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Transgenic stem cells replace almost total organ

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By Anette Breindl
Senior Science Editor

Transgenic skin stem cells combined with improved tissue engineering methods have enabled an almost total autologous skin transplant for a 7-year-old child with junctional epidermolysis bullosa (JEB), a rare genetic disease that causes the top layer of skin, the epidermis, to separate from the underlying dermis.

The success of the procedure has returned the young patient from life in an intensive care unit under chronic morphine treatment in 2015 back into his family and onto the soccer field. It has also provided proof of principle that "transgenic stem cells can regenerate an entire solid tissue," Michele De Luca told reporters at a press conference announcing the findings, which were also published in the Nov. 9, 2017, issue of Nature.

The treated child "went back to a normal life including school and sports," he added.

De Luca is a professor of biochemistry at the Italian University of Modena and Reggio Emilia's Center for Regenerative Medicine, and the senior author of the Nature paper reporting the case.

Tobias Rothoeft, a pediatrician at the Ruhr University Bochum where the surgeries were performed, elaborated that the child "now behaves like his healthy siblings... if he gets any bruises, they tend to just heal, like bruises do."

It is important to recognize that JEB is a rare subset of a rare disease, and has several features that make transplants more feasible than in burn victims, which are the largest group of patients that could benefit from skin transplants. Critically, epidermolysis bullosa (EB) patients have a healthy dermal layer, while in severe burns, both the epidermis and the dermis are damaged.

Even for JEB patients, the procedure can be used to replace external skin but not internal mucosa, which suffer from the same anchoring problem. De Luca told BioWorld that this is due to surgical issues, not an inability to make the necessary tissue, and Rothoeft added that for the patient whose case is being described in Nature, internal mucosa have not been a clinical issue.

The procedure's success showed that it is possible to successfully generate enough skin for an essentially complete replacement of the body's largest organ.

Almost two years after the series of surgeries that replaced 80 percent of the child's skin, the transgenic stem cells in the transplanted skin "behave like they are supposed to. [They] keep making epidermis as a normal physiological situation," including in response to injury, De Luca said.

Those cells were taken from a small part of the patient's own skin that was not blistering, and engineered to contain a normal copy of the gene for laminin beta 3 (LAMB3), which the child lacked. Laminins are a group of proteins that anchor the top epidermal and deep dermal layer of the skin to each other.

The team also performed molecular analyses of the keratinocytes, the skin cells produced by the stem cells, both before and after transplantation, that allowed them to address safety concerns as well as an open question in the stem cell field, namely, what type of cells are responsible for the long-term regeneration of the epidermis.

The upper skin layer regenerates, on average, monthly, and so after transplantation, the engineered skin cells have to be able to sustain that regeneration.

There are several different pools of progenitor cells that are potential candidates for that regenerative job. The team showed that in their work, clusters of one particular type of long-lived stem cell, called a holoclone, were the source of the vast majority of the repeated cycles of regeneration that were undergone by the transplanted skin.

The important lesson for transplant surgeons is that "we have to make sure that defined population [of] long-lived stem cells is preserved" within grafts, De Luca said.

The team also analyzed the cells for any sign of insertional mutagenesis, which occurs when the viral vector that delivers a transgene integrates into the cellular DNA at a location that affects cell growth, for example, by disrupting a tumor suppressor.

"Insertional mutagenesis is something that has clearly been shown in the hematopoietic system," De Luca said.

An early gene therapy trial that restored an immune gene to boys with X-related severe combined immunodeficiency (X-SCID) went from triumph to tragedy when three of the treated boys developed leukemia as a result of insertional mutagenesis.

Even quite similar trials, though, have not seen comparable rates of insertional mutagenesis, and De Luca said that in skin transplants, "all the preclinical data we have point to a lack of toxicity," and in clinical work on skin grafts, clinicians have applied "on the order of" 100 million transduced cells, and never observed carcinoma.

If such carcinoma did form, it would be removable by local surgery – unlike leukemia, which is by its nature a systemic disease.

Medical intervention, he acknowledged, always means balancing risks and benefits. But for a potentially fatal disease like JEB, that balance is "in my mind, in favor of the therapy."