Unrelated cord blood transplantation after reduced toxicity conditioning with mesenchymal stromal cell co-infusion in patients with severe epidermolysis bullosa

Epidermolysis bullosa (EB) is a heterogeneous group of inherited diseases
characterized by trauma-induced blistering and erosions of the skin and mucous
membranes. There are over 25 different subtypes of highly variable clinical
severity caused by mutations in 17 different genes.

Severe generalized recessive dystrophic EB (RDEB) is caused by absence or
severely reduced type VII collagen (Col7), a constituent of the anchoring
fibrils and an important protein involved in the adhesion of the basement
membrane to the underlying dermis. The total loss of Col7 is caused by null
mutations in the COL7A1 gene encoding for the collagenous polypeptide type
Col7. Besides mutation analysis, other clues to diagnosis are electron
microscopy, in which absence or reduction of anchoring fibrils is seen, and
immunofluorescence antigen mapping, in which (almost) complete absence of the
monoclonal antibody LH7:2 staining against Col7 is seen. Diagnosis can be made
in most cases within 24 hours by IF antigen mapping. Patients with RDEB suffer
from lifelong generalized painful blistering, erosions and granulation tissue.
Due to mucosal involvement, patients also develop extracutaneous complications,
such as mitten deformity of the hands (pseudosyndactyly), esophageal
strictures, anemia, ectropion, short tongue, severe caries, painful open skin
wounds, and multiple cutaneous squamous cell carcinomata. Patients with RDEB
survive to adulthood and decease within the third to fifth decade of life
despite optimal multidisciplinary care, mostly by fatal metastasis of skin
cancer. Severe RDEB is a disease with such an expected poor life quality of
hopeless, unbearable suffering, that some parents request euthanasia in the
neonatal period.

To date, the care of patients with the most severe epidermolysis bullosa forms
has been palliative and supportive. Since no curative treatment is available,
treatment is restricted to individual wounds. Recently in addition to various
experimental settings, there has been clinical evidence that (allogeneic)
hematopoietic stem cell transplantation (HSCT) influences the natural phenotype
(natural course) of the disease significantly by engraftment of a substantial
percentage of donor cells in the skin / mucosa. The substantial sustained
engraftment (median 20%) of donor cells were from hematopoietic (CD45+) as well
as from non-hematopoietic/non-endothelial (CD45-/CD34-) donor cells. In the
small group of transplanted patients there is definitely an indication that the
percentage of engrafted donor cells, as well as having 2 wild type genes of the
donor in the skin correlates with the correction of the phenotype: the higher
the percentage of donor cells- the higher the phenotypic correction. This
landmark paper published in the NEJM, describes the clinical outcome of 7
patients, who had a heterozygous donor, and illustrates a worse clinical effect
in comparison to the non-mutated donor. This was recently confirmed in a larger
series (n=18) of EB patients transplanted in the same institute (personal
communication with Dr. Jakub Tolar, Minnesota.) Furthermore, this is in line
with the long-term outcome results of HSCT in patients with lysosomal storage
diseases: full donor chimerism with a donor containing 2 wild-type genes is
associated with the highest enzyme levels and therefore the best long term
outcome.

The above mentioned clinical evidence is hopefully the first step to more
sophisticated and *targeted stem cell therapies* for EB resulting in better
correction of the disease. For these future therapies targeting cells towards
skin engraftment and differentiation into type VII collagen producing cells;
cord blood transplantation may be an interesting cell source. Over the last
decade classical HSCT has become much safer and more effective also due to the
emergence of cord blood (CB). CB has many advantages above the conventional
bone marrow grafts (e.g. prompt availability, less stringent HLA-typing
criteria, less graft-versus-host disease (GvHD) notwithstanding a stronger
graft-versus-leukaemia effect, but is also suggested having more
trans-differentiation abilities because it contains younger pluripotent stem
cells. Long term outcomes in patients transplanted with unrelated cord blood
for lysosomal storage disease appears better then in those transplanted with
the more conventional sources i.e. bone marrow and peripheral blood. The prompt
availability (transplant possibility <4weeks of life), lower GvHD rates as well
trans-differentiation abilities makes cord blood an interesting cell source for
transplantation in EB as well as a platform for future adjuvant stem cell based
therapies.


Mesenchymal stromal cells (MSC), also known as mesenchymal stem cells are
non-hematopoietic cells that reside in the bone marrow stroma. In vitro studies
showed that MSC have an important interaction with hematopoietic stem cells
(HSC). MSC express ligands for surface molecules of HSC, form cell clusters
with HSC in co-culture experiments and secrete cytokines necessary for HSC
homing, proliferation and differentiation. The speculated role of MSC in the
bone marrow has been examined in vivo, and it was observed that
co-transplantation of MSC with HSC promotes engraftment of the donor cells.
Besides this supportive role of MSC for HSC growth and differentiation, the
main benefit comes from their immunomodulatory ability. Results suggest that
MSC promote a shift from a pro-inflammatory environment to an anti-inflammatory
environment. Infusion of mesenchymal stroma cells thus offers adjuvancy to two
very important aspects of the procedure. Firstly, the additional
anti-inflammatory effect will help reduce toxicity of conditioning prior to
procedure, minimizing the chance of GvHD. This in turn facilitates not only
proper engraftment of transplanted cord blood, but will allow for better
correction of the disease phenotype itself through decreased inflammation and
improved wound healing

We propose a phase II study (intervention) in patients with severe generalized
recessive dystrophic EB receiving reduced toxicity conditioning chemotherapy
followe by cord blood transplantation with co-infusion of mesenchymal stromal
cell units. Next to safety (transplantation - related mortality), the outcomes
(biology, halting of progression of severe physical deformities) will be
compared with the natural course of the disease.

Phase II intervention study

Reduced toxicity chemotherapy conditioning followed by cord blood
transplantation with co-infusion of mesenchymal stromal cells for severe
generalized recessive dystrophic EB

The burden of the therapy is associated with HSCT itself (less likely to be
associated with the MSC infusions). Cord blood stem cells will be from an
unrelated donor, while the mesenchymal stroma cells will be from 3rd party
donors. Despite a reduced toxicity myeloablation will be used, EB patients, due
to the nature of EB and it*s characterizing chronic wounds, will be of
increased susceptiblity for GvHD. MSC infusion may potentially impact this
susceptibility, due to the immune-modulation potential. All other acts,
measurements, follow-up and level of care are similar to off-study patients
undergoing allogeneic HSCT.

Potential Risks:
1) Potentially risk of aGVHD and cGVHD resulting in death
2) Infections associated with allogeneic HSCT resulting in death
3) Death due to transplantation associated mortality (e.g. due to 1,2)
4) Passagere worsening of skin disease due to effects of conditioning (toxicity)
Potential Benefits:
1) Enhanced wound healing and reduction of pain with
potentially less scar formation as a result of protein expression in the skin
2) Clinical improvement of severe EB that is expected or has become a misery
3) Potential prevention/prolongation of onset of *mitten deformity* by
RDEB-sev-gen patients
4) Better *quality of life* due to less pain, less disease burden on a
psychosocial level*