Anti-AAV Antibodies as an Obstacle to Cardiac AAV Gene Therapy

近年来，腺相关病毒（AAV）载体作为一种有前途的基因传递载体， 包括心血管疾病在内的一系列疾病。尽管早期的结果令人鼓舞， CUPID（心脏病经皮基因治疗钙上调）试验，使用 编码肌浆钙ATP酶SERCA2a的AAV血清型1载体不能满足其两个要求， 主要和次要终点。这些结果是令人惊讶的，因为在HF的猪模型中， AAV1.SERCA2a改善心脏功能。初步结果表明，令人失望的结果是， 这是由于AAV1.SERCA2a不能有效地递送SERCA2a基因。一个可能的解释 差的基因递送是用体外NAb未检测到针对AAV1的中和抗体（NAb 这些NAb与CUPID试验中使用的测定结果一致，但这些NAb阻止了转导。在本申请的目标1中，我们将 在猪HF模型中测试了以下假设：极低水平的NAb，其只能通过 更灵敏的体内NAb测定法，可以阻止AAV1.SERCA2a的转导和治疗功效。 相反，如果体外NAb检测足够灵敏，我们将确定最大NAb水平， 仍然与AAV1.SERCA2a（在猪中）的有效转导和治疗功效相容。 CUPID试验的阴性结果的另一种解释是AAV 1显示种特异性表达。 嗜性，即AAV 1可以有效地扩增猪而不是人心肌细胞。 为了将心脏AAV基因治疗引入临床，分离AAV变体将是至关重要的，1）可以 有效地刺激人心脏细胞和2）显示出对NAb增加的抗性。这种隔离 变体是Aim 2的目标。 不幸的是，似乎不太可能有效地使人心肌细胞增殖的AAV变体 并且还对针对天然存在的AAV血清型的非常高水平的NAb具有抗性的细胞， 与世隔绝因此，在目标3中，我们将测试通过血浆置换从血液中消耗NAb的方法 结合用具有固定化AAV颗粒的柱的免疫吸附。 随着该提案的成功完成，我们将确定体外NAb检测是否适用于 足够敏感以用作递送AAV的心脏AAV基因治疗试验的排除标准 通过冠状动脉内输注，或者如果必须使用更灵敏的体内测定。我们将有分离的新型AAV 具有人心肌细胞向性和对NAb的抗性增加的变体。最后，我们将有 确定了血浆置换结合AAV柱上的免疫吸附是否可用于消除 血液样本这些参数在未来基因治疗试验的设计和执行中至关重要， 心血管疾病

Recently, Adeno-Associated Virus (AAV)-based vectors have emerged as promising gene delivery vehicles for a wide array of diseases, including cardiovascular disorders. Despite early encouraging results, the CUPID (Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease) trial using an AAV serotype 1 vector encoding the sarcoplasmatic calcium ATPase SERCA2a failed to meet both its primary and secondary endpoints. These results were surprising because in porcine models of HF AAV1.SERCA2a improved cardiac function. Preliminary results suggest that the disappointing outcome was due to a failure of AAV1.SERCA2a to deliver efficiently the SERCA2a gene. One possible explanation for the poor gene delivery is that neutralizing antibodies (NAbs) against AAV1 were not detected with the in vitro NAb assay used in the CUPID trial, but that these NAbs prevented transduction. In Aim 1 of this application we will test in a porcine HF model the hypothesis that extremely low levels of NAbs, which can only be detected by a more sensitive in vivo NAb assay, can prevent transduction and therapeutic efficacy of AAV1.SERCA2a. Conversely, if in vitro NAb assays are sufficiently sensitive, we will determine the maximal NAb levels that are still compatible with efficient transduction and therapeutic efficacy of AAV1.SERCA2a (in pigs). An alternative explanation for the negative results of the CUPID trial is that AAV1 displays specie-specific tropism, i.e. that AAV1 can efficiently transduce pig but not human cardiomyocytes. To bring cardiac AAV gene therapy to the clinic, it will be critical to isolate AAV variants that 1) Can efficiently transduce human cardiac cells and 2) Show increased resistance to NAbs. The isolation of such variants is the goal of Aim 2. Unfortunately, it seems unlikely that AAV variants that can efficiently transduce human cardiomyocytes and that are also resistant to very high levels of NAbs against the naturally occurring AAV serotypes can be isolated. Therefore, in Aim 3, we will test an approach to deplete NAbs from the blood by plasmapheresis coupled with immunadsorption with columns with immobilized AAV particles. With the successful completion of this proposal, we will have established whether an in vitro NAb assay is sensitive enough to serve as an exclusion criterion for cardiac AAV gene therapy trials where AAV is delivered by intracoronary infusion, or if a more sensitive in vivo assay must be used. We will have isolated novel AAV variants with tropism for human cardiomyocytes and increased resistance to NAbs. Finally, we will have established whether plasmapheresis coupled with immunadsorption on AAV columns can be used to deplete NAbs from blood. These parameters will be critical in the design and execution of future gene therapy trials for cardiovascular diseases.