Vectors For Cancer Gene Therapy Research Articles

NGR-bearing human adenovirus type 5 infects cells in flotillin- or caveolin-mediated manner depending on the NGR insertion site

Human adenoviruses represent attractive candidates for the design of cancer gene therapy vectors. Modification of adenovirus tropism by incorporating a targeting ligand into the adenovirus capsid proteins allows retargeting of adenovirus towards the cells of interest. Human adenovirus type 5 (HAdV-C5) bearing NGR containing peptide (CNGRCVSGCAGRC) inserted into the fiber (AdFNGR) or the hexon (AdHNGR) protein demonstrated an increased transduction of endothelial cells showing expression of aminopeptidase N, also known as CD13, and αvβ3 integrin both present on tumor vasculature, indicating that NGR-bearing adenoviruses could be used as tools for anti-angiogenic cancer therapy. Here we investigated how AdFNGR and AdHNGR infect cells lacking HAdV-C5 primary receptor, coxsackie and adenovirus receptor, and we showed that both AFNGR and AdHNGR enter cells by dynamin- and lipid raft-mediated endocytosis, while clathrin is not required for endocytosis of these viruses. We present evidence that productive infection of both AdFNGR and AdHNGR involves lipid rafts, with usage of flotillin-mediated cell entry for AdFNGR and limited role of caveolin in AdHNGR transduction efficiency. Lipid rafts play important role in angiogenesis and process of metastasis. Therefore, the ability of AdFNGR and AdHNGR to use lipid raft-dependent endocytosis, involving respectively flotillin- or caveolin-mediated pathway, could give them an advantage in targeting tumor cells lacking HAdV-C5 primary receptor.

HydrAd: A Helper-Dependent Adenovirus Targeting Multiple Immune Pathways for Cancer Immunotherapy.

Simple SummarySolid tumors are highly immunosuppressive and develop multiple inhibitory mechanisms that must be targeted simultaneously for successful cancer immunotherapy. Adenoviral vectors are promising cancer gene therapy vectors due to their inherent ability to stimulate multiple immune pathways. Adenoviruses are well characterized, and their genomes are easily manipulated, allowing for therapeutic transgene expression. Oncolytic adenoviruses are engineered to replicate specifically in malignant cells, resulting in cancer cell lysis. However, oncolytic adenoviral vectors have limited transgene capacity. Helper-dependent adenoviral vectors have been developed with the capability of expressing multiple transgenes through removal of all viral coding sequences. We have developed a helper-dependent platform for cancer immunotherapy and demonstrate expression of up to four functional transgenes. This platform allows us to target tumors with specific inhibitory pathways using our library of immunomodulatory transgenes in a mix-and-match approach for a synchronized cancer immunotherapy strategy.For decades, Adenoviruses (Ads) have been staple cancer gene therapy vectors. Ads are highly immunogenic, making them effective adjuvants. These viruses have well characterized genomes, allowing for substantial modifications including capsid chimerism and therapeutic transgene insertion. Multiple generations of Ad vectors have been generated with reduced or enhanced immunogenicity, depending on their intended purpose, and with increased transgene capacity. The latest-generation Ad vector is the Helper-dependent Ad (HDAd), in which all viral coding sequences are removed from the genome, leaving only the cis-acting ITRs and packaging sequences, providing up to 34 kb of transgene capacity. Although HDAds are replication incompetent, their innate immunogenicity remains intact. Therefore, the HDAd is an ideal cancer gene therapy vector as its infection results in anti-viral immune stimulation that can be enhanced or redirected towards the tumor via transgene expression. Co-infection of tumor cells with an oncolytic Ad and an HDAd results in tumor cell lysis and amplification of HDAd-encoded transgene expression. Here, we describe an HDAd-based cancer gene therapy expressing multiple classes of immunomodulatory molecules to simultaneously stimulate multiple axes of immune pathways: the HydrAd. Overall, the HydrAd platform represents a promising cancer immunotherapy agent against complex solid tumors.

Cancer gene therapy goes viral: viral vector platforms come of age.

BackgroundSince the advent of viral vector gene therapy in 1990s, cancer treatment with viral vectors promised to revolutionize the field of oncology. Notably, viral vectors offer a unique combination of efficient gene delivery and engagement of the immune system for anti-tumour response. Despite the early potential, viral vector-based cancer treatments are only recently making a big impact, most prominently as gene delivery devices in approved CAR-T cell therapies, cancer vaccines and targeted oncolytic therapeutics. To reach this broad spectrum of applications, a number of challenges have been overcome – from our understanding of cancer biology to vector design, manufacture and engineering. Here, we take an overview of viral vector usage in cancer therapy and discuss the latest advancements. We also consider production platforms that enable mainstream adoption of viral vectors for cancer gene therapy.ConclusionsViral vectors offer numerous opportunities in cancer therapy. Recent advances in vector production platforms open new avenues in safe and efficient viral therapeutic strategies, streamlining the transition from lab bench to bedside. As viral vectors come of age, they could become a standard tool in the cancer treatment arsenal.

Abstract 1710: A T-SIGn cancer gene therapy virus expressing an EpCAM-targeted bispecific T-cell activator stimulates T-cells and induces tumor cell death in patient-derived organotypic non-small cell lung cancer (NSCLC) spheroids ex vivo

Abstract Tumor-Specific Immuno-Gene (T-SIGn) cancer gene therapy vectors, which are based on the oncolytic adenovirus enadenotucirev, carry a gene cassette encoding combinations of human transgenes under control of the virus major late promoter (MLP) which restricts expression to tumor cells. These vectors can be dosed intravenously for delivery to tumors and selective expression of therapeutic proteins locally within the tumor microenvironment. Because these viruses only infect human tumor cells, in vivo studies are limited to human tumor xenografts in immunodeficient mice. Recently, we have optimized a 3D microfluidic system for evaluating response to immunotherapies using patient-derived organotypic tumor spheroids (PDOTS). PDOTS contain stromal as well as autologous myeloid and lymphoid cell populations and respond to PD-1 blockade in short-term ex vivo culture. As an alternative preclinical approach for evaluating activity and mechanistic underpinnings of T-SIGn viruses expressing human immunomodulatory transgenes, we have been developing the PDOTS ex vivo culture model. Here we report on the visualization of oncolytic virus uptake, replication in tumor cells and activation of T cells with a T-SIGn virus encoding an EpCAM-targeted bispecific T-cell activator (NG-611) within NSCLC PDOTS. Surgical NSCLC cases from St. Elizabeth's Medical Center, collected under an IRB approved protocol, were processed in PDOTS and assessed for EpCAM expression and number of T cells. Five different samples were treated with NG-611 (mixed with a GFP expressing vector) at concentrations ranging from 2x109 to 2x106 vp/mL final volume of culture. Virus infection was shown after 3 and 5 days of culture by dose-dependent expression of GFP reporter transgene driven by a CMV promoter, and virus replication in tumor cells was confirmed by using GFP expressed under the virus MLP promoter. Upregulation of IFNγ and Granzyme B in culture media indicated a strong activation of T-cells via expression of the bispecific T-cell activator. Cytotoxic T-cell activation also led to dose-dependent cell killing in NSCLC spheroids as shown by life/death analysis. Having established this proof of concept for functionality of T-SIGn viruses in PDOTS, ongoing studies are evaluating the immunomodulatory activities of a series of T-SIGn viruses expressing human IL-12 with or without other cytokine/chemokines. Citation Format: Moataz Noureddine, Katy West, Elizabeth Isaacson, Sylvia Alarcon, Christopher S. Lathan, John Wain, David A. Barbie, Cloud P. Paweletz, Elena Ivanova, Brian R. Champion. A T-SIGn cancer gene therapy virus expressing an EpCAM-targeted bispecific T-cell activator stimulates T-cells and induces tumor cell death in patient-derived organotypic non-small cell lung cancer (NSCLC) spheroids ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1710.

Abstract 1582: T-SIGn cancer gene therapy and anti-EGFR CAR-T cells synergize in combination therapy to clear A549 lung tumor xenografts and lung metastases in NSG mice

Abstract Chimeric antigen receptor (CAR) T cell therapy is an effective treatment for hematologic malignancies but has demonstrated limited efficacy in solid tumors. This is largely attributed to the immunosuppressive tumor microenvironment (TME) that impairs the ability of CAR-T cells to infiltrate, become activated, and kill tumor cells. Virotherapy is another emerging cancer treatment that has so far shown only limited clinical efficacy as a monotherapy for solid tumors. Importantly, in addition to their ability to directly kill tumor cells, viral vectors can also be leveraged to deliver transgenes to “reprogram” the TME from an immunosuppressive to a pro-inflammatory environment. We hypothesized that IV-deliverable T-SIGn cancer gene therapy vectors based on the oncolytic adenovirus enadenotucirev, which carry a transgene cassette encoding human immunomodulatory molecules to aid recruitment and activation of T-cells, could enhance the anti-tumor activity of human anti-EGFR CAR T cells in vivo. To test this hypothesis, NSG mice implanted subcutaneously with human lung cancer A549 xenografts, were treated intravenously with the T-SIGn virus followed by treatment with human anti-EGFR CAR-T cells. This treatment regimen demonstrated clearance of the xenograft and pulmonary metastases, whereas either therapy alone showed either no efficacy or delayed disease progression. When xenograft transcript analysis was performed, robust expression of the virus-encoded transgenes, as well as an upregulation of transcripts involved in type I IFN responses (e.g. IRF1, SOCS1), antigen presentation (e.g. HLA.DRA, TAP1), T cell activation (IFNγ, GrB), and inflammation (e.g. CXCL9, TNFSF10) was measured in the xenografts of animals treated with combination therapy, indicating a reprogramming of the TME to more effectively recruit and/or enhance T-cell responses. In addition, the combination treatment also dramatically reduced the number of A549 tumor cell micrometastases detectable in the lungs. Since both agents were delivered systemically, this observation may reflect direct therapeutic activity at the metastatic sites. Taken together, these results indicate that combination therapy with a T-SIGn vector and CAR T cells holds great potential to be efficacious in humans with solid tumors. Citation Format: Olmo Sonzogni, Daniel Zak, Katy West, Rochelle Lear, Alice Muntzer, Brian R. Champion, James B. Rottman. T-SIGn cancer gene therapy and anti-EGFR CAR-T cells synergize in combination therapy to clear A549 lung tumor xenografts and lung metastases in NSG mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1582.

Towards Clinical Implementation of Adeno-Associated Virus (AAV) Vectors for Cancer Gene Therapy: Current Status and Future Perspectives.

Adeno-associated virus (AAV) vectors have gained tremendous attention as in vivo delivery systems in gene therapy for inherited monogenetic diseases. First market approvals, excellent safety data, availability of large-scale production protocols, and the possibility to tailor the vector towards optimized and cell-type specific gene transfer offers to move from (ultra) rare to common diseases. Cancer, a major health burden for which novel therapeutic options are urgently needed, represents such a target. We here provide an up-to-date overview of the strategies which are currently developed for the use of AAV vectors in cancer gene therapy and discuss the perspectives for the future translation of these pre-clinical approaches into the clinic.

Improving NK1R-targeted gene delivery of stearyl-antimicrobial peptide CAMEL by conjugating it with substance P

Specificity is a crucial condition that hampers the application of non-viral vectors for cancer gene therapy. In a previous study, we developed an efficient gene vector, stearyl-CAMEL, using N-terminal stearylation of the antimicrobial peptide CAMEL. Substance P (SP), an 11-residue neuropeptide, rapidly enters cells after binding to the neurokinin-1 receptor (NK1R), which is expressed in many cancer cell lines. In this study, the NK1R-targeted gene vector stearyl-CMSP was constructed by conjugating SP to the C-terminus of stearyl-CAMEL. Our results indicated that stearyl-CMSP displayed significant transfection specificity for NK1R-expressing cells compared with that shown by stearyl-CAMEL. Accordingly, the stearyl-CMSP/p53 plasmid complexes had significantly higher antiproliferative activity against HEK293-NK1R cells than they did against HEK293 cells, while the stearyl-CAMEL/p53 plasmid complexes did not show this specificity in antiproliferative activity. Consequently, conjugation of the NK1R-targeted ligand SP is a simple and successful strategy to construct efficient cancer-targeted non-viral gene vectors.

Adenovirus and Immunotherapy: Advancing Cancer Treatment by Combination.

Gene therapy with viral vectors has significantly advanced in the past few decades, with adenovirus being one of the most commonly employed vectors for cancer gene therapy. Adenovirus vectors can be divided into 2 groups: (1) replication-deficient viruses; and (2) replication-competent, oncolytic (OVs) viruses. Replication-deficient adenoviruses have been explored as vaccine carriers and gene therapy vectors. Oncolytic adenoviruses are designed to selectively target, replicate, and directly destroy cancer cells. Additionally, virus-mediated cell lysis releases tumor antigens and induces local inflammation (e.g., immunogenic cell death), which contributes significantly to the reversal of local immune suppression and development of antitumor immune responses (“cold” tumor into “hot” tumor). There is a growing body of evidence suggesting that the host immune response may provide a critical boost for the efficacy of oncolytic virotherapy. Additionally, genetic engineering of oncolytic viruses allows local expression of immune therapeutics, thereby reducing related toxicities. Therefore, the combination of oncolytic virus and immunotherapy is an attractive therapeutic strategy for cancer treatment. In this review, we focus on adenovirus-based vectors and discuss recent progress in combination therapy of adenoviruses with immunotherapy in preclinical and clinical studies.

Delivery of CRISPR/Cas systems for cancer gene therapy and immunotherapy.

The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems are efficient and versatile gene editing tools, which offer enormous potential to treat cancer by editing genome, transcriptome or epigenome of tumor cells and/or immune cells. A large body of works have been done with CRISPR/Cas systems for genetic modification, and 16 clinical trials were conducted to treat cancer by ex vivo or in vivo gene editing approaches. Now, promising preclinical works have begun using CRISPR/Cas systems in vivo. However, efficient and safe delivery of CRISPR/Cas systems in vivo is still a critical challenge for their clinical applications. This article summarizes delivery of CRISPR/Cas systems by physical methods, viral vectors and non-viral vectors for cancer gene therapy and immunotherapy. The prospects for the development of physical methods, viral vectors and non-viral vectors for delivery of CRISPR/Cas systems are reviewed, and promising advances in cancer treatment using CRISPR/Cas systems are discussed.

Acylation of the antimicrobial peptide CAMEL for cancer gene therapy

Obtaining ideal gene delivery vectors is still a major goal in cancer gene therapy. CAMEL, a short hybrid antimicrobial peptide, can kill cancer cells by membrane lysis. In this study, we constructed a series of non-viral vectors by attaching fatty acids with different chain lengths to the N-terminus of CAMEL. Our results showed that the cellular uptake and transfection efficiency of acyl-CAMEL started to significantly increase from a chain length of 12 carbons. C18-CAMEL was screened for gene delivery because it had the highest transfection efficiency. Surprisingly, C18-CAMEL/plasmid complexes displayed strong endosomal escape activity after entering cells via endocytosis. Importantly, C18-CAMEL could deliver p53 plasmids to cancer cells and significantly inhibited cell proliferation by the expression of p53. In addition, the C18-CAMEL/p53 plasmid complexes and the MDM2 inhibitor nutlin-3a showed significantly synergistic anticancer activity against MCF-7 cells expressing wild-type p53. Conclusively, our study demonstrated that conjugation of stearic acid to antimicrobial peptides is a simple and successful approach for constructing efficient and economical non-viral vectors for cancer gene therapy.

Effect of Relaxin Expression from an Alginate Gel-Encapsulated Adenovirus on Scar Remodeling in a Pig Model.

PurposeRelaxin (RLX) is a transforming growth factor-β1 (TGF-β1) antagonist that is believed to function as a potent collagen re-arranger and a major suppressor of extracellular matrix components. Adenoviruses (Ads) are accepted vectors for cancer gene therapy. However, repeated treatments of Ad are limited by short-term biological activity in vivo. The efficacy of sustained RLX expression to scar remodeling was assessed using an injectable alginate gel-matrix system.Materials and MethodsPig scar tissue was treated with relaxin-expressing Ad loaded in alginate gel (gel/Ad-RLX). Surface areas, color, and pliability of scars were compared, and various factors influencing scar formation and collagen arrangement were analyzed.ResultsGel/Ad-RLX decreased scar size, color index, and pliability. Immunohistochemistry showed decreased levels of major extracellular matrix proteins in the gel/Ad-RLX-treated group. Furthermore, treatment with gel/Ad-RLX reduced expression of tissue inhibitor of metalloproteinase-1 and alpha-smooth muscle actin and markedly increased expression of matrix metalloproteinase-1 in pig scar tissues. Gel/Ad-RLX also significantly downregulated TGF-β1 and upregulated TGF-β3 mRNAs in pig scar tissues.ConclusionThese results support a prominent role for RLX in scar remodeling and suggest that gel/Ad-RLX may have therapeutic effects on scar formation.

Targeting of Cellular Organelles by Fluorescent Plasmid DNA Nanoparticles.

The development of a suitable delivery system and the targeting of intracellular organelles are both essential for the success of drug and gene therapies. The conception of fluorescent ligands, displaying targeting specificity together with low toxicity, is an emerging and reliable tool to develop innovative delivery systems. Biocompatible BSA or pDNA/ligand nanoparticles were synthesized by a coprecipitation method and were shown to display adequate sizes and morphology for delivery purposes, and positive surface charges. Additionally, these fluorescent vectors can target specific intracellular organelles. In vitro transfection mediated by BSA or pDNA based carriers can result in the accumulation of BSA in the cytosol, lysosomes, and mitochondria or the expression of the plasmid-encoded protein, respectively. Moreover, the therapeutic effect of pDNA/ligand vectors in cancer gene therapy instigates further research aiming clinical translation.

Cell-Surface Integrins and CAR Are Both Essential for Adenovirus Type 5 Transduction of Canine Cells of Lymphocytic Origin.

Adenoviruses are the most widely used vectors in cancer gene therapy. Adenoviruses vectors are well characterized and are easily manipulated. Adenovirus serotype 5 (Ad5) is the most commonly used human serotype. Ad5 internalization into host cells is a combined effect of binding of Ad5 fiber knob with the coxsackie virus and adenovirus receptor (CAR) and binding of RGD motifs in viral penton to cell surface integrins (αvβ3, αvβ5). Ad5’s wide range of host-cell transduction and lack of integration into the host genome have made it an excellent choice for cancer therapeutics. However, Ad5 has limited ability to transduce cells of hematopoietic origin. It has been previously reported that low or no expression of CAR is a potential obstacle to Ad5 infection in hematopoietic origin cells. In addition, we have previously reported that low levels of cell surface integrins (αvβ3, αvβ5) may inhibit Ad5 infection in canine lymphoma cell lines. In the current report we have examined the ability of an Ad5 vector to infect human (HEK293) and canine non-cancerous (NCF and PBMC), canine non-hematopoietic origin cancer (CMT28, CML7, and CML10), and canine hematopoietic origin cancer (DH82, 17–71, OSW, MPT-1, and BR) cells. In addition, we have quantified CAR, αvβ3 and αvβ5 integrin transcript expression in these cells by using quantitative reverse transcriptase PCR (q-RT-PCR). Low levels of integrins were present in MPT1, 17–71, OSW, and PBMC cells in comparison to CMT28, DH82, and BR cells. CAR mRNA levels were comparatively higher in MPT1, 17–71, OSW, and PBMC cells. This report confirms and expands the finding that low or absent expression of cell surface integrins may be the primary reason for the inability of Ad5-based vectors to transduce cells of lymphocytic origin and some myeloid cells but this is not true for all hematopoietic origin cells. For efficient use of Ad5-based therapeutic vectors in cancers of lymphocytic origin, it is important to address the defects in cell surface integrins.

Oncolytic vaccinia virus inhibits human hepatocellular carcinoma MHCC97-H cell proliferation via endoplasmic reticulum stress, autophagy and Wnt pathways.

Hepatocellular carcinoma (HCC) is a highly lethal malignancy. Vaccinia virus (VV) possessed many inherent advantages with respect to being engineered as a vector for cancer gene therapy, although the mechanism of action remains to be explored further. We constructed a thymidine kinase gene insertional inactivated VV, named VV-Onco, and then tested its effects on cell viability, apoptosis and colony formation ability in a highly metastatic human hepatocellular carcinoma cell line MHCC97-H, and also investigated the potential cell signal pathways involved in this action. VV-Onco induced strong cytotoxicity and apoptosis and also inhibited the colony formation of MHCC97-H cells. The tumor cell apoptosis induced by VV-Onco is likely mediated via endoplasmic reticulum stress, autophagy and Wnt signaling pathways. The downregulation of survivin and c-Myc may also play a role in VV-Onco induced cell death. The results of the present study provide new insights into the mechanisms of VV-induced tumor cell death. The engineered recombinant VV containing optimized therapeutic transgenes may represent a new avenue for cancer gene therapy. Copyright © 2016 John Wiley & Sons, Ltd.

Adeno-associated virus (AAV) vectors in cancer gene therapy

Gene delivery vectors based on adeno-associated virus (AAV) have been utilized in a large number of gene therapy clinical trials, which have demonstrated their strong safety profile and increasingly their therapeutic efficacy for treating monogenic diseases. For cancer applications, AAV vectors have been harnessed for delivery of an extensive repertoire of transgenes to preclinical models and, more recently, clinical trials involving certain cancers. This review describes the applications of AAV vectors to cancer models and presents developments in vector engineering and payload design aimed at tailoring AAV vectors for transduction and treatment of cancer cells. We also discuss the current status of AAV clinical development in oncology and future directions for AAV in this field.

PiggyBac as a novel vector in cancer gene therapy: current perspective.

Selection of suitable delivery system is one of the crucial aspects in gene therapy that determines the efficiency of gene therapy. The past two decades have witnessed extensive efforts for finding safe and efficient vectors to overcome the limitations of viral vectors. The utilization of DNA transposon-based vectors for gene therapy has emerged as a promising non-viral alternative. DNA 'cut-and-paste' is one of the main mechanisms of genome engineering by transposon elements. However, the lack of an efficient transposition system has limited the utilization of transposon vectors in mice and mammalian systems. PiggyBac (PB) is known as a highly efficient DNA transposon originally isolated from Trichoplusia ni as an alternative to Sleeping Beauty (SB). It has been shown that PB can be functional in various species including mammalian systems. This vector could overcome some limitations of other vectors in cancer gene therapy. Some advantages of PB include the capacity for integration into the genome and providing a stable expression, capacity to harbor 10 and 9.1 kb of foreign DNA into the host genome, without a significant reduction in their transposition activity and display non-overlapping targeting preferences. However, to advance PB to clinical applications, some obstacles still require to be overcome to improve its safety and efficiency. Hence, it seems that this vector could open new horizons in gene and cancer therapy.

Retroviral Vectors for Cancer Gene Therapy.

Advances in molecular technologies have led to the discovery of many disease-related genetic mutations as well as elucidation of aberrant gene and protein expression patterns in several human diseases, including cancer. This information has driven the development of novel therapeutic strategies, such as the utilization of small molecules to target specific cellular pathways and the use of retroviral vectors to retarget immune cells to recognize and eliminate tumor cells. Retroviral-mediated gene transfer has allowed efficient production of T cells engineered with chimeric antigen receptors (CARs), which have demonstrated marked success in the treatment of hematological malignancies. As a safety point, these modified cells can be outfitted with suicide genes. Customized gene editing tools, such as clustered regularly interspaced short palindromic repeats-CRISPR-associated nucleases (CRISPR-Cas9), zinc-finger nucleases (ZFNs), or TAL-effector nucleases (TALENs), may also be combined with retroviral delivery to specifically delete oncogenes, inactivate oncogenic signaling pathways, or deliver wild-type genes. Additionally, the feasibility of retroviral gene transfer strategies to protect the hematopoietic stem cells (HSC) from the dose-limiting toxic effects of chemotherapy and radiotherapy was demonstrated. While some of these approaches have yet to be translated into clinical application, the potential implications for improved cellular replacement therapies to enhance and/or support the current treatment modalities are enormous.

Bio-inspired polymer envelopes around adenoviral vectors to reduce immunogenicity and improve in vivo kinetics

Adenoviral vectors have attracted substantial interest for systemic tumor gene therapy, but further work is needed to reduce their immunogenicity and alter their biodistribution before they can be used in the clinic. Here we describe a bio-inspired, cleavable PEGylated β-cyclodextrin-polyethyleneimine conjugate (CDPCP) that spontaneously coats adenovirus in solution. This cleavable PEG coating reduces the innate and adaptive immunogenicity of adenovirus particles, as well as improves their biodistribution away from the liver and into the tumor. Insertion of a matrix metalloproteinase substrate sequence into the conjugate allows PEG cleavage at the tumor site, simultaneously reducing liver biodistribution and increasing transgene expression in tumors, thereby avoiding the “PEG dilemma”. Cationic β-cyclodextrin–PEI not only provides electrostatic attraction to promote envelope attachment to the viral capsid, but it also improves vector internalization and transduction after PEG cleavage. These results suggest that CDPCP may help expand the use of adenoviral vectors in cancer gene therapy. Statement of significanceThe synthesized β-cyclodextrin–PEI-MMP-cleavable-PEG polymer (CDPCP), held great potential for gene therapy when applied for adenovirus coating. The β-cyclodextrin–PEI provided a powerful electrostatic attraction to attach the whole polymer onto the viral capsid, while the MMPs-cleavable PEG reduced innate and adaptive immunogenicity and improved the biodistribution of adenovirus vectors due to the tumor-specific enzyme triggered PEG cleavage. More importantly, an ingenious cooperation between the two components could solve the PEG dilemma. The CDPCP/Ad complexes exhibited a comprehensive and valued profile to be a candidate vector for future tumor gene therapy, we believe the current investigation on this kind of biomaterial may be of particular interest to the readership of Acta biomaterialia.

Using a magnetic field to redirect an oncolytic adenovirus complexed with iron oxide augments gene therapy efficacy

Adenovirus (Ad) is a widely used vector for cancer gene therapy but its therapeutic efficacy is limited by low coxsackievirus and adenovirus receptor (CAR) expression in tumors and non-specifically targeted infection. Ad infectivity and specificity can be markedly improved by creating Ad-magnetic nanoparticles cluster complexes and directing their migration with an external magnetic field (MGF). We electrostatically complexed GFP-expressing, replication-incompetent Ad (dAd) with PEGylated and cross-linked iron oxide nanoparticles (PCION), generating dAd-PCION complexes. The dAd-PCION showed increased transduction efficiency, independent of CAR expression, in the absence or presence of an MGF. Cancer cell killing and intracellular oncolytic Ad (HmT)-PCION replication significantly increased with MGF exposure. Site-directed, magnetically-targeted delivery of the HmT-PCION elicited significantly greater therapeutic efficacy versus treatment with naked HmT or HmT-PCION without MGF in CAR-negative MCF7 tumors. Immunohistochemical tumor analysis showed increased oncolytic Ad replication in tumors following infection by HmT-PCION using an MGF. Whole-body bioluminescence imaging of tumor-bearing mice showed a 450-fold increased tumor-to-liver ratio for HmT-PCION with, versus without, MGF. These results demonstrate the feasibility and potential of external MGF-responsive PCION-coated oncolytic Ads as smart hybrid vectors for cancer gene therapy.

362. Utilization of Magnetic Field To Redirect Oncolytic Adenovirus Complexed With Iron Oxide for Augmented Therapeutic Efficacy

Adenovirus (Ad) is a widely used vector for cancer gene therapy but its therapeutic efficacy is limited on the lacked expression of the coxsackievirus and adenovirus receptor (CAR) in tumor tissues as well as specificity of targeted infection. Ad infectivity and specificity can be markedly improved by creating Ad-magnetic nanoparticles cluster complexes and subjecting them to an external magnetic field. First, we characterized the PEGylated and cross-linked iron oxide nanoparticles (PCION) through NMR, transmission electron microscope, and cytotoxicity assay. Then, we electrostatically complexed GFP-expressing replication-incompetent Ad (dE1/GFP, or dAd) with PCION, generating dAd/PCION complex. dAd/PCION showed increased transduction efficiency, independent of CAR in the absence or presence of an external magnetic field (MGF). The size distribution and zeta potential of the polyelectrolyte complex (dAd/PCION) were shown 182.3 and 24.6, respectively, compared with naked Ad (74.6 and -22.6). Due to the magnetic properties of the clustered magnetic nanoparticles, we attributed the markedly enhanced transduction efficiency to be primarily mediated by the MGF. Cancer cell killing effect was significantly increased Hmt/PCION+MGF system compared to that of HmT or HmT/PCION, independent of CAR expression. Importantly, in MCF7 tumor xenograft models, the magnetofection-mediated HmT/PCION complex displayed significantly enhanced anti-tumor efficacy in the presence of an external magnetic field by 2.74- and 2.10- fold than cancer cell killing effects in the absence of an MGF and Ad alone, respectively. These results demonstrate that magnetofection-mediated oncolytic Ad infection is able to overcome CAR-dependent infectivity while producing higher antitumor efficacy.