Disorders of Copper Transport

门克斯病（MD）是一种以低脑铜为特征的铜运输x连锁遗传疾病，可导致婴儿神经变性和过早死亡。MD是由铜转运基因ATP7A的突变引起的。目前的治疗方法仅限于皮下铜注射，对携带突变等位基因的患者尤其有效，这些突变等位基因不能完全消除ATP7A的功能，并且在出生时被发现（N Engl J Med 2008 358:605-614）。对于完全丧失功能突变的患者，铜的外周递送不能有效地穿过血脑屏障。因此，需要替代的治疗方法。在不能通过腹腔或静脉注射铜拯救的小鼠经典MD （atp7amo-br）模型中，我们正在研究两种脑定向治疗方法：1)早期脑室内注射氯化铜，2)早期脑室内注射表达缩小大小的人ATP7A， rsATP7A-AAV的重组腺相关病毒血清5型（AAV5）载体。

Menkes disease (MD) is a X-linked genetic disorder of copper transport characterized by low brain copper that results in infantile neurodegeneration and premature death. MD is caused by mutations in a copper transporter gene, ATP7A. Current treatment is limited to subcutaneous copper injections and is especially effective in patients with mutant alleles that do not completely abrogate ATP7A function, and who are identified near birth (N Engl J Med 2008 358:605-614). For patients with complete loss-of-function mutations, copper delivered peripherally does not efficiently cross the blood-brain barrier. Thus, alternative treatment approaches are needed. In a murine model of classical MD (atp7amo-br) which cannot be rescued by intraperitoneal or intravenous copper, we are studying two brain-directed therapies: 1) early intracerebroventricular injection of copper chloride, and 2) early intracerebroventricular injection of a recombinant adeno-associated virus serotype 5 (AAV5) vector expressing a reduced size human ATP7A, rsATP7A-AAV. We developed a genotyping assay that identifies mutant pups before clinical signs, and administered 50 ng copper chloride to affected mice by intracerebroventricular injection within 3 days of birth. The dose was extrapolated from a maximum tolerated dose in adult rats that we determined previously (Molec Genet Metab 2007 91:30-36). Treatment modestly extended survival in mutant males and increased mean brain cortex copper levels in mutant mice . In an alternative approach, we are studying the expression and effects of rsATP7A-AAV delivered by intracerebroventricular injection. The transgene product begins at methionine 461 of the Menkes ATP7A and includes the fifth and sixth copper-binding domains. In mice receiving the AAV vector, we documented preliminary evidence of improved brain catechol ratios reflecting enhanced activity of dopamine-beta-hydroxylase, a copper-dependent enzyme. Our preliminary data indicate that intracerebroventricular delivery of copper and rsATP7A-AAV results in distribution throughout the atp7a<mo-br> brain. Whereas both treatments appeared to improve catecholamine biosynthesis in mutant brains, the effect of copper was more dramatic in this very small initial sample. Dose refinements, or combination therapy, may be required to optimize biochemical and clinical outcomes in this mouse model. Brain-directed therapies may ultimately provide a useful approach for treatment of MD patients with severe mutations.