Controlled Release Systems for DNA Delivery

Adapting controlled release technologies to the delivery of DNA has the potential to overcome extracellular barriers that limit gene therapy. Controlled release systems can enhance gene delivery and increase the extent and duration of transgene expression relative to more traditional delivery methods (e.g., injection). These systems typically deliver vectors locally, which can avoid distribution to distant tissues, decrease toxicity to nontarget cells, and reduce the immune response to the vector. Delivery vehicles for controlled release are fabricated from natural and synthetic polymers, which function either by releasing the vector into the local tissue environment or by maintaining the vector at the polymer surface. Vector release or binding is regulated by the effective affinity of the vector for the polymer, which depends upon the strength of molecular interactions. These interactions occur through nonspecific binding based on vector and polymer composition or through the incorporation of complementary binding sites (e.g., biotin–avidin). This review examines the delivery of nonviral and viral vectors from natural and synthetic polymers and presents opportunities for continuing developments to increase their applicability

Incorporation of Polyethylene Glycol into Self-Assembled Monolayers Enhances Substrate-Mediated Gene Delivery by Nonspecifically- Bound Complexes

Developing systems capable of controlled and efficient gene transfer is a fundamental goal of biotechnology, with applications including functional genomics, gene therapy, and tissue engineering. Substrate-mediated delivery, also termed solid phase delivery, describes the immobilization of DNA, complexed with nonviral vectors, to a biomaterial or substrate through specific or nonspecific interactions. Cells cultured on the substrate are exposed to elevated DNA concentrations within the local microenvironment, which enhances transfection. We investigated transfection resulting from DNA complexes immobilized to a substrate through specific interactions introduced through complementary functional groups on the vector and surface or through nonspecific interactions. Self-assembled monolayers (SAMs) of alkanethiols on gold were used to provide a controlled surface to investigate transfection following specific and non-specific immobilization. DNA, complexed with polyethylenimine (PEI), was immobilized to SAMs through nonspecific mechanisms or covalently linked to SAMs presenting appropriate functional groups through a fraction of the functional groups available on the PEI present in the complex. Nonspecific immobilization of DNA complexes and subsequent transfection was mediated by the hydrophilicity and ionization of the substrate, while covalent tethering resulted in immobilized quantities similar to nonspecific conditions, but provided no transfection. Subsequent studies incorporated polyethylene glycol (PEG)-terminated alkanethiols into the SAMs to reduce nonspecific complex adsorption. Covalent tethering of complexes to PEG/carboxylic acid monolayers resulted in statistically less complex immobilization and no transfection. Nonspecific immobilization to monolayers containing 40% PEG resulted in statistically less DNA complexes immobilized, but substantially greater transfection. Cell adhesion was not affected at this percentage of PEG. Similarly, the addition of Pluronic block copolymers (of polyethylene oxide and polypropylene oxide) to surfaces also enhanced transfection. We hypothesize that the presence of PEG in the monolayer may better preserve complex conformation upon binding to substrates, thereby enhancing the activity of substrate-mediated delivery of DNA complexes

Cellular Arrays (US Patent Application)

The present invention relates to characterizing transcription within cells. In particular, the present invention provides transfected cell arrays (e.g., two-dimensional and/or three-dimensional arrays) and systems, kits and methods utilizing the same (e.g., for transcriptional activity characterization). Compositions and methods of the present invention find use in, among other things, research, drug discovery and clinical (e.g., diagnostic, preventative and therapeutic) applications

Bioluminescence Imaging for Assessment and Normalization in Transfected Cell Arrays

Transfected cell arrays (TCAs) represent a high-throughput technique to correlate gene expression with functional cell responses. Despite advances in TCAs, improvements are needed for the widespread application of this technology. We have developed a TCA that combines a two-plasmid system and dual-bioluminescence imaging to quantitatively normalize for variability in transfection and increase sensitivity. The two-plasmids consist of: (i) normalization plasmid present within each spot, and (ii) functional plasmid that varies between spots, responsible for the functional endpoint of the array. Bioluminescence imaging of dual-luciferase reporters (renilla, firefly luciferase) provides sensitive and quantitative detection of cellular response, with minimal post-transfection processing. The array was applied to quantify estrogen receptor α (ERα) activity in MCF-7 breast cancer cells. A plasmid containing an ERα-regulated promoter directing firefly luciferase expression was mixed with a normalization plasmid, complexed with cationic lipids and deposited into an array. ER induction mimicked results obtained through traditional assays methods, with estrogen inducing luciferase expression 10-fold over the antiestrogen fulvestrant or vehicle. Furthermore, the array captured a dose response to estrogen, demonstrating the sensitivity of bioluminescence quantification. This system provides a tool for basic science research, with potential application for the development of patient specific therapies

Bioluminescence Imaging for Assessment and Normalization in Transfected Cell Arrays

Transfected cell arrays (TCAs) represent a high-throughput technique to correlate gene expression with functional cell responses. Despite advances in TCAs, improvements are needed for the widespread application of this technology. We have developed a TCA that combines a two-plasmid system and dual-bioluminescence imaging to quantitatively normalize for variability in transfection and increase sensitivity. The two-plasmids consist of: (i) normalization plasmid present within each spot, and (ii) functional plasmid that varies between spots, responsible for the functional endpoint of the array. Bioluminescence imaging of dual-luciferase reporters (renilla, firefly luciferase) provides sensitive and quantitative detection of cellular response, with minimal post-transfection processing. The array was applied to quantify estrogen receptor α (ERα) activity in MCF-7 breast cancer cells. A plasmid containing an ERα-regulated promoter directing firefly luciferase expression was mixed with a normalization plasmid, complexed with cationic lipids and deposited into an array. ER induction mimicked results obtained through traditional assays methods, with estrogen inducing luciferase expression 10-fold over the antiestrogen fulvestrant or vehicle. Furthermore, the array captured a dose response to estrogen, demonstrating the sensitivity of bioluminescence quantification. This system provides a tool for basic science research, with potential application for the development of patient specific therapies

Motor Outcomes After Neonatal Arterial Ischemic Stroke Related to Early MRI Data in a Prospective Study

OBJECTIVE: We aimed to correlate early imaging data with motor outcomes in a large, homogeneous, cohort of infants with neonatal (diagnosed before 29 days of life) arterial ischemic stroke (AIS).METHODS: From a prospective cohort of 100 children with neonatal AIS, we analyzed the MRI studies performed within the 28 first days of life for 80 infants evaluated at 2 years of age. The relationships between infarction location and corticospinal tract (CST) involvement and motor outcomes were studied RESULTS: Seventy-three infarctions involved the middle cerebral artery (MCA) territory. Of those, 50 were superficial infarctions, 5 deep infarctions, and 18 mixed infarctions. The CST was involved in 24 cases. Nineteen patients with MCA infarctions (26% [95% confidence interval: 16%–34%]) developed hemiplegia. Mixed infarctions (P < .0001) and CST involvement (P < .0001) were highly predictive of hemiplegia. In contrast, 88% of children with isolated superficial MCA infarctions did not exhibit impairment. CONCLUSIONS: Accurate prediction of motor outcomes can be obtained from early MRI scans after neonatal AIS. The absence of involvement of the CST resulted in normal motor development in 94% of cases. CST involvement resulted in congenital hemiplegia in 66% of cases

Controlled surface-associated delivery of genes and oligonucleotides

A system and methods for controlled gene delivery comprising condensed nucleic acids complexed with polylinkers, wherein the complexes are covalently and/or noncovalently bound to the surface of a substrate capable of supporting cell adhesion. The gene delivery system achieves temporal and spatial control of nucleic acid delivery to a target cell or cells through control of complex density on the surface of the support substrate, and reversibility of the attachment of the polylinker to the support substrate. The system and method of the invention can be used to create spatial patterns of gene expression, and in tissue engineering, high throughput screening, and gene therapy applications. What is claimed is: 1. A method for increasing transgene expression, comprising making a controlled nucleic acid delivery system, said system comprising forming nucleic acid polylinker complexes capable of being delivered to cells cultured on a support substrate, wherein said complexes are formed prior to being covalently or non-covalently immobilized to the surface of a support substrate, and wherein said method comprises: a) contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex, said complex being formed prior to attachment to a support substrate; and b) immobilizing the nucleic acid-polylinker complex to a support substrate; and wherein said cells are added to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate. 2. The method of claim 1, further comprising modification of the support substrate with serum prior to addition of the nucleic acid-polylinker complex, and wherein said modification allows for an increase in transgene expression. 3. The method of claim 1, wherein the extent of transgene expression is dependent upon substrate modification and complex formation. 4. The method of claim 1, wherein said nucleic acid polylinker complexes are polyplexes or lipoplexes. 5. The method of claim 1, wherein said support substrate is polystyrene, gold, hyaluronic acid collagen hydrogels or polylactide-co-glycolide (PLG). 6. The method of claim 2, wherein said substrate modification is made by treatment with serum. 7. The method of claim 1, wherein delivery of the nucleic acid-polylinker complexes to cells occurs from a polystyrene surface treated with serum, and wherein said delivery results in similar or greater percentage of transfected cells relative to bolus delivery. 8. The method of claim 1, said method further comprising release of the nucleic acid from the nucleic acid-polylinker complexes, wherein said release is maximized when the support substrate is treated with serum. 9. A method for increasing transgene expression, comprising making a controlled nucleic acid delivery system, said system comprising forming nucleic acid polylinker complexes capable of being delivered to cells cultured on a support substrate, wherein said complexes are covalently or non-covalently immobilized to the surface of a support substrate, and wherein said method comprises: a) contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; and c) adding cells to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein the release of nucleic acid from the nucleic acid-polylinker complexes is further enhanced when the support substrate containing the complexes is treated with serum or is incubated in conditioned medium. 10. The method of claim 2, wherein the delivery of the nucleic acid-polylinker complexes to cells from a serum-modified support substrate results in higher cellular association of the nucleic acid-polylinker complexes with the support substrate. 11. A method for increasing transgene expression, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex, wherein said complex is formed prior to addition to a support substrate; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate. 12. The method of claim 11, wherein said support substrate comprises a biodegradable or non-biodegradable material. 13. The method of either one of claim 1 or 11, wherein said complexes are formed prior to attachment to the solid support substrate. 14. The method of claim 12, wherein said biodegradable material is a hydrogel and said non-biodegradable material is polystyrene or gold. 15. The method of claim 12, wherein said hydrogel comprises a mixture of hyaluronic acid and collagen. 16. A method for increasing transgene expression, wherein said method promotes transfection of primary cells, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein the biodegradable material is a hydrogel and the non-biodegradable material is polystyrene or gold, and wherein the hvdrogel comprises a mixture of hyaluronic acid and collagen. 17. The method of either one of claim 1 or 11, wherein said nucleic acid polylinker complexes are immobilized to the support substrate using biotin and avidin, or an avidin derivative, or by non-specific adsorption. 18. The method of claim 17, wherein said avidin derivative is streptavidin or neutravidin. 19. The method of any one of claim 11–15, wherein the method further comprises controlling the size of the nucleic acid polylinker complex by regulating the salt content during complex formation. 20. The method of claim 19, wherein controlling the size of said complex formation is accomplished by the presence or absence of salt during the formation of the complexes, wherein the forming of large diameter complexes in the presence of salt results in increased transgene expression, and wherein the forming of small diameter complexes in the absence of salt results in a greater percentage of cells being transfected. 21. The method of claim 19, wherein the salt is sodium chloride. 22. A method for increasing transgene expression, comprising the steps of: a) making a controlled nucleic acid delivery system by contacting a nucleic acid with a polylinker to form a nucleic acid-polylinker complex; b) immobilizing the nucleic acid-polylinker complex to a support substrate; wherein said immobilizing is accomplished by covalent or non-covalent means, and c) adding the cells into which transgene expression is desired to the support substrate after immobilization of the nucleic acid-polylinker complex to the support substrate, wherein said method further comprises release of the nucleic acid from the substrate, wherein the release is optimized by using conditioned medium. 23. The method of claim 11, wherein said method further comprises biotinylation of said complex to enhance release of said complex from said substrate. 24. The method of either of claim 1 or 11, wherein the nucleic acid is DNA, RNA or an oligonucleotide. 25. The method of claim 24, wherein said oligonucleotide is an antisense oligonucleotide or a catalytic RNA capable of interfering with the expression of a gene. 26. The controlled nucleic acid delivery system of either of claim 1 or 11, wherein the polylinker is a cationic polymer, cationic lipid, cationic protein, or cationic peptide

Carcass characteristics and beef quality of young grass-fed Angus x Salers bovines

To characterize carcass and meat attributes, such as beef eating quality in specific farming conditions, 31 young grass-fed crossbred Angus x Salers cattle in two farming systems (a mono-cattle system versus a mixed system with beef cattle and sheep) were used in this study. Three muscle cuts (striploin—m. longissimus dorsi et thoracis; bolar blade—m. triceps brachii caput longum; internal flank plate—m. obliquus internus abdominis) were used for consumer eating quality testing and striploin was used for panelist eating quality assessment, and objective measurements [Warner–Bratzler shear force (WBSF) and fatty acid (FA) and antioxidant contents]. Results indicated that the farming system had no impact on carcass characteristics or meat quality, but it tended to affect FA content, which is likely explained by between-system differences in animal maturity (assessed by ossification score). Animal gender had significant effects on three eating quality traits evaluated by untrained consumers, with higher flavor liking, overall liking, and overall meat eating quality (MQ4) scores in females than in males. Additionally, FA contents were correlated with sensory quality traits to varying extents: consumer-scored tenderness, flavor, and overall liking were mainly positively correlated with ω-3 and ω-6 polyunsaturated fatty acid (PUFA) contents, and panelist-evaluated tenderness and abnormal flavor were more positively correlated with total lipids, saturated fatty acid (SFA), and monounsaturated fatty acid (MUFA) contents. Overall, this study showed that specific grass-fed crossbred Angus x Salers cattle can produce lean meat rich in ω-3 PUFAs with a low ω-6/ω-3 ratio and with “better than average” beef eating quality

Secreted metabolome of porcine blastocysts encapsulated with in \u3ci\u3ein vitro\u3c/i\u3e 3D alginate hydrogel culture systems under going morphological changes provides insights into specific mechanisms involved in the initiation of porcine conceptus elongation

Context. The exact mechanisms regulating the initiation of porcine conceptus elongation are not known due to the complexity of the uterine environment. Aims. To identify contributing factors for initiation of conceptus elongation in vitro, this study evaluated differential metabolite abundance within media following culture of blastocysts within unmodified alginate (ALG) or Arg-Gly-Asp (RGD)-modified alginate hydrogel culture systems. Methods. Blastocysts were harvested from pregnant gilts, encapsulated within ALG or RGD or as non-encapsulated control blastocysts (CONT), and cultured. At the termination of 96 h culture, media were separated into blastocyst media groups: non-encapsulated control blastocysts (CONT); ALG and RGD blastocysts with no morphological change (ALG− and RGD−); ALG and RGD blastocysts with morphological changes (ALG+ and RGD+) and evaluated for non-targeted metabolomic profiling by liquid chromatography (LC)–mass spectrometry (MS) techniques and gas chromatography– (GC–MS). Key results. Analysis of variance identified 280 (LC–MS) and 1 (GC–MS) compounds that differed (P \u3c 0.05), of which 134 (LC–MS) and 1 (GC–MS) were annotated. Metabolites abundance between ALG+ vs ALG−, RGD+ vs RGD−, and RGD+ vs ALG+ were further investigated to identify potential differences in metabolic processes during the initiation of elongation. Conclusions. This study identified changes in phospholipid, glycosphingolipid, lipid signalling, and amino acid metabolic processes as potential RGD-independent mechanisms of elongation and identified changes in lysophosphatidylcholine and sphingolipid secretions during RGD-mediated elongation. Implications. These results illustrate changes in phospholipid and sphingolipid metabolic processes and secretions may act as mediators of the RGD-integrin adhesion that promotes porcine conceptus elongation