Michael D. Pungente

Aspects of nonviral gene therapy: Correlation of molecular parameters with lipoplex structure and transfection efficacy in pyridinium-based cationic lipids

This study seeks correlations between the molecular structures of cationic and neutral lipids, the lipid phase behavior of the mixed-lipid lipoplexes they form with plasmid DNA, and the transfection efficacy of the lipoplexes. Synthetic cationic pyridinium lipids were co-formulated (1:1) with the cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and these lipids were co-formulated (3:2) with the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. All lipoplex formulations exhibited plasmid DNA binding and a level of protection from DNase I degradation. Composition-dependent transfection (beta-galactosidase and GFP) and cytotoxicity was observed in Chinese hamster ovarian-K1 cells. The most active formulations containing the pyridinium lipids were less cytotoxic but of comparable activity to a Lipofectamine 2000™ control. Molecular structure parameters and partition coefficients were calculated for all lipids using fragment additive methods. The derived shape parameter values correctly correlated with observed hexagonal lipid phase behavior of lipoplexes as derived from small-angle X-ray scattering experiments. A transfection index applicable to hexagonal phase lipoplexes derived from calculated parameters of the lipid mixture (partition coefficient, shape parameter, lipoplex packing) produced a direct correlation with transfection efficiency.

Novel Cationic Carotenoid Lipids as Delivery Vectors of Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO)-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO) AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs). The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).

Novel macrocyclic and acyclic cationic lipids for gene transfer: Synthesis and in vitro evaluation

The synthesis and in vitro evaluation of four cationic lipid gene delivery vectors, characterized by acyclic or macrocyclic, and saturated or unsaturated hydrophobic regions, is described. The synthesis employed standard protocols, including ring-closing metathesis for macrocyclic lipid construction. All lipoplexes studied, formulated from plasmid DNA and a liposome composed of a synthesized lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol as co-lipid, exhibited plasmid DNA binding and protection from DNase I degradation, and concentration dependent cytotoxicity using Chinese hamster ovary-K1 cells. The transfection efficiency of formulations with cholesterol outperformed those with DOPE, and in many cases the EPC/cholesterol control, and formulations with a macrocyclic lipid (+/- 10:1) outperformed their acyclic counterparts (+/- 3:1).

Polyene-based cationic lipids as visually traceable siRNA transfer reagents.

Cationic lipids are promising non-viral vectors for the cellular delivery of nucleic acids. Important considerations for the development of new delivery vectors are enhanced uptake efficiency, low toxicity and traceability. Traceable gene transfer systems however typically require the inclusion of a labeled excipient, and highly sensitive imaging instrumentation to detect the presence of the label. Recently, we reported the synthesis and characterization of colored, polyene cationic phospholipidoids composed of a rigid, polyenoic acid of predetermined dimension (C20:5 and C30:9) paired with flexible saturated alkyl chains of varying lengths (12:0, 14:0, 16:0, 18:0, 20:0 carbons). Herein, the potential of these cationic phospholipids as siRNA carriers was evaluated through standard liposomal formulations in combination with a neutral helper lipid DOPE. The polyene-based lipids were compared with a standard cationic lipid for siRNA-delivery into luciferase expressing HR5-CL11 cells. Within the series of lipids screened, knockdown results indicated that polyene cationic phospholipids paired with longer saturated alkyl chains are more effective as gene transfer agents, and perform comparably with the commercial lipid EPC. Furthermore, the chromophore associated with the polyene chain allowed tracking of the siRNA delivery using direct observation. The polyene lipoplexes were tracked on both a macroscopic and microscopic level either as a single-component or as a multi-component lipoplex formulation. When combined with a reference EPC, effective knockdown and tracking abilities were combined in a single preparation.

Novel cationic polyene glycol phospholipids as DNA transfer reagents—Lack of a structure-activity relationship due to uncontrolled self-assembling processes

Cationic glycol phospholipids were synthesized introducing chromophoric, rigid polyenoic C20:5 and C30:9 chains next to saturated flexible alkyl chains of variable lengths C6-20:0. Surface properties and liposome formation of the amphiphilic compounds were determined, the properties of liposome/DNA complexes (lipoplexes) were established using three formulations (no co-lipid, DOPE as a co-lipid, or cholesterol as a co-lipid), and the microstructure of the best transfecting compounds inspected using small angle X-ray diffraction to explore details of the partially ordered structures of the systems that constitute the series. Transfection and cytotoxicity of the lipoplexes were evaluated by DNA delivery to Chinese hamster ovary (CHO-K1) cells using the cationic glycerol phospholipid 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) as a reference compound. The uncontrollable self-association of the molecules in water resulted in aggregates and liposomes of quite different sizes without a structure-property relationship. Likewise, adding DNA to the liposomes gave rise to unpredictable sized lipoplexes, which, again, transfected without a structure-activity relationship. Nevertheless, one compound among the novel lipids (C30:9 chain paired with a C20:0 chain) exhibited comparable transfection efficiency and toxicity to the control cationic lipid EPC. Thus, the presence of a rigid polyene chain in this best performing achiral glycol lipid did not have an influence on transfection compared with the chiral glycerolipid reference ethyl phosphocholine EPC with two flexible saturated C14 chains.

Cationic Polyene Phospholipids as DNA Carriers for Ocular Gene Therapy

Recent success in the treatment of congenital blindness demonstrates the potential of ocular gene therapy as a therapeutic approach. The eye is a good target due to its small size, minimal diffusion of therapeutic agent to the systemic circulation, and low immune and inflammatory responses. Currently, most approaches are based on viral vectors, but efforts continue towards the synthesis and evaluation of new nonviral carriers to improve nucleic acid delivery. Our objective is to evaluate the efficiency of novel cationic retinoic and carotenoic glycol phospholipids, designated C20-18, C20-20, and C30-20, to deliver DNA to human retinal pigmented epithelium (RPE) cells. Liposomes were produced by solvent evaporation of ethanolic mixtures of the polyene compounds and coformulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol). Addition of DNA to the liposomes formed lipoplexes, which were characterized for binding, size, biocompatibility, and transgene efficiency. Lipoplex formulations of suitable size and biocompatibility were assayed for DNA delivery, both qualitatively and quantitatively, using RPE cells and a GFP-encoding plasmid. The retinoic lipoplex formulation with DOPE revealed a transfection efficiency comparable to the known lipid references 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) and GeneJuice. The results demonstrate that cationic polyene phospholipids have potential as DNA carriers for ocular gene therapy.

Synthesis, self-assembling and gene delivery potential of a novel highly unsaturated, conjugated cationic phospholipid.

The synthesis and self-assembling properties of a model compound in a new class of cationic phospholipids with a highly unsaturated conjugated fatty acid are described. In addition, the potential of this new lipid as a nucleic acid carrier was evaluated through lipoplex formulations employing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as helper lipid with and without the polycationic peptide protamine, together with a plasmid DNA (pDNA). Lipoplexes composed of this novel unsaturated lipid exhibited pDNA binding and protection from DNase I degradation when formulated with protamine. The new cationic lipid revealed transfection efficiency comparable to the commercial reference 1,2-dimyristoyl-sn-glycero-3-ethylphophocholine (EPC) in Chinese hamster ovary-K1 (CHO-K1) cells and performed equally to the standard reference Lipofectamine 2000 when the formulation included protamine.

Next generation macrocyclic and acyclic cationic lipids for gene transfer: Synthesis and in vitro evaluation.

Previously we reported the synthesis and in vitro evaluation of four novel, short-chain cationic lipid gene delivery vectors, characterized by acyclic or macrocyclic hydrophobic regions composed of, or derived from, two 7-carbon chains. Herein we describe a revised synthesis of an expanded library of related cationic lipids to include extended chain analogues, their formulation with plasmid DNA (pDNA) and in vitro delivery into Chinese hamster ovarian (CHO-K1) cells. The formulations were evaluated against each other based on structural differences in the hydrophobic domain and headgroup. Structurally the library is divided into four sets based on lipids derived from two 7- or two 11-carbon hydrophobic chains, C7 and C11 respectively, which possess either a dimethylamine or a trimethylamine derived headgroup. Each set includes four cationic lipids based on an acyclic or macrocyclic, saturated or unsaturated hydrophobic domain. All lipids were co-formulated with the commercial cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC) in a 1:1 molar ratio, along with one of two distinct neutral co-lipids, cholesterol or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in an overall cationic-to-neutral lipid molar ratio of 3:2. Binding of lipid formulations with DNA, and packing morphology associated with the individual lipid-DNA complexes were characterized by gel electrophoresis and small angle X-ray diffraction (SAXD), respectively. As a general trend, lipoplex formulations based on mismatched binary cationic lipids, composed of a shorter C7 lipid and the longer lipid EPC (C14), were generally associated with higher transfection efficiency and lower cytotoxicity than their more closely matched C11/EPC binary lipid formulation counterparts. Furthermore, the cyclic lipids gave transfection levels as high as or greater than their acyclic counterparts, and formulations with cholesterol exhibited higher transfection and lower cytotoxicity than those formulated with DOPE. A number of the lipid formulations with cholesterol as co-lipid performed as well as, or better than Lipofectamine 2000™ and EPC, the two positive controls employed in these studies. These results suggest that our novel cyclic and acyclic cationic lipid vectors are effective nonviral gene transfer agents that warrant further investigation.

Cationic Lipid-Based Nucleic Acid Vectors.

The delivery of nucleic acids into cells remains an important laboratory cell culture technique and potential clinical therapy, based upon the initial cellular uptake, then translation into protein (in the case of DNA), or gene deletion by RNA interference (RNAi). Although viral delivery vectors are more efficient, the high production costs, limited cargo capacity, and the potential for clinical adverse events make nonviral strategies attractive. Cationic lipids are the most widely applied and studied nonviral vectors; however, much remains to be solved to overcome limitations of these systems. Advances in the field of cationic lipid-based nucleic acid (lipoplex) delivery rely upon the development of robust and reproducible lipoplex formulations, together with the use of cell culture assays. This chapter provides detailed protocols towards the formulation, delivery, and assessment of in vitro cationic lipid-based delivery of DNA.