Team:Nanjing NFLS/Design

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• Home
• Project
  • Description
  • Background
  • Design
  • Experiment
  • Demonstrate
  • Notebook
  • Vision
• Parts
• Safety
• Model
• Team
  • Team Members
  • Attributions
  • Collaborations
• Human Practices
  • Overview
  • Integrated Human Practices
  • Public Engagement
• Judging

Design

• “Trojan Horse”
• “AND” GATE
• CITHA systems
  • HBsAg expression system
  • Specific regulation system
• References

Cancer immunotherapy with neoantigen could avoid the huge side effects and high recurrence of traditional cancer therapies. To bypass the task of individualizing treatments for the different neoantigens of each individual, and to tackle the problems of immune tolerance, we designed our neoantigen system to make sure that this treatment is as universalized as possible, and exclusively target tumor cells through a series of checking mechanism.

“Trojan Horse”

Through prediction and analyses of Immune Epitope Database (IEDB) on immunogenicity, we choose Hepatitis B Surface Antigen (HBsAg) as the “Trojan Horse” Antigen since the protein is not pathogenic but immunogenic. If humoral immunity provoked, the body starts to produce neutralizing antibodies HBsAb, which indicates the termination of HBV infection. Therefore, we add membrane localization sequence SCP1 to localize HBsAg on cell membrane, therefore this protein can be expressed on the cell membrane rather than cytoplasm. To ensure our system can specificly target tumor cells, we employ Human Telomerase Reverse Transcriptase (hTERT), a tumor specific promoter, which is specially high expressed in tumor cells. Furthermore, hTERT’s wide expression in various tumor cells allows us to universalize our Trojan Horse Antigen therapy from liver cancer to other types of cancer in the future. Thus, hTERT-HBsAg-SCP1 concludes the initial design of our project.

“AND”GATE

Despite the fact that hTERT promoter has over 300 times higher driven efficiency in tumor cells than in normal cells, there is still a possibility of it activating HBsAg synthesis in normal cells. The consequences of this might be devastating, so we want to add a regulation system to our project. To ensure our system target exclusively the tumor cells, we construct a regulation system through employing the AND Gate logic conjunction. Through the “AND” Gate, a “True” output results only if all the inputs to the gate are “True”. If none or not all inputs are “True”, a “False” output would result.

Figure 1 mechanism of AND GATE

The two inputs of our “AND” Gate are microRNA (miRNA) and competing endogenous RNA (ceRNA). MiRNA silences mRNA via base-pairing with complementary sequences within mRNA molecules, thereby  repressing the synthesis of HBsAg. CeRNA, through competing for targeted miRNAs, reduces miRNA’s repression on mRNA. We constructed the miRNA in order to regulate the neoantigen in the downsteam of “Trojan Horse Antigen” gene segment, and we further designed ceRNA counterparts in the second plasmid.

A Hepatocellular Carcinoma (HCC) specific promoter, High Up-regulated in Liver Cancer (HULC), is also incorporated into the second plasmid. These two specific promoters form our “AND” gate which will only open when the two promoters are activated, which is in the HCC cells. Therefore, when both plasmids are present, HBsAg synthesis starts exclusively in HCC cells.

CITHA systems

Figure 2 Schematic diagram of CITHA

As shown in Figure 2, our system can regulate the expression of HBsAg by miRNA and ceRNA, thereby specifically killing tumor cells. We divided the system into the HBsAg expression system (Plasmid 1) and the specific regulation system (Plasmid 2).

HBsAg expression system—Plasmid 1

The plasmid 1 contains tumor-specific promoter hTERT(BBa_K2624000), the Trojan Horse Antigen HBsAg gene region(BBa_K2989001), membrane localization sequence SCP1(BBa_K2989001), and the first input of “AND” Gate, miRNA(BBa_K2989002, BBa_K2989003, BBa_K2989004). This plasmid is turned “off” under most circumstances

Figure 3 HBsAg expression system

hTERT promoter

The hTERT promoter is a tumor-specific promoter and is used as the promoter in HBsAg expression system. Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase^[1], which determines the activity of telomerase, and is highly activated in most tumor cells. There was hTERT expression in all hepatocellular carcinoma (HCC), of which 74% were strongly expressed.^[2] The upstream regulatory region of the hTERT gene has strong promoter activity and is only expressed in telomerase-positive immortalized and cancer cell lines.^[3]

 

HBsAg

Hepatitis B surface antigen (HBsAg) is our “Trojan Horse”, which expressed on the liver cancer cells and cause immune response. It is an outer membrane protein of HBV encoded by HBV. Hepatitis B surface antigen is the main immunogen of host protective immunity, which can stimulate the body to produce neutralizing antibodies, also known as protective antibodies HBsAb. It is generally believed that the appearance of HBsAb^[4] indicates the termination of HBV infection.

 

Membrane localization sequence SCP1

SCP1 helps to locate HBsAg on the cell memberane. The serine/threonine phosphatase (PSP) in the protein contains three major families: phosphorylated protein phosphoprotein esterases (PPPs), metal-dependent phosphatases (PPMs), and aspartate-based phosphatases. Among them, SCP1^[5] belongs to the third family, aspartate-based phosphatase. It is highly conserved from fruit flies to mammals. The sequence of the 31 to 55 position of SCP1 has a membrane localization effect in various cell lines, such as human embryonic kidney cell HEK293T, human colon cancer cell DLD1, and human breast cancer epithelial cell MCF7.^[6] Therefore, we chose this sequence to localize the HBsAg on the surface of the tumor cells.

miRNA^[7]

miRNA is used as one input in AND GATE. A microRNA (miRNA) is a small non-coding RNA molecule (found in plants, animals and some viruses, that functions in RNA silencing  and post-transcriptional regulation of gene expression. miRNAs function via base-pairing with complementary sequences within mRNA molecules. As a result, these mRNA molecules are silenced, by one or more of the following processes: (1) Cleavage of the mRNA strand into two pieces; (2) Destabilization of the mRNA through shortening of its poly(A) tail; (3) Less efficient translation of the mRNA into proteins by ribosomes. Here, we utilize miRNA to silence HBsAg gene. Based on the chosen HBsAg segment, we further designed three miRNAs to target HBsAg: miR-HBsAg-70, miR-HBsAg-125, and miR-HBsAg-90.

Specific regulation system—Plasmid 2

The plasmid 2 contains the second input of “AND” Gate, ceRNA（BBa_K2989005, BBa_K2989006, BBa_K2989007）and a HCC specific promoter - HULC. This plasmid acts as the switch to turn plasmid 1 “on”.

Figure 4 Specific regulation system

These two plasmids together form our CITHA system and can exert specific tumor-killing effects under the control of AND GATE.

HULC promoter

HULC is a liver cancer-specific lncRNA that is Highly Up-regulated in Liver Cancer (HULC) and is one of the most up-regulated genes in HCC. In 76% of hepatocellular carcinoma tissues, the expression level of HULC was up-regulated by 33 times ^[8] compared with normal liver tissue. This enables HULC to be a HCC specific promoter.

CeRNA^[9]

CeRNA is aother input in AND GATE. The interaction of the miRNA seed region with mRNA is not unidirectional, but that the pool of mRNAs, transcribed pseudogenes, long noncoding RNAs (lncRNA), circular RNA(circRNA) compete for the same pool of miRNA thereby regulating miRNA activity. These competitive endogenous RNAs (ceRNAs) act as molecular sponges for a microRNA through their miRNA binding sites (also referred to as miRNA response elements, MRE), thereby de-repressing all target genes of the respective miRNA family. We selected our ceRNA segments for the three miRNA bind site through modeling and predicted their binding ability.

 

References

[1]Soder AI , Hoare SF , M uir S , et al .Amplificatio n, increased dosage and in situ expression of the telomerase RNA gene in human cancer [J].Oncogene , 1997;14(9):1013 -1021 .

[2] T oshikuni N , Nouso K , Higashi T , et al.Expression of telomerase -associated pro tein 1 and telomerase reverse transcriptase in hepatocellular carcinoma[J] .Br J Cancer , 2000 ;82 (4):833-837 .

[3] Horikaw a Ⅰ , Cable PL , A fshari C , et al.Cloning and characterization of the promo ter regio n of human telomer ase reverse transcriptase gene[J] .Cancer Res, 1999;59(4):826 -830

[4]Krugman S, Giles J P, Hammond J. Hepatitis virus: effect of heat on the infectivity and antigenicity of the MS-1 and MS-2 strains [J]. Journal of Infectious Diseases, 1970, 122(5): 432-436.

[5] YEO M, LIN P S, DAHMUS M E, et al. A novel RNA polymerase II C-terminal domain phosphatase that preferentially dephosphorylates serine 5 [J]. J Biol Chem, 2003, 278(28): 26078-85.

[6] 廖鹏. 磷酸酯酶SCP1调控c-Myc蛋白去磷酸化及相关生物学功能的研究和SCP1细胞膜定位的发现及其棕榈酰化修饰的研究 [D]; 华东师范大学, 2012.

[7] Fabian MR, Sonenberg N, Filipowicz W (2010). Regulation of mRNA translation and stability by microRNAs. Annual Review of Biochemistry. 79: 351–79. doi:10.1146/annurev-biochem-060308-103103. PMID 20533884.

[8] Tschernatsch M, Guelly C, Moustafa T. Characterization of HULC, a novel gene with striking up-regulation in hepatocellular carcinoma, as noncoding RNA.[J]. Gastroenterology, 2007, 132（1）:330-42.

[9]Tay Y, Kats L, Salmena L, Weiss D, Tan SM, Ala U, Karreth F, Poliseno L, Provero P, Di Cunto F, Lieberman J, Rigoutsos I, Pandolfi PP (October 2011). Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs. Cell. 147 (2): 344–57. doi:10.1016/j.cell.2011.09.029. PMC 3235920. PMID 22000013.