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Characterization of an anti-Human Papillomavirus (HPV) agent

抗人乳头瘤病毒 (HPV) 药物的表征

基本信息

批准号：
9891919
负责人：
Asok Antony
金额：
--
依托单位：
RLR VA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9891919
关键词：
Affinity American Binding Proteins Biological Assay Biological Products Cancer Model Capsid Capsid Proteins Cells Development Dose Effectiveness Elements Episome Exhibits Folic Acid Folic Acid Deficiency Gel Generations Genital system Genitourinary system Genomics HPV-High Risk Heterogeneous-Nuclear Ribonucleoproteins Histology Human Papilloma Virus Vaccine Human Papillomavirus Human papilloma virus infection Human papillomavirus 16 Human papillomavirus 18 Human papillomavirus 6 Implant In Vitro Individual Infection L2 viral capsid protein Legal patent Low risk HPV Malignant Neoplasms Messenger RNA Modeling New Agents Newly Diagnosed Nuclear Nucleotides Nude Mice Oncogenic Oropharyngeal Proteins Publishing RNA RNA-Protein Interaction Reporter Ribonucleoproteins Risk Role Safety Sexual Partners Sexual Transmission Standardization Teenagers Testing Therapeutic Time Tissue Donors Tissues Veterans Viral Virion Virus Xenograft Model density experimental study high risk implantation in vivo in vivo Model innovation keratinocyte malignant oropharynx neoplasm mouse model mutant novel particle prevent protein expression response subcutaneous transmission process tumor viral transmission

项目摘要

Despite the advent of effective anti-Human Papillomavirus (HPV) vaccines, there are no biological agents to reliably prevent ~80 million Americans from transmitting their infectious HPV viral particles to sexual partners. Earlier we determined that the post-translational homocysteinylation of an mRNA-binding protein (heterogenous nuclear ribonucleoprotein-E1, hnRNP-E1) can transform hnRNP-E1 into a moiety with high affinity for a HPV16 57-nucleotide (nt) RNA cis-element under conditions of folate deficiency; this interaction led to a profound inhibition of both HPV16 L1 and L2 viral capsid proteins that are essential for HPV16- encapsidation (and infectivity). We have patented a powerful mutant of hnRNP-E1 [DomPos-E1(C293S)] that functions like homocysteinylated-hnRNP-E1 under folate-replete conditions. Because DomPos-E1(C293S)] has such a strong likelihood to eliminate HPV16 viral capsid proteins and thereby function as an anti-HPV agent, we wish to test its therapeutic potential both in vitro and in our novel HPV16-xenograft model in Beige Nude mice. In Specific Aim 1 we will compare effects of the interaction of DomPos-E1(C293S)-protein [relative to control wild-type(wt)-like-E1(G292A)-proteins] with HPV16 57-nt cis-element in eliminating HPV16 L1 and L2 viral capsid protein expression. We will also extend these studies to assess the interaction of DomPos-E1(C293S) with similar cis-elements from low risk HPV6 and 11 and high-risk types (HPV18, 31, 33, 45, 52, 58). Next, we will confirm the greater impact of DomPos-E1(C293S)- over control wt-like-E1(G292A)- expression in reducing HPV16 L1 and L2 after stable transduction into HPV16-harboring keratinocytes that are also transformed into HPV16-organotypic rafts; then we can evaluate the extent in reduction of infectious HPV16 viral particles in 18- day old rafts and whether there is any increase in genomic integration by amplified capsid-less HPV16 episomes. In Specific Aim 2, we will subcutaneously implant these DomPos-E1(C293S)- or control wt-like- E1(G292A))- expressing rafts in Beige Nude mice using our recently published model. This will allow us to assess the relative effects of DomPos-E1(C293S)- over control wt-like-E1(G292A) in reducing both HPV16 viral capsid proteins and infectious viral particles of HPV16 over the ensuing 8 weeks in vivo; evaluating if this reduces the capacity of the implanted HPV16-raft to auto-infect itself; determining changes in genomic integration by amplified capsid-less HPV16 episomes; and in prolonging the expected time of rafts to develop HPV16-cancers. In Specific Aim 3, we will determine if DomPos-E1(C293S) is significantly more effective than control wt- like-E1(G292A) in preventing transmission of HPV16 to adjacent tissue. We will adapt our in vivo model to assess HPV16-infectivity wherein the effectiveness of transmission of infectious HPV16 from one donor tissue to an uninfected recipient tissue is assessed over 8 weeks in Beige Nude mice. These studies will help determine if DomPos-E1(C293S) or its mutant derivatives can be moved forward as first-in-class agents to help reduce transmission of infectious HPV16 viral particles from an infected host.

尽管出现了有效的抗人乳头瘤病毒（HPV）疫苗，但没有生物制剂可可靠地防止约8000万美国人将其感染性HPV病毒颗粒传播给性伴侣。早先我们确定mRNA结合蛋白的翻译后同型半胱氨酸化（异质核核糖核蛋白-E1，hnRNP-E1）可以将hnRNP-E1转化为具有高表达的部分。叶酸缺乏条件下对HPV 16 57-核苷酸（nt）RNA顺式元件的亲和力;这种相互作用导致HPV 16 L1和L2病毒衣壳蛋白的显著抑制，所述病毒衣壳蛋白是HPV 16- 感染性（Infectivity）。我们已经为hnRNP-E1 [DomPos-E1（C293 S）]的强大突变体申请了专利，在叶酸充足的条件下，其功能类似于同型半胱氨酸化hnRNP-E1。因为DomPos-E1（C293 S）]具有如此强消除HPV 16病毒衣壳蛋白并由此作为抗HPV剂的可能性，我们希望在Beige裸小鼠的体外和我们的新型HPV 16异种移植模型中测试其治疗潜力。在具体目标1中，我们将比较DomPos-E1（C293 S）-蛋白质[相对于对照野生型（wt）样E1（G292 A）蛋白]与HPV 16 57-nt顺式元件在消除HPV 16 L1和L2中的作用病毒衣壳蛋白表达。我们还将扩展这些研究，以评估DomPos-E1（C293 S）与低危型HPV 6和11以及高危型HPV（HPV 18、31、33、45、52、58）的顺式元件相似。接下来我们将证实DomPos-E1（C293 S）-相对于对照wt-样-E1（G292 A）-表达在降低 HPV 16 L1和L2在稳定转导到携带HPV 16的角质形成细胞中后，也转化为 HPV 16-器官型筏;然后我们可以评估在18- 18个器官型筏中感染性HPV 16病毒颗粒减少的程度。日龄的筏以及是否存在通过扩增的无帽HPV 16附加体的基因组整合的任何增加。在特定目标2中，我们将皮下植入这些DomPos-E1（C293 S）-或对照wt-样- E1（G292 A））表达筏在米色裸鼠使用我们最近发表的模型。这将使我们能够评估 DomPos-E1（C293 S）-相对于对照wt-样-E1（G292 A）在减少HPV 16病毒衣壳蛋白质和感染性病毒颗粒的HPV 16在随后的8周内体内;评估这是否减少了植入的HPV 16筏自身感染的能力;通过以下方式确定基因组整合的变化：扩增的无衣壳HPV 16附加体;以及延长筏发展HPV 16癌症的预期时间。在具体目标3中，我们将确定DomPos-E1（C293 S）是否比对照wt. 类似于E1（G292 A），防止HPV 16传播到邻近组织。我们将调整我们的体内模型， HPV 16-感染性，其中感染性HPV 16从一个供体组织传播到另一个供体组织的有效性在BeigeNude小鼠中评估未感染的受体组织8周。这些研究将有助于确定DomPos-E1（C293 S）或其突变衍生物是否可以向前推进作为一流的试剂，帮助减少感染性HPV 16病毒颗粒从受感染宿主的传播。