Revision as of 02:18, 20 October 2016

• BACKGROUND
• EXPERIMENTS
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• FUTURE WORK
  Circuit Design Clinical Applications
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Clinical Applications

Developing a diagnostic

Endometriosis is a complex disease – any diagnostic needs to account for a variety of factors. We opted for a three-pronged approach, sensing progesterone resistance and miRNA dysfunction while maintaining temporal specificity. Each of these components represents a key hallmark of the disease, and when put together can create a sensitive diagnostic tool.

Implementing a Diagnostic

We initially contemplated creating an in-vivo diagnostic, but changed our minds after speaking with Professor Linda Griffith (MIT). Realistically, inserting a genetic circuit into a human (likely through the use of a viral vector) is many years away from being a possibility due to major safety concerns.

Implementing an in-vitro diagnostic that can be used on an endometrial biopsy is more feasible. An endometrial biopsy is a 5-15 minute procedure that can be done in a doctor’s office without anesthesia and is commonly used when testing for endometrial cancer. While not pain-free, endometrial biopsies are considerably less invasive than laparoscopic surgery, the current diagnostic method for endometriosis.

Due to the nature of the procedure, the cells collected are eutopic endometrial cells, not the ectopic cells associated with endometriosis. However, the literature suggests that the eutopic endometrium of women with endometriosis displays different molecular markers than that of healthy women. Notably, the eutopic endometrium of patients with endometriosis displays progesterone-resistance – a key identifier we used in designing our circuit [1]. Additionally, there are differences in miRNA profiles between the eutopic endometrium of healthy women and that of women with endometriosis [2][3].

Of course, an in-vitro sample does not experience the natural hormone cycle. However, the tissue from the biopsy can be dosed with estrogen and progesterone to simulate the menstrual cycle, taking advantage of our proposed circuit architecture. We have characterized our promoters by dosing cells with progesterone and estrogen and have had success.

An in-vitro approach is considerably safer than using a viral vector for in-vivo diagnosis. The literature supports the notion that the molecular markings of eutopic endometrial cells can be used to identify patients with and without endometriosis. Dosing tissue from an endometrial biopsy with hormones allows us to test these molecular differences and use our diagnostic in an in-vitro setting. With a less invasive diagnostic method, we hope that the diagnostic process can be expedited.

References
[1] Burney et al. Endocrinology 148 (2007)
[2] Joshi et al. Hum Reprod. 30:2881-91 (2015)
[3] Burney et al. Mol Hum Reprod. 15:625-631 (2009)

Synthetic Biology & Medicine - How SynBio Approaches Can Transform Diagnostics

Our approach of using a genetic circuit to sense diseased cells presents possibilities beyond endometriosis. Principles of synthetic biology can be applied to develop gene and cell therapies/diagnostics for a number of diseases [1]. In order to implement gene therapies safely and effectively, there must be therapeutic specificity as well as control over these synthetic systems.

Using biomarkers to enhance therapeutic specificity is one way to ensure that gene circuits are only activated at the correct time and location. miRNA profiles, which we took advantage of in our diagnostic, are one way to differentiate between diseased and healthy states. miRNA dysregulation has been observed in different cancers [2]. Additionally, synthetic biology tools can be used to improve the specificity of cancer immunotherapies, creating engineered T-cells with the ability to bind multiple antigens [3].

Small molecule control of gene circuits allows for an added level of safety and potentially better patient outcomes. Once the therapy has been delivered to the patient, the clinician can have control over the system to fine-tune its activity through the use of small-molecules or biologics. Through controlled dosages and monitoring, these compounds (which have relatively short half-lives) can be used safely to ensure the therapeutic is at an appropriate activity level [4].

As building, testing, and delivering complex gene circuits continues to become more efficient and reliable, synthetic biology has the potential to develop new diagnostics and therapeutics. Our approach of using a genetic circuit to sense diseased cells emphasizes our belief that medicine is a promising direction for synthetic biology.

References
[1] Lienert et al. Nature Reviews Molecular Cell Biology 15:95-107 (2014)
[2] Xie et al. Science 333:1307-11 (2011)
[3] Roybal et al. Cell 167:419-432.e16 (2016)
[4] Gitzinger et al. Proc Natl Acad Sci USA 106:10638-43 (2009)