Basic Biology III
$1 468 800
This proposal seeks ways to measure and minimize damage to the chromosomes and DNA of cells to be used for stem cell therapy. These cells are called iPSCs, which stands for induced pluripotent stem cells, and they serve as stem cells for replacing cells that have been lost due to disease. In order to make iPSCs, we and others use normal cells from the body (called somatic cells) and use genetic methods to push them into being stem-cell like. This is a promising approach because we can use the patient's own cells for this. However, we do not know how much genetic damage that we cause when we manipulate and then push the cells to become stem cells. We must have ways to measure and decide whether any genetic damage is excessive. Excessively damaged iPSCs could be quite dangerous because they might not behave normally, and they could even become tumors. The goals here are: 1. Measure the amount of genetic damage in the iPSCs compared to the cells from which they are created (Aim 1A). It turns out that it is not trivial to measure the amount of genetic damage across the entire human genome. Our research team has a remarkably strong background in genetic and biochemical measurement of chromosomal and DNA damage. 2. Measure the amount of genetic damage in iPSCs after gene correction procedures. Gene correction will be a central step in using human stem cells to cure disease. For example, if a person has a disease, then, often, one of their genes predisposes to this disease. In these cases, it is helpful if we can correct that one gene before making the iPSCs or after making them. Our team is the most experienced in the world in human gene correction. In Aim 1B, C and Aim 2, we assess the amount of genetic damage that arises during the gene correction process. 3. Develop gentler ways of handling somatic cells and the iPSCs derived from them, so as to keep genetic damage to a minimum (Aim 3). Cells of our body are normally accustomed to seeing only about 3% oxygen in the dissolved gas around them when in our tissues. This is much lower than the 20% oxygen in room air and in the liquids (including laboratory solutions) that we use to wash and nourish such cells in the laboratory. Too much oxygen results in oxidation of the molecules that compose our cells, including the DNA that makes up our genome. We will develop methods that help the iPSC cells (or their somatic cell precursors) to make proteins that help protect them from high oxygen and oxidative conditions (Aim 3A). We will also develop reagents to add to the liquid in which we grow the cells in the laboratory to protect them from the oxidative environment (Aim 3B). We will also examine how the highly oxidative conditions affect the gene correction process (Aim 3C). Genetic quality is a critical aspect of stem cells, including iPSCs. This project will lay the foundation for minimizing any unwanted genetic damage.
Statement of Benefit to California:
The research proposed here will benefit the state of California in at least three major ways. First, the research here will move the application of stem cells to human disease forward. The progress that we will make will provide a firm foundation for assessing the genetic quality and genetic problems with various preparations of iPS cells. It will also generate better methods for preserving iPS cells in a genetically undamaged state. Second, the work here will advance biotechnology on stem cells within California. Third, the most immediate impact of this work will be to fund employment within the health research sector within California. The companies to which we planning to send samples or with who we are collaborating are all located in California (see Letters of Collaboration).
Project Synopsis: This proposal seeks to measure the level of genetic change that occurs during the process of generating human induced pluripotent stem cells (hiPSC) and in cells that have undergone gene targeting via homologous recombination (HR), with the overall goal to develop ways to minimize mutational load. To accomplish this goal, the applicants propose, in Aim 1, to perform several types of genome analyses, including whole genome sequencing, to determine the level of genetic and epigenetic change present in hiPSC as compared to the somatic cells from which they were derived. The mutational load acquired under different hiPSC derivation methods and following targeted gene corrections will be assessed. In Aim 2, they plan to perform a very similar analysis, starting with patient somatic cells. The goal of Aim 3 is to assess whether experimental reduction of oxidative stress alleviates mutational load in hiPSC and alters gene targeting efficiency. Significance and Innovation: - The proposed project addresses a major problem in the stem cell field, since understanding the extent of genetic change acquired in hiPSC, which may have detrimental consequences for cell behavior, is critical for the use of these cells for cell-based therapies. - Although the application of whole genome sequencing and other genome-wide studies to hiPSC is not greatly innovative, the goal of understanding the extent of secondary genetic changes that occur while performing targeted HR, and whether the mutational load is different when gene correction is performed in somatic cells or after reprogramming, is quite innovative. - The vast majority of this project is descriptive in nature, although the experiments proposed in Aim 3, albeit not truly mechanistic, intend to validate genes or pathways that may be involved in the acquisition of mutations in cultured cells. - The rationale for the project is logical and scientifically sound. Feasibility and Experimental Design: - Being able to generate, grow, characterize and genetically manipulate large numbers of hiPSC lines is critical for the success of this proposal. The absence of this expertise on the team and the lack of preliminary data in this regard call the feasibility of this project into question. - It is unclear if all the reagents for the numerous different reprogramming protocols are in hand, and whether the very large scope of this proposal can be achieved in the proposed time frame. - Since each hiPSC line originates from a single somatic cell, any observed genetic alteration may represent a rare occurrence already present in a potentially heterogeneous parental cell population rather than an acquisition during reprogramming or during gene targeting. Bulk analysis of the parental cell population, as proposed, will mask existing mutations present in rare cells, and is therefore not a valid experimental design. To overcome this problem, a reviewer suggested to compare the mutational load between several hiPSC lines and several somatic subclones, each derived from a different single cell within the same starting somatic cell population. - The ability to identify differences between two genomes critically depends on the quality of the biostatistical analyses. This application provides no detail in this regard, nor any detail regarding the challenging whole genome sequencing approach. - It is not clear if a study as proposed can be adequately powered using current technology and available resources to properly address the question. Principal Investigator (PI) and Research Team: - The PI is among the world’s leading researchers in the area of DNA repair and somatic mutations with numerous publications in top tier journals. - The assembled team has considerable experience with some of the technology relevant to this proposal. - The research team lacks critical expertise in generating and handling hiPSC. Although a well-regarded stem cell researcher provides a letter of support, that person’s role appears to be purely consultative in nature. Similarly it was not clear if expertise in HR in human will be available. - Computational biology support is provided through a core facility, but neither the nature nor the cost of this support is mentioned. No personnel are dedicated to biostatistical analyses, a critical omission in light of the cutting edge analyses that will be required to support whole genome sequencing efforts Responsiveness to the RFA: - The proposed research appropriately addresses the goals and objectives of the RFA in that it seeks to understand the genomic stability of hiPSC and to identify molecular mechanisms that reduce mutagenesis while performing gene correction on reprogrammed cells. - To achieve maximum benefit to science, the data (raw files and derived sequence files) should be deposited publicly. Human subjects protocols should be developed that allow this.