Antitumor Gene Therapy Research Articles

Co-delivery of siRNA and cisplatin via electrospun Nanofibrous membranes for synergistic treatment of malignant melanoma

Tumor recurrence and metastasis remain formidable challenges in clinical oncology. Although surgery is an effective treatment for early-stage solid tumors, residual cancer cells can lead to subsequent recurrence or metastasis. Conventional treatments for melanoma, such as anti-tumor medications and gene therapy, have distinct limitations. The rapid systemic distribution of anti-tumor drugs poses a significant challenge, often resulting in notable side effects and inadequate drug concentrations at the tumor site. Melanoma (MM), a deadly form of skin cancer, is known for its high mortality rate. In this study, we propose a novel strategy for treating MM by combining the controlled release of chemotherapeutic drugs encapsulated within Metal-Organic Frameworks (MOFs) and liposomes with gene therapy targeting Minichromosome Maintenance Proteins 4 (MCM4) using electrospinning and surface modification techniques. In vitro and in vivo results confirmed that this hierarchical membrane system can effectively deliver therapeutic MCM4 siRNA and release cisplatin to inhibit tumor growth. Furthermore, we demonstrated that MCM4 silencing promoted the sensitivity of melanoma cells to ferroptosis both in vitro and in vivo. The proposed strategy, by allowing for a controlled and sustained release of medication, could alleviate the challenges in drug delivery and aid in prevent tumor recurrence.

Functionalized polyesters based on valerolactones and [12]aneN3 as effective non-viral gene vectors in HepG2 cells

Block polyesters with BODIPY and [12]aneN3 moieties showed efficient cell-specific nucleic acid expression and anti-tumor gene therapy in vitro.

Recent advances in DNA nanomaterials for cancer diagnosis and treatment

Abstract The elucidation of the structural characteristics of deoxyribonucleic acid (DNA) by Watson and Crick, the advent of polymerase chain reaction technology, and the development of primer-synthesis methods, enable DNA to be replicated and transformed in an increasingly easy manner, such that it is among the most directed and evolving nanomaterials in molecular biology. Traditional cancer diagnosis methods are somewhat limited and are unable to meet existing needs; consequently, DNA nanostructures with wide ranging properties are receiving increasingly more attention because a number of DNA materials have been shown to exhibit diagnostic properties. Some DNA materials can functionally alter the biological behavior of cells, such as cell migration, cell proliferation, cell differentiation, autophagy, and anti-inflammatory effects. Some single-stranded DNA or ribonucleic acid with secondary structure through self-pairing, called aptamer, systematic evolution of ligands by exponential enrichment (SELEX), have targeting capabilities and can be applied to tumor targeted diagnosis and therapy. Several DNA nanomaterials with three-dimensional nanostructures and stable structures are being investigated as drug carrier systems to act on a variety of antitumor drugs or gene therapy agents. This article reviews the use of functionalized DNA nanostructures in cancer diagnosis and treatment applications. Various biosensors and carriers based on DNA nanostructures are introduced and chemical reactions related to the preparation of the carrier and the signal transduction mechanism of the sensor are discussed. Efficient carrier/biosensor platforms based on various structural developments and current DNA nanomaterial developments are also presented.

Abstract 692: OAd-mediated SA-4-1BBL fused to HPV-16 E7 elicits specific antitumor efficacy in syngeneic mouse lung cancer model

Abstract Introduction: Human papillomaviruses (HPVs) are responsible for about 25% of cancer cases worldwide. HPV-16 E7 antigen is a tumor-associated antigen (TAA) commonly expressed in HPV-induced tumors; however, it has low immunogenicity. The interaction of 4-1BBL with its receptor induces pleiotropic effects on innate, adaptive, and regulatory immunity and, if fused to TAAs in DNA vaccines, can improve the antitumor response; however, there is low transfection and antitumor efficiency. Oncolytic virotherapy is promising for antitumor gene therapy as it can be selectively replicated in tumor cells, inducing cell lysis, and furthermore, tumor cell debris can be taken in by immune cells to potentiate antitumor responses. Methods: The AdenoQuick 2.0 system was used to construct three OAds, expressing 1) SP-SA-E7-4-1BBL, 2) SA-E7-4-1BBL or 3) SP-SA-41BBL. The expression of transgenes was confirmed by western blot and subcellular localization of SP-SA-E7-4-1BBL was evaluated by immunofluorescence. The recombinant OAds-mediated cancer killing effect was evaluated by MTT assay and crystal violet staining, The recombinant OAds therapeutic efficacy was evaluated in a C57BL/6 mice bearing subcutaneous TC-1 lung tumor. Tumor growth and survival were monitored for a period of twenty-eight days. Results: In this study, we expressed the immunomodulatory molecule SA-4-1BBL fused to E7 on an oncolytic adenovirus (OAd) system. In vitro infection of TC-1 tumor cells and NIH-3T3 non-tumor cells with SA/E7/4-1BBL OAd demonstrated that only tumor cells are selectively destroyed. Moreover, protein expression is targeted to the endoplasmic reticulum in both cell lines when a signal peptide (SP) is added. Finally, in an HPV-induced cancer murine model, the therapeutic oncolytic activity of OAd can be detected, and this can be improved when fused to E7 and SP. Conclusions: In this study, we report the design of an oncolytic adenovirus expressing the SP/SA/E7/4-1BBL fusion gene, which is capable of infecting murine cancer and normal cells, and the expressed protein was able to be targeted to the endoplasmic reticulum. Most importantly, OAd induced a cell killing effect that is specific to cancer cells. Moreover, OAd treatment induced a potent antitumor effect in a mouse TC-1 cancer model. Citation Format: Jorge G. Gomez-Gutierrez, Rodolfo Garza-Morales, Alejandra G. Martinez-Perez, Jose J. Perez-Trujillo, Odila Saucedo-Cardenas, Maria J. Loera-Arias, Roberto Montes-de-Oca-Luna. OAd-mediated SA-4-1BBL fused to HPV-16 E7 elicits specific antitumor efficacy in syngeneic mouse lung cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 692.

PIL-12 delivered by polymer based nanovector for anti-tumor genetherapy

Finding more effective and safe non-viral vectors to transfer genes into cancer cells has become the key of immune gene therapy for cancer. Herein a triblock compound MPEG2000–PDLLA4000–MPEG2000 modified by cationic liposome DOTAP was used as a non-viral vector DOTAP/MPEG2000–PDLLA4000–MPEG2000 (DMPM) to effectively transfer interleukin (IL)-12 plasmid (pIL-12) into tumor tissue. IL-12 produced by transfected tumor cells successfully inducing lymphocyte proliferation and promoting interferon-γ (IFN-γ) secretion, which resulted in tumor cells death. The ability of DMPM to transfer pIL-12 and the immune effect induced by IL-12 in cells had been explored. The anti-tumor effect, mechanism and safety of pIL-12/DMPM in mice cancer model were investigated in this study. Our results showed that the pIL-12 transferred by DMPM was highly expressed both in CT26 cells and B16-F10 cells. IL-12 expressed in the culture supernatant of transfected tumor cells stimulated lymphocyte proliferation and promoted IFN-γ secretion. The experimental result confirmed that pIL-12/DMPM therapy significantly reduced tumor growth in mice model. We designed the nanocomposite DMPM to deliver pIL-12 for cancer treatment and explored its therapeutic efficacy and the underlying anti-tumor mechanism. Our study suggested pIL-12 loaded by DMPM complex would be an effective strategy for cancer treatment.

Applications of nanocomposites based on zeolitic imidazolate framework-8 in photodynamic and synergistic anti-tumor therapy.

Due to the limitations resulting from hypoxia and the self-aggregation of photosensitizers, photodynamic therapy (PDT) has not been applied clinically to treat most types of solid tumors. Zeolitic imidazolate framework-8 (ZIF-8) is a common metal–organic framework that has ultra-high porosity, an adjustable structure, good biocompatibility, and pH-induced biodegradability. In this review, we summarize the applications of ZIF-8 and its derivatives in PDT. This review is divided into two parts. In the first part, we summarize progress in the application of ZIF-8 to enhance PDT and realize theranostics. We discuss the use of ZIF-8 to avoid the self-aggregation of photosensitizers, alleviate hypoxia, increase the PDT penetration depth, and combine PDT with multi-modal imaging. In the second part, we summarize how ZIF-8 can achieve synergistic PDT with other anti-tumor therapies, including chemotherapy, photothermal therapy, chemodynamic therapy, starvation therapy, protein therapy, gene therapy, and immunotherapy. Finally, we highlight the challenges that must be overcome for ZIF-8 to be widely applied in PDT. To the best of our knowledge, this is the first review of ZIF-8-based nanoplatforms for PDT.

The Nanosystems Involved in Treating Lung Cancer.

Even though there are various types of cancer, this pathology as a whole is considered the principal cause of death worldwide. Lung cancer is known as a heterogeneous condition, and it is apparent that genome modification presents a significant role in the occurrence of this disorder. There are conventional procedures that can be utilized against diverse cancer types, such as chemotherapy or radiotherapy, but they are hampered by the numerous side effects. Owing to the many adverse events observed in these therapies, it is imperative to continuously develop new and improved strategies for managing individuals with cancer. Nanomedicine plays an important role in establishing new methods for detecting chromosomal rearrangements and mutations for targeted chemotherapeutics or the local delivery of drugs via different types of nano-particle carriers to the lungs or other organs or areas of interest. Because of the complex signaling pathways involved in developing different types of cancer, the need to discover new methods for prevention and detection is crucial in producing gene delivery materials that exhibit the desired roles. Scientists have confirmed that nanotechnology-based procedures are more effective than conventional chemotherapy or radiotherapy, with minor side effects. Several nanoparticles, nanomaterials, and nanosystems have been studied, including liposomes, dendrimers, polymers, micelles, inorganic nanoparticles, such as gold nanoparticles or carbon nanotubes, and even siRNA delivery systems. The cytotoxicity of such nanosystems is a debatable concern, and nanotechnology-based delivery systems must be improved to increase the bioavailability, biocompatibility, and safety profiles, since these nanosystems boast a remarkable potential in many biomedical applications, including anti-tumor activity or gene therapy. In this review, the nanosystems involved in treating lung cancer and its associated challenges are discussed.

Covalent organic framework nanoparticles for anti-tumor gene therapy

Covalent organic framework nanoparticles for anti-tumor gene therapy

An Oncolytic Adenovirus Encoding SA-4-1BBL Adjuvant Fused to HPV-16 E7 Antigen Produces a Specific Antitumor Effect in a Cancer Mouse Model.

Human papillomaviruses (HPVs) are responsible for about 25% of cancer cases worldwide. HPV-16 E7 antigen is a tumor-associated antigen (TAA) commonly expressed in HPV-induced tumors; however, it has low immunogenicity. The interaction of 4-1BBL with its receptor induces pleiotropic effects on innate, adaptive, and regulatory immunity and, if fused to TAAs in DNA vaccines, can improve the antitumor response; however, there is low transfection and antitumor efficiency. Oncolytic virotherapy is promising for antitumor gene therapy as it can be selectively replicated in tumor cells, inducing cell lysis, and furthermore, tumor cell debris can be taken in by immune cells to potentiate antitumor responses. In this study, we expressed the immunomodulatory molecule SA-4-1BBL fused to E7 on an oncolytic adenovirus (OAd) system. In vitro infection of TC-1 tumor cells and NIH-3T3 non-tumor cells with SA/E7/4-1BBL OAd demonstrated that only tumor cells are selectively destroyed. Moreover, protein expression is targeted to the endoplasmic reticulum in both cell lines when a signal peptide (SP) is added. Finally, in an HPV-induced cancer murine model, the therapeutic oncolytic activity of OAd can be detected, and this can be improved when fused to E7 and SP.

A Review of Mesoporous Silica Nanoparticle Delivery Systems in Chemo-Based Combination Cancer Therapies.

Chemotherapy is an important anti-tumor treatment in clinic to date, however, the effectiveness of traditional chemotherapy is limited by its poor selectivity, high systemic toxicity, and multidrug resistance. In recent years, mesoporous silica nanoparticles (MSNs) have become exciting drug delivery systems (DDS) due to their unique advantages, such as easy large-scale production, adjustable uniform pore size, large surface area and pore volumes. While mesoporous silica-based DDS can improve chemotherapy to a certain extent, when used in combination with other cancer therapies MSN based chemotherapy exhibits a synergistic effect, greatly improving therapeutic outcomes. In this review, we discuss the applications of MSN DDS for a diverse range of chemotherapeutic combination anti-tumor therapies, including phototherapy, gene therapy, immunotherapy and other less common modalities. Furthermore, we focus on the characteristics of each nanomaterial and the synergistic advantages of the combination therapies. Lastly, we examine the challenges and future prospects of MSN based chemotherapeutic combination therapies.

Functional Self-Assembled DNA Nanohydrogels for Specific Telomerase Activity Imaging and Telomerase-Activated Antitumor Gene Therapy.

Engineering a functional nanoplatform that integrates dynamic monitoring of endogenous biomarkers and a stimuli-activated therapeutic mode is promising for early diagnosis and treatment of cancers. In this study, we developed an intelligent DNA nanohydrogel with specific targeting capability that can be stimuli-activated for both in vitro telomerase detection and in vivo telomerase-triggered gene therapy. The DNA nanohydrogel was formed simply by the self-assembly of two Y-shaped DNA units and a double-stranded DNA linker labeled with fluorophores and loaded with therapeutic siRNA. When intracellular telomerase was overexpressed, the DNA nanohydrogel collapsed owing to the prolongation of the telomeric primer at the terminal sequence of one of the Y-shaped DNA units. As a result, the quenched fluorescence due to fluorescence resonance energy transfer (FRET) of the DNA nanohydrogel recovered and the trapped siRNA was released, enabling the accurate detection and imaging of intracellular telomerase activity as well as effective gene therapy of tumors. Benefiting from the great biocompatibility, specificity, and stimuli-responsive property, the developed DNA nanoplatform provides a new opportunity for precise cancer diagnosis and treatment as well as other biological applications.

CRISPR/Cas9-Mediated OC-2 Editing Inhibits the Tumor Growth and Angiogenesis of Ovarian Cancer.

Ovarian cancer is the leading cancer-related cause of death in women worldwide. It is of great relevance to understand the mechanism responsible for tumor progression and identify unique oncogenesis markers for a higher chance of preventing this malignant disease. The high-expression OC-2 gene has been shown to be a potential candidate for regulating oncogenesis and angiogenesis in ovarian cancer. Hence, we wished to investigate the impact of OC-2 gene on ovarian cancer aggressiveness. CRISPR/Cas9, a gene editing tool, allows for direct ablation of OC-2 at the genomic level, and we successfully generated OC-2 KO cell lines from SKOV3 and CAOV3 cells. In an apoptosis assay, OC-2 KO induced the apoptosis activation of tumor cells, with the up-regulation of Bax/Caspase-8 and the down-regulation of Bcl-2. Consequently, the proliferation, migration, and invasion of OC-2 KO cell lines were significantly inhibited. Assays of qRT-PCR and Western blotting showed that the expression levels of pro-angiogenic growth factors VEGFA, FGF2, HGF, and HIF-1α and the activation of Akt/ERK pathways were significantly down-regulated at the loss of OC-2. In the xenograft model, OC-2 KO potently suppressed the subcutaneous tumor growth, with the inhibition exceeding 56%. The down-regulation of CD31 and relevant pro-angiogenic growth factors were observed in OC-2 KO tumor tissues. Taken together, OC-2 depletion negatively regulated the ovarian cancer progression possibly by apoptosis activation and angiogenesis inhibition. This work revealed a pivotal regulator of apoptosis and angiogenesis networks in ovarian cancer, and we applied the CRISPR/Cas9 system to the transcription factor pathway for developing a broad-acting anti-tumor gene therapy.

A dual-functional buformin-mimicking poly(amido amine) for efficient and safe gene delivery

Biguanides (i.e. metformin, phenformin and buformin) are antidiabetic drugs with potential antitumor effects. Herein, a polycationic polymer, N,N′-bis(cystamine)acrylamide-buformin (CBA-Bu), containing multiple biodegradable disulphide bonds and buformin-mimicking side chains was synthesised. CBA-Bu was equipped with high efficiency and safety profile for gene delivery, meanwhile exhibiting potential antitumor efficacy. As a gene vector, CBA-Bu was able to condense plasmid DNA (pDNA) into nano-sized (<200 nm), positively-charged (>30 mV) uniform polyplexes that were well resistant to heparin and DNase I. Due to the reduction responsiveness of the disulphide bonds, CBA-Bu/pDNA polyplexes could release the loaded pDNA in the presence of dithiothreitol, and induce extremely low cytotoxicity in NIH/3T3 and U87 MG cells. The transfection results showed that CBA-Bu had a cellular uptake efficiency comparable to 25 kDa PEI, while a significantly higher gene expression level. Additionally, CBA-Bu had a lower IC50 value than its non-biguanide counterpart in two cancer cell lines. Furthermore, CBA-Bu could activate AMPK and inhibit mTOR pathways in U87 MG cells, a mechanism involved in the antitumor effect of biguanides. Taken together, CBA-Bu represented an advanced gene vector combining desirable gene delivery capability with potential antitumor activity, which was promising to achieve enhanced therapeutic efficacy in antitumor gene therapy.

A review of potential applications of MR-guided focused ultrasound for targeting brain tumor therapy.

Magnetic resonance-guided focused ultrasound (MRgFUS) has been used extensively to ablate brain tissue in movement disorders, such as essential tremor. At a lower energy, MRgFUS can disrupt the blood-brain barrier (BBB) to allow passage of drugs. This focal disruption of the BBB can target systemic medications to specific portions of the brain, such as for brain tumors. Current methods to bypass the BBB are invasive, as the BBB is relatively impermeable to systemically delivered antineoplastic agents. Multiple healthy and brain tumor animal models have suggested that MRgFUS disrupts the BBB and focally increases the concentration of systemically delivered antitumor chemotherapy, immunotherapy, and gene therapy. In animal tumor models, combining MRgFUS with systemic drug delivery increases median survival times and delays tumor progression. Liposomes, modified microbubbles, and magnetic nanoparticles, combined with MRgFUS, more effectively deliver chemotherapy to brain tumors. MRgFUS has great potential to enhance brain tumor drug delivery, while limiting treatment toxicity to the healthy brain.

Effects of recombinant human adenovirus-p53 on the regression of hepatic fibrosis.

Hepatic fibrosis is a scarring process that may progress to hepatic cirrhosis and even hepatic carcinoma if left untreated. Hepatic stellate cells (HSCs) play essential roles in the development of hepatic fibrosis. The tumor suppressor protein p53 is a transcription factor that is involved in cell proliferation, cell cycle regulation, apoptosis and DNA repair. Recombinant human adenovirus-p53 (Ad-p53) has been demonstrated to act as a promising antitumor gene therapy in various types of cancer. However, there is limited infomration regarding the therapeutic effect of Ad-p53 on the regression of hepatic fibrosis. In order to examine the underlying molecular mechanism responsible for the effects of Ad-p53 on HSCs, a rat model of hepatic fibrosis was established and HSC-T6 cells were cultured under different conditions. The expression of p53, transforming growth factor (TGF-β1) and α-smooth muscle actin (α-SMA), which is a marker of activated HSCs, was detected by immunohistochemical assays and RT-qPCR. In vitro, five different concentrations (1×106, 5×106, 1×107, 2×107 and 5×107 PFU/ml) of Ad-p53 were selected for use in the MTT assay to analyze the proliferation of HSCs at 0, 24, 48 and 72 h. Flow cytometric analysis was applied to determine the effect of three different concentrations of Ad-p53 (5×106, 1×107 and 2×107 PFU/ml) on the cell cycle and the apoptosis of HSC-T6 cells at 24 and 48 h. The results of immunohistochemical studies and RT-qPCR showed that Ad-p53 upregulated the expression of p53, and downregulated the expression of TGF-β1 and α-SMA. The MTT assay revealed that when treated with various doses of Ad-p53, the proliferation of HSCs was inhibited within a certain range of concentrations and time periods. Analysis of flow cytometric data showed that Ad-p53 arrested the cell cycle in G1 phase and significantly induced apoptosis. Taken together, these findings suggest that Ad-p53 promotes apoptosis and inhibits the proliferation of HSCs in a time- and dose-dependent manner by modulating the expression of p53, TGF-β1 and α-SMA.

A nanogel with passive targeting function and adjustable polyplex surface properties for efficient anti-tumor gene therapy

A dual responsive nanogel with tuneable polyplex properties was finely prepared. Its highin vivo/vitrogene transfection ability and passive cellular targeting function strongly promoted intratumor accumulation and tumor inhibition.

OFFICIAL MEDICATIONS FOR ANTI-TUMOR GENE THERAPY

This is a review of modern literature data of official medications for anti-tumor gene therapy as well as of medications that finished clinical trials. The article discusses the concept of gene therapy, the statistical analysis results of initiated clinical trials of gene products, the most actively developing directions of anticancer gene therapy, and the characteristics of anti-tumor gene medications. Various delivery systems for gene material are being examined, including viruses that are defective in replication (Gendicine™ and Advexin) and oncolytic (tumor specific conditionally replicating) viruses (Oncorine™, ONYX-015, Imlygic®). By now three preparations for intra-tumor injection have been introduced into oncology clinical practice: two of them – Gendicine™ and Oncorine™ have been registered in China, and one of them – Imlygic® has been registered in the USA. Gendicine™ and Oncorine™ are based on the wild type p53 gene and are designed for treatment of patients with head and neck malignancies. Replicating adenovirus is the delivery system in Gendicine™, whereas oncolytic adenovirus is the vector for gene material in Oncorine™. Imlygic® is based on the recombinant replicating HSV1 virus with an introduced GM–CSF gene and is designed for treatment of melanoma patients. These medications are well tolerated and do not cause any serious adverse events. Gendicine™ and Oncorine™ are not effective in monotherapy but demonstrate pronounced synergism with chemoand radiation therapy. Imlygic® has just started the post marketing trials.

Combined antitumor gene therapy with herpes simplex virus-thymidine kinase and short hairpin RNA specific for mammalian target of rapamycin.

A proof-of-concept study is presented using dual gene therapy that employed a small hairpin RNA (shRNA) specific for mammalian target of rapamycin (mTOR) and a herpes simplex virus-thymidine kinase (HSV-TK) gene to inhibit the growth of tumors. Recombinant adeno-associated virus (rAAV) vectors containing a mutant TK gene (sc39TK) were transduced into HeLa cells, and the prodrug ganciclovir (GCV) was administered to establish a suicide gene-therapy strategy. Additionally, rAAV vectors expressing an mTOR-targeted shRNA were employed to suppress mTOR-dependent tumor growth. GCV selectively induced death in tumor cells expressing TK, and the mTOR-targeted shRNA altered the cell cycle to impair tumor growth. Combining the TK-GCV system with mTOR inhibition suppressed tumor growth to a greater extent than that achieved with either treatment alone. Furthermore, HSV-TK expression and mTOR inhibition did not mutually interfere with each other. In conclusion, gene therapy that combines the TK-GCV system and mTOR inhibition shows promise as a novel strategy for cancer therapy.

Optimization of T-cell Reactivity by Exploiting TCR Chain Centricity for the Purpose of Safe and Effective Antitumor TCR Gene Therapy.

Adoptive transfer of T cells redirected by a high-affinity antitumor T-cell receptor (TCR) is a promising treatment modality for cancer patients. Safety and efficacy depend on the selection of a TCR that induces minimal toxicity and elicits sufficient antitumor reactivity. Many, if not all, TCRs possess cross-reactivity to unrelated MHC molecules in addition to reactivity to target self-MHC/peptide complexes. Some TCRs display chain centricity, in which recognition of MHC/peptide complexes is dominated by one of the TCR hemi-chains. In this study, we comprehensively studied how TCR chain centricity affects reactivity to target self-MHC/peptide complexes and alloreactivity using the TCR, clone TAK1, which is specific for human leukocyte antigen-A*24:02/Wilms tumor 1(235-243) (A24/WT1(235)) and cross-reactive with B*57:01 (B57). The TAK1β, but not the TAK1α, hemi-chain possessed chain centricity. When paired with multiple clonotypic TCRα counter-chains encoding TRAV12-2, 20, 36, or 38-2, the de novo TAK1β-containing TCRs showed enhanced, weakened, or absent reactivity to A24/WT1(235) and/or to B57. T cells reconstituted with these TCRα genes along with TAK1β possessed a very broad range (>3 log orders) of functional and structural avidities. These results suggest that TCR chain centricity can be exploited to enhance desired antitumor TCR reactivity and eliminate unwanted TCR cross-reactivity. TCR reactivity to target MHC/peptide complexes and cross-reactivity to unrelated MHC molecules are not inextricably linked and are separable at the TCR sequence level. However, it is still mandatory to carefully monitor for possible harmful toxicities caused by adoptive transfer of T cells redirected by thymically unselected TCRs.

Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine.

We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine “Max” (PEI “Max”), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI “Max”/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI “Max”/polyanion small ternary complexes with high transfection efficiency. DNA/PEI “Max”/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.