Characterization of an anti-Human Papillomavirus (HPV) agent

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HPV16 L1和L2病毒衣壳蛋白的显著抑制，所述病毒衣壳蛋白是HPV16- 壳体化（和传染性）。我们已经为hnRNP-E1 [DomPos-E1（C293S）]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（C293S）]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[相对于 对照野生型（wt）样E1（G292A）蛋白]与HPV16 57-nt顺式元件在消除HPV16 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（C293S） 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（C293S）-相对于对照wt-样-E1（G292A）-表达在降低 HPV16 L1和L2在稳定转导到携带HPV16的角质形成细胞中后，也转化为 HPV16-器官型筏;然后我们可以评估在18 - 18个器官型筏中感染性HPV16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HPV16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（C293S）-或对照wt-样- E1（G292A））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（C293S）-相对于对照wt-样-E1（G292A）在减少HPV16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HPV16附加体;以及延长筏发展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. 类似于E1（G292A），防止HPV16传播到邻近组织。我们将调整我们的体内模型， HPV16-感染性，其中感染性HPV16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（C293S）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HPV16病毒颗粒从受感染宿主的传播。

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.