Vijaya Gopal

Recombinant fusion proteins TAT‐Mu, Mu and Mu‐Mu mediate efficient non‐viral gene delivery

The inherent ability of certain peptides or proteins of viral, prokaryotic and eukaryotic origin to bind DNA was used to generate novel peptide‐based DNA delivery protocols. We have developed a recombinant approach to make fusion proteins with motifs for DNA‐binding ability, Mu and membrane transduction domains, TAT, and tested them for their DNA‐binding, uptake and transfection efficiencies. In one of the constructs, the recombinant plasmid was designed to encode the Mu moiety of sequence MRRAHHRRRRASHRRMRGG in‐frame with TAT of sequence YGRKKRRQRRR to generate TAT‐Mu, while the other two constructs, Mu and Mu‐Mu, harbor a single copy or two copies of the Mu moiety.

Designed multi-domain protein as a carrier of nucleic acids into cells

Protein-based nucleic acid carriers offer attractive possibilities to enhance in vitro and in vivo gene delivery to combat diseases. A multi-domain fusion protein, namely TAT-NLS-Mu, designated as TNM, has been designed, cloned, heterologously expressed in E. coli and purified to homogeneity by affinity chromatography. The recombinant chimera TNM harbors three epitopes, a cell-penetrating (TAT) domain, a nuclear localization domain comprising of three nuclear localization sequence (NLS) motifs in tandem and a DNA-binding (Mu) domain. Complexes prepared by combining plasmid DNA with TNM (DP) transfect MCF-7, COS, CHO and HepG2 cells. Ternary complexes prepared with DNA, protein and cationic lipid (DPL) resulted in ~5-7 fold enhancement in reporter gene expression over the DP alone. Treatment of cells with chloroquine during transfection, with DP complexes, resulted in remarkable increases in reporter gene expression suggesting the involvement of endosomal compartments in the uptake process. Interestingly, DPL prepared with Lipofectin or 1, 2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) exhibited enhanced transfection in the presence of serum in MCF-7 and HepG2 cells. Microinjection of DP complexes, with and without NLS sequence, into the cytoplasm and nucleus of smooth muscle cells (SMC) indicated that the presence of NLS sequence in protein carrier significantly enhanced transgene expression. Together the data suggest that modular design of proteins is a promising method to develop gene delivery carriers and also the role of NLS epitopes in mediating nuclear transfer of DNA complexes into various cell types.

Correlation between the DNA supercoiling and the initiation of transcription by Escherichia coli RNA polymerase in vitro: role of the sequences upstream of the promoter region.

Binding of Escherichia coli RNA polymerase and the abortive initiation of transcription at the A2 promoter ofbacteriophage T7, separately cloned in pBR322, was found to be strongly dependent on the degree of supercoiling of the plasmid. Supercoiling does not seem to play any role in the initiation of transcription at the T7A1 promoter under identical conditions. Plasmid containing T7A2 promoter was found to be less amenable to S1 nuclease in comparison to that having T7A1. Sequence comparison reveals a high G/C content upstream to the −35 region of T7A2 which by extra duplex stability probably renders the initiation of transcription more dependent on the state of supercoiling of the template.

Cationic lipids for gene delivery in vitro and in vivo

Certain disease states can be corrected by using nucleic acids as therapeutic agents. To achieve this, nucleic acids must be delivered into the affected cells efficiently. At the core of a successful gene therapy protocol is the design of the nucleic acid carrier. Cationic lipids, as one of the gene delivery systems, have a wide potential in delivering nucleic acids both in vivo and in vitro. They are synthetic in origin and, hence, can be produced in required quantities and are biologically safe. Significant inputs from synthetic chemists in the recent past have resulted in the exploration of cationic lipids with very interesting functionalities. Transfection efficiencies of cationic lipids are comparable to viral-mediated transfection in vitro. However, viral-based methods for gene delivery in vivo are comparatively more efficient. Current understanding of lipid-mediated transfection is partially due to incomplete characterisation of the lipoplex, poor understanding of cell biology of transfection and cell type variations in transfection efficiencies. The published patents and research demonstrates the need for incorporation of the biological information in the design of the gene delivery formulations. In this review, the cell biological aspects critical for lipid-mediated transfection are emphasised. The parameters that influence the colloidal stability of the lipoplexes, cell biological processes relevant to gene delivery, such as cell association/uptake, cytoplasmic stability of the DNA and nuclear import, are discussed. The main focus of this review is patents published in the last 5 years.

Fluorescence spectroscopy analysis of active and regulatory sites of RNA polymerase

Publisher Summary This chapter discusses the fluorescence spectroscopy analysis of active and regulatory sites of RNA polymerase. The chapter divides the primary method of fluorescence spectroscopy into two categories to understand both the functional and the structural aspects of Escherichia coli (E. Coli) RNA polymerase. First, it deals with the extrinsic probe labeling of the substrate and enzyme, and then discusses the different important conclusions that can be drawn from such studies. Next, the time-resolved emission spectroscopic analysis of tryptophan fluorescence and the structure-function relationship of a transcription factor are discussed. The chapter discusses the syntheses and characterization of (γ-AmNS) ATP and UTP. The labeling of E. coli RNA polymerase and its subunits is discussed. The E. coli RNA polymerase has five subunits that are essential for the catalytic functions of the enzyme. Most of the active site labeling of the enzyme has been carried out at the substrate level. The only subunit that has been studied, with the help of fluorescence spectroscopy in some detail, is the regulatory subunit of E. coli RNA polymerase or the subunit.

Structural changes in DNA mediated by cationic lipids alter in vitro transcriptional activity at low charge ratios

Lipid/DNA complexes or Lipoplexes have been characterized by various biochemical and biophysical methods to understand the physical basis of transfection. Here we have addressed the effect of cationic liposomes, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), on transcription of DNA templates in vitro. Transcriptional activity of DNA-dependent RNA polymerase at DNA templates complexed with the cationic lipid varied as a function of charge ratio of lipid/DNA. At low charge ratios of 0.3:1 lipid/DNA and up to 1:1, we observed stimulation in transcription, while at higher charge ratios of lipid/DNA 3:1, complete inhibition in the activity occurred. Cetyl tri-methyl ammonium bromide (CTAB), a cationic detergent, and polyethylenimine (PEI), a cationic polymer, also bring about similar changes although to a lesser extent. The stimulation in transcription motivated us to probe into the molecular nature of the lipid/DNA interactions by absorbance spectroscopy and circular dichroism (CD). Upon interaction with lipids, hyperchromicity and susceptibility to micrococcal nuclease has increased, which suggests that the DNA was partially denatured. On complexation with the cationic lipid (DOTAP), the magnitude of the positive band in CD spectra decreased, accompanied with a red shift, as a function of charge ratio. Results from spectroscopic and enzyme assays suggest that at low charge ratios DNA may be partially unwound.

Gene delivery into human cancer cells by cationic lipid-mediated magnetofection

In this study, a combination of magnetic nanoparticles (MNPs) together with cationic lipid N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium chloride formulated with colipid cholesterol, upon magnetofection, enhanced DNA uptake into human glioblastoma-astrocytoma, epithelial-like cell line U-87 MG, hepatocellular carcinoma Hep G2, cervical cancer HeLa and breast cancer MDA-MB-231 cells. Having confirmed this, we monitored uptake of plasmid DNA mediated by ternary magnetoplexes by fluorescence microscopy, flow cytometry and reporter gene expression assays in the presence and absence of a magnetic field. Our observations clearly indicate enhanced transfection efficiency in vitro, upon magnetofection, in the presence of serum as seen from β-Gal reporter gene expression. The observed activity in serum suggests the suitability of MNPs for in vivo applications. Further, we measured the transverse relaxation time (T2) and obtained T2-weighted MRI images of treated U-87 MG cells. T2 determined for MNP-VP-Me22 and MNP-VP-Et22 corresponds to 22.6±0.8 ms and 36.0±2.1 ms, respectively, as compared to 47±1.7 ms for control, suggesting their applicability in molecular imaging. Our results collectively highlight the potential of lipid-based approach to augment magnetic-field guided-gene delivery using MNPs and additionally towards developing intracellular molecular probes for magnetic resonance imaging.

Synthesis and transfection efficiency of cationic oligopeptide lipids: Role of linker

In the design of new cationic lipids for gene transfection, the chemistry of linkers is widely investigated from the viewpoint of biodegradation and less from their contribution to the biophysical properties. We synthesized two dodecyl lipids with glutamide as the backbone and two lysines to provide the cationic headgroup. Lipid 1 differs from Lipid 2 by the presence of an amide linkage instead of an ester linkage that characterizes Lipid 2. The transfection efficiency of lipoplexes with cholesterol as colipid was found to be very high with Lipid 1 on Chinese Hamster Ovary (CHO) and HepG2 cell lines, whereas Lipid 2 has shown partial transfection efficiency on HepG2 cells. Lipid 1 was found to be stable in the presence of serum when tested in HepG2 and CHO cells albeit with lower activity. Fluorescence-based dye-binding and agarose gel-based assays indicated that Lipid 1 binds to DNA more efficiently than Lipid 2 at charge ratios of >1:1. The uptake of oligonucleotides with Lipid 1 was higher than Lipid 2 as revealed by confocal microscopy. Transmission electron microscopy (TEM) images reveal distinct formation of liposomes and lipoplexes with Lipid 1 but fragmented and unordered structures with Lipid 2. Fusion of Lipids 1 and 2 with anionic vesicles, with composition similar to plasma membrane, suggests that fusion of Lipid 2 was very rapid and unlike a fusion event, whereas the fusion kinetics of Lipid 1 vesicles was more defined. Differential scanning calorimetry (DSC) revealed a high T(m) for Lipid 1 (65.4 °C) while Lipid 2 had a T(m) of 23.5 °C. Surface area-pressure isotherms of Lipid 1 was less compressible compared to Lipid 2. However, microviscosity measured using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed identical values for vesicles made with either of the lipids. The presence of amide linker apparently resulted in stable vesicle formation, higher melting temperature, and low compressibility, while retaining the membrane fluid properties suggesting that the intermolecular hydrogen bonds of Lipid 1 yielded stable lipoplexes of high transfection efficiency.

Liposomes for targeting hepatocellular carcinoma: Use of conjugated arabinogalactan as targeting ligand

Present study investigates the potential of chemically modified (Shah et al., 2013) palmitoylated arabinogalactan (PAG) in guiding liposomal delivery system and targeting asialoglycoprotein receptors (ASGPR) which are expressed in hepatocellular carcinoma (HCC). PAG was incorporated in liposomes during preparation and doxorubicin hydrochloride was actively loaded in preformed liposomes with and without PAG. The liposomal systems with or without PAG were evaluated for in vitro release, in vitro cytotoxicity, in vitro cell uptake on ASGPR(+) cells, in vivo pharmacokinetic study, in vivo biodistribution study, and in vivo efficacy study in immunocompromised mice. The particle size for all the liposomal systems was below 200 nm with a negative zeta potential. Doxorubicin loaded PAG liposomes released significantly higher amount of doxorubicin at pH 5.5 as compared to pH 7.4, providing advantage for targeted tumor therapy. Doxorubicin in PAG liposomes showed superior cytotoxicity on ASGPR(+) HepG2 cells as compared to ASGPR(-), MCF7, A549, and HT29 cells. Superior uptake of doxorubicin loaded PAG liposomes as compared to doxorubicin loaded conventional liposomes was evident in confocal microscopy studies. Higher AUC in pharmacokinetic study and higher deposition in liver was observed for PAG liposomes compared to conventional liposomes. Significantly higher tumor suppression was noted in immunocompromised mice for mice treated with PAG liposomes as compared to the conventional liposomes. Targeting ability and superior activity of PAG liposomes is established pre-clinically suggesting potential of targeted delivery system for improved treatment of HCC.

Bioinspired peptides as versatile nucleic acid delivery platforms.

Non-viral gene therapy approaches have strongly established the utility of peptides as integral constituents of delivery platforms devised for efficient transfer of therapeutic molecules into cells. Among these, cell-penetrating peptides (CPPs), encompassing a family of short peptide sequences and their chimeric derivatives, have gained versatility through the addition of de novo peptide ligands primarily to facilitate cell-specific nucleic acid delivery in vitro and in vivo. The review illustrates the structural requirements of a noteworthy peptide TAT-PTD and other derivatives chiefly to exemplify their implication in gene therapy. An overview of the emerging concept and recent explorations will be presented through unique examples which form a facet in nanotechnology-based cancer therapy. Finally the basis for the utility of CPPs in plants will be discussed in view of its biotechnological potential.