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，但没有生物制剂可以 可靠地防止约8000万美国人将他们的传染性HPV病毒颗粒传播给性伴侣。 早些时候，我们确定了一个mRNA结合蛋白的翻译后同型半胱氨酸化 (异源核糖核蛋白-E_1，hnRNP-E_1)能将hnRNP-E_1转化为高表达的部分。 叶酸缺乏条件下HPV1657-核苷酸(NT)RNA顺式元件的亲和力；这种相互作用 导致对HPV16L1和L2病毒衣壳蛋白的深刻抑制，这是HPV16必不可少的 包裹性(和传染性)。我们已经申请了一种强大的hnRNP-E1突变体[DomPos-E1(C293S)]的专利 功能类似于同型半胱氨酸化的hnRNP-E1在叶酸充足的条件下。因为DomPos-E1(C293S)]具有 如此强大的可能性消除HPV16病毒衣壳蛋白，从而发挥抗HPV试剂的作用，我们 希望在体外和我们的新型HPV16-异种移植模型中测试其在Beige裸鼠中的治疗潜力。 在特定目标1中，我们将比较DomPos-E1(C293S)-蛋白质[相对于 用HPV1657-NT顺式元件控制野生型(Wt)样-E1(G292a)蛋白清除HPV16L1和L2 病毒衣壳蛋白的表达。我们还将扩展这些研究，以评估DomPos-E1(C293S)的相互作用 来自低危型HPV6和11型以及高危型(HPV18、31、33、45、52、58)的相似顺式元件。接下来，我们 将证实DomPos-e1(C293S)-控制wt-like-e1(G292a)-在减少表达方面的更大影响 HPV16L1和L2稳定地转导到携带HPV16的角质形成细胞后，也转化为 HPV16-器官型木筏；然后我们可以评估传染性HPV16病毒颗粒在18-15岁时减少的程度。 以及扩增的无衣壳HPV16附体是否会增加基因组的整合度。 在特定的目标2中，我们将这些DomPos-E1(C293S)-或对照wt-like- E1(G292A)-使用我们最近发表的模型在米色裸鼠体内表达筏。这将使我们能够评估 DomPos-E1(C293S)超对照wt-like-E1(G292a)对HPV16病毒衣壳蛋白的相对抑制作用 在接下来的8周内体内感染HPV16的蛋白质和感染病毒颗粒；评估这是否会降低 植入的HPV16-RAFT自我感染的能力；通过以下方式确定基因组整合的变化 扩增的无衣壳的HPV16异构体；以及延长RAFT发展为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-1显著更有效- Like-E1(G292A)在防止HPV16传播到邻近组织中的作用。我们将调整我们的体内模型来评估 HPV16的感染性，其中传染性HPV16从一个供体组织传播到一个供体组织的有效性 在米色裸鼠身上，对未感染的受者组织进行了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，帮助减少受感染宿主的传染性HPV16病毒颗粒的传播。

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.