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

罕见的遗传性皮肤病包括一个大的和不同的疾病组。虽然这些疾病只影响普通人群中的少数患者，但它们往往发生在出生时或生命早期，通常是慢性的、严重的，有时危及生命。患者及其家属的生活质量因疾病的身体表现以及缺乏或丧失自主性而受到严重影响。目前，针对这些严重疾病的临床治疗方法仅限于对症治疗。针对遗传性皮肤病，已经开发了不同的基因治疗方法，而基因修饰的皮片移植是这些疾病最有希望的基因治疗方法。在这种疗法中，一个正确的基因被添加到患者的皮肤干细胞中，然后在实验室中生长，形成一个完整的皮肤片，移植回患者体内。我们已经对严重和使人衰弱的皮肤病内瑟顿综合征这样做了，并且I期临床试验显示没有不良并发症，具有校正的基因表达和功能。我们现在正计划将这种治疗平台用于其他遗传性皮肤病，如大疱性表皮病。然而，皮肤片在实验室中的生长时间延长（超过40至50天）阻碍了这种疗法在进一步临床应用中的应用。我们NS试验的研究表明，干细胞的初始低扩增是实验室中皮肤片生长缓慢的原因。我们已经找到了解决这个问题的可能办法。我们建议用一种名为Rho相关激酶抑制剂（ROCKi）的药物处理从一小片皮肤组织中获得的皮肤细胞，以加速皮肤细胞，特别是皮肤干细胞的生长。通过这种方式，我们希望减少制造蒙皮所需的时间。我们将在从人类皮肤样品新鲜获得的皮肤细胞中测试ROCKi。将在细胞培养物上评估有和没有ROCKi处理的皮肤干细胞的速度、行为和特征。干细胞的行为将在人源化小鼠皮肤移植模型中进一步评估，其中人类皮肤干细胞可以生长并存活数月。我们将在单细胞水平上进一步检查未处理的和ROCKi处理的皮肤细胞的命运，以确定处理后细胞群的变化。在单细胞研究中，我们将监测数千个单个细胞中的基因表达，并从含有混合细胞的培养物中鉴定不同的细胞群。通过比较ROCKi处理前后的干细胞在数量、种类、比例等方面的差异，回答ROCKi是否具有在不改变皮肤干细胞特性的情况下高效扩增皮肤干细胞的潜力，从而大幅缩短皮肤片的制备时间，确保临床应用安全。这项拟议的工作与患有罕见遗传性皮肤病且无有效治疗方法的患者直接相关。如果我们成功，我们将在皮肤干细胞培养中加入ROCKi，以有效地产生基因校正的皮肤片，以治疗严重和破坏性的皮肤病，如大疱性表皮病。

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.