Jaesung Kim

Evaluation of E1B gene-attenuated replicating adenoviruses for cancer gene therapy

Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. For the aim of improving adenoviral vectors for cancer gene therapy, we have constructed genetically attenuated adenoviral vectors with different combinations of E1B genes and investigated the possibility of enhanced oncolytic and replication effects of these engineered replication-competent adenoviruses. We show here that the cytolytic potency of each gene-attenuated replicating adenovirus differed significantly depending on the presence or deletion of E1B 55 kDa and E1B 19 kDa function. More specifically, among the constructed vectors (Ad-ΔE1B19, Ad-ΔE1B55, Ad-ΔE1B19/55, and Ad-wt), E1B 19 kDa–inactivated adenovirus (Ad-ΔE1B19) was the most potent against all tumor cells tested, inducing the largest-sized plaques and marked CPE. Further, cells infected with either Ad-ΔE1B19 or E1B19/55 kDa–deleted adenovirus (Ad-ΔE1B19/55) showed complete cell lysis with disintegrated cellular structure, whereas cells infected with Ad-wt maintained intact cellular and nuclear membrane with properly structured organelles. TUNEL and DNA fragmentation assay also revealed that the Ad-ΔE1B19 or Ad-ΔE1B19/55 adenovirus-infected cells showed more profound induction of apoptosis in comparison to wild-type adenovirus-infected cells. The presence of E1B 55 kDa gene was required for efficient viral replication and deletion of E1B 19 kDa function in replicating adenovirus-induced apoptosis, leading to increased cytopathic effects. Moreover, Ad-ΔE1B19 adenovirus showed a better antitumor effect than other E1B-attenuated adenoviruses. Taken together, the replicating adenoviruses deleted in E1B 19 kDa function may serve as an improved vector for anticancer gene therapy in combination with apoptosis-inducing modalities such as chemotherapeutic agents and radiation therapy.

Active targeting of RGD-conjugated bioreducible polymer for delivery of oncolytic adenovirus expressing shRNA against IL-8 mRNA

Even though oncolytic adenovirus (Ad) has been highlighted in the field of cancer gene therapy, transductional targeting and immune privilege still remain difficult challenges. The recent reports have noted the increasing tendency of adenoviral surface shielding with polymer to overcome the limits of its practical application. We previously reported the potential of the biodegradable polymer, poly(CBA-DAH) (CD) as a promising candidate for efficient gene delivery. To endow the selective-targeting moiety of tumor vasculature to CD, cRGDfC well-known as a ligand for cell-surface integrins on tumor endothelium was conjugated to CD using hetero-bifunctional cross-linker SM (PEG)(n). The cytopathic effects of oncolytic Ad coated with the polymers were much more enhanced dose-dependently when compared with that of naked Ad in cancer cells selectively. Above all, the most potent oncolytic effect was assessed with the treatment of Ad/CD-PEG(500)-RGD in all cancer cells. The enhanced cytopathic effect of Ad/RGD-conjugated polymer was specifically inhibited by blocking antibodies to integrins, but not by blocking antibody to CAR. HT1080 cells treated with Ad/CD-PEG(500)-RGD showed strong induction of apoptosis and suppression of IL-8 and VEGF expression as well. These results suggest that RGD-conjugated bioreducible polymer might be used to deliver oncolytic Ad safely and efficiently for tumor therapy.

Cell penetrating peptide conjugated bioreducible polymer for siRNA delivery.

The primary cardiomyocyte-specific peptide (PCM) and the cell-penetrating peptide (CPP), HIV-Tat (49-57), were incorporated into the polymer, cystamine bisacrylamide-diaminohexane (CBA-DAH), to increase the delivery of RNAi to target cells, specifically cardiomyocytes. Interestingly, the impact of PCM and Tat conjugation on cellular uptake and transfection efficiency was greater in H9C2 rat cardiomyocytes than in NIH 3T3 cells. We examined the potential for siRNA targeting SHP-1 or Fas to inhibit the apoptosis of cardiomyocytes under hypoxic conditions using PCM and Tat-modified poly(CBA-DAH), (PCM-CD-Tat). To evaluate for efficacy in inhibiting apoptosis, either Fas siRNA/polymer or SHP-1 siRNA/polymer were transfected into cardiomyocytes treated under hypoxic and serum-deprived conditions. After incubation under hypoxic conditions, treatment with either the SHP-1 siRNA complex or the Fas siRNA complex resulted in an increase in cell viability and a reduction in LDH-cytotoxicity. The cells transfected with either of the siRNA polyplexes had a lower incidence of apoptosis as demonstrated by Annexin V-FITC/PI staining. Both the SHP-1 siRNA/PCM-CD-Tat complex and the Fas siRNA/PCM-CD-Tat complex warrant further investigation as therapeutic agents to inhibit the apoptosis of cardiomyocytes.

Enhancing the therapeutic efficacy of adenovirus in combination with biomaterials

With the reason that systemically administered adenovirus (Ad) is rapidly extinguished by innate/adaptive immune responses and accumulation in liver, in vivo application of the Ad vector is strictly restricted. For achieving to develop successful Ad vector systems for cancer therapy, the chemical or physical modification of Ad vectors with polymers has been generally used as a promising strategy to overcome the obstacles. With polyethylene glycol (PEG) first in order, a variety of polymers have been developed to shield the surface of therapeutic Ad vectors and well accomplished to extend circulation time in blood and reduce liver toxicity. However, although polymer-coated Ads can successfully evacuate from a series of guarding systems in vivo and locate within tumors by enhanced permeability and retention (EPR) effect, the possibility to entering into the target cell is few and far between. To endow targeting moiety to polymer-coated Ad vectors, a diversity of ligands such as tumor-homing peptides, growth factors or antibodies, have been introduced with avoiding unwanted transduction and enhancing therapeutic efficacy. Here, we will describe and classify the characteristics of the published polymers with respect to Ad vectors. Furthermore, we will also compare the properties of variable targeting ligands, which are being utilized for addressing polymer-coated Ad vectors actively.

Antitumoral effects of recombinant adenovirus YKL-1001, conditionally replicating in α-fetoprotein-producing human liver cancer cells

Selectively replicating recombinant adenovirus has emerged as a novel strategy for the treatment of incurable human cancers. One of the major characteristics of hepatocellular carcinoma is the transcriptional reactivation of alpha-fetoprotein (AFP). In this study, we evaluated the liver cancer-specific oncolytic potential of E1B 55kDa-deleted recombinant adenovirus (YKL-1001), which retained other E1 genes driven by the AFP promoter. Transient transfection study using luciferase indicated the selective activation of the AFP promoter only in human liver cancer cells secreting AFP (HepG2, Hep3B). YKL-1001 induced both cytopathic effects exclusively in AFP-positive liver cancer cells and the growth inhibition of pre-established Hep3B xenografts. Finally, hematoxylin-eosin staining and the immunohistochemistry to the adenoviral hexon showed a large distributed necrotic area and this implied a wide spread of YKL-1001. Therefore, the present study demonstrated that YKL-1001 holds significant promise as an oncolytic agent for hepatocellular carcinoma.

Oncolytic potential of E1B 55 kDa-deleted YKL-1 recombinant adenovirus: Correlation with p53 functional status

YKL‐1, E1B 55 kDa‐deleted recombinant adenovirus vector, capable of harboring a transgene casette of up to 4.9 kb, was newly constructed by reintroducing E1A and E1B 19 kDa into E1/E3‐deleted adenoviral vector with a homologous recombination in E. coli. Virus replication and cytotoxicity were dramatically attenuated in all 3 different types of normal human cells. In contrast, YKL‐1 efficiently replicated and induced cytotoxicity in most cancer cells, especially Hep3B and C33A cells with an inactivating p53 mutation. However, both H460 and HepG2 exhibited intermediate sensitivity to YKL‐1, which was between that of Hep3B or C33A and normal human cells. The YKL‐1 and DNA damaging agent, camptothecin effectively induced p53 in H460 and HepG2 as well as in normal cells. Furthermore, YKL‐1 effectively prohibited both Hep3B and C33A tumor growth in nu/nu mice in a dose‐dependent manner. H/E staining and TUNEL assay indicated a largely distributed necrotic area and apoptosis on its periphery. This study, therefore, indicates that YKL‐1, possesses promising potential as an oncolytic adenoviral vector, which acts partially in a p53‐dependent manner. Int. J. Cancer 88:454–463, 2000.

Ultrasound-assisted siRNA delivery via arginine-grafted bioreducible polymer and microbubbles targeting VEGF for ovarian cancer treatment.

The major drawback hampering siRNA therapies from being more widely accepted in clinical practice is its insufficient accumulation at the target site mainly due to poor cellular uptake and rapid degradation in serum. Therefore, we designed a novel polymeric siRNA carrier system, which would withstand serum-containing environments and tested its performance in vitro as well as in vivo. Delivering siRNA with a system combining an arginine-grafted bioreducible polymer (ABP), microbubbles (MBs), and ultrasound technology (US) we were able to synergize the advantages each delivery system owns individually, and created our innovative siRNA-ABP-MB (SAM) complexes. SAM complexes show significantly higher siRNA uptake and VEGF protein knockdown in vitro with serum-containing media when compared to naked siRNA, and 25k-branched-polyethylenimine (bPEI) representing the current standard in nonviral gene therapy. SAM complexes activated by US are also able to improve siRNA uptake in tumor tissue resulting in decelerating tumor growth in vivo.

Oncolytic adenovirus expressing IL-23 and p35 elicits IFN-γ- and TNF-α-co-producing T cell-mediated antitumor immunity.

Cytokine immunogene therapy is a promising strategy for cancer treatment. Interleukin (IL)-12 boosts potent antitumor immunity by inducing T helper 1 cell differentiation and stimulating cytotoxic T lymphocyte and natural killer cell cytotoxicity. IL-23 has been proposed to have similar but not overlapping functions with IL-12 in inducing Th1 cell differentiation and antitumor immunity. However, the therapeutic effects of intratumoral co-expression of IL-12 and IL-23 in a cancer model have yet to be investigated. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral inoculation of oncolytic adenovirus co-expressing IL-23 and p35, RdB/IL23/p35. Intratumoral administration of RdB/IL23/p35 elicited strong antitumor effects and increased survival in a murine B16-F10 syngeneic tumor model. The levels of IL-12, IL-23, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were elevated in RdB/IL23/p35-treated tumors. Moreover, the proportion of regulatory T cells was markedly decreased in mice treated with RdB/IL23/p35. Consistent with these data, mice injected with RdB/IL23/p35 showed massive infiltration of CD4+ and CD8+ T cells into the tumor as well as enhanced induction of tumor-specific immunity. Importantly, therapeutic mechanism of antitumor immunity mediated by RdB/IL23/p35 is associated with the generation and recruitment of IFN-γ- and TNF-α-co-producing T cells in tumor microenvironment. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-23 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.

Therapeutic efficacy of a systemically delivered oncolytic adenovirus - biodegradable polymer complex.

Despite great efforts to develop a more effective oncolytic adenovirus (Ad) for eradicating tumors, in vivo application via systemic administration is strictly limited to local injection due to host immune responses by Ad surface proteins and liver accumulation by the inherent nature of the Ad. In the last decade, numerous techniques using synthetic polymers have widely emerged to shield the exterior of therapeutic Ad vectors for systemic delivery. We developed a cationic polymer linked with polyethylene glycol for systemically delivering oncolytic Ad. The increased transduction efficiency and oncolytic effect of the Ad vectors physically coated with the polymer were evaluated, showing the optimal size (130 nm) of the Ad/polymer complex for systemic administration and prolonged stability of the Ad/polymer complex. Marked tumor growth suppression of the oncolytic Ad delivered by the polymer through systemic injection was observed in HT1080 and A549 xenograft models. The masking effect of the Ad surface by the polymer elicited evasion of innate adaptive immune responses and the tumor-to-liver ratio of the complex was significantly elevated 1229-fold greater than that of a naked Ad. These results demonstrate that the potential system of oncolytic Ad complexed with the biodegradable polymer may be useful for developing therapeutic vector systems via systemic delivery.

Linearized oncolytic adenoviral plasmid DNA delivered by bioreducible polymers

As an effort to overcome limits of adenovirus (Ad) as a systemic delivery vector for cancer therapy, we developed a novel system using oncolytic Ad plasmid DNA with two bioreducible polymers: arginine-grafted bioreducible poly(disulfide amine)polymer (ABP) and PEG5k-conjugated ABP (ABP5k) in expectation of oncolytic effect caused by progeny viral production followed by replication. The linearized Ad DNAs for active viral replication polyplexed with each polymer were able to replicate only in human cancer cells and produce progeny viruses. The non-immunogenic polymers delivering the DNAs markedly elicited to evade the innate and adaptive immune response. The biodistribution ratio of the polyplexes administered systemically was approximately 99% decreased in liver when compared with naked Ad. Moreover, tumor-to-liver ratio of the Ad DNA delivered by ABP or ABP5k was significantly elevated at 229- or 419-fold greater than that of naked Ad, respectively. The ABP5k improved the chance of the DNA to localize within tumor versus liver with 1.8-fold increased ratio. In conclusion, the innovative and simple system for delivering oncolytic Ad plasmid DNA with the bioreducible polymers, skipping time-consuming steps such as generation and characterization of oncolytic Ad vectors, can be utilized as an alternative approach for cancer therapy.