| NSF Org: |
CBET Division of Chemical, Bioengineering, Environmental, and Transport Systems |
| Recipient: |
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| Initial Amendment Date: | May 27, 2014 |
| Latest Amendment Date: | May 27, 2014 |
| Award Number: | 1403214 |
| Award Instrument: | Standard Grant |
| Program Manager: |
Steven Peretti
speretti@nsf.gov (703)292-4201 CBET Division of Chemical, Bioengineering, Environmental, and Transport Systems ENG Directorate for Engineering |
| Start Date: | August 1, 2014 |
| End Date: | December 31, 2017 (Estimated) |
| Total Intended Award Amount: | $173,614.00 |
| Total Awarded Amount to Date: | $173,614.00 |
| Funds Obligated to Date: |
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| History of Investigator: |
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| Recipient Sponsored Research Office: |
800 E LANCASTER AVE VILLANOVA PA US 19085-1603 (610)519-4220 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
800 East Lancaster Avenue Villanova PA US 19085-1603 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): |
Cellular & Biochem Engineering, Cross-BIO Activities, Systems and Synthetic Biology |
| Primary Program Source: |
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| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.041 |
ABSTRACT
CBET - 1404084/1403214
Rege/Elmer
DNA or gene delivery to mammalian cells has several applications in biotechnology and medicine. However, the activation of foreign genes (transgenes), and subsequent efficacy of protein expression from them, is often hindered by defense mechanisms inside the host cell. For example, cells are able to control transgene protein expression by either burying existing genes inside condensed chromosomes in the nucleus in order to turn them off, or by relaxing chromosomes in order to expose genes and activate them. A family of 'epigenetic' enzymes present within cells typically controls these processes of gene regulation; enzymes that activate genes are generally classified as activators, while those that turn genes off are called repressors. In the proposed research, the investigators will study the role of these epigenetic enzymes in regulating the expression from delivered transgenes. Furthermore, tools from the emerging field of synthetic biology will be employed to design novel DNA sequences and activator enzymes that enhance protein expression from delivered transgenes. Findings from these studies will have a transformative impact on biotechnology processes and medical treatments (e.g. gene therapy) that utilize DNA or gene delivery to mammalian cells. Outreach from this project will reach K-12 students through a PCR-based molecular biology project facilitated by the Quanta program at ASU. The proposed outreach will be designed to increase participation of students from underrepresented populations in STEM activities in Arizona and Pennsylvania.
In the same way that words are neatly organized in the pages of books in a library, cellular genes are condensed by histone proteins to form chromosomes in the nucleus. Epigenetic regulation, mediated by several enzymes, plays a key role in controlling gene expression inside cells. Since recent work has shown that epigenetic mechanisms can silence viral genes, it is likely that epigenetics may also regulate the expression of foreign genes (i.e. transgenes) that are delivered in the form of plasmid DNA to mammalian cells. The goal of this project is to manipulate intracellular epigenetic enzymes in order to enhance transgene expression by (1) using drugs to inhibit repressors and to prevent the deactivation of transgenes, (2) adding designed DNA sequences to the plasmids in order to recruit activator enzymes, and (3) using principles from synthetic biology in order to design and validate novel epigenetic activator enzymes that can selectively bind transgenes and enhance their expression. These approaches will deepen our understanding of epigenetic mechanisms that regulate protein expression from foreign transgenes (plasmid DNA) in human cells, leading to increased efficacy of gene therapy treatments in medicine, and cellular engineering approaches in biotechnology.
This award is co-funded by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division and by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.
PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
Even if a therapeutic gene is successfully delivered to a cell nucleus, its expression may still be limited by epigenetic mechanisms that silence the transgene. For example, once a gene reaches the nucleus, it may be bound by histone proteins that tightly pack it into heterochromatin or recruit repressor proteins that block transcription. In either case, although the gene is present in the nucleus, it is unable to be transcribed into mRNA (and subsequently translated into a therapeutic protein that could treat the specific genetic disorder).
In this study, we sought to enhance gene expression by adding binding motifs (i.e. short DNA sequences that host cell proteins bind) for several different epigenetic enzymes either upstream or downstream of human (EF1a) and viral (CMV) promoters. Screening these plasmids revealed that gene (e.g., luciferase) expression was enhanced 10-fold by the addition of a CCAAT motif just upstream of the EF1a promoter to recruit the nuclear transcription factor Y (NF-Y), while inserting a CCCTC-binding factor (CTCF) motif downstream of the EF1a promoter enhanced expression 14-fold. In contrast, these motifs did not significantly affect expression from the CMV promoter. This lack of enhancement may be due to the fact that the CMV promoter sequence already contains NF-Y and CTCF motifs, such that the addition of more NF-Y or CTCF motifs is unnecessary. At any rate, ChIP assays confirmed that NF-Y and CTCF bound to the motifs that were added to the EF1a promoter, but the presence of NF-Y and CTCF did not significantly affect the levels of histone acetylation (H3K9ac) or methylation (H3K9me3).
Overall, these result show that transgene expression from the EF1a promoter can be significantly increased with motifs that recruit NF-Y or CTCF. It may be possible to use this improved EF1a promoter to increase the expression of therapeutic genes and the overall efficacy of gene therapy treatments.
Last Modified: 08/17/2018
Modified by: Jacob Elmer
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