Tools and Technologies I
Human embryonic stem cells hold great potential for treating multiple human diseases, including cancer. However, our knowledge of the precise patterns of gene expression in stem cells is limited by very low efficiency techniques for introducing reporters into the gene(s) being investigated. Such knowledge can inform us of the state of differentiation of the cell being examined and also how these cells function in comparison to other cells in our bodies. Cancer may have an origin in cells with stem cell-like properties or even in stem cells themselves. For prostate cancer, although we have isolated a precise cancer-promoting genetic event, we cannot yet generate accurate models for study because of our inability to efficiently transfer large- sized DNAs into lineage-specific stem/precursor cells, such as prostate stem cells. In this proposal we provide a new technology for generating stem cells to study changes in gene expression during development and for generating accurate molecular models of human disease, such as prostate cancer. For over 2 years, we have been developing a radically new approach for transferring nuclei and large DNAs into stem/precursor cells. Our innovative device is called BLAST (Biophotonic Laser-Assisted cell Surgery Tool) and consists of metallic nanoparticles attached to the tip of a micropipette that is used to deliver materials into cells. This pipette is placed a close, fixed distance from a stem cell membrane by a robotic controller on a microscope stage. A pulse of non-damaging laser light rapidly heats the nanoparticles so that they generate precise local membrane holes for gene delivery (and in the future nuclear transfer). For soft/fragile stem cells, conventional microinjection is lethal due to excess trauma from piercing the pipette through the cell membrane, and an electrical-based method (electroporation) is very low efficiency for large DNAs. BLAST addresses 4 stated objectives from the CIRM as a tool for (1) more effective non-viral gene transduction, (2) a method for developing accurate reporter genes, (3) a potential approach for higher efficiency gene targeting, and (4) a potential method for tracking stem cells and their derivatives. We have assembled a team of recognized experts in optics and electronics, cancer, stem cell biology, and cell reprogramming to lead this effort. In proof-of-principle studies, BLAST successfully introduced DNA into 3 cell types with a survival rate of ~40-50%. In 3 Specific Aims we will further develop BLAST, generate stem cell gene-reporter lines, and introduce a large prostate cancer-specific genetic alteration. OUR PROJECT GOAL is to generate stable, genetically modified stem cells and lineage-specific stem/precursor cells containing large DNAs to study gene regulation and generate more accurate molecular models of human disease, such as prostate cancer.
Statement of Benefit to California:
Our proposal will benefit California by providing a new approach for inserting large DNAs into stem cells, which will allow precise interrogation of patterns of gene expression and the development of more accurate models of human disease. This work will support the California peoples' and taxpayers' commitments to individualized medical treatments of the near future through enhanced knowledge of appropriate targets for therapy and new insights into pathogenetic mechanisms of disease. Our work could also be applied to somatic cell nuclear transfer (SCNT) at a later stage of development, to further accelerate the push to personalized medical treatments for Californians and for the nation. Our approach will provide information to many of California's biotechnology and pharmaceutical companies in the burgeoning stem cell industry, whose success will propel hiring and increased economic prosperity for the state. Our work will provide additional information to patient advocates, ethicists, and even (eventually) medical geneticists to help select the optimal course for developing and modifying stem cell usage policies and infrastructure within California. In sum, the added knowledge provided by enhancing our capabilities in constructing accurate molecular models of human disease and detailed interrogations of gene expression patterns will have tangible health and economic impact on California, its academic institutions and biotechnology/pharmaceutical companies, and the rest of the nation as California and its people move forward with personalized medicine during the 21st century.
The goal of this proposal is to develop a new tool for the genetic engineering of human embryonic stem cells (hESC). The approach employs a novel laser-mediated technique to deliver large DNA fragments or bacterial artificial chromosomes (BACs) into cells. The technique employs laser pulses to induce localized holes in the plasma membrane that facilitate the transport of DNA fragments from outside the cell. The applicant proposes to perfect the technology and use it to generate stable, genetically-modified hESC. Reviewers agreed that the potential impact of the proposed work is high, since gene transfer methodologies that deliver large DNA molecules into cells could overcome a major hurdle in the stable transfection of hESC. Applications of the technique include the creation of induced pluripotent stem cells and engineering hESC with various disease traits. This method could significantly advance the field. Reviewers had serious concerns about the feasibility of the proposed approach. Preliminary data supporting the feasibility are limited, and no data on application of the technique to stem cells were presented. A proof-of-principle experiment employed an established cell line that is highly amenable to transfection, and the results may not be applicable to hESC. Justification for the cell types chosen for the proposed study was lacking and information comparing the new technique with other non-needle DNA delivery methods was not included. In addition, there was insufficient information about the number of cells that can be handled or rate at which they can be transduced with the technology. Finally, it was noted that the goal of delivering large DNA molecules seem to contradict the premise of this proposal to create smaller holes (submicron) since the DNA radius will be larger than a micron. The PI has expertise in cancer biology and is one of the inventors of the novel technology that is the focus of the proposal. The research team appears well qualified and has a good record of interaction. Reviewers were concerned that the PI is already heavily committed on a number of other research projects. Overall, reviewers felt the potential impact of the proposal is high but were unconvinced of the unique abilities of this technology to impact stem cells.