SOURCES= The Scientist, Nature, UC San Diego
A synthetic genetic circuit programmed into an attenuated Salmonella enterica subspecies can be used to systemically deliver an anti-tumor toxin into mice with cancer. The circuit allows the bacterial cells inside a tumor to synchronously self-destruct by lysis, releasing the toxin directly in the tumor
Researchers at the University of California San Diego and the Massachusetts Institute of Technology (MIT) have come up with a strategy for using synthetic biology in therapeutics. The approach enables continual production and release of drugs at disease sites in mice while simultaneously limiting the size, over time, of the populations of bacteria engineered to produce the drugs
“This impressive study represents a big step towards one of the great dreams of synthetic biology: rationally programming cells, in this case bacteria, to exhibit complex, dynamic, and beneficial behaviors in a host organism,” Michael Elowitz, whose Caltech lab builds synthetic genetic circuits and who was not involved in the work, wrote in an email to The Scientist.
Bert Vogelstein, a cancer geneticist at the Johns Hopkins’ Sidney Kimmel Cancer Center in Baltimore who also was not involved in the work, agreed. “This paper describes a highly innovative strategy employing synthetic biology to weaponize bacteria and shows that these bacteria can be used to slow the growth of tumors growing in mice,” he wrote in an email to The Scientist.
Schematic of the microfluidic device used to co-culture engineered bacteria and cancer cells (top). Live co-culture of engineered bacteria and cancer cells immediately before and after the synchronized lysis event, showing cancer cell death (bottom left, right, respectively).
JEFF HASTY, UC SAN DIEGO
The idea of using bacteria as a cancer therapy is more than 100 years old. “And attempts to engineer bacteria for therapeutic and other purposes are also many,” said Shibin Zhou, an associate professor of oncology at Johns Hopkins, who penned an accompanying perspective. “What is new here is a genetic circuit that allows synchronized production and release of a toxin in repeated cycles by the engineered bacteria.”
Jeff Hasty, a professor of biology and bioengineering at UCSD, and his colleagues had previously developed a simple genetic circuit in Escherichia coli consisting of a positive feedback loop. The circuit produces a small molecule autoinducer—AHL, which is secreted by the bacteria and diffuses to neighboring cells—as well as a green fluorescent protein (GFP) reporter from the same promoter. Another downstream gene under the same promoter encodes a protein that degrades AHL, forming a negative feedback loop. The circuit allows the cells to go through multiple cycles of GFP bursts that can be viewed under the microscope when the cell population reaches a critical mass and produces enough AHL.
For the present study, the team modified the circuit to include a gene expressing an anti-tumor toxin—haemolysin E, which accumulates inside the cell—and a gene for a bacteriophage protein that lyses bacteria. Once the AHL reaches a critical level, the bacteriophage lysis protein is expressed, kick-starting a negative feedback loop, allowing the cells to go through a cycle of growth followed by lysis when a population threshold is reached, leaving behind only a few surviving cells.
The researchers challenged cervical tumor cells in a microfluidic device with the engineered bacteria and saw that, following bacterial lysis, the tumor cells were killed, about 111 minutes after the initial GFP activity. The team also created similar circuits but substituted haemolysin E with either a gene to activate murine T cells or to trigger tumor apoptosis.
Injecting the bacteria expressing the immune-activating gene directly into the tumor was the most effective in deterring malignant growth in a colorectal cancer mouse model, the researchers found.
Next, Hasty and colleagues orally administered a mixture of all three bacterial strains to mice with colorectal tumor metastases, and simultaneously treated the animals with the chemotherapy drug 5-fluorouracil (5-FU). The team found that this combination increased overall survival of the mice compared to either chemotherapy or the bacterial strains alone.
The researchers found that the population of bacteria cycled within the mice for 18 days. While the orally injected bacteria are spread systemically, the anaerobic bacteria are likely able to infiltrate and grow within the anaerobic tumor where standard drug penetration is poor, according to Hasty. “The toxin-producing bacteria are likely killing tumor cells from behind enemy lines, so to speak.”
“The combination therapy didn’t result in tumor eradication,” noted Zhou. “But the idea that you can engineer this type of circuit is really forward-thinking and a proof of concept that could be applied not only to cancer, but also to other diseases.”
Elowitz agreed. “Even this relatively simple genetic circuit built out of just a handful of genes can be amazingly powerful. As we learn to design and construct genetic circuitry better, it should become possible to program cells to perform an almost unlimited variety of computations and behaviors.”
One major question, according to Elowitz, is how to reliably get the synthetic bacteria to efficiently home to the diseased tissue or organ while not harming healthy tissue.
Hasty’s team is now working to develop a modified strain that, rather than cycling through growth and lysis phases on its own, can be killed and mopped by the delivery of a subsequent dose of the bacteria.
The widespread view of bacteria as strictly pathogenic has given way to an appreciation of the prevalence of some beneficial microbes within the human body. It is perhaps inevitable that some bacteria would evolve to preferentially grow in environments that harbour disease and thus provide a natural platform for the development of engineered therapies. Such therapies could benefit from bacteria that are programmed to limit bacterial growth while continually producing and releasing cytotoxic agents in situ. Here we engineer a clinically relevant bacterium to lyse synchronously at a threshold population density and to release genetically encoded cargo. Following quorum lysis, a small number of surviving bacteria reseed the growing population, thus leading to pulsatile delivery cycles. We used microfluidic devices to characterize the engineered lysis strain and we demonstrate its potential as a drug delivery platform via co-culture with human cancer cells in vitro. As a proof of principle, we tracked the bacterial population dynamics in ectopic syngeneic colorectal tumours in mice via a luminescent reporter. The lysis strain exhibits pulsatile population dynamics in vivo, with mean bacterial luminescence that remained two orders of magnitude lower than an unmodified strain. Finally, guided by previous findings that certain bacteria can enhance the efficacy of standard therapies11, we orally administered the lysis strain alone or in combination with a clinical chemotherapeutic to a syngeneic mouse transplantation model of hepatic colorectal metastases. We found that the combination of both circuit-engineered bacteria and chemotherapy leads to a notable reduction of tumour activity along with a marked survival benefit over either therapy alone. Our approach establishes a methodology for leveraging the tools of synthetic biology to exploit the natural propensity for certain bacteria to colonize disease sites.