Team:Hong Kong HKU/Description

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                              Our team recieved the Silver medal at the Giant Jamboree in 2019!  
                              Thank you to all of you who supported us throughout the whole competition!

Project Description





Background

DNA nanostructure are known for their malleability in target selection with the usage of aptamers, and also stability to nuclease attacks due to 3D hindrance. In our project, we aim to construct DNA nanostructure and engineered salmonella targeting cancer stem cells. Which without eradication, would lead to relapses of tumours.

Despite there are existing chemical synthesis methods for single stranded DNA for therapeutic DNA nanostructure, the production of nano drug carrier (NDC) is still expensive and time consuming. The usage of ETHERNO system (HKU 2018 iGEM) aims to provide a functional basis for in vivo synthesis of DNA nanostructure, which would path way to increasing the utility toolbox available in co-culture systems in future projects. Improvement of co-culture systems could allow us to utilise engineered microbes in the tumour micro-environment or even in the cancer cell cytoplasm. With NDC directly synthesised in the tumour in vivo, it could also act as "drug sponge" due to binding of free drugs onto these NDC, increased specificity and also increased safety by less damage to normal cells could be achieved. Other NDC design could also include antisense RNA or DNA designs to down-regulate specific pathways. However, it is not within the scope of our project.

Cancer pharmacotherapy has long been yielding considerable research interests, with the great advancements in drug screening and target identification. Yet the progress is greatly impeded by drug resistance and low metabolic activity of cancer stem-cells, rendering many chemotherapy drugs, like Doxorubicin, ineffective.

Moreover, Doxorubicin, a conventional anti-cancer drug that suppresses proliferation in a variety of cancer cells, often entails with various life-threatening side effects, including cardiomyopathy [1-2], and there is thus an urge to fabricate a novel pharmacological platform to both increase the specificity of the drug, as well as combination treatment to lower drug resistance in cancer stem cells.

Evidence has been emerging that the attenuated salmonella strains display a compelling intrinsic antitumor activity, penetrating and colocalizing the hypoxic region of tumor [3]. Strain used in the project, SL7207 favours accumulation in tumour micro-environment instead of other body parts, and exhibits invasion and endosomal escape mechanisms, making it a great vaccination and gene delivery system [4]. After interviewing with experts, we further increased the specificity and decreasing the off target effect, by conjugating NDC to Salmonella using a salmonella aptamer in the DNA nanostructure design. With the increasing focus on cancer stem cell treatments and DNA nanostructure therapeutics, we herein demonstrate a novel system with high programmability and specificity.






1. Mass production of DNA tetrahedron

Our DNA nanostructure is a tetrahedron composed of just four ssDNA. The vertex of each ssDNA consists of one aptamer which includes SYL3C (targeting liver cancer stem cell epithelial cell adhesion molecule), AS1411 (targeting cancer cell surface nucleolin). And in different situations, like to be used in animal models, also includes ST1 and APT33 (targeting Salmonella surface antigen). We employedETHERNO, system by 2018 HKU igem team, for in-vivo functional DNA nanostructure synthesis. The strands are synthesised by the reverse transcription system, where HIV reverse transcriptase (HIV-RT) and Murine Leukemia Virus Reverse Transcriptase (MLRT) together, enable efficient reverse transcription and mass production of the ssDNA strands in E. coli. The strands can be further extracted and purified for in vivo assembly into functional nanostructures for doxorubicin (Dox) loading and targeted treatment of liver cancer.

We also presents a system which utilises the malleability of DNA nano-drug carriers. Aptamer could be changed using an easy cloning system which involves type IIS restriction enzymes. The system allows the input of different aptamers into the NDC strands with different therapeutic targets, like easily changeable bacterial and specific cell type targeting.

2. Multifunctional Genetically-Modified Salmonella Typhimurium

Salmonella Typhimurium (Strain SL7207) was employed as a vaccination strain in this project. This genetically modified salmonella serves three major purposes:
(i) To deliver our Dox-intercalated DNA nanostructure into cancer cells;
(ii) To enhance Dox efficacy by minimizing the stemness of cancer stem cells by amiRNA construct delivery;
(iii) To enhance the safety of clinical purpose via decrease of escaped bacteria;

Doxorubicin relies on the intercalation of genome DNA, and impedes cell function during replication [1]. Reduced stemness of cancer cell would be reported via doxorubicin induced cytotoxicity [5]. The salmonella was transformed with plasmid carrying reporter GFP gene and amiRNA targeting stemness genes STAT3, SOX4 and SOX9. Those genes were transcribed into miRNA. They then bound to target mRNA. This triggered RNA silencing and downregulation of STAT3, SOX4 and SOX9 mRNA in cancer stem cell.

A better co-culture system was also developed by incorporating a single biobrick into salmonella strain, which is a flagellar transcription factor. The flagella of Salmollela was over-expressed in favor of its mobility, that considerably enhance the efficiency of Dox-nanostructure delivery, as well as the accumulation inside the tumour [6]. Moreover, compared to traditional liposomal and viral gene delivery, bacterial delivery could provide higher motility, specificity, long lasting effect as bacteria will colonise inside tumour, as well as provides endurance to extreme conditions like pH and hypoxic conditions.

3. Gene Delivery

Liver cancer stem cell line Huh7 was utilised as a model system in this project, due to its high expression of cancer stem cells (60%) compared to HepG2 and some other cell lines [6-7]. Spheroids of huh7 could provide a good modelling for cancer stem cell targeting due to its large population of cancer stem cells, but also includes a moderate population of normal cancer cells.

Evidence has showed discrepancy of drug response between monolayer and spheroid cell culture [8]. To reduce discrepancy between cultures and real situations, further mimicking the physiological niche of cancer cells including the hypoxic and acidic tumour environment, we modified a spheroid co-culture system.

This not only provides a better modelling, but would also bring the benefit of not using animal modelling due to ethics and animal cruelty. Which animal modelling has been used for nearly all the researches in the past for therapeutic Salmonella strains, as a mature co-culture system was not previously been developed. It would provide an usable alternative to animal modelling which was not viable without a maturely developed co-culturing protocol.

Our dual delivery system with both salmonella and DNA tetrahedron can provide manifold benefits that certify both efficacy and specificity. In vivo synthesis system enables mass production of the nanostructure. Capability of Salmonella Typhimurium growing in both aerobic and anaerobic environment expands its range of usage, from small to large tumours [9-10]. Taken all the characteristics together, our system provides a more efficient and specific alternative to existing therapy.


Workflow Demonstration







What could our system achieve:


DNA in vivo Synthesis Parts


The parts were improved from 2018 iGEM system ETHERNO, with an addition of interchangeable aptamer system. The parts can provide E. Coli or other prokaryotes with:

  1. The ability to synthesise ssDNA in vivo.
  2. The opportunity of synthesising DNA nanostructure at tumour site, providing more utility and flexibility to bacterial cancer therapeutics.
  3. Higher safety for chemotherapy, as DNA intercalates on the nanostructure carrier, decreasing unspecific spreading to non-cancer body tissue.
  4. Specific delivery via aptamers to cancer cells/cancer stem cells. Interchangeable aptamer system also increases the flexibility of system for other diseases.



flhDC Transcription Factor Parts

The parts can contribute to Salmonella therapeutic with:

  1. Increasing safety of clinical application, and specificity of bacteria to tumour site, avoiding spread of bacteria to other body tissue.
  2. 10-fold higher colonisation of spheroids, allowing the development of better co-culture model.
  3. Increasing gene delivery rate and frequency via higher colonisation.



eSIBR Cassette: amiRNA Parts


The parts can function in mammalian cells (when delivered with a vector, bacterial or liposomal or other methods) by:

  1. Knock down any desired mRNA via customised insertion into the empty eSIBR cassette biobrick.
  2. Multiple mRNA knockdown via a single plasmid construct.
  3. Stem-cell targeting via knock down of STAT3, SOX4, SOX9. Decreasing drug resistance and stemness, for the use in combination for efficient chemotherapy.
  4. Safe method, for not involving any sort of toxin mediated therapy. Eliminating side effects.



The Whole System


The combination of all the parts could:

  1. Provide a safe and efficient clinical method for cancer treatment.
  2. Eliminate the use of mouse model for ethical purposes.
  3. Dual approach via chemotherapy and gene therapy (mRNA knockdown).
  4. Dual specificity via aptamer conjugation and bacterial tumour residing property.
  5. Cheap andefficient method, since liposomal gene delivery is expensive, and virus based delivery is unstable in tumour microenvironment. Bacterial could inhabit in tumour environment bringing consistent effect.



Reference
  1. Shafei, A., El-Bakly, W., Sobhy, A., Wagdy, O., Reda, A., Aboelenin, O., ... & Ellithy, M. (2017). A review on the efficacy and toxicity of different doxorubicin nanoparticles for targeted therapy in metastatic breast cancer. Biomedicine & Pharmacotherapy, 95, 1209-1218.
  2. O’Brien, M. E., Wigler, N., Inbar, M. C. B. C. S. G., Rosso, R., Grischke, E., Santoro, A., ... & Orlandi, F. (2004). Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX™/Doxil®) versus conventional doxorubicin for first-line treatment of metastatic breast cancer. Annals of oncology, 15(3), 440-449.
  3. Hernández-Luna, M. A., & Luria-Pérez, R. (2018). Cancer Immunotherapy: Priming the Host Immune Response with Live Attenuated Salmonella enterica. Journal of immunology research, 2018.
  4. Yu, Xia et al. “Attenuated Salmonella typhimurium delivering DNA vaccine encoding duck enteritis virus UL24 induced systemic and mucosal immune responses and conferred good protection against challenge.” Veterinary research vol. 43,1 56. 6 Jul. 2012, doi:10.1186/1297-9716-43-56
  5. Kimura, O., et al., Characterisation of the epithelial cell adhesion molecule (EpCAM)+ cell population in hepatocellular carcinoma cell lines. Cancer Sci, 2010. 101(10): p. 2145-55.
  6. Yamashita, T., et al., Discrete nature of EpCAM+ and CD90+ cancer stem cells in human hepatocellular carcinoma. Hepatology, 2013. 57(4): p. 1484-1497.
  7. Raman V, Van Dessel N, O'Connor OM, Forbes NS. The motility regulator flhDC drives intracellular accumulation and tumor colonization of Salmonella. J Immunother Cancer. 2019 Feb 12;7(1):44. doi: 10.1186/s40425-018-0490-z.
  8. Dubbelboer IR, Pavlovic N, Heindryckx F, Sjögren E, Lennernäs H.Liver Cancer Cell Lines Treated with Doxorubicin under Normoxia and Hypoxia: Cell Viability and Oncologic Protein Profile. Cancers (Basel). 2019 Jul 20;11(7). pii: E1024. doi: 10.3390/cancers11071024.
  9. Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA. Spheroid-based drug screen: considerations and practical approach. Nat Protoc. 2009;4(3):309-24. doi: 10.1038/nprot.2008.226.
  10. Patyar, S., Joshi, R., Byrav, D. P., Prakash, A., Medhi, B., & Das, B. K. (2010). Bacteria in cancer therapy: a novel experimental strategy. Journal of biomedical science, 17(1), 21.





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