A pilot in vitro and in vivo study for molecular characterization of epidermal stem cells and quality and safety assessment of epidermal cultures for combined cell and gene therapy
Project Recent breakthroughs in regenerative medicine, in particular cell and gene therapies based oncultures of somatic and pluripotent stem cells, have generated increased demand to explorenew tools able to focus on QUALITY and SAFETY of stem cells.
For more than half a century, cultures of autologous keratinocytes have been used to preparegrafts that can permanently restore severe epidermal defects, such as life-threatening massivefull thickness burns and more recently gene therapy of genetic skin disorders such asEpidermolysis Bullosa (EB). Notwithstanding appropriate clinical procedures, a criticalevaluation of clinical successes and failures unveiled that the essential features for a durableepidermal graft are the presence of an adequate number of keratinocytes stem cells (KSC),tailored gene correction approaches and a deep knowledge of the pathogenic disease. Clonaltracing performed on the regenerated transgenic epidermis has formally shown that the humanepidermis is sustained solely by self-renewing KSC, that continuously generates pools oftransient amplifying progenitors (TA). To detect KSC in epidermal grafts, the only availableassay is the clonal analysis, a cumbersome and lengthy procedure. Thus, a thoroughcharacterization of molecular pathways governing self-renewal in KSC at single cell level iscrucial for clinical translation. Although, it has been proven γRV efficacy and safety inkeratinocytes context, some concerns remain unsolved. The γRV safety issues are associatedwith the risk of insertional mutagenesis and improvements have been reached through thedevelopment of self-inactivating (SIN)-γRV. Albeit, no insertional mutagenesis has beendetected in ex-vivo skin gene therapy, its in-depth knowledge requires additional biosafetycharacterization especially in light of its use in a wider clinical trial application.
To highlight and define the intricate pathways governing epithelial stem cells, we propose to usea multi-omics approach to evaluate the QUALITY and SAFETY of normal and gene-correctedKSC for cell and gene therapy. We will accurately define a fully comprehensive atlas mappingboth the transcriptional and proteomic profile of KSC and TA in cell cultures derived fromhealthy donors and EB patients, before and after gene correction. Data deriving from thefollowing analysis will define a KSC-specific and EB signature. Thanks to the classification ofexclusive molecular patterns, in vitro and in vivo bio-safety assays will be developed todemonstrate the safety of γRV for gene therapy. A strong scientific rationale and an integratednetwork of the expertise shared between UNIMORE, UNITO, UNIBO, will lead to innovativeimprovement in the translational application of stem cells.
The project can be intended as a proof of concept. This pilot study is conducted on a small-scale basis to ascertain the feasibility and the data collected should be cross-validated for theiruse in cell and gene therapy application.