Specific Aims

There is approximately a 39% chance for any given human to develop a cancer, and about 6.5% of those
cases are pancreatic cancer. Although it is not a substantial percentage, it is quite lethal; about 72% of
diagnosed pancreatic cancer patients will die within the first year of diagnosis. The cancer spreads rapidly and
is often hard to detect early on due to the pancreas being difficult to see since it sits behind the stomach. The
current treatments for pancreatic cancer are standard: surgery (if the cancer is in the pancreatic head),
radiation and chemotherapy. These treatments may be somewhat effective, but in order to reduce the lethality
of this disease, a more targeted therapy to eradicate this type of cancer must be created, a treatment that
attacks the cancers stem cells and prevents a tumor from reinvigorating itself. Currently, a major obstacle in
the field is that creating a targeted therapy is very difficult to accomplish because it is difficult to find genes
specific only to tumor cells, and even more difficult to find genes specific only to cancer stem cells.

Cancer stem cells are a distinct population of cells that have all of the multipotent properties of stem cells, and
proliferate into tumor cells, and they are necessary for the formation and upkeep of tumors. Pancreatic cancer
tumors have cancer stem cells. There are only a small number of cells in a tumor that are cancer stem cells,
but they sustain the population of tumor cells. Cancer stem cells usually have distinct surface markers and can
asymmetric cell division that allows them to divide into one stem cell and one progenitor cell. The most relevant
aspect of cancer stem cells is that they are resistant to standard cancer treatments.

To solve the problem, we will develop a strategy in order to evaluate many different genes known to be
expressed in pancreatic cancer stem cells. This strategy will allow us to evaluate whether the genes explored
will have any effect on cancer stem cell growth, and whether they affect other pancreatic tissue. If we can find
some examples of genes that affect stem cell growth, we can attempt to explore whether multiple genes being
turned off simultaneously would affect the health of the stem cells. If we can positively affect the destruction of
the stem cells, we can attempt to induce cancer in a pancreas organoid, to simulate a more physiologically
relevant system in which to test this hypothesis. We would induce cancer and attempt to dismantle the stem
cells by shutting off the genes only found in the cancer stem cells. This is supported by the fact that other
targeted therapies have been shown to be highly effective in destroying cancers, and this process can possibly
lead to a faster and more accurate result. Thus, finding the genes specific to the cancer stem cells will provide
us with a breakthrough in making targeted therapies for pancreatic cancer.

Aim I. Insert a dead Cas9 complex, with a specific guide RNA, into pluripotent stem cells. A repressor
domain will be fused to a dead Cas9, and inserted into the pluripotent stem cells along with a specific guide
RNA. The guide RNA will match the sequence for a specific gene that is known to be expressed in pancreatic
cancer, and will lead the dCas9 to the gene and instead of cutting the DNA, the dCas9 will just repress the
gene. This strategy will allow us to investigate the effects of turning certain single genes off known to be
necessary for pancreatic cancer stem cell survival.

Aim 2. Insert a dead Cas9 complex, with specific guide RNAs for multiple genes, into pluripotent stem
cells. The same exact protocol will be followed as in Aim I, however multiple genes will be targeted for
downregulation at the same time, so multiple guide RNAs will be added to the pluripotent stem cells. This
strategy will allow us to investigate the effects of turning multiple genes off in the stem cell system.

Aim 3. Create a functional pancreatic organoid, induce cancer and observe the progression. Creating
an organoid of the pancreas using the modified induced pluripotent stem cells, we would test the function of
the genes turned off by the dead Cas9. Once an organoid has formed, cancer would be induced, and the stem
cells would be monitored to determine whether tumor growth is suppressed by the genes turned off by the
dead Cas9.

The expected outcomes of this proposal are to be able to understand the combined effects of turning off
different genes known to be expressed in pancreatic cancer stem cells. These results will aid in developing
targeted treatments for pancreatic cancer, as well as provide implications for creating treatments for other
cancers as well.