RNA Technology to Regenerate and Repair Alveolar Bone Defects.

microRNA-200a (miR-200a) targets multiple signaling pathways that are involved in osteogenic differentiation and bone development. However, its therapeutic function in osteogenesis and bone regeneration remains unknown. In this study, we use in vitro and in vivo models to investigate the molecular function of miR-200a overexpression and miR-200a inhibition using a plasmid-based miR inhibitor system (PMIS) on osteogenic differentiation and bone regeneration. Inhibition of miR-200a using PMIS-miR-200a significantly increased osteogenic biomarkers of human embryonic palatal mesenchyme cells and promoted bone regeneration in rat tooth socket defects. In rat maxillary M1 molar extractions, the supporting tooth structures were removed with an implant drill to yield a 3-mm defect in the alveolar bone. A collagen sponge was inserted into the open alveolar defect and PMIS-miR-200a plasmid DNA was added to the sponge and the wound sutured to protect the sponge and close the defect. It was important to remove the existing tooth supporting structure, which can influence alveolar bone regeneration. The alveolar bone was regenerated in 4 wk. The collagen sponge acts to stabilize and deliver the PMIS-miR-200a DNA to cells entering the sponge in the bone defect. We show that mesenchymal stem cells expressing CD90 and Stro-1 enter the sponges, take up the DNA, and express PMIS-miR-200a. PMIS-miR-200a initiates a bone regeneration program in transformed cells in vivo. In vitro inhibition of miR-200a was found to upregulate Wnt and BMP signaling activity as well as Runx2, OCN, Lef-1, Msx2, and Dlx5 associated with osteogenesis. Liver and blood toxicity testing of PMIS-miR-200a-treated rats showed no increase in several biomarkers of liver disease. These results demonstrate the therapeutic function of PMIS-miR-200a for rapid bone regeneration. Furthermore, the studies were designed to demonstrate the ease of use of PMIS-miR-200a in solution and applied using a syringe in the clinic through a simple one-time application.

Efficient expression of stabilized mRNA PEG-peptide polyplexes in liver.

The expression efficiency in liver following hydrodynamic delivery of in vitro transcribed mRNA was improved 2000-fold using a codon-optimized mRNA luciferase construct with flanking 3' and 5' human ß-globin untranslated regions (UTR mRNA) over an unoptimized mRNA without ß-globin UTRs. Nanoparticle UTR mRNA polyplexes were formed using a novel polyacridine polyethylene glycol (PEG) peptide, resulting in an additional 15-fold increase in expression efficiency in the liver. The combined increase in expression for UTR mRNA PEG-peptide polyplexes was 3500-fold over mRNA lacking UTRs and PEG-peptide. The expression efficiency of UTR mRNA polyplex was 10-fold greater than the expression from an equivalent 1 µg dose of pGL3. Maximal expression was maintained from 4 to 24 h. Serum incubation established the unique ability of the polyacridine PEG-peptide to protect UTR mRNA polyplexes from RNase metabolism by binding to double-stranded regions. UTR mRNA PEG-peptide polyplexes are efficient nonviral vectors that circumvent the need for a nuclear uptake, representing an advancement toward the development of a targeted gene delivery system to transfect liver hepatocytes.

The uptake mechanism of PEGylated DNA polyplexes by the liver influences gene expression.

Two uptake mechanisms were identified for PEGylated DNA polyplex biodistribution to the liver. At a low polyplex dose, a rapid-uptake mechanism dominates, resulting in 60% capture by liver in 5 min, due to a saturable receptor-mediated process. Rapid-uptake led to the fast metabolism of polyplexes by liver (t1/2 = 2.1 h), correlating with a 1-µg pGL3 polyplex dose losing full transfection competency after 4 h in the liver. Dose escalation of either polyplex or poly(ethylene glycol) (PEG) peptide led to the saturation of rapid-uptake and revealed a delayed-uptake mechanism for polyplexes by liver. Delayed-uptake was characterized by the slower liver accumulation of 40% of the polyplex dose over 40 min, followed by slow metabolism (t1/2 = 15 h) and an extended time (12 h) for a 1-µg pGL3 polyplex dose, remaining fully transfection competent in the liver. The delayed-uptake mechanism is consistent with polyplexes crossing liver fenestrated endothelial cells to reach steady state in the space of Disse. The results describe how to control polyplex biodistribution to liver to avoid rapid-uptake and metabolism, in favor of delayed-uptake, to preserve polyplex transfection competency in the liver for up to 12 h.

High-affinity PEGylated polyacridine peptide polyplexes mediate potent in vivo gene expression.

Polyethylene glycol (PEG)ylated polyacridine peptides bind to plasmid DNA with high affinity to form unique polyplexes that possess a long circulatory half-life and are hydrodynamically (HD)-stimulated to produce efficient gene expression in the liver of mice. We previously demonstrated that acridine-modified lysine (Acr) in (Acr-Lys)(6)-Cys-PEG(5kDa) stabilizes a 1-µg pGL3 dose for up to 1 h in the circulation, resulting in HD-stimulated (saline only) gene expression in the liver, equivalent in magnitude to direct-HD dosing of 1 µg of pGL3. In this study, we report that increasing the spacing of Acr with either four or five Lys residues markedly increases the stability of PEGylated polyacridine peptide polyplexes in the circulation allowing maximal HD-stimulated expression for up to 5 h post DNA administration. Co-administration of a decoy dose of 9 µg of non-expressing DNA polyplex with 1 µg of pGL3 polyplex further extended the HD-stimulated expression to 9 h. This structure-activity relationship study defines the PEGylated polyacridine peptide requirements for maintaining fully transfection competent plasmid DNA in the circulation for 5 h and provides an understanding as to why polyplexes or lipoplexes prepared with polyethylenimine, chitosan or Lipofectamine are inactive within 5 min following intravenous dosing.

Metabolically stabilized long-circulating PEGylated polyacridine peptide polyplexes mediate hydrodynamically stimulated gene expression in liver.

A novel class of PEGylated polyacridine peptides was developed that mediate potent stimulated gene transfer in the liver of mice. Polyacridine peptides, (Acr-X)(n)-Cys-polyethylene glycol (PEG), possessing 2-6 repeats of Lys-acridine (Acr) spaced by either Lys, Arg, Leu or Glu, were Cys derivatized with PEG (PEG(5000 kDa)) and evaluated as in vivo gene transfer agents. An optimal peptide of (Acr-Lys)(6)-Cys-PEG was able to bind to plasmid DNA (pGL3) with high affinity by polyintercalation, stabilize DNA from metabolism by DNAse and extend the pharmacokinetic half-life of DNA in the circulation for up to 2 h. A tail vein dose of PEGylated polyacridine peptide pGL3 polyplexes (1 µg in 50 µl), followed by a stimulatory hydrodynamic dose of normal saline at times ranging from 5 to 60 min post-DNA administration, led to a high level of luciferase expression in the liver, equivalent to levels mediated by direct hydrodynamic dosing of 1 µg of pGL3. The results establish the unique properties of PEGylated polyacridine peptides as a new and promising class of gene delivery peptides that facilitate reversible binding to plasmid DNA, protecting it from DNase in vivo resulting in an extended circulatory half-life, and release of transfection-competent DNA into the liver to mediate a high-level of gene expression upon hydrodynamic boost.

The proteasome metabolizes peptide-mediated nonviral gene delivery systems.

The proteasome is a multisubunit cytosolic protein complex responsible for degrading cytosolic proteins. Several studies have implicated its involvement in the processing of viral particles used for gene delivery, thereby limiting the efficiency of gene transfer. Peptide-based nonviral gene delivery systems are sufficiently similar to viral particles in their size and surface properties and thereby could also be recognized and metabolized by the proteasome. The present study utilized proteasome inhibitors (MG 115 and MG 132) to establish that peptide DNA condensates are metabolized by the proteasome, thereby limiting their gene transfer efficiency. Transfection of HepG2 or cystic fibrosis/T1 (CF/T1) cells with CWK18 DNA condensates in the presence of MG 115 or MG 132 resulted in significantly enhanced gene expression. MG 115 and MG 132 increased luciferase expression 30-fold in a dose-dependent manner in HepG2 and CF/T1. The enhanced gene expression correlated directly with proteasome inhibition, and was not the result of lysosomal enzyme inhibition. The enhanced transfection was specific for peptide DNA condensates, whereas Lipofectamine- and polyethylenimine-mediated gene transfer were significantly blocked. A series of novel gene transfer peptides containing intrinsic GA proteasome inhibitors (CWK18(GA)n, where n=4, 6, 8 and 10) were synthesized and found to inhibit the proteasome. The gene transfer efficiency mediated by these peptides in four different cell lines established that a GA repeat of four is sufficient to block the proteasome and significantly enhance the gene transfer. Together, these results implicate the proteasome as a previously undiscovered route of metabolism of peptide-based nonviral gene delivery systems and provide a rationale for the use of proteasome inhibition to increase gene transfer efficiency.

Bone regeneration in a rat cranial defect with delivery of PEI-condensed plasmid DNA encoding for bone morphogenetic protein-4 (BMP-4).

Gene therapy approaches to bone tissue engineering have been widely explored. While localized delivery of plasmid DNA encoding for osteogenic factors is attractive for promoting bone regeneration, the low transfection efficiency inherent with plasmid delivery may limit this approach. We hypothesized that this limitation could be overcome by condensing plasmid DNA with nonviral vectors such as poly(ethylenimine) (PEI), and delivering the plasmid DNA in a sustained and localized manner from poly(lactic-co-glycolic acid) (PLGA) scaffolds. To address this possibility, scaffolds delivering plasmid DNA encoding for bone morphogenetic protein-4 (BMP-4) were implanted into a cranial critical-sized defect for time periods up to 15 weeks. The control conditions included no scaffold (defect left empty), blank scaffolds (no delivered DNA), and scaffolds encapsulating plasmid DNA (non-condensed). Histological and microcomputed tomography analysis of the defect sites over time demonstrated that bone regeneration was significant at the defect edges and within the defect site when scaffolds encapsulating condensed DNA were placed in the defect. In contrast, bone formation was mainly confined to the defect edges within scaffolds encapsulating plasmid DNA, and when blank scaffolds were used to fill the defect. Histomorphometric analysis revealed a significant increase in total bone formation (at least 4.5-fold) within scaffolds incorporating condensed DNA, relative to blank scaffolds and scaffolds incorporating uncondensed DNA at each time point. In addition, there was a significant increase both in osteoid and mineralized tissue density within scaffolds incorporating condensed DNA, when compared with blank scaffolds and scaffolds incorporating uncondensed DNA, suggesting that delivery of condensed DNA led to more complete mineralized tissue regeneration within the defect area. This study demonstrated that the scaffold delivery system encapsulating PEI-condensed DNA encoding for BMP-4 was capable of enhancing bone formation and may find applications in other tissue types.

An improved large-scale synthesis of PEG-peptides for gene delivery.

Polyethylene glycol (PEG)-peptides are under development as components of nonviral gene delivery systems. Several earlier reports have demonstrated that covalent attachment of PEG to the surface of peptide condensed DNA particles blocks non-specific biodistribution during gene targeting. In this study, we report an improved large-scale synthesis and purification of a DNA condensing PEG-peptide used for gene delivery. The new method takes advantage of low-pressure cation-exchange chromatography to isolate dimeric Cys-Trp-Lys(18). The dimeric peptide was reduced and directly conjugated with PEG-maleimide resulting in PEG-Cys-Trp-Lys(18). The PEG-peptide was purified by low-pressure chromatography affording 50 mumol (400 mg) quantities of PEG-peptide in >95% purity. The approach offers the advantage of avoiding preparative high-performance liquid chromatography (HPLC) purifications of polylysine peptides to increase yield and capacity.

Cross-linked low molecular weight glycopeptide-mediated gene delivery: relationship between DNA metabolic stability and the level of transient gene expression in vivo.

DNA co-condensates were formed by reacting [125I]DNA with an admixture of a high-mannose glycopeptide (Man9-CWK(18)) and either of two poly(ethylene glycol) peptides (PEG-VS-CWK(18) or PEG-SS-CWK(18)) followed by cross-linking with 6-50 mol equiv of glutaraldehyde. [125I]DNA co-condensates were administered intravenously in mice to determine the influence of peptide DNA formulation parameters on specific targeting to Kupffer cells. Optimal targeting to Kupffer cells required the combined use of 50 mol % Man9-CWK(18) and PEG-CWK(18) to mediate specific recognition by the mannose receptor to Kupffer cells. The cellular uptake of cross-linked Man9-CWK(18)/PEG-CWK(18) DNA co-condensates was receptor mediated since Kupffer cell targeting was inhibited by pre-administration of Man-bovine serum albumin (BSA) but not BSA. An optimized formulation targeted 60% of the dose to the liver, with 80% of the liver-targeted DNA localized to Kupffer cells. Cross-linking with either 6, 15, or 50 mol equiv of glutaraldehyde led to a corresponding decrease in the metabolism rate of DNA in liver as measured by half-live- of 4, 6, and 39 h, respectively. Tail vein dosing of 50 microg of DNA co-condensates cross-linked with 6 mol equiv of glutaraldehyde produced detectable levels of human alpha1-antitrypsin in blood after 12 h, which peaked at day six and persisted for 10 days. The level of human alpha1-antitrypsin was elevated two-fold each day when dosing with DNA co-condensates cross-linked with 15 mol equiv of glutaraldehyde, revealing a correlation between the metabolic stability of the DNA in liver and level of gene expression. In addition to possessing greater metabolic stability, DNA co-condensates cross-linked with 50 mol equiv of glutaraldehyde, but lacking a targeting ligand, avoided rapid liver uptake and possessed a prolonged pharmacokinetic half-life, providing insight into a means to target DNA condensates to peripheral tissues.

Thiosugar nucleotide analogues: synthesis of uridine 5'-(2,3,6-tri-O-acetyl-4-S-acetyl-4-thio-alpha-D-galactopyranosyl diphosphate).

The synthesis of a novel uridine diphosphate galactose (UDP-Gal) analog, (UDP-2,3,6-tri-O-acetyl-4-S-acetyl-4-thio-alpha-D-galactopyranose) (10) is described. Compound 10 contains a sulfur in the place of oxygen at the 4-position of the galactose moiety. Compound 10 represents a protected form of a novel sugar nucleotide analog that can potentially be used during chemoenzymatic synthesis to modify complex oligosaccharides.

Evolution of cross-linked non-viral gene delivery systems.

Developing a non-viral gene delivery system that functions in vivo raises the challenge of finding solutions to efficiently deliver DNA to the cell surface that are also compatible with the efficient release of DNA into the cytosol. The stability, particle size and charge of DNA polyplexes and lipoplexes may be optimized to mediate efficient in vitro transfection only to find that different properties are necessary for successful in vivo transfection. Despite their versatility and improved safety, non-viral gene vectors still lack appreciable in vivo transfection efficiency compared to viral vectors. An emerging theme in recent studies is the use of cross-linking to achieve balance between the stability of polyplexes and lipoplexes in the blood and the controlled release of DNA in the cytosol. This review evaluates the evolution of cross-linking strategies aimed at transiently stabilizing non-viral gene delivery systems.

Low molecular weight disulfide cross-linking peptides as nonviral gene delivery carriers.

Cross-linking peptides have been developed by inserting multiple Cys residues into a 20 amino acid condensing peptide that polymerizes through disulfide bond formation when bound to DNA resulting in small, highly stable DNA condensates that mediate efficient in vitro gene transfer [McKenzie et al. (2000) J. Biol. Chem. 275, 9970-9977]. In the present study, a minimal peptide of four Lys and two terminal Cys residues was found to substitute for Cys-Trp-(Lys)(17)-Cys, resulting in DNA condensates with similar particle size and gene expression in HepG2 cells. Substitution of His for Lys residues resulted in an optimal peptide of Cys-His-(Lys)(6)-His-Cys that, in addition to the attributes described above, also provided buffering capacity to enhance in vitro gene expression in the absence of chloroquine. The reported structure-activity relationships systematically explore peptides with combinations of Lys, Cys, and His residues resulting in low molecular weight peptides with improved gene transfer properties.

Strategies for maintaining the particle size of peptide DNA condensates following freeze-drying.

The particle size of peptide DNA condensates were studied after freeze-drying and rehydration as a function of sugar excipient, concentration, pH, DNA concentration, and peptide condensing agent. In the absence of an excipient, freeze-dried 50 microg/ml AlkCWK(18) (iodoacetic acid alkylated Cys-Typ-Lys(18)) DNA condensates formed large fibrous flocculates on rehydration. Of the sugars tested as lyoprotectants, sucrose proved most effective at preserving particle size during rehydration. The addition of 5 wt/vol% sucrose preserved a mean particle diameter of less than 50 nm during rehydration of AlkCWK(18) DNA condensates prepared at DNA concentrations up to 200 microg/ml; however, higher DNA concentrations led to the formation of insoluble fibrous flocculates. Substitution of polyethylene glycol (PEG)-CWK(18) as a DNA condensing peptide eliminated the need for sucrose, resulting in peptide DNA condensates that retained particle size when rehydrated in water or normal saline at concentrations up to 5 mg/ml. The results suggest that sucrose functions primarily as a bulking agent during freeze-drying that only preserves the particle size of AlkCWK(18) DNA condensates up to a maximum concentration of 200 microg/ml. Alternatively, the steric layer created on the surface of PEG-CWK(18) DNA condensates provides far more efficient lyoprotection, preserving their particle size at a concentration of 5 mg/ml without a bulking agent.

A potent new class of reductively activated peptide gene delivery agents.

A new class of peptide gene delivery agents were developed by inserting multiple cysteine residues into short (dp 20) synthetic peptides. Substitution of one to four cysteine residues for lysine residues in Cys-Trp-Lys(18) resulted in low molecular weight DNA condensing peptides that spontaneously oxidize after binding to plasmid DNA to form interpeptide disulfide bonds. The stability of cross-linked peptide DNA condensates increased in proportion to the number of cysteines incorporated into the peptide. Disulfide bond formation led to a decrease in particle size relative to control peptide DNA condensates and prevented dissociation of peptide DNA condensates in concentrated sodium chloride. Cross-linked peptide DNA condensates were 5-60-fold more potent at mediating gene expression in HepG2 and COS 7 cells relative to uncross-linked peptide DNA condensates. The enhanced gene expression was dependent on the number of cysteine residues incorporated, with a peptide containing two cysteines mediating maximal gene expression. Cross-linking peptides caused elevated gene expression without increasing DNA uptake by cells, suggesting a mechanism involving intracellular release of DNA triggered by disulfide bond reduction. The results establish cross-linking peptides as a novel class of potent gene delivery agents that enhance gene expression through a new mechanism of action.

Synthesis of homogeneous glycopeptides and their utility as DNA condensing agents.

Two glycopeptides were synthesized by attaching purified glycosylamines (N-glycans) to a 20 amino acid peptide. Triantennary and Man9 Boc-tyrosinamide N-glycans were treated with trifluoroacetic acid to remove the Boc group and expose a tyrosinamide amine. The amine group was coupled with iodoacetic acid to produce N-iodoacetyl-oligosaccharides. These were reacted with the sulfhydryl group of a cysteine-containing peptide (CWK18), resulting in the formation of glycopeptides in good yield that were characterized by 1H NMR and ESIMS. Both glycopeptides were able to bind to plasmid DNA and form DNA condensates of approximately 110 nm mean diameter with zeta potential of +31 mV. The resulting homogeneous glycopeptide DNA condensates will be valuable as receptor-mediated gene-delivery agents.

Biodistribution, metabolism, and in vivo gene expression of low molecular weight glycopeptide polyethylene glycol peptide DNA co-condensates.

The biodistribution, metabolism, cellular targeting, and gene expression of a nonviral peptide DNA gene delivery system was examined. (125)I-labeled plasmid DNA was condensed with low molecular weight peptide conjugates and dosed i.v. in mice to determine the influence of peptide DNA formulation parameters on specific gene targeting to hepatocytes. Optimal targeting to hepatocytes required the combined use of a triantennary glycopeptide (Tri-CWK(18)) and a polyethylene glycol-peptide (PEG-CWK(18)) to mediate specific recognition by the asialoglycoprotein receptor and to reduce nonspecific uptake by Kupffer cells. Tri-CWK(18)/PEG-CWK(18) DNA co-condensates were stabilized and protected from metabolism by glutaraldehyde crosslinking. An optimized formulation targeted 60% of the dose to the liver with 80% of the liver targeted DNA localized to hepatocytes. Glutaraldehyde crosslinking of DNA condensates reduced the liver elimination rate from a t((1/2)) of 0.8 to 3.6 h. An optimized gene delivery formulation produced detectable levels of human alpha1-antitrypsin in mouse serum which peaked at day 7 compared to no expression using control formulations. The results demonstrate the application of formulation optimization to improve the targeting selectivity and gene expression of a peptide DNA delivery system.

Tissue targeting of multivalent GalNAc Le(x) terminated N-glycans in mice.

N-Linked biantennary and triantennary oligosaccharides containing multiple terminal GalNAc Le(x) (GalNAcss1-4[Fuc-alpha1-3]GlcNAc) determinants were radioiodinated and their pharmacokinetics, biodistribution, and hepatic cellular localization were determined in mice. Pharmacokinetic analysis revealed GalNAc Le(x) biantennary and triantennary oligosaccharides had a similar mean residence time and steady-state volume of distribution but differed in their total body clearance rate due a shorter alpha half-life for GalNAc Le(x) triantennary. Biodistribution and whole-body-autoradiography studies revealed that both GalNAc Le(x) terminated biantennary and triantennary oligosaccharides predominately targeted to the liver, which accumulated 72% and 79% of the dose 30 min after administration, respectively. Separation of mouse liver parenchymal from non-parenchymal cells demonstrated both N-glycans were almost exclusively (94%) taken up by the parenchymal cells. By comparison, GalNAc terminated biantennary and triantennary N-glycans accumulated in the liver with a targeting efficiency of 73% and 81%, respectively. It is concluded that GalNAc and GalNAc Le(x) terminated N-glycans are recognized in vivo with equivalent affinity by the murine hepatic asialoglycoprotein receptor.

Xanthosoma sagittifolium tubers contain a lectin with two different types of carbohydrate-binding sites.

An unusual lectin possessing two distinctly different types of carbohydrate-combining sites was purified from tubers of Xanthosoma sagittifolium L. by consecutive passage through two affinity columns, i.e. asialofetuin-Sepharose and invertase-Sepharose. SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and gel filtration chromatography of the purified lectin showed that the X. sagittifolium lectin is a heterotetrameric protein composed of four 12-kDa subunits (alpha(2)beta(2)) linked by noncovalent bonds. The results obtained by quantitative precipitation and hapten inhibition assays revealed that the lectin has two different types of carbohydrate-combining sites: one type for oligomannoses, which preferentially binds to a cluster of nonreducing terminal alpha1,3-linked mannosyl residues, and the other type for complex N-linked carbohydrates, which best accommodates a non-sialylated, triantennary oligosaccharide with N-acetyllactosamine (i.e. Galbeta1,4GlcNAc-) or lacto-N-biose (i.e. Galbeta1,3GlcNAc-) groups at its three nonreducing termini.