Comparative gene transfer efficiency of low molecular weight polylysine DNA-condensing peptides

In a previous report (M.S. Wadhwa et al . (1997) Bioconjugate Chem. 8, 81–88), we synthesized a panel of polylysine-containing peptides and determined that a minimal repeating lysine chain of 18 residues followed by a tryptophan and an alkylated cysteine residue (AlkCWK 18 ) resulted in the formation of optimal size (78 nm diameter) plasmid DNA condensates that mediated efficient in vitro gene transfer. Shorter polylysine chains produced larger DNA condensates and mediated much lower gene expression while longer lysine chains were equivalent to AlkCWK 18 . Surprisingly, AlkCWK 18 (molecular weight 2672) was a much better gene transfer agent than commercially available low molecular weight polylysine (molecular weight 1000–4000), despite its similar molecular weight. Possible explanations were that the cysteine or tryptophan residue in AlkCWK 18 contributed to the DNA binding and the formation of small condensates or that the homogeneity of AlkCWK 18 relative to low molecular weight polylysine facilitated optimal condensation. To test these hypotheses, the present study prepared AlkCYK 18 and K 20 and used these to form DNA condensates and conduct in vitro gene transfer. The results established that DNA condensates prepared with either AlkCYK 18 or K 20 possessed identical particle size and mediated in vitro gene transfer efficiencies that were indistinguishable from AlkCWK 18 DNA condensates, eliminating the possibility of contributions from cysteine or tryptophan. However, a detailed chromatographic and electrospray mass spectrometry analysis of low molecular weight polylysine revealed it to possess a much lower than anticipated average chain length of dp 6. Thus, the short chain length of low molecular weight polylysine explains its inability to form small DNA condensates and mediate efficient gene transfer relative to AlkCWK 18 DNA condensates. These experiments further emphasize the need to develop homogenous low molecular weight carrier molecules for nonviral gene delivery.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74767/1/j.1399-3011.1999.00104.x.pd

Long-Term In Vivo Gene Expression via Delivery of PEI–DNA Condensates from Porous Polymer Scaffolds

Nonviral delivery vectors are attractive for gene therapy approaches in tissue engineering, but suffer from low transfection efficiency and short-term gene expression. We hypothesized that the sustained delivery of poly(ethylenimine) (PEI)-condensed DNA from three-dimensional biodegradable scaffolds that encourage cell infiltration could greatly enhance gene expression. To test this hypothesis, a PEI-condensed plasmid encoding β-galactosidase was incorporated into porous poly(lactide-co-glycolide) (PLG) scaffolds, using a gas foaming process. Four conditions were examined: condensed DNA and uncondensed DNA encapsulated into PLG scaffolds, blank scaffolds, and bolus delivery of condensed DNA in combination with implantation of PLG scaffolds. Implantation of scaffolds incorporating condensed β-galactosidase plasmid into the subcutaneous tissue of rats resulted in a high level of gene expression for the entire 15-week duration of the experiment, as exemplified by extensive positive staining for β-galactosidase gene expression observed on the exterior surface and throughout the cross-sections of the explanted scaffolds. No positive staining could be observed for the control conditions either on the exterior surface or in the cross-section at 8- and 15-week time points. In addition, a high percentage (55–60%) of cells within scaffolds incorporating condensed DNA at 15 weeks demonstrated expression of the DNA, confirming the sustained uptake and expression of the encapsulated plasmid DNA. Quantitative analysis of β-galactosidase gene expression revealed that expression levels in scaffolds incorporating condensed DNA were one order of magnitude higher than those of other conditions at the 2- week time point and nearly two orders of magnitude higher than those of the control conditions at the 8- and 15-week time points. This study demonstrated that the sustained delivery of PEI–condensed plasmid DNA from PLG scaffolds led to an in vivo long-term and high level of gene expression, and this system may find application in areas such as bone tissue engineering.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63281/1/hum.2005.16.609.pd

Metabolic stability of glutaraldehyde cross-linked peptide dna condensates

The stability of peptide DNA condensates was examined after introducing glutaraldehyde to cross-link surface amine groups. A 20 amino acid peptide (CWK 18 ) was used to condense DNA into small (70 nm) condensates. The reaction between glutaraldehyde and peptide DNA condensates was indirectly monitored using a fluorescence-based assay to establish reaction completion in 4–5 h when using glutaraldehyde-to-peptide ratios of 1 to 4 mol equiv. Higher levels of glutaraldehyde cross-linking led to significant increases in particle size. The improved stability imparted by glutaraldehyde cross-linking was demonstrated by the increased resistance of DNA condensates to shear stress induced fragmentation. The cross-linked condensates were also significantly more resistant to in vitro metabolism by serum endonucleases. A decrease in the magnitude of transient gene expression was determined for cross-linked DNA condensates which also resulted in a 10-day steady-state expression when cross-linking with 4 mol equiv of glutaraldehyde. The results suggest that cross-linking DNA condensates may provide a means to alter the time course of transient gene expression by inhibiting DNA metabolism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34498/1/1_ftp.pd

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 [ 125 I]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. [ 125 I]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 Μg of DNA co-condensates cross-linked with 6 mol equiv of glutaraldehyde produced detectable levels of human Α 1 -antitrypsin in blood after 12 h, which peaked at day six and persisted for 10 days. The level of human Α 1 -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. © 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:2010–2022, 2001Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34505/1/1152_ftp.pd

Formulation of highly soluble poly(ethylene glycol)-peptide DNA condensates

Two poly(ethylene glycol) (PEG)-peptides were synthesized and tested for their ability to bind to plasmid DNA and form soluble DNA condensates with reduced spontaneous gene expression. PEG-vinyl sulfone or PEG-orthopyridyl disulfide were reacted with the sulfhydryl of Cys-Trp-Lys 18 (CWK 18 ) resulting in the formation of nonreducible (PEG-VS-CWK 18 ) and reducible (PEG-SS-CWK 18 ) PEG- peptides. Both PEG-peptides were prepared on a micromole scale, purified by RP-HPLC in >80% yield, and characterized by 1H NMR and MALDI-TOF. PEG-peptides bound to plasmid DNA with an apparent affinity that was equivalent to alkylated (Alk)CWK 18 , resulting in DNA condensates with a mean diameter of 80–90 nm and ζ (zeta) potential of +10 mV. The particle size of PEG-peptide DNA condensates was constant throughout the DNA concentration range of 0.05–2 mg/mL, indicating these to be approximately 20-fold more soluble than AlkCWK 18 DNA condensates. The spontaneous gene transfer to HepG2 cells mediated by PEG-VS-CWK 18 DNA conden- sates was over two orders of magnitude lower than PEG-SS-CWK 18 DNA condensates and three orders of magnitude lower than AlkCWK 18 DNA condensates. PEG-VS-CWK 18 efficiently blocked in vitro gene transfer by reducing cell uptake. The results indicate that a high loading density of PEG on DNA is necessary to achieve highly soluble DNA condensates that reduce spontaneous in vitro gene transfer by blocking nonspecific uptake by HepG2 cells. These two properties are important for developing targeted gene delivery systems to be used in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34495/1/10_ftp.pd

Fabrication and in vitro testing of polymeric delivery system for condensed DNA

Polyethylenimine (PEI) was combined with plasmid DNA and freeze dried following the addition of sucrose as a lyoprotectant and pore-forming agent. Freeze-dried PEI DNA condensates were dry mixed with granular polylactideglycolic acid (PLGA) then compression molded and sponged to encapsulated PEI DNA. A measurement of the elastic modulus indicated that 91 wt% sucrose substituted for 95 wt% sodium chloride as a porogen, resulting in PLGA sponges with a mechanical modulus of 100 kPa. The PEI DNA was retained (80%) within PLGA sponges prepared with sucrose during the leaching and subsequent 2-week release studies, whereas sodium chloride PLGA sponges caused the premature release (100%) of PEI DNA within 2 days. In vitro gene transfer studies with PEI DNA PLGA sponges established that adherent and infiltrating fibroblasts expressed reporter gene for 15 days compared with the short, 3-day expression mediated by direct gene of PEI DNA on cells in culture. The results demonstrate an approach to encapsulate condensed DNA in a PLGA sponge for the purpose of retaining DNA within the matrices and creating efficient gene transfer during tissue engineering. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 1384–1392, 2003Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34432/1/20036_ftp.pd

Acetaminophen Combinations Protect Against Iron-Induced Cardiac Damage in Gerbils

This study tested if acetaminophen, N-methyl-D-glucamine dithiocarbamate (NMGDTC), deferoxamine, and combinations of these agents reduce excess iron content, prevent iron-induced pathology, reduce cardiac arrhythmias, and reduce mortality in iron-overloaded gerbils. Eight groups of 16 gerbils received iron dextran injections (ferric hydroxide dextran complex, 120 mg/kg, ip) or saline solution (controls) twice/wk for 8 wk. The 8 groups were treated every Monday, Wednesday, and Friday with one of the following: saline control, acetaminophen, 150 mg/kg, ip), acetaminophen (150 mg/kg, po), deferoxamine, 83 mg/kg, ip), NMGDTC (200 mg/kg, ip), or combinations of acetaminophen (75 mg/kg) with deferoxamine (42 mg/kg, each ip, separately) or acetaminophen (75 mg/kg) with NMGDTC (100 mg/kg, each ip, separately). The treatments were given 4 hr after each iron injection on days when both iron administration and treatment occurred during iron overloading (8 wk) and were continued 4 wk thereafter. Echocardiography (ECHO) was used to evaluate iron-induced cardiac changes and detect arrhythmias. Acetaminophen and NMGDTC, or combinations thereof, reduced cardiac and hepatic excess iron content as measured by inductively coupled plasma atomic emission spectrometry (ICP-AES). Acetaminophen was effective whether administered po or ip. Acetaminophen treatment had a positive inotropic effect on cardiac function. Acetaminophen-deferoxamine combination conferred equal cardioprotection as acetaminophen or deferoxamine alone, was equally able to remove hepatic iron, and was superior to either acetaminophen or deferoxamine in removing cardiac iron from iron-overloaded gerbils. Acetaminophen-NMGDTC combination was also effective in removing cardiac and hepatic iron and protecting against iron-induced cardiac damage. ECHO evaluation of iron-overloaded, untreated gerbils demonstrated a high incidence of cardiac arrhythmias, usually PVCs (10/16 = 63%), and mortality prior to completion of the experiment (4/16 = 25%). All treatments except deferoxamine, alone, reduced the incidence of cardiac arrhythmias and deaths. All treatments reduced iron-induced increases in hepatic and cardiac weights. This study demonstrates injection alternates that are equally or more effective than deferoxamine injections and shows oral acetaminophen to be effective in treatment of iron-overload and associated cardiac complications

In vivo gene transfer using sulfhydryl cross-linked PEG-peptide/glycopeptide DNA co-condensates

Recent interest in sulfhydryl cross-linked nonviral gene delivery systems, designed to trigger the intracellular release of DNA, has inspired studies to establish their utility in vitro . To determine if this concept can be extrapolated to in vivo gene delivery, sulfhydryl cross-linking peptides (dp 20), derivatized with either an N-glycan or polyethylene glycol (PEG), were used to generate sulfhydryl cross-linked gene formulations. The biodistribution, metabolism, cell-type targeting, and gene expression of sulfhydryl cross-linked PEG-peptide/glycopeptide DNA co-condensates were examined following i.v. dosing in mice. Optimal targeting to hepatocytes was achieved by condensing 125 I-DNA with an add-mixture of 10 mol % triantennary glycopeptide, 5 mol % PEG-peptide, and 85 mol % backbone peptide. Four backbone peptides were substituted into the formulation to examine the influence of peptide metabolism and disulfide bond strength on the rate of DNA metabolism and the level of gene expression in vivo . The half-life of DNA in liver was extended from 1 to 3 h using a backbone peptide composed of d -amino acids, whereas substituting penicillamine for cysteine failed to further increase the metabolic stability of DNA. Optimized gene delivery formulations transiently expressed secreted alkaline phosphatase in mouse serum for 12 days. The results suggest that disulfide bond reduction in liver hepatocytes proceeds rapidly, followed by peptide metabolism, ultimately limiting the metabolic half-life of sulfhydryl cross-linked DNA condensates in vivo . © 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:1174–1185, 2003Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34509/1/10384_ftp.pd

Reduced cellularity of bone marrow in multiple sclerosis with decreased MSC expansion potential and premature ageing in vitro

Background: Autologous bone-marrow-derived cells are currently employed in clinical studies of cell-based therapy in multiple sclerosis (MS) although the bone marrow microenvironment and marrow-derived cells isolated from patients with MS have not been extensively characterised. Objectives: To examine the bone marrow microenvironment and assess the proliferative potential of multipotent mesenchymal stromal cells (MSCs) in progressive MS. Methods: Comparative phenotypic analysis of bone marrow and marrow-derived MSCs isolated from patients with progressive MS and control subjects was undertaken. Results: In MS marrow, there was an interstitial infiltrate of inflammatory cells with lymphoid (predominantly T-cell) nodules although total cellularity was reduced. Controlling for age, MSCs isolated from patients with MS had reduced in vitro expansion potential as determined by population doubling time, colony-forming unit assay, and expression of β-galactosidase. MS MSCs expressed reduced levels of Stro-1 and displayed accelerated shortening of telomere terminal restriction fragments (TRF) in vitro. Conclusion: Our results are consistent with reduced proliferative capacity and ex vivo premature ageing of bone-marrow-derived cells, particularly MSCs, in MS. They have significant implication for MSC-based therapies for MS and suggest that accelerated cellular ageing and senescence may contribute to the pathophysiology of progressive MS. </jats:sec