WO2017214449A1 - Inhibition of e5 in hpv-infected cells - Google Patents

Abstract

The present invention relates to the inhibition of the E5 gene in HPV-infected cells. The inventors of the present disclosure have determined that inhibition of E5 provides numerous therapeutic benefits in the treatment of HPV infections, lesions, and HPV-related cancers. Notably, inhibition of E5 enables knockdown of oncogenes E6 and E7, which are responsible for various aspects of HPV pathology. The methods and associated compositions of the invention may be directed to HPV-infected tissues of the anus, cervix, and other sites. The scope of the invention further includes a novel anal epithelial cell line for enabling HPV research.

Description

Title: Inhibition of E5 in HPV-Infected Cells

CROSS-RELATED APPLICATIONS: This application claims the benefit of priority to United States Provisional Application Serial Number 62/347,720, entitled "Treatment of HPV- associated neoplasias by inhibition of E5," filed June 9, 2016, the contents of which are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT: This invention was made with government support under grant nos.

CA121947 and P30 CA082103 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING: The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 5, 2017, is named UCSF033PCT_SL.txt and is 941 bytes in size.

Background of the Invention

The human papillomavirus (HPV) exists in over a hundred different forms. The HPV virus may infect cutaneous and mucosal epithelial cells of the anus, cervix and oral cavity, as well as surrounding regions. Infection rates are high, with millions of persons infected each year. Infection by certain forms of the virus may result in the formation of lesions having mild to severe dysplasia, with some of the latter lesions progressing to invasive epithelial cancer. Cervical cancer, for example, is caused almost exclusively by HPV infection, and is the second most common cancer in women worldwide. Likewise, anal cancer is driven by HPV infection, and the incidence of anal cancer is increasing steadily in the general population.

While anti-HPV vaccines now available, currently there is no effective treatment for HPV infection once it has occurred. Accordingly, there remains a need in the art for novel treatments that can inhibit HPV infection, eliminate of HPV-associated lesions, and prevent HPV lesions from developing into cancers.

Summary of the Invention

Several HPV genes are implicated in HPV pathology. HPV oncogenes E6 and E7 have previously been implicated in HPV-associated cancers. E6 activity inhibits the tumor suppressor p53, while E7 inhibits the tumor-suppressing retinoblastoma protein. These activities unfavorably modify the cell cycle and drive pathogenic replication of HPV-infected cells.

Meanwhile, the role of the HPV E5 gene in cancer initiation and progression has been unclear, with mixed reports as to whether the gene is present or active at all in HPV neoplasias.

The inventors of the present disclosure have determined that E5 plays a key role in HPV infection, lesion formation, and progression to cancer. Importantly, the inventors of the present disclosure have developed a novel model of precancerous HPV infection which has allowed elucidation E5's role in carcinogenesis. Further, the inventors of the present disclosure have demonstrated that E5 inhibition provides an effective means of halting several pathological aspects of HPV infection.

In one aspect, the scope of the invention is directed to methods of inhibiting HPV E5 activity. Inhibition of E5 activity will induce multiple biological effects in HPV-infected cells, for example, inhibiting carcinogenesis, inducing regression of lesions, preventing lesion formation, and treating HPV-associated cancers.

In another aspect, the scope of the invention is directed to novel agents for the inhibition of E5 activity in HPV-infected cells.

In another aspect, the scope of the invention is directed to novel cell lines which may be used in HPV research and in the development and testing of therapeutics directed to HPV infection. Brief Description of the Drawings.

Figure 1. Figure 1 depicts in vitro collagen invasion assay results. Invasive capacity was measured in cultured cells wherein SiRNA knockdown of E5 (compared to control cells "C") was performed, with some treatments having E6/E7 rescue by transfection with a plasmid encoding E6 and E7 proteins ("E67", with control plasmid as "C"). Exogenous EGF was applied in some treatments (+) and omitted in a control treatment (-).

Figure 2A, 2B, 2C, 2D, and 2E. The expression of HPV 16 genes E2 (Figure 2D), E5 (Figure 2A), E6 (Figure 2B) and E7 (Figure 2C) was measured in control cells (siRNA = C), cells wherein E5 was knocked down (siRNA = E5) and cells wherein E6 and E7 were knocked down (siRNA = E6/7). In Figure 2E, the rate of AKC2 proliferation was measured in control cells (siRNA = C), cells wherein E6 and E7 were knocked down (siRNA = E6/7), and in cells wherein E5 was knocked down (siRNA = E5).

Figure 3A, 3B, and 3C. Figure 3A depicts the expression of E5, E6, and E7 in control cells (plasmid =C) and cells wherein E6 and E7 expression was rescued by an E6 and E7 expressing plasmid (plasmid = E6/7). The effects were compared in cells having normal E5 (siRNA = C) and in cells wherein E5 expression was knocked down (siRNA = E5). Figure 3B depicts p- EGFR abundance in cells having normal E5 (siRNA = C) and in cells wherein E5 expression was knocked down (siRNA = E5) and cells wherein E6 and E7 expression was rescued by an E6 and E7 expressing plasmid (plasmid = E6/7) and in control cells with no E6/E7 rescue (plasmid = C). The E6/E7 expression plasmid was applied in 1000 ng and 2000 ng treatments. Figure 3C depicts total EGFR in cells having normal E5 (siRNA = C), in cells wherein E5 expression was knocked down (siRNA = E5) and cells wherein E6 and E7 expression was rescued by an E6 and E7 expressing plasmid (plasmid = E6/7), and in control cells with no E6/E7 rescue (plasmid = C). The E6/E7 expression plasmid was applied at 1000 ng.

Detailed Description of the Invention.

General Context of the Invention. The invention is directed to several methods and compositions for the inhibition of E5 in cells infected with the HPV virus. As used herein, "HPV" or the "HPV virus" will refer to any HPV virus genotype, wherein the HPV viral genotype expresses an E5 gene. In one embodiment, the HPV virus comprises a "high-risk" HPV variant, as known in the art. In one embodiment, the HPV virus comprises HPV 16. In another embodiment, the HPV virus comprises HPV 18. In other embodiments, the HPV virus comprises an HPV virus of a genotype selected from 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.

Various methods of the invention are directed to the treatment of HPV-infected cells. As used herein, HPV-infected cells include any epithelial cells infected with an HPV virus. The HPV infected cell may comprise a keratinized epithelial cell or a mucosal epithelial cell. HPV- infected cells, as referred to herein, will also refer to dysplastic epithelial cells, for example, dysplastic and epithelial cells of squamous intraepithelial lesions, for example, as characterized by genetic or histopathological markers. Intraepithelial lesions, or "lesions" as used herein, may encompass squamous intraepithelial lesions, as known in the art, including both low-grade and high-grade squamous intraepithelial lesions.

HPV-infected cells will further refer to certain precancerous cells and cancer cells, wherein the such cells are derived from HPV-infected epithelium and wherein such cells express an E5 gene. HPV-infected cancer cells, as referred to herein, include cells from tumors which have arisen in the aforementioned epithelial tissues, as well as such cancer cells that have subsequently infiltrated into neighboring tissues or which have metastasized to other organs such as the lymph nodes, liver, lungs, bone, or brain.

The HPV-infected cells may be present in any epithelial cell layer of the body, including, for example, epithelial cells of the anus and perianal region, the genitals and perigenital region, the cervix, orofacial regions, and other areas afflicted by HPV infection.

Various embodiments of the invention are directed to the treatment of HPV-infected cells in a subject. In many embodiments, the subject is a human subject in need of treatment, for example, a patient having an HPV infection. In other embodiments, the subject may comprise an animal subject, such as a veterinary subject or test animal. In some contexts, the scope of the invention extends to the treatment of isolated, cultured, and xenografted cells. The several methods of the invention are directed to inhibition of E5 expression. As used herein, E5 will refer to any E5 open reading frame present in an HPV virus. In an HPV-infected cell, it will be understood that reference to E5 will encompass the specific E5 sequences present in the particular HPV strain causing the infection. E5 nucleic acid and protein sequences are known in the art and may be readily retrieved from publicly available genetic databases, such as GenBank. As dictated by context, reference to "E5" herein may encompass any of a viral E5 open reading frame sequence (whether present episomally or integrated into the host genome); a transcribed E5 mRNA sequence, or a translated E5 protein. Accordingly, as used herein, reference to "inhibiting E5" or "inhibition of E5" will encompass any of: the disruption of E5 genomic sequences; a reduction in the transcription of E5 mRNA's; a reduction in the translation of E5 proteins; and a reduction in the biological activity of E5 proteins. A "reduction" is determined relative to levels of transcription, translation, or activity in like, untreated cells.

Various embodiments of the invention encompass the administration of a treatment. In many implementations, the treatment will comprise the administration of an agent. It will be understood that the agent is to be administered in a pharmaceutically effective amount. Where reference is made to the administration of compositions, it is understood that compositions may be formulated with any number of excipients, carriers, delivery vehicles, etc., as known in the art. Unless specified, it will be understood that the methods described herein encompass various delivery methods, including systemic delivery as well as localized delivery to region of the infected cells, for example by oral administration, intravenous administration, intraperitoneal injection, or topical administration.

Certain embodiments set forth below are directed to denoted nucleic acid sequences (e.g., SEQ ID NO: 1 and SEQ ID NO: 2). It will be understood that reference to a specific sequence encompasses variants of the denoted sequence, including subsequences thereof, deletion or insertion mutants thereof, variants comprising one or more nucleic acid substitutions, and sequences comprising substitutions with non-natural nucleic acids and/or nucleic acid analogs.

Biological Effects of E5 Inhibition in HPV-infected Cells. The HPV virus expresses the E2 protein in its latent phase, with E2 maintaining the HPV genome in its episomal plasmid form. The HPV virus also expresses oncogenes E6 and E7. The E6 and E7 genes play a key role in the progression of HPV infection to cancer. The HPV E6 protein binds and inactivates the tumor suppressor protein p53, leading to chromosomal instability. The impairment of DNA repair mechanisms by E6 leads to the accumulation of genetic mutations and may be the driving force behind development of high-grade intraepithelial lesions and their progression to cancer.

Meanwhile, the HPV E7 protein disrupts the tumor-suppressing functions of the retinoblastoma protein. The activities of E6 and E7 thus unfavorably modify the cell cycle, creating an increased risk for carcinogenesis.

Meanwhile, the HPV E5 protein is also known to be expressed during HPV infection. There have been inconsistent reports regarding E5's expression with respect to E6 and E7 activity in HPV-associated squamous cell cancers, and the role of E5 is poorly understood in HPV-associated carcinogenesis and cancer. The majority of studies report that E5 has weak transforming capabilities, mostly enhancing E6- and E7-driven carcinogenesis. One of the established roles of E5 is its ability to enhance epidermal growth factor (EGFR) ligand-receptor activation. Several studies show that E5 increases EGFR ligand receptor activation primarily through preventing degradation of EGFR, but some studies indicate that E6 and/or E7 are also involved in EGFR dysregulation. In summary, the current understanding of E5's role in HPV infection and cancer is not well defined. E5 is generally believed to augment the oncogenic effects of E6 and E7.

The inventors of the present disclosure have advantageously determined that E5 inhibition can effectively halt neoplastic progression in HPV infected cells. Utilizing a novel in vitro model of anal cancer pathogenesis, the inventors of the present disclosure have demonstrated for the first time that E5 inhibition shuts down the activity of oncogenes E6 and E7, reduces expression of the E2 gene, restores normal EGFR activity, and reduces the invasiveness and proliferation rates of HPV-infected cells.

Therefore, the discoveries disclosed herein provide the art with a novel means of treating HPV infection, inhibiting HPV carcinogenesis, and treating HPV-associated cancers.

In a first aspect, the scope of the invention is directed to methods and agents for the inhibition of E5 in HPV-infected cells. In one implementation, the method of the invention comprises a method of inducing one or more biological effects in an HPV-infected cell by the inhibition of E5.

In another embodiment, the scope of the invention comprises a method of treating HPV- associated processes in HPV-infected cells. The HPV-associated process may include any pathological process or the manifestation of any pathological condition caused or augmented by HPV infection. The HPV-associated process may include, for example: HPV infection; E6 expression; E7 expression; E2 expression; progression of HPV-infected cells in carcinogenesis; progression of HPV-infected cells to intraepithelial lesions; progression of low-grade intraepithelial lesions to to high-grade intraepithelial lesions; manifestation of intraepithelial lesions, proliferation of HPV-infected cells, invasion of HPV-infected cells into non-infected tissues, manifestation of HPV-associated cancer, progression of HPV-associated cancer (e.g. progression of tumors of a cancer stage to higher order stages); growth of HPV-associated tumors; and the spread of HPV-associated tumors.

In another aspect, the scope of the invention is directed to methods and agents for inhibiting the oncogenes E6 and/or E7 in HPV-infected cells. E6 and E7, as referred to herein, mean the E6 and E7 open reading frames of an HPV virus, respectively. "Inhibiting" E6 encompasses disrupting expression of the E6 gene, reducing transcription of the E6 gene, reducing translation of the E6 gene, or reducing the biological activity of the E6 protein. In one embodiment, inhibition of Inhibition of E6 may be assessed as a reduction of E6 transcription or translation, or by an increase in the activity of p53. Likewise, "inhibiting" E7 encompasses disrupting expression of the E7 gene, reducing transcription of the E7 gene, reducing translation of the E7 gene, or reducing the biological activity of the E7 protein. Inhibition of E7 may be assessed as a reduction of E7 transcription or translation, or by an increase in the activity of retinoblastoma protein. "Reduction" and "increase" are assessed relative to like, untreated cells.

In another aspect, the scope of the invention is directed to methods and agents for the inhibition of the E2 gene in HPV-infected cells. Inhibition of E2 encompasses disrupting expression of the E2 gene, reducing transcription of the E2 gene, reducing translation of the E2 gene, or reducing the biological activity of the E2 protein. In another aspect, the scope of the invention is directed to methods and agents for the inhibition of EGFR activity, and/or the restoration of normal EGFR activity in HPV-infected cells. The inventors of the present disclosure have determined that EGFR overexpression in neoplastic HPV-infected cells is driven by E5, and that inhibition of E5 can reduce EGFR expression and activity, for example, restoring normal levels EGFR activity (e.g., levels found in uninfected cells). Inhibition of EGFR, as used herein, refers to any reduction of EGFR expression or activity, and may encompass a reduction in EGFR expression, a reduction in EGFR abundance on the cell surface, or a reduction in EGFR-mediated processes.

In another aspect, the scope of the invention is directed to methods and agents for reducing the proliferative capacity of HPV-infected cells. The inventors of the present disclosure have demonstrated a role for E5-driven proliferation of HPV-infected cells, likely mediated by EGFR activity. The scope of the invention encompasses any reduction in the proliferative rate of HPV-infected cells by the inhibition of E5.

In another aspect, the scope of the invention is directed to methods and agents for reducing the invasive capacity of HPV-infected cells. The inventors of the present disclosure have demonstrated that the inhibition of E5 in HPV-infected cells reduces the severity and incidence of the invasive phenotype. Reducing the invasive capacity of HPV-infected cells encompasses any method or treatment that decreases the invasive phenotype, for example, as measured by infiltration rates, transwell assay, and other measures of the invasive phenotype. Such invasive phenotype may be observed in high-grade intraepithelial cells, precancerous cells, and cancers cells derived from HPV-infected cells. The scope of the invention further encompasses inhibiting the spread of HPV-infected cells to non-infected tissues.

In another aspect, the scope of the invention is directed to methods and agents for inhibiting carcinogenesis in HPV-infected cells. Carcinogenesis, as used herein, refers to the progression of HPV-infected cells from infected but histologically normal cells to low grade intraepithelial lesion cells to high grade intraepithelial lesions to precancerous cells to cancer cells. Inhibition of carcinogenesis encompasses any slowing or halting this progression.

In another aspect, the scope of the invention is directed to methods of eliminating or reducing the severity of intraepithelial lesions. Eliminating or reducing the severity of intraepithelial lesions encompasses any treatment which reduces the size of intraepithelial lesions, eliminates intraepithelial lesions, prevents the manifestation of intraepithelial lesions, inhibits the progression of HPV-infected cells to intraepithelial lesions, inhibits the progression of low-grade intraepithelial lesions to high-grade intraepithelial lesions, inhibits the invasion of non-infected tissues by intraepithelial lesions, reduces the degree of dysplasia observed in intraepithelial lesions, or which otherwise alleviates the dysplastic phenotype in HPV-infected cells.

In another aspect, the scope of the invention is directed to methods of treating HPV- associated cancers. Treatment of an HPV-associated cancer encompasses treatment of any cancer derived from HPV-infected cells, wherein the cancer cell expresses E5, for example anal cancer or cervical cancer. Treatment of such cancers encompasses any treatment which eliminates such cancers, prevents the manifestation of such cancers, reduces the proliferation rate of the cancer, reduces the invasiveness of the cancer, reduces tumor size, inhibits the progression of the cancer to higher order stages, reduces the spread of the cancer, or reduces any other measures of cancer severity.

In one aspect, the scope of the invention is directed to agents which inhibit E5. In one implementation, the scope of the invention encompasses an E5-inhibiting agent for use as a medicament. In one implementation, the scope of the invention encompasses the use of an E5 inhibiting agent for the manufacture of a medicament. In one implementation, the scope of the invention encompasses the use of an E5 inhibiting agent for the manufacture of a medicament for the treatment of HPV-associated processes in HPV-infected cells. In one implementation, the scope of the invention encompasses an E5 -inhibiting agent for use in the treatment of HPV- associated processes in HPV-infected cells.

In the various methods set forth above, the HPV-infected cell may comprise any HPV- infected epithelial cell or epithelial-derived cell. For example, in various embodiments, the cell selected from a cell of the anus, the cervix, and the oropharyngeal tissues. The HPV-infected cells may comprise cells infected with any high-risk HPV serotype, for example, HPV 16, HPV 18, or HPV 31. Methods and Associated Compositions of Matter. The various methods disclosed herein are directed to inhibiting E5 in HPV-infected cells. In most implementations, the various methods encompass the administration of an E5-inhibiting agent which causes or enhances the inhibition of E5 in the treated cells.

In some implementations, the agent is a small molecule. In some implementations, the agent is a protein, for example, an antibody or intrabody. In some implementations, the agent comprises a nucleic acid, such as an aptamer directed to the E5 protein or a regulatory nucleic acid construct which reduces the expression of E5. In some implementations, the agent comprises a composition which is a hybrid of two or more of the foregoing composition types.

In one aspect, the scope of the invention encompasses methods and associated agents which accomplish the silencing of E5. Silencing of E5, as used herein, refers to any process which inhibits the expression of E5. Exemplary methods include methods which harness host RNA interference (RNAi) mechanisms for the degradation of a target gene's transcripts.

In a first embodiment, the method of the invention comprises the knockdown of E5 by small interfering RNA (siRNA) constructs. Native RNAi is initiated when long double stranded RNAs are processed by into 21-24 nucleotide double stranded siRNAs with 2-base 3' overhangs. The resulting siRNA duplexes are then incorporated into the effector complex known as RNA- induced silencing complex (RISC), where the antisense or guide strand of the siRNA guides RISC to recognize and cleave complementary target mRNA sequences. As known in the art, exogenously applied siRNA' s which are complementary to a target gene may be administered to a cell and will be taken up by native RISC complexes, leading to the degradation of the target gene's transcripts.

Accordingly, in one embodiment, the invention comprises a method of inhibiting E5 in an HPV-infected cell by the administration of siRNA 's complementary to an E5 transcript sequence expressed in the HPV-infected cell. In a related embodiment, the invention is a composition of matter comprising an siRNA construct targeted to an HPV E5 transcript. In one embodiment, the siRNA targets an HPV 16 E5. In another embodiment, the siRNA comprises a guide (antisense) strand comprising SEQ ID NO: 2 (UUA UCC ACA AUA GUA AUA CCA TT) and a sense strand comprising SEQ ID NO: 1 (UGG UAU UAC UAU UGU GGA UAA TT).

In a related method, the invention encompasses a method of inhibiting E5 in an HPV- infected cell, comprising the administration of a gene construct coding for a short hairpin RNA (shRNA), wherein the shRNA is complementary to an E5 transcript of the HPV infecting the cell. When the gene construct is transcribed, the resulting transcript is eventually processed by dicer and loaded into RISC. The antisense (guide) strand directs RISC to bind and degrade the targeted E5 transcript. In one embodiment, the method comprises targeting HPV 16 E5. In one embodiment, the method comprises administration of an shRNA wherein the shRNA comprises a guide sequence comprising SEQ ID NO: 2 and a sense sequence of SEQ ID NO: 1, for example, comprising the complementary (non-overhang) portions of the sequences of SEQ ID NO: l and SEQ ID NO: 2, for example nucleotides 1-21 of SEQ ID NO: 1 and nucleotides 1-21 of SEQ ID NO: 2. The scope of the invention further encompasses compositions of matter, including shRNA sequences directed to E5, and expression vectors coding for such shRNA's. In one embodiment, the shRNA's comprises SEQ ID NO: 1 and SEQ ID NO: 2 or nucleotides 1-21 of SEQ ID NO: 1 and nucleotides 1-21 of SEQ ID NO: 2.

In another embodiment, the E5 inhibition method comprises the administration of a gene construct coding for a clustered regularly interspaced short palindromic repeats (CRISPR)— CRISPR-associated nuclease system (CRISPR/Cas) system. In this system, a gene construct coding for the Cas9 DNA endonuclease and coding for a guide RNA (gRNA) can be administered to the HPV-infected cells. The gRNA is complementary to a coding region of the target E5 gene. Once the gRNA and Cas9 are expressed in cells, the gRNA will direct Cas9 to bind to the target sequence and introduce a double-strand break. Repair of the break by native DNA repair mechanisms will result in indel mutations, eliminating E5 gene expression. The scope of the invention encompasses CRISPR/Cas guide RNA's wherein such guide RNA's are complementary to an HPV E5 gene. In one embodiment, the HPV E5 gene is an HPV 16 E5 gene. In one embodiment, the guide RNA comprises SEQ ID NO: 1 and/or SEQ ID NO: 2, or a subsequence thereof, for example, nucleotides 1-21 of SEQ ID NO: 1 and/or nucleotides 1-21 of SEQ ID NO: 2. In another embodiment, the method of inhibiting E5 comprises the administration a zinc finger nucleases (ZFNs) directed to target E5 sequences.

In another embodiment, the method of inhibiting E5, comprises the administration of a transcription activator-like effector nucleases (TALENs) directed to E5 sequences.

In another embodiment, the method of inhibiting E5 comprises the administration of an agent which selectively targets E5 protein for degradation by ubiquitin-dependent proteolysis. The native ubiquitin-dependent proteolysis system employs the El ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin-protein ligase. As known in the art, elements of the E3 ligase can be engineered to redirect the substrate specificity of the

degradation machinery to a selected protein target. Accordingly, in one embodiment, the method of the invention comprises the administration of an engineered E3, or gene construct coding therefore, to HPV-infected cells wherein the engineered E3 promotes the degradation of E5.

Agent Delivery. It will be understood that the agents employed in the practice of the invention, for example, siRNA's, ZFN's, TALEN's, and CRISPR/Cas constructs, will be utilized with appropriate delivery systems and/or in appropriate delivery vehicles or pharmaceutically acceptable carriers. Therapeutic constructs must access the target cells, enter the target cells, and function in the target cells, while evading degradation by nucleases, proteases, immune cells, etc. and while avoiding immunogenic responses. Expression vectors, such as AAV vectors and others known in the art, must access the target cells and successfully express their payload gene constructs. Such delivery challenges may be addressed by methods and compositions known in the art of drug and/or gene delivery.

In one implementation, nucleic acid constructs coding for therapeutic constructs (e.g. coding for siRNA's, ZNF's, TALENs, etc.), may be delivered as incorporated with viral vectors, cationic liposomes, cationic polymers, cell penetrating peptides, or any other gene delivery method known in the art. Likewise, therapeutic species themselves (e.g. ZNF proteins, siRNA's) may be delivered to target cells in association with materials that facilitate their delivery to and action within the target cells. For example, siRNA's targeting E5 may be incorporated into cyclodextrin polymer-based nanoparticles, adamantane-PEG nanoparticles, adamantane-PEG- transferrin constructs, or cationic or ionizable liposomes. In one embodiment, the therapeutic constructs of the invention are delivered systemically (e.g. by intravenous administration). However, in many embodiments, topical delivery of E5- targeting agents is utilized, as HPV-infected lesions and HPV-associated lesions, and cancers are typically present at or near epithelial surfaces which are often readily accessible to topical agents.

Depending on the location of the target tissues, topical administration of E5-targeting therapeutic constructs may have to overcome physical barriers present in the target region. For example, tight junctions present in epithelial layers may form a significant barrier for targeting of HPV-infected cells. In the skin, the stratum corneum also forms a significant barrier. Topical administration may be improved by the use of pharmaceutically acceptable carriers known in the art which aid in the penetration of the epithelial layers. For example, transdermal siRNA's delivery has been demonstrated using cationic elastic liposomes, or peptides such as Tat, AT1002, or SPACE peptide. For the treatment of epithelial surfaces, mucus may present a barrier to the penetration of therapeutic agents. In such case, therapeutic agents may be combined with materials that enhance transmucosal delivery. For example, effective

transmucosal siRNA delivery has been demonstrated with chitosan and hydrogel carriers.

Physical treatment of the target region may enhance the effectiveness of topical delivery. Exemplary methods of enhancing topical delivery through physical means include iontophoresis, microneedle injection, and ultrasound treatment. Hydrodynamic injection may likewise be used.

E5 -targeting therapeutic constructs may be emulsified, suspended, or otherwise admixed with creams, salves, adhesives, and other materials that enable stable topical delivery. E5- targeting therapeutic constructs may be incorporated into adhesive patches or other bodies which promote localized and/or extended release of the therapeutic moiety at the target site.

Appropriate delivery methods and vehicles may be selected depending on the particular location of the cells or tissues to be treated. For example, agent-eluting patches may be used in the topical administration of E5-targeting therapeutic agents where accessible. Agent eluting-suppositories may be used in the treatment of HPV-associated neoplasms of the anus. Agent-eluting rings or suppositories may be used in the treatment of vaginal or cervical targets. HPV Epithelial Anal Cell Line. In one aspect, the scope of the invention encompasses an anal epithelial cell line having an integrated HPV genome. In one embodiment, the anal epithelial cell line is derived by transfecting a normal parental HPV-negative primary anal epithelial cell with a full-length HPV 16 genome, or a truncated genome which retains HPV pathological functions. In one embodiment, the parental anal cell line is the AKp cell line. In one embodiment, the HPV genome is an HPV 16 genome. In one embodiment, the HPV genome is the HPV 16 W12 genome. In alternative embodiments, the HPV genome is an HPV 18 genome or a genome from another high-risk HPV genotype. In one embodiment, the HPV anal epithelial cell line of the invention is the AKC2 cell line.

The HPV-infected anal epithelial cells of the invention may comprise isolated cells, cell cultures, and xenografted cells. In one embodiment, the scope of the invention encompasses the use of HPV-infected anal epithelial cells to screen for or test the efficacy of anti-HPV treatments.

Examples. Here a novel in vitro model of anal cancer pathogenesis is provided using the first HPV 16-transformed anal epithelial cell line, known as AKC2 cells. AKC2 cells express all three HPV 16 oncogenes (E5, E6 and E7) from an integrated HPV- 16 genome. Here it is demonstrated that targeting E5 with E5-specific siRNAs in AKC2 cells leads to 99% reduction of all three oncogenes as well the E2 replication gene. In addition, rescue of E6 and E7 expression confirmed that E5 alone drives EGFR overexpression/activation and EGFR-mediated invasion of AKC2 cells. Coupled with detection of E5 expression in HPV16-positive anal squamous cell carcinomas, it is concluded that E5 plays a significant role in anal cancer progression and provides a therapeutic target for treatment of HPV 16-ssociated anal HSIL or cancer.

HPV- 16 positive anal SCC contain transcripts for E5, E6 and E7. There is a lack of studies that characterize viral oncogene expression in HPV 16-positive anal biopsies and there have been inconsistent reports with detection of E5 expression alongside E6 and E7 in HPV-associated squamous cell cancers (SCCs). Therefore, it was sought to determine if all three viral oncogenes were expressed in HPV 16-positve anal cancer biopsies.

To investigate HPV 16 oncogene expression total RNA was extracted from formalin-fixed sections of four HPV 16 positive anal SCCs. HPV-specific genotyping of anal biopsies was performed as known in the art. RNA from a HPV- 18 and HPV-33-positive anal SCC was also extracted, which was included as negative controls for HPV 16-specific expression. HPV 16 E5, E6 and E7 expression was detected alongside two internal controls (i.e. PGKl and RPLPO) using qPCR Sybr green method. HPV 16 E5, E6 and E7 expression were detected in all four HPV 16- positive anal SCCs. It was thus demonstrated that all three viral oncogenes including E5 are expressed in anal cancers and all contribute to HPV 16 associated anal carcinogenesis.

Establishment and characterization of a novel HPV- 16 positive (E5, E6 and E7) anal epithelial cell line. In addition, HPV 16-associated anal pathogenesis has been largely understudied due to a lack of permanent HPV-positive anal cell lines that phenotypically model anal cancer progression. Here, the AKC2 cell line was established, one of the first known permanent HPV 16-positive (E5, E6 and E7)-positive anal cell lines, by transfecting normal HPV-negative primary anal epithelial cells with the pEF3-99 plasmid that contains the full-length 8KB HPV 16 W12 genome. AKC2 was passaged over 400 times and can be cultured in standard media (DMEM/10%FBS) without supplemented growth factors or an irradiated feeder layer.

At current passage, AKC2 cells exhibit a typical monolayer epithelial morphology with predominantly cobblestone-like colonies and are positive for pan-keratin staining. Similar to the HPV 16-positive anal SCC biopsies, expression in the AKC2 cells of HPV 16 E5, E6 and E7 as well E2 was detected by Sybr green qPCR methods. Both the HPV 16-positive CaSki cell line (cervical) and the SCC90 cell line (oral) were used as positive controls for HPV 16 oncogene expression and the HPV-negative oral cancer line SCC1 was used as a negative control. E7 protein was also detected in AKC2 cells and p53 expression levels were reduced in AKC2 cells relative to the AKp anal parental cells, verifying E6 protein expression. LI expression was not detected in AKC2 by qPCR or dot blot analysis, consistent with maintenance of the HPV- 16 genome in AKC2 cells in an integrated form without an intact LI gene.

AKC2 cells have a poorly differentiated and invasive phenotvpe in three-dimensional raft culture. Since AKC2 cells expressed E5, E6 and E7, similar to the anal cancer biopsies, these cells were used to model HPV 16-associated anal carcinogenesis. Three-dimensional organotypic raft culture of HPV-positive cell lines is an in vitro system for studying HPV- associated pathogenesis within an intact epithelium. HPV 16-positive cancer lines can model invasive carcinoma when propagated in raft culture. Previous studies have also shown that raft cultures of episomal HPV-positive cell lines can mimic different lesion grades (i.e. LSIL and HSIL) when stained with established HPV biomarkers such as MCM and pi 6. MCM, a surrogate marker of E7 expression and pi 6, also associated with E7 overexpression, are typically expressed throughout the entire epithelium of HPV 16-positive high-grade lesions and cancers.

Here it was shown that the AKp HPV-negative parental cells produced only a few layers in raft culture, had a normal differentiation pattern with late terminal differentiation in the upper layers indicated by positive K10/13 staining. The AKp raft cultures were negative for both MCM and pi 6 staining. In contrast, HPV 16-positive AKC2 raft cultures resembled a high-grade lesion with invasive phenotype based on H&E, MCM and pi 6 staining. H&E analysis of AKC2 raft cultures showed that AKC2 cells formed poorly differentiated layers throughout the entire raft culture where a subset of cells invaded through a collagen dermal equivalent. AKC2 raft cultures were also negative for keratin 10/13, consistent with a poorly differentiated phenotype. In addition, MCM and p 16 expression were detected throughout the entire AKC2 raft culture and in invading cells.

Since invasion can be indicative of transformation and tumorigenic potential, the anchorage independent growth of AKC2 cells in soft agar was measured. For comparison, similar assays using CaSki cells were performed. It was found that AKC2 cells produced colonies in soft agar that were comparable to CaSki cells, both in size and in numbers. These data demonstrate that AKC2 could phenotypically represent an early transition phase from precancer to cancer based on its poorly differentiated and invasive phenotype. This provided a unique opportunity to investigate the contribution of HPV 16 oncogene expression towards the invasive phenotype.

EGFR overexpression promotes EGF/EGFR-induced invasion of AKC2 cells. Recent studies have shown that EGFR can be overexpressed in HPV-associated anal cancer biopsies. It is also well established that overexpression of EGFR promotes invasion of a wide range of cell types. There have been inconsistent reports with detection of E5 expression alongside E6 and E7 in HPV-associated squamous cell cancers (SCCs). It was therefore concluded that there is a high likelihood that all three viral oncogenes including E5 are expressed in anal cancers and all contribute to HPV 16 associated anal carcinogenesis. E5, E6 and E7 expression have all been linked to EGFR overexpression.

It was determined whether total EGFR (T-EGFR) and the active phosphorylated form of EGFR (p-EGFR) were overexpressed in AKC2 cells and if EGFR overexpression contributed to their invasive phenotype. The AKp anal parental cells and early (plO), mid (pi 05) and late (p 450) passage AKC2 cells were starved for 24-hours and then stimulated with lOng/mL EGF for 15 minutes and 30 minutes without the addition of serum or additional growth factors. AKC2 cells of early passage (plO), which similar to AKp cells were maintained in low-calcium full- supplemented growth factor media. These were compared with AKC2 cells that were maintained in high calcium non-growth factor supplemented media to show that changes in EGFR expression were due to HPV 16 and not due to differences in cell culture media.

Unstimulated AKC2 cells of all three passages contained approximately five (in AKC2 plO and pl05) to seven (in AKC2 p450) times more T-EGFR compared with AKp cells. It was also found that p-EGFR levels decreased in AKp cells from 15 to 30 minutes EGF treatment whereas p-EGFR increased from 15 to 30 minutes in all three AKC2 cell passages.

To see if EGF alone (i.e. without the addition of serum or other growth factors) could induce invasion of AKC2 cells, the invasive potential of the AKp and AKC2 cells was measured using an in vitro collagen invasion assay. Equal numbers of previously serum-starved cells AKp or AKC2 cells were seeded onto collagen-coated membranes and invasion levels were measured 24 hours post-seeding. It was found that AKC2 cells had a higher basal level of invasion compared with AKp cells. There was a substantial increase in the number of invasive AKC2 cells compared with the AKp following exposure to lOng/mL EGF whereas a significant increase in AKp invasion was not detected.

Finally, to show that overexpression of p-EGFR played a role in AKC2 invasion AKC2 cells were treated with the EGFR tyrosine kinase inhibitor, gefitinib. Treatment of AKC2 cells with luM of gefitinib for 24 or 48 hours did not decrease cell viability and reduced p-EGFR levels of AKC2 close to basal levels. To determine if treatment with gefitinib also decreased EGF/EGFR-induced invasion, AKC2 cells were pre-treated with luM gefitinib for 24 hours during serum starvation and then seeded in equal numbers of non-treated and treated cells on both control non-coated inserts and in vitro collagen coated inserts. Non-coated inserts were used to control for changes in proliferation or other effects during seeding that might confound the collagen invasion results. Using EGF as a chemoattractant, percent invasion was calculated comparing the number of cells that invaded the collagen-coated insert after 24 hours of incubation with the number of cells that grew on the control membrane. Treatment of AKC2 cells with gefitinib decreased the percent invasion close to basal levels consistent with a role for total EGFR/p-EGFR over-expression in AKC2 invasion.

Targeting E5 by transfecting E5-specifc siRNAs leads to global knockdown of HPV 16 genes and normalizes EGFR expression levels. Having established that EGFR overexpression played a significant role in AKC2 invasion, next it was determined which HPV 16 oncogenes contributed to EGFR overexpression. To examine the individual roles of E5, E6 and E7 in EGFR

overexpression in AKC2 cells, AKC2 cells were transfected with individual siRNAs to knock down each viral oncogene. Consistent with previous studies, initial attempts to knock down E6 and E7 individually were unsuccessful due to the fact that HPV 16 contains a polycistronic genome. Therefore, a cocktail of E6/E7-specific siRNAs was used to ensure a high knockdown efficiency of both oncogenes. In addition, individual cultures were transfected with equal concentrations of E5-specific siRNAs as well as a scrambled control siRNA. Total RNA was harvested from each well 72 hours post-transfection and HPV 16 E5, E6 and E7 expression was measured using Sybr Green qPCR methods.

Transfection with E6/E7 siRNAs resulted in an 83% decrease in both E6 and E7 expression (Figure 2B and Figure 2C). In addition, transfection of E6/E7-specific siRNAs also resulted in a 65% decrease in E5 expression (Figure 2A) and an 84% decrease in E2 expression (Figure 2D), relative to controls. Transfection with E5-specific siRNAs also resulted in a global knockdown of E2, E5, E6 and E7 expression and with a knockdown efficiency of 99% in all four genes relative to controls (Figure 2A, 2B, 2C and 2D). A larger increase in p53 protein was detected (i.e. a direct measurement of E6 protein loss of function), a larger decrease in E7 protein, and lower proliferation rates in cells transfected with E5-specific siRNAs, compared with cells transfected with control or E6/E7-specific siRNAs (Figure 2E). Transfecting both CaSki cells and SCC-90 cells with E5-specific siRNAs resulted in global knockdown of HPV 16. To determine if reduction in E6/E7 or E5 expression resulted in a decrease in total EGFR and p-EGFR, AKC2 cells were transfected with either control, E6/E7 or E5 siRNAs. Lysates were harvested 72 hours post-transfection following 24 hours of serum starvation and then 30 minutes of induction with lOng/mL EGF. A decrease in total EGFR or p-EGFR levels was not detected in EGF-induced cultures transfected with E6/E7 siRNAs relative to controls. However, transfection with E5 siRNAs reduced total EGFR and p-EGFR expression to control levels.

Overall, it was found that targeting the HPV 16 genome with E5-specific siRNAs led to near complete global knockdown of HPV 16 and EGFR normalization thus demonstrating the therapeutic benefit of targeting E5 in anal cancers.

E5 but not E6/E7 plays a role in p-EGFR over-expression and EGF/EGFR-induced invasion. Since E5 siRNAs led to reduced expression of E5, E6 and E7, next it was sought to determine their relative importance in EGFR overexpression and downstream EGF/EGFR-induced invasion. E6/E7 rescue experiments were performed in AKC2 cells, while also knocking down E5 expression. AKC2 cells were first transfected with either a control promoter-less plasmid (pGL3-Basic) or the pB-actin E6/E7 expression plasmid. Twenty- four hours later, they were transfected with either a scrambled control or E5 -specific siRNA. It should be noted that E5 siRNAs only targeted endogenous E6/E7 but not exogenous E6/E7 from the overexpression plasmid. Total RNA was harvested 72 hours post-plasmid transfection and 48 hours post-siRNA transfection, and E5, E6 and E7 expression was measured by Sybr Green qPCR methods.

Transfection with the E6/E7 plasmid followed by control siRNA (E6/E7/C) increased both E6 and E7 expression relative to control (C/C) without affecting E5 expression (Figure 3A).

Transfection of E6/E7 plasmid followed by E5 siRNA was able to rescue E6/E7 expression above control (C/C) and E5 knockdown cultures (C/E5), whereas E5 expression was reduced to levels comparable to E5-knockdown cultures (C/E5).

To next determine if E5 played a role in EGFR overexpression/p-EGFR activation during E6/E7 rescue, the identical rescue transfection protocol was performed and AKC2 lysates were harvested 72 hours post-plasmid transfection. AKC2 cells were transfected with 1000 ng and 2000 ng of E6/E7 expression plasmid in order to sufficiently visualize the increase in E7 protein during rescue. E6 protein was not detected due to lack of a suitable E6 antibody. However, it was found that E7 protein levels incrementally increased above control levels in AKC2 cultures transfected with E6/E7 expression plasmid (Figure 3B). In addition, it was found that overexpression of E6/E7 did not restore p-EGFR levels in cultures with reduced E5 levels (E67/E5) to control levels (Figure 3B), consistent with a role for E5 alone in p-EGFR

overexpression. Due to the fact that the E6/E7 rescue experiment could only be performed using a staggered (plasmid/siRNA) transfection leaving a 48-hour E5-knockdown phase, a slight decrease in total EGFR levels was observed in both E5-knockdown (C E5) and E6/E7 rescue cultures (E67/E5) (Figure 3C). However, reductions in activated p-EGFR, were easily observed as early as 48 hours post-E5 siRNA transfection. A more pronounced reduction in total EGFR protein was seen at 72 hours post-siRNA transfection, likely due to an extended protein degradation time.

To determine if E5 alone played a role in EGF/EGFR-induced invasion, an identical rescue transfection experiment was performed using the invasion model and percent invasion into collagen was measured over 24 hours. Overexpression of E6/E7 in cultures with reduced E5 expression (E6/7/E5) did not increase invasion above control E5 knockdown cultures (C/E5) (Figure 1) consistent with significant role for E5 in HPV 16-associated anal carcinogenesis through modulation of EGFR overexpression.

All patents, patent applications, and publications cited in this specification are herein incorporated by reference to the same extent as if each independent patent application or publication was specifically and individually indicated to be incorporated by reference. The disclosed embodiments are presented for purposes of illustration and not limitation. While the invention has been described with reference to the described embodiments thereof, it will be appreciated by those of skill in the art that modifications can be made to the structure and elements of the invention without departing from the spirit and scope of the invention as a whole.

Claims

Claims. What is claimed is:

Claim 1. A method of inducing a biological effect in HPV-infected cells, comprising administering to the cells an agent which inhibits HPV E5.

Claim 2. The method of Claim 1 , wherein the biological effect is selected from the group consisting of: inhibiting E6; inhibiting E7, knocking down E6 expression, knocking down E7 expression, knocking down E2 expression, inhibiting EGFR activity; inhibiting carcinogenesis in HPV-associated cells; inhibiting intraepithelial lesion formation; reducing intraepithelial lesion size; inhibiting the progression of HPV-infected cells to intraepithelial lesions; inhibiting the progression of low grade intraepithelial lesions to high-grade intraepithelial lesions inhibiting HPV- infected cell proliferation rates; inhibiting the invasion of non-infected tissues by HPV- infected cells; and inhibiting HPV-associated cancer.

Claim 3. The method of Claim 1 , wherein the cells are selected from the group consisting of: cells present in an HPV-associated intraepithelial lesion; precancerous HPV-infected cells; and HPV-associated cancer cells.

Claim 4. The method of Claim 1 , wherein the cells are epithelial or epithelial-derived cells of the anus, perianal region, genitals, perigenital region, cervix, or orofacial region.

Claim 5. The method of Claim 1 , wherein the cells are infected with a high-risk HPV genotype.

Claim 6. The method of Claim 5, wherein the high-risk HPV genotype is HPV 16 or HPV 18.

Claim 7. The method of Claim 1 , wherein the inhibition of E5 comprises one or more effects selected from the group consisting of: disruption of E5 gene sequences; a reduction in E5 transcription; a reduction in E5 translation; and a reduction in the activity of the E5 protein.

Claim 8. The method of Claim 1 , wherein the agent is selected from the group consisting of: a small molecule inhibitor of E5; an antibody or intrabody directed to E5; an ap tamer directed to the E5 protein; an siRNA construct which targets one or more E5 transcripts; an shRNA which targets one or more E5 transcripts; a CRISPR/Cas9 construct directed to the E5 gene sequence; a TALEN directed to the E5 gene sequence; a ZNF directed to the E5 gene sequence; and a ubiquitin ligase E3 protein directed to the E5 protein.

Claim 9. The method of Claim 8, wherein the agent is an siRNA construct comprising a guide strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 1.

Claim 10. The method of Claim 1, wherein the therapeutic agent is administered topically.

Claim 11. The method of Claim 10, wherein the therapeutic agent is administered in a cream, salve, patch, ring, or suppository.

Claim 12. A composition for the inhibition of E5 in HPV-infected cells, comprising a composition of matter selected from the group consisting of: a small molecule inhibitor of E5; an antibody or intrabody directed to E5; an aptamer directed to the E5 protein; an siRNA construct which targets one or more E5 transcripts; an shRNA which targets one or more E5 transcripts; a CRISPR/Cas9 construct directed to the E5 gene sequence; a TALEN directed to the E5 gene sequence; a ZNF directed to the E5 gene sequence; and a ubiquitin ligase E3 protein directed to the E5 protein.

Claim 13. The composition of Claim 12, wherein the agent is an siRNA construct comprising a guide strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 1.

Claim 14. The E5-inhibiting agent of Claim 12 for the use as a medicament.

Claim 15. The E5-inhibiting agent of Claim 14, wherein the medicament is for the treatment of an HPV-associated process.

Claim 16. The E5-inhibiting agent of Claim 15, wherein the HPV-associated process is selected from the group consisting of HPV infection; E6 expression; E7 expression; E2 expression; progression of HPV-infected cells in carcinogenesis; progression of HPV-infected cells to intraepithelial lesions; progression of low-grade intraepithelial lesions to to high-grade intraepithelial lesions; manifestation of intraepithelial lesions, proliferation of HPV-infected cells, invasion of HPV-infected cells into non-infected tissues, manifestation of HPV-associated cancer, progression of HPV-associated cancer; growth of HPV-associated tumors; and the spread of HPV- associated tumors.

Claim 17. A method of manufacturing a medicament, wherein the medicament is manufactured using the E5 -inhibiting agent of Claim 12.

Claim 18. The method of Claim 17, wherein the medicament is for the treatment of an HPV-associated process in HPV-infected cells.

Claim 19. The method of Claim 18, wherein the HPV-associated process is selected from the group consisting of HPV infection; E6 expression; E7 expression; E2 expression; progression of HPV-infected cells in carcinogenesis; progression of HPV-infected cells to intraepithelial lesions; progression of low-grade intraepithelial lesions to to high-grade intraepithelial lesions; manifestation of intraepithelial lesions, proliferation of HPV-infected cells, invasion of HPV-infected cells into non-infected tissues, manifestation of HPV-associated cancer, progression of HPV-associated cancer; growth of HPV-associated tumors; and the spread of HPV- associated tumors.

Claim 20. The E5-inhibiting agent of Claim 12 for use in the treatment of an HPV-associated process in HPV-infected cells.

Claim 21. The E5-inhibiting agent of Claim 20, wherein the HPV-associated process is selected from the group consisting of HPV infection; E6 expression; E7 expression; E2 expression; progression of HPV-infected cells in carcinogenesis; progression of HPV-infected cells to intraepithelial lesions; progression of low-grade intraepithelial lesions to to high-grade intraepithelial lesions; manifestation of intraepithelial lesions, proliferation of HPV-infected cells, invasion of HPV-infected cells into non-infected tissues, manifestation of HPV-associated cancer, progression of HPV-associated cancer; growth of HPV-associated tumors; and the spread of HPV- associated tumors.

Claim 22. A cell, comprising a primary anal epithelial cell transfected with an HPV genome.

Claim 23. The cell of Claim 12, wherein the primary anal epithelial cell is an AKp cell transfected with an HPV 16 genome.