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Lipid Nanovesicle Platforms for Hepatocellular Carcinoma Precision Medicine Therapeutics: Progress and Perspectives

Lipid Nanovesicle Platforms for Hepatocellular Carcinoma Precision Medicine Therapeutics:... ORGANOGENESIS 2024, VOL. 20, NO. 1, 1–17 https://doi.org/10.1080/15476278.2024.2313696 REVIEW Lipid Nanovesicle Platforms for Hepatocellular Carcinoma Precision Medicine Therapeutics: Progress and Perspectives a,b a Brandon M. Lehrich and Evan R. Delgado a b Division of Experimental Pathology, Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; Medical Scientist Training Program, University of Pittsburgh, Pittsburgh, Pennsylvania, USA ABSTRACT ARTICLE HISTORY Received 6 July 2023 Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality globally. Revised 4 January 2024 HCC is highly heterogenous with diverse etiologies leading to different driver mutations potentiat- Accepted 30 January 2024 ing unique tumor immune microenvironments. Current therapeutic options, including immune checkpoint inhibitors and combinations, have achieved limited objective response rates for the KEYWORDS majority of patients. Thus, a precision medicine approach is needed to tailor specific treatment Cell therapy; exosomes; options for molecular subsets of HCC patients. Lipid nanovesicle platforms, either liposome- extracellular vesicles; (synthetic) or extracellular vesicle (natural)-derived present are improved drug delivery vehicles hepatocellular carcinoma; lipid nanovesicles; precision which may be modified to contain specific cargos for targeting specific tumor sites, with a natural medicine affinity for liver with limited toxicity. This mini-review provides updates on the applications of novel lipid nanovesicle-based therapeutics for HCC precision medicine and the challenges asso- ciated with translating this therapeutic subclass from preclinical models to the clinic. Introduction immune checkpoint inhibitors (ICIs). Despite ICIs demonstrating improved OS of roughly six Hepatocellular Carcinoma (HCC) is a growing months over TKIs, such as Sorafenib, the benefit global public health burden. HCC is the sixth is still marginal with only 25–30% response rates most common cancer globally, with > 900,000 5–7 in patients. cases each year, and the third highest in cancer- Therefore, novel targeted therapies used in related mortality, with > 800,000 deaths conjunction with immunotherapy, in a precision- each year. HCC typically follows a sequalae of medicine based approach, may overcome HCC chronic liver disease, with the main etiologies therapeutic resistance to ICIs in molecular subsets including Hepatitis B and C virus (HBV & of patients. Lipid nanovesicle platforms, either HCV) infection, alcohol-related disease, steatotic liposome- (synthetic) or extracellular vesicle (nat- liver diseases (SLD) (e.g., metabolic dysfunction ural)-derived, have demonstrated promise as drug associated SLD [MASLD], diabetes mellitus, obe- delivery vehicles to the liver and for targeted cancer sity), and toxin exposure (e.g., cigarette smoke, agents. These nanocarriers are ideal drug delivery aflatoxin, liver fluke). HCC has a dismal prog- vehicles which may be functionalized to harbor nosis with a 5-year overall survival (OS) rate of specific cargo molecules and “home” to specific ~ 15–20% and <18 months median survival with current therapeutic paradigms. Very few patients tumor sites, with native affinity for liver with lim- 8,9 are diagnosed at early stages where surgical resec- ited toxicity. This mini-review discusses HCC tion/transplantation is feasible and nearly molecular subclasses and current treatment para- curative. In fact, the vast majority of patients digms, along with applications of novel lipid nano- are diagnosed with advanced disease, limiting vesicle-based therapeutics for HCC precision their options to systemic agents, including tyro- medicine with a focus on naturally-derived nano- sine kinase inhibitors (TKIs) and, more recently, vesicle formulations, and the challenges associated CONTACT Brandon M. Lehrich brandon.lehrich@pitt.edu Division of Experimental Pathology, Department of Pathology, University of Pittsburgh School of Medicine, M246 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15232, USA; Evan R. Delgado evd7@pitt.edu Division of Experimental Pathology, Department of Pathology, University of Pittsburgh, S423 200 Lothrop St, Pittsburgh, Pennsylvania 15232, USA © 2024 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. 2 B. M. LEHRICH AND E. R. DELGADO with translating this new therapeutic subclass from Additionally, the inflamed class can be further sub- preclinical models to the clinic. divided into either immune-active or immune- exhausted, with the immune-active subclass repre- senting high adaptive immunity gene expression Genetic heterogeneity and immunologic with improved survival and reduced rates of recur- landscape rence. The immune exhausted subclass demon- strates activated stroma and immunosuppressive Over the last decade, next-generation sequencing gene set signatures. Overall, these classification technologies have been utilized to profile genetic systems illustrate how tumor genetics drive both drivers of HCC, guiding the path toward precision tumoral heterogeneity and specific tumor microen- medicine therapeutics. Our current understanding vironments, which may be differentially susceptible of the HCC genomic landscape includes major to various systemic agents, and thus, may require somatic mutations in TERT (~50%; telomere main- tailored treatment options for patients informed by tenance; promoter mutation and gain-of-function tissue and/or liquid biopsy. [GOF]), TP53 (~30%; cell cycle control; missense/ nonsense; loss-of-function [LOF]), CTNNB1 (~30%; Wnt/β-catenin signaling; missense; GOF), Current treatment modalities and patient ARID1A (~10%; chromatin remodeling; truncat- selection ing/missense; LOF), and TSC2 (~10%; cell growth; 10,11 deletions; LOF). Less common molecular dri- For advanced HCC, current standard of care has vers include FGF19 (~10%), AXIN1 (~6%), MYC shifted from the use of TKIs toward ICIs in the last 10,11 (~6%), APC (~5%), and MET (~2%). Some of decade. ICIs are monoclonal antibodies which these mutations may not be mutually exclusive; block the interaction between immune checkpoint however, mutations in Wnt/β-catenin pathway molecules (e.g., programmed death-ligand 1 members and TP53 tend to be mutually exclusive [PDL1] on tumor cells interacting with pro- events. This dichotomy also forms the foundation grammed cell death protein 1 [PD1] on T cells) for defining the various molecular subclasses of potentiating cytotoxic CD8+ T cell mediated tumor HCC described here. cell killing. The IMbrave 150 trial demonstrated The two main molecular classification systems 19.2 months median survival with atezolizumab proposed are the G1-G6 system by Boyault et al. (anti-PDL1 antibody) plus bevacizumab (anti- and the S1-S3 subgroups by Hoshida et al. Briefly, VEGF antibody) compared to 13.4 months median G1-G3 and S1-S2 subclasses represent proliferative/ survival with sorafenib (TKI). Also, the poorly differentiated tumors associated with chro- HIMALAYA trial demonstrated 16.4 months med- mosomal instability, high HBV viral load, and TP53 ian survival with the ICI combination of tremeli- mutations, while G5-G6 and S3 subclasses represent mumab (anti-CTLA4 antibody) plus durvalumab non-proliferative/well-differentiated tumors asso- (anti-PDL1 antibody) compared to 13.7 months ciated with chromosomal stability, alcohol/HCV/ median survival with sorafenib. Moreover, the 13,14 NASH-driven HCC, and CTNNB1 mutations. CARES-310 trial demonstrated 22.1 months med- More recently, HCC can be classified into inflamed ian survival with camrelizumab (anti-PD1 anti- (Hoshida S1-S2 subgroups) or non-inflamed body) plus the VEGFR2-targeted TKI rivoceranib 14,15 (Hoshida S3) subgroups. The inflamed class of compared to 15.2 months median survival with HCC (~25% of patients) demonstrates increased sorafenib. However, despite the improved OS in expression of gene signatures related to immune ICI treated patients, response rates overall remain infiltration (i.e., cytotoxic T cells, tertiary lymphoid relatively low with only 25–30% of patients achiev- structures [TLS], IFN alpha and gamma signaling, ing objective response rates (ORR). Low ORRs are and chemokines CXCL9, CXCL10), high immune poorly understood but have been linked to patient checkpoint immunohistochemical expression, tumor microenvironments with low tumor- CTNNB1-mutated depleted, and enrichment of infiltrating effector T lymphocyte density, high amplification in q13 locus (CCND1, FGF19). regulatory T cell density, and high expression of ORGANOGENESIS 3 oncofetal genes. Thus, to improve these response include small unilamellar vesicles (<100 nm), large rates, an individualized treatment approach is war- unilamellar (>100 nm), and multilamellar vesicles ranted to guide therapeutic selection based on (>500 nm), with the former two more typically used underlying genetic alterations. This may be aided for nanomedicine applications. Excellent reviews by tissue or liquid biopsy for key drivers of HCC elsewhere discuss preparation methodologies (e.g., tumorigenesis. However, an improved under- reverse-phase evaporation, freeze-thaw method, standing of which genetic drivers influence the vaporization technique, and others) of nanoliposome 28,29 immune microenvironment resistant to ICI formulations. Briefly, the phospholipid character- response is warranted for screening, along with istics (e.g., degrees of unsaturation, quantity of fatty needing an expanded arsenal of drugs targeting acid moieties, and others) and the number of choles- these underlying pathways to be used in conjunc- terol molecules can affect the membrane 30,31 tion with ICIs. configuration. Further modifications to the nano- On a molecular basis, the Wnt/β-catenin pathway liposomal structure include the addition of either 32 33 has been the most prominently studied pathway to polyethylene glycol or surface ligands, which evaluate ICI resistance, yet controversy remains avoids host immune system elimination and whether all mutations in the pathway decrease improves cellular targeting, respectively. For cellular immune infiltration to the same degree, and thus uptake, nanovesicles are internalized typically 16, 21, 22 ICI resistance. Moreover, despite studies through endocytosis or phagocytosis, with nanovesi- demonstrating the feasibility of prospective tissue cle structure influencing which mechanistic process. genotyping to identify clinically actionable driver Efficient perfusion of the liver through its dual blood mutations, very few patients receive personalized supply mediates optimal delivery, and lipid nanove- therapeutic intervention. The major driver muta- sicle uptake is augmented due to its fenestrated 23; tions in HCC are currently not actionable there- endothelium. Additionally, opsonization by ApoE fore, efforts should be made to identify and stratify facilitates low-density lipoprotein (LDL) receptor patients which may respond to current druggable (LDLR)-mediated uptake into hepatocytes (“endo- targets, including FGF19/FGFR4, VEGF, TSC1/2, genous targeting”), while engineering 24,25 and MET inhibitors. Although none of these N-acetylgalactosamine (GalNAc)-PEG-lipid on the targets have shown clinical responses, these molecu- nanovesicle surface can target the asialoglycoprotein lar events may be co-occurring in the background of receptor (ASGPR) on hepatocytes (“exogenous tar- strong drivers (e.g., TP53, CTNNB1), and thus geting”), with both options providing efficient deliv- 35,36 a combination of therapeutics may need to be even- ery to the liver. This well-characterized ApoE- tually employed. Thus, further studies are needed in LDLR endogenous hepatocyte targeting mechanism clinically relevant animal models to determine the is the route by which Patisiran, the first FDA differential response of ICIs in combination with approved siRNA-based drug, facilitates its end- targeted therapy approaches in unique molecular organ targeting to the liver and mechanism of subsets of HCC. action. The remainder of this section will discuss the applications of nanoliposomes as targeted drug delivery vehicles in various preclinical models of Synthetic lipid nanovesicle drug delivery HCC as potential precision medicine therapeutic platforms for HCC platforms (Figure 1). Synthetic lipid nanovesicles have conventionally been The realization of using lipid nanovesicles as nanoliposome-based formulations containing dis- a targeted therapy delivery vehicle for liver cancer tinct molecular entities, including either RNA inter- in humans was first achieved in 2013 by Tabernero ference (RNAi) technologies or chemotherapeutic and colleagues in their phase I study. This lipid drugs. Nanoliposomes typically size range between nanovesicle (ALN-VSP) encapsulated siRNAs tar- 10 nm to 200 nm in diameter and are composed of geting vascular endothelial growth factor (VEGF) a phospholipid bilayer with or without cholesterol, and kinesin spindle protein (KSP) to treat patients resulting in an aqueous interior and an outer hydro- with liver metastases. Tumor regression was phobic exterior. The main types of nanoliposomes achieved in nearly 50% of the patients in the trial. 4 B. M. LEHRICH AND E. R. DELGADO Figure 1. Schematic representation of nanoliposome and extracellular vesicle loading strategies, cellular uptake mechanisms of these drug delivery vehicles, and clinical parameters to monitor for toxicity in patients. Figure made in BioRender. These results, demonstrating the safety, tolerability, 3 components: 1) SP94 peptide (specific to HCC ability to achieve target downregulation in the liver, cells), 2) YSK05 lipid (increased cytotoxic effects and short-term clinical responses underscore the and limited endosomal escape), and 3) specific importance and potential of using lipid nanovesicles phosphatidylcholine/cholesterol ratio (improves for HCC therapy. liposome stability). They demonstrated both Nanoliposomes encapsulating RNA interference in vitro and in vivo that their nanoliposome had (RNAi) platforms, such as small interfering RNAs specific uptake to HCC cells over normal hepato- (siRNAs), microRNAs (miRNAs), or messenger cytes, potentiated sorafenib’s effects, and resulted 44,45 RNAs (mRNAs) have been administered as drug in profound tumor regressions (~70%). delivery systems in preclinical models of HCC with Additionally, Woitok et al. delivered siRNA target- considerable success in terms of safety, tolerability, ing Jun N-terminal kinase-2 (Jnk2), known to and treatment response. Various groups have affect fibrosis progression, in lipid nanovesicle to attempted to use RNAi to either target oncogenic mice with chronic liver disease and demonstrated genes involved in cell cycle regulation and cell decreased HCC premalignant nodules and a shift proliferation/death pathways, or directly inhibit in the immune microenvironment of the diseased driver mutations deemed to be traditionally liver. Moreover, targeting key cellular pathways “undruggable.” For reviews on how RNAi plat- in HCC with siRNAs has also been feasible as forms are processed following cellular uptake, we demonstrated by the work from Fitamant and refer the interested reader to the following colleagues. They delivered nanovesicles contain- 39–42 reviews. An example of directly targeting ing siRNA to Yes-associated protein 1 (YAP), a key oncogenic factors is illustrated by work from downstream transcriptional co-activator of Hippo Younis colleagues where they encapsulated both signaling, resulting in tumor regression through a siRNA to midkine (MK; a gene involved in directing hepatocyte differentiation to normal many cellular pathways including apoptosis and hepatocyte-like cells. Other groups have also deliv- angiogenesis and the chemotherapeutic, sorafe- ered nanoliposomes containing siRNAs targeting 48 49 nib, into a nanoliposome functionalized to contain PD-L1, T cell immunoglobulin mucin-3 (Tim- ORGANOGENESIS 5 3; immune checkpoint molecule), vascular release of chemotherapeutic agents through stimuli endothelial growth factor (VEGF; angiogenic fac- responses. Examples of this include using either 51 64 65,66 tor), alpha-fetoprotein (AFP; biomarker for temperature sensitive, pH responsive, photo- HCC), cyclo-oxygenase-2 (COX-2; important sensitive, magnetic-sensitive, or ultrasound-guided for prostaglandin synthesis in inflammatory pro- lipids. In terms of temperature-sensitive lipids, 53 64 cesses), hypoxia inducible factor 1 subunit alpha Peng et al. utilized PF127 (copolymer) which 6− 6 (HIF1a), or RNA N methyladenosine (m A) has temperature-sensitive properties and aids in reader protein YTHDF1 either alone or in com- degrading the nanoliposome following photother- bination with chemotherapeutics. Moreover, mal conversion of IR-780 (a near-infrared [NIR] miRNAs can be packaged into nanoliposomes to dye) also contained on the nanoliposome surface. target specific cellular pathways. For example, This combination of PF127 and IR-780 allowed for Zhao et al. loaded miR-375 and sorafenib in nano- efficient doxorubicin and sorafenib release at the liposomes to hinder autophagic processes and tumor site in vivo. Also, as illustrated by Li et al., reduce tumor burden. Lastly, mRNAs may also interchanging the nanoliposome bilayer to include be packaged into nanovesicles for HCC therapy. the cationic lipid (2E)-4-(dioleostearin)-amino Lai et al. demonstrated that delivery of IL-12 -4-carbonyl-2-butenonic (DC), can allow for direct mRNA in nanovesicles reduced tumor burden tumor cell internalization upon conformational and prolonged survival of transgenic MYC- change in the acidic tumor microenvironment, induced HCC mice. This effect was also asso- and subsequently release its cargo in the acidified ciated with a shift toward a more anti-tumor endosome. This allowed for reduced drug toxicity immune microenvironment with increases in and targeting of tumor cells over normal hepato- T helper cells and IFNγ expression. Similar cytes. Overall, the lipid composition can allow for effects were seen with mRNA for OX40L encapsu- improved pharmacokinetics and tumor cell lated nanovesicles. Overall, lipid nanoparticles internalization. provide an efficient platform to deliver both che- motherapeutics and gene therapy at subtoxic doses Extracellular vesicle-based drug delivery 44,53 with high efficiency and stability. platforms for HCC As previously discussed, modifying the outer shell of the nanoliposome can improve the delivery effi- Extracellular vesicles (EVs) are lipid nanovesicles ciency and targeting to the desired end organ. For (50 nm to >2000 nm) which are spontaneously pro- targeting HCC cells specifically, various groups have duced by nearly all mammalian cells and released functionalized nanoliposomes to target CXCR4 high into extracellular fluid as part of autocrine, para- expressing cells given its sorafenib resistance crine, and endocrine cell-to-cell signaling mechanisms. These studies have demonstrated circuits. There are various EV subclasses, includ- reduced toxicity with targeted nanoparticles and ing exosomes (derived from endosomal membrane synergistic effects when combined with chemothera- trafficking machinery), microvesicles (outward 58–60 pies, such as a sorafenib. Additionally, GalNAc- plasma membrane blebbings), and apoptotic conjugated nanovesicles have demonstrated consid- bodies (from apoptotic processes). All EVs contain erable success in highly relevant animal models of cargos comprising various membrane and soluble molecular subsets of HCC with the nanoliposomes proteins, nucleic acid species, and metabolites, encapsulating siRNAs to oncogenic drivers, such as which are specific to their cell of origin. Once 61,62 CTNNB1. Also, the lipid configuration and released into the extracellular milieu, EVs travel inclusion of PEG/mannose into the membrane can systemically until they make contact with and fuse also affect targeting to different liver cell types. with their target cell plasma membrane through Therefore, using targeting molecules on nanolipo- various endocytic or phagocytic mechanisms. some surface can improve the efficiency of tumor The natural ability for EVs to avoid immune sys- cell transfection and diminish off-target effects. tem clearance, systemically travel to end organs, Moreover, another strategy is modifying the and package cargos within lipid bilayers has made lipid composition of the liposome for controllable them an attractive tool for drug delivery. Through 6 B. M. LEHRICH AND E. R. DELGADO 61, 62, 82 the use of nanomedicine platforms, EV mimetics with others previously mentioned provide are being translated to the clinic as novel drug direct evidence that therapeutically targeting onco- delivery vehicles. Various researchers have devel- genic mutations with siRNAs are effective approaches oped different EV mimetic technologies, either to treat HCC. And, using EVs may have improved through modifying parental cells (e.g., stem cells, RNA delivery efficiency, unique targeting capabilities, fibroblasts, immune cells) and isolating their EVs and enhanced biocompatibility compared to syn- 83–85 for delivery, or ex vivo loading of cargo compo- thetic nanovesicle platforms. nents into EVs. This section will explore applica- Similar to siRNAs, miRNAs packaged into EVs tions of EV mimetics for HCC precision medicine offer another platform to target actively proliferating in preclinical models (Figure 1), and we refer to the cancer cells. Many miRNAs have been implicated in reader to excellent reviews detailing techniques HCC pathogenesis, including miR-21, miR-125b, used for preparation of EV-based therapeutics, miR-155, and miR-221/222. Particularly, miR- 70–74 including their isolation and purification. 125b down-regulation is associated with worse overall The main class of EV mimetics utilized for HCC survival. Baldari and colleagues isolated EVs (via targeted therapy are siRNA- encapsulated EVs, which polymer-based methods) from adipose-derived stro- target specific mRNAs encoding oncogenic signaling mal cells (ADSCs) engineered to express miR-125b proteins. Various groups have identified target genes, with a unique “ExoMotif” sequence that increases which when suppressed, may synergize with ICIs. release of miR-125b into EVs. These EVs were One target is CD38, a transmembrane protein delivered in vitro to HepG2 and HuH-7 cells and which is aberrantly expressed in many tumors and reduced cell proliferation, along with expression of associated with a pro-inflammatory tumor microen- p53 signaling pathway components. In another vironment, and has been shown to be associated ICI study, Mahati and colleagues loaded mesenchymal 75,76 response. EVs isolated from bone marrow stem cell (MSC)-derived EVs with miR-26a (via elec- mesenchymal stem cells packaged with siRNA to troporation) and observed impaired cell proliferation CD38 (via electroporation) reduced HCC tumor bur- and migration in vitro, along with reduced tumor den, metastatic potential, repolarized macrophages burden in subcutaneous HCC models. Lastly, from M2 (immunosuppressive) to M1 (pro- Ellipilli and colleagues demonstrated that combined inflammatory) phenotype, and improved ICI Paclitaxel and miR-122 (liver specific miRNA; response. Other genes/pathways identified which reduced levels shown in HCC) administration within have been targeted with siRNAs packaged in EVs, GalNAc-EVs reduced tumor burden in multiple mice include components of the ferroptosis pathway xenograft HCC models. Complementary to RNAi, (GPX4 and DHODH), cell cycle regulation another strategy for EV therapeutics includes exogen- 78 79 (CDK1), JAK/STAT pathway (STAT6), and ous or endogenous small molecule and protein load- NFkB pathway (p50 subunit). Rather than directly ing. Exogenous protein loading of EVs has been targeting translation of molecules displayed on tumor excellently reviewed elsewhere, but includes techni- cell surface mediating immunosuppression, another ques such as mixing, sonication, electroporation, approach is targeting the underlying genetic mutation freeze-thaw cycles, and extrusion, with sonication 91–94 of the tumor cell. Matusda and colleagues designed an and extrusion being the most efficient. siRNA targeting CTNNB1 delivered within EVs. Monoclonal antibodies, nanobodies, and various Using the Met/β-catenin mouse model (which repre- cytokines can even be packaged into EVs to target sents ~ 10% of human HCC), they remarkably specific immune checkpoint molecules to induce 95,96 demonstrated that delivery of milk-derived EVs native immune activity. However, these techni- 92,97 encapsulating siRNA to CTNNB1 (using transfection ques may damage the membrane integrity of EVs. techniques) reduced tumor burden, in part through Endogenous protein loading into EVs is a novel tech- reversing the immunosuppressive tumor microenvir- nique which hijacks cell signaling cascades to load onment driven by β-catenin, which allowed for particular payloads into EVs, which can be isolated, synergy with ICIs. Another group utilized a similar and subsequently administered as therapeutics. platform, but functionalized the EVs to target Different groups have utilized the ability of FK506 EpCAM-positive HCC cells. These studies along binding protein (FKBP) and FKBP12–rapamycin- ORGANOGENESIS 7 binding (FRB) domain to heterodimerize following a therapeutic source of EVs are ADSCs. Wu and 98,99 rapamycin administration. The FRB domain is colleagues revealed that ADSC-EVs (isolated via fused to the protein of interest via a GGSGG linker, ultracentrifugation of culture media) decreased hepa- and the FKBP domain is fused to a canonical EV tic fibrosis and glutamine synthetase levels, suggesting protein (e.g., CD81 or CD63) via the that this may have therapeutic potential in subsets of N-myristoylation sequence to facilitate protein entry HCC. Moreover, another cell type which has into EVs. Cell lines can be modified to express these demonstrated promise are dendritic cell (DC)- fusion proteins and EVs can be isolated and delivered derived EVs. The pathogenesis of CTNNB1- 98,99 in vivo for effective protein delivery. Small mole- mutated HCC involves defective recruitment of cule/chemotherapeutic agent packaging into EVs DCs, likely making DC-EVs an interesting plat- have demonstrated potential, including the use of form as an HCC therapeutic. Lu and colleagues sys- 100 101 102 doxorubicin, norcantharidin, and sorafenib. temically administered DC-EVs in three different Additionally, Cas9 ribonucleoprotein can be pack- HCC models and observed shifts in the tumor micro- aged into EVs and delivered in vivo to liver, offering environment such as increases in cytotoxic CD8 103–105 avenues for HCC gene therapy. Overall, these T-cells and fewer immunosuppressive T regulatory methods of protein/small molecule packaging are cells, which associated with tumor regression. appealing options for therapeutic delivery to liver. Lastly, M1 macrophages-derived EVs loaded with In the last two decades, recombinant Adeno- docosahexaenoic acid have been shown to induce associated viruses (AAVs) have been explored as ferroptosis and reduce tumor burden in orthotopic gene delivery vehicles for cancer due to their ability HCC models. Therefore, EVs isolated from allo- to target many cell types and long-lasting gene geneic sources have intrinsic capabilities to alter expression. More recently, EVs have been tumor cell survival and growth. However, autolo- shown to be associate with isolated AAVs (termed gous-derived EVs may have improved tumor target- “vexosomes”) during virus isolation from cell- ing properties. Villa et al. illustrated that EVs derived culture media. These vexosomes have become an from blood plasma of cancer patients had selective 107,108 alternate gene delivery vehicle. Moreover, uptake into associated patient-derived xenograft vexosomes protect AAVs from antibody neutrali- (PDX) mouse models. Therefore, autologous EV zation, a major issue for AAV in vivo translation. sources may be another pipeline for manufacture Khan et al. isolated AAV6-derived vexosomes (via with improved tumor-specific targeting properties. ultracentrifugation) containing an inducible cas- pase 9 (iCasp9), which upon delivery with Challenges in good manufacturing practices for a prodrug (AP20187), results in impaired HCC nanovesicle therapeutics cell proliferation in vitro and tumor cell death in vivo via apoptosis. Overall, vexosomes are Many of the challenges of translating nanovesicle another gene therapy-based EV mimetic technol- therapeutics are shared between synthetic and ogy which are highly efficient delivery vehicles, natural platforms; however, this section will require lower therapeutic doses than AAVs, and focus on the nuances associated with translating are not cumbersome to manufacture. EV-based therapeutics. The first consideration is Lastly, EVs isolated from allogeneic or autologous isolation purity. Current clinical Good cell sources are another therapeutic option for HCC. Manufacturing Processes (GMP) of therapeutic Kim and colleagues have demonstrated that EVs iso- EVs may lead to downstream isolation of con- lated from natural killer (NK) cells, which contain taminants (e.g., viral) from cell culture proteins important for mediating immunogenic cell supernatants. For regulatory agency approval death, can functionally impair HCC growth in vitro of EVs, a complete biochemical characterization and in vivo. These NK-EVs (isolated via ultracen- is required for biologics, which remains incom- trifugation) express granzyme B, FasL, and TRAIL plete due to technological limitations and EV and mediate apoptosis through inducing caspase-3, isolation best practices. Additionally, given 7, 8, and 9 upon internalization in tumor cells. EVs are a cell-free therapy, the mechanisms of Additionally, another cell type with promise as cellular uptake/targeting, cargo delivery/release, 8 B. M. LEHRICH AND E. R. DELGADO and an understanding of the precise bioactive and Oncology clinical trials implementing 117,118 nanovesicle platforms nonactive components are unclear. Whether the membrane lipids/proteins, or the The translation of lipid nanoparticles and EVs to proteins/nucleic acids in the lumen, or both, con- clinical practice as HCC therapies has not moved tribute to the intended therapeutic effect is not swiftly. Currently, EVs are being studied as diag- determined. Therefore, extensive functional nostic biomarkers for HCC to detect initial diag- assays, “–omic,” and imaging platforms are nosis, response to therapy, and disease needed to fully elucidate and differentiate the 125 126, 127 recurrence using DNA mutations or physiochemical properties and bioactivity of 128 129 methylation patterns, mRNA /miRNA EVs. The International Society for Extracellular 130 131, 132 signatures, or proteins encapsulated in Vesicles (ISEV) has established guidelines for their lumen. This section will briefly cover in- clinical GMP of therapeutic EVs. human studies in oncology which has successfully The second consideration is cellular source and translated nanovesicle therapeutic platforms to the cell culture ecosystems of therapeutic EVs. As dis- clinic. To investigate whether lipid nanovesicles cussed in the previous section, cellular sources of were actively being translated into clinical trials, therapeutic EVs for cancer can include either stem we surveyed the clinicaltrials.gov website to search cells, immune cells, or nonparenchymal/stromal for active or terminated trials. A review of the cells. Each of these cell types require different culture clinicaltrials.gov website for clinical trials related methods and release differing quantities of EVs. to “cancer” and “exosomes” yielded 132 studies, Additionally, cell culture practices of these cell with 7 unique studies focusing on therapeutic types typically include utilizing fetal bovine serum applications (Table 1). Additionally, a review for (FBS) as a culture media supplement, which presents clinical trials related to “cancer” and “nanovesicle” challenges due to introducing FBS-derived EVs into yielded 12 studies, with 7 unique studies focusing the pool of cell culture-derived EVs. Simply, this on therapeutic applications (Table 1). Overall, contamination means that upon isolation of EVs there are few trials investigating the therapeutic from the cell culture supernatant, the final EV frac- potential of lipid nanovesicle platforms in HCC tion will contain both EVs from the FBS and the space. Notably, Omega Therapeutics is leading cultured cells. To circumvent these issues, the use their phase I/II MYCHELANGELO™ trial of EV-depleted FBS or serum-free culture condi- (NCT05497453) evaluating OTX-2002 as mono- tions have been proposed, with each providing therapy or in combination with HCC standard of their own inherent limitations, including cell death, care (TKIs or ICIs), which is an mRNA therapeutic incomplete elimination of FBS-derived EVs, and encapsulated in lipid nanovesicle which decreases 120,121 changes to cellular differentiation/state. c-MYC gene expression through modifying the Moreover, when culturing cells, the passage number, c-Myc transcript via epigenetic modulation. cell seeding density, and timing of media harvest can They most recently (September 2023) have contribute to heterogeneity in cultured cells, and described preliminary results in 8 patients and 117,122 thus EVs isolated. observed on-target effects with associated decreases The third consideration is the scale of manufac- in c-MYC gene expression. This signals the tran- turing. For mass production of EVs, unique culture sition of siRNA/mRNA lipid nanovesicle therapeu- systems are needed, such as stacked culture vessels tics from the preclinical to clinical realm to target 117,118 or bioreactors. Also, each EV isolation proto- traditionally “undruggable” oncogenic drivers to be col (e.g., ultracentrifugation, precipitation, size- used in conjunction with standard of care agents exclusion chromatography, and filtration) present (i.e., TKIs or ICIs). differences in efficiency, quantity, purity, and quality of final EV formulations. For example, although centrifugation-based approaches improve EV pur- Conclusions and future perspectives ity, this is at the expense of cost and time. Lastly, with high-volume manufacturing, evaluating differ- Lipid nanovesicles are next-generation drug deliv- ences in batches is also important to consider. ery vehicles swiftly becoming part of the oncologist ORGANOGENESIS 9 Table 1. Clinical trials registered on clinicaltrials.Gov website for use of lipid nanovesicles and extracellular vesicles in oncology. Active or Name Identifier Stage Location Clinical Setting Agent(s) Utilized Completed Lipid Nanovesicle Based Therapeutics A Phase I First in Human Study to NCT05267899 Phase I Valkyrie Clinical Any solid tumor WGI-0301 is a lipid nanoparticle Active Evaluate the Safety, Tolerability, Trials (Los containing Akt-1 antisense and Pharmacokinetics of WGI- Angeles) oligonucleotide 0301 in Patients With Advanced Innovative Solid Tumors Clinical Research Institute (Whittier, CA) Dose Escalation and Efficacy Study NCT03323398 Phase I Multi-site Relapsed/ mRNA-2416 is a lipid nanoparticle Terminated of mRNA-2416 for Intratumoral ModernaTx Refractory Solid containing mRNA encoding for Injection Alone and in Tumors or OX40L Combination With Durvalumab Lymphoma for Participants With Advanced Malignancies TKM 080301 for Primary or NCT01437007 Phase I National Primary liver TKM-080301 is a lipid nanoparticle Completed Secondary Liver Cancer Institutes of cancer of liver containing siRNA against PLK1 Health metastases (polo-like kinase-1) Clinical Center Dose Escalation Study of mRNA- NCT03739931 Phase I Multi-site Relapsed/ mRNA-2752 is a lipid nanoparticle Active, 2752 for Intratumoral Injection to ModernaTx Refractory Solid containing mRNA encoding for Recruiting Participants in Advanced Tumors or OX40L, IL-23, and IL-36 g Malignancies Lymphoma Phase I, Multicenter, Dose Escalation NCT02110563 Phase I Multi-site Solid Tumors DCR-MYC is a lipid nanoparticle Terminated Study of DCR-MYC in Patients Dicerna Multiple Myeloma containing siRNA to MYC With Solid Tumors, Multiple Pharmaceuticals Non-Hodgkins oncogene Myeloma, or Lymphoma Lymphoma Pancreatic Neuroendocrine Tumors PNET NHL First-in-Human Study of INT-1B3 in NCT04675996 Phase I Multi-site Solid Tumor INT-1B3 is a lipid nanoparticle Active, Patients With Advanced Solid InteRNA containing miRNA-193a-3p Recruiting Tumors A Phase 1/2 Study to Evaluate OTX- NCT05497453 Phase I/ Multi-site HCC OTX-2002 is a mRNA therapeutic Active, 2002 in Patients With II Omega called an Omega epigenomic Recruiting Hepatocellular Carcinoma and Therapeutics controller which modulates MYC Other Solid Tumor Types Known gene expression; tested as for Association With the MYC monotherapy and in combination Oncogene (MYCHELANGELO I) with standard of care Extracellular Vesicle Based Therapeutics Study Investigating the Ability of NCT01294072 Phase I University of Colon Cancer Curcumin alone in capsule form Active, Plant Exosomes to Deliver Louisville (Arm 1), Curcumin combined with Recruiting Curcumin to Normal and Colon Hospital plant exosomes (Arm 2), or No Cancer Tissue intervention (Arm 3) Trial of a Vaccination With Tumor NCT01159288 Phase II Gustave Roussy, Lung Cancer Vaccine with tumor antigen-loaded Completed Antigen-loaded Dendritic Cell- Cancer exosomes derived from dendritic derived Exosomes (CSET 1437) Campus, cells Grand Paris Edible Plant Exosome Ability to NCT01668849 Phase I James Graham Head and Neck Plant (grape) exosomes to prevent Completed Prevent Oral Mucositis Associated Brown Cancer oral mucositis typically observed With Chemoradiation Treatment Cancer following chemoradiation of Head and Neck Cancer Center, University of Louisville An Open, Dose-escalation Clinical NCT05559177 Phase I Fudan Bladder Cancer Chimeric exosomal vaccines Active, Study of Chimeric Exosomal University prepared from autologous Recruiting Tumor Vaccines for Recurrent or Pudong sources from differentiated blood Metastatic Bladder Cancer Medical monocytes to antigen presenting Center cells (Continued) 10 B. M. LEHRICH AND E. R. DELGADO Table 1. (Continued). Active or Name Identifier Stage Location Clinical Setting Agent(s) Utilized Completed A Study of exoASO-STAT6 (CDK-004) NCT05375604 Phase I City of Hope Hepatocellular CDK-004 is a STAT6 antisense Active, not in Patients With Advanced National carcinoma and oligonucleotide in cell-derived recruiting Hepatocellular Carcinoma (HCC) Medical liver metastases exosomes and Patients With Liver Center Metastases From Either Primary Memorial Sloan Gastric Cancer or Colorectal Kettering Cancer (CRC) Cancer Center Sarah Cannon Research Institute Codiak Biosciences Antisense102: Pilot Immunotherapy NCT02507583 Phase I Thomas Glioma IGF-1 R/AS ODN is an Insulin-like Completed for Newly Diagnosed Malignant Jefferson growth factor receptor-1 Glioma University antisense oligonucleotide in Hospital exosomes derived from malignant glioma cells iExosomes in Treating Participants NCT03608631 Phase I MD Anderson Metastatic Exosomes derived from Active, With Metastatic Pancreas Cancer Cancer Pancreatic mesenchymal stromal cells with Recruiting With KrasG12D Mutation Center Cancer siRNA to KrasG12D mutation armamentarium. Compared to the administration respectively. Also, these nanovesicles are opsonized of “naked” drug, encapsulated drug within lipid by ApoE and recognized by the hepatocyte LDLR nanovesicles allows for reduced toxicity, improved for efficient targeting. Or functionalization of the biocompatibility, and improved in vivo efficacy nanovesicle may allow for directed cell-type through enhanced delivery to end-organ and target specificity. cell internalization. Several studies have illumi- There are distinct advantages and disadvantages nated the potential of lipid nanovesicles, both syn- of each platform (Table 2). To improve the transla- thetic and natural, as drug delivery platforms in tion of this new EV class of biologics to the clinic, preclinical models and in patients, with several there are several technical challenges, including companies licensing these technologies from aca- improving isolation techniques, component charac- 70,117 demia and translating their products to the clinic. terization, and manufacturing. Additionally, an These platforms are ideal drug delivery vehicles for enhanced understanding of the factors lending treating various liver pathologies, including cancer, toward high biocompatibility of EVs may augment due to the liver’s inherent dual blood supply and the development and translation of synthetic fenestrated endothelium to allow for efficient sys- nanovesicles. Despite these challenges, the future temic administration and hepatocyte delivery, is bright for nanovesicle therapeutic applications in Table 2. Advantages and disadvantages of different nanovesicle platforms for liver cancer. Advantages Disadvantages Nanoliposomes Endogenous targeting to liver via ApoE-LDLR uptake mechanism May have premature clearance by immune system before reaching end-organ Exogenous targeting to liver via GalNAc (and others) Cell-type specificity is challenged by vesicle size and membrane receptor components functionalization Can selectively encapsulate specific nucleic acid species of choice Scale-up manufacturing may be issue with high-cost Formulations already FDA approved for various liver pathologies Long term durability and bioactivity of the encapsulated payload Extracellular Vesicles Enhanced biocompatibility compared to nanoliposomes May contain other bioactive components not otherwise appreciated contributing to therapeutic effect Less off-target toxicity compared to nanoliposomes Isolation techniques may result in impurities May have improved cell-type targeting based on parental source of GMP standards not well established for industry mass production EVs derived Improved ability to evade host immune clearance compared to Lack of predictable and precise sizing may hamper translation as hepatocyte targeting nanoliposomes needs <200 nm ORGANOGENESIS 11 oncology, particularly EVs, and as technology in 2020 and predictions to 2040. 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Lipid Nanovesicle Platforms for Hepatocellular Carcinoma Precision Medicine Therapeutics: Progress and Perspectives

Organogenesis , Volume 20 (1): 1 – Dec 31, 2024

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Abstract

ORGANOGENESIS 2024, VOL. 20, NO. 1, 1–17 https://doi.org/10.1080/15476278.2024.2313696 REVIEW Lipid Nanovesicle Platforms for Hepatocellular Carcinoma Precision Medicine Therapeutics: Progress and Perspectives a,b a Brandon M. Lehrich and Evan R. Delgado a b Division of Experimental Pathology, Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; Medical Scientist Training Program, University of Pittsburgh, Pittsburgh, Pennsylvania, USA ABSTRACT ARTICLE HISTORY Received 6 July 2023 Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality globally. Revised 4 January 2024 HCC is highly heterogenous with diverse etiologies leading to different driver mutations potentiat- Accepted 30 January 2024 ing unique tumor immune microenvironments. Current therapeutic options, including immune checkpoint inhibitors and combinations, have achieved limited objective response rates for the KEYWORDS majority of patients. Thus, a precision medicine approach is needed to tailor specific treatment Cell therapy; exosomes; options for molecular subsets of HCC patients. Lipid nanovesicle platforms, either liposome- extracellular vesicles; (synthetic) or extracellular vesicle (natural)-derived present are improved drug delivery vehicles hepatocellular carcinoma; lipid nanovesicles; precision which may be modified to contain specific cargos for targeting specific tumor sites, with a natural medicine affinity for liver with limited toxicity. This mini-review provides updates on the applications of novel lipid nanovesicle-based therapeutics for HCC precision medicine and the challenges asso- ciated with translating this therapeutic subclass from preclinical models to the clinic. Introduction immune checkpoint inhibitors (ICIs). Despite ICIs demonstrating improved OS of roughly six Hepatocellular Carcinoma (HCC) is a growing months over TKIs, such as Sorafenib, the benefit global public health burden. HCC is the sixth is still marginal with only 25–30% response rates most common cancer globally, with > 900,000 5–7 in patients. cases each year, and the third highest in cancer- Therefore, novel targeted therapies used in related mortality, with > 800,000 deaths conjunction with immunotherapy, in a precision- each year. HCC typically follows a sequalae of medicine based approach, may overcome HCC chronic liver disease, with the main etiologies therapeutic resistance to ICIs in molecular subsets including Hepatitis B and C virus (HBV & of patients. Lipid nanovesicle platforms, either HCV) infection, alcohol-related disease, steatotic liposome- (synthetic) or extracellular vesicle (nat- liver diseases (SLD) (e.g., metabolic dysfunction ural)-derived, have demonstrated promise as drug associated SLD [MASLD], diabetes mellitus, obe- delivery vehicles to the liver and for targeted cancer sity), and toxin exposure (e.g., cigarette smoke, agents. These nanocarriers are ideal drug delivery aflatoxin, liver fluke). HCC has a dismal prog- vehicles which may be functionalized to harbor nosis with a 5-year overall survival (OS) rate of specific cargo molecules and “home” to specific ~ 15–20% and <18 months median survival with current therapeutic paradigms. Very few patients tumor sites, with native affinity for liver with lim- 8,9 are diagnosed at early stages where surgical resec- ited toxicity. This mini-review discusses HCC tion/transplantation is feasible and nearly molecular subclasses and current treatment para- curative. In fact, the vast majority of patients digms, along with applications of novel lipid nano- are diagnosed with advanced disease, limiting vesicle-based therapeutics for HCC precision their options to systemic agents, including tyro- medicine with a focus on naturally-derived nano- sine kinase inhibitors (TKIs) and, more recently, vesicle formulations, and the challenges associated CONTACT Brandon M. Lehrich brandon.lehrich@pitt.edu Division of Experimental Pathology, Department of Pathology, University of Pittsburgh School of Medicine, M246 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15232, USA; Evan R. Delgado evd7@pitt.edu Division of Experimental Pathology, Department of Pathology, University of Pittsburgh, S423 200 Lothrop St, Pittsburgh, Pennsylvania 15232, USA © 2024 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. 2 B. M. LEHRICH AND E. R. DELGADO with translating this new therapeutic subclass from Additionally, the inflamed class can be further sub- preclinical models to the clinic. divided into either immune-active or immune- exhausted, with the immune-active subclass repre- senting high adaptive immunity gene expression Genetic heterogeneity and immunologic with improved survival and reduced rates of recur- landscape rence. The immune exhausted subclass demon- strates activated stroma and immunosuppressive Over the last decade, next-generation sequencing gene set signatures. Overall, these classification technologies have been utilized to profile genetic systems illustrate how tumor genetics drive both drivers of HCC, guiding the path toward precision tumoral heterogeneity and specific tumor microen- medicine therapeutics. Our current understanding vironments, which may be differentially susceptible of the HCC genomic landscape includes major to various systemic agents, and thus, may require somatic mutations in TERT (~50%; telomere main- tailored treatment options for patients informed by tenance; promoter mutation and gain-of-function tissue and/or liquid biopsy. [GOF]), TP53 (~30%; cell cycle control; missense/ nonsense; loss-of-function [LOF]), CTNNB1 (~30%; Wnt/β-catenin signaling; missense; GOF), Current treatment modalities and patient ARID1A (~10%; chromatin remodeling; truncat- selection ing/missense; LOF), and TSC2 (~10%; cell growth; 10,11 deletions; LOF). Less common molecular dri- For advanced HCC, current standard of care has vers include FGF19 (~10%), AXIN1 (~6%), MYC shifted from the use of TKIs toward ICIs in the last 10,11 (~6%), APC (~5%), and MET (~2%). Some of decade. ICIs are monoclonal antibodies which these mutations may not be mutually exclusive; block the interaction between immune checkpoint however, mutations in Wnt/β-catenin pathway molecules (e.g., programmed death-ligand 1 members and TP53 tend to be mutually exclusive [PDL1] on tumor cells interacting with pro- events. This dichotomy also forms the foundation grammed cell death protein 1 [PD1] on T cells) for defining the various molecular subclasses of potentiating cytotoxic CD8+ T cell mediated tumor HCC described here. cell killing. The IMbrave 150 trial demonstrated The two main molecular classification systems 19.2 months median survival with atezolizumab proposed are the G1-G6 system by Boyault et al. (anti-PDL1 antibody) plus bevacizumab (anti- and the S1-S3 subgroups by Hoshida et al. Briefly, VEGF antibody) compared to 13.4 months median G1-G3 and S1-S2 subclasses represent proliferative/ survival with sorafenib (TKI). Also, the poorly differentiated tumors associated with chro- HIMALAYA trial demonstrated 16.4 months med- mosomal instability, high HBV viral load, and TP53 ian survival with the ICI combination of tremeli- mutations, while G5-G6 and S3 subclasses represent mumab (anti-CTLA4 antibody) plus durvalumab non-proliferative/well-differentiated tumors asso- (anti-PDL1 antibody) compared to 13.7 months ciated with chromosomal stability, alcohol/HCV/ median survival with sorafenib. Moreover, the 13,14 NASH-driven HCC, and CTNNB1 mutations. CARES-310 trial demonstrated 22.1 months med- More recently, HCC can be classified into inflamed ian survival with camrelizumab (anti-PD1 anti- (Hoshida S1-S2 subgroups) or non-inflamed body) plus the VEGFR2-targeted TKI rivoceranib 14,15 (Hoshida S3) subgroups. The inflamed class of compared to 15.2 months median survival with HCC (~25% of patients) demonstrates increased sorafenib. However, despite the improved OS in expression of gene signatures related to immune ICI treated patients, response rates overall remain infiltration (i.e., cytotoxic T cells, tertiary lymphoid relatively low with only 25–30% of patients achiev- structures [TLS], IFN alpha and gamma signaling, ing objective response rates (ORR). Low ORRs are and chemokines CXCL9, CXCL10), high immune poorly understood but have been linked to patient checkpoint immunohistochemical expression, tumor microenvironments with low tumor- CTNNB1-mutated depleted, and enrichment of infiltrating effector T lymphocyte density, high amplification in q13 locus (CCND1, FGF19). regulatory T cell density, and high expression of ORGANOGENESIS 3 oncofetal genes. Thus, to improve these response include small unilamellar vesicles (<100 nm), large rates, an individualized treatment approach is war- unilamellar (>100 nm), and multilamellar vesicles ranted to guide therapeutic selection based on (>500 nm), with the former two more typically used underlying genetic alterations. This may be aided for nanomedicine applications. Excellent reviews by tissue or liquid biopsy for key drivers of HCC elsewhere discuss preparation methodologies (e.g., tumorigenesis. However, an improved under- reverse-phase evaporation, freeze-thaw method, standing of which genetic drivers influence the vaporization technique, and others) of nanoliposome 28,29 immune microenvironment resistant to ICI formulations. Briefly, the phospholipid character- response is warranted for screening, along with istics (e.g., degrees of unsaturation, quantity of fatty needing an expanded arsenal of drugs targeting acid moieties, and others) and the number of choles- these underlying pathways to be used in conjunc- terol molecules can affect the membrane 30,31 tion with ICIs. configuration. Further modifications to the nano- On a molecular basis, the Wnt/β-catenin pathway liposomal structure include the addition of either 32 33 has been the most prominently studied pathway to polyethylene glycol or surface ligands, which evaluate ICI resistance, yet controversy remains avoids host immune system elimination and whether all mutations in the pathway decrease improves cellular targeting, respectively. For cellular immune infiltration to the same degree, and thus uptake, nanovesicles are internalized typically 16, 21, 22 ICI resistance. Moreover, despite studies through endocytosis or phagocytosis, with nanovesi- demonstrating the feasibility of prospective tissue cle structure influencing which mechanistic process. genotyping to identify clinically actionable driver Efficient perfusion of the liver through its dual blood mutations, very few patients receive personalized supply mediates optimal delivery, and lipid nanove- therapeutic intervention. The major driver muta- sicle uptake is augmented due to its fenestrated 23; tions in HCC are currently not actionable there- endothelium. Additionally, opsonization by ApoE fore, efforts should be made to identify and stratify facilitates low-density lipoprotein (LDL) receptor patients which may respond to current druggable (LDLR)-mediated uptake into hepatocytes (“endo- targets, including FGF19/FGFR4, VEGF, TSC1/2, genous targeting”), while engineering 24,25 and MET inhibitors. Although none of these N-acetylgalactosamine (GalNAc)-PEG-lipid on the targets have shown clinical responses, these molecu- nanovesicle surface can target the asialoglycoprotein lar events may be co-occurring in the background of receptor (ASGPR) on hepatocytes (“exogenous tar- strong drivers (e.g., TP53, CTNNB1), and thus geting”), with both options providing efficient deliv- 35,36 a combination of therapeutics may need to be even- ery to the liver. This well-characterized ApoE- tually employed. Thus, further studies are needed in LDLR endogenous hepatocyte targeting mechanism clinically relevant animal models to determine the is the route by which Patisiran, the first FDA differential response of ICIs in combination with approved siRNA-based drug, facilitates its end- targeted therapy approaches in unique molecular organ targeting to the liver and mechanism of subsets of HCC. action. The remainder of this section will discuss the applications of nanoliposomes as targeted drug delivery vehicles in various preclinical models of Synthetic lipid nanovesicle drug delivery HCC as potential precision medicine therapeutic platforms for HCC platforms (Figure 1). Synthetic lipid nanovesicles have conventionally been The realization of using lipid nanovesicles as nanoliposome-based formulations containing dis- a targeted therapy delivery vehicle for liver cancer tinct molecular entities, including either RNA inter- in humans was first achieved in 2013 by Tabernero ference (RNAi) technologies or chemotherapeutic and colleagues in their phase I study. This lipid drugs. Nanoliposomes typically size range between nanovesicle (ALN-VSP) encapsulated siRNAs tar- 10 nm to 200 nm in diameter and are composed of geting vascular endothelial growth factor (VEGF) a phospholipid bilayer with or without cholesterol, and kinesin spindle protein (KSP) to treat patients resulting in an aqueous interior and an outer hydro- with liver metastases. Tumor regression was phobic exterior. The main types of nanoliposomes achieved in nearly 50% of the patients in the trial. 4 B. M. LEHRICH AND E. R. DELGADO Figure 1. Schematic representation of nanoliposome and extracellular vesicle loading strategies, cellular uptake mechanisms of these drug delivery vehicles, and clinical parameters to monitor for toxicity in patients. Figure made in BioRender. These results, demonstrating the safety, tolerability, 3 components: 1) SP94 peptide (specific to HCC ability to achieve target downregulation in the liver, cells), 2) YSK05 lipid (increased cytotoxic effects and short-term clinical responses underscore the and limited endosomal escape), and 3) specific importance and potential of using lipid nanovesicles phosphatidylcholine/cholesterol ratio (improves for HCC therapy. liposome stability). They demonstrated both Nanoliposomes encapsulating RNA interference in vitro and in vivo that their nanoliposome had (RNAi) platforms, such as small interfering RNAs specific uptake to HCC cells over normal hepato- (siRNAs), microRNAs (miRNAs), or messenger cytes, potentiated sorafenib’s effects, and resulted 44,45 RNAs (mRNAs) have been administered as drug in profound tumor regressions (~70%). delivery systems in preclinical models of HCC with Additionally, Woitok et al. delivered siRNA target- considerable success in terms of safety, tolerability, ing Jun N-terminal kinase-2 (Jnk2), known to and treatment response. Various groups have affect fibrosis progression, in lipid nanovesicle to attempted to use RNAi to either target oncogenic mice with chronic liver disease and demonstrated genes involved in cell cycle regulation and cell decreased HCC premalignant nodules and a shift proliferation/death pathways, or directly inhibit in the immune microenvironment of the diseased driver mutations deemed to be traditionally liver. Moreover, targeting key cellular pathways “undruggable.” For reviews on how RNAi plat- in HCC with siRNAs has also been feasible as forms are processed following cellular uptake, we demonstrated by the work from Fitamant and refer the interested reader to the following colleagues. They delivered nanovesicles contain- 39–42 reviews. An example of directly targeting ing siRNA to Yes-associated protein 1 (YAP), a key oncogenic factors is illustrated by work from downstream transcriptional co-activator of Hippo Younis colleagues where they encapsulated both signaling, resulting in tumor regression through a siRNA to midkine (MK; a gene involved in directing hepatocyte differentiation to normal many cellular pathways including apoptosis and hepatocyte-like cells. Other groups have also deliv- angiogenesis and the chemotherapeutic, sorafe- ered nanoliposomes containing siRNAs targeting 48 49 nib, into a nanoliposome functionalized to contain PD-L1, T cell immunoglobulin mucin-3 (Tim- ORGANOGENESIS 5 3; immune checkpoint molecule), vascular release of chemotherapeutic agents through stimuli endothelial growth factor (VEGF; angiogenic fac- responses. Examples of this include using either 51 64 65,66 tor), alpha-fetoprotein (AFP; biomarker for temperature sensitive, pH responsive, photo- HCC), cyclo-oxygenase-2 (COX-2; important sensitive, magnetic-sensitive, or ultrasound-guided for prostaglandin synthesis in inflammatory pro- lipids. In terms of temperature-sensitive lipids, 53 64 cesses), hypoxia inducible factor 1 subunit alpha Peng et al. utilized PF127 (copolymer) which 6− 6 (HIF1a), or RNA N methyladenosine (m A) has temperature-sensitive properties and aids in reader protein YTHDF1 either alone or in com- degrading the nanoliposome following photother- bination with chemotherapeutics. Moreover, mal conversion of IR-780 (a near-infrared [NIR] miRNAs can be packaged into nanoliposomes to dye) also contained on the nanoliposome surface. target specific cellular pathways. For example, This combination of PF127 and IR-780 allowed for Zhao et al. loaded miR-375 and sorafenib in nano- efficient doxorubicin and sorafenib release at the liposomes to hinder autophagic processes and tumor site in vivo. Also, as illustrated by Li et al., reduce tumor burden. Lastly, mRNAs may also interchanging the nanoliposome bilayer to include be packaged into nanovesicles for HCC therapy. the cationic lipid (2E)-4-(dioleostearin)-amino Lai et al. demonstrated that delivery of IL-12 -4-carbonyl-2-butenonic (DC), can allow for direct mRNA in nanovesicles reduced tumor burden tumor cell internalization upon conformational and prolonged survival of transgenic MYC- change in the acidic tumor microenvironment, induced HCC mice. This effect was also asso- and subsequently release its cargo in the acidified ciated with a shift toward a more anti-tumor endosome. This allowed for reduced drug toxicity immune microenvironment with increases in and targeting of tumor cells over normal hepato- T helper cells and IFNγ expression. Similar cytes. Overall, the lipid composition can allow for effects were seen with mRNA for OX40L encapsu- improved pharmacokinetics and tumor cell lated nanovesicles. Overall, lipid nanoparticles internalization. provide an efficient platform to deliver both che- motherapeutics and gene therapy at subtoxic doses Extracellular vesicle-based drug delivery 44,53 with high efficiency and stability. platforms for HCC As previously discussed, modifying the outer shell of the nanoliposome can improve the delivery effi- Extracellular vesicles (EVs) are lipid nanovesicles ciency and targeting to the desired end organ. For (50 nm to >2000 nm) which are spontaneously pro- targeting HCC cells specifically, various groups have duced by nearly all mammalian cells and released functionalized nanoliposomes to target CXCR4 high into extracellular fluid as part of autocrine, para- expressing cells given its sorafenib resistance crine, and endocrine cell-to-cell signaling mechanisms. These studies have demonstrated circuits. There are various EV subclasses, includ- reduced toxicity with targeted nanoparticles and ing exosomes (derived from endosomal membrane synergistic effects when combined with chemothera- trafficking machinery), microvesicles (outward 58–60 pies, such as a sorafenib. Additionally, GalNAc- plasma membrane blebbings), and apoptotic conjugated nanovesicles have demonstrated consid- bodies (from apoptotic processes). All EVs contain erable success in highly relevant animal models of cargos comprising various membrane and soluble molecular subsets of HCC with the nanoliposomes proteins, nucleic acid species, and metabolites, encapsulating siRNAs to oncogenic drivers, such as which are specific to their cell of origin. Once 61,62 CTNNB1. Also, the lipid configuration and released into the extracellular milieu, EVs travel inclusion of PEG/mannose into the membrane can systemically until they make contact with and fuse also affect targeting to different liver cell types. with their target cell plasma membrane through Therefore, using targeting molecules on nanolipo- various endocytic or phagocytic mechanisms. some surface can improve the efficiency of tumor The natural ability for EVs to avoid immune sys- cell transfection and diminish off-target effects. tem clearance, systemically travel to end organs, Moreover, another strategy is modifying the and package cargos within lipid bilayers has made lipid composition of the liposome for controllable them an attractive tool for drug delivery. Through 6 B. M. LEHRICH AND E. R. DELGADO 61, 62, 82 the use of nanomedicine platforms, EV mimetics with others previously mentioned provide are being translated to the clinic as novel drug direct evidence that therapeutically targeting onco- delivery vehicles. Various researchers have devel- genic mutations with siRNAs are effective approaches oped different EV mimetic technologies, either to treat HCC. And, using EVs may have improved through modifying parental cells (e.g., stem cells, RNA delivery efficiency, unique targeting capabilities, fibroblasts, immune cells) and isolating their EVs and enhanced biocompatibility compared to syn- 83–85 for delivery, or ex vivo loading of cargo compo- thetic nanovesicle platforms. nents into EVs. This section will explore applica- Similar to siRNAs, miRNAs packaged into EVs tions of EV mimetics for HCC precision medicine offer another platform to target actively proliferating in preclinical models (Figure 1), and we refer to the cancer cells. Many miRNAs have been implicated in reader to excellent reviews detailing techniques HCC pathogenesis, including miR-21, miR-125b, used for preparation of EV-based therapeutics, miR-155, and miR-221/222. Particularly, miR- 70–74 including their isolation and purification. 125b down-regulation is associated with worse overall The main class of EV mimetics utilized for HCC survival. Baldari and colleagues isolated EVs (via targeted therapy are siRNA- encapsulated EVs, which polymer-based methods) from adipose-derived stro- target specific mRNAs encoding oncogenic signaling mal cells (ADSCs) engineered to express miR-125b proteins. Various groups have identified target genes, with a unique “ExoMotif” sequence that increases which when suppressed, may synergize with ICIs. release of miR-125b into EVs. These EVs were One target is CD38, a transmembrane protein delivered in vitro to HepG2 and HuH-7 cells and which is aberrantly expressed in many tumors and reduced cell proliferation, along with expression of associated with a pro-inflammatory tumor microen- p53 signaling pathway components. In another vironment, and has been shown to be associated ICI study, Mahati and colleagues loaded mesenchymal 75,76 response. EVs isolated from bone marrow stem cell (MSC)-derived EVs with miR-26a (via elec- mesenchymal stem cells packaged with siRNA to troporation) and observed impaired cell proliferation CD38 (via electroporation) reduced HCC tumor bur- and migration in vitro, along with reduced tumor den, metastatic potential, repolarized macrophages burden in subcutaneous HCC models. Lastly, from M2 (immunosuppressive) to M1 (pro- Ellipilli and colleagues demonstrated that combined inflammatory) phenotype, and improved ICI Paclitaxel and miR-122 (liver specific miRNA; response. Other genes/pathways identified which reduced levels shown in HCC) administration within have been targeted with siRNAs packaged in EVs, GalNAc-EVs reduced tumor burden in multiple mice include components of the ferroptosis pathway xenograft HCC models. Complementary to RNAi, (GPX4 and DHODH), cell cycle regulation another strategy for EV therapeutics includes exogen- 78 79 (CDK1), JAK/STAT pathway (STAT6), and ous or endogenous small molecule and protein load- NFkB pathway (p50 subunit). Rather than directly ing. Exogenous protein loading of EVs has been targeting translation of molecules displayed on tumor excellently reviewed elsewhere, but includes techni- cell surface mediating immunosuppression, another ques such as mixing, sonication, electroporation, approach is targeting the underlying genetic mutation freeze-thaw cycles, and extrusion, with sonication 91–94 of the tumor cell. Matusda and colleagues designed an and extrusion being the most efficient. siRNA targeting CTNNB1 delivered within EVs. Monoclonal antibodies, nanobodies, and various Using the Met/β-catenin mouse model (which repre- cytokines can even be packaged into EVs to target sents ~ 10% of human HCC), they remarkably specific immune checkpoint molecules to induce 95,96 demonstrated that delivery of milk-derived EVs native immune activity. However, these techni- 92,97 encapsulating siRNA to CTNNB1 (using transfection ques may damage the membrane integrity of EVs. techniques) reduced tumor burden, in part through Endogenous protein loading into EVs is a novel tech- reversing the immunosuppressive tumor microenvir- nique which hijacks cell signaling cascades to load onment driven by β-catenin, which allowed for particular payloads into EVs, which can be isolated, synergy with ICIs. Another group utilized a similar and subsequently administered as therapeutics. platform, but functionalized the EVs to target Different groups have utilized the ability of FK506 EpCAM-positive HCC cells. These studies along binding protein (FKBP) and FKBP12–rapamycin- ORGANOGENESIS 7 binding (FRB) domain to heterodimerize following a therapeutic source of EVs are ADSCs. Wu and 98,99 rapamycin administration. The FRB domain is colleagues revealed that ADSC-EVs (isolated via fused to the protein of interest via a GGSGG linker, ultracentrifugation of culture media) decreased hepa- and the FKBP domain is fused to a canonical EV tic fibrosis and glutamine synthetase levels, suggesting protein (e.g., CD81 or CD63) via the that this may have therapeutic potential in subsets of N-myristoylation sequence to facilitate protein entry HCC. Moreover, another cell type which has into EVs. Cell lines can be modified to express these demonstrated promise are dendritic cell (DC)- fusion proteins and EVs can be isolated and delivered derived EVs. The pathogenesis of CTNNB1- 98,99 in vivo for effective protein delivery. Small mole- mutated HCC involves defective recruitment of cule/chemotherapeutic agent packaging into EVs DCs, likely making DC-EVs an interesting plat- have demonstrated potential, including the use of form as an HCC therapeutic. Lu and colleagues sys- 100 101 102 doxorubicin, norcantharidin, and sorafenib. temically administered DC-EVs in three different Additionally, Cas9 ribonucleoprotein can be pack- HCC models and observed shifts in the tumor micro- aged into EVs and delivered in vivo to liver, offering environment such as increases in cytotoxic CD8 103–105 avenues for HCC gene therapy. Overall, these T-cells and fewer immunosuppressive T regulatory methods of protein/small molecule packaging are cells, which associated with tumor regression. appealing options for therapeutic delivery to liver. Lastly, M1 macrophages-derived EVs loaded with In the last two decades, recombinant Adeno- docosahexaenoic acid have been shown to induce associated viruses (AAVs) have been explored as ferroptosis and reduce tumor burden in orthotopic gene delivery vehicles for cancer due to their ability HCC models. Therefore, EVs isolated from allo- to target many cell types and long-lasting gene geneic sources have intrinsic capabilities to alter expression. More recently, EVs have been tumor cell survival and growth. However, autolo- shown to be associate with isolated AAVs (termed gous-derived EVs may have improved tumor target- “vexosomes”) during virus isolation from cell- ing properties. Villa et al. illustrated that EVs derived culture media. These vexosomes have become an from blood plasma of cancer patients had selective 107,108 alternate gene delivery vehicle. Moreover, uptake into associated patient-derived xenograft vexosomes protect AAVs from antibody neutrali- (PDX) mouse models. Therefore, autologous EV zation, a major issue for AAV in vivo translation. sources may be another pipeline for manufacture Khan et al. isolated AAV6-derived vexosomes (via with improved tumor-specific targeting properties. ultracentrifugation) containing an inducible cas- pase 9 (iCasp9), which upon delivery with Challenges in good manufacturing practices for a prodrug (AP20187), results in impaired HCC nanovesicle therapeutics cell proliferation in vitro and tumor cell death in vivo via apoptosis. Overall, vexosomes are Many of the challenges of translating nanovesicle another gene therapy-based EV mimetic technol- therapeutics are shared between synthetic and ogy which are highly efficient delivery vehicles, natural platforms; however, this section will require lower therapeutic doses than AAVs, and focus on the nuances associated with translating are not cumbersome to manufacture. EV-based therapeutics. The first consideration is Lastly, EVs isolated from allogeneic or autologous isolation purity. Current clinical Good cell sources are another therapeutic option for HCC. Manufacturing Processes (GMP) of therapeutic Kim and colleagues have demonstrated that EVs iso- EVs may lead to downstream isolation of con- lated from natural killer (NK) cells, which contain taminants (e.g., viral) from cell culture proteins important for mediating immunogenic cell supernatants. For regulatory agency approval death, can functionally impair HCC growth in vitro of EVs, a complete biochemical characterization and in vivo. These NK-EVs (isolated via ultracen- is required for biologics, which remains incom- trifugation) express granzyme B, FasL, and TRAIL plete due to technological limitations and EV and mediate apoptosis through inducing caspase-3, isolation best practices. Additionally, given 7, 8, and 9 upon internalization in tumor cells. EVs are a cell-free therapy, the mechanisms of Additionally, another cell type with promise as cellular uptake/targeting, cargo delivery/release, 8 B. M. LEHRICH AND E. R. DELGADO and an understanding of the precise bioactive and Oncology clinical trials implementing 117,118 nanovesicle platforms nonactive components are unclear. Whether the membrane lipids/proteins, or the The translation of lipid nanoparticles and EVs to proteins/nucleic acids in the lumen, or both, con- clinical practice as HCC therapies has not moved tribute to the intended therapeutic effect is not swiftly. Currently, EVs are being studied as diag- determined. Therefore, extensive functional nostic biomarkers for HCC to detect initial diag- assays, “–omic,” and imaging platforms are nosis, response to therapy, and disease needed to fully elucidate and differentiate the 125 126, 127 recurrence using DNA mutations or physiochemical properties and bioactivity of 128 129 methylation patterns, mRNA /miRNA EVs. The International Society for Extracellular 130 131, 132 signatures, or proteins encapsulated in Vesicles (ISEV) has established guidelines for their lumen. This section will briefly cover in- clinical GMP of therapeutic EVs. human studies in oncology which has successfully The second consideration is cellular source and translated nanovesicle therapeutic platforms to the cell culture ecosystems of therapeutic EVs. As dis- clinic. To investigate whether lipid nanovesicles cussed in the previous section, cellular sources of were actively being translated into clinical trials, therapeutic EVs for cancer can include either stem we surveyed the clinicaltrials.gov website to search cells, immune cells, or nonparenchymal/stromal for active or terminated trials. A review of the cells. Each of these cell types require different culture clinicaltrials.gov website for clinical trials related methods and release differing quantities of EVs. to “cancer” and “exosomes” yielded 132 studies, Additionally, cell culture practices of these cell with 7 unique studies focusing on therapeutic types typically include utilizing fetal bovine serum applications (Table 1). Additionally, a review for (FBS) as a culture media supplement, which presents clinical trials related to “cancer” and “nanovesicle” challenges due to introducing FBS-derived EVs into yielded 12 studies, with 7 unique studies focusing the pool of cell culture-derived EVs. Simply, this on therapeutic applications (Table 1). Overall, contamination means that upon isolation of EVs there are few trials investigating the therapeutic from the cell culture supernatant, the final EV frac- potential of lipid nanovesicle platforms in HCC tion will contain both EVs from the FBS and the space. Notably, Omega Therapeutics is leading cultured cells. To circumvent these issues, the use their phase I/II MYCHELANGELO™ trial of EV-depleted FBS or serum-free culture condi- (NCT05497453) evaluating OTX-2002 as mono- tions have been proposed, with each providing therapy or in combination with HCC standard of their own inherent limitations, including cell death, care (TKIs or ICIs), which is an mRNA therapeutic incomplete elimination of FBS-derived EVs, and encapsulated in lipid nanovesicle which decreases 120,121 changes to cellular differentiation/state. c-MYC gene expression through modifying the Moreover, when culturing cells, the passage number, c-Myc transcript via epigenetic modulation. cell seeding density, and timing of media harvest can They most recently (September 2023) have contribute to heterogeneity in cultured cells, and described preliminary results in 8 patients and 117,122 thus EVs isolated. observed on-target effects with associated decreases The third consideration is the scale of manufac- in c-MYC gene expression. This signals the tran- turing. For mass production of EVs, unique culture sition of siRNA/mRNA lipid nanovesicle therapeu- systems are needed, such as stacked culture vessels tics from the preclinical to clinical realm to target 117,118 or bioreactors. Also, each EV isolation proto- traditionally “undruggable” oncogenic drivers to be col (e.g., ultracentrifugation, precipitation, size- used in conjunction with standard of care agents exclusion chromatography, and filtration) present (i.e., TKIs or ICIs). differences in efficiency, quantity, purity, and quality of final EV formulations. For example, although centrifugation-based approaches improve EV pur- Conclusions and future perspectives ity, this is at the expense of cost and time. Lastly, with high-volume manufacturing, evaluating differ- Lipid nanovesicles are next-generation drug deliv- ences in batches is also important to consider. ery vehicles swiftly becoming part of the oncologist ORGANOGENESIS 9 Table 1. Clinical trials registered on clinicaltrials.Gov website for use of lipid nanovesicles and extracellular vesicles in oncology. Active or Name Identifier Stage Location Clinical Setting Agent(s) Utilized Completed Lipid Nanovesicle Based Therapeutics A Phase I First in Human Study to NCT05267899 Phase I Valkyrie Clinical Any solid tumor WGI-0301 is a lipid nanoparticle Active Evaluate the Safety, Tolerability, Trials (Los containing Akt-1 antisense and Pharmacokinetics of WGI- Angeles) oligonucleotide 0301 in Patients With Advanced Innovative Solid Tumors Clinical Research Institute (Whittier, CA) Dose Escalation and Efficacy Study NCT03323398 Phase I Multi-site Relapsed/ mRNA-2416 is a lipid nanoparticle Terminated of mRNA-2416 for Intratumoral ModernaTx Refractory Solid containing mRNA encoding for Injection Alone and in Tumors or OX40L Combination With Durvalumab Lymphoma for Participants With Advanced Malignancies TKM 080301 for Primary or NCT01437007 Phase I National Primary liver TKM-080301 is a lipid nanoparticle Completed Secondary Liver Cancer Institutes of cancer of liver containing siRNA against PLK1 Health metastases (polo-like kinase-1) Clinical Center Dose Escalation Study of mRNA- NCT03739931 Phase I Multi-site Relapsed/ mRNA-2752 is a lipid nanoparticle Active, 2752 for Intratumoral Injection to ModernaTx Refractory Solid containing mRNA encoding for Recruiting Participants in Advanced Tumors or OX40L, IL-23, and IL-36 g Malignancies Lymphoma Phase I, Multicenter, Dose Escalation NCT02110563 Phase I Multi-site Solid Tumors DCR-MYC is a lipid nanoparticle Terminated Study of DCR-MYC in Patients Dicerna Multiple Myeloma containing siRNA to MYC With Solid Tumors, Multiple Pharmaceuticals Non-Hodgkins oncogene Myeloma, or Lymphoma Lymphoma Pancreatic Neuroendocrine Tumors PNET NHL First-in-Human Study of INT-1B3 in NCT04675996 Phase I Multi-site Solid Tumor INT-1B3 is a lipid nanoparticle Active, Patients With Advanced Solid InteRNA containing miRNA-193a-3p Recruiting Tumors A Phase 1/2 Study to Evaluate OTX- NCT05497453 Phase I/ Multi-site HCC OTX-2002 is a mRNA therapeutic Active, 2002 in Patients With II Omega called an Omega epigenomic Recruiting Hepatocellular Carcinoma and Therapeutics controller which modulates MYC Other Solid Tumor Types Known gene expression; tested as for Association With the MYC monotherapy and in combination Oncogene (MYCHELANGELO I) with standard of care Extracellular Vesicle Based Therapeutics Study Investigating the Ability of NCT01294072 Phase I University of Colon Cancer Curcumin alone in capsule form Active, Plant Exosomes to Deliver Louisville (Arm 1), Curcumin combined with Recruiting Curcumin to Normal and Colon Hospital plant exosomes (Arm 2), or No Cancer Tissue intervention (Arm 3) Trial of a Vaccination With Tumor NCT01159288 Phase II Gustave Roussy, Lung Cancer Vaccine with tumor antigen-loaded Completed Antigen-loaded Dendritic Cell- Cancer exosomes derived from dendritic derived Exosomes (CSET 1437) Campus, cells Grand Paris Edible Plant Exosome Ability to NCT01668849 Phase I James Graham Head and Neck Plant (grape) exosomes to prevent Completed Prevent Oral Mucositis Associated Brown Cancer oral mucositis typically observed With Chemoradiation Treatment Cancer following chemoradiation of Head and Neck Cancer Center, University of Louisville An Open, Dose-escalation Clinical NCT05559177 Phase I Fudan Bladder Cancer Chimeric exosomal vaccines Active, Study of Chimeric Exosomal University prepared from autologous Recruiting Tumor Vaccines for Recurrent or Pudong sources from differentiated blood Metastatic Bladder Cancer Medical monocytes to antigen presenting Center cells (Continued) 10 B. M. LEHRICH AND E. R. DELGADO Table 1. (Continued). Active or Name Identifier Stage Location Clinical Setting Agent(s) Utilized Completed A Study of exoASO-STAT6 (CDK-004) NCT05375604 Phase I City of Hope Hepatocellular CDK-004 is a STAT6 antisense Active, not in Patients With Advanced National carcinoma and oligonucleotide in cell-derived recruiting Hepatocellular Carcinoma (HCC) Medical liver metastases exosomes and Patients With Liver Center Metastases From Either Primary Memorial Sloan Gastric Cancer or Colorectal Kettering Cancer (CRC) Cancer Center Sarah Cannon Research Institute Codiak Biosciences Antisense102: Pilot Immunotherapy NCT02507583 Phase I Thomas Glioma IGF-1 R/AS ODN is an Insulin-like Completed for Newly Diagnosed Malignant Jefferson growth factor receptor-1 Glioma University antisense oligonucleotide in Hospital exosomes derived from malignant glioma cells iExosomes in Treating Participants NCT03608631 Phase I MD Anderson Metastatic Exosomes derived from Active, With Metastatic Pancreas Cancer Cancer Pancreatic mesenchymal stromal cells with Recruiting With KrasG12D Mutation Center Cancer siRNA to KrasG12D mutation armamentarium. Compared to the administration respectively. Also, these nanovesicles are opsonized of “naked” drug, encapsulated drug within lipid by ApoE and recognized by the hepatocyte LDLR nanovesicles allows for reduced toxicity, improved for efficient targeting. Or functionalization of the biocompatibility, and improved in vivo efficacy nanovesicle may allow for directed cell-type through enhanced delivery to end-organ and target specificity. cell internalization. Several studies have illumi- There are distinct advantages and disadvantages nated the potential of lipid nanovesicles, both syn- of each platform (Table 2). To improve the transla- thetic and natural, as drug delivery platforms in tion of this new EV class of biologics to the clinic, preclinical models and in patients, with several there are several technical challenges, including companies licensing these technologies from aca- improving isolation techniques, component charac- 70,117 demia and translating their products to the clinic. terization, and manufacturing. Additionally, an These platforms are ideal drug delivery vehicles for enhanced understanding of the factors lending treating various liver pathologies, including cancer, toward high biocompatibility of EVs may augment due to the liver’s inherent dual blood supply and the development and translation of synthetic fenestrated endothelium to allow for efficient sys- nanovesicles. Despite these challenges, the future temic administration and hepatocyte delivery, is bright for nanovesicle therapeutic applications in Table 2. Advantages and disadvantages of different nanovesicle platforms for liver cancer. Advantages Disadvantages Nanoliposomes Endogenous targeting to liver via ApoE-LDLR uptake mechanism May have premature clearance by immune system before reaching end-organ Exogenous targeting to liver via GalNAc (and others) Cell-type specificity is challenged by vesicle size and membrane receptor components functionalization Can selectively encapsulate specific nucleic acid species of choice Scale-up manufacturing may be issue with high-cost Formulations already FDA approved for various liver pathologies Long term durability and bioactivity of the encapsulated payload Extracellular Vesicles Enhanced biocompatibility compared to nanoliposomes May contain other bioactive components not otherwise appreciated contributing to therapeutic effect Less off-target toxicity compared to nanoliposomes Isolation techniques may result in impurities May have improved cell-type targeting based on parental source of GMP standards not well established for industry mass production EVs derived Improved ability to evade host immune clearance compared to Lack of predictable and precise sizing may hamper translation as hepatocyte targeting nanoliposomes needs <200 nm ORGANOGENESIS 11 oncology, particularly EVs, and as technology in 2020 and predictions to 2040. J Hepatol. 2022;77 (6):1598–606. doi:10.1016/j.jhep.2022.08.021 . advances, these roadblocks will only become sur- 2. Singal AG, Lampertico P, Nahon P. Epidemiology and passed and push these biologics toward clinical surveillance for hepatocellular carcinoma: new trends. practice. For translation of EV therapeutics, lessons J Hepatol. 2020;72(2):250–61. doi:10.1016/j.jhep.2019. may be learned from some of the hurdles overcome 08.025 . by those involved in translating nanoliposome 3. Llovet JM, Kelley RK, Villanueva A, Singal AG, formulations. For example, for nanoliposomes, Pikarsky E, Roayaie S, Lencioni R, Koike K, Zucman- Rossi J, Finn RS. et al. Hepatocellular carcinoma. Nat great detail was undertaken to understand how the Rev Dis Primers. 2021;7(1):6. doi:10.1038/s41572-020- composition of ionizable lipids, various active drug 00240-3 . loading techniques, and the cholesterol composition 4. Galle PR, Forner A, Llovet JM, Mazzaferro V, in the membrane affected drug stability, and thus Piscaglia F, Raoul J-L, Schirmacher P, Vilgrain V, enhanced in vivo activity. Additionally, the size of European Association for the Study of the Liver. the nanovesicle plays an important role in the ability Electronic address eee, European association for the study of the L. EASL clinical practice guidelines: man- to target the liver (and specific cell-type), with stu- agement of hepatocellular carcinoma. J Hepatol. dies concluding <100 nm is ideal for hepatocyte 136,138 2018;69(1):182–236. doi:10.1016/j.jhep.2018.03.019 . delivery. Interrogation of all these different 5. Cheng AL, Qin S, Ikeda M, Galle PR, Ducreux M, tunable characteristics of nanovesicles for EV- Kim TY, Lim HY, Kudo M, Breder V, Merle P. et al. based drug delivery vehicles will ultimately improve Updated efficacy and safety data from IMbrave150: their translatability to the clinic. Atezolizumab plus bevacizumab vs. sorafenib for unre- sectable hepatocellular carcinoma. J Hepatol. 2022;76 (4):862–73. doi:10.1016/j.jhep.2021.11.030 . Acknowledgments 6. Bejjani AC, Finn RS. Hepatocellular carcinoma: pick the winner—tyrosine kinase inhibitor versus immuno- The authors would like to acknowledge the University of oncology agent–based combinations. J Clin Oncol. Pittsburgh School of Medicine Cell Therapy Course directed 2022;40(24):2763–73. doi:10.1200/JCO.21.02605 . by Dr. Alejandro Soto-Gutierrez and Dr. Diana Metes. The 7. Abou-Alfa GK, Lau G, Kudo M, Chan SL, Kelley RK, authors would also like to acknowledge support from Furuse J, Sukeepaisarnjaroen W, Kang Y-K, Van Dao T, Dr. Satdarshan P. Monga. De Toni EN. et al. Tremelimumab plus durvalumab in unresectable hepatocellular carcinoma. NEJM Evidence. 2022;1(8):EVIDoa2100070. doi:10.1056/ Disclosure statement EVIDoa2100070 . 8. 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Journal

OrganogenesisTaylor & Francis

Published: Dec 31, 2024

Keywords: Cell therapy; exosomes; extracellular vesicles; hepatocellular carcinoma; lipid nanovesicles; precision medicine

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