Rosenke Kyle, Leventhal Shanna, Moulton Hong M, Hatlevig Susan, Hawman David, Feldmann Heinz, Stein David A
bioRxiv. 2020 Sep 30:2020.09.29.319731. doi: 10.1101/2020.09.29.319731.
SARS-CoV-2 is the causative agent of COVID-19 and a pathogen of immense global public health importance. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense agents composed of a phosphordiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking.
Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5'-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method and viral growth was measured with quantitative RT-PCR and TCID infectivity assays.
PPMO designed to base-pair with sequence in the 5'-terminal region or the leader transcription regulatory sequence-region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titers by up to 4-6 log10 in cell cultures at 48-72 hours post-infection, in a non-toxic and dose-responsive manner.
The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further pre-clinical development.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）是冠状病毒病（COVID-19）的病原体，对全球公共卫生具有极其重要的意义。迫切需要开发创新的直接作用抗病毒药物来应对这种病毒。肽缀合吗啉代寡聚物（PPMO）是一种反义药物，由与细胞穿透肽共价连接的磷二酰胺吗啉代寡聚物组成。PPMO进入细胞无需递送辅助，并且能够通过序列特异性空间位阻降低靶向RNA的表达。
合成了5种针对SARS-CoV-2 5'非翻译区基因组RNA序列设计的PPMO和1种随机序列的阴性对照PPMO。评估每种PPMO对未感染细胞活力的影响及其对Vero-E6细胞培养物中SARS-CoV-2复制的抑制作用。用基于ATP的方法评估细胞活力，用定量逆转录聚合酶链反应（RT-PCR）和组织培养感染剂量（TCID）感染性测定法测量病毒生长。
设计与SARS-CoV-2基因组RNA 5'末端区域或前导转录调控序列区域中的序列碱基配对的PPMO非常有效，在感染后48 - 72小时的细胞培养物中，以无毒且剂量反应性的方式将病毒滴度降低多达4 - 6个对数10。
数据表明PPMO有能力有效且特异性地抑制SARS-CoV-2生长，是进一步临床前开发的有前景的候选药物。
