Development of targeted recombinant polymers that can deliver siRNA to the cytoplasm and plasmid DNA to the cell nucleus.

One of the major limitations to effective siRNA delivery is the lack of a siRNA-specific delivery system. Currently, the same delivery systems that are used for plasmid DNA (pDNA) delivery to the cell nucleus are used for siRNA delivery to the cytoplasm. To fill this gap, the objective of this study was to design a biopolymer that can be programmed via its amino acid sequence to deliver siRNA specifically to cytoplasm. For pDNA delivery, a nuclear localization signal (NLS) was added to the biopolymer structure to facilitate active translocation of the genetic material towards nucleus. The biopolymers were complexed with pEGFP and GFP-siRNA and used to transfect SKOV-3 (HER2+) cells. The intracellular trafficking of the nanoparticles was also monitored in real-time and live cells. The results demonstrated that the biopolymer with NLS is a suitable carrier for pDNA delivery but not siRNA delivery. Conversely, the biopolymer without NLS was suitable for siRNA delivery to the cytoplasm but not pDNA to the cell nucleus. The potential use of the designed biopolymer for combination therapy of cancer cells with gene (thymidine kinase) and siRNA (BCL2) was also examined in SKOV-3 cancer cells.

HSV-TK/GCV cancer suicide gene therapy by a designed recombinant multifunctional vector.

The objective of this research was to evaluate the efficacy of a recombinant nonviral vector for targeted delivery of a thymidine kinase (TK) suicide gene to xenograft SKOV-3 tumors. The vector was genetically engineered and used to condense the TK gene into particles of less than 100 nm. The nanoparticles were used to transfect and kill SKOV-3 cancer cells in combination with ganciclovir (GCV) in vitro. The results demonstrated that the vector could effectively kill up to 80% of the SKOV-3 cancer cells. In the next step, the ability of the vector to deliver the TK suicide gene to xenograft tumors of SKOV-3 was studied. The results demonstrated that the vector could transfect tumors and result in significant tumor size reduction during the period that GCV was administered. Administration of GCV for at least 3 weeks post transfection was of paramount importance. These results illustrate the therapeutic efficacy and application of a designed recombinant nonviral vector in cancer gene therapy. FROM THE CLINICAL EDITOR: A recombinant nonviral vector is used to deliver a suicide thymidine kinase gene under gancylovir control in vitro to SKOV-3 cancer cells with 70% efficiency. Follow on testing in a xenograft tumor demonstrated tumor reduction persisting for three weeks.

Development of recombinant cationic polymers for gene therapy research.

Cationic polymers created through recombinant DNA technology have the potential to fill a void in the area of gene delivery. The recombinant cationic polymers to be discussed here are amino acid based polymers synthesized in E. coli with the purpose to not only address the major barriers to efficient gene delivery but offer safety, biodegradability, targetability and cost-effectiveness. This review helps the readers to get a better understanding about the evolution of recombinant cationic polymers; and the potential advantages that they could offer over viral and synthetic non-viral vectors for gene delivery. It also discusses some of the major challenges that must be addressed in future studies to turn recombinant polymers into clinically effective gene delivery systems. Recent advances with the biopolymer design suggest that this emerging new class of gene delivery systems has the potential to address some of the major barriers to efficient, safe and cost-effective gene therapy.

Perspectives in vector development for systemic cancer gene therapy.

Gene therapy is perceived as a revolutionary technology with the promise to cure almost any disease, provided that we understand its genetic basis. However, enthusiasm has rapidly abated as multiple clinical trials have failed to show efficacy. The limiting factor seems to be the lack of a suitable delivery system to carry the therapeutic genes to the target tissue safely and efficiently. Therefore, advancements in cancer gene therapy in general depend on the development of novel vectors with maximum therapeutic efficacy at the target site and minimal toxicity to normal tissues. This mini-review highlights both the major fortes and the unique challenges associated with the state-of-the-art gene carriers currently being used in cancer gene therapy.

Development of a genetically engineered biomimetic vector for targeted gene transfer to breast cancer cells.

A biomimetic vector was genetically engineered to contain at precise locations (a) an adenovirus mu peptide to condense pDNA into nanosize particles, (b) a synthetic cyclic peptide to target breast cancer cells and enhance internalization of nanoparticles, (c) a pH-responsive synthetic fusogenic peptide to disrupt endosome membranes and facilitate escape of the nanoparticles into the cytosol, and (d) a nuclear localization signal from human immunodeficiency virus for microtubule mediated transfer of genetic material to the nucleus. The vector was characterized using physicochemical and biological assays to demonstrate the functionality of each motif in the vector backbone. The results demonstrated that the vector is able to condense plasmid DNA into nanosize particles (<100 nm), protect pDNA from serum endonucleases, target ZR-75-1 breast cancer cells and internalize, efficiently disrupt endosome membranes, exploit microtubules to reach nucleus and mediate gene expression. The therapeutic potential of the vector was evaluated by complexing with plasmid DNA encoding TRAIL (pTRAIL) and transfecting ZR-75-1 cells. The results demonstrated that up to 62% of the ZR-75-1 breast cancer cells can be killed after administration of pTRAIL in complex with the vector.

Biosynthesis and characterization of a novel genetically engineered polymer for targeted gene transfer to cancer cells.

A novel multi-domain biopolymer was designed and genetically engineered with the purpose to target and transfect cancer cells. The biopolymer contains at precise locations: 1) repeating units of arginine and histidine to condense pDNA and lyse endosome membranes, 2) a HER2 targeting affibody to target cancer cells, 3) a pH responsive fusogenic peptide to destabilize endosome membranes and enhance endosomolytic activity of histidine residues, and 4) a nuclear localization signal to enhance translocation of pDNA towards the cell nucleus. The results demonstrated that the biopolymer was able to condense pDNA into nanosize particles, protect pDNA from serum endonucleases, target HER2 positive cancer cells but not HER2 negative ones, efficiently disrupt endosomes, and effectively reach the cell nucleus of target cells to mediate gene expression. To reduce potential toxicity and enhance biodegradability, the biopolymer was designed to be susceptible to digestion by endogenous furin enzymes inside the cells. The results revealed no significant biopolymer related toxicity as determined by impact on cell viability.

A designer biomimetic vector with a chimeric architecture for targeted gene transfer.

Designer biomimetic vectors are genetically engineered biomacromolecules that are designed to mimic viral characteristics in order to overcome the cellular barriers associated with the targeted gene transfer. The vector in this study was genetically engineered to contain at precise locations: a) four tandem repeating units of N-terminal domain of histone H2A to condense DNA into stable nanosize particles suitable for cellular uptake, b) a model targeting motif to target HER2 and enhance internalization of nanoparticles, and c) a pH-responsive synthetic fusogenic peptide to disrupt endosome membranes and promote escape of the nanoparticles into the cytosol. The results demonstrate that a fully functional, multi-domain, designer vector can be engineered to target cells with high specificity, overcome the biological barriers associated with targeted gene transfer, and mediate efficient gene transfer.

Evaluation of the effect of vector architecture on DNA condensation and gene transfer efficiency.

The objective of this study was to evaluate the effect of vector architecture on DNA condensation, particle stability, and gene transfer efficiency. Two recombinant non-viral vectors with the same amino acid compositions but different architectures, composed of lysine-histidine (KH) repeating units fused to fibroblast growth factor, were genetically engineered. In one vector lysine residues were dispersed (KHKHKHKHKK)(6)-FGF2, whereas in the other they were in clusters (KKKHHHHKKK)(6)-FGF2. Organization of lysine residues in this manner was inspired by the sequence of DNA condensing motifs that exist in nature (e.g., histones) where lysine residues are organized in clusters. These two constructs were compared in terms of DNA condensation and gene transfer efficiency. It was observed that the construct with KH units in clusters was able to condense pDNA into more stable particles with sizes <150 nm making them suitable for cellular uptake via receptor mediated endocytosis. This in turn resulted in five times higher transfection efficiency for the cKH-FGF2. This study demonstrates that in targeted non-viral gene transfer, the vector architecture plays as significant a role as its amino acid sequence. Thus, in the design of the non-viral vectors (synthetic or recombinant) this factor should be considered of paramount importance.