Salmonella typhimurium A1-R

Attenuated Salmonella strains are currently being explored as either cancer adjuvant or monotherapy. Anticancer properties of Salmonella are mostly immune-mediated. It essentially increases the “signal” coming from the tumor microenvironment by adding its own antigens and other immunostimulatory signals to the cancer cells after colonizing it. The end-result is enhanced immune system recruitment in the microenvironment hence augmented cancer cell death. At a more local level, it also directly competes for resources with the cancer cells. (1) Attenuation is necessary as wild-type Salmonella is too pathogenic such that the strength of the immune system wrought upon the cancer cells has too much collateral on the host. Attenuation seeks to limit this collateral and, ideally, eliminate it entirely. (2

Data from a phase 1 clinical trial (3) utilizing intravenous administration of attenuated Salmonella revealed several potential pitfalls. It would seem that colonization of the tumor microenvironment is not as potent as demonstrated in murine cells. Mechanisms underlying this difference have yet to be explored. However, once Salmonella colonizes the tumor cells, elimination becomes minimal, but not enough Salmonella colonizes the tumor microenvironment to provide ample anticancer properties. In other words, not enough missiles reach the target to ensure satisfactory destruction. Hence, our design adds additional anticancer properties through anticancer peptide release. One area in which VNP20009 did not fail, however, was safety. Intravenous infusion was remarkably tolerated by trial patients. Fever, hypotension, thrombocytopenia, anemia, persistent bacteremia, low WBC count (only observed at lower dose levels), hypophosphatemia, hyperbilirubinemia, diarrhea, vomiting, nausea, and elevated alkaline phosphatase and transaminases were observed, but none worsened clinical outcomes of the patients nor severely complicated the patients’ conditions.

Initially, we planned to utilize VNP20009, the strain used in the only phase 1 clinical trial. However, upon further analysis of alternative attenuated strains, we concluded that A1-R is the best option. As mentioned, VNP20009 does not demonstrate efficacious anticancer activity. So following our initial design philosophy of increasing payload, we found that A1-R was more potent than VPN20009. (4) The same study also found that A1-R in-vivo safety was superior. Considering the acceptable safety of VNP20009 in human subjects, we assessed A1-R as even a safer option. With these considerations, A1-R was decided to be the strain of choice for this system.

Buforin IIb

Bacterial-mediated cancer treatment (BMCT) is a quite recent development in the search for a cure for cancer. It uses different attenuated bacteria to target tumors and exert various antitumour effects. Among the many bacteria used in BMCT, one of them is Salmonella enterica servoar Typhimurium. This bacteria species is capable of targeting and colonizing tumors. It can be used to directly kill tumor cells or deliver anti-tumor agents. (1) The method that our team used is using S. typhimurium to deliver anti-tumor agents, more specifically Buforin IIb

Buforin IIb is an antimicrobial peptide with a helix-hinge-helix structure derived from histone 2A which was isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans.(6) This peptide contains 21 amino acids and exhibits a strong antimicrobial activity against a variety of microorganisms. It has a unique mechanism of action such as rapidly crossing bacterial membranes without lysing the cells. It kills bacteria by interacting with intracellular macromolecules. (7)

Buforin IIb is cytotoxic to cancer cells, but not to normal proliferating cells thus making it safer to use in a cancer treatment. Buforin also requires gangliosides on cell surface for internalization, something a cancer cell is abundant of, thus making it incredibly selective. Buforin IIb induces apoptosis to cancer cells by activating pro-caspase 9 and pro-caspase 3 which initiates mitochondria-dependent apoptosis, thus suppressing the progression of cancer. (8)

We had several other anticancer peptide candidates (Chrysophsin-1,2,3, UM-6, and HPRP-A1-TAT). Buforin IIb was singled out as it was the only peptide with thorough documentation on its cell specificity. It is documented to selectively act in negatively charged cells, a common property of cancer cells. This means that the peptides will not be wasted on healthy cells and, subsequently, healthy cells will not suffer destruction. This specificity increases the safety profile of our system as Salmonella already activates a systemic response even when attenuated. (8,10) In addition, buforin IIb is also more well-documented in literature than the other candidates as well. This abundance will assist the team in troubleshooting any problems that might arise.

6xHistag

A polyhistidine tag, commonly referred to as a His tag, is a collection of several histidine molecules used as a "tag" or marker that is attached to a protein. Histidine has a natural tendency to chelate with metal ions, making it easily bind to metals like nickel. In the case of recombinant proteins, the His tag is introduced into the protein through genetic engineering, typically consisting of two hexahistidine motifs. The purification of His-tagged proteins is achieved by exploiting their strong affinity for metal ions, a process known as affinity chromatography. Ni-NTA (Nickel-nitrilotriacetic acid) is often employed for this purpose, as it relies solely on the chelating site and is thus compatible with both the native and denatured protein structures. Following binding to the metal ions, the protein is subsequently washed with a specific solution, which may include compounds such as imidazole, EDTA, or conditions with a pH range of 5.9 to 4.5. This purification process ensures the isolation of the target protein with the His tag intact, allowing for further research or applications in various fields of study. (11)

p53

If the body is a complex, self-regulating machine that performs quality assurance checks every single time on every part of itself, then cancer cells are the “bug” of the software that enables certain parts to be detached and work independently, stealing the resources meant for the surrounding parts and disrupting the whole mechanism. Early signs of cancer cells are continuously checked, detected, and destroyed in an unending process of regulation.

The main agent that suppresses cancer cells from being “born” is the p53 gene, (12) the principal cellular responder to various stress signals such as oncogene activation, DNA damage, hypoxia, reactive oxygen species, and many more. Upon activation, tumor suppressor p53 will elicit a lot of cellular responses that induce arrested cell cycle, senescence, apoptosis, and even ferroptosis

Mutation of p53 gene in a cell is crucial to the formation of cancer cells, being the most commonly mutated gene in human cancers. (12) Mutated p53 leads to unexpressed self-destruction sequence, allowing cancer cells to live and proliferate. As such, p53 mutation is an integral part in cancer cell detection of the recent era. This ubiquity also extends to our cancer of choice. Cervical cancer cells have shown to reliably have mutated p53 proteins as a key part of their pathophysiology. (13,14) Therefore, p53 is a viable marker to detect cervical cancer cells.

In this project, we exploit the tendency of cancer cells to express mutated p53 as a genetic marker to denote the tumor microenvironment. A genetic sensor detecting mutated p53 is the basis of our system to selectively release our payload augmentation in an environment in which it can be most efficacious.

SCD derived p53-repressible promoter

Prior testing by Mircetic J et al (15) revealed genetic elements sensitive towards wild-type p53 derived from p53 regulated genes. The elements were sensitive not only to the absence or presence of p53, but could also differentiate between wild-type and mutated p53. We decided to use an SCD-derived element as it is wild-type p53 repressible and does not repress downstream expression when exposed to mutant p53. Additionally, this sensor has the impressive ability to respond to an environment expressing both wild-type and mutated p53 by repressing downstream genes. This sensor has also been proven to function in-vivo as well. Effectively, this becomes the sensor to trigger the detonation of our additional warhead. Subsequently, we would like to also introduce this promoter into iGEM parts database.

Pspv2

Pspv2 (Part:BBa K112706) is a constitutive promoter compatible in Salmonella. It will be used to constitutively express wild-type p53. Wild-type p53 will be how our system recognises a “neutral” environment i.e. not a cancer cell and keeps expression of buforin IIb off. Competitive inhibition of wild-type p53 by mutated p53 once Salmonella colonizes the cancer cells will alleviate the repression and allow free expression of buforin IIb.

Other genetic components

The other genetic components of the system were taken from iGEM parts database. These components are:

• Spacer 02 (Part: BBa K3831026):
• Spacer_7 (Part: BBa K3831015):
• Double terminator (B0010-B0012) (Part: BBa B0015):
• RBS by Elowitz (Part: BBa B0034):

Among the available spacers in the iGEM parts registry, Spacer 02 and Spacer_7 were chosen because they are the shortest and have the lowest GC content. Length and GC content were a concern brought by the gene synthesis process (Twist Bioscience) Our initial designs used a wider variety of spacers which resulted in genes that are incompatible for synthesis. Therefore, spacer selection was tweaked as it was the only genetic element easily swappable to meet the GC and length criteria. Meanwhile, the double terminator was selected for reliability and RBS for expression strength

We also utilized a rigid spacer for 6xHis tags not found in the iGEM parts database. The spacer functions to ensure that the tags do not interfere or to minimize any interference towards protein folding. Simulated protein folding yielded inconclusive results, therefore we chose to err on the side of caution and include the spacer. Subsequently, we would like to add this spacer to the iGEM parts registry.

pFPV25

pFPV25 was a gift from Raphael Valdivia (Addgene plasmid # 20667 ; http://n2t.net/addgene:20667 ; RRID:Addgene_20667). It is one of few commercially available expression vectors tested with Salmonella typhimurium with ample documentation and performs well in acidic conditions which lines up with our target tumor microenvironment. (16)

Drug delivery nanoparticles

Salmonella Typhimurium, a well-studied engineered facultative anaerobe, has been extensively modified for various purposes, including serving as carriers for oral vaccine delivery. This approach offers significant advantages over traditional intravenous vaccination methods, as it is not only cost-effective but also less toxic, also explored the use of synthetic polymer-coated live bacteria to target and damage cancer cells, incorporating multiple functionalities to enhance tumor-targeting efficiency, as demonstrated by Xiang et al. Moreover, the concept of "microbotics" has emerged, wherein bacteria are employed as vehicles for delivering nanoparticles and cargo. This approach leverages the invasive properties of bacteria for the delivery of nucleic acids and other payloads. Building on these previous studies, our work aims to coat Salmonella with nanoparticles formed from cationic polymers and DNA. This innovative approach is designed to overcome multiple challenges associated with oral DNA vaccine delivery. Cationic polymers have proven to be promising non-viral gene delivery vectors, as they can spontaneously form nanoscale polymer/DNA complexes (polyplexes) through electrostatic interactions. One notable advantage of cationic polymers is their strong buffering capacity, which enables them to escape from endo/lysosomes within cells through a phenomenon known as the "proton sponge" effect. Calcium carbonate (CaCO3) nanoparticles will serve as carriers for drug delivery. When these nanoparticles are administered orally and enter the body, they will disperse and eventually reach the target cancer cells. Subsequently, the drug carried by the nanoparticles will be released, entering the nucleus of the cancer cells and effectively treating the cancer. (19)

Further attenuation

As previously enumerated, A1-R is an excellent Salmonella strain for use in cancer therapeutics. It is widely studied and therefore its properties well-documented. However, it is also true that our team did not fully explore further modifications to the A1-R strain to create novel strains which have even better and tailored functions for our purposes. For example, knockout of inner membrane ring and basal body component FliF or the flagellum-specific ATPase complex FliHIJ of the flagellar machinery in Salmonella provides better efficacy in treating cancer cells, despite a loss of flagella intuitively meaning attenuated ability for colonization and reduced immune response. (2) Furthermore, the issue of rapid intravascular clearance found in the phase 1 clinical trial (3) was sidestepped. If investigated and proper modifications are made to solve this issue, our increased payload along with more thorough colonization increases efficacy even further. In conclusion, given the opportunity, further modifications to the A1-R strain should be investigated to increase its efficacy as an anticancer drug.

Tumor-positive genetic sensor

Our current genetic sensor relies on a change of substance instead of absolute detection of a marker. While the study on which we base our genetic sensor designed the p53 responsive elements as a method of detecting p53 deficient or mutant cells, the genetic sensor effectively detects wild-type p53 and its absence instead of mutant p53. This is why our design relies on competitive inhibition of the sensor with mutant p53 to function. This introduces an additional failure point which has to be taken into account when troubleshooting results. If a genetic sensor exists which activates directly on mutant p53 or another cancer cell genetic marker is found with a genetic sensor that detects it directly, it would make for a handsome replacement of our genetic sensor granted it actually experimentally succeeds.

In-vivo testing

A fairly obvious avenue left to be explored is in-vivo testing. Despite extrapolating safety profiles, it remains that our chosen strain has not been human tested and our system has no safety experiments. It is essential that the safety of A1-R expressing buforin IIb through our system is assessed in-vivo that it may provide a baseline expectation for human tests.