Exploring the Rules and Therapeutic Promise of Exosome-Mediated Intercellular Communication

Exploring the Rules and Therapeutic Promise of Exosome-Mediated Intercellular Communication

Michelle Marcus

PhD Proposal

May 2013

Summary

Intercellular 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 Aims

1. 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 bacteriophagecoat 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 RNAs2.

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 cargoareunknown.

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, becausemTOR 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 apoptosisin 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 Significance

Exosomes

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 antigensto 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 (FigureA1). 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 death34. 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.

RNAinterference-based prostate cancer therapies

RNAi 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 rapidRES 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.