Immunologic Aspects of In Utero or Neonatal AAV-Based Gene Therapy

描述(申请人提供)：通过基因转移技术引入和表达正常蛋白质可能会导致中和免疫反应的发展，因为在患有蛋白质遗传性疾病的人中，正常蛋白质可能被识别为非自我。基因治疗的成功依赖于对蛋白质产品的免疫耐受性或无效性的发展；实现这一点可能会对人类分泌或细胞内蛋白质疾病以及神经肌肉疾病的治疗产生影响，在这些疾病中可能会产生抑制性抗体；对于目前的基于蛋白质的治疗和未来的基因治疗试验，这仍然是一个重要且尚未解决的问题。虽然小鼠一直是大多数免疫学家的实验模型，对小鼠免疫反应的研究也产生了重要的见解，但这些模型未能预测基因治疗对人类的疗效是一个主要限制。因此，目前的建议将研究在免疫系统个体发育与人类相似的非人灵长类动物中，AAV载体表达的异种转基因编码蛋白的免疫应答，并检验从宫内或新生儿早期开始接触这些持久性抗原将导致诱导免疫耐受的假设。此外，将表达异种因子VIII的AAV应用于猴胎儿和新生儿，作为血友病A基因治疗的模型，以防止抑制因子VIII的发展，这是本领域研究的治疗目标。初步数据：我们的研究小组给新生的猴子注射了表达卵清蛋白的AAV，这是一种免疫原性和特征良好的蛋白质，并证明了对这种异种蛋白缺乏免疫反应，而对AAV的保护性免疫仍保持不变；出生后随后给药的AAV有可能在没有对异种蛋白的体液反应的情况下增强表达，但确实需要AAV血清型转换。在目标1中，AAV将在子宫内和新生非人灵长类动物中接种，以研究对外来转基因编码蛋白免疫无应答的机制。在目标2中，我们将评估病毒载体在宫内和新生儿体内的免疫应答。最后，我们将评估对异种因子VIII的免疫反应，作为血友病A基因治疗和抑制物形成预防的模型。这些研究将确定是否有一种临床可接受的方法通过启动转基因表达来建立对免疫原性蛋白的耐受性，而免疫个体发育可能不会在非人类灵长类动物模型中完全发展，从而复制人类干预的策略。

DESCRIPTION (provided by applicant): The introduction and expression of a normal protein by gene transfer techniques may result in the development of neutralizing immune responses as the normal protein may be recognized as non-self in those with inherited disorders of proteins. The success of gene therapy is dependent on the development of a state of immunological tolerance or anergy to protein products; achieving this could have an impact on the treatment of human disorders of secretory or intracellular proteins and neuromuscular disorders where inhibitory antibodies can develop; this remains an important and unresolved issue with present day protein-based therapy and future gene therapy trials. While mice have been the experimental model for most immunologists and the study of the murine immune response has yielded important insights, the failure of these models to predict the efficacy of gene therapy in humans is a major limitation. Accordingly, the current proposal will study the immune response to xenogenic transgene-encoded proteins expressed by AAV vectors in the nonhuman primate where the ontogeny on the immune system is similar to humans, and test the hypothesis that exposure to these persistent antigens, beginning either in utero or in the early neonatal period, will result in the induction of immunological tolerance. In addition, AAV expressing xenogenic factor VIII will be administered to the monkey fetus and neonate as a model for hemophilia A gene therapy to prevent inhibitor development to factor VIII, the treatment goal for this area of investigation. Preliminary data: Our research group has administered AAV expressing ovalbumin, an immunogenic and well- characterized protein, to newborn monkeys and have demonstrated a lack of immune response to this xenogenic protein while protective immunity to AAV has remained intact; augmented expression was possible with subsequent postnatal AAV administration without a humoral response to the xenogenic protein but did require AAV serotype switching. In Aim 1, AAV will be administered in utero and to neonatal nonhuman primates to study the mechanism of immune unresponsiveness to the foreign transgene-encoded protein. In Aim 2, we will assess the immune response to the viral vector administered in utero and the neonate. Finally, we will assess immune responses to the xenogenic factor VIII as a model for hemophilia A gene therapy and inhibitor formation prevention. These studies will determine if a clinically acceptable method to establish tolerance to immunogenic proteins is possible by initiating transgene expression while immunologic ontogeny may not be completely developed in a nonhuman primate model that could replicate a strategy for human intervention.