Gene Therapy for Canine X-linked SCID

描述(由申请人提供)：X连锁严重联合免疫缺陷(XSCID)和大多数免疫和血液学疾病的遗传治疗将需要转导多能、自我更新的造血干细胞(HSCs)，而不是它们的祖细胞，以实现持久生产经过基因校正的细胞和持久的免疫重建。根据转导效率的不同，经过基因修正的HSCs不仅要与有缺陷的宿主细胞竞争，还要与大量未修正的HSCs竞争。在XSCID中，表达正常的共同伽马链(Gamma C)基因的T细胞具有选择性优势，这意味着转导少量未成熟的祖细胞可以在临床上产生大量的成熟T细胞。然而，B细胞谱系缺乏选择性优势意味着正常B细胞的重建可能依赖于大量未成熟的祖细胞和/或HSCs的转导。最近成功的人类XSCID逆转录病毒基因治疗试验的结果表明，转导细胞的数量将对治疗后免疫重建的质量和持久性至关重要。尽管所有成功治疗的患者在治疗后T细胞水平正常，但他们几乎没有发展出基因校正的B细胞。然而，年龄最大的患者表现出T细胞数量的下降，这引发了对T细胞重建持久性的质疑。此外，其中一名患者最近由于基因治疗而患上了T细胞白血病。这种竞争性更新的总体假设是，增加经过基因校正的HSCs和/或祖细胞的数量将导致免疫重建的质量和持久性的改善。一般的预测是，提供最高HSC转移率的载体将产生更多的B细胞和更好的体液功能，以及产生更持久的T细胞重建。因此，建议研究的主要焦点是评估改善基因传递以增加基因校正细胞数量的策略。将特别强调慢病毒载体，与逆转录病毒载体不同，它可以转导非周期细胞，这应该会导致更多的基因校正的造血干细胞。我们还将评估使用非清髓性条件处理或体内选择基因校正细胞来增强基因校正细胞的选择优势的策略。由于更高的转导效率和/或更强的转导细胞植入可能导致更多的转基因插入增加插入突变的潜在风险，我们将进行纵向整合位点分析，以确定克隆是否随着时间的推移而变化，并对与转基因整合和表达相关的副作用进行风险评估。犬XSCID与人类XSCID具有相同的免疫学表型，使其成为进行这些研究的理想大动物模型。

DESCRIPTION (provided by the applicant): The genetic treatment of X-linked severe combined immunodeficiency (XSCID) and most immune and hematological disorders will require the transduction of pluripotent, self-renewing hematopoietic stem cells (HSCs) rather than their progenitors in order to achieve enduring production of genetically corrected cells and durable immune reconstitution. Gene-corrected HSCs will have to compete not only with the defective host cells but also with a significant number of uncorrected HSCs depending upon the transduction efficiency. In XSCID, the selective advantage of T cells expressing a normal common gamma chain (gamma c) gene means that transduction of small numbers of immature progenitor cells may produce clinically significant numbers of mature T cells. However, the lack of a selective advantage in the B cell lineage means that reconstitution of normal B cells may depend on transduction of a large number of immature progenitors and/or HSCs. The results of the recent successful retroviral gene therapy trial for human XSCID suggest that the number of transduced cells will be important for the quality and durability of immune reconstitution following treatment. Although all successfully treated patients developed normal levels of T cells following treatment, they developed few, if any, gene-corrected B cells. However the oldest patient is showing decline in the number of T cells raising questions of the durability of T cell reconstitution. In addition, one of the patients recently developed a T cell leukemia as a result of the gene therapy. The overall hypothesis of this competitive renewal is that increasing the number of genetically corrected HSCs and/or progenitors will result in improved quality and durability of immune reconstitution. The general prediction is that the vectors that give the highest rate of HSC transduction will produce more B cells and better humoral function as well as produce a more durable T cell reconstitution. Therefore, the major focus of the proposed studies is to evaluate strategies for improving gene delivery to increase the number of gene-corrected cells. Special emphasis will be placed upon lentiviral vectors that, unlike retroviral vectors, can transduce non-cycling cells that should result in increased numbers of gene-corrected HSCs. We will also evaluate strategies for enhancing the selective advantage of gene corrected cells using either nonmyeloablative conditioning or in vivo selection of genetically corrected cells. Since higher transduction efficiencies and/or enhanced engraftment of transduced cells may lead to an overall higher number of transgene insertions increasing the potential risk of insertional mutagenesis, we will perform longitudinal integration site analyses to determine whether clonality changes over time and for risk assessment of side effects related to transgene integration and expression. Canine XSCID has an identical immunologic phenotype as human XSCID making it an ideal large-animal model in which to perform these studies.