Gene therapy found to generate C7 protein needed in RDEB in study
Stem cells used in mouse model taken from blister fluid from patients
Gene therapy applied to stem cells — ones derived from blister fluid collected from people with recessive dystrophic epidermolysis bullosa (RDEB) — successfully generated C7 protein within the skin layers of RDEB model mice.
Production of C7, which is lacking in RDEB, worked best when modified cells were applied directly to blisters, particularly those occurring in the early stages of the disease or as flat sheets of cells to heal chronic ulcers over wider skin areas.
“We successfully developed a minimally invasive and highly efficient ex vivo [outside the body] gene therapy for RDEB,” the researchers wrote, noting the treatment was shown to be effective “in the RDEB mouse model for both early blistering skin and advanced ulcerative lesions.”
Novel gene therapy for RDEB uses cells from blister fluid
RDEB, a severe form of epidermolysis bullosa, is caused by mutations in the COL7A1 gene, which encodes part of the type VII collagen (C7) protein.
Because C7 is responsible for tethering two key skin layers called the epidermis and dermis, defects in the gene leave skin fragile and prone to blisters, scarring, and ulcers.
Researchers in Japan previously had discovered that a protein called HMGB1, released from RDEB lesions, acted as a signal to attract mesenchymal stromal cells (MSCs) from bone marrow and boost tissue regeneration. RDEB blister fluid also contained high levels of HMGB1, suggesting that MSCs are recruited to blister fluid to promote tissue repair.
“These results encouraged us to develop a novel RDEB gene therapy targeting blister-accumulating MSCs,” the researchers wrote, noting that these cells “have anti-inflammatory and fibrosis-limiting activities in addition to tissue-regenerative potential.”
The first step was to investigate whether regenerating MSCs could be efficiently collected from the blister fluid of RDEB patients.
Blister fluid mixed with MSC growth media generated fibroblast-like cells, connective tissue cells that produce collagen. Cells obtained from RDEB blister fluid met the criteria for MSCs and were similar to those collected from bone marrow. Named blister fluid-derived MSCs (Bf-MSCs) by researchers, they were reproducibly collected regardless of blister volume, patient age, and level of C7.
Next, the team genetically modified the Bf-MSCs to produce C7, then injected cell suspensions into the blister cavity created on the skin of newborn mice lacking C7.
Four weeks after injection, C7 was found in the dermal–epidermal junction or DEJ, located near the injection site of the entire blister area. Microscopy imaging revealed the formation of anchoring fibrils — the structures that connect the epidermis with the dermis. Moreover, C7 production was 42.4% of that seen in normal human skin.
Long-term engraftment studies showed the transplanted Bf-MSCs, injected into the skin, remained detectable for 141 days, or longer than four months. No evidence of tumor formation at the site of cell transplant was detected, regardless of whether cells were genetically modified.
“These results indicated that ex vivo gene therapy using C7-Bf-MSCs enables sustained C7 expression, without [any gain of malignant properties],” the team wrote.
Potential for use in many different lesion types
The scientists then applied a sheet of C7-Bf-MSCs to the dermal skin layer surface, which was exposed by peeling the epidermis or outer skin layer of newborn mice with C7 deficiency. Cell sheets are complete layers of cells, including supporting structures, growth factor receptors, and other cell surface proteins.
Results showed C7 was secreted and distributed continuously along the DEJ 10 days after treatment. The length of the C7 deposit was significantly longer than that achieved by into-the-skin (subcutaneous) injection.
Likewise, with cell sheets, C7 production was 70.6% of normal human skin, higher than skin injections at 26.3%. Microscopic analysis revealed a large number of mature anchoring fibrils.
“Therefore, C7-Bf-MSCs can supply C7 to a wide area of the DEJ even when applied as a cell sheet,” the team wrote, adding, “Bf-MSC sheets may yield better results than cell suspensions because of long-term cell engraftment and preservation of MSC function.”
As our treatment targets blistered regions, it can be applied in the early stages of RDEB.
Because the method they used to genetically modify Bf-MSCs would not translate to clinical settings, the researchers tested whether a lentivirus vector could deliver a functioning copy of the COL7A1 gene to Bf-MSC cell sheets. Consistently, four weeks after applying these new sheets under the mouse epidermis, C7 was detected at the DEJ at levels 58% of normal.
“As our treatment targets blistered regions, it can be applied in the early stages of RDEB,” the scientists wrote. “In addition, the use of cell sheets could be adapted to chronic ulcers, indicating their potential for use in many different types of skin lesions in RDEB.”
According to the team, these findings show the potential of this gene therapy for treating the rare disorder.
“This study proposes an entirely different ex vivo approach to gene therapy for RDEB developed based on the knowledge of the mechanisms underlying injury-induced regeneration,” they concluded.