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Engineering a Cure

Kristie Nybo, Ph.D.

When first introduced, gene therapy promised cures for many devastating diseases, but researchers watched their plans crumble in the face of technical challenges. Can stem cells and new genome engineering nucleases restore hope?

Over the course of his career, pediatric oncologist Jakub Tolar has counseled hundreds of patients and their families, but one visit in particular stands out. In his care were two young brothers, both of whom had been diagnosed with recessive dystrophic epidermolysis bullosa (RDEB), an incurable and often fatal disease that presents with widespread skin blisters shortly after birth and progresses towards malfunction of internal organs such as the lungs, heart, and kidneys. RDEB causes significant pain and mutilating skin damage, and at that time, no treatment existed except wound care. Faced with this future, the boys’ grieving mother pleaded for Tolar to use stem cell therapy to treat her sons.

Type VII Collagen Expression and Skin Healing after Haematopoietic Cell Transplantation. Clinical photographs from the same individual before and after haematopoietic cell transplantation. Source: J. Tolar, J. E. Wagner, Experimental dermatology 21, 896 (2012).

Jakub Tolar helped cure a patient with an incurable disease using genome engineering and stem cell technology. Source: University of Minnesota

Charles Gersbach recently used TALENs without a repair template to correct a mutation causing Duchenne Muscular Dystrophy (DMD). Source: Duke University Photography

“In the case of doctoring, I have always been very careful to listen to the mother or father. The parents usually know much, much more—not only about their kid, but other things too—because of the intensity of the tragedy of having a kid with an incurable disease,” Tolar said.

Because RDEB is caused by a mutation in a single gene, COL7A1 in this case, restoring the correct gene product has the potential to cure the disease. As he pondered this mother’s request, Tolar, who now serves as director of the University of Minnesota (UM) Stem Cell Institute and associate professor in the Pediatric Blood and Marrow Transplant program, began to think that stem cells just might be the answer.

Fingers or TALENs?

To develop a stem cell–based therapy for RDEB, Tolar’s team focused on bone marrow transplantation, an area where they had significant experience. “Bone marrow transplant is not only the first stem cell therapy that has been used for more than 40 years now, but it really is the first and original gene therapy. It just happens that we are using cells from a healthy donor as a delivery for that correct gene,” Tolar said.

Successful preclinical laboratory studies led to clinical trials that established bone marrow transplant as the only systemic treatment for RDEB. Still, transplants come with significant physical side effects from necessary chemotherapy and immunosuppression treatments, as well as the risk for immune response or rejection of the donor cells. So Tolar and his group kept a close watch on developments in the field, hoping for a safer option for their patients.

The introduction of TALENs for genome engineering was just the technology Tolar’s group were waiting for. Like zinc finger nucleases (ZFN), TALENs combine a nuclease with a sequence-specific DNA binding domain that can be precisely targeted. Tolar turned to Mark Osborn, an assistant professor in the Division of Blood and Marrow Transplantation at UM, in the hope of developing a gene correction therapy for RDEB.

In deciding between a ZFN or TALEN approach, Osborn first considered the ability to target a nuclease to a particular mutation in the COL7A1 gene. “The TALENs were much more flexible in their targeting capacity, which was a critical aspect since most patients with RDEB have a specific mutation that is unique to them,” he said. While ZFNs have been studied more extensively, TALENs offered a couple of decided advantages: each domain recognizes a single nucleotide, making it easier to target specific sequences compared to ZFN, where each zinc finger recognizes a set of three nucleotides, and unlike ZFN, TALEN binding is not dependent on surrounding DNA sequences.

At Tolar's request, Dan Voytas, a UM colleague, created a nuclease to cut the DNA near a mutation in the COL7A1 gene in cells taken from a patient with RDEB. Osborn and Tolar demonstrated that this TALEN corrected the gene mutation. They then converted the modified cells into induced pluripotent stem cells (iPSCs). These cells promoted skin formation including the correct COL7A1 protein in mice.

“The hope is to be able to use the patient’s own cells, correct their specific mutation, and then give them back to them to mitigate any transplant related issues,” Osborn explained.


TALEN-mediated correction of RDEB in patient cells required delivering not only a nuclease specific for a particular mutation, but also a nucleic acid template to provide the corrected gene sequence. TALENs cut both strands of the DNA, leading the cell to repair the break, either by homologous recombination (HR) using a donor template, as used for RDEB, or nonhomologous end joining (NHEJ).

NHEJ repair occurs when the cell simply re-ligates broken DNA strands together. The process is stochastic and error-prone, often leading to small insertions or deletions at the DNA breakpoint. As such, NHEJ has been used by researchers mainly for disrupting genes or deleting segments of DNA.

For diseases caused by gene mutations, often a donor template and HR will be required to repair the damage. Charles Gersbach, assistant professor of Biomedical Engineering at Duke University, and his graduate student David Ousterout recently used TALENs without a repair template to correct a mutation causing Duchenne Muscular Dystrophy (DMD) through NHEJ.

"Duchenne Muscular Dystrophy is somewhat of a unique disease because you do not need the whole protein to be restored in order to get a therapeutic effect," Gersbach said. DMD results from mutations that disrupt the reading frame of the dystrophin gene, often introducing premature stop codons near exon 51. The result is muscle wasting and, eventually, death. Gersbach's group engineered and tested a series of TALENs targeting this region and found that deletions resulting from NHEJ could restore the reading frame, leading to expression of functional dystrophin.

"Why not use homologous recombination?" Gersbach asked, "Well, for one, [NHEJ] is easier. Homologous recombination requires a piece of DNA in the mix and that requires making that piece of DNA; it requires delivering that piece of DNA; and it requires making sure that extra piece of DNA is safe and isn't going to do anything else to the genome." Additionally, certain cell types that do not divide, such as those found in muscle fibers, have little to no capacity for HR.

Safety First

In addition to verifying the safety of the DNA or RNA template delivered with the nuclease, other obstacles must be overcome before these genome engineered cells can be delivered to patients.

"The biggest issues are delivery and efficiency," Gersbach said. His group is currently studying the effects of injecting DMD corrected cells into mouse models. "That would be a model of stem cell-based therapy, but we are also interested in taking nucleases and delivering them in vivo into the muscle tissue."

Osborn's biggest concern right now is the extent to which nucleases can be trusted to cut only their specific target sequences. "Each gene target is going to have a differential on- and off-target profile based on other regions of the genome, so it will have to be determined for each set of reagents what that profile is."

Osborn and Tolar's group collaborated with Christof von Kalle at the German Cancer Research Center in Heidelberg, Germany to conduct extensive sequencing studies on the specificity of the TALENs they created to correct the mutation causing RDEB. Three off-target cut sites were detected, but each was at least 10 kb away from any coding sequences. "I think that what will end up being the gold standard will be the deep sequencing analysis that we did with our collaborators in Germany," Osborn said. "Each reagent is going to have to have that level of stringency, in my opinion, to reach the clinical realm."

Gersbach's group also used sequencing to determine the off-target profile of the TALENs they generated to restore the reading frame of the dystrophin gene; these showed precise targeting and a safe profile as well.

Both groups are working to improve the efficiency of the nuclease activity and the procedure for preparing the cells. Osborn would like to develop a method for gene correction that does not depend on an antibiotic marker to detect HR. This would remove one more step, decreasing the time the cells remain in culture and reducing the potential for cytogenetic mutations to occur.

Despite these challenges, Tolar, Osborn, and Gersbach are optimistic about the future for genome engineering using ZFNs, TALENs, and the newly developed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nuclease system, as well as the ability to eventually move these techniques into the clinic.

"When I was in graduate school more than 20 years ago, everyone thought that gene therapy was just the answer for everything," Tolar said. "Conceptually, I think that gene therapy is still the goal. It goes after the cause of the disease rather than the side effects of the disease. It has a unique potential for curing the disease rather than just ameliorating some of the manifestations of the disease. So the goal is sound."

And that goal is quickly moving to the clinic. For example, Sangamo Biosciences is currently conducting clinical trials of a ZFN-based stem cell therapy for human immunodeficiency virus (HIV). "This isn't just fairy tales in Petri dishes. Actually, people right now are getting treated with cells modified by these types of nucleases," Gersbach said. In fact, there are two additional ZFN-based therapies in clinical trial right now, one for treating diabetic neuropathy and the other for treating recurrent glioblastoma multiforme.

While it will require months to years to work through the technical and regulatory challenges needed to bring TALEN-based stem cell treatments for RDEB and DMD to clinical trial, these demonstrations of correcting genetic disease in human cells are significant steps forward.

"The exciting thing for us is that it truly is a personalized gene medicine approach. The cells were obtained from an individual patient at our institution and were brought to the laboratory. We utilized this powerful gene targeting tool to correct them," Osborn said.


1. Osborn, M. J., C. G. Starker, A. N. McElroy, B. R. Webber, M. J. Riddle, L. Xia, A. P. Defeo, R. Gabriel, M. Schmidt, C. Von Kalle, D. F. Carlson, M. L. Maeder, J. K. Joung, J. E. Wagner, D. F. Voytas, B. R. Blazar, and J. Tolar. 2013. TALEN-based gene correction for epidermolysis bullosa. Molecular therapy : the journal of the American Society of Gene Therapy 21(6):1151-1159.

2. Ousterout, D. G., P. Perez-Pinera, P. I. Thakore, A. M. Kabadi, M. T. Brown, X. Qin, O. Fedrigo, V. Mouly, J. P. Tremblay, and C. A. Gersbach. 2013. Reading frame correction by targeted genome editing restores dystrophin expression in cells from duchenne muscular dystrophy patients. Molecular therapy : the journal of the American Society of Gene Therapy (June).