Novel nonviral vectors for gene delivery: Synthesis and applications

Summary We have synthesized novel cationic lipids for gene delivery bearing an ester bond between the lipid moiety and the polyamine head. We have found that an intramolecular rearrangement occurs during purification of one of the products. The rearrangement led to a cyclic lipopolyamine which was active for DNA gene transfer. The formation of cyclization products depends on the spacer found between the lipid and the polyamine. The introduction of arginine in the linker position prevents the formation of cyclic products. Linear as well as cyclic analogues showed high-efficiency gene transfer when tested in vitro for luciferase gene expression as compared to dioctadecylamidoglycyl spermine or lipofectamine and also in vivo in the Lewis lung carcinoma model. The introduction of arginine in the linker position promoted increased transfection activity, demonstrating that a diversity of linkers, such as short peptides or glycosides, can be introduced into cationic lipids for targeted gene transfer.     

Methylthioadenosine phosphorylase regulates ornithine decarboxylase by production of downstream metabolites

The gene encoding methylthioadenosine phosphorylase (MTAP), the initial enzyme in the methionine salvage pathway, is deleted in a variety of human tumors and acts as a tumor suppressor gene in cell culture (Christopher, S. A., Diegelman, P., Porter, C. W., and Kruger, W. D. (2002) Cancer Res. 62, 6639-6644). Overexpression of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is frequently observed in tumors and has been shown to be tumorigenic in vitro and in vivo. In this paper, we demonstrate a novel regulatory pathway in which the methionine salvage pathway products inhibit ODC activity. We show that in Saccharomyces cerevisiae the MEU1 gene encodes MTAP and that Meu1delta cells have an 8-fold increase in ODC activity, resulting in large elevations in polyamine pools. Mutations in putative salvage pathway genes downstream of MTAP also cause elevated ODC activity and elevated polyamines. The addition of the penultimate salvage pathway compound 4-methylthio-2-oxobutanoic acid represses ODC levels in both MTAP-deleted yeast and human tumor cell lines, indicating that 4-methylthio-2-oxobutanoic acid acts as a negative regulator of polyamine biosynthesis. Expression of MTAP in MTAP-deleted MCF-7 breast adenocarcinoma cells results in a significant reduction of ODC activity and reduction in polyamine levels. Taken together, our results show that products of the methionine salvage pathway regulate polyamine biosynthesis and suggest that MTAP deletion may lead to ODC activation in human tumors.     

Polyamines and novel polyamine conjugates interact with DNA in ways that can be exploited in non-viral gene therapy

As a part of our continuing studies on 'Polyamines and their role in human disease' we are investigating how polyamines, and especially how novel polyamine conjugates, interact with DNA. We are studying how these conjugates interact with circular plasmids in order to produce nanometre-sized particles suitable for transfecting cells. Our considerations of structure--activity relationships (SAR) within naturally occurring and synthetic polyamines have shown the significance of the inter-atomic distances between the basic nitrogen atoms. As these atoms are typically fully protonated under physiological conditions, they exist in equilibrium as polyammonium ions. The covalent addition of a lipid moiety, typically one or two alkyl or alkenyl chains, or a steroid, allows much greater efficiency in DNA condensation and in the cellular transfection achieved. Thus efficient DNA condensation and subsequently drug delivery (i.e. with DNA as the drug) can be brought about using novel polyamine conjugates. Taking further advantage of the functionalization of specific steroids (e.g. cholesterol and certain bile acids), we have designed and prepared novel fluorescent molecular probes as tools to throw light on the problematic steps in non-viral gene delivery which still impede efficient gene therapy. Thus, the current aims of our research are to understand, design and prepare small-molecule lipopolyamines for non-viral gene therapy (NVGT). The rational design and practical preparation of non-symmetrical polyamine carbamates and amides, based on steroid templates of cholesterol and the bile acid lithocholic acid as the lipid moiety, provides fluorescent molecular probes that condense DNA. These novel lipopolyamine conjugates mimic the positive charge distribution found in the triamine spermidine and the tetra-amine spermine alkaloids. After optimizing their SAR, these fluorescent probes will be useful in monitoring gene delivery in NVGT.     

Incorporation of oxyethylene units between hydrocarbon chain and pseudoglyceryl backbone in cationic lipid potentiates gene transfection efficiency in the presence of serum.

Four novel cationic lipids with different numbers of oxyethylene units at the linkage region between the pseudoglyceryl backbone and the hydrocarbon chains have been synthesized and used as mixtures with 1,2-dioleoyl-L-alpha-glycero-3-phosphatidyl ethanolamine (DOPE) for liposome-mediated gene transfection. Incorporation of different numbers of oxyethylene (-CH(2)CH(2)O-) units between long hydrocarbon chain at the C-1 and C-2 positions of the pseudoglyceryl skeleton improved the transfection efficiency considerably compared to the one in which the chains were connected via simple ether links. A pronounced improvement in the gene transfer efficiency was observed with the unsymmetrical cationic lipid 3 in which the long hydrocarbon at the C-1 position of the pseudoglyceryl segment is connected via two (-CH(2)CH(2)O-) units. Notably, the transfection ability of lipid 3 with DOPE in the presence of serum was significantly greater than LIPOFECTAMINE. This suggests that introduction of oxyethylene units between long hydrocarbon chains at the C-1 and C-2 positions of the pseudoglyceryl skeleton provides a novel strategy to achieve efficient gene transfer, especially in conditions where the presence of serum is critical.     

Optimization of cationic lipid mediated gene transfer: structure-function,physico-chemical,and cellular studies

The rationale design aimed at the enhancement of cationic lipid mediated gene transfer is discussed. These improvements are based on the straight evaluation of the structure-activity relationship and on the introduction of new structures. Much attention have been given to the supramolecular structures of the lipid/DNA complexes, to the effect of serum on gene transfer and to the intracellular trafficking of the lipoplexes. Finally new avenue using reducible cationic lipids has been discussed.     

Synthesis of cholesteryl polyamine carbamates: pK(a) studies and condensation of calf thymus DNA

Novel polyamine carbamates have been designed and prepared from cholesterol. Our synthesis uses an orthogonal protection strategy based upon trifluoroacetyl and Boc-protecting groups. These unsymmetrical polyamine carbamates have been prepared from symmetrical (e.g., spermine and thermine) polyamines. Detailed interpretations of (1)H and (13)C NMR spectroscopic data led to the unambiguous assignment of these polyamine carbamates. These target conjugates contain a variety of positive charges distributed along methylene chains. Their pK(a)s have been determined potentiometrically for conjugates substituted with up to five amino functional groups. Condensation of calf thymus DNA into particles was monitored using light scattering at 320 nm. Salt-dependent binding affinity for calf thymus DNA was determined using an ethidium bromide fluorescence quenching assay. These cholesteryl polyamine carbamates are models for lipoplex formation with respect to gene delivery (lipofection), a key first step in gene therapy.     

OPTIMIZATION OF CATIONIC LIPID MEDIATED GENE TRANSFER: STRUCTURE-FUNCTION,PHYSICO-CHEMICAL,AND CELLULAR STUDIES

ABSTRACT

The rationale design aimed at the enhancement of cationic lipid mediated gene transfer is discussed. These improvements are based on the straight evaluation of the structure-activity relationship and on the introduction of new structures. Much attention have been given to the supramolecular structures of the lipid/DNA complexes, to the effect of serum on gene transfer and to the intracellular trafficking of the lipoplexes. Finally new avenue using reducible cationic lipids has been discussed.     

Glycolipid intermembrane transfer is accelerated by HET-C2, a filamentous fungus gene product involved in the cell-cell incompatibility response

Among filamentous fungi capable of mycelial growth, het genes play crucial roles by regulating heterokaryon formation between different individuals. When fusion occurs between fungal mycelia that differ genetically at their het loci, the resulting heterokaryotic cells are quickly destroyed. It is unclear how het gene products of Podospora anserina trigger heterokaryon incompatibility. One unexplored possibility is that glycosphingolipids play a role because the het-c2 gene encodes a protein that displays 32% sequence identity and an additional 30% similarity to the mammalian glycolipid transfer protein. Here, P. anserina protoplasts containing wild-type het-c2 genes were shown to have greater glycosphingolipid transfer activity than protoplasts with disrupted het-c2 genes, a condition previously linked to altered cell compatibility following hyphal fusion. The observed glycolipid transfer activity could not be accounted for by nonspecific lipid transfer protein activity. Direct assessment showed that purified, recombinant HET-C2 accelerates the intermembrane transfer of glycolipid in vitro, but that the HET-C2 activity is mitigated much less by negatively charged membranes than the mammalian glycolipid transfer protein. The findings are discussed within the context of HET-C2 being a member of an emerging family of ancestral sphingolipid transfer proteins that play important roles in cell proliferation and accelerated death.     

Spermidine regulates Vibrio cholerae biofilm formation via transport and signaling pathways

Vibrio cholerae , the causative agent of the devastating diarrheal disease cholera, can form biofilms on diverse biotic and abiotic surfaces. Biofilm formation is important for the survival of this organism both in its natural environment and in the human host. Development of V. cholerae biofilms are regulated by complex regulatory networks that respond to environmental signals. One of these signals, norspermidine, is a polyamine that enhances biofilm formation via the NspS/MbaA signaling system. In this work, we have investigated the role of the polyamine spermidine in regulating biofilm formation in V. cholerae . We show that spermidine import requires PotD1, an ortholog of the periplasmic substrate-binding protein of the spermidine transport system in Escherichia coli . We also show that deletion of the potD1 gene results in a significant increase in biofilm formation. We hypothesize that spermidine imported into the cell hinders biofilm formation. Exogenous spermidine further reduces biofilm formation in a PotD1-independent, but NspS/MbaA-dependent, manner. Our results suggest that polyamines affect biofilm formation in V. cholerae via multiple pathways involving both transport and signaling networks.     

Biosynthesis of lipid A precursors in Escherichia coli. A membrane-bound enzyme that transfers a palmitoyl residue from a glycerophospholipid to lipid X

Certain phosphatidylglycerol-deficient mutants of Escherichia coli accumulate two fatty acylated monosaccharides related to lipid A biosynthesis that have been identified as 2,3-diacylglucosamine 1-phosphate (lipid X) and triacylglucosamine 1-phosphate (lipid Y) (Raetz, C. R. H. (1984) Rev. Infect. Dis. 6, 463-472). Lipid Y has the same structure as lipid X, except that it bears an additional palmitoyl moiety, esterified to the 3-OH of the N-linked R-3-hydroxymyristoyl residue. We now describe a membrane-associated system for the enzymatic conversion of lipid X to lipid Y. Removal of glycerophospholipids form such membranes by washing with cold ethanol abolishes the activity. The system can be reactivated by the addition of exogenous phospholipids dispersed as mixed micelles with Triton X-100. When reconstituted in this manner, the formation of lipid Y is strictly dependent upon a glycerophospholipid donor bearing a palmitoyl residue in the sn-1 position. The enzyme system does not utilize palmitoyl coenzyme A or palmitoyl acyl carrier protein. It does not catalyze efficient transfer of fatty acids differing from palmitate by only one carbon atom. In contrast, the enzyme has relatively little specificity for the polar headgroup of the phospholipid donor, and it also appears to utilize a disaccharide precursor of lipid A as an alternative palmitoyl acceptor. Since the in vitro synthesis of lipid Y proceeds with a high yield, we have isolated the product and verified its structure by 1H NMR spectroscopy and mass spectrometry. The transesterification reaction that converts lipid X to lipid Y may be a model for the enzymatic synthesis of other acyloxyacyl structures, known to occur in mature lipid A.     

Scanning the Topography of Polyamine Blocker Binding in an Inwardly Rectifying Potassium Channel

Steeply voltage-dependent inward rectification of Kir (inwardly rectifying potassium) channels arises from blockade by cytoplasmic polyamines. These polycationic blockers traverse a long (>70 Å) pore, displacing multiple permeant ions, en route to a high affinity binding site that remains loosely defined. We have scanned the effects of cysteine modification at multiple pore-lining positions on the blocking properties of a library of polyamine analogs, demonstrating that the effects of cysteine modification are position- and blocker-dependent. Specifically, introduction of positively charged adducts results in two distinct phenotypes: either disruption of blocker binding or generation of a barrier to blocker migration, in a consistent pattern that depends on both the length of the polyamine blocker and the position of the modified cysteine. These findings reveal important details about the chemical basis and specific location of high affinity polyamine binding.     

Effect of reduced arginine decarboxylase activity on salt tolerance and on polyamine formation during salt stress in Arabidopsis thaliana

Polyamines have been suggested to play an important role in stress protection. However, attempts to determine the function of polyamines have been complicated by the fact that, dependent on the conditions, polyamine contents increase or decrease during stress. To determine the importance of polyamine formation during salt stress, we analysed polyamine contents and salt tolerance in two Arabidopsis thaliana mutants, spe1-1 and spe2-1 (Watson et al. Plant J 13: 231–239, 1998), with reduced activity of arginine decarboxylase (EC 4.1.1.19), an important enzyme in polyamine synthesis. Polyamines accumulated in wild-type plants (Col-0 and Ler-0) that were pre-treated with 100 m M NaCl before transfer to 125 m M NaCl, but not in plants that were directly transferred to 125 m M NaCl without prior treatment with 100 m M NaCl. This shows that polyamine accumulation depends on acclimation to salinity. The salt treatment that induced polyamine accumulation in wild-type plants did not lead to polyamine accumulation in the spe1-1 and spe2-1 mutants. Decreased fresh weight, chlorophyll content and photosynthetic efficiency indicated that the spe1-1 mutant was more severely affected by salt stress than its wild type, Col-0. In the spe2-1 mutant decreased salt tolerance compared to its wild type, Ler-0, became apparent as bleaching under severe salt stress. The present results demonstrate that decreased polyamine formation due to lower arginine decarboxylase activity leads to reduced salt tolerance.     

Functional characterization of GumK, a membrane-associated beta-glucuronosyltransferase from Xanthomonas campestris required for xanthan polysaccharide synthesis

Xanthomonas campestris is a Gram-negative bacterium that produces an exopolysaccharide known as xanthan gum. Xanthan is involved in a variety of biological functions, including pathogenesis, and is widely used in the industry as thickener and viscosifier. Although the genetics and biosynthetic process of xanthan are well documented, the enzymatic components have not been examined and no data on glycosyltransferases have been reported. We describe the functional characterization of the gumK gene product, an essential protein for xanthan synthesis. Immunoblots and complementation studies showed that GumK is a 44-kDa protein associated to the membrane fraction. This value corresponds to the expected molecular mass for GumK encoded by an extended open reading frame than proposed from previous genetic data and in X. campestris published complete genome. The protein was expressed in Escherichia coli cells. The purified protein catalyzed the transfer of a glucuronic acid residue from UDP-glucuronic acid to mannose-alpha-1,3-glucose-beta-1,4-glucose-P-P-polyisoprenyl with formation of a glucuronic acid-beta-mannose linkage. We examined the acceptor substrate specificity. GumK was unable to use the trisaccharide acceptor freed from the pyrophosphate lipid moiety. Replacement of the natural lipid moiety by phytanyl showed that the catalytic function could proceed with glucuronic acid transfer. These results suggest the enzyme does not show specificity for the lipidic portion of the acceptor. GumK showed diminished activity when tested with 6-O-acetyl-mannose-alpha-1,3-glucose-beta-1,4-glucose-P-P-polyisoprenyl, a putative intermediate in the synthesis of xanthan. This could indicate that acetylation of the internal mannose takes place after the formation of the GumK product.     

Combination of adeno-associated virus and adenovirus vectors expressing bone morphogenetic protein-2 produces enhanced osteogenic activity in immunocompetent rats

We have previously shown that gene therapy using adeno-associated virus (AAV) carrying bone morphogenetic proteins (BMPs) is a promising strategy for new bone formation in vivo in SD rats. However, it had a relatively low transduction efficiency. We investigate here whether enhanced osteogenic activity can be achieved without eliciting a severe immune response, using a cocktail of AAV-BMP2 and adenovirus (Ad)-BMP2 as a vector system. The muscles of SD rats were injected with either AAV-BMP2, Ad-BMP2, or an AAV-BMP2/Ad-BMP2 cocktail, and the in vivo bone formation was determined at eight weeks post-injection. Radiographic examination demonstrated that the addition of a low level of Ad-BMP2 to AAV-BMP2 produced significantly higher new bone formation than the use of AAV-BMP2 alone. Histological and immunohistological analysis revealed an enlarged bone-forming area and a long-term BMP2 expression, without pronounced infiltration of lymphocytes. Our results provide the first evidence that the introduction of a low level of adenovirus in vivo in immunocompetent subjects can greatly enhance AAV-mediated gene transfer, without inducing severe immune responses. This cocktail vector system may offer an attractive way of improving the efficiency of AAV-based gene delivery.     

Efficient calf thymus DNA condensation upon binding with novel bile acid polyamine amides

Polyamine amides have been prepared from lithocholic and cholic acids (5beta-colanes) by acylation of tri-Boc-protected tetraamines spermine and thermine. These designed ligands for DNA are polyammonium ions at physiological pH. In NMR spectra, they display 14N-1H 1J = 51 Hz, 1:1:1 triplets, due to the symmetry of the R14NH(3)+ cations. The binding affinities of these conjugates for calf thymus DNA were determined using an ethidium bromide fluorescence quenching assay and compared with spermine and polylysine. DNA-binding affinities were dependent upon both salt concentration and the hydrophobicity or intermolecular bonding (facial effects) of the lipid moieties in these conjugates. Light scattering at 320 nm was used to determine DNA condensation and particle formation. The observed self-assembly phenomena are discussed with respect to DNA charge neutralization and DNA bending with loss of ethidium cation intercalation sites, ultimately leading to DNA condensation. These polyamine amides are models for lipoplex formation with respect to gene delivery (lipofection), a key first step in gene therapy.     

A new triantennary galactose-targeted PEGylated gene carrier, characterization of its complex with DNA, and transfection of hepatoma cells

Nonviral gene vectors remain inefficient in vivo largely because of their rapid clearance from the circulation and also their nonspecific association with the extracellular matrix. To overcome such drawbacks, cationic lipoplexes are now frequently coated with hydrophilic polymers such as PEGs to reduce nonspecific interactions, and ligands are also linked to their surface to obtain cell-specific gene transfer. In view of the development of vectors for systemic gene delivery, we have designed and studied lipoplexes that carry a triantennary galactosyl ligand attached to the distal end of a (PEG)45-conjugated lipid. We incorporated this targeted PEGylated lipid into lipoplexes using two strategies of formulation, i.e., using either preformed micelles or liposomes. We demonstrated that the incorporation of PEG chains stabilized lipoplexes and masked, but only partially, the positive charges exposed on the surface of the particles. We have also shown that incorporation into lipoplexes of a lipidated PEG chain, bearing a ligand at its distal end, yielded particles that exhibited an accessible ligand throughout the whole range of cationic lipid to DNA ratios. We obtained a targeted transfection in HepG2 cells with one of the formulations. Our results strengthen the validity of using a ligand carried by a long PEG spacer arm for targeted gene transfer.     

Trypanosoma cruzi epimastigotes lack ornithine decarboxylase but can express a foreign gene encoding this enzyme.

Trypanosoma cruzi, a pathogenic protozoan causing Chagas disease, lacks ornithine decarboxylase (ODC), the enzyme catalyzing the first step of polyamine biosynthetic pathway in eukaryotic cells. Our results indicate that the auxotrophy for diamines of T. cruzi epimastigotes is due to the absence of an active ODC gene in these parasites and not to the inability for the expression of this gene. The introduction of an exogenous complete coding region from Crithidia fasciculata ODC gene inserted in an expression vector specific for trypanosomatids induces the normal expression of the foreign genetic information allowing the transformed T. cruzi to overcome the exogenous polyamine requirement for growth. The enzyme expressed in the transformed parasites has shown a considerably extended metabolic stability. The loss of ODC activity in T. cruzi might be related to the parasite adaptation to the intracellular stages of its life cycle.     

Variable expression of hypercholesterolemia in Apolipoprotein E2* (Arg136 --> Cys) heterozygotes

In the process of population screening for apo E gene polymorphism with the PCR and subsequent restriction analysis, we identified a female who demonstrated heterozygosity for an unusual restriction fragment caused by the loss of a CfoI restriction site. Sequence analysis of the apo E gene was performed and a carrier of the mutant allele with C --> T substitution at cDNA position 3817 was identified, which caused an Arg136 --> Cys change. The first-line relatives have been screened for this rare mutation with PCR and restriction analysis of PCR products. The complete lipoprotein parameters have been determined in the probands family. In the family, only one child had the same mutant allele as his mother had. The proband (7.49 mmol/l) with her siblings had hypercholesterolemia and a high body mass index (BMI 31.6 kg/m2). By contrast, her son had a normal lipid spectrum with normal BMI. We described the mutation apo E2* (Arg136 --> Cys) in a family with elevated lipid levels, but there was no confirmation of the connection between this mutation and type III hyperlipoproteinemia or hyperlipoproteinemia at all. In the case of this mutation, other factors (mainly genetic) are important for the development of lipid metabolism disorders.