkinson’s Disease. In Viral Vector Approaches in Neurobiology and Brain Diseases (pp. 181-191). Humana Press, Totowa, NJ.

[17]https://2015.igem.org/Team:Manchester-Graz/Description

[18]https://2017.igem.org/Team:Austin_UTexas_LASA/Description

[19]Yao, L., Ye, Y., Mao, H., Lu, F., He, X., Lu, G., & Zhang, S. (2018). MicroRNA-124 regulates the expression of MEKK3 in the inflammatory pathogenesis of Parkinson’s disease. Journal of neuroinflammation, 15(1), 13.

[20]Doxakis, E. (2010). Post-transcriptional regulation of alpha-synuclein expression by mir-7 and mir-153. Journal of Biological Chemistry, jbc-M109.

[21]Weinberg, M. S., & Wood, M. J. (2009). Short non-coding RNA biology and neurodegenerative disorders: novel disease targets and therapeutics. Human molecular genetics, 18(R1), R27-R39.

[22]Ramachandran, P. S., Keiser, M. S., & Davidson, B. L. (2013). Recent advances in RNA interference therapeutics for CNS diseases. Neurotherapeutics, 10(3), 473-485.

[23]https://2009.igem.org/Team:NTU-Singapore/Project

[24]https://2014.igem.org/Team:ITESM-CEM/Project

[25]Zhu, H., Cao, M., Mirandola, L., Figueroa, J. A., Cobos, E., Chiriva-Internati, M., & Hermonat, P. L. (2014). Comparison of efficacy of the disease-specific LOX1-and constitutive cytomegalovirus-promoters in expressing interleukin 10 through adeno-associated virus 2/8 delivery in atherosclerotic mice. PLoS One, 9(4), e94665.

[26]Levin, M. C., Lidberg, U., Jirholt, P., Adiels, M., Wramstedt, A., Gustafsson, K., ... & Olofsson, S. O. (2012). Evaluation of macrophage-specific promoters using lentiviral delivery in mice. Gene therapy, 19(11), 1041.

[27]Kang, W. S., Kwon, J. S., Kim, H. B., Jeong, H. Y., Kang, H. J., Jeong, M. H., ... & Ahn, Y. (2014). A macrophage-specific synthetic promoter for therapeutic application of adiponectin. Gene therapy, 21(4), 353.

[28]Aouadi, M., Tesz, G. J., Nicoloro, S. M., Wang, M., Chouinard, M., Soto, E., ... & Czech, M. P. (2009). Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation. Nature, 458(7242), 1180.

[29]Flach, R. J. R., Skoura, A., Matevossian, A., Danai, L. V., Zheng, W., Cortes, C., ... & Vangala, P. (2015). Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis. Nature communications, 6, 8995.

[30]Manichaikul, A., Wang, Q., Shi, Y. L., Zhang, Z., Leitinger, N., & Shi, W. (2011). Characterization of Ath29, a major mouse atherosclerosis susceptibility locus, and identification of Rcn2 as a novel regulator of cytokine expression. American Journal of Physiology-Heart and Circulatory Physiology, 301(3), H1056-H1061.

[31]Hermonat PL (2014) Improving AAV Gene Therapy: Graduating From Transgene Expression “Everywhere, All The Time” To “Disease-Specific”. Clon Transgen 3:e114. doi:10.4172/2168-9849.1000e114

Drug delivery system

Introductions

RNA interference (RNAi) is an endogenous, ubiquitous, evolutionarily conserved and powerful method for regulating gene expression, which can cause the gene encoding messenger RNA (mRNA) degradation^[1]. In this case, a medication in transcription level can be achieved. RNAi is highly effective in silencing the target gene that regulates the specific biological/pathological pathway^[2]. Several in vitro and in vivo studies have shown that every human disease with over-expression of disease causing gene(s) is a potential target for RNAi-based therapeutics ^[3].

Cancer is a genetic disease of stepwise deregulation of cell death mechanisms and cell proliferation. The growth phenotype of cancer cells differs from normal cells due to proto-oncogene mutation, aberrant genetic activation, amplification, over-expression or deletion of tumor suppressor^[1]. RNAi is being explored as a way to inhibit the expression of genes involved in oncogenesis. However, when it comes to cancer therapy, specifically targeting tumor cells is an important direction^[1]. In our RNAi system, cancer specific target and promoters are designed which means that the strategy can selectively cause destruction of cancer cells without affecting normal cells. However, the efficiency of our system in vivo is also an issue that need to be concerned otherwise our gene device may not reach cancer cells to do its job. What’s more, our gene therapy strategy, if ever to become transgenic drugs, involves multiple genes transferring with two plasmids. There’s an “AND” gate system which protects the normal cells from damaging, however undesirable effects like mutagenesis lead by encoded proteins or RNA products alone (although happens relatively low in normal cells) may happens, these are indeed problems to consider.

In this case, to further improve our project for future application when it comes into a drug, if possible, two methods which allow us to target tumor tissue in vivo are designed.

Drug delivery based on ERP

Enrich the drugs in or near tumor is vital when it comes to targeting the cancer cells. Liposome is commonly used as carrier for drug delivery^[4], and some of them are in macromolecular size.

The major goal of liposomal drug delivery is to deliver the therapeutic agent preferentially to the tumor site through the enhanced permeability and retention (EPR) effect ^[5]. There are seven barriers in the development of cancer selective macromolecular drugs and steps to be overcome, first of which is vascular wall and blood cirrculation. ERP effect is of prime importance for this step because drug extravasation occurs in a tumor-selective manner^[6].

ERP was first proposed by Yasuhiro in 1986^[7], which is now becoming the most important pharmacokinetic principle at the first step of the design of macromolecular drugs or nanomedicines^[8]. Clearance of macromolecules and lipids from tumor is so impaired that they remain in the tumor interstitium for along time which enables the drugs to be permanent. This phenomenon has been characterized and termed the tumor-selective EPR effect of macromolecules and lipidic particles^[9]. The EPR concept is now regarded as a ‘‘gold standard’’ in the design of new anticancer agents^[10].

The disease we choose to apply on, Hepatocellular carcinoma(HCC), manifest a high vascular density, so it tend to have a relatively increased EPR effect^[8]. By containing our two plasmids system in a liposome size ranges between 1-200nm(the size of nanomedicines) it can enrich in the tumor owing to EPR effect and thus, having a better selectivity on HCC.

Drug delivery based on Ligand-targeted liposome

Passive delivery like the delivery based on EPR effect is simple, yet its effect varies depending on a patient's pathological and physiological characteristics and clinical condition, for instance, when a patient's systolic blood pressure is low side of about 90 mm Hg instead of 120–130 mm Hg, the hydro-dynamic force pushing blood from the luminal side of a vessel into tumor tissue becomes significantly low, which results in a low EPR ^[8].

Aside from preferential accumulation in tumors, nano-particle-based drug delivery systems can also enhance the pharmacokinetic profile of therapeutic agents. Specifically, the use of polyethylene glycol (PEG) ‘stealth’ coatings greatly enhances blood circulation times by allowing the liposome to evade immune detection^[12].

Nanomedicine, particularly liposomal drug delivery, has expanded considerably over the past few decades, and several liposomal drugs are already providing improved clinical outcomes. Liposomes have now progressed beyond simple, inert drug carriers and can be designed to be highly responsive in vivo, with active targeting, increased stealth, and controlled drug-release properties. Ligand-targeted liposomes (LTLs) have the potential to revolutionize the treatment of cancer^[13].

In order to achieve maximum efficacy, controlled release of therapeutic agents from liposomes at the tumor site is essential^[14]. Liposomes, can be surface functionalized with targeting ligands to enhance the selective targeting of tumors ^[15][16]. The grafting of targeting ligands to the liposome surface can further enhance tumor targeting and facilitate intracellular uptake after the liposome reaches the tumor interstitium, which indicates that a specific HCC binding protein(like HCC specific antibody) modified liposome will have a promising efficacy.

Macrophage signal regulatory protein-α (SIRPα) interacts with HCC surface molecule CD47 and resulted in low macrophage response towards HCC^[17]. By binding the CD47 specific antibodies (CD47mAbs) on the surface of liposome which carries our two plasmids system, not only our system can have a more efficacy drug release, immune response towards HCC will also be increased since the interaction between SIRPα and CD47 is blocked.

© 2018 Huandi Xu, CPU_CHINA

[1] Yang, W. Q., Zhang, Y. (2012). Rnai-mediated gene silencing in cancer therapy. Expert Opinion on Biological Therapy, 12(11), 1495.

[2] Weiler, J., Hunziker, J., Hall, J. (2006). Anti-mirna oligonucleotides (amos): ammunition to target mirnas implicated in human disease?. Gene Therapy, 13(6), 496-502.

[3] Ren, Y. J., & Zhang, Y. (2014). An update on rna interference-mediated gene silencing in cancer therapy. Expert Opin Biol Ther, 14(11), 1581-1592.

[4] https://en.wikipedia.org/wiki/Transfection

[5] Maeda, H. (2012) Macromolecular therapeutics in cancer treatment: the EPR effect and beyond. J. Control. Release 164, 138–144

[6] Maeda, H., Nakamura, H., & Fang, J. (2013). The epr effect for macromolecular drug delivery to solid tumors: improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Advanced Drug Delivery Reviews, 65(1), 71-79.

[7] Matsumura, Y., & Maeda, H. (1986). A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Research, 46(12 Pt 1), 6387.

[8] Maeda, H. (2015). Toward a full understanding of the epr effect in primary and metastatic tumors as well as issues related to its heterogeneity. Advanced Drug Delivery Reviews, 91, 3-6.

[9] Maeda, H. (2001). The enhanced permeability and retention (epr) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul, 41(1), 189-207.

[10] Maeda, H., & Matsumura, Y. (1989). Tumoritropic and lymphotropic principles of macromolecular drugs. Crit Rev Ther Drug Carrier Syst, 6(3), 193-210.,

[11] Noble, G. T., Stefanick, J. F., Ashley, J. D., Kiziltepe, T., & Bilgicer, B. (2014). Ligand-targeted liposome design: challenges and fundamental considerations. Trends in Biotechnology, 32(1), 32-45.

[12] Allen, T.M. and Cullis, P.R. (2013) Liposomal drug delivery systems:From concept to clinical applications. Adv. Drug Deliv. Rev. 65, 36–48

[14] Barenholz, Y. (Chezy) (2012) Doxil1 — The first FDA-approved nano-drug: lessons learned. J. Control. Release 160, 117–134

[15] Sapra, P. and Allen, T.M. (2003) Ligand-targeted liposomal anticancer drugs. Prog. Lipid Res. 42, 439–462

[16] Ruoslahti, E. (2012) Peptides as targeting elements and tissue penetration devices for nanoparticles. Adv. Mater. 24, 3747–3756