Metal elements are essential components of approximately half of all cellular proteins, and approximately one-third of all known enzymes thus far are metalloenzymes. Several cellular proteins and enzymes undoubtedly impact the transduction efficiency of recombinant adeno-associated virus (AAV) vectors, but the precise role of metal ions in this process has not been studied in detail. In the present studies, we systematically evaluated the effects of all 10 essential metal ions (calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, potassium, sodium, and zinc) on the transduction efficiency of AAV vectors. We report herein that five essential metal ions (iron, magnesium, manganese, molybdenum, and sodium) had little to no effect, and calcium strongly inhibited the transduction efficiency of AAV2 vectors. Whereas copper and potassium increased the transduction efficiency by ∼5-fold and ∼2-fold, respectively, at low concentrations, both essential metals were strongly inhibitory at higher concentrations. Calcium also inhibited the transduction efficiency by ∼3-fold. Two metal ions (cobalt and zinc) increased the transduction efficiency up to ∼10-fold in a dose-dependent manner. The combined use of cobalt and zinc resulted in more than an additive effect on AAV2 vector transduction efficiency (∼30-fold). The transduction efficiency of AAV serotypes 1 through 6 (AAV1–AAV6) vectors was also augmented by zinc. Similarly, the transduction of both single-stranded (ss) and self-complementary (sc) AAV3 vectors was enhanced by zinc. Zinc treatment also led to a dose-dependent increase in expression of a therapeutic protein, the human clotting factor IX (hF.IX), mediated by scAAV3 vectors in a human hepatic cell line. This simple strategy of essential metal ion-mediated enhancement may be useful to lower the dose of AAV vectors for their optimal use in human gene therapy.

金属元素是大约一半细胞蛋白质的必需成分，迄今为止所有已知酶中大约三分之一是金属酶。几种细胞蛋白和酶无疑会影响重组腺相关病毒（AAV）载体的转导效率，但金属离子在此过程中的确切作用尚未得到详细研究。在本研究中，我们系统地评估了所有 10 种必需金属离子（钙、钴、铜、铁、镁、锰、钼、钾、钠和锌）对 AAV 载体转导效率的影响。我们在此报道，五种必需金属离子（铁、镁、锰、钼和钠）几乎没有影响，而钙强烈抑制 AAV2 载体的转导效率。虽然铜和钾在低浓度下分别将转导效率提高了约 5 倍和约 2 倍，但两种必需金属在较高浓度下均具有强烈的抑制作用。钙还将转导效率抑制约 3 倍。两种金属离子（钴和锌）以剂量依赖性方式将转导效率提高约 10 倍。钴和锌的联合使用对 AAV2 载体转导效率产生了超过累加的效果（约 30 倍）。锌也增强了 AAV 血清型 1 至 6 (AAV1–AAV6) 载体的转导效率。同样，锌增强了单链 (ss) 和自互补 (sc) AAV3 载体的转导。锌治疗还导致人肝细胞系中由 scAAV3 载体介导的治疗蛋白、人凝血因子 IX (hF.IX) 的表达呈剂量依赖性增加。 这种必需金属离子介导的增强的简单策略可能有助于降低 AAV 载体的剂量，使其在人类基因治疗中得到最佳应用。