Next-Generation Anticancer Peptides: Engineering, Nanotheranostics and Clinical Translation

Nanotheranostics · 2026-04-16

Anticancer peptides (ACPs) have emerged as a transformative class of next-generation therapeutics that bridge molecular precision with multifunctional tunability. Unlike many conventional small molecule chemotherapeutics, ACPs offer intrinsic selectivity toward malignant cells through preferential membrane targeting, immunomodulation, and disruption of oncogenic signalling pathways. Advances in peptide engineering, including sequence optimization, incorporation of non-natural amino acids, cyclization, stapling, PEGylation, and structure-activity relationship-guided refinement, have substantially improved their stability, potency, and pharmacokinetic performance. Parallel progress in nanotechnology has further expanded the translational potential of ACPs by enabling controlled release, cancer cell specific targeting, and multimodal theranostic integration. Lipid nanoparticles, solid lipid nanoparticles, polymeric systems, dendrimers, mesoporous silica nanoparticles, and stimuli-responsive platforms now provide multiple and combinatorial strategies to overcome biological barriers, enhance intracellular delivery, and minimize systemic toxicity. Emerging concepts such as enzyme-activated nanocarriers, ligand-directed precision delivery, and light- or pH-responsive systems are redefining and energizing the spatial and temporal control of peptide therapeutics research fields. Despite encouraging preclinical and early clinical progress, including FDA-approved peptide-based agents and peptide receptor radionuclide therapies, challenges related to stability, immunogenicity, manufacturing scalability, and regulatory harmonization remain significant. This review highlights current advances in ACP discovery, molecular engineering, and nanotheranostic integration, and outlines a roadmap for advancing peptide-based precision oncology. Collectively, next-generation ACP platforms hold promise to reshape cancer therapy by integrating targeted cytotoxicity, immune activation, and real-time imaging within a single modular framework.

Design, development, and evaluation of gene therapeutics specific to KSHV-associated diseases

Molecular Therapy Oncology · 2025-09-05 · 4 citations

-TK injection/GCV showed no detectable side effects. Our proof-of-concept studies highlight a promising strategy for targeting cancers driven by herpesviruses.

Design, development, and evaluation of gene therapeutics specific to KSHV-associated diseases

bioRxiv (Cold Spring Harbor Laboratory) · 2025-02-19

Abstract Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS) and two human lymphoproliferative diseases: primary effusion lymphoma and AIDS-related multicentric Castleman’s disease. KSHV-encoded latency-associated nuclear antigen (LANA) is expressed in KSHV-infected cancer cells and is responsible for maintaining viral genomes in infected cells. Thus, LANA is an attractive target for therapeutic intervention for KSHV-associated diseases. Here, we devised a cancer gene therapy vector using the adeno-associated virus (AAV), which capitalizes the LANA’s function to maintain terminal repeat (TR) containing circular genome in latently infected cells and the TR’s enhancer function for KSHV inducible gene promoters. By including two TR copies with a lytic inducible gene promoter (TR2 -OriP ), we prepared an AAV vector, which expresses an engineered thymidine kinase (TK) selectively in KSHV-infected cells. Ganciclovir (GCV), an anti-herpesvirus drug, effectively eradicated multiple KSHV-infected cells that include iPSC-derived epithelial colony-forming cells, but not non-KSHV-infected counterparts in the presence of AAV8-TR2 -OriP -TK. In addition, AAV8-TR2 -OriP -TK prevents KSHV virion production from reactivated cells, spreading KSHV infections from reactivated cells. Anti-cancer drugs, known to reactivate KSHV, stimulated TK expression from the vector and, therefore, synergized with AAV8 TR2 -OriP -TK to induce KSHV-infected cancer cell death. Finally, the AAV8-TR2 -OriP -TK with GCV completely diminished KSHV-infected cancer cells in the xenograft tumor model. The new cancer gene therapeutics should augment the current clinical protocol for KS.

Studies on Gene Enhancer with KSHV mini-chromatin

bioRxiv (Cold Spring Harbor Laboratory) · 2025-03-24 · 2 citations

Abstract Kaposi’s sarcoma-associated herpesvirus (KSHV) genome contains a terminal repeats (TR) sequence. Previous studies demonstrated that KSHV TR functions as a gene enhancer for inducible lytic gene promoters. Gene enhancers anchor bromodomain-containing protein 4 (BRD4) at specific genomic region, where BRD4 interacts flexibly with transcription-related proteins through its intrinsically disordered domain and exerts transcription regulatory function. Here, we generated recombinant KSHV with reduced TR copy numbers and studied BRD4 recruitment and its contributions to the inducible promoter activation. Reducing the TR copy numbers from 21 (TR21) to 5 (TR5) strongly attenuated viral gene expression during de novo infection and impaired reactivation. The EF1α promoter encoded in the KSHV BAC backbone also showed reduced promoter activity, suggesting a global attenuation of transcription activity within TR5 latent episomes. Isolation of reactivating cells confirmed that the reduced inducible gene transcription from TR-shortened DNA template and is mediated by decreased efficacies of BRD4 recruitment to viral gene promoters. Separating the reactivating iSLK cell population from non-responders showed that reactivatable iSLK cells harbored larger LANA nuclear bodies (NBs) compared to non-responders. The cells with larger LANA NBs, either due to prior transcription activation or TR copy number, supported KSHV reactivation more efficiently than those with smaller LANA NBs. With auxin-inducible LANA degradation, we confirmed that LANA is responsible for BRD4 occupancies on latent chromatin. Finally, with purified fluorescence-tagged proteins, we demonstrated that BRD4 is required for LANA to form liquid-liquid phase-separated dots. The inclusion of TR DNA fragments further facilitated the formation of larger BRD4-containing LLPS in the presence of LANA, similar to the “cellular enhancer dot” formed by transcription factor-DNA bindings. These results suggest that LANA binding to TR establishes an enhancer domain for infected KSHV episomes. The strength of this enhancer, regulated by TR length or transcription memory, determines the outcome of KSHV replication. Importance Gene enhancers are genomic domains that regulate frequency and duration of transcription burst at gene promoters, with BRD4 playing a critical role in their enhancer functions. KSHV latent mini-chromosome also contains an enhancer domain made with multiple copies of 801 bp identical repeat DNA fragments, terminal repeats. Here, we utilized manipulable mini-scale chromatins with convenient inducible KSHV reactivation to systematically examine the association between enhancer strength and the outcome of inducible promoter activation. This study illustrated the amount of BRD4 recruitment at the enhancer associated with frequencies of BRD4 distribution to the inducible promoters during KSHV reactivation and, therefore, KSHV lytic replication. Recruitment of BRD4 to the TR is specifically regulated by KSHV latent protein, LANA. KSHV evolves clever enhancer elements designed to be regulated by the KSHV own latent protein, LANA.

KSHV TR deletion episomes uncover enhancer–promoter dynamics in gene regulation

PLoS Pathogens · 2025-12-04

Kaposi's sarcoma-associated herpesvirus (KSHV) genome contains a terminal repeats (TR) sequence. Previous studies demonstrated that KSHV TR functions as a gene enhancer for inducible lytic gene promoters. Gene enhancers anchor bromodomain-containing protein 4 (BRD4) at specific genomic region, where BRD4 interacts flexibly with transcription-related proteins through its intrinsically disordered domain and exerts transcription regulatory function. Here, we generated recombinant KSHV with reduced TR copy numbers and studied BRD4 recruitment and its contributions to the inducible promoter activation. Reducing the TR copy numbers from 21 (TR21) to 5 (TR5) strongly attenuated viral gene expression during de novo infection and impaired reactivation. The EF1α promoter encoded in the KSHV BAC backbone also showed reduced promoter activity, suggesting a global attenuation of transcription activity within TR5 latent mini-chromatin. Isolation of reactivating cells confirmed that the reduced inducible gene transcription from TR-shortened DNA template is mediated by decreased efficacy of BRD4 recruitment to viral gene promoters. Separating the reactivating iSLK cell population from non-responders showed that reactivatable iSLK cells harbored larger LANA nuclear bodies (NBs) compared to non-responders. The cells with larger LANA NBs, either due to prior transcription activation or TR copy number, supported KSHV reactivation more efficiently than those with smaller LANA NBs. With auxin-inducible LANA degradation, we confirmed that LANA is responsible for BRD4 occupancies on latent chromatin. Finally, with purified fluorescence-tagged proteins, we demonstrated that BRD4 is required for LANA to form liquid-liquid phase-separated dots. The inclusion of TR DNA fragments further facilitated the formation of larger BRD4-containing LLPS with LANA as similar to the "cellular enhancer dot" formed by transcription factor-DNA bindings. These results suggest that LANA TR binding establishes an enhancer domain for infected KSHV episomes. The strength of this enhancer, regulated by TR length or transcription memories from prior activation, determines the degree of KSHV lytic replication.

Kaposi’s sarcoma-associated herpesvirus terminal repeat regulates inducible lytic gene promoters

Journal of Virology · 2024-01-19 · 20 citations

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

Kaposi’s sarcoma-associated herpesvirus (KSHV) LANA prevents KSHV episomes from degradation

Journal of Virology · 2024-01-19 · 16 citations

Protein knockdown with an inducible degradation system is a powerful tool for studying proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-auxin-inducible degron (mAID) tag at the LANA N-terminus with KSHV bacterial artificial chromosome 16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-LANA. Incubation with 5-phenyl-indole-3-acetic acid, a derivative of natural auxin, rapidly degraded LANA within 1.5 h. In contrast to our hypothesis, depletion of LANA alone did not trigger lytic reactivation but rather decreased inducible lytic gene expression when we stimulated reactivation with a combination of ORF50 protein expression and sodium butyrate. Decreased overall lytic gene induction seemed to be associated with a rapid loss of KSHV genomes in the absence of LANA. The rapid loss of viral genomic DNA was blocked by a lysosomal inhibitor, chloroquine. Furthermore, siRNA-mediated knockdown of cellular innate immune proteins, cyclic AMP-GMP synthase (cGAS) and simulator of interferon genes (STING), and other autophagy-related genes rescued the degradation of viral genomic DNA upon LANA depletion. Reduction of the viral genome was not observed in 293FT cells that lack the expression of cGAS. These results suggest that LANA actively prevents viral genomic DNA from sensing by cGAS-STING signaling axis, adding novel insights into the role of LANA in latent genome maintenance.IMPORTANCESensing of pathogens' components is a fundamental cellular immune response. Pathogens have therefore evolved strategies to evade such cellular immune responses. KSHV LANA is a multifunctional protein and plays an essential role in maintaining the latent infection by tethering viral genomic DNA to the host chromosome. We adopted the inducible protein knockdown approach and found that depletion of LANA induced rapid degradation of viral genomic DNA, which is mediated by innate immune DNA sensors and autophagy pathway. These observations suggest that LANA may play a role in hiding KSHV episome from innate immune DNA sensors. Our study thus provides new insights into the role of LANA in latency maintenance.

Editorial: Viruses and cancer

Virology · 2024-12-09

A LANA peptide inhibits tumor growth by inducing CHD4 protein cleavage and triggers cell death

Cell chemical biology · 2024-11-01 · 6 citations

) of 14 nM. Pre-treatment with VGN73 enhanced monocyte differentiation into macrophages and globally altered the repertoire of activated genes in U937 cells. Furthermore, the introduction of the peptide into the cancer cells induced caspase-mediated CHD4 cleavage, triggered cell death, and inhibited tumor growth in a xenograft mouse model. The VGN73 may facilitate cell differentiation therapy.

An atlas of chromatin landscape in KSHV-infected cells during de novo infection and reactivation

Virology · 2024-06-19 · 12 citations

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus with a double-stranded DNA capable of establishing latent infection in the host cell. During latency, only a limited number of viral genes are expressed in infected host cells, and that helps the virus to evade host immune cell response. During primary infection, the KSHV genome is chromatinized and maintained as an episome, which is tethered to the host chromosome via Latency Associated Nuclear Antigen (LANA). The KSHV episome undergoes the same chromatin modification with the host cell chromosome and, therefore, is regulated by various epigenetic modifications, such as DNA methylation, histone methylation, and histone acetylation. The KSHV genome is also organized in a spatiotemporal manner by forming genomic loops, which enable simultaneous and coordinated control of dynamic gene transcription, particularly during the lytic replication phase. The genome-wide approaches and advancing bioinformatic tools have increased the resolution of studies on the dynamic transcriptional control and our understanding of KSHV latency-lytic switch regulation. We will summarize our current understanding of the epigenetic gene regulation on the KSHV chromatin.