Characterisation and acceleration of keratinocyte stem cell culture - Rho-associated kinase inhibitor treatment and epithelial sheet gene therapy

Rare genetic skin disease comprises a large and diverse group of diseases. While these diseases affect a small number of patients from the general population, they often occur at birth or early in life and are generally chronic, severe and sometimes life-threatening. The quality of life of patients and their families is severely compromised by the impact of the disease physical manifestations and the lack or loss of autonomy. Clinical options for these devastating conditions are currently limited to symptomatic treatments.

Different gene therapies have been developed for genetic skin conditions, and gene modified skin sheet graft is the most promising gene therapy for these conditions. In this therapy, a correct gene is added into patients' skin stem cells, which are then grown in the laboratory to form an entire skin sheet that is grafted back to the patient. We have done this for the severe and debilitating skin conditions Netherton syndrome and a phase I clinical trial showed no adverse complications, with the corrected gene expression and function. We are now planning to bring this therapy platform to other genetic skin diseases such as epidermolysis bullosa. However, the prolonged growth time of skin sheets in the laboratory (more than 40 to 50 days) hinders the application of this therapy for further clinical applications.

Studies from our NS trial have suggested that initial low expansion of stem cells is the cause for the slow growth of the skin sheets in the laboratory. We have found a potential solution to this problem. We propose to treat skin cells obtained from a small piece of skin tissue with a medicine known as Rho-associated kinase inhibitor (ROCKi) to speed up skin cells growth, in particular skin stem cells. In this way, we expect to reduce the time needed to manufacture skin sheets.

We will test ROCKi in skin cells freshly obtained from human skin samples. The speed, behaviour and features of skin stem cells with and without ROCKi treatment will be assessed on cell cultures. The stem cell behaviour will further evaluated in a humanised mouse skin graft model in which human skin stem cells can grow and survive for months. We will further examine the fate of non-treated and ROCKi treated skin cells at single cell level to determine the cell populations changes following treatment. In the single cell study, we will monitor the gene expressions in thousands individual single cells and identify the distinct cell populations from cultures containing mixed cells. Differences in the number, type and proportion of cell populations between non-treated and ROCKi treated stem cells will be identified.

We will thus answer whether the ROCKi has the potential to efficiently expand skin stem cells without changing skin stem cells' characteristics, significantly reducing the preparation time of skin sheets that are safe to use in clinical applications.

The proposed work is directly related to patients who suffer from rare genetic skin diseases with no effective treatment available. If we are successful, we will include ROCKi in skin stem cell cultures to efficiently produce gene corrected skin sheet to treat severe and devastating skin conditions such as epidermolysis bullosa.

We propose to use the Rho-associated kinase inhibitor (ROCKi) to efficiently expand keratinocyte stem cells (KSCs) in vitro. Increased KSCs would then produce sufficient numbers of keratinocytes to accelerate the manufactory of epithelial sheet. To determine the potential of ROCKi treated keratinocytes including KSCs for clinical application, we will examine i) the optimal ROCKi exposure time in the culture; ii) the characteristics of KSCs in ROCKi treated cultures; and iii) whether acceleration of sheet culture is achievable.

Holoclone formation assay will be used to determine KSCs expansion efficiency. The characteristics of KSCs, in particular their clonogenicity and fate will also be analysed. Clonogenicity of KSCs with and without ROCKi treatment will be assessed in cell model in vitro and in a human:murine chimera skin graft model in vivo. The fate of KSCs in non-treated and ROCKi treated cultures will be examined at single cell transcriptome resolution.

Transcriptome changes will be examined in thousands of cells under different conditions (non-treated and ROCKi treated) and culture times (primary and passaged cells). Unbiased cell population separation for these cell cultures will be performed using changes in the cells transcriptomes. Finally, we will test the potential acceleration of ROCKi treated cultures based on the criteria used in our previous phase I trial.

Thus, we will assess the suitability of ROCKi treated cells for clinical application from the evidence gathered in this study.

We propose to use the ROCKi for the efficient expansion of keratinocyte stem cells (KSCs) from freshly isolated heterogeneous skin cell populations in vitro. Increased numbers of KSCs would then differentiate into the numerous and necessary keratinocyte cell populations for forming epithelial sheets in a much shorter time, facilitating their production for clinical application. Because gene-modified epithelial sheet therapy offers the best prospect of disease amelioration in genetic skin disorders, the timely production of epithelial sheets would offer a personalised and effective clinical treatment to patients with untreatable genetic skin conditions currently under the care of the NHS, for example, dystrophic epidermolysis bullosa. In addition, we will provide new insight into how ROCKi treatment contributes to stem cell growth and differentiation, opening the door to future improvements in the culture of KSCs for the benefit of patients with skin diseases.