A phase I/II randomized, double-blinded, placebo-controlled trial of a self-amplifying Covid-19 mRNA vaccine.

Coronavirus disease-19 (Covid-19) pandemic have demonstrated the importantance of vaccines in disease prevention. Self-amplifying mRNA vaccines could be another option for disease prevention if demonstrated to be safe and immunogenic. Phase 1 of this randomized, double-blinded, placebo-controlled trial (N = 42) assessed the safety, tolerability, and immunogenicity in healthy young and older adults of ascending levels of one-dose ARCT-021, a self-amplifying mRNA vaccine against Covid-19. Phase 2 (N = 64) tested two-doses of ARCT-021 given 28 days apart. During phase 1, ARCT-021 was well tolerated up to one 7.5 µg dose and two 5.0 µg doses. Local solicited AEs, namely injection-site pain and tenderness were more common in ARCT-021vaccinated, while systemic solicited AEs, mainly fatigue, headache and myalgia were reported in 62.8% and 46.4% of ARCT-021 and placebo recipients, respectively. Seroconversion rate for anti-S IgG was 100% in all cohorts, except for the 1 µg one-dose in younger adults and the 7.5 µg one-dose in older adults. Anti-S IgG and neutralizing antibody titers showed a general increase with increasing dose, and overlapped with titers in Covid-19 convalescent patients. T-cell responses were also observed in response to stimulation with S-protein peptides. Taken collectively, ARCT-021 is immunogenic and has favorable safety profile for further development.

Corazonin Neurons Contribute to Dimorphic Ethanol Sedation Sensitivity in Drosophila melanogaster.

Exposure to alcohol has multiple effects on nervous system function, and organisms have evolved mechanisms to optimally respond to the presence of ethanol. Sex differences in ethanol-induced behaviors have been observed in several organisms, ranging from humans to invertebrates. However, the molecular mechanisms underlying the dimorphic regulation of ethanol-induced behaviors remain incompletely understood. Here, we observed sex differences in ethanol sedation sensitivity in Drosophila Genome Reference Panel (DGRP) lines of Drosophila melanogaster compared to the absence of dimorphism in standard laboratory wildtype and control lines. However, in dose response experiments, we were able to unmask dimorphic responses for the control mutant line w 1118 by lowering the testing ethanol concentration. Notably, feminization of the small population of Corazonin (Crz) neurons in males was sufficient to induce female-like sedation sensitivity. We also tested the role of the transcription factor apontic (apt) based on its known expression in Crz neurons and its regulation of sedation responses. Interestingly, loss of function apt mutations increased sedation times in both males and females as compared to controls. No significant difference between male and female apt mutants was observed, suggesting a possible role of apt in the regulation of dimorphic ethanol-induced responses. Thus, our results shed light into the mechanisms regulating sex-differences in ethanol-induced behaviors at the cellular and molecular level, suggesting that the genetic sex in a small neuronal population plays an important role in modulating sex differences in behavioral responses to ethanol.

A general protocol for the expression and purification of the intact transmembrane transporter FeoB.

Ferrous iron (Fe2+) transport is an essential process that supports the growth, intracellular survival, and virulence of several drug-resistant pathogens, and the ferrous iron transport (Feo) system is the most important and widespread protein complex that mediates Fe2+ transport in these organisms. The Feo system canonically comprises three proteins (FeoA/B/C). FeoA and FeoC are both small, accessory proteins localized to the cytoplasm, and their roles in the Fe2+ transport process have been of great debate. FeoB is the only wholly-conserved component of the Feo system and serves as the inner membrane-embedded Fe2+ transporter with a soluble G-protein-like N-terminal domain. In vivo studies have underscored the importance of Feo during infection, emphasizing the need to better understand Feo-mediated Fe2+ uptake, although a paucity of research exists on intact FeoB. To surmount this problem, we designed an overproduction and purification system that can be applied generally to a suite of intact FeoBs from several organisms. Importantly, we noted that FeoB is extremely sensitive to excess salt while in the membrane of a recombinant host, and we designed a workflow to circumvent this issue. We also demonstrated effective protein extraction from the lipid bilayer through small-scale solubilization studies. We then applied this approach to the large-scale purifications of Escherichia coli and Pseudomonas aeruginosa FeoBs to high purity and homogeneity. Lastly, we show that our protocol can be generally applied to various FeoB proteins. Thus, this workflow allows for isolation of suitable quantities of FeoB for future biochemical and biophysical characterization.

A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB.

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe2+ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein-protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen-deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein-protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA-NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.

A single dose of self-transcribing and replicating RNA-based SARS-CoV-2 vaccine produces protective adaptive immunity in mice.

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4+ T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 µg and 10 µg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.

The Siderophore Transporter Sit1 Determines Susceptibility to the Antifungal VL-2397.

VL-2397 (previously termed ASP2397) is an antifungal, aluminum-chelating cyclic hexapeptide with a structure analogous to that of ferrichrome-type siderophores, whereby replacement of aluminum by iron was shown to decrease the antifungal activity of this compound. Here, we found that inactivation of an importer for ferrichrome-type siderophores, termed Sit1, renders Aspergillus fumigatus resistant to VL-2397. Moreover, expression of the endogenous sit1 gene under the control of a xylose-inducible promoter (to uncouple sit1 expression from iron repression) combined with C-terminal tagging with a fluorescent protein demonstrated localization of Sit1 in the plasma membrane and xylose-dependent VL-2397 susceptibility. This underlines that Sit1-mediated uptake is essential for VL-2397 susceptibility. Under xylose-induced sit1 expression, VL-2397 also retained antifungal activity after replacing aluminum with iron, which demonstrates that VL-2397 bears antifungal activity independent of cellular aluminum importation. Analysis of sit1 expression indicated that the reduced antifungal activity of the iron-chelated VL-2397 is caused by downregulation of sit1 expression by the imported iron. Furthermore, we demonstrate that defects in iron homeostatic mechanisms modulate the activity of VL-2397. In contrast to A. fumigatus and Candida glabrata, Saccharomyces cerevisiae displays intrinsic resistance to VL-2397 antifungal activity. However, expression of sit1 from A. fumigatus, or its homologue from C. glabrata, resulted in susceptibility to VL-2397, which suggests that the intrinsic resistance of S. cerevisiae is based on lack of uptake and that A. fumigatus, C. glabrata, and S. cerevisiae share an intracellular target for VL-2397.

First-in-Human Phase 1 Study To Assess Safety, Tolerability, and Pharmacokinetics of a Novel Antifungal Drug, VL-2397, in Healthy Adults.

VL-2397 is an antifungal drug with a novel mechanism of action, rapid fungicidal in vitro activity, and potent in vivo activity against Aspergillus fumigatus, including azole-resistant strains. VL2397-101, a phase 1 first-in-human, randomized, double-blind, placebo-controlled dose-escalation study, was conducted in healthy adults to determine the safety, tolerability, and pharmacokinetics (PK) of single and multiple ascending intravenous (i.v.) doses of VL-2397. All dosing cohorts were fully enrolled; all subjects completed the safety follow-up. A safety committee reviewed the safety data for each dosing cohort prior to recommending the initiation of each subsequent cohort. No serious adverse events (SAEs) occurred; the majority of treatment-emergent adverse events (TEAEs) were mild and self-limited. The most common drug-related TEAEs were infusion site reactions. No clinically concerning trends were noted in vital signs, electrocardiograms, physical examinations, or safety laboratory results. Following single infusions of VL-2397, the overall and maximum exposures rose less than proportionally with increasing doses from 3 mg to 1,200 mg as indicated by area under the concentration-time curve over 24 h (AUC24) and maximum concentration (Cmax). No signs of VL-2397 accumulation were observed following i.v. infusions of 300, 600, and 1,200 mg every 24 h (q24h) for 7 days. Renal elimination played a major role in total body clearance, with up to 47% of unmetabolized drug in urine 24 h after administration at single doses of >30 mg. Overall, VL-2397 dosing in the study appeared to be safe and well tolerated in the healthy subjects. The safety profile, consistent PK, and lack of drug accumulation support further development of VL-2397 in patients with invasive aspergillosis.

Population Pharmacokinetic Modeling of VL-2397, a Novel Systemic Antifungal Agent: Analysis of a Single- and Multiple-Ascending-Dose Study in Healthy Subjects.

VL-2397, a novel, systemic antifungal agent, has potent in vitro and in vivo fungicidal activity against Aspergillus species. Plasma concentrations from a phase 1 study were used to construct a population pharmacokinetic (PPK) model for VL-2397. Healthy subjects aged 18 to 55 years received single doses of VL-2397, ranging from 3 to 1,200 mg, multiple daily doses of 300, 600, or 1,200 mg for 7 days, or 300 mg three times/day for 7 days followed by 600 mg daily for 21 days. Plasma samples were collected throughout the dosing intervals. Sixty-six subjects provided 1,908 concentrations. Drug concentrations over time were increased less than dose proportionally for doses above 30 mg. Dose-normalized concentrations plotted over time did not overlap. A 3-compartment nonlinear saturable binding model fit the data well. Clearance increased with dose, and mean values ranged from 0.4 liters/h at 3 mg to 8.5 liters/h at 1,200 mg. Mean volume in the central compartment ranged from 4.8 to 6.9 liters across doses. In the first 24 h, once-daily dosing results in a rapid decrease in concentrations by hour 16 to approximately 1 mg/liter, regardless of dose, with slow clearance over time. Administration of 300 mg every 8 h achieved concentrations above 1 mg/liter over an entire 24-h period. There was a significant relationship between body surface area and clearance. The data suggest that VL-2397 has nonlinear saturable binding kinetics. Protein binding is the likely primary source of the nonlinearity. The PPK model can now be used to optimize dosing by bridging the kinetics to efficacious pharmacodynamic targets.

Preclinical evaluation of Vaxfectin-adjuvanted Vero cell-derived seasonal split and pandemic whole virus influenza vaccines.

Increasing the potency and supply of seasonal and pandemic influenza vaccines remains an important unmet medical need which may be effectively accomplished with adjuvanted egg- or cell culture-derived vaccines. Vaxfectin, a cationic lipid-based adjuvant with a favorable safety profile in phase 1 plasmid DNA vaccines trials, was tested in combination with seasonal split, trivalent and pandemic whole virus, monovalent influenza vaccines produced in Vero cell cultures. Comparison of hemagglutination inhibition (HI) antibody titers in Vaxfectin-adjuvanted to nonadjuvanted vaccinated mice and guinea pigs revealed 3- to 20-fold increases in antibody titers against each of the trivalent influenza virus vaccine strains and 2- to 8-fold increases in antibody titers against the monovalent H5N1 influenza virus vaccine strain. With the vaccine doses tested, comparable antibody responses were induced with formulations that were freshly prepared or refrigerated at conventional 2-8°C storage conditions for up to 6 mo. Comparison of T-cell frequencies measured by interferon-gamma ELISPOT assay between groups revealed increases of between 2- to 10-fold for each of the adjuvanted trivalent strains and up to 22-fold higher with monovalent H5N1 strain. Both trivalent and monovalent vaccines were easy to formulate with Vaxfectin by simple mixing. These preclinical data support further testing of Vaxfectin-adjuvanted Vero cell culture vaccines toward clinical studies designed to assess safety and immunogenicity of these vaccines in humans.

Vaccination with Vaxfectin(®) adjuvanted SIV DNA induces long-lasting humoral immune responses able to reduce SIVmac251 Viremia.

We evaluated the immunogenicity and efficacy of Vaxfectin(®) adjuvanted SIV DNA vaccines in mice and macaques. Vaccination of mice with Vaxfectin(®) adjuvanted SIV gag DNA induced higher humoral immune responses than administration of unadjuvanted DNA, whereas similar levels of cellular immunity were elicited. Vaxfectin(®) adjuvanted SIVmac251 gag and env DNA immunization of rhesus macaques was used to examine magnitude, durability, and efficacy of humoral immunity. Vaccinated macaques elicited potent neutralizing antibodies able to cross-neutralize the heterologous SIVsmE660 Env. We found remarkable durability of Gag and Env humoral responses, sustained during ~2 y of follow-up. The Env-specific antibody responses induced by Vaxfectin(®) adjuvanted env DNA vaccination disseminated into mucosal tissues, as demonstrated by their presence in saliva, including responses to the V1-V2 region, and rectal fluids. The efficacy of the immune responses was evaluated upon intrarectal challenge with low repeated dose SIVmac251. Although 2 of the 3 vaccinees became infected, these animals showed significantly lower peak virus loads and lower chronic viremia than non-immunized infected controls. Thus, Vaxfectin(®) adjuvanted DNA is a promising vaccine approach for inducing potent immune responses able to control the highly pathogenic SIVmac251.

Vaxfectin adjuvant improves antibody responses of juvenile rhesus macaques to a DNA vaccine encoding the measles virus hemagglutinin and fusion proteins.

DNA vaccines formulated with the cationic lipid-based adjuvant Vaxfectin induce protective immunity in macaques after intradermal (i.d.) or intramuscular (i.m.) delivery of 0.5 to 1 mg of codon-optimized DNA encoding the hemagglutinin (H) and fusion (F) proteins of measles virus (MeV). To characterize the effect of Vaxfectin at lower doses of H+F DNA, rhesus macaques were vaccinated twice with 20 µg of DNA plus Vaxfectin i.d., 100 µg of DNA plus Vaxfectin i.d., 100 µg of DNA plus Vaxfectin i.m. or 100 µg of DNA plus phosphate-buffered saline (PBS) i.m. using a needleless Biojector device. The levels of neutralizing (P = 0.036) and binding (P = 0.0001) antibodies were higher after 20 or 100 µg of DNA plus Vaxfectin than after 100 µg of DNA plus PBS. Gamma interferon (IFN-γ)-producing T cells were induced more rapidly than antibody, but were not improved with Vaxfectin. At 18 months after vaccination, monkeys were challenged with wild-type MeV. None developed rash or viremia, but all showed evidence of infection. Antibody levels increased, and IFN-γ- and interleukin-17-producing T cells, including cells specific for the nucleoprotein absent from the vaccine, were induced. At 3 months after challenge, MeV RNA was detected in the leukocytes of two monkeys. The levels of antibody peaked 2 to 4 weeks after challenge and then declined in vaccinated animals reflecting low numbers of bone marrow-resident plasma cells. Therefore, Vaxfectin was dose sparing and substantially improved the antibody response to the H+F DNA vaccine. This immune response led to protection from disease (rash/viremia) but not from infection. Antibody responses after challenge were more transient in vaccinated animals than in an unvaccinated animal.

A Vaxfectin(®)-adjuvanted HSV-2 plasmid DNA vaccine is effective for prophylactic and therapeutic use in the guinea pig model of genital herpes.

Here we describe studies in the guinea pig model of genital herpes to evaluate a novel plasmid DNA (pDNA) vaccine encoding the HSV-2 glycoprotein D and UL46 and UL47 genes encoding tegument proteins VP11/12 and VP 13/14 (gD2/UL46/UL47), formulated with a cationic lipid-based adjuvant Vaxfectin(®). Prophylactic immunization with Vaxfectin(®)-gD2/UL46/UL47 significantly reduced viral replication in the genital tract, provided complete protection against both primary and recurrent genital skin disease following intravaginal HSV-2 challenge, and significantly reduced latent HSV-2 DNA in the dorsal root ganglia compared to controls. We also examined the impact of therapeutic immunization of HSV-2 infected animals. Here, Vaxfectin(®)-gD2/UL46/UL47 immunization significantly reduced both the frequency of recurrent disease and viral shedding into the genital tract compared to controls. This novel adjuvanted pDNA vaccine has demonstrated both prophylactic and therapeutic efficacy in the guinea pig model of genital herpes and warrants further development.

In vivo anti-tumor effect of expressing p14ARF-TAT using a FGF2-targeted cationic lipid vector.

PURPOSE: To develop an efficient and safe strategy to introduce a therapeutic gene into target cells in vivo for cancer therapy. The overall efficiency is based on proper selection of the delivery vector and expressed protein. METHODS: A plasmid coding for a specific cytotoxic fusion peptide, p14ARF-TAT, was evaluated in a xenograft mouse tumor model. The expressed peptide consisted of three domains, a secretory signal, a membrane permeability segment and a cytotoxic fragment. Gene expression was verified in U87-MG cells by Western blot and cytotoxicity confirmed with CyQuant assay. To improve the delivery, a FGF2 targeting peptide, MQLPLATC, was incorporated into the vector, which was evaluated using a luciferase-expressing plasmid. RESULTS: The luciferase activity in vitro was two-fold higher with the targeted formulations, and cytotoxicity was three-fold higher with expression of the p14ARF-TAT protein. A murine xenograph model of human glioma (U87-MG cells) tumors was used to address in vivo activity. FGF2-targeted lipoplexes demonstrated increased tumor volume reduction as compared to non-targeted formulations. RT-PCR and Western blot of tumor homogenizes indicated p14ARF-TAT expression in tumors along with other tissues. CONCLUSION: p14ARF-TAT was cytotoxic and is a promising approach when combined with an efficient targeting.

Vaxfectin: a versatile adjuvant for plasmid DNA- and protein-based vaccines.

IMPORTANCE OF THE FIELD: Many vaccines require the use of an adjuvant to achieve immunity. So far, few adjuvants have advanced successfully through clinical trials to become part of licensed vaccines. Vaxfectin® (Vical, CA, USA) represents a next-generation adjuvant with promise as a platform technology, showing utility with both plasmid DNA (pDNA) and protein-based vaccines. AREAS COVERED IN THIS REVIEW: This review describes the chemical, physical, preclinical and clinical development of Vaxfectin for pDNA-based vaccines. Also included is the preclinical development of Vaxfectin-adjuvanted protein- and peptide-based vaccines. WHAT THE READER WILL GAIN: The reader will gain knowledge of vaccine adjuvant development from bench to bedside. TAKE HOME MESSAGE: Vaxfectin has effectively boosted the immune response against a range of pDNA-expressed pathogenic antigens in preclinical models extending from rodents to non-human primates. In the clinic, Vaxfectin-adjuvanted pDNA-based H5N1 influenza vaccines have been shown to be well tolerated and to result in durable immune responses within the predicted protective range reported for protein-based vaccines.

In vitro cytotoxic activity of cationic paclitaxel nanoparticles on MDR-3T3 cells.

Cationic paclitaxel nanoparticles were developed and the possible delivery mechanism was explored by cellular uptake studies. In vitro cytotoxicity of paclitaxel-loaded nanoparticles was evaluated with NIH-3T3 cells and multidrug resistant MDR-3T3 cells (with active P-glycoprotein). The IC(50)s of paclitaxel nanoparticles, liposomal paclitaxel, and Taxol((R)) on NIH-3T3 cells were 0.7 microg/mL, 3.0 microg/mL, and 3.6 microg/mL, respectively, and on MDR-3T3 cells changed to 1.4 microg/mL, 4.4 microg/mL, and 7.3 microg/mL respectively. After addition of verapamil (nonspecific P-glycoprotein inhibition), the IC(50)s on MDR-3T3 cells changed to 0.3 microg/mL, 0.7 microg/mL, and 1.5 microg/mL, respectively. The cellular uptake study of NBD-DOPE labeled nanoparticles by MDR-3T3 cells showed more cellular associated fluorescence than neutral liposomes (EPC/cholesterol). The cellular uptake was not affected by verapamil. Fluorescent nanoparticle-encapsulated 10-nonyl bromide acridine orange also demonstrated an enhanced uptake compared to neutral liposomes. The cellular uptake was increased after verapamil's addition. The cellular uptake of formulations with increased positive charges and the competition of free cationic lipid GL89 demonstrated that the positive charge of the particles enhanced the cellular uptake. In conclusion, although the cationic paclitaxel nanoparticle is susceptible to P-glycoprotein efflux, it is still a promising delivery system for paclitaxel, because of enhanced uptake, which resulted in significantly increased cytotoxicity.

Regulation of adult hematopoietic stem cells fate for enhanced tissue-specific repair.

The ability to control the differentiation of adult hematopoietic stem cells (HSCs) would promote development of new cell-based therapies to treat multiple degenerative diseases. Systemic injection of NaIO(3) was used to ablate the retinal pigment epithelial (RPE) layer in C57Bl6 mice and initiate neural retinal degeneration. HSCs infected ex vivo with lentiviral vector expressing the RPE-specific gene RPE65 restored a functional RPE layer, with typical RPE phenotype including coexpression of another RPE-specific marker, CRALBP, and photoreceptor outer segment phagocytosis. Retinal degeneration was prevented and visual function, as measured by electroretinography (ERG), was restored to levels similar to that found in normal animals. None of the controls (no HSCs, HSCs alone and HSCs infected with lentiviral vector expressing LacZ) showed these effects. In vitro gene array studies demonstrated that infection of HSC with RPE65 increased adenylate cyclase mRNA. In vitro exposure of HSCs to a pharmacological agonist of adenylate cyclase also led to in vitro differentiation of HSCs to RPE-like cells expressing pigment granules and the RPE-specific marker, CRALBP. Our data confirm that expression of the cell-specific gene RPE65 promoted fate determination of HSCs toward RPE for targeted tissue repair, and did so in part by activation of adenylate cyclase signaling pathways. Expression by HSCs of single genes unique to a differentiated cell may represent a novel experimental paradigm to influence HSC plasticity, force selective differentiation, and ultimately lead to identification of pharmacological alternatives to viral gene delivery.

Development of peptide-targeted lipoplexes to CXCR4-expressing rat glioma cells and rat proliferating endothelial cells.

A peptide analog, 4-fluorobenzoyl-RR-(L-3-(2-naphthyl)alanine)-CYEK-(L-citrulline)-PYR-(L-citrulline)-CR, covalently linked to a phospholipid, was used for targeting a lipid-based gene delivery vehicle to CXCR4(+)-cells. Characterization of transfection activity was done in vitro using a transformed rat glioma cell line (RG2) that expresses CXCR4. The substitution of the targeting lipid at increasing mole percentages in the place of helper lipids yielded a progressive increase in reporter gene expression, reaching a maximum of 2.5 times the control value at 20 mol% of ligand. The substitution of helper lipids with cysteine-derivatized phospholipid analog or phosphatidylethanolamine resulted in a progressive decrease in transfection activity, with complete inactivation of the complex occurring at 20 mol%. A DNA dose-response with 10 mol% of lipopeptide reduced the effective DNA dose at least fivefold with regard to the number of transfected cells and >20-fold with regard to the amount of gene expression. Gene transfer to rat endothelial cells was studied in the context of an arterial organ culture. Mesenteric arteries were cannulated and maintained in culture for up to 4 days. CXCR4 cell-surface expression on endothelial cells was induced after overnight incubation with vascular endothelial growth factor (VEGF). Gene transfer studies showed that only the peptide-targeted lipoplexes transfected the endothelium, and only after CXCR4 had been induced with VEGF. These results demonstrate that non-viral transfection complexes can be targeted to cells expressing CXCR4, and that gene transfer is dependent upon cell surface receptor expression levels.

IGF binding protein-3 regulates hematopoietic stem cell and endothelial precursor cell function during vascular development.

We asked whether the hypoxia-regulated factor, insulin-like growth factor binding protein-3 (IGFBP3), could modulate stem cell factor receptor (c-kit+), stem cell antigen-1 (sca-1+), hematopoietic stem cell (HSC), or CD34+ endothelial precursor cell (EPC) function. Exposure of CD34+ EPCs to IGFBP3 resulted in rapid differentiation into endothelial cells and dose-dependent increases in cell migration and capillary tube formation. IGFBP3-expressing plasmid was injected into the vitreous of neonatal mice undergoing the oxygen-induced retinopathy (OIR) model. In separate studies, GFP-expressing HSCs were transfected with IGFBP3 plasmid and injected into the vitreous of OIR mice. Administering either IGFBP3 plasmid alone or HSCs transfected with the plasmid resulted in a similar reduction in areas of vasoobliteration, protection of the developing vasculature from hyperoxia-induced regression, and reduction in preretinal neovascularization compared to control plasmid or HSCs transfected with control plasmid. In conclusion, IGFBP3 mediates EPC migration, differentiation, and capillary formation in vitro. Targeted expression of IGFBP3 protects the vasculature from damage and promotes proper vascular repair after hyperoxic insult in the OIR model. IGFBP3 expression may represent a physiological adaptation to ischemia and potentially a therapeutic target for treatment of ischemic conditions.

Feasibility of weekly HIV drug delivery to enhance drug localization in lymphoid tissues based on pharmacokinetic models of lipid-associated indinavir.

PURPOSE: Compare the simulated pharmacokinetics of lipid-associated and soluble indinavir (IDV) to determine the potential for greater control of virus replication in the lymphoid tissues. METHODS: Two-compartment mathematical models were developed to simulate the human pharmacokinetics of soluble and lipid-associated forms of IDV in the central compartment and the lymphoid tissue. The lipid-associated IDV model was then used to determine the minimum dosing schedule needed to attain central or lymph drug concentrations comparable to the soluble form. RESULTS: Association of IDV to lipid nanoparticles has a favorable half-life and tissue distribution and allows comparable minimum drug concentration in the lymph (where the majority of viral replication occurs) to be achieved with a dosing schedule of every 95.5 h (approximately 4 days). CONCLUSIONS: Presuming pharmacodynamics of lipid-associated IDV are similar to soluble IDV, estimations based on the proposed kinetic model suggest the novel delivery system could have a tremendous impact on the current standard of HIV treatment, particularly for therapy targeted to clear virus sanctuaries in lymphoid tissues. With less frequent and more effective dosing, lipid-associated indinavir delivery as an adjunct to conventional antiviral therapy could lead to better suppression of viral replication, increased immunological benefit, and fewer treatment failures.

Formation of plasmid-based transfection complexes with an acid-labile cationic lipid: characterization of in vitro and in vivo gene transfer.

PURPOSE: This study tests the hypothesis that gene transfer efficiency may be improved through the use of transiently stable transfection complexes that degrade within endosomal compartments and promote plasmid escape into the cytosol. METHOD: An acid labile cationic lipid, O-(2R-1,2-di-O-(1'Z, 9'Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), was designed, synthesized, and tested for enhanced gene transfer activity relative to non-labile controls. RESULTS: The O-alkenyl chains of BCAT were completely hydrolyzed after 4 h incubation in pH 4.5 buffer at 25 degrees C. Addition of BCAT to plasmid DNA in 40% ethanol followed by ethanol evaporation yielded transfection complexes that transfected several cell types in the presence of fetal calf serum and without the need of a helper lipid. Transfection complexes prepared from BCAT displayed higher luciferase expression than the corresponding DCAT complexes (an acid-insensitive derivative of BCAT) for all cell types tested. Uptake studies showed that this increase was not due to a difference in the amount of DNA being delivered. FAGS analysis for GFP expression showed that BCAT transfection complexes yielded 1.6 more transfected cells and 20% higher log mean fluorescence than DCAT transfection complexes. In vivo gene transfer was demonstrated in subcutaneous tumor-bearing mice by systemic administration of a 60 microg plasmid dose. Expression was observed in the lungs and in the tumor, with the highest activity being observed in the lungs. CONCLUSIONS: Our results show that increased transfection can be obtained by coupling the cationic headgroup to the hydrophobic amphiphilic tails via acid-labile bonds. Acid-catalyzed release of the alkyl chains should facilitate dissociation of the cationic lipid headgroup from the plasmid, thus accelerating one of the rate-limiting steps in cationic lipid mediated transfection.