Arash Hatefi

Gene transfer efficiency of high primary amine content, hydrophobic, alkyl-oligoamine derivatives of polyethylenimine

In this study, a series of alkyl-oligoamine derivatives of low-toxicity 10 kDa polyethylenimine (PEI) were synthesized to enhance the hydrophobicity of PEI while preserving most of its primary amine content. PEI was reacted with a series of omega-bromoalkylcarboxylic acids with different chain lengths (2-bromoacetic, 6-bromohexanoic, 10-bromodecanoic and 16-bromohexadecanoic acids) to modify hydrophobicity followed by coupling to various oligoamines (spermine, spermidine, ethylendiamine or diethylentriamine) to partially restore primary amine density. These modifications were designed to influence hydrophobic-hydrophilic balance as well as maintain the proton sponge effect in order to create an efficient vector with low toxicity. Ethidium bromide exclusion assays and dynamic light scattering studies showed that the modified PEIs could bind to plasmid DNA and form nanoparticles in the range of 100 nm. The transfection efficiency of modified PEIs complexed with a luciferase reporter gene (pCMV-luc) in N2A murine neuroblastoma cells was increased to a level comparable to that of 25,000 Da PEI. These results indicate that hydrophobic modification of low-toxicity PEI without reduction in primary amine content is an effective strategy for improving transfection efficiency of polycation-based non-viral vectors while maintaining low toxicity.

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.

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.

Modified polyethyleneimine with histidine-lysine short peptides as gene carrier.

There are several strategies that can be utilized to improve transfection efficiency while reducing the cytotoxicity of polyethyleneimine (PEI) as a promising non-viral gene delivery vector. In this study, we evaluated the potential use of lysine–histidine (KH) peptides in modifying the PEI 10 kDa structure and enhancing its efficiency while maintaining low toxicity of PEI. PEI 10 kDa was modified with 6-bromohexanoic acid (alkyl) to increase its lipophilicity. Then, ethylenediamine (EDA) was attached to the carboxylic groups of PEI-hexanoate to restore the primary amines of PEI. Subsequently, six different KH short peptides were conjugated to PEIs and evaluated for the effect of the KH sequence on vector transfection efficiency and cytotoxicity. The transfection efficiency of PEI-peptides complexed with a luciferase reporter gene (pRLCMV) in Neuro-2A murine neuroblastoma cells showed that the PEI conjugated to KHHHKKHHHK peptide had a significantly higher rate of gene transfection efficiency in comparison with other KH peptides. This peptide was conjugated to PEI-alkyl and PEI-alkyl-EDA and significant improvement in efficiency with minimal cytotoxicity was observed. The results obtained suggest that the sequence and content of KH peptides will have a significant impact on the transfection efficiency of modified PEI 10 kDa.

Progress and problems with the use of suicide genes for targeted cancer therapy

Among various gene therapy methods for cancer, suicide gene therapy attracts a special attention because it allows selective conversion of non-toxic compounds into cytotoxic drugs inside cancer cells. As a result, therapeutic index can be increased significantly by introducing high concentrations of cytotoxic molecules to the tumor environment while minimizing impact on normal tissues. Despite significant success at the preclinical level, no cancer suicide gene therapy protocol has delivered the desirable clinical significance yet. This review gives a critical look at the six main enzyme/prodrug systems that are used in suicide gene therapy of cancer and familiarizes readers with the state-of-the-art research and practices in this field. For each enzyme/prodrug system, the mechanisms of action, protein engineering strategies to enhance enzyme stability/affinity and chemical modification techniques to increase prodrug kinetics and potency are discussed. In each category, major clinical trials that have been performed in the past decade with each enzyme/prodrug system are discussed to highlight the progress to date. Finally, shortcomings are underlined and areas that need improvement in order to produce clinical significance are delineated.

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.

Design, engineering and preparation of a multi-domain fusion vector for gene delivery.

Peptide based gene carriers are among the most promising non-viral vectors for gene delivery to eukaryotic cells. We have engineered a new fusion peptide using recombinant technology with the purpose of overcoming the cell barriers to gene delivery. A His- tagged multi-domain peptide was expressed in Escherichia coli BL21 (DE3) pLysS and purified using Ni-NTA resin. The fusion peptide is composed of two repeats of truncated histone H1 peptide to condense pDNA, a fusogenic peptide to disrupt endosome membranes and a nuclear localization signal to enhance translocation of pDNA towards nucleus. The results demonstrated that the vector can effectively condense plasmid DNA into nanoparticles with average sizes of 200 nm. The fusogenic peptide in the vector structure also showed membrane disruptive effect in the endosomal pH. Overall, the transfection efficiency of the vector demonstrated that it holds great promise as a nontoxic and effective non-viral gene carrier.

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

UNLABELLED 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.

Advances with the use of bio-inspired vectors towards creation of artificial viruses

Importance of the field: In recent years, there has been a great deal of interest in the development of recombinant vectors based on biological motifs with potential applications in gene therapy. Several such vectors have been genetically engineered, resulting in biomacromolecules with new properties that are not present in nature. Areas covered in this review: This review briefly discusses the advantages and disadvantages of the current state-of-the-art gene delivery systems (viral and non-viral) and then provides an overview on the application of various biological motifs in vector development for gene delivery. Finally, it highlights some of the most advanced bio-inspired vectors that are designed to perform several self-guided functions. What the reader will gain: This review helps the readers get a better understanding about the history and evolution of bio-inspired fusion vectors with the potential to merge the strengths of both viral and non-viral vectors in order to create efficient, safe and cost-effective gene delivery systems. Take home message: With the emergence of new technologies such as recombinant bio-inspired vectors, it may not take long before non-viral vectors are observed that are not just safe and tissue-specific, but even more efficient than viral vectors.