Tolerance Induction by Neonatal Gene Delivery

描述（由申请人提供）：对载体和转基因产物的抑制性免疫反应的发展仍然是长期纠正遗传疾病的主要障碍。为了解决这个问题，我们建议在免疫系统成熟的早期传递和表达基因，以实现对治疗性基因产物和基因传递载体的耐受性。这也将使与围产期发病率和死亡率有关的遗传疾病能够得到先发制人的治疗。在这种情况下，小鼠和人类免疫系统之间的个体发生的一个重要差异是，T细胞成熟主要发生在小鼠出生后的早期，而在人类发育的妊娠中期。因此，出生后小鼠在出生后3周内的免疫成熟度代表了一个更相关的临床前模型，用于测试围产期基因传递策略和诱导人类免疫耐受。因此，本修订提案的总体目标是制定策略，以诱导对AAV载体介导的递送和转基因在新生小鼠中的表达的免疫耐受。在整个研究过程中，我们将(1)比较新生儿初次分娩和成人二次再感染高效AAV 1和8血清型载体后基因表达的生物分布、水平和持续时间；(2)监测对载体和细胞内报告细胞荧光素酶或分泌治疗性蛋白因子IX的免疫反应；(3)确定生命最初3周内诱导耐受的关键时期。我们还将研究AAV 1和AAV 8组织趋向性的差异是否会影响免疫反应，以及给药途径（静脉或IP给药）是否会影响正常和血友病动物对人因子IX的免疫反应。最后，通过过继性转移和免疫消耗实验，我们将在血友病动物中确定可能影响新生儿基因传递和成年aav -小鼠F.IX载体再攻毒后基因表达持续或消失的特定免疫机制。的相关性。这些研究将定义免疫个体发生的一个时期，病毒基因传递载体可以反复注射和表达，而不会引起限制治疗性基因表达的抑制性免疫反应。此外，对于许多遗传性疾病，在围产期进行干预以纠正这种疾病，将限制或消除这种疾病不可挽回的、往往是致命的后果的发展。

DESCRIPTION (provided by applicant): Development of inhibitory immune responses to vector and transgene products continues to be a major impediment to long-term correction of genetic disorders. To circumvent this problem, we propose to deliver and express genes early in the maturation of the immune system, with the goal of achieving tolerance to therapeutic gene products and gene delivery vectors. This would also enable pre-emptive treatment of genetic Disorders with perinatal morbidity and mortality. In this context, one of the important differences in ontogeny between mouse and human immune systems is that T cell maturation primarily occurs early during the postnatal period in the mouse, but during mid-gestation in human development. Thus, the'immunological maturity of postnatal mice within the first 3 weeks of life represents a more relevant preclinical model to test strategies for perinatal gene delivery and induction of immunotolerance in humans. Therefore, the overall goal of this revised proposal is to develop strategies for induction of immunotolerance to AAV vector- mediated delivery and expression of transgenes in neonatal mice. Throughout, we will (1) compare bio- distribution, level, and duration of gene expression after primary neonatal delivery and secondary adult re- challenge with highly efficient AAV 1 and 8 serotype vectors, (2) monitor immune responses to the vectors and an intracellular reporter, luciferase, or a secreted therapeutic protein, Factor IX, and (3) define the critical period in the first 3 weeks of life when induction of tolerance is feasible. We will also examine whether differences in AAV 1 vs. 8 tissue tropism affect immune response, and whether the route of administration (IV or IP delivery) affects immune responses to human Factor IX in normal and hemophilic animals. Finally, through adoptive transfer and immunodepletion experiments, we will identify specific immune mechanisms that may influence the persistence or abrogation of gene expression following neonatal gene delivery and adult re-challenge with AAV-murine F.IX vectors in hemophilic animals. RELEVANCE. These studies will define a period in immune ontogeny when viral gene delivery vectors can be repeatedly injected and expressed without eliciting inhibitory immune responses that limit therapeutic gene expression. Furthermore, for many genetic disorders, intervention in the perinatal period to correct the disorder would limit or abrogate the development of irrevocable, often lethal consequences of the disease.