Methods
Summary
CRISPR technology has enabled the manipulation of cells by deletion of DNA and/or introduction of novel genes. The first stage of this project will be to use this technology to manipulate and tag certain cells of the immune system (from commercial sources). Those cells will be identified as "enabled cells" Under a number of different conditions, those enabled cells will be tested in parallel with non-treated cells for their ability to kill cancer cells in cell culture. Both the immune and cancer cells will be from mice. This will be tested under various conditions designed to mimic the situation in the mouse as much as possible.
Success in this part of the project will lead to the next stage which will be in two parts. The first part will be to examine the lifetime of the enabled cells when introduced back into mice that do not have cancer. Do the enabled cells survive for a longer or shorter time in the mice. Normally, cells of the immune system have a defined lifetime after which they die. Replacement of immune cells is a normal phenomenon that ensures a continuous supply of immune cells. Analysis of the lifetime of the enabled cells will allow for a course of injections that will enable a continuous supply of enough cells to fight cancer while assuring that the enabled cells do not present a potential hazard because of their persistence in the mice.
The second part of this approach will then be to use the results of the first part to inject mice with cancer cells and then, at various times that are well established, to inject enabled cells in a schedule determined by the first part described above. The mouse cancer model will be one that examines both the growth of a tumour and its spread to other organs. This is a very well established model and I have worked with this model in the past.
Challenges
The first challenge is to create the enable cells. How to evaluate the percentage of cells that have been changed by the procedure? Does this need to be 10% or 90%. The cells also will be "tagged" with a marker that will allow them to be identified in blood samples. The cells will therefore need to have multiple alterations that can be done with a single CRISPR manipulation. The goal should be at least 75% The conditions of the experiment will be manipulated in order to achieve this goal. The next challenge is to create the conditions for cells in culture so that a correct comparison can be made between natural and enable cells. The normal cells should be able to initially kill some cancer cells but then to be inhibited from further activity, allowing the cancer cells to grow. The enabled cells should continue to kill cancer cells and, in the correct ratio, either control the numbers of cancer cells or to eliminated them. The challenge is to manipulate the conditions so as to enable the cancer cells to selectively inhibit the normal immune cells. This will be achieved through manipulation of the growth conditions in the culture medium
Next, the challenge is to measure the numbers of enabled cells in the mice. This should be relatively minor by taking blood samples and looking for the enabled cells that have been tagged.
Pre Analysis Plan
My hypothesis is that, by manipulating the genome of certain white blood cells, they will become resistant to the inhibitory effects of cancer cells and will continue to recognise them as cancerous and destroy them. In order to support this hypothesis, the following data and outcomes will be analysed.
1. The work in cell culture will assess the extent to which the enabled versus normal white blood cells can survive in co-culture with cancer cells, and whether or not they can kill those cancer cells. This will entail the counting of cancer cells in active growth and analysis of any decrease in number of those cancer cells. Specifically, do they decline under conditions where normal while blood have no effect but where enabled cells do have an effect. the hypothesis also suggests that quiescent or growing cells should be recognised and destroyed and so this will be tested under growth versus non-growth conditions. The analysis will determine whether or not all the cancer cells will be eliminated, with time. This might not be possible in cell culture, however the parameters will be examined to see if any decline in cancer cell survival could be extrapolated to their elimination. If non-dividing cancer cells are not destroyed, this can also be beneficial in terms of controlling any growth of a cancer in the patient. Finally in this part of the project, the hypothesis is that cancer cells will not develop resistance to the enabled cells. This will be analysed by looking at any surviving cancer cells, and culturing them separately until they enter a growth phase and then re-challenging with enable cells. If no resistance has developed, the enabled cells should again destroy the cells to a similar extent as previously.
2. The hypothesis that enabled cells will destroy cancer cells in culture will also be extended to examining their effect in mouse models of cancer. The first model will be a melanoma model that has been established for many years, where cancer cells are injected into mice and migrate to the lung where they form colonies. By testing the model whereby about 100 lung colonies are expected, and where enabled cells are injected, two scenarios will be tested. First, when enabled cells are injected along with the cancer cells, how many lung colonies will become established and how much longer do they survive.. The best outcome would be none, however substantial decreases (e.g 10) would also be acceptable. This is only a preventable option and likely would not represent a good clinical use and outcome. The second scenario is when the colonies are allowed to become establish,. Now when the enabled cells are injected, can they reduced the number and/or size of the colonies. A good potential for clinical utility would be substantial reductions in numbers and/or size in colonies with a single administration of enabled cells, along with better survival of the mice.
Further work can be done in this model with repeated administrations of enabled cells to determine if they improve upon the initial results.
It is expected that enabled cells would have a similar survival time (10-14 days) that normal cells would have in the body. This will be assessed and if there is a different survival compared with normal cells, this will be assessed. The hypothesis is that enabled cells will have a similar lifespan as normal cells so that the potential of repeated administration is possible and that adverse effects of the enabled cells within the body will be minimized.
Protocols
This project has not yet shared any protocols.
