Herzog Roland W, Fields Paul A, Arruda Valder R, Brubaker Jeff O, Armstrong Elina, McClintock Darryl, Bellinger Dwight A, Couto Linda B, Nichols Timothy C, High Katherine A
Department of Pediatrics and Pathology, University of Pennsylvania Medical Center and Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Hum Gene Ther. 2002 Jul 20;13(11):1281-91. doi: 10.1089/104303402760128513.
Intramuscular injection of an adeno-associated virus (AAV) vector has resulted in vector dose-dependent, stable expression of canine factor IX (cF.IX) in hemophilia B dogs with an F.IX missense mutation (Herzog et al., Nat. Med. 1999;5:56-63). The use of a species-specific transgene allowed us to study risks and characteristics of antibody formation against the therapeutic transgene product. We analyzed seven dogs that had been injected at a single time point at multiple intramuscular sites with varying vector doses (dose per kilogram, dose per animal, dose per site). Comparison of individual animals suggests an increased likelihood of inhibitory anti-cF.IX (inhibitor) development with increased vector doses, with dose per site showing the strongest correlation with the risk of inhibitor formation. In six of seven animals, such immune responses were either absent or transient, and therefore did not prevent sustained systemic expression of cF.IX. Transient inhibitory/neutralizing anti-cF.IX responses occurred at vector doses of 2 x 10(12)/site, whereas a 6-fold higher dose resulted in a longer lasting, higher titer inhibitor. Anti-cF.IX was efficiently blocked in an eighth animal that was injected with a high vector dose per site, but in addition received transient immune suppression. Inhibitor formation was characterized by synthesis of two IgG subclasses and in vitro proliferation of lymphocytes to cF.IX antigen, indicating a helper T cell-dependent mechanism. Anti-cF.IX formation is likely influenced by the extent of local antigen presentation and may be avoided by limited vector doses or by transient immune modulation.
在患有FIX错义突变的乙型血友病犬中，肌肉注射腺相关病毒（AAV）载体已导致犬FIX（cF.IX）呈载体剂量依赖性稳定表达（Herzog等人，《自然医学》，1999年；5：56 - 63）。使用物种特异性转基因使我们能够研究针对治疗性转基因产物产生抗体的风险和特征。我们分析了7只犬，这些犬在多个肌肉部位的单个时间点接受了不同载体剂量（每千克剂量、每只动物剂量、每个部位剂量）的注射。个体动物的比较表明，随着载体剂量增加，产生抑制性抗cF.IX（抑制剂）的可能性增加，其中每个部位的剂量与抑制剂形成风险的相关性最强。在7只动物中的6只中，此类免疫反应要么不存在，要么是短暂的，因此并未阻止cF.IX的持续全身表达。在载体剂量为2×10¹²/部位时出现短暂的抑制性/中和性抗cF.IX反应，而高出6倍的剂量则导致持续时间更长、滴度更高的抑制剂。在第8只每个部位接受高载体剂量注射但还接受了短暂免疫抑制的动物中，抗cF.IX被有效阻断。抑制剂的形成表现为两种IgG亚类的合成以及淋巴细胞对cF.IX抗原的体外增殖，表明这是一种辅助性T细胞依赖性机制。抗cF.IX的形成可能受局部抗原呈递程度的影响，并且可以通过限制载体剂量或短暂免疫调节来避免。
