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：
10454760
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/10454760
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 Genitalia 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，但还没有生物制剂可以预防可靠地防止约 8000 万美国人将传染性 HPV 病毒颗粒传播给性伴侣。早些时候我们确定 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转化为具有高活性的部分在叶酸缺乏的情况下，对 HPV16 57 核苷酸 (nt) RNA 顺式元件的亲和力；这种互动导致对 HPV16 L1 和 L2 病毒衣壳蛋白的深度抑制，这些蛋白对于 HPV16 至关重要衣壳化（和传染性）。我们已获得 hnRNP-E1 强大突变体 [DomPos-E1(C293S)] 的专利，该突变体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(C293S)]有消除 HPV16 病毒衣壳蛋白并从而发挥抗 HPV 作用的可能性如此之大，我们希望在体外和我们的米色裸鼠新型 HPV16 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(C293S)-蛋白质相互作用的影响[相对于控制野生型（wt）样-E1（G292A）-蛋白]与HPV16 57-nt顺式元件消除HPV16 L1和L2 病毒衣壳蛋白表达。我们还将扩展这些研究以评估 DomPos-E1(C293S) 的相互作用具有来自低风险 HPV6 和 11 以及高风险类型（HPV18、31、33、45、52、58）的相似顺式元件。接下来，我们将证实 DomPos-E1(C293S)- 相对于对照 wt-like-E1(G292A)- 表达在减少 HPV16 L1 和 L2 稳定转导至携带 HPV16 的角质形成细胞后，这些角质形成细胞也转化为 HPV16-器官型筏；那么我们可以评估 18-18 种感染性 HPV16 病毒颗粒的减少程度日龄筏以及扩增的无衣壳 HPV16 附加体是否会增加基因组整合。在具体目标 2 中，我们将皮下植入这些 DomPos-E1(C293S)-或对照 wt-like- E1(G292A))-使用我们最近发布的模型在米色裸鼠中表达筏。这将使我们能够评估 DomPos-E1(C293S)-相对于对照wt-like-E1(G292A)在减少HPV16病毒衣壳方面的相对效果随后 8 周体内 HPV16 的蛋白质和感染性病毒颗粒；评估这是否会减少植入的 HPV16 筏自我感染的能力；确定基因组整合的变化扩增无衣壳 HPV16 附加体；以及延长筏发生 HPV16 癌症的预期时间。在具体目标 3 中，我们将确定 DomPos-E1(C293S) 是否比对照 wt 显着更有效 like-E1(G292A) 可以防止 HPV16 传播到邻近组织。我们将调整我们的体内模型来评估 HPV16-感染性，其中感染性 HPV16 从一个供体组织传播到另一个组织的有效性在 Beige Nude 小鼠中对未感染的受体组织进行了 8 周的评估。这些研究将有助于确定 DomPos-E1(C293S) 或其突变衍生物是否可以向前推进作为一流的药物，有助于减少感染性 HPV16 病毒颗粒从受感染宿主的传播。