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

近年来，基于腺相关病毒(AAV)的载体已成为一种很有前途的基因传递载体 治疗多种疾病，包括心血管疾病。尽管早期的结果令人鼓舞，但 Cupid(心脏病基因治疗经皮给药上调钙)试验使用 编码肌浆钙ATPase SERCA2a的AAV血清1型载体未能同时满足其 主端点和次要端点。这些结果令人惊讶，因为在猪的心力衰竭模型中 AAV1.SERCA2a可改善心功能。初步结果表明，令人失望的结果是 由于AAV1.SERCA2a不能有效传递SERCA2a基因。一种可能的解释是 较差的基因传递是用体外NAB没有检测到抗AAV1的中和抗体(NAB) 丘比特试验中使用的化验，但这些NaB阻止了转导。在本应用程序的目标1中，我们将 在猪心衰模型中测试极低水平的NaBS的假设，这只能通过 体内NAB检测较为灵敏，可预防AAV1.SERCA2a的转导和治疗效果。 相反，如果体外NAB检测足够灵敏，我们将确定符合以下条件的最大NAB水平 仍然符合AAV1.SERCA2a的有效转导和治疗效果(在猪)。 丘比特试验阴性结果的另一种解释是AAV1显示物种特异性 趋向性，即AAV1能有效转导猪心肌细胞，但不能转导人心肌细胞。 为了将心脏AAV基因疗法引入临床，关键是要分离出能够 有效地转导人心肌细胞；2)对NaBS表现出更高的耐药性。与世隔绝 变种是目标2的目标。 不幸的是，AAV变种似乎不太可能有效地转导人类心肌细胞 对自然产生的AAV血清型具有很高水平的NAB也具有抵抗力 与世隔绝。因此，在目标3中，我们将测试一种通过血浆置换从血液中清除nabs的方法。 结合免疫吸附与固定化AAV颗粒的柱。 随着这项建议的成功完成，我们将确定体外NAB检测是否 足够敏感，足以作为心脏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试。我们将会有孤立的小说AAV 对人心肌细胞有趋向性的突变体，并增加了对nabs的抗性。最后，我们将拥有 确定血浆置换和免疫吸附在AAV柱上是否可以用于耗尽 血中的纳布。这些参数将在未来基因治疗试验的设计和执行中发挥关键作用 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.