WO2017214449A1 - Inhibition de e5 dans des cellules infectées par le virus du papillome humain (vph) - Google Patents

Inhibition de e5 dans des cellules infectées par le virus du papillome humain (vph) Download PDF

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WO2017214449A1
WO2017214449A1 PCT/US2017/036630 US2017036630W WO2017214449A1 WO 2017214449 A1 WO2017214449 A1 WO 2017214449A1 US 2017036630 W US2017036630 W US 2017036630W WO 2017214449 A1 WO2017214449 A1 WO 2017214449A1
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hpv
cells
expression
infected cells
infected
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Joel Palefsky
Erin WECHSLER
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • HPV human papillomavirus
  • the human papillomavirus 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.
  • 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.
  • 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.
  • 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.
  • the scope of the invention is directed to novel agents for the inhibition of E5 activity in HPV-infected cells.
  • 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 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 (-).
  • the E6/E7 expression plasmid was applied in 1000 ng and 2000 ng treatments.
  • the E6/E7 expression plasmid was applied at 1000 ng.
  • HPV or the “HPV virus” will refer to any HPV virus genotype, wherein the HPV viral genotype expresses an E5 gene.
  • the HPV virus comprises a "high-risk” HPV variant, as known in the art.
  • the HPV virus comprises HPV 16.
  • the HPV virus comprises HPV 18.
  • the HPV virus comprises an HPV virus of a genotype selected from 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.
  • 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 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 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.
  • 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.
  • the subject is a human subject in need of treatment, for example, a patient having an HPV infection.
  • the subject may comprise an animal subject, such as a veterinary subject or test animal.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions may be formulated with any number of excipients, carriers, delivery vehicles, etc., as known in the art.
  • 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.
  • 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.
  • 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.
  • 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.
  • HPV E5 protein is also known to be expressed during HPV infection.
  • 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.
  • EGFR epidermal growth factor
  • 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.
  • 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.
  • the scope of the invention is directed to methods and agents for the inhibition of E5 in HPV-infected cells.
  • 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.
  • 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.
  • HPV infection HPV infection
  • E6 expression E7 expression
  • 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 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the scope of the invention is directed to methods and agents for inhibiting carcinogenesis in HPV-infected cells.
  • Carcinogenesis 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.
  • 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.
  • 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.
  • the scope of the invention is directed to agents which inhibit E5.
  • the scope of the invention encompasses an E5-inhibiting agent for use as a medicament.
  • the scope of the invention encompasses the use of an E5 inhibiting agent for the manufacture of a medicament.
  • 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.
  • the scope of the invention encompasses an E5 -inhibiting agent for use in the treatment of HPV- associated processes in HPV-infected cells.
  • the HPV-infected cell may comprise any HPV- infected epithelial cell or epithelial-derived cell.
  • 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.
  • the agent is a small molecule.
  • the agent is a protein, for example, an antibody or intrabody.
  • 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.
  • the agent comprises a composition which is a hybrid of two or more of the foregoing composition types.
  • 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.
  • RNAi RNA interference
  • 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.
  • RISC RNA- induced silencing complex
  • 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.
  • 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.
  • the invention is a composition of matter comprising an siRNA construct targeted to an HPV E5 transcript.
  • the siRNA targets an HPV 16 E5.
  • 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).
  • 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.
  • shRNA short hairpin RNA
  • 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.
  • the method comprises targeting HPV 16 E5.
  • 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.
  • 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.
  • 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.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • CRISPR/Cas CRISPR-associated nuclease system
  • gRNA guide RNA
  • 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.
  • the scope of the invention encompasses CRISPR/Cas guide RNA's wherein such guide RNA's are complementary to an HPV E5 gene.
  • the HPV E5 gene is an HPV 16 E5 gene.
  • 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.
  • the method of inhibiting E5 comprises the administration a zinc finger nucleases (ZFNs) directed to target E5 sequences.
  • ZFNs zinc finger nucleases
  • the method of inhibiting E5 comprises the administration of a transcription activator-like effector nucleases (TALENs) directed to E5 sequences.
  • TALENs transcription activator-like effector nucleases
  • 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.
  • elements of the E3 ligase can be engineered to redirect the substrate specificity of the
  • 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.
  • nucleic acid constructs coding for therapeutic constructs 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.
  • 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.
  • siRNA's targeting E5 may be incorporated into cyclodextrin polymer-based nanoparticles, adamantane-PEG nanoparticles, adamantane-PEG- transferrin constructs, or cationic or ionizable liposomes.
  • the therapeutic constructs of the invention are delivered systemically (e.g. by intravenous administration).
  • 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.
  • topical administration of E5-targeting therapeutic constructs may have to overcome physical barriers present in the target region.
  • tight junctions present in epithelial layers may form a significant barrier for targeting of HPV-infected cells.
  • 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.
  • transdermal siRNA's delivery has been demonstrated using cationic elastic liposomes, or peptides such as Tat, AT1002, or SPACE peptide.
  • 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.
  • 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.
  • 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.
  • the parental anal cell line is the AKp cell line.
  • the HPV genome is an HPV 16 genome.
  • the HPV genome is the HPV 16 W12 genome.
  • the HPV genome is an HPV 18 genome or a genome from another high-risk HPV genotype.
  • the HPV anal epithelial cell line of the invention is the AKC2 cell line.
  • HPV-infected anal epithelial cells of the invention may comprise isolated cells, cell cultures, and xenografted cells.
  • 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.
  • AKC2 cells express all three HPV 16 oncogenes (E5, E6 and E7) from an integrated HPV- 16 genome.
  • E5 HPV 16 oncogenes
  • E6 E7
  • E5-specific siRNAs E5-specific siRNAs
  • rescue of E6 and E7 expression confirmed that E5 alone drives EGFR overexpression/activation and EGFR-mediated invasion of AKC2 cells.
  • 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.
  • SCCs HPV-associated squamous cell cancers
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • MCM and p 16 expression were detected throughout the entire AKC2 raft culture and in invading cells.
  • AKC2 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.
  • T-EGFR total EGFR
  • p-EGFR active phosphorylated form of EGFR
  • p-EGFR active phosphorylated form of EGFR
  • 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.
  • AKp and AKC2 cells were 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • pGL3-Basic control promoter-less plasmid
  • E6/E7 expression plasmid Twenty- four hours later, they were transfected with either a scrambled control or E5 -specific siRNA.
  • 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.
  • 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).

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Abstract

La présente invention concerne l'inhibition du gène E5 dans les cellules infectées par le VPH. Les inventeurs de la présente invention ont déterminé que l'inhibition de E5 offre de nombreux avantages thérapeutiques dans le traitement d'infections par le VPH, de lésions et de cancers associés au VPH. En particulier, l'inhibition de E5 permet l'inactivation des oncogènes E6 et E7, qui sont responsables de divers aspects de la pathologie du VPH. Les méthodes et les compositions associées selon l'invention peuvent être dirigées vers des tissus infectés par le VPH de l'anus, du col de l'utérus et d'autres sites. L'invention concerne en outre une nouvelle lignée de cellules épithéliales anales permettant la recherche du VPH.
PCT/US2017/036630 2016-06-09 2017-06-08 Inhibition de e5 dans des cellules infectées par le virus du papillome humain (vph) WO2017214449A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004002416A2 (fr) * 2002-06-26 2004-01-08 The Penn State Research Foundation Procedes et substances pour traiter des infections au virus du papillome humain
US20040235171A1 (en) * 2001-07-17 2004-11-25 Milner Ann Josephine Silencing of gene expression by sirna
US20140256799A1 (en) * 2013-02-28 2014-09-11 City Of Hope REPLICATION CAPABLE rAAV VECTORS ENCODING INHIBITORY siRNA AND METHODS OF THEIR USE

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040235171A1 (en) * 2001-07-17 2004-11-25 Milner Ann Josephine Silencing of gene expression by sirna
WO2004002416A2 (fr) * 2002-06-26 2004-01-08 The Penn State Research Foundation Procedes et substances pour traiter des infections au virus du papillome humain
US20140256799A1 (en) * 2013-02-28 2014-09-11 City Of Hope REPLICATION CAPABLE rAAV VECTORS ENCODING INHIBITORY siRNA AND METHODS OF THEIR USE

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Title
OH, J.-M. ET AL.: "Human papillomavirus E5 protein induces expression of the EP 4 subtype of prostaglandin E2 receptor in cyclic AMP response element-dependent pathways in cervical cancer cells", CARCINOGENESIS, vol. 30, no. 1, 2009, pages 141 - 149, XP055449054 *
RAMAMOORTHY, S. ET AL.: "Detection of multiple human papillomavirus genotypes in anal carcinoma", INFECTIOUS AGENTS AND CANCER, vol. 5, no. 17, 2010, pages 1 - 5, XP021081870 *

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