2008 Progress Report – Executive Summary

A-1: Vision

CCC is dedicated to first use system level control to direct the behavior of cells toward a desired fate for therapeutic purposes and then to study the responses of nano-cellular components in the signaling network using a smart Petri dish system.

A-2: Strategy

CCC will focus its effort on developing rapid search platforms for potent drug cocktails. This approach places CCC in a strategic position to efficiently move along the pathway toward medicine and to understanding the system response of nano-cellular molecules under multiple drug stimulations to explore new combinatory drugs.

A-3: Plan

Based on the strategy, CCC will realize its vision by setting the plan as follows:

A-3-a: Rapid Search for Potent Drug Cocktails

Bacteria and viruses evolve rapidly and can become resistant to a particular drug. If we apply multiple drugs against infectious agent, it can alleviate the drug resistance problem. The best known use of drug cocktails has been in the fight against HIV, the virus that causes AIDS. During two years, 1995-1997, the AIDS death rate dropped 60%. However, not many diseases have been treated using drug cocktails.

It has been a difficult challenge for researchers to determine the optimal dose of individual drugs to use in combination. For instance, a researcher testing 10 different concentrations of six drugs in every possible arrangement would be faced with 1 million potential combinations.

The feedback control scheme is to first start with an arbitrary combination of drug dosages. Quantitative output marker(s) are chosen and to indicate the outcome of the system, cell or patent, under drug cocktail stimulation. The output will be fed into a search algorithm for deciding the next combinatory drug dosages for possible better system response toward desired fate.

With a feedback control scheme, we have demonstrated the ability to home in to the most potent drug combinations in tens of searches instead of performing all one million trials. In an inhibiting viral infection experiment, the CCC team has found that total inhibition of the virus occurred at much lower drug doses than would be necessary if the drugs were used alone; in fact, the concentrations of the drugs were about 10 percent of that required when used individually (Wong et al, PNAS 2008).

A-3-b: Pathway to Medicine

We will use FDA approved drugs for applying drug cocktail studies to cellular and animal experiments. Once the optimized drug cocktail is identified, we can move quickly towards clinical tests. Our approach is a universal methodology that can be applied to many disease treatments. We have chosen infection, cancer and regenerative medicine as our targets.

Infectious diseases: The rapid search for potent combinatory drugs can be applied to diseases infected by either viruses or bacteria. Our current focus is to study infection of Herpes Simplex Virus 1 (HSV-1). HSV-1 has strong clinical relevance due to its high degree of prevalence in human diseases. Available therapeutics against this pathogen is only marginally effective and the suppression of disease reactivation remains elusive. Furthermore, since all available pharmaceutical agents target one enzyme of the virus, strains resistant to a particular drug have emerged for various infections.

Cancer: Signal transduction defects originate within the genome feedback onto the genome to assist in the generation and/or permissivity of complementary lesions that are required to drive the carcinogenic process forward. Collection of cellular components in the signaling pathway network participates in the process. The process may be interrupted by identifying key participants and purposeful introduction of offsetting signals or blockade of the signaling pathway. Drug combinations can be designed to modify a group of selected pathways that offset these signals or blockades. We are studying alterations in key signal transduction pathways, including Jak and Ras mutations, Pten deficiency, and dysregulated expression of TCL1. They all function as primary lesions in the development of a wide array of cancers, including leukemia/lymphoma and prostate cancer.

Stem cell studies for regenerative medicine: Among many important challenges of advancing stem cell for regenerative medicine, optimizing EB mediated ES cell differentiation is chosen as our first task. We have demonstrated that hydrophobic surfaces do not require fetal bovine serum, a potential source of pathogens, to promote the formation of desired-size EBs. In addition, our studies hold true for the human EB model. These two observations were key requirements to obtain high quality ES. The next step is to develop feedback control scheme for directing the differentiation of ES amenable for therapeutic applications.

A-3-c: Smart Petri Dish for Studying Network Responses of Nano Cellular Components

Exploring and monitoring the behavior of nano-cellular molecules are important for understanding the overall network response subjected to multiple external stimulations and for providing opportunities to develop new drugs. The challenge is to fully reveal the molecular behavior without missing critical information. Expression level measurements quantify the numbers of molecules in cells and provide useful information, but lose critical information of their interactions in the network of pathways. We are developing microfluidics-based phosphorylation arrays which monitor the activities and interactions of cellular components on a predetermined time course. In addition, we will be able to directly measure phosphorylation activities in living cell, once the tunable nano-plasmonic resonator (TNPR) is developed. Furthermore, the cells under investigation will be arranged in arrays by opto-electronic tweezers (OET) which allow us to perform single cell based studies.

Microfluidic circuitry: In the late 1980s, micro-electro-mechanical-system (MEMS) technologies emerged and enabled us to manufacture micron scale mechanical devices. A micron size device matches the size of a cell and facilitates the direct handling of cells. Fluid handling is necessary in all areas of biotechnology. Cells are cultured in fluid. Reagents are transported by fluid. By using microfluidic circuitries, it greatly facilitates the advancements of medical diagnostic and therapeutic studies.

TNPR: During the past decade, many functional nano-devices have been developed by the advancement of nanotechnologies. A nanometer thick sandwich structure, tunable nanoplasmonic resonator can achieve high level of surface-enhanced Raman scattering (SERS) signal. With the size of about 50 nm, it can be inserted into living cell and provide real-time monitoring of a large number of the cellular functions.

OET based cell processor: The invention of optoelectronic tweezers (OET) technology allows us to optically-control cells in a very flexible way. Essentially, we can manipulate the positions of a large array of cells in a time varying fashion as we need. Currently, we are integrating OET with electroporation techniques that have the potential to dramatically increase the throughput of single-cell-level electroporation.

The integration of these modalities forms a smart Petri dish which will enable us to perform real time sensing, processing information and applying stimulations.

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