Targeting lentiviral vectors to modified hES derived dendritic cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00210
ICOC Funds Committed: 
$0
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
We hypothesize that human embryonic stem cells represent a potentially scalable source of human dendritic cells that could be used to treat a wide array of diseases including HIV/AIDS and cancer. In fact dendritic cell based therapies have shown some promise in early trials, but they are largely limited by the numbers of cells available for treatment protocols. We propose here to develop a potentially unlimited source of human dendritic cells from human embryonic stem cells. More importantly, we propose to modify these cells to express a novel cell surface molecule that can be used for targeted delivery of specific proteins. Ultimately we envision using these cells to direct primary immune responses. The studies described in this SEED proposal are directed towards HIV specific immune responses, but would be widely applicable to other pathogen-based diseases, as well to tumor associated antigens.
Statement of Benefit to California: 
Dendritic cell immunization, dendritic cell-based gene therapies and immunotherapies are being explored as treatments for a number of diverse human diseases, particularly in settings that are refractory to conventional therapies. Unfortunately, these protocols are directly limited by the ability to generate sufficient quantities of dendritic cells ex vivo. Human embryonic stem cells represent a potentially scalable source of dendritic cells that could be used in these settings. For instance, they could be used for the prevention and treatment of pathogen based diseases such as HIV/AIDS, Hepatitis C and Influenza. In addition, development of a source of self renewing dendritic cells could dramatically advance patient specific protocols for the treatment of cancer. Taken together these diseases and their treatments impact Californians personally and economically. The development of improved treatment and prevention modalities by harnessing the potential of human embryonic stem cells would represent a major benefit to the lives of all Californians.
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