life sciences

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GRANT PROPOSAL/WORK PLAN: EXAMPLE (Please note: this example proposal is longer than the page limit you have been provided with. However, it should give you an indication of the format/structure of a proposal. Please follow the guidelines you have been provided with).

1. Project title (not exceeding 120 characters)*

Novel RNA aptamer molecules selected against Human Papillomavirus 16 E6 with the potential to induce apoptosis in cervical cancer cells.

(117 characters)

2. Abstract of proposed research project (not exceeding 750 characters)*

High-risk Human Papillomaviruses (HR HPVs) such as HPV16 infect epithelial cells including those of the genital tract and cause many cancers such as cervical cancer; due to the expression of E6 and E7 oncoproteins that attenuate tumour suppressor proteins p53 and pRb respectively. Cervical cancer affects millions of women and current treatment and diagnosis is limited. RNA aptamers are small, non-coding RNAs that bind proteins with high affinity and specificity. This research aims to screen RNA aptamers specific to E6 to determine if expression allows cervical cancer cells to undergo apoptosis. Aptamer sequences will be cloned and transfected into cells prior to analysis of apoptosis using propidium iodide and annexin V staining. This work may indicate the usefulness of RNA aptamers in the study of diseases and HR HPVs and improve diagnosis and treatment of HPV16 cancers.

(748 characters)

3. Description of research project (in no more than 6 sides of A4 describe the proposed research project providing a case for support. Font size Ariel 11pt or higher, 2 cm margins)

The case for support must describe:

a) A summary of the aims and objectives of the project and the importance of the research being proposed (UP TO 1 A4 page)

b) Relevant background to the project (UP TO 2 A4 pages) c) A Detailed plan of research; this should list each specific objective identified in (a) above

with a description of how it will be achieved experimentally

d) Justification of any resources requested e) Impact of the proposed research; this should identify who will benefit from the research,

how they will benefit and the methods or activities through which you will communicate and engage with those who benefit.

f) Any ethical considerations; this should identify any ethical permissions you may require (d-f UP to 1 A4 page)

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a) A summary of the aims and objectives of the project and the importance of the research being proposed (UP TO 1 A4 page)

Aims/objectives 1. To examine a range of candidate RNA aptamer molecules previously identified through in vitro

selection to HPV16 E6

2. To transfect DNA sequences encoding the chosen aptamers into a number of cell types including cells of; a more easily transfected cell line such as 293T cells, a cervical cancer cell line such as SiHa cells and a control cell line such as HaCaT cells

3. To determine if any of the RNA aptamer molecules expressed are capable of inducing apoptosis within cervical cancer cells i.e. the consequences of RNA aptamer(s) binding to E6

4. To help determine the course of future work with the aptamers

Importance of the research HPV16 infection causes roughly 50% of cervical cancers (Braaten and Laufer, 2008; Clifford et al., 2003), a disease that affects millions of women worldwide. Approximately 493,000 cases of cervical cancer are diagnosed and 233,000 deaths reported annually across the globe as a result of the disease (Parkin et al., 2005). Research in this field has the potential to significantly impact the lives of many women, along with the lives of individuals affected by other cancers resulting from HPV16 infection, such as those of the penis, vagina and head and neck carcinomas (Boulet et al., 2007; Gillison, 2004). This is because the aptamers under investigation may have applications as therapeutic and/or diagnostic agents in the future; in the treatment and/or identification of HPV16 infection that can cause cancers within a number of tissues.

This research may aid and encourage the development of aptamers to bind various proteins implicated in other diseases that lack suitable therapies such as Alzheimer’s disease, proteins of additional HPV strains, or, HPV16 viral proteins that are not as well characterised, such as oncoprotein E5. This could lead to developments in treating other diseases, other HPV infections (such as HPV18 also implicated in cervical cancer) and would allow the study of effects on HPV16 cervical cancer proliferation in the absence of a viral oncoprotein that is poorly understood, in comparison to E6 and E7 oncoproteins.

Additionally, scientists in the field of cervical cancer research would be able to build on the findings if they were promising, for instance, by studying the interactions of aptamers of interest in vivo. Future research may also involve expression of E6 and E7 aptamers of interest within the same cervical cancer cells, with the hope of inhibiting the actions of both major viral oncoproteins.

This research is therefore important to further knowledge in the field of cervical cancer research through the development of potential techniques for diagnosis and treatment of HPV16 infection. Findings would also apply to a number of different cancers resulting from HPV16 infection and techniques may be utilised for the diagnosis and treatment of other HPV infections, along with other forms of cancer and additional diseases with suitable proteins to target.

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b) Relevant background to the project (UP TO 2 A4 pages) Human Papillomaviruses Papillomaviruses were first described in 1907 (Ciuffo, 1907) and are a family of small, non- enveloped, double-stranded DNA viruses that are highly species specific. More than 150 types of human papillomaviruses (HPVs) exist, spanning five different genera (de Villiers et al., 2004). The alpha genus consists of around 40 different HPVs that infect the anogenital tract (Byg et al., 2012). These HPV infections represent the most common sexually transmitted viral infection (Markowitz et al., 2007) and can result in a range of abnormalities; from genital warts to cancers. HPVs of the alpha genus are classified as low-risk (LR) or high-risk (HR) according to the tendency towards malignant transformation of infected cells (Lorincz et al., 1992). LR types include HPV6 and 11 and HR types include HPV16, 18, 31, 33 and 45; these HPVs are associated with a number of epithelial cancers including those of the vagina, anus, vulva, penis, oropharynx, head, neck and mainly, the cervix (Blanchette and Branton, 2008; Boulet et al., 2007). Cervical cancers account for 12% of female malignancies corresponding to the second most prevalent cancer among women worldwide (Parkin et al., 2005), with around 500,000 cases diagnosed each year, one third of which are fatal (de Villiers, 1989). 99% of cases are the result of HR HPV infection (Boulet et al., 2007) and HPV16 is the most prevalent, responsible for over 50% of cervical cancers (Braaten and Laufer, 2008; Clifford et al., 2003). However, in most cases, HPV infections are transient and cleared by the immune system within 12-18 months (Richardson et al., 2003). It is thought that the major risk factor for malignancy is the persistent infection of HR HPVs (Markowitz et al., 2007), as this permits the accumulation of genetic abnormalities that allow transformation of cells and cancer development. For instance, HPV infections that lead to cervical cancer usually persist for decades.

Viral genome and life cycle The genomes of HPVs are circular, roughly 8kb in size, encode eight genes and consist of three main regions; an upstream long control region (LCR), an early region and a late region (see figure 1) (Burk et al., 2009; Ganguly and Parihar, 2009).

HPVs infect epithelial cells or keratinocytes within the basal layer of epithelium in the genital tract following wounding, where the virus synchronises its life cycle to the changes in differentiation that occur in host cells (Bodily and Laimins, 2010). Following entry and uncoating the viral genome is replicated episomally within the cell nucleus to roughly 100 copies (Moody and Laimins, 2010).

As host keratinocytes differentiate and leave the basal layer episomes are copied to 1000s within the cells and viruses are assembled (Bodily and Laimins, 2010). These virions are then released into the surrounding tissue as cells undergo desquamation.

Oncoproteins The cellular interactions of E5, E6 and E7 proteins promote tumourigenesis in persistent HPV16 infection. Each of these oncoproteins interferes with a number of signalling pathways. The mechanisms underlying the action of E5 are not fully elucidated, yet it is thought to modulate cell signalling to enhance that of growth factors such as epidermal growth factor (Bouvard et al., 1994) and prevent Fas- and TRAIL-mediated apoptosis (Kabsch and Alonso, 2002) to enhance immortalisation of cells by E6 and E7 and result in transformation and anchorage-dependent growth. The interactions of E6 and E7 are much more understood, and both are known to interact

Figure 1. The HPV16 circular genome. Transcription of six early genes; E1, E2, E4, E5, E6 and E7, and two late genes; L1 and L2 is regulated by the LCR (Fehrmann and Laimins, 2003). E1 and E2 are involved in initiation and regulation of viral replication, E4 and E5 are thought to be involved in late viral functions, E5, E6 and E7 are oncoproteins that ensure the maintenance of the viral genome, while, L1 and L2 proteins have structural roles, forming the icosahedral capsid of the virus (Bodily and Laimins, 2010).

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with a range of cellular proteins. Primarily, E6 binds with E6AP to the tumour suppressor protein p53 to result in its ubiquitination and proteosomal degradation (Scheffner et al., 1993; Scheffner et al., 1990). E6 is also known to interact with proteins involved in regulating transcription and DNA replication, apoptosis and immune evasion, epithelial differentiation, cell-cell adhesion, polarity and proliferation and DNA repair processes (Garnett and Duerksen-Hughes, 2006). The major interaction of E7 is binding to the tumour suppressor protein pRb to inhibit binding of E2F to pRb and allow cell cycle progression (Dyson et al., 1989). E7 binding to pRb can also result in ubiquitin- mediated degradation of the tumour suppressor protein (Fehrmann and Laimins, 2003). Additional E7 interactions take place with various proteins involved in cell cycle control, transcription regulation and other cellular functions (Garnett and Duerksen-Hughes, 2006).

E6 and E7 viral proteins therefore attenuate two major tumour suppressor proteins. Consequently, apoptosis and DNA repair pathways are repressed in infected cells, allowing survival of cells with damaged DNA as p53 is absent, while, replication and proliferation of cells is unregulated without the pRb protein. Ultimately, cells undergo proliferation, immortalisation and malignant transformation following expression and deregulation of E6 and E7 as viral genes integrate into the host genome (Boulet et al., 2007) and mutations accumulate. These oncoproteins have synergistic functions and both are required to induce cervical cancer formation in vivo (Howie et al., 2009). Treatment Although there are methods available to detect abnormalities that may lead to cervical cancer and prevent cervical cancer; the Pap smear and recently developed prophylactic vaccines to HPVs 16 and 18 respectively; treatment is currently limited to removal of lesions, through surgery or cryotherapy (D’Abramo and Archambault, 2011). There is therefore a need to develop novel therapies and targeting oncoproteins E5, E6 and E7 with small molecules such as RNA aptamers has great potential.

RNA aptamers RNA was first found to be involved in catalysis in the 1980s (Kruger et al., 1982). Subsequently, in 1990, three separate groups of researchers developed methods to select and isolate short RNA and DNA molecules in vitro, to bind specific targets (Ellington and Szostak, 1990; Robertson and Joyce, 1990; Tuerk and Gold, 1990). The RNA molecules produced are short, single-stranded non- coding RNA aptamers that today are frequently created by automated SELEX (systemic evolution of ligands by exponential enrichment), based on those methods. SELEX involves rounds (usually 5-20) of identification and amplification of nucleotides that bind with high affinity to a chosen target (usually a protein) from libraries of up to 1015unique sequences (Blank and Blind, 2005; Burnett and Rossi, 2012). The synthetic aptamer molecules produced are usually 12-30 nucleotides in length and fold into precise, stable three dimensional structures that possess high affinity and specificity to the selected target and bind through complementary shape interactions (Bouchard et al., 2010; Ireson and Kelland, 2006).

Frequently referred to as ‘chemical antibodies’, aptamers are potent inhibitors of protein function, non-toxic and non-immunogenic, efficient and cost effective, can be selected against any type of molecule/molecular complex and easily produced and manipulated within the laboratory; for example, to increase biostability, modified bases can be added to prevent nuclease degradation (Blank and Blind, 2005). Aptamers are therefore advantageous over traditional antibodies.

Aptamers are highly versatile and can be applied to many fields such as therapeutics. The first RNA aptamer approved for clinical use was Macugen in 2004, to treat age-related macular degeneration (Bunka et al., 2010; Gragoudas et al., 2004). This success has fuelled the development of many more therapeutic aptamers, and a number are currently in clinical trials.

RNA aptamers have previously been selected against HPV16 E7, with promising results including loss of E7 from and apoptosis of cervical cancer cells (Nicol et al., 2011; Nicol et al., in press). E6 will now be targeted in the same way in the hope of blocking the protein. This would prevent numerous cellular interactions including binding to and degradation of p53, and ultimately, cervical cancer progression. Blocking E6 would also allow induction of apoptosis in HPV16 infected cells.

c) A Detailed plan of research; this should list each specific objective identified in (a) above with a description of how it will be achieved experimentally

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1) To examine a range of candidate RNA aptamer molecules previously identified through in vitro selection to HPV16 E6

The aptamers under investigation will be chosen from a pool of aptamers previously identified via SELEX to the HPV16 E6 oncoprotein and 10-12 will be studied in human cells.

2) To transfect DNA sequences encoding the chosen aptamers into a number of cell types including cells of; a more easily transfected cell line such as 293T cells, a cervical cancer cell line such as SiHa cells and a control cell line such as HaCaT cells

293T cells are derived from a human embryonic kidney cell line and are widely utilised and relatively easy to culture and transfect. Use of this cell line would allow assessment of transfection efficiency and could indicate the half lives of aptamers prior to use of other mammalian cells. These cells would also act as a control for cell type, helping to ensure any observations following aptamer expression are specific to cervical cancer cells. SiHa cells are from a human cervical carcinoma cell line resulting from HPV16 infection, possessing chromosomal insertion of HPV16 sequences and therefore expressing E6 and E7 oncoproteins, while HaCaT cells are from an immortal human keratinocyte cell line and represent normal epithelial cells (i.e. found in the cervix and infected by HPV16 in cervical cancer) expressing p53 and pRb. As these cells do not express E6 or E7, they can act as a control to confirm any findings are specific to cervical cancer cells and do not apply to normal epithelial cells found in the cervix.

These mammalian cell lines will be cultured appropriately; in media such as Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with components including foetal calf serum (FCS) and antibiotics such as penicillin and streptomycin. This would provide optimum growth conditions for the cells and prevent contamination of cultures.

As cells are being cultured, cloning of RNA aptamer cDNA sequences will take place into mammalian expression vectors such as pSUPER (Oligoengine). This plasmid vector possesses a polymerase III HI-RNA gene promoter, unique BgIII and HindIII/Xhol restriction sites, ampicillin resistance and is available with puromycin or neomycin resistance genes (see figure 2). pSUPER therefore initiates transcription of mammalian genes and increases the ease of cloning and selection of cells possessing the recombinant vector produced. Other suitable vectors include pSIREN and pSILENCER. pSUPER would however be preferred and has been utilised to successfully introduce shRNA (Chen et al., 2013; Zhang et al., 2012), siRNA (Niu et al., 2012) and microRNAs (Li et al., 2012) into mammalian cells.

Prior to insertion into the vector, DNA sequences encoding RNA aptamers may be amplified using PCR, and restriction sites incorporated flanking the DNA, such as BglII and HindIII sites recommended for use with pSUPER. Both vector and aptamer sequences can then be cut with these restriction enzymes to yield complementary, cohesive ends, and ligated to form a

Figure 2. pSUPER vector possessing neomycin resistance (Oligoengine, 2004).

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recombinant vector. Transformation of competent bacterial cells such as Escherichia coli DH5α cells with recombinant vector would allow determination of transformation efficiency in comparison to positive (vector without insert) and negative (water only) controls and selection of transformed bacterial cells containing the desired recombinant plasmid vector with the antibiotic ampicillin. Presence of the insert can then be confirmed using PCR and sequencing.

Once the presence of insert has been confirmed in the vector, transfection of mammalian cells using a method such as electroporation or lipofection can take place, including transfection of a positive control vector and a negative control/mock transfection. Stable cell selection will follow, using neomycin or puromycin depending on the variant of pSUPER utilised, with two non- transfected controls; one that is subjected to antibiotic selection (when all these cells are killed selection is complete) and one that is not (to control for cell viability).

Following transfection of aptamers into the cells, expression levels may be determined using a method such as quantitative RT-PCR, with nucleic acid primers complementary to regions within the aptamer sequences.

3) To determine if any of the aptamer molecules expressed are capable of inducing apoptosis within cervical cancer cells i.e. the consequences of RNA aptamer(s) binding to E6 If one or more of the aptamers selected for study is able to bind E6 and prevent its binding to and degradation of p53, p53 would then be able to induce apoptosis in the cervical cancer cells. In order to determine whether or not cells are undergoing apoptosis, a number of assays could be carried out. Cells may be stained with propidium iodide (PI), an intercalating agent and fluorescent molecule that stains DNA within cells and is generally only taken up by cells that are dying. Analysis of stained cells can be achieved using flow cytometry. PI staining indicates cell death, however, alone is unable to distinguish between apoptotic and necrotic cells. Staining with annexin V and PI prior to flow cytometric analysis would help to confirm cells undergoing apoptosis, as annexin V detects the externalisation of phosphatidylserine protein.

Transfection of sequences encoding a control aptamer such as staurosporine, or a randomly synthesised aptamer into cells and study of apoptosis would ensure that findings were not the result of the vector used, but of the aptamer(s).

4) To help determine the course of future work with the aptamers Future work would then involve studying the binding activities of any RNA aptamers of interest identified from the apoptosis assays to E6 in vitro and resulting affects on p53 binding and E6 levels, along with the nature of any interactions and results on cell proliferation. Decreases of both the amount of p53 bound to E6 and levels of E6 protein would be expected in the presence of successful aptamers.

Aptamer binding assays may be undertaken with a fusion protein such as GST-E6 following expression and purification in cells such as BL21 (DE3) cells. RNA immunoprecipitation experiments would allow study of aptamer and E6 interactions, co-immunoprecipitation studies would help determine the E6 and p53 interactions before and after transfection and affinity chromatography such as GST pull-down assays with immunoblotting would help characterise any interactions. Immunoblotting could also determine E6 levels and cervical cancer cell proliferation assays may be carried out using a system such as xCELLigence (Roche, UK).

In addition, interactions should be studied within other cervical carcinoma cell lines such as; C33A cells that do not express any HPV DNA sequences (control), CaSki cells that express HPV16 DNA sequences (experimental) and HeLa cells that express HPV18 DNA sequences (control).

d) Justification of any resources requested The proposed investigation will be undertaken at the University of Leeds, in collaboration with Dr Nicola Stonehouse who possesses both wide ranging knowledge and experience of working with

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RNA aptamers and various viruses including HPV16 (Nicol et al., 2011; Nicol et al., in press; Wetherill et al., 2012).

The resources needed include; cells lines, culture medium, vector, restriction enzymes, and reagents required to express RNA aptamers in cervical cancer cells and study the ability of cells to induce apoptosis. All of the equipment required is accessible within the laboratory.

e) Impact of the proposed research; this should identify who will benefit from the research, how they will benefit and the methods or activities through which you will communicate and engage with those who benefit.

The major beneficiary of this research is women worldwide currently diagnosed with cervical cancer resulting from HPV16 infection, along with individuals with other forms of cancer associated with HPV16 infection, and those infected in the future, as these individuals may be able to receive more effective diagnosis and treatment. Other scientists within the field of cervical cancer research resulting from HPV16 and other high-risk HPV types such as HPV18 may benefit from the research if results are promising through the methods utilised and a boost in morale. These researchers may also want to further their own work by building on the findings, while scientists in the fields of other forms of cancer and disease research may benefit, again, from the approach utilised such as the use of aptamers.

Findings could therefore have a greater impact than cervical cancer alone as the techniques could be applied to other cancers associated with HPV16 and other HPV infections, and potentially, additional forms of cancer and diseases that may or may not involve viral infection, as aptamers can be applied to block almost any target. Thus, this research has the potential to impact women worldwide, the general public and the scientific community.

Any significant developments will be communicated to the scientific community by attending conferences both in the United Kingdom and abroad to present and discuss findings, publishing in peer-reviewed journals and also in an electronic journal to allow easy access to the data. Findings could be communicated to patients by making the information available on educational websites such as MEDLINEplus. Finally, the general public may be notified through scientific media such as magazines, and public media via press releases together with the University of Leeds press offices.

f) Any ethical considerations; this should identify any ethical permissions you may require (d-f UP to 1 A4 page)

The planned experimental procedure involves the use of various cell lines, cloning of sequences encoding RNA aptamers, transfection into cells and analysis via apoptosis assays. Ethical permissions are therefore not required.

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