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大组不同的疾病。虽然这些疾病影响普通人群中的一小部分患者，但它们通常发生在出生或生命早期，通常是慢性的、严重的，有时甚至危及生命。由于疾病、身体表现和缺乏或丧失自主性的影响，患者及其家人的生活质量受到严重影响。目前针对这些破坏性疾病的临床选择仅限于对症治疗。针对遗传性皮肤疾病已经开发了不同的基因疗法，而基因修饰的皮片移植是针对这些疾病最有前途的基因疗法。在这种疗法中，将正确的基因添加到患者的皮肤干细胞中，然后在实验室中培养这些干细胞，形成完整的皮片，然后移植回患者身上。我们对严重和衰弱的皮肤疾病Netherton综合征进行了这一治疗，I期临床试验没有显示任何不良并发症，基因表达和功能都得到了纠正。我们现在正计划将这个治疗平台带到其他遗传性皮肤病，如大疱性表皮松解症。然而，皮片在实验室中的生长时间延长(超过40至50天)阻碍了这种疗法的进一步临床应用。我们的NS试验研究表明，干细胞最初的低扩增是实验室皮片生长缓慢的原因。我们已经找到了这个问题的潜在解决方案。我们建议用一种名为Rho相关激酶抑制物(ROKI)的药物来治疗从一小块皮肤组织中获得的皮肤细胞，以加速皮肤细胞的生长，特别是皮肤干细胞。通过这种方式，我们希望减少生产皮肤床单所需的时间。我们将在新鲜从人类皮肤样本中获得的皮肤细胞中测试ROKI。使用和不使用Rocki治疗的皮肤干细胞的速度、行为和特征将通过细胞培养进行评估。干细胞的行为将在人源化的小鼠皮肤移植模型中进一步评估，在该模型中，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研究未经处理和经ROKI处理的皮肤细胞的命运，以确定治疗后细胞群的变化。在单细胞研究中，我们将监测数千个单个单细胞中的基因表达，并从含有混合细胞的培养物中识别出不同的细胞群体。我们将确定未经治疗的干细胞和经ROKI治疗的干细胞在数量、类型和比例上的差异。因此，我们将回答ROKI是否具有在不改变皮肤干细胞特性的情况下有效地扩增皮肤干细胞的潜力，从而显著缩短临床应用中安全使用的皮肤片的准备时间。这项拟议的工作与患有罕见遗传性皮肤病但没有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ROKI，以有效地生产经过基因修正的皮肤片，以治疗严重和破坏性的皮肤疾病，如大疱性表皮松解症。

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.