Exploring the Rules and Therapeutic Promise of Exosome-Mediated

Intercellular CommunicationMichelle Marcus

PhD Proposal

May 2013

SummaryIntercellular communication is essential to proper functioning of many biological processes.

Recent evidence has now established that one form of intercellular communication occurs via the secretion of extracellular lipid vesicles. In particular, much interest has been devoted to exosomes, 30-200 nm diameter vesicles of endosomal origin. Exosomes are capable of transferring proteins and RNAs from the cytoplasm of an exosome producing cell to the cytoplasm of a recipient cell where these RNAs and proteins have an effect on recipient cell behavior. Proteins are packaged into exosomes via incorporation of endosomal membrane proteins into the exosome membrane. The loading of cytosolic protein and RNA into exosomes is not well understood, and in the case of RNA appears to occur by a mixture of mass-action driven loading of highly abundant cytoplasmic species as well as specific targeting of particular RNAs into exosomes. Because exosomes play a wide variety of roles in communication during health and disease, understanding how this communication is controlled is of great interest and may also be valuable for developing exosome-based therapeutics. I aim to develop a quantitative framework for understanding RNA loading into exosomes.

The ability of exosomes to deliver RNA and protein, protected from degradation and immune recognition in vivo, makes them potentially attractive delivery vehicles for therapeutic RNA and protein. Exosomes can also be modified to display targeting peptides – allowing for enhanced uptake by specific target recipient cells. Exosomes have also been validated as immunologically compatible in humans. I aim to develop exosomes for delivery of therapeutic RNAi to prostate cancer cells to inhibit proliferation and induce apoptosis. Furthermore, based on native ability of exosomes to be taken up preferentially by macrophages in vivo, I also aim to develop exosomes for delivery of RNA capable of altering the behavior of anti-inflammatory macrophages associated with prostate cancer progression.

To achieve these aims, exosomes will be isolated from cells via centrifugation and visualized via transmission electron microscopy (TEM). I will use standard protein and RNA detection techniques (Western blotting, qRT-PCR) to study the contents of exosomes and the impact of exosome-mediated RNA delivery on the protein and RNA content of recipient cells.

Specific Aims1. Develop a quantitative framework to elucidate the rules governing RNA incorporation into exosomes. Rationale: The degree to which RNAs are packaged into exosomes based on their expression level versus an active sorting mechanism is unknown, and a quantitative framework capable of separately assessing these two effects is needed. Objectives. (i) To target RNAs to exosomes, I will engineer RNAs to display a bacteriophage coat protein binding motif. I will also engineer the RNA-binding coat protein to traffic to the lumen of exosomes via fusion to the lumenal side of an exosome-associated protein, Lamp2b1. The coat protein will bind the engineered RNA and target it to the lumen of exosomes. I predict that this strategy will enable targeted incorporation of RNA into exosomes. (ii) Using this system, I will map out how degree of targeting (expression level of Lamp2b-coat protein) and RNA expression level affect exosomal RNA levels. To describe and understand the factors impacting RNA packaging into exosomes, I will develop a computational model of RNA loading into exosomes. I predict that RNA incorporation into exosomes is a saturable process and that increasing either RNA expression or degree of targeting will increase RNA level in exosomes, up to a maximum. This maximum incorporation is expected to be higher for sRNAs than for mRNAs, because exosomes are enriched in small RNAs 2. 2. Engineer exosomes to deliver therapeutic RNA to prostate cancer cells. Rationale: Exosomes are known to deliver functional miRNA and mRNA to recipient cells2,3, and have been used to deliver siRNA to neural cells1 and miRNA to breast cancer cells4 in mice. In both cases, exosomal display of a targeting ligand was required to achieve specificity for target cells in vivo1,4. Targeted exosomes may be widely applicable for delivering therapeutic RNA. The impact of specific targeting ligand/cell surface receptor pairs on efficiency of exosome uptake by recipient cells and the efficacy of different types of RNA cargo are unknown. Objectives. (i) I will develop exosomes for uptake by prostate cancer cells by displaying on the exosome surface a previously characterized peptide (IPLVVPL5) that binds hepsin, a surface receptor overexpressed in prostate cancer. I will determine how hepsin expression level affects exosome uptake by and delivery of exosomal contents to recipient prostate cancer cells. I expect enhanced binding of exosomes to surface receptors will increase exosome uptake via receptor-mediated endocytosis. (ii) I will use exosomes to package and deliver shRNA or shRNA-miR for knockdown of mTOR in prostate cancer cells, because mTOR knockdown inhibits the growth of prostate cancer cells and xenograft tumors6,7, and induces apoptosis of prostate cancer cells in a mouse prostate cancer model8. I predict that each form of RNAi will have varying degrees of efficacy in recipient cells. I will determine which form is most effective at mTOR knockdown and inhibition of proliferation/induction of apoptosis in prostate cancer cells. Aim 3. Use exosomes to direct macrophage polarization and inhibit cancer development. Rationale. Exosomes injected into mice are cleared by the reticuloendothelial system (RES)1,4, and exosomes are enriched in phosphatidylserine, which is known to induce macrophage uptake9. Thus, exosomes may be efficient vehicles for influencing macrophage (and tumor associated macrophage) behavior. Tumor associated macrophages are important in prostate cancer resistance to therapy, so altering macrophage phenotype may be able to inhibit prostate cancer proliferation and migration. Objectives. (i) I will use exosomes to deliver RNA that alters the polarization of macrophages from M2 (anti-inflammatory, associated with tumor progression) to M1 (pro-inflammatory). I will use exosomes to package RNAi against Bcl-3, a known NF-κB inhibitor induced by IL-10 signaling10. (ii) I will determine if exosome modulation of macrophage polarization is sufficient to reverse cancer-cell mediated polarization to M2 in a prostate cancer – macrophage coculture model, and determine the effect on cancer cell proliferation and migration.

Background and SignificanceExosomes

Discussion of exosomes and exosomal cargo is similar to discussion in a review article by Michelle Marcus and Joshua Leonard11.

Secreted extracellular vesicles are emerging as important new features of the expanding landscape of intercellular communication. Extracellular vesicles were first observed by Trams et al. in 1981 as particles that were shed from neoplastic cell lines and carried membrane-bound enzymes 12. The authors noted that secreted extracellular vesicles could be taken up by recipient cells and presciently predicted that extracellular vesicles represented a physiological method for transferring information between cells, likening extracellular vesicles to liposomes used to package and deliver therapeutic molecules. A subset of extracellular vesicles in the 30-200 nanometer diameter range, known as exosomes, were subsequently found to play a number of important roles in intercellular signaling, including shedding of obsolete proteins during reticulocyte maturation 13, presentation of antigens to T cells 14, activation of B and T cell proliferation 15, and induction of immune rejection of murine tumors, presumably by delivery or presentation of tumor antigens to the immune system16.

Exosomes have been discovered in the supernatants of a wide variety of cells in culture, and are present in all human bodily fluids, suggesting that they can be produced by any type of cell 17. Exosomes are the extracellular equivalent of intraluminal vesicles (ILVs). ILVs are formed when the limiting membrane of an endosome buds inward, forming an internal vesicle (Figure A1). Endosomes containing ILVs are known as multivesicular endosomes or multivesicular bodies (MVBs). Although some MVBs traffic along the endosomal pathway towards the lysosome, other MVBs back fuse with the plasma membrane, releasing their contents, including ILVs, into the extracellular space. ILVs that have been released into the extracellular space are known as exosomes. Exosomes are therefore topologically equivalent to cells, encapsulating cellular cytoplasmic contents in the exosomal lumen and presenting membrane protein domains on the exosomal exterior that correspond to domains presented at the cell surface and in the lumen of the endoplasmic reticulum 18. Exosomal cargo

Exosomes are enriched in particular cellular proteins, including the tetraspanins CD63, CD9, and CD81, ESCRT related proteins Alix and Tsg101, MHCI, and heat shock proteins 17. Exosomes derived from immune cells are also enriched in MHCII and costimulatory molecules 19. Sorting of proteins into exosomes is somewhat understood. The protein content of exosomes is thought to be composed mainly of endosomal and recycled plasma membrane proteins20,21, however highly expressed cytoplasmic proteins are also found to be packaged by exosomes22, likely due to the high probability of incorporating proteins present at high concentrations in the cytoplasm into the lumen of the exosome. Proteins present in exosomes play a functional role in recipient cells. Exosomal proteins play a role in adhesion to and uptake by recipient cells 23, participate in transcriptional regulation 24, and bind recipient cell receptors to modulate signaling pathways 25,26.

Exosomes also package cellular RNAs and protect them from degradation 2, such that exosomes isolated from serum contain RNA that represents a subset of the RNA present in the exosome-producing cells 27. Exosomal mRNA is expressed in recipient cells 2, and exosomal microRNA (miRNA) inhibits gene expression in recipient cells 3. Interestingly, the RNA profile found in exosomes is distinct from that of their cells of origin2,3,28 and differs between closely related cells, for example between mature and immature bone marrow derived dendritic cells3, and between healthy and cancerous prostate cells29. This suggests that RNA loading into exosomes is at least in part a regulated process. To date, one such mechanism of regulated RNA loading into exosomes has been discovered. Bolukbasi et al. identified two features – a miR-1289 binding site and a core

“CTGCC” motif – that are enriched in the 3’UTRs of a large proportion of mRNAs found in glioblastoma- and melanoma-derived exosomes. Replacing the 3’ UTR of eGFP with a 25 nucleotide sequence containing the miR-1289 binding site and the “CTGCC” motif added was sufficient to increase eGFP mRNA incorporation into HEK293T exosomes by 2-fold compared to untagged eGFP mRNA. Overexpression of miR-1289 further increased the incorporation of the construct 6-fold compared to the untagged eGFP mRNA. This increase in exosome targeting depended on the presence of the miR-1289 binding site, as mutation of this site abrogated enrichment of the mRNA in exosomes 30. This is the first discovery of a mechanism for loading of RNA into exosomes, yet the picture remains incomplete. For example, it is still unclear how miR-1289 directs mRNA loading into exosomes. Furthermore, the incorporation of miRNAs and other non-coding RNAs into exosomes is not explained by this mechanism. Additionally, while loading of RNA into exosomes is in part a regulated process, the observation that overexpression of RNA is sufficient to incorporate miRNA 4,29,31, chemically modified 3’ benzen-pyridine miRNA 32, shRNA 31, and mRNA 22,33 into exosomes suggests that to some extent RNA loading into exosomes is a mass-action driven process, in which highly concentrated cytoplasmic RNAs are incorporated into exosomes. Significance of Aim 1

Because exosomes are produced naturally by most cells in the body and natively transport biological information between cells, it is possible that exosomes are well-suited to delivery of therapeutic molecules as well. However, application of exosomes as therapeutic delivery vehicles is limited by our lack of knowledge about the contents of exosomes produced in various contexts and their impacts on recipient cells. In particular, exosomal RNA has been observed to have a variety of both beneficial and detrimental effects on recipient cells. Therefore, understanding the rules governing cargo incorporation into exosomes is a question of both scientific interest and therapeutic importance. The goal of Aim 1 of my proposal is to develop a quantitative framework that will enable a better understanding of how RNAs are packaged into exosomes; specifically, to elucidate the RNA expression levels and targeting molecule levels at which RNA is loaded into exosomes non-specifically, by mass-action driven forces, or specifically, by targeted incorporation of the specific RNA into exosomes. Understanding how targeting and mass-action interact to load RNA into exosomes will offer new insights into how to enhance or prevent the packaging of specific RNA molecules into exosomes. Prostate cancer

According to the National Cancer Institute, prostate cancer is the most commonly diagnosed cancer in the United States, and the fourth most common cause of cancer-related death 34. The human prostate consists of three regions, the peripheral, central and transition zones. Prostate cancer is an adenocarcinoma that develops in the peripheral zone35. Prostate cancer develops from prostatic intraepithelial neoplasia (PIN), characterized by proliferation and de-differentiation of cells lining the prostatic glandular units (acini) 35. This primary tumor is non-lethal in the majority of prostate cancer cases, with most prostate cancer deaths stemming from the development of metastatic disease36. The primary sites of prostate cancer metastasis are bone, lung, and lymph nodes 37. Conventional prostate cancer therapies

The first line of prostate cancer treatments include active surveillance, prostatectomy, androgen ablation (because prostate cancer initially depends on androgen receptor signaling), and radiation therapy, or a combination of these38. The blockage of androgen receptor signaling in ablation therapy prevents the production of growth factors and vascular endothelial growth factor (VEGF) that support the proliferation of cancer cells36. This therapy is often followed by the development of hormone resistant disease through the overexpression of the androgen receptor, mutations in the

androgen receptor that allow for ligand-independent signaling, or activation of alternative signaling pathways that allow cancer progression36.

Chemotherapy is used to treat metastatic prostate cancer if androgen ablation is not effective39. The main chemotherapeutic agents used include cytotoxic drugs, epidermal and insulin-like growth factor receptor inhibitors, and bone-protective agents39,40. Chemotherapeutic agents are continuing to be developed for prostate cancer. Promising drug candidates in clinical trials include inhibitors of Hsp90 (overexpressed in prostate cancer cells), receptor tyrosine kinases (including MET and VEGFR2, both of which are overactive in prostate cancer), histone deacetylases, and platelet-derived growth factor receptor, as well as improved androgen ablation drugs36,40,41. Clinical trials are also investigating the use of the immunosuppressant drug rapamycin to block mammalian target of rapamycin (mTOR) – mediated induction of cell division40. Despite the wide range of chemotherapeutic options for prostate cancer treatment, chemotherapy is plagued by the development of drug resistance, fueling the need for further innovation in prostate cancer treatment. RNA interference-based prostate cancer therapiesRNAi candidates

Prostate cancer therapies based on RNAi are being developed as a complement to traditional chemotherapeutic agents. RNAi-based drugs are capable of inhibiting a wider range of targets than small molecule inhibitors, can be rapidly engineered for specificity and efficacy, and are easily synthesized. For these reasons, developing RNAi therapeutics is an attractive alternative to the development of new small molecule drugs. Two antisense oligonucleotide therapies are currently in advanced phase clinical trials for use in treatment of hormone resistant prostate cancer36. OGX-011 is an antisense oligonucleotide that knocks down expression of clusterin, a stress-induced chaperone implicated in suppressing prostate cancer cell death in response to androgen ablation, chemotherapy, and radiation42. OGX-427 is an antisense oligonucleotide that knocks down expression of Hsp27, which is implicated in preventing apoptosis due to androgen ablation, and driving epithelial-to-mesenchymal transition (increasing the probability of metastasis). In addition to these therapies in clinical trials, RNAi-based therapies for prostate cancer are continuing to be developed in preclinical studies. For example, lipoplex-mediated delivery of Atu027, an siRNA against protein kinase N3, involved in angiogenesis, decreased prostate cancer tumor volume and metastases volumes in an orthotropic mouse model of prostate cancer43. RNAi delivery strategies

Though RNAi drugs show promise for treatment of prostate cancer, their use is limited by challenges in delivery to the tumor site. Small RNAs are unstable in systemic circulation due to the presence of RNases in serum, as well as rapid RES and renal clearance. Furthermore, the hydrophobicity of cell membranes creates a barrier blocking intracellular delivery of highly negatively charged RNAs44. One solution to this problem is chemical modification of the RNA backbone, to increase resistance to degradation and decrease charge. However, this can interfere with cellular processing including assembly of RISC, and the rules governing which modifications can be tolerated are not fully established45. Another option is to deliver expression cassettes coding for production of siRNA (via expression of shRNA or shRNA-miR) by lentiviral or adeno-associated viral vectors. However, viral vectors suffer from immune recognition, precluding multiple administrations of the drug. Furthermore, lentiviral vectors are also known to cause insertional mutagenesis resulting from disruption of genes or promoters at the site of genomic integration. A third option is to encapsulate RNA in a nanoparticle carrier. Common carriers include liposomes (artificial phospholipid vesicles), lipoplexes (cationic lipid-nucleic acid complexes formed through electrostatic interactions), and dendrimers (branched cationic polymers)44. In order to decrease liposome and lipoplex clearance from circulation, decrease particle aggregation, and prevent deposition of negatively charged serum proteins (rendering the particle ineffective at

intracellular delivery), lipid nanoparticles are often coated with polyethylene glycol (PEG)46. However, PEGylation of lipid nanoparticles can result in adverse immunological events such as binding of complement proteins or production of IgM against PEG47. Furthermore, cationic lipids and polymers can induce interferon responses, pulmonary inflammation, and can cause changes in gene expression independent of the RNA drug44. Finally, cationic liposomes have low nucleic acid delivery efficiencies compared to viral delivery48. Alternative nanoparticle carriers are being developed using inorganic materials, especially gold nanoparticles and carbon nanotubes49,50. Application in vivo can be toxic, but appears to be dose-dependent47,48. New types of nanoparticle nucleic acid carriers are being developed based on biological polymers (including chitosan, alginate, collagen, and cyclodextrins), and biological nanoparticles (including HDL and exosomes). These biologically-based delivery vehicles show promise in animal models, based on absence of toxicity or immune responses to the particles1,44.

Aside from protecting RNA from degradation and rapid clearance, nanoparticle RNA carriers would ideally mediate accumulation of the therapeutic RNA in the target cells. When administered systemically, nanoparticles tend to accumulate in the spleen, liver and kidneys51. In the case of cancer, a degree of passive targeting does occur. This is thought to be due to the enhanced permeability and retention (EPR) effect, in which nanoparticles accumulate at tumor vasculature, where the nanoparticles can then be taken up by cancer cells52. However, this passive targeting can be improved by the display of ligand that bind surface receptors on the cancer cell and enhance uptake via receptor-mediated endocytosis. In some cases, targeted nanoparticles can effectively deliver therapeutics to the target cells while untargeted nanoparticles do not1,4,53-55. Finally, unlike utilizing the EPR effect, the strategy of targeting cancer-specific markers has the capacity to deliver therapeutics to sites of incipient metastasis. To achieve enhanced uptake only in cancer cells, one must find markers that are present at high levels on cancer cells but not on healthy cells. In practice, the specificity for cancer cells is not complete, but it is possible to choose surface receptors that are overexpressed on cancer cells and expressed at low levels on healthy cells. In prostate cancer, the most common target is prostate-specific membrane antigen (PSMA)51,53,56,57. Despite the popularity of PSMA as a target for prostate cancer therapies, PSMA expression, as detected in patient serum samples, is highly heterogeneous within both healthy individuals and prostate cancer patients58. Serum PSMA levels increase with age, and serum PSMA levels are not significantly different between early stage prostate cancer patients and patients with non-cancerous prostate conditions, including prostatitis and benign prostate hyperplasia58. While serum PSMA levels may not exactly reflect PSMA levels on the surface of prostate cells, there is still a need to validate the specificity of PSMA-targeted therapies for prostate cancer cells, as well as the ability for PSMA-targeted therapies to be effective in early stages of prostate cancer, in which PSMA levels are not as high as in later stages. Additionally, gene expression profile studies of human prostate cancer, BPH, and healthy prostate tissues have revealed other promising biomarkers of prostate cancer that are consistently upregulated across a wide variety of samples in cancer, but not non-cancerous tissues59-

61. Hepsin, a transmembrane serine protease, was repeatedly observed to be uniquely upregulated in prostate cancer59-61. Furthermore, a hepsin targeting peptide ligand (IPLVVPL) has been discovered that mediates enhanced uptake of IPLVVPL conjugated nanoparticles by human prostate cancer cells in vitro, and causes accumulation of IPLVVPL conjugated nanoparticles in hepsin-expressing human xenograft tumors in mice5.Significance of Aim 2

Exosomes are natively equipped with the ability to deliver functional RNA cargo between cells. When engineered to display a rabies viral glycoprotein peptide, systemically administered exosomes successfully delivered siRNA to the brain in mice1. Similarly, systemically administered exosomes displaying a synthetic peptide ligand for EGFR delivered miRNA to breast cancer tumor

cells in mice4. In human clinical trials, exosomes were used as a vaccine to deliver tumor-associated antigens, and repeated administration of exosomes was well tolerated62-64. These results suggest that exosomes could be effective delivery vehicles for RNA therapeutics in humans. The goal of Aim 2 of my proposal is to use exosomes displaying the IPLVVPL targeting ligand to deliver RNAi against mTOR to human prostate cancer cells. The choice of mTOR as an initial target for RNAi therapy is based on the observation that mTOR knockdown can inhibit the growth of prostate cancer cells and xenograft tumors6,7, as well as the current clinical trial investigation of rapamycin as a treatment to inhibit mTOR-mediated cell division in prostate cancer40. Aim 2 will investigate both the efficacy of the IPLVVPL-hepsin targeting strategy, and the efficacy of delivering various forms of RNAi via exosomes. In addition to developing a new therapeutic modality for treating cancer, Aim 2 will further our understanding of the properties of exosomes that can be harnessed for therapeutic purposes, paving the way for future therapeutic applications of exosomes. Macrophage polarization

Macrophages are versatile cells capable of adopting different phenotypes in response to different environmental signals. Macrophages become functionally “polarized” towards a pro-inflammatory (M1) state in response to pathogen associated markers or inflammatory cytokines such as IFNγ and IL1β.65 Macrophages become polarized towards an anti-inflammatory (M2) state in response to glucocorticoid hormones, vitamin D3, or anti-inflammatory cytokines including IL-4, IL-13, and IL-1065,66. Upon activation (for example by lipopolysaccharide, LPS), polarized macrophages execute distinct gene expression programs. M1 macrophages are characterized by high expression levels of pro-inflammatory genes, such as TNFα, inducible nitric oxide synthase (iNOS – which creates NO from arginine), IL-12, IL-1β, and IL-666. Conversely, M2 macrophages are characterized by high expression levels of anti-inflammatory genes, including TGFβ1, arginase (which inhibits NO production), IL-1 receptor antagonist, and IL-1066. Despite the vast gene expression differences between M1 and M2 macrophages, macrophage phenotype is plastic, such that polarized macrophages can be converted from M1 to M2 via incubation with IL-4 or from M2 to M1 via incubation with LPS and IFNγ67,68. Conversion from M2 to M1 polarization may not even require M1-inducing signals, as simple removal of IL-10 can alter macrophage gene expression from M2-like to M1-like. This may be mediated by concomitant reduction in Bcl-3, an NF-κB inhibitor69, (Yishan Chuang, Leonard lab, unpublished data). Similarly, removal of M2-like tumor-associated macrophages from the immunosuppressive tumor microenvironment (discussed below) also resulted in a change from M2-like gene expression to M1-like gene expression70. These observations suggest that removing the influence of IL-10 can cause the “re-polarization” of M2 into M1 macrophages. Tumor-associated macrophages

Macrophages are a major component of the tumor microenvironment, and these tumor-associated macrophages (TAMs) are programmed by signals from the tumor, such as IL-10 and TGF-β, to adopt an M2-like phenotype66. In prostate cancer, M2 polarization may be promoted by a variety of cytokines, including COX2, IL-4, IL-6, and IL-10, though the exact pathways are not fully understood71-73. TAMs support tumor progression in a variety of ways, including production of VEGF (to promote angiogenesis), matrix metalloproteinases (MMPs), serine proteases, cathepsins and collagens (to remodel the extracellular matrix and promote invasion), and immunosuppressive cytokines (inhibiting CD8+ T cell tumor cell killing)74. TAMs in prostate cancer tissue are associated with tumor recovery from radiotherapy75 and from androgen ablation76. The tumor-supportive activities of TAMs are complex and involve interactions not only between the tumor and macrophages, but also between macrophages and other tumor-infiltrating immune cells. However, in vitro culture of macrophages and cancer cells can reveal the most prominent tumor-supporting interactions between TAMs and cancer cells. This approach recapitulates tumor-induced polarization of macrophages, as it has been observed that coculture of macrophages with ovarian

cancer cells77, conditioned media from cervical cancer cells78, or conditioned media (CM) from pancreatic cancer cells68 resulted in macrophages adopting an M2 phenotype in vitro. This approach also reveals that TAMs can increase metastatic potential of cancer cells. Coculture of TAMs with ovarian cancer cells increased invasiveness of ovarian cancer cells as measured by migration through a modified Boyden chamber79. CM from macrophages induced to M2 phenotype (by cytokines or by pancreatic cancer cell CM) induced migration of pancreatic cancer cells in a transwell migration assay. In contrast, CM from cytokine-induced M1 macrophages did not induce cancer cell migration68. In a model using human prostate cancer and macrophage cell lines, coculture of phenotypically M2 macrophages with prostate cancer cells upregulated the expression of urokinase plasminogen activated receptor (uPAR). uPAR binds the serine protease uPA to mediate extracellular matrix remodeling necessary for metastasis80. These observations reveal that M2 macrophages can induce cancer cell metastatic potential.Significance of Aim 3

Prostate cancer death is generally caused by progression to metastatic disease. TAMs play a role in supporting prostate cancer resistance to radio- and chemotherapy and enhancing metastatic potential, so therapeutically intervening at the level of TAMs may be an effective way of preventing prostate cancer progression. Because systemically injected exosomes can easily access macrophages, (and could be targeted to TAMs through TAM markers, such as p3281), exosomes could be easily adapted to deliver therapeutic RNA to TAMs. In particular, the delivery of RNAi against Bcl-3 could attenuate IL-10 induced M2 polarization. Thus, exosomes may be capable of altering polarization of macrophages from M2 to M1, and this re-polarization may reduce TAM-induced cancer cell migration.

Experimental Design and MethodsAim 1Preparation of exosomes.

Exosomes can be isolated from conditioned cell culture media or bodily fluids by differential centrifugation, filtration paired with centrifugation, high-performance liquid chromatography (HPLC) paired with centrifugation, adsorption to antibody-coated beads, or polymer-based precipitation82,83. For the purpose of this project, differential centrifugation has been chosen for exosome isolation because it results in high exosome yield and purity80,81.

My exosome preparation protocol is modified for increased yield from protocols in the literature 82,84. I have modified the protocol in the following ways: increasing the ultracentrifugation acceleration from 100,000xg to 120,416xg (maximum rotor acceleration in a swinging bucket rotor), and increasing the ultracentrifugation time from 70 minutes to 150 minutes. HEK293FT cells are used as a source of exosomes. Exosomes are then characterized for structure by TEM, for quantity by NanoSight, for protein by Western blotting, and for RNA by qRT-PCR. NanoSight measures nanoparticle concentration and size distribution based on detecting laser scattering of nanoparticles and measuring their diffusion rates to calculate their size. Although this method underestimates the concentration of particles <50nm in diameter, it can provide an estimate of particle concentration and size distribution. Following the above protocol, I observed exosomes with a structure and size distribution consistent with what has been reported in the literature1,82,85 (Figure A2). I did not observe exosomes in exosome-free media that was not conditioned with cells.

Engineering the incorporation of an RNA-binding bacteriophage coat protein into exosomes.

The most common method for engineering the incorporation of a “cargo” protein or peptide into exosomes is via genetic fusion of the cargo-encoding gene to the gene encoding a protein known to localize to exosomes. To date, there have been no studies aiming to localize proteins to the exosome lumen. However, studies focusing on localization of proteins to exosomes without designating the topology of these proteins have (likely) achieved lumenal protein targeting via fusions to the exosomal marker CD6386 (both termini should be lumenal based on the topology of CD63 in endosomal membranes), or via creation of oligomeric membrane anchored proteins that can be secreted in exosomes (the incorporated proteins are cytoplasmic, and thus should be in the exosome lumen)87. However, this method is not ideal for targeting an RNA binding protein to the exosome lumen, the function of which will likely be adversely affected by oligomerization. Therefore, I decided to target my RNA-binding proteins to exosomes through fusion to an exosomal protein. Because I plan to target proteins or peptides to both the exosome interior (Aim 1) and exterior (Aim 2), I did not choose to use CD63. Instead, I initially began with a transmembrane lysosomal protein, Lamp2b, which has been found in exosomes and can tolerate peptide fusions to both its N and C termini1. The N-terminus of Lamp2b is on the exterior of the exosome, and the C-terminus is on the interior1. Therefore, fusion of an RNA-binding protein to the C-terminus of Lamp2b should result in targeting of the RNA-binding protein to the lumen of the exosome. To test whether or not it was possible to fuse a protein to the C-terminus of Lamp2b and achieve localization of that protein to exosomes, I initially fused GFP to the C-terminus of Lamp2b. An Ala-Ser flexible spacer was included between the C-terminus of Lamp2b and the N-terminus of GFP. To determine if Lamp2b-GFP was expressed, I transfected HEK293FT cells with the construct and with DsRed (as a reporter for transfection efficiency), harvested cell lysate 60h post transfection, and did a Western blot for GFP. As a negative control, I used cell lysate from cells transfected with DsRed only. Lamp2b-GFP was expressed and appeared to be approximately 100kDa, which is consistent with the expected size of the protein (72-143 kDa, depending on the degree of glycosylation of Lamp2b88) (Figure 1A). I observed the localization of Lamp2b-GFP via microscopy (Figure 1C). When co-expressed in HEK293FT cells with a cell membrane localized mCherry-CD4 fusion protein (Nichole Daringer, Leonard lab, Figure 1B), I observed that Lamp2b-GFP was localized within distinct puncta within the cell, consistent with localization to a membrane compartment. These puncta overlapped somewhat, but not completely, with mCherry-CD4. While mCherry-CD4 was present in projections of the cell

Figure 1: (A) 20μg total protein loaded per lane. Cells transfected with: 1. DsRed only 2. DsRed, Lamp2b-GFP 3. DsRed, GFP-Lamp2b. (B) mCherry-CD4 localization compared to cytosolic mCherry. (C) GFP-Lamp2b + mCherry-CD4, or (D) Lamp2b-GFP + mCherry-CD4. Insets in C & D show GFP and mCherry channels, and overlays with brightfield. C & D were captured using the same fluorescent microscope settings (40x zoom) such that fluorescence is comparable.

membrane, consistent with surface membrane localization, Lamp2b-GFP was not, consistent with localization to a distinct membrane compartment (Figure 1C). Furthermore, fusion of GFP to the N-terminus of Lamp2b resulted in extremely faint fluorescent signal in cells (Figure 1D), which could be caused by the topology of Lamp2b in endosomal compartments. If GFP-Lamp2b localized to endosomal compartments, the N-terminus of Lamp2b would be present in a low pH environment, and the GFP chromphore would be protonated, resulting in loss of fluorescence89. This evidence encouraged investigating the presence of Lamp2b-GFP and GFP-Lamp2b in exosomes via Western blot. I did a Western blot against GFP in exosomes from HEK293FT cells transfected with Lamp2b-GFP or GFP-Lamp2b and DsRed (as a reporter for transfection efficiency). As a negative control, I used exosomes from cells that had been transfected with DsRed only. However, I observed a non-specific band at approximately 100kDa in exosome samples stained with either of two different GFP antibodies (ab290, AbCam, or MMS-118P, Covance). A Lamp2b antibody has since become available that I can use to investigate, but at the time, I developed new constructs containing an HA tag. I observed the presence of GFP-Lamp2b-HA in exosomes derived from cells transfected with DsRed and GFP-Lamp2b-HA, but not in exosomes derived from cells transfected with DsRed only (Figure 2). Unexpectedly, HA-Lamp2b-GFP was not observed in cells or exosomes. When assayed with a GFP Western blot, HA-Lamp2b-GFP was present in cells, suggesting that the HA tag may be cleaved or degraded.

The cellular expression and apparent exosomal localization of the Lamp2b/GFP fusion proteins prompted me to attempt localizing RNA binding proteins to exosomes. Two bacteriophage coat proteins are commonly used to tether proteins to RNA: MS2 coat protein, and a peptide comprising the first 22 amino acids of the λN coat protein. The MS2 coat protein has been fused to both the N and C termini of various proteins to tether these proteins to RNA, whereas the λN peptide has only been fused to the N termini of proteins90. Therefore, it is unknown whether fusion of the λN peptide to the C-terminus of Lamp2b will retain the ability of the λN peptide to bind its cognate protein. However, the λN peptide is much less bulky than the MS2 coat protein (which also must dimerize to bind RNA – discussed below), and may be more amenable to fusion to a membrane protein without disrupting trafficking of that protein. Therefore, I fused the λN peptide to the C-terminus of Lamp2b with a Gly-Ala-Ser spacer between the C-terminus of Lamp2b and the N-terminus of the λN peptide. The MS2 coat protein binds to its cognate MS2 RNA stem-loop (MS2 loop) as an antiparallel dimer91. Genetic fusion of two MS2 monomers is sufficient to create a synthetic dimer capable of binding its cognate RNA91. The MS2 coat protein can also oligomerize further to form the MS2 bacteriophage capsid, and this oligomerization results in decreased affinity for the MS2 loop92. However, mutation of the coat protein by removing amino acids 67-79 (which form a flexible loop necessary for capsid formation) results in the production of MS2 coat protein mutants (d1FG mutant) with enhanced RNA binding ability. Another mutation in combination with d1FG, V29I, further increases the affinity of MS2 coat protein for its cognate RNA92. Based on these observations, I fused two d1FG/V29I mutant MS2 coat protein monomers to the C-terminus of Lamp2b with a Gly-Ala-Ser spacer between the C-terminus of Lamp2b and the N-terminus of the mutant MS2 coat protein dimer. For tracking localization to exosomes, I added a Gly-Ser-Gly spacer followed by an HA tag to the C-terminus of the MS2 coat protein. I transfected HEK293FT cells with DsRed and the Lamp2b-MS2 dimer-HA fusion protein and observed expression of the

Figure 2: 20μg protein loaded per lane, but presence of lipids can interfere with BCA assay accuracy, so CD63 marker protein is used as a loading control .Exosomes from cells transfected with DsRed plus: 1. GFP-Lamp2b-HA 2. IPLVVPL-Lamp2b-MS2-HA 3. Lamp2b-MS2-HA 4. None (negative control)

protein in cells, as well as localization in exosomes via Western blot (Figure 2). As a negative control, cells were transfected with DsRed only and the cell and exosome fractions were also Western blotted for the presence of HA. Design of cargo RNA.

Because I expect RNA size to play an important role in small RNA incorporation into exosomes, I have designed small non-coding RNAs to be transcribed from the RNA polymerase III based U6 promoter. Use of RNA polymerase III allows for precise termination of the transcript after a run of 4-6 Ts93,94. The maximum length of transcript that can be made from the U6 promoter is unknown. This has influenced the design of the cargo RNA: the MS2 loop or BoxB loop (binding stem-loop for the λN peptide) is at the 5’ end of the transcript. A qRT-PCR target (a fragment of GFP – an RNA sequence that should not overlap with any native sequences) is at the 3’ end of the transcript. Thus, I will only be able to detect full length transcripts by qRT-PCR. To begin, I have created ~200 nucleotide constructs with either high affinity mutant of the MS2 loop (MS2 dimer Kd = 2.86*10-11M)90, the wild type MS2 loop (MS2 dimer Kd = 1.49*10-9M)90, the BoxB loop (λN Kd = 1.3*10-9M)90, or no RNA stem-loop. I will insert successively longer regions of RNA between the loop and the qRT-PCR target, creating 400 and 600bp small RNA constructs. For each construct, I will test its expression in cells prior to quantifying its incorporation into exosomes. To incorporate larger RNAs into exosomes, such as longer non-coding and mRNAs, I will use the RNA polymerase II based CMV promoter. I will not be able to precisely control RNA length, due to variable polyA tail lengths, but I can observe differences at a more coarse-grained level.

To test incorporation of miRNA into exosomes, I will create artificial miRNAs in which the loop region of the miRNA is replaced with the loop region of the MS2 loop or the BoxB loop. The guide and passenger strands of the miRNA will be modified such that detection of the modified miRNA can be distinguished from the native miRNA. It is possible to alter the guide and passenger strands of an miRNA without affecting its ability to be processed and knock down gene expression, as evidenced by the development of shRNA-miR3095. It is also possible to change the sequence of the miRNA loop without affecting processing and knockdown ability of shRNA-miR3096. MS2 dimer or λN peptide binding to these modified miRNA should still occur, because only the loop sequences of these stem-loops are essential for binding, and despite extra stem sequence, the loop sequence will still be available for binding90. I will modify miR30, which has been proven flexible for modification95,96. It is unclear whether mature or pre-miRNA are naturally incorporated into exosomes, or if both are incorporated. I will measure the native incorporation of mature and pre-miRNA into exosomes and determine the degree to which pre-miRNA can be targeted to exosomes. Determining incorporation of RNA into exosomes.

To test the incorporation of RNA into exosomes, I harvest exosomes as described previously. I lyse the exosomes and their producer cells in Trizol© and proceed with RNA purification via Trizol©. This method yields the entire spectrum of RNA present in cells and exosomes, unlike kit-based methods which have a bias towards small or large RNAs. The isolated RNA is then reverse transcribed. For small non-coding RNA (miRNA discussed in more detail below), I will use a gene-specific primer. mRNA is reverse-transcribed with an oligo dT primer. For qPCR, cDNA samples are measured as well as a sample that was not reverse transcribed (no RT control), and the copy number of each RNA (with no RT control subtracted) is determined based on standard curve calibration. The level of cargo RNA in exosomes and cells is normalized to GAPDH level to account for differences in RNA quantity and quality between samples. I can detect GAPDH mRNA in exosomes (Figure A3A). As a control to determine the degree to which targeting is playing a role in incorporation of RNA into exosomes, I will measure the incorporation of the no loop RNA construct. To test the incorporation of artificial miRNA into exosomes, reverse transcription of miRNA will be performed according to a previously described protocol97. For pre-miRNA, the

RNA is denatured, a gene-specific primer is annealed, and then reverse transcriptase is added, to allow amplification of stable hairpins. For mature miRNA, I will use a primer that anneals to the guide strand of the miRNA and adds sequence from the passenger strand such that a stem-loop is created during reverse transcription. qPCR primers for the miRNA will be placed such that one primer is on the guide strand and the other on the passenger strand of the stem. As a comparison to see if targeting alters the mature to pre-miRNA ratio in exosomes, I will also measure the incorporation of an shRNA-miR that does not contain the MS2 or BoxB loop sequence.

I have observed that transfection of HEK293FT cells with a vector encoding the cargo RNA results in high levels of DNA contamination of exosome samples (no RT samples amplify in qPCR). The reason for this observation is unknown but may be due to transfection particles sticking to exosomes in conditioned media. The isolation of DNA with exosomes isolated by ultracentrifugation has been observed previously (informal communication by Leileata Russo at Exosomes and Microvesicles conference). To prevent DNA contamination of exosomes, I therefore will create lentiviral vectors encoding all cargo RNA constructs along select stably transduced HEK293FT cells from which I can isolate exosomes. I have created stable cell lines expressing the previously described ~200 nucleotide constructs at either high or low transduction levels and assessed the expression in these cell lines by qRT-PCR (Figure A3B). The cargo RNAs are incorporated into exosomes and the levels in exosomes roughly correspond to the levels in cells (Figure A3C). This indicates that cellular expression level plays an important role in determining which cargo RNAs are incorporated into exosomes.

Mapping the effects of targeting and expression level on RNA loading into exosomes.

I initially added targeting proteins Lamp2b-MS2 and Lamp2b-λN peptide via transfection of the stable cell lines expressing the cognate cargo RNAs. Transfection of the RNA binding protein does not significantly alter cargo RNA expression levels in the cells (Figure 3A). (Significance is tested by a student’s t test with a p-value of 0.05). An exception to this observation is the significant decrease in cargo RNA expression level when Lamp2b-λN peptide is transfected into cells transduced at a high expression level with BoxB loop cargo RNA. This decrease is not observed when Lamp2b-λN peptide is transfected into cells transduced at a low expression level with BoxB loop cargo RNA, suggesting that transfection of the Lamp2b-λN peptide is not the cause of this effect. Further

validation is required to confirm these results as I have observed significant fluctuations in cellular levels of cargo RNA in other experiments. I also measured levels of cargo RNA in exosomes (Figure 3B). Addition of the RNA binding proteins has a significant impact on copy number of the cargo RNA in some exosome samples, suggesting that an interaction is occurring. However, the impact is not consistent. One possible explanation for

Figure 3. Cargo RNA levels in cells (A) and exosomes (B) normalized to GAPDH. Detected with qRT-PCR for GFP fragment

these trends is that there may be a narrow range of expression levels of cargo RNA and RNA binding protein that result in increased incorporation of the cargo RNA in exosomes. For example, when cargo RNA is highly expressed, mass action may be a sufficient driving force to package the cargo RNA into exosomes at a maximum level. In this case, addition of the Lamp2b-RNA binding protein may not be able to increase incorporation of RNA into exosomes. However, at low expression levels of the cargo RNA, the Lamp2b-RNA binding protein could have an effect, targeting a greater percentage of cargo RNA to exosomes (this trend is observed in the high affinity MS2 loop cargo RNA). In this experiment, expression of the RNA binding protein is not tightly controlled (it depends on transfection efficiency, which differs between plates of cells). Because not 100% of Lamp2b-RNA binding proteins will localize to exosomes, there may be concentrations at which excess Lamp2b-RNA binding protein binds and sequesters cargo RNA in an endosomal compartment that never becomes an exosome. This may be the cause for the significant decrease in exosome incorporation in the wild-type MS2 loop cargo at high expression and the BoxB loop cargo at low expression. At this point, repeating these experimental results has revealed that the observed trends can change between experiments, emphasizing the need for more fine-tuned control over the expression of cargo RNA and Lamp2b-RNA binding protein. Therefore, I will transduce cells expressing cargo RNA with an inducible (tet-ON promoter) Lamp2b-RNA binding protein vector, allowing for fine-tuning of Lamp2b-RNA binding protein expression. I will select clonal populations of the transduced cell lines to generate a range of cargo RNA expression levels. I will then analyze exosomal RNA copy # over a range of Lamp2b-RNA binding protein and cargo RNA expression levels to map out the regimes in which one observes targeting driven incorporation of RNA into exosomes, and the regimes in which one observes mass action driven incorporation of RNA into exosomes. This strategy will be repeated for each type of RNA to be investigated. Computational model.

I will generate a model to predict and understand the different regimes in which expression level versus targeting play a more important role in RNA incorporation into exosomes, and examine whether or not these regimes change based on RNA type. Because I expect stochastic fluctuations in RNA and targeting protein levels to play a role in determining exosome incorporation, I will create a stochastic chemical kinetics model based on ordinary differential equations describing the rates of RNA binding protein production, degradation, and trafficking to compartments that become exosomes, and RNA production, degradation, mass-action driven exosome incorporation, and binding-protein driven exosome incorporation. I will calibrate my model using experimental data and known constants (e.g. binding affinities) and perform sensitivity analysis to determine which parameters play the largest role in determining RNA incorporation into exosomes. This may inform future experiments to examine the effects of the most important parameters. Expected results and alternative strategies.

My results so far demonstrate that I can target RNA binding bacteriophage coat proteins to exosomes. I can also express cargo RNA at different levels in stable cell lines and detect incorporation of cargo RNA into exosomes. It appears that co-expression of Lamp2b-RNA binding protein with cargo RNA can play a role in determining incorporation levels of RNA into exosomes. However, this has not been proven. Furthermore, it is possible that the use of Lamp2b as a targeting protein is not the best choice, since it may lead to sequestration of the coat protein-cargo RNA complex in endosomal compartments that do not go on to become exosomes. Fusion to the lumenal side of other exosomal proteins, such as CD63, may be better for targeting proteins to the interior of exosomes. In the case that this targeting system is not effective, I will take an alternative approach to mapping the effects of targeting and expression level on RNA loading into exosomes. It has been previously shown that the presence of a miR-1289 binding site and a core “CTGCC” motif in the 3’ UTR of mRNA can increase incorporation of that mRNA into exosomes, and that this targeted

incorporation increases with increasing miR-1289 expression30. While this strategy has not yet been used to target small RNAs to exosomes, the presence of these motifs in the RNA itself could potentially result in targeting of small RNAs to exosomes. Regardless, tuning miR-1289 expression and expression of cargo mRNAs containing the miR-1289 binding site/CTGCC motif would allow me to compare the effects of targeting and expression level on RNA loading into exosomes. This data would also allow me to create a computational model of mRNA loading into exosomes. Aim 2Engineering the display of the hepsin-targeting IPLVVPL peptide on the surface of exosomes.

There are many possible protein fusions that can be used to display a targeting ligand on the surface of exosomes. Because I may want to use methods developed in Aim 1 to incorporate therapeutic RNA into prostate cancer-targeted exosomes, the use of Lamp2b for both incorporating RNA and targeting uptake by prostate cancer cells may be beneficial. The use of one multifunctional targeting protein would ensure that the RNA incorporation and hepsin-targeting functions are encapsulated in the same exosome because they are on the same protein.

I began by fusing the IPLVVPL hepsin-binding peptide to Lamp2b-MS2-HA protein (because I knew it successfully localizes to exosomes, and to assess the possibility for creating a multifunctional protein discussed above). A Western blot against HA revealed that cells transfected with DsRed and IPLVVPL-Lamp2b-MS2 dimer-HA expressed IPLVVPL-Lamp2b-MS2 dimer-HA at the expected size (~120 kDa, expected 66-139 kDa depending on Lamp2b glycosylation). Exosomes produced by these cells also contained IPLVVPL-Lamp2b-MS2 dimer-HA. Cells transfected with DsRed only and the exosomes isolated from those cells did not have IPLVVPL-Lamp2b-MS2 dimer-HA (Figure 2). Because I previously observed loss of an N-terminal HA tag from HA-Lamp2b-GFP, I must verify that the IPLVVPL peptide is not lost from the N-terminus of Lamp2b. Alvarez-Erviti et al. successfully fused the Flag peptide to the N-terminus of Lamp2b and found Flag in exosomes1, so I expect that the sequence of the N-terminal tag may be important for determining whether or not it is preserved. Alvarez-Erviti et al. had an Ala-Arg spacer at the N-termini of all of their Lamp2b fusion proteins, and this may play a role in stability of the N-terminus. To test this, I will create HA-IPLVVPL-Lamp2b fusion proteins with and without the Ala-Arg spacer and Western blot for HA in cells and exosomes. If the spacer is capable of maintaining the HA tag on the N-terminus, I expect that it can also maintain the IPLVVPL peptide on the N-terminus. I may need to add longer flexible spacers between the N-terminal amino acids and the IPLVVPL peptide to preserve its binding to hepsin.Measuring exosome uptake by hepsin-expressing prostate cancer cells.

I will determine whether or not the presence of IPLVVPL-Lamp2b in exosomes increases uptake of these exosomes by hepsin-expressing LNCaP5 prostate cancer cells by comparing the uptake of IPLVVPL-Lamp2b exosomes to that of untargeted exosomes. Exosome uptake can be monitored by labeling of exosomes with a lipophilic dye (PKH67 is often used to label exosomes and track their uptake by recipient cells4,24,98). Exosomes with or without IPLVVPL-Lamp2b will be incubated with prostate cancer cells for 1-2 hours, as described98. Cells will be washed to remove unbound exosomes and fluorescence will be measured by flow cytometry. To assess how expression affects uptake efficiency of IPLVVPL-Lamp2b exosomes, I will transduce non-hepsin expressing PC35 cells with a lentiviral vector expressing hepsin from a tet-ON inducible promoter. I will measure hepsin expression and exosome uptake in transduced PC3 cells at a range of hepsin induction levels. Measuring the efficacy of exosome-mediated RNAi delivery to prostate cancer cells.

I expect that different forms of RNAi may have different efficacies in recipient cells, based on differences in processing in exosome producing cells, loading efficiency into exosomes, and processing in recipient cells. I will focus my investigation on the differences in RNAi efficacy between shRNAs and shRNA-miRs. If I find in Aim 1 that targeting can skew the RNA profile in

exosomes towards more unprocessed RNA (pre-miRNA, shRNA that has not been cleaved by Dicer), I will investigate the differences in RNAi efficacy between pre- and mature shRNA or shRNA-miR. I will use a previously validated mTOR shRNA sequence99 and use this sequence to design and shRNA-miR targeting mTOR. I will validate these new constructs by transfecting them into HEK293FT cells and measuring mTOR mRNA levels by qRT-PCR and mTOR protein levels by Western blot. I will use scrambled versions of the shRNA and shRNA-miR as negative controls. Once these are validated, I will incubate exosomes carrying these RNAs with prostate cancer cells for 24-48 hours based on similar assays that have been previously reported4,84 (the time points may be changed if I suspect that earlier or later time points are more relevant). I will measure mTOR mRNA knockdown by qRT-PCR, and mTOR protein knockdown by Western blot. To measure the effect of mTOR knockdown on prostate cancer cell apoptosis, I will wash cells to remove unbound exosomes and stain with Annexin V to mark cells going through early apoptosis. I will include a propidium iodide (PI) stain to exclude cells going through necrosis (as marked by loss of membrane integrity and uptake of PI)100. As a negative control, the same measures will be made in cells receiving exosomes carrying scrambled shRNA or shRNA-miR. Because exosome membranes are enriched in phosphatidyl serine, the marker detected by Annexin V, the negative control will be particularly useful in determining any Annexin V staining resulting from the presence of exosomes in the sample. Exosome delivery of shRNA/shRNA-miR will be compared in all these assays to lentiviral transduction of the shRNA/shRNA-miR to determine the difference in efficacy between exosomal delivery and direct shRNA/shRNA-miR expression. Expected results and alternative strategies.

My results demonstrate that I can fuse IPLVVPL to Lamp2b-MS2 dimer-HA and that the fusion protein is present in exosomes, although the integrity of the N-terminal sequence is not yet verified. If I am unable to display IPLVVPL on the N-terminus of Lamp2b, I will try fusion to other previously used fusion partners show to target peptides to exosomes (for example, lactadherin C1C2101). Although I will then depend on the presence of IPLVVPL-lactadherin and Lamp2b-RNA binding protein in the same exosome to achieve targeted prostate cancer cell uptake and targeted cargo RNA incorporation, it is not unreasonable to believe that these proteins will colocalize in some exosomes. To verify this, I will construct a Flag-IPLVVPL-lactadherin C1C2 protein and pull down exosomes on anti-Flag beads as previously described1. I will then do a Western blot on these exosomes to look for Lamp2b-MS2 dimer-HA.

Previous research has validated the IPLVVPL peptide as an effective ligand for targeting uptake by hepsin-expressing cells5. However, in the context of exosomes this interaction may not be effective for enhancing uptake by recipient cells. Although not the ideal target, I can instead display a PSMA targeting peptide Saupw-1 selected for specificity to PSMA via phage display102.

There are many examples in the literature of exosomes delivering functional miRNA and shRNA to recipient cells4,22,29,31,32. Based on this evidence I do not expect that it will be difficult to package or deliver shRNA or shRNA-miR into exosomes. I will investigate whether different sequences or structures (for example using different miRNA backbones for shRNA-miR design) can improve incorporation into exosomes and delivery to recipient cells. Aim 3Measuring the efficacy of exosome-mediated RNA delivery to macrophages.

I will use the THP-1 human macrophage cell line. I will design Bcl-3 shRNA and shRNA-miRs based on previously reported Bcl-3 siRNA sequences103 and test their efficacy (in comparison to scrambled controls) at knocking down Bcl-3 mRNA levels in IL-10 stimulated macrophages. As previously described for Aim 2, I will validate the efficacy of the Bcl-3 shRNA and shRNA-miR when expressed in macrophages. Also as previously described for Aim 2, I will produce exosomes containing Bcl-3 or control shRNA or shRNAmiR, incubate them with macrophages, and determine

the efficacy of knockdown of Bcl-3 in recipient macrophages. As a negative control, exosomes packaging GFP mRNA will be used. Exosomes will be delivered to macrophages as described in Aim 2. Exosome delivery of RNA will be compared to transduction of the RNA to determine the difference in efficacy between exosomal delivery and direct RNA expression. Assaying macrophage polarization.

I will incubate M2 macrophages (treated with IL-10) with exosomes delivering RNAi constructs for Bcl-3 or control scrambled RNAi as described in Aim 2. Macrophages will be activated with LPS and polarization will be assayed by gene expression, looking for classic M1 and M2 gene expression profiles (TNFα, iNOS, IL-12 for M1, and TGFβ1, ArgI, and IL-10 for M266). Coculture of prostate cancer cells and macrophages and assay of prostate cancer cell migration.

Prostate cancer and macrophages will be cocultured and prostate cancer migration will be measured by a method that has been described in the literature79,104. Briefly, prostate cancer cells will be seeded in the upper well of a modified Boyden chamber, which is separated from the lower well (which contains media) by a Matrigel-coated polycarbonate membrane. Macrophages are seeded into transwell inserts which are cell-impermeable and inserted into the upper well of the Boyden chamber. Prostate cancer invasiveness will be measured as the percentage of cells that migrate through the Matrigel-polycarbonate membrane. The coculture will be established for 24 hours, which has been shown to be sufficient for inducing M2 phenotype77. After 24 hours, shRNA/shRNA-miR against Bcl-3 containing and negative control exosomes described above will be added to the macrophage media. Macrophage phenotype and prostate cancer invasion will be measured 24-48 hours post addition of exosomes (although the relevant time points may be altered and earlier and later time points will be tested as well). Expected results and alternative strategies.

If expression of RNAi against Bcl-3 is not capable of re-polarizing macrophages, other targets in the IL-10 signaling pathway or other M2 signaling molecules (such as TGFβ1) will be investigated.

It is possible that coculture of macrophages with prostate cancer cells will prevent repolarization of macrophages from M2 to M1. In this case, I can compare gene expression profiles between macrophages cultured with and without prostate cancer cells to determine the differences in response to exosome RNA delivery in each condition to learn how the prostate cancer cells prevent macrophage repolarization. It is also possible that prostate cancer cells will not induce M2 polarization through IL-10 signaling, as the cytokine profile of prostate cancer cells is not fully understood. In that case, I can use melanoma cells, which are known to secrete IL-10 to modulate immune cells105. (Our lab is also currently set up to study macrophage – melanoma interactions).

Prostate cancer cells have previously been shown to increase migration in response to signals from M2 macrophages, but if I cannot recapitulate this result, I can measure other readouts of the impact that M2 macrophages have on prostate cancer cells, such as resistance to chemotherapy, and assess how this changes when macrophages are re-polarized from M2 to M1.

Expected TimelineSee Figure A4.

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Appendix

Figure A1: Exosome production. Exosomes incorporate membrane components from the plasma and endosomal membranes, cytoplasmic proteins and RNA. Plasma membrane proteins reach exosomes via endocytosis into the endosomes followed by invagination of the endosomal membrane to form intraluminal vesicles (intracellular precursors of exosomes). An endosome containing many such intraluminal vesicles is termed a multivesicular body. Upon invagination of the endosomal membrane, endosomal membrane proteins also get incorporated into intraluminal vesicles. During invagination, cytoplasmic contents including RNA and proteins are engulfed into the lumen of the intraluminal vesicles. Upon backfusion of the multivesicular body with the plasma membrane, intraluminal vesicles are released into the extracellular space and are then termed exosomes.

Figure A2: (A) Exosomes were visualized by TEM. Exosomes were fixed in 2% PFA. 30μL of fixed exosomes were applied to a Formvar coated grid for 30 minutes. The grid was then washed in PBS, washed in water, a stained with 2% uranyl acetate for 10 seconds. TEM was performed at 80 kV. (B) Exosome size distribution by NanoSight. Nanoparticles are detected as points of scattered light from a 405nm laser beam. Scatter points are tracked over 60s and size is calculated based diffusion.

Figure A3: RNA detection in cells and exosomes. (A) GAPDH mRNA is detected in exosomes derived from cells transduced with different RNA cargoes. (B) Successful transduction of cargo RNA at high and low transduction levels into cells as detected by qRT-PCR for the GFP fragment of the cargo RNA. (C) Successful detection of cargo RNA in exosomes as detected by qRT-PCR for the GFP fragment of the cargo RNA. Amount in exosomes correlates to amount in cells.

Objectives Year 1 Year 2 Year 3 Year 4Aim 1 – Testing of stable cell lines at various RNA expression/targeting levelsAim 1 – Computational Model, Experimental ValidationAim 2 – Achieve targeted exosome uptake of exosomes by CaP cellsAim 2 – Assess exosome-delivered mTOR RNAi efficacyAim 3 – Assess re-polarization of macrophages via exosome RNA deliveryAim 3 – Assess exosome-mediated re-polarization and inhibition of CaP invasion in coculture model

Figure A4: Expected Timeline