We have been focused on developing homologous recombination as a method to efficiently modify the mammalian genome for both therapeutic purposes and research purposes. While homologous recombination has been a staple of mouse genetics through its use in murine embryonic stem cells (Smithies, Cappechi and Evans were awarded the Nobel Prize in Medicine for developing this methodology), its low relative and absolute frequency has precluded its use in somatic mammalian cells. An important discovery in the mid 1990’s by Jasin, Wilson, Choulika and their colleagues was that a double-strand break introduced into the genomic target could stimulate gene targeting by homologous recombination at that locus by 1000-fold. In 2003 we showed that a double-strand break could stimulate gene targeting by 10,000-50,000 fold (to an absolute frequency of 5%). Moreover, in 2003 we were the first to show that an engineered nuclease designed to target a specific sequence could stimulate gene targeting by homologous recombination to a similar extent. In these experiments we used zinc finger nucleases (ZFNs), an engineered protein in which a zinc finger DNA binding domain is fused to a nuclease domain. In collaboration with Sangamo Biosciences in 2005 we demonstrated that ZFNs could stimulate genome editing in a gene associated with a genetic disease in close to 20% of cells. The 2003 and 2005 results have proven to be the foundations of the new field of nuclease based genome editing.
The reason a double-strand break stimulates genome editing to such a degree is that it activates the cell’s own repair machinery. The cell has two basic ways of repairing a double-strand break: non-homologous end-joining (NHEJ) or homologous recombination. NHEJ functionally “stitches” the two ends of broken DNA together. While NHEJ is mostly error-free, it can occasionally result in insertion or deletions of DNA at the site of the break resulting in mutations at a specific site in the genome (NHEJ mediated genome editing). Homologous recombination uses an undamaged homologous piece of DNA as a template to repair the break in a “copy and paste” type mechanism. By providing an appropriately designed donor DNA, precise small and large modifications can be made to the genome (homologous recombination mediated genome editing).
We focused on designing high quality ZFNs for a number of years and used several different methods to do so. When TALENs were discovered we compared TALENs that we made in a week to ZFNs that we had taken months to engineer at the same target site and have found that TALENs have both greater activity and greater specificity at every target we have compared. We thank the Voytas lab for sharing their reagents and help with TALEN engineering when we were just learning this new platform. We have now adopted TALENs as our platform for the specific genome editing work described below although will be comparing the new CRISPR platform to TALENs in the near future.
Sickle cell disease and b-thalassemia are genetic diseases of the blood caused by mutations in the b-globin gene (b-hemoglobinopathies). Our goal is to develop homologous recombination based gene therapy in order to cure these diseases. We spent many years engineering zinc finger nucleases (ZFNs) to target the human b-globin gene but now have adopted TAL effector nucleases (TALENs) as our platform for engineered nucleases. Using our b-globin TALENs we can achieve gene targeting rates of ~20% in cell lines and are currently studying the efficiency of targeting in human hematopoietic stem and progenitor cells. We perform our work on the b-hemoglobinopathies as part of a multi-institutional, multi-lab project funded as a Nanomedicine Development Center form the NIH. The other institutions include Georgia Tech, Emory, Medical College of Georgia, Harvard, Cold Spring Harbor Laboratories, University of Pennsylvania and the University of Washington.
Mutations in the IL2RG gene and the ADA gene are two of the most common causes of severe combined immunodeficiency (SCID). Patients with SCID are unable to develop T-cells and as a consequence will die of infections in the first years of life. Patients with SCID are treated by hematopoietic stem cell transplantation although there are a number of retroviral/lentiviral based gene therapy clinical trials currently accruing patients. Our goal is develop a homologous recombination based approach to gene therapy for these diseases. Just as with the hemoglobinopathies we had made ZFNs to target the IL2RG and ADA genes but have found that TALENs are substantially more active and show less cytotoxicity than ZFNs and have adopted TALENs as our platform moving forward.
For the IL2RG gene we have developed a pair of TALENs and broad set of targeting constructs. Using these reagents we are achieving high rates of genome editing at the endogenous locus in cell lines and are working on optimizing the frequency of gene targeting in human CD34+ cells and their subsequent differentiation into T-cells and transplantation into NSG mice.
We have made similar progress targeting exon 1 of the human ADA gene and have a similar experimental path to the IL2RG gene.
This project is funded by the NIH/NIAID and the goal of this project, as it is with the sickle cell disease/b-thalassemia project is to develop a homologous recombination based approach to gene therapy to cure children with SCID.
Huntington’s disease, myotonic dystrophy, and spinocerebellar ataxia are all devastating neurologic diseases caused by the expansion of triplet repeats. In fact, each of these diseases is caused by the expansion of the “CAG” triplet repeat. We have engineered nucleases to target the CAG expansions for each of these diseases and have shown that these nucleases can cause selective contraction of expanded repeats. We are interested in using these nucleases to understand the mechanism of triplet repeat instability, developing isogenic cell lines to study the pathogenesis of these diseases, and hopefully novel therapeutic approaches.
HIV is the virus that causes acquired immunodeficiency syndrome (AIDS). Prior to the development of combination drug therapy (highly active anti-retroviral therapy (HAART)), HIV would cause progressive destruction of the immune system causing patients to be susceptible to a wide range of infections that people with intact immune systems usually handle without serious difficulty. That is, patients get sick from the destruction of the immune system not from the virus itself. Our goal is to develop “genetic HAART” by using synthetic biology principles to engineer the immune system to be resistant to HIV destruction. In contrast to pharmacologic HAART, which focuses on protecting the immune system by limiting viral replication, our strategy is focused on directly protecting the immune system from HIV. Our general approach is to use nuclease mediated homologous recombination to introduce anti-HIV genes into the genome of T-cells and hematopoietic stem and progenitor cells (HSPCs) and simultaneously mutating the CCR5 gene, one of the co-receptors for HIV. We have completed the proof-of-concept studies in T-cell lines and are now in the process of refining our approach and applying it to primary T-cells and HSPCs.
While our interests in developing homologous recombination arose from a desire to have a precise method of correcting disease-causing mutations, we have also recognized that the precision of homologous recombination can also be a powerful tool in synthetic biology. In particular we are interested in using homologous recombination to engineer cells to secrete therapeutic proteins, that is, to create cell based protein bioreactor’s that can serve to deliver continuous doses of therapeutic proteins/peptides in vivo. We have shown that we can engineer primary fibroblasts to secrete a variety of proteins, including growth hormone and platelet derived growth factor. Moreover, these cells can then be transplanted and exert local beneficial biologic effects. We are excited about the general applicability of being able to engineer cells to secrete specified amounts of therapeutic proteins and use these engineered cells to clinical effect.
One of the success stories in cancer biology is the transformation of pediatric acute lymphoblastic leukemia (ALL) from a universally fatal disease to one in which >80% of patients are cured. Despite this success, there remain certain subtypes of ALL that continue to have lower cure rates even with the development of highly aggressive, multi-modal chemotherapy with contemporary supportive care. Infant ALL, for example, is one such sub-type of leukemia and is often characterized by specific translocations involving the MLL gene. We are using nuclease mediated genome editing to engineer cells to have these specific translocations with the goal of developing improved models of the pathogenesis of the disease.
While our focus has been on developing nuclease mediated genome editing by homologous recombination for translational purposes, we also are interested in both developing improved methods of genome editing and using genome editing as a research tool. Thus, we are always interested in contrasting our current methods of genome editing, both in terms of nuclease architecture and targeting strategy, with newer approaches. In addition, we have an ongoing interest in using genome editing as a tool to study important biologic processes both in our own lab and in collaboration with others.