The advent of custom built DNA binding domains fused to restriction enzymes changed the landscape of biomedical research and heralded a new age in gene therapy. This fact is highlighted by Phase I clinical trials in which zinc finger nucleases are being used to knockout the CCR5 receptor in CD34+ T cells of HIV infected individuals to generate HIV-resistant T cells (clinical trial #NCT00842634). In addition to targeted gene knockout, other biomedical applications of the designer nucleases are gene correction and gene activation. However, gene correction requires homologous recombination following a DNA cut - a very inefficient process - and current strategies for stable gene activation require constitutive expression of an ectopic protein, limitations that are likely to hinder therapeutic efficacy. In contrast to endogenous gene editing or genetic activation, I propose to develop a platform for epigenetic reprogramming of any locus of interest. As a proof-of-concept for this technology, I propose to engineer site-specific DNA binding module fusions with DNA demethylating enzymes for epigenetic induction of fetal hemoglobin (HbF) for therapy of sickle cell disease (SCD). SCD is caused by mutation of the adult b-globin gene which forces red blood cells to sickle and occlude small blood vessels leading to exquisite pain and a wide range of medical complications. Fetal hemoglobin is normally silenced at birth by DNA methylation of the g-globin locus as adult hemoglobin increases. Because small increases in HbF can cure this disease, SCD is an optimal application for developing epigenetic re-programming technologies. The major advantage of epigenetic engineering is that successful DNA demethylation of CpGs on both strands will lead to durable HbF-induction, not requiring continuous expression of ectopic proteins. This highly-innovative strategy has no precedent in the SCD literature. Development of this technology will lead to potent and durable

Public Health Relevance

Genetic engineering using custom built DNA binding domains holds enormous and untapped medical potential. Epigenetic engineering may be an even more powerful means to activate or repress endogenous gene expression because transient modification of DNA and histones is expected to remain stable over many cell divisions. Experiments in this proposal will develop an epigenetic reprogramming toolkit that can potentially be applied to any locus using induction of fetal gamma globin for treatment of sickle cell disease as a proof-of-concept.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
NIH Director’s Pioneer Award (NDPA) (DP1)
Project #
5DP1DK105602-04
Application #
9342865
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Bishop, Terry Rogers
Project Start
2014-09-18
Project End
2019-08-30
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
4
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
076580745
City
Boston
State
MA
Country
United States
Zip Code
02215
Xiong, Tina; Meister, Glenna E; Workman, Rachael E et al. (2017) Targeted DNA methylation in human cells using engineered dCas9-methyltransferases. Sci Rep 7:6732