Generation of Inherited Disease Human Embryonic Stem Cell Lines

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Disease Focus: 
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
The development of human embryonic stem (hES) cell lines that carry a disease causing mutation can provide insight into the mechanisms underlying disease progression as well as into the development of therapies that can ameliorate that pathology. The primary goal of this proposal will be the development of novel hES cell lines from embryos that will manifest a given genetic disease upon further development. This will be achieved following two distinct approaches. The first will be through the identification of embryos that are homozygous for a given mutation and the generation of novel cell lines from these embryos. These embryos will be identified by preimplantation genetic diagnosis (PDG). The afflicted embryos will then be grown according to established protocols known to generate human ES cell lines. The second approach will involve the generation of disease associated homozygote cell lines through a technique that will specifically modify a gene sequence and introduce a disease associated mutation. This technique, small fragment homologous replacement (SFHR), has been shown to be effective at modifying DNA sequences in human cells. SFHR-mediated changes are caused by small DNA fragments (SDFs) that are introduced into the cells. The SDFs are effectively the same as the gene target sequences except for the changes to be introduced. Initial studies will target genes on the X-chromosome of normal male hES cells that carry only one X-chromosome. The genetic diseases that are anticipated to be served by this proposal include, but are not limited to, cystic fibrosis, sickle cell disease, ?- and ?-thalassemia, Duchenne’s and Becker muscular dystrophy, X chromosome-linked severe combined immune deficiency (SCID-X1), spinal muscular atrophy (SMA), Guacher’s disease, Fanconi anemia, and Lesch-Nyhan syndrome.
Statement of Benefit to California: 
This proposal will provide benefit to the citizens of California by increase our knowledge of the basis of genetic diseases and by providing a means to develop new and more effective therapies for these diseases. This project is focused on improving the health and well being of the citizens of California and could have far reaching positive implications for health and economic factors influencing the quality of life for the citizens of this state.
Progress Report: 
  • Human embryonic stem cells contain roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of human genome. While many of these retroelements have been silenced during evolution by crippling mutations, many remain active and capable of jumping to new chromosomal locations potentially producing disease-causing mutations or cancer. In tissues, mobility of these elements is suppressed by DNA methylation, which inactivates expression of the retroelement RNAs. In sharp contrast, embryonic stem cells exhibit very dynamic changes in DNA methylation, where the methylation patterns are gained and lost at high rates. During periods of low DNA methylation, retroelement RNA expression likely increases. Accordingly, hESCs must deploy other defensive strategies in order to maintain genomic integrity. Recent studies have identified the APOBEC3 family of genes (A3A-A3H) as powerful antiviral factors. These A3s interrupt the conversion of viral RNA into DNA (reverse transcription), a key step also employed by retroelements for their successful retrotransposition. We hypothesized that one or more of the APOBECs function as guardians of genome integrity in hESCs. In the last two years we have found that six out of the seven human A3 genes located in a tandem array on chromosome 22 are expressed in hESCs. A3A, which in prior studies was suggested to exert the greatest anti-retroelement effects, surprisingly is not expressed in hESCs. Further, we find that the A3 proteins decrease when pluripotent cells differentiate into somatic cells suggesting an important function of these A3 proteins in pluripotent hESCs. We established a LINE1 retrotransposition assay in hESCs that allows us to visualize genetic jumping of this class of “marked” retroelements via flow cytometry. Using this assay we have found that LINE1 elements effectively jump in hESCs. To test our central hypothesis, namely that A3 proteins guard the genome in hESCs, we have established experimental conditions for RNAi knock-down of all expressed A3 genes. By combining the knock-down and the retrotransposition assay we demonstrated that the knock-down of one member of the A3 protein family leads to a 3.5-fold increase in LINE1 retrotranspositon. This finding highlights a protective role for the A3 family of cytidine deaminases that helps safeguard the genome integrity of hESCs.

© 2013 California Institute for Regenerative Medicine