Mondo Erica, Moser Richard, Gao Guangping, Mueller Christian, Sena-Esteves Miguel, Sapp Ellen, Pfister Edith, O'Connell Denice, Takle Kendra, Erger Kirsten E, Liu Wanglin, Conlon Thomas J, DiFiglia Marian, Gounis Matthew J, Aronin Neil
Department of Medicine and RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA.
Department of Neurosurgery, University of Massachusetts Medical School, Worcester, MA, USA.
J Huntingtons Dis. 2018;7(4):309-319. doi: 10.3233/JHD-180302.
Transgenic sheep are currently the only large animal model of Huntington's disease expressing full-length mutant human huntingtin. These transgenic sheep provide an opportunity to test adeno associated virus (AAV) therapies directly targeting the huntingtin gene. A recent study demonstrated that self-complementary (sc) AAV with artificial miRNA against human huntingtin reduced mutant human huntingtin in caudate and putamen after a single injection near the internal capsule.
To identify an AAV serotype among AAVrh8, AAV9 and AAVrh10 with the highest neuronal uptake and distribution, with no obvious cell loss in the neostriatum of the sheep.
We tested AAVrh8, AAV9 and AAVrh10 by stereotactic direct unilateral injection into the neostriatum of sheep, near the internal capsule. Four weeks after administration, we examined the viral spread and neuronal uptake of each serotype of AAV containing GFP. We compared single stranded (ss) and scAAVs. Further, we measured the distribution of AAVrh8 and AAV9 to a variety of tissues outside the brain.
Sc AAV9 had the best combination of neuronal uptake and distribution throughout the neostriatum. scAAVrh10 demonstrated good spread, but was not taken up by neurons. scAAVrh8 demonstrated good spread, but had less neuronal uptake than AAV9. Six hours after convection-enhanced administration to the neostriatum, both AAVrh8 and AAV9 viral genomes were detected in blood, saliva, urine, feces and wool. By four weeks, viral genomes were detected in wool only. Administration of AAVrh8, AAV9 and AAVrh10 was not associated with loss of neostriatal, medium spiny neuron number as measured by DARPP32 immunohistochemistry.
Altogether, we found scAAV9 had the best neuronal uptake and spread, showed no loss of neurons at one-month post-injection, and was not measurable in body fluids one month after injection. This information will guide future clinical experiments requiring brain injection of AAV for therapeutics for gene or miRNA deliveries in sheep transgenic for the human huntingtin gene.
转基因绵羊是目前唯一表达全长突变型人类亨廷顿蛋白的亨廷顿病大型动物模型。这些转基因绵羊为测试直接靶向亨廷顿基因的腺相关病毒（AAV）疗法提供了机会。最近一项研究表明，携带针对人类亨廷顿蛋白的人工微小RNA的自互补（sc）AAV在靠近内囊单次注射后，可降低尾状核和壳核中的突变型人类亨廷顿蛋白水平。
在AAVrh8、AAV9和AAVrh10中鉴定出神经元摄取和分布最高、且绵羊新纹状体中无明显细胞丢失的AAV血清型。
我们通过立体定向直接单侧注射到绵羊靠近内囊的新纹状体中，来测试AAVrh8、AAV9和AAVrh10。给药四周后，我们检查了每种含绿色荧光蛋白（GFP）的AAV血清型的病毒扩散和神经元摄取情况。我们比较了单链（ss）和scAAV。此外，我们测量了AAVrh8和AAV9在脑外多种组织中的分布。
ScAAV9在整个新纹状体的神经元摄取和分布方面具有最佳组合。scAAVrh10显示出良好的扩散，但未被神经元摄取。scAAVrh8显示出良好的扩散，但神经元摄取比AAV9少。在对新纹状体进行对流增强给药6小时后，在血液、唾液、尿液、粪便和羊毛中均检测到AAVrh8和AAV9病毒基因组。到四周时，仅在羊毛中检测到病毒基因组。通过DARPP32免疫组织化学检测，注射AAVrh8、AAV9和AAVrh10与新纹状体中型棘状神经元数量的减少无关。
总体而言，我们发现scAAV9具有最佳的神经元摄取和扩散，在注射后一个月未显示神经元丢失，且在注射一个月后体液中无法检测到。这些信息将指导未来在人类亨廷顿基因转基因绵羊中进行需要脑内注射AAV进行基因或微小RNA递送治疗的临床试验。
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