Assessing efficacy and safety of retro- and lentiviruses as gene therapy vectors in human embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Embryonic stem cells (ESCs) are derived from the inner cell contents of the pre-implanted embryo. These nascent cells not only can divide and perpetuate themselves (self-renewal) but are also capable of generating almost every body cell type, including muscle, heart, skin, bone, and brain cells. Because of these special properties, human ESCs (hESCs) offer the possibility of treating various human diseases, and can serve as an invaluable experimental model to study early events in human developmental biology. Many potential applications involving hESCs require reproducible and safe methods for delivering genes. A gene delivery vehicle that is based upon retroviruses represents the most efficient strategy to stably introduce therapeutic genes into hESCs. However, this process currently involves insertion of the gene randomly at many possible sites within the host cell genome. A chance insertion into regulatory or coding region of an important host gene may have significant consequences to hES or hES-derived cells, and thus the effects of insertion site location in hESCs need to be fully investigated. Our broad, long-term objective is to access the efficacy and safety of using retroviruses as gene delivery vehicles in hESCs. The Specific Aims of this proposal are (i) to perform a comprehensive, genome-wide analysis of insertion sites for retroviral gene delivery vehicles in hESCs, and (ii) to determine the role of insertion site location upon the activity of the introduced gene and the onversion of hESCs to more mature cell types. Understanding the insertion site preference of retroviral vectors in hESCs is critical for evaluating the efficacy and safety of using these vectors for diagnostic and therapeutic purposes. The above information gained from the proposed study may reveal retroviral factors and host conditions that can affect insertion site selection, and thus guide strategies for engineering retroviral vectors that can safely and effectively manipulate the hESC.
Statement of Benefit to California: 
Because of human embryonic stem cells’ (hESCs) special properties to self-perpetuate and to mature to form any cell type, hESCs offer the possibility of treating various human diseases. Many potential diagnostic and therapeutic applications for hESCs require genetic manipulation of hESCs using retroviral vectors. The proposed study is aimed at investigating, and ultimately improving the safety and efficacy of delivering genes into hESCs by retroviral vectors, an important consideration in successfully bringing stem cell treatments to a clinical setting. The study will therefore benefit the State of California in terms of lowering health costs and in treating its citizens who suffer from a wide range of different diseases, including stroke and heart disease, diabetes, cancer, neurological disorders, burns, autoimmune disease, muscular dystrophy, and AIDS.
Progress Report: 
  • Full clinical potential of human ES (hES) cell therapy can be achieved when one can grow hES cells effectively while maintaining full pluripotency. We have focused on developing stem cell culture media by which we can maintain pluripotency of human ES (hES) cells. It is critical to determine and develop a chemically defined media that are animal product-free and feeder cell-free conditions so that the media can be standardized throughout stem cell research and in clinical situations.
  • One major recombinant protein component we will use in developing chemically-defined media is a set of TGF-beta signaling ligands, receptor domains, and ligand-specific antagonists. We have established a new method of generating a diverse array of these ligands, including BMPs, Activins, inhibin, and their heteromeric ligands of the BMP/Activin class ligands. Some of these heteromeric ligands possess their signaling properties unlike their homodimeric counterparts. These reagents include Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, and their derivatives. These reagents have been engineered by chimeric recombination. They were also further modified by site-specific mutagenesis, and by combinatorial heterodimeric assembly to create and modify protein-specific binding affinity to their binding counterparts. Several of these reagents are now available as recombinant protein in sufficient quantity for large-scale screening for media composition.
  • To establish the functional characteristics and optimal culture combinations using these new reagents, we have used an established hES cell, H9. We have cultured H9 cells in various compositions of culture media containing some of the engineered reagent and followed expression of several differentiation markers to monitor for pluripotency of hES cells, and also for their differentiation-guiding and pluripotency-maintaining abilities. We have first examined effect of aforementioned reagents: Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, BMP3 S28A mutant, in our standard culture media mTeSR condition, which does contain bFGF, for proliferation and differentiation of hES cells. In these assays, hES cell line H9 was cultured and reagents were added at varying concentration (1-100 ng per ml) over 1-5 days culture period. Reagents were added in new media during the course of cell culture. We have used morphological change and the presence of markers as a means to follow the differentiation. Ectoderm markers are Nestin, Cdx2; Mesoderm by Brachyury, HBZ; Endoderm markers by CXCR4, Sox17, Gata4, HBF4 alpha, Gata6, AFP. Two BMPs had pronounced effects in inducing cells to endoderm. We have followed up by analyzing the efficiency using FACS. Up to 60% of cells have undergone to endoderm-marked cells. With the availability of a cell sorter, we evaluated pluripotency by means of proliferation rate, morphology, fluorescent signal in the reporter lines by visual inspection and FACS, then we further characterized the factors by real-time PCR for stem cell markers and karyotyping.
  • It is known that high concentration of FGF can suppress the action of BMPs, so we planned to repeat the experiments in mTeSR media with lowered levels of FGF to re-evaluate the effects of BMPs on cell differentiation abilities. After these tests were completed, we established a protocol performing these assays in high-throughput manner. We are currently in the process of writing this work for publication (Valera et al., in preparation).
  • Towards the development of chemically defined culture media to maintain pluripotency, we have then tested various newly-engineered reagent to replace a protein component in TeSR media. We have established a combination of protein factors known to maintain established hES cells without using nonhuman products except human albumin, which include basic fibroblast growth factors (bFGF), and a bone morphogenetic protein derivative known as AB2008. We have termed this new media as CAV media. We are currently in the process of writing this work for publication (Valera et al., in preparation).

© 2013 California Institute for Regenerative Medicine