Lisjak Michela, De Caneva Alessia, Marais Thibaut, Barbon Elena, Biferi Maria Grazia, Porro Fabiola, Barzel Adi, Zentilin Lorena, Kay Mark A, Mingozzi Federico, Muro Andrés F
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Inserm UMRS974, Centre of Research in Myology (CRM), Institut de Myologie, Sorbonne Université, Paris, France.
Front Genome Ed. 2022 Mar 11;4:785698. doi: 10.3389/fgeed.2022.785698. eCollection 2022.
Many inborn errors of metabolism require life-long treatments and, in severe conditions involving the liver, organ transplantation remains the only curative treatment. Non-integrative AAV-mediated gene therapy has shown efficacy in adult patients. However, treatment in pediatric or juvenile settings, or in conditions associated with hepatocyte proliferation, may result in rapid loss of episomal viral DNA and thus therapeutic efficacy. Re-administration of the therapeutic vector later in time may not be possible due to the presence of anti-AAV neutralizing antibodies. We have previously shown the permanent rescue of the neonatal lethality of a Crigler-Najjar mouse model by applying an integrative gene-therapy based approach. Here, we targeted the human coagulation factor IX (hFIX) cDNA into a hemophilia B mouse model. Two AAV8 vectors were used: a promoterless vector with two arms of homology for the albumin locus, and a vector carrying the CRISPR/SaCas9 and the sgRNA. Treatment of neonatal P2 wild-type mice resulted in supraphysiological levels of hFIX being stable 10 months after dosing. A single injection of the AAV vectors into neonatal FIX KO mice also resulted in the stable expression of above-normal levels of hFIX, reaching up to 150% of the human levels. Mice subjected to tail clip analysis showed a clotting capacity comparable to wild-type animals, thus demonstrating the rescue of the disease phenotype. Immunohistological analysis revealed clusters of hFIX-positive hepatocytes. When we tested the approach in adult FIX KO mice, we detected hFIX in plasma by ELISA and in the liver by western blot. However, the hFIX levels were not sufficient to significantly ameliorate the bleeding phenotype upon tail clip assay. Experiments conducted using a AAV donor vectors containing the eGFP or the hFIX cDNAs showed a higher recombination rate in P2 mice compared to adult animals. With this study, we demonstrate an alternative gene targeting strategy exploiting the use of the CRISPR/SaCas9 platform that can be potentially applied in the treatment of pediatric patients suffering from hemophilia, also supporting its application to other liver monogenic diseases. For the treatment of adult patients, further studies for the improvement of targeting efficiency are still required.
许多先天性代谢缺陷需要终身治疗，在涉及肝脏的严重情况下，器官移植仍然是唯一的治愈性治疗方法。非整合型腺相关病毒（AAV）介导的基因治疗已在成年患者中显示出疗效。然而，在儿科或青少年患者中进行治疗，或在与肝细胞增殖相关的情况下进行治疗，可能会导致游离病毒DNA迅速丢失，从而影响治疗效果。由于存在抗AAV中和抗体，后期再给予治疗载体可能无法实现。我们之前已经证明，通过应用基于整合基因治疗的方法，可以永久性挽救克里格勒-纳贾尔小鼠模型的新生儿致死性。在此，我们将人凝血因子IX（hFIX）cDNA靶向到乙型血友病小鼠模型中。使用了两种AAV8载体：一种无启动子载体，其具有与白蛋白基因座同源的两个臂，另一种载体携带CRISPR/SaCas9和sgRNA。对新生P2野生型小鼠进行治疗后，给药10个月后hFIX水平超出生理水平且保持稳定。对新生FIX基因敲除小鼠单次注射AAV载体也导致hFIX水平稳定表达且高于正常水平，达到人类水平的150%。接受尾尖剪试验的小鼠显示出与野生型动物相当的凝血能力，从而证明了疾病表型得到挽救。免疫组织学分析显示存在hFIX阳性肝细胞簇。当我们在成年FIX基因敲除小鼠中测试该方法时，通过ELISA在血浆中以及通过蛋白质印迹法在肝脏中检测到了hFIX。然而，hFIX水平不足以在尾尖剪试验中显著改善出血表型。使用含有eGFP或hFIX cDNA的AAV供体载体进行的实验表明，与成年动物相比，P2小鼠的重组率更高。通过这项研究，我们展示了一种利用CRISPR/SaCas9平台的替代基因靶向策略，该策略有可能应用于治疗患有血友病的儿科患者，也支持其应用于其他肝脏单基因疾病。对于成年患者的治疗，仍需要进一步研究以提高靶向效率。
