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Identification of ebola glycoprotein mutants that exhibit increased transduction efficiencySandersfeld, Lindsay Marie 01 December 2009 (has links)
Gene delivery via lentiviruses can yield long term expression of transgenes. Specificity of host cell targeting by viral vectors occurs primarily through viral glycoprotein (GP)/cellular receptor interactions. Ebola virus (EBOV) GP has broad tropism for a variety of cell types making this viral GP a potentially useful reagent for delivery of gene therapy. However, titers of EBOV GP pseudotyped lentiviruses are insufficient for practical use in clinical applications. Enhancement of EBOV-GP pseudotyped titers by as little as half a log might yield clinically applicable titers.
In an alanine scanning study, we identified 19 residues in EBOV-GP1 that increased transduction efficiency two to three fold. When mapped onto the crystal structure of EBOV GP, these residues were primarily located at the interface of GP1/GP2 suggesting these residue substitutions may confer conformational changes in the protein structure thereby enhancing transduction efficiency. To determine if combinations of these alanine substitutions might further enhance transduction, we have introduced the changes into EBOV GP in a stepwise manner. To date, introduction of some combinations of alanine substitutions resulted in as much as an eight-fold increase in transduction over WT GP, this being our super mutant combination, whereas other combinations eliminated transduction.
Identification of 5 additional mutations via 3D modeling of the glycoprotein uncovered an additional mutation in GP2, located at the GP1/GP2 interface, which also enhances EBOV GP transduction. Transduction of cell lines important for gene therapy including hepatocytes and porcine airway cells confirmed an enhancement in transduction as well. Other cell populations, specifically fibroblasts and renal cells, were also transduced but enhanced transduction was not observed indicating this phenomenon may be cell type specific. The in vivo studies were inconclusive because no expression was detected from any of the EBOV GP pseudovirions. Even expression of the positive control, GP64 particles, waned after 3 weeks post inoculation indicating insufficient quantities or poor quality pseudovirions were used. These EBOV GPs should prove useful for future gene therapy studies by providing an alternate glycoprotein that is as effective as GP64 at producing high titer lentiviral vectors.
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Novel gene transfer vector targeted high affinity IL-2 receptor bearing cell /Leung, Chung-wai. January 2001 (has links)
Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references.
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Effective DNA delivery mediated by pH responsive peptidesChan, Fu-lun., 陳賦麟. January 2012 (has links)
Non-viral vectors have been used to deliver therapeutic genes to treat different diseases. There are a variety of non-viral vectors such as liposomes, cationic polymers and peptides. Among all, pH responsive peptides showed excellent DNA transfection efficiency in many types of cell. These peptides are capable of changing their structural conformation as pH decreases, adopting a disordered structure which can destabilize endosomal membrane and therefore enhancing the release of DNA from endosomes into cytosol.
Traditional pH responsive histidine-rich peptides showed good DNA transfection efficiency and low toxicity to the cells when compared with other non-viral vectors. However, their low pKa value restricted these peptides to be protonated only at late endosomal stage, in which DNA is extremely susceptible to endosomal degradation. This hindered the DNA to be released to the cytosol efficiently and therefore reduced DNA transfection efficiency. In response to this, it is of great interest to probe into the insertion of either 2,3-diaminopropionic acid (Dap) or methylated-2,3-diaminopropionic acid Dap(Me) to the peptide as alternative pH sensitive components. The pKa values for both Dap and Dap(Me) peptides are higher than that of histidine. It is anticipated that the higher pKa value, the protonation of peptide could be happened at an earlier stage of endosomal maturation. Such protonation of peptide destabilizes the endosome membrane rapidly, causing the release of DNA to the cytosol effectively and hence improving DNA transfection efficiency.
In this experiment, LADap(Me)4-L1 peptide was the optimal candidate within the series. It showed good DNA transfection efficiency and cell viability in A549 cells among all Dap and Dap(Me) peptides. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences
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Functional characterization of cell cycle-related kinase in glioblastoma and development of gene delivery systemXu, Zhenhua, 许振华 January 2011 (has links)
Cell cycle-related kinase (CCRK) is a 42 KDa serine/threonine protein kinase
homologous to Cdk1, 2 and 7. Previous work has shown that CCRK regulates cell
cycle transition by phosphorylating Cdk2 and Rb. More importantly, it was found
that CCRK was a candidate oncogene in both glioblastoma multiform (GBM) and
human colorectal cancer. However, the mechanistic role of CCRK in
tumorigenicity is still not completely understood.
In the first part of this thesis, I found that casein kinas II beta (CKIIβ) was one
of proteins that interact with CCRK using the high-throughput yeast-two-hybrid
analysis. Then I confirmed their interaction by co-immunoprecipitation. CCRK
phosphorylated CKIIβ at Ser-209 in a cell cycle-dependent manner. The
phosphorylation of CKIIβ by CCRK enhanced the activity of CKII holoenzyme,
protected CKIIβ against proteasome degradation, and facilitated CKIIβ
translocation into the nucleus in U-87 MG and U-373 MG GBM cells. Importantly,
CCRK de-sensitized GBM cells to the cytotoxic effect of three chemotherapy
drugs, whereas knockdown of CCRK by siRNA reduced chemoresistance.
Functionally, CKIIβ is responsible for CCRK-mediated inhibition of apoptosis, as
suppression of CKIIβ by siRNA or CKIIβ inhibitor could re-sensitize cells to the
cytotoxic effect of cisplatin in both wild type and CCRK-overexpressing U-87
MG cells. In vivo studies also showed that stable over-expression of CCRK
increased tumor growth and decreased the anti-tumor efficacy of cisplatin in a
nude mice GBM xenograft model. These results provide the first evidence that
phosphorylation of CKIIβ is a new mechanism by which CCRK confers tumor
growth and drug resistance to GBM cells.
In the second part of this thesis I described a novel polymer, mPPS-FA,
synthesized as a potential gene transfer vector. To complete mPPS-FA, folic acid
was conjugated to a backbone (named mPPS) consisting of a copolymer of methyl
PEG-2000, PEI-600 and sebacoyl chloride. 1H-NMR, FT-IR and UV spectroscopy
were used to characterize the structure of mPPS-FA. It was revealed that
mPPS-FA holds the ability to bind plasmid DNA yielding positively charged
particles (polyplexes). Dynamic light scattering (DLS) and TEM techniques were
used to study the size and morphology of the formed mPPS-FA/DNA
nanocomplexes. Cytotoxicity of the mPPS-FA/DNA nanoparticles was also
evaluated on B16-F0, U87MG, CHO-1 and Ho-8910 cells. The ability of
mPPS-FA to deliver EGFP plasmid to melanoma B16-F0, U87, CHO-1, Ho-8910
and A549 cells was investigated in vitro as compared to the lipid-based
transfection agent LipofectamineTM2000 and Linear PEI 22KDa (L-PEI 22KDa). I
found that mPPS-FA/DNA complexes yielded the highest GFP transfection
efficiency in B16-F0, U87, CHO-1 and Ho-8910 cells, which all highly express
folate receptors (FR), at an mPPS-FA/DNA ratio (w/w) of 15. Furthermore, the
transfection of mPPS-FA/DNA complexes in CHO-1 cells could be significantly
competed and blocked by the free folic acid molecules. All together, mPPS-FA
showed the highest efficiency in vitro and the potential to be developed as a
nonviral gene carrier. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
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The Goodwillie tower of free augmented algebras over connective ring spectraPancia, Matthew 10 February 2015 (has links)
Let R be a connective ring spectrum and let M be an R-bimodule. In this paper
we prove several results that relate the K-theory of R⋉M and T[superscript M, subscript R] to a “topological Witt vectors” construction W(R; M), where R ⋉ M is the square-zero extension of R by M and T [superscript M, subscript R] is the tensor algebra on M. Our main results include a desciption
of the Taylor tower of K(R ⋉ (−)) and the derived functor of K̃(TR(−)) on the category
of R-bimodules in terms of the Taylor tower of W(R;−). W(R;−) has an easily described Taylor tower, given explicitly by Lindenstrauss and McCarthy in [17]. Our main results serve as generalizations of the results for discrete rings in [17, 18] and also extend the computations by Hesselholt and Madsen [15] showing that π₀(TR(R; p)) is isomorphic to the p-typical Witt vectors over R when R a commutative ring. / text
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Progress toward a combined bacterial and viral gene delivery system for mammalian cellsSimper, Melissa Sue 28 August 2008 (has links)
Not available / text
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Novel gene transfer vector targeted high affinity IL-2 receptor bearing cell梁頌偉, Leung, Chung-wai. January 2001 (has links)
published_or_final_version / Medicine / Master / Master of Philosophy
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Genetic re-targeting and de-targeting of adenovirus type 5 in order to create vectors for gene therapy /Myhre, Susanna, January 2007 (has links)
Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 5 uppsatser.
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Characterisation of DNA damage inducible responses and repair in human cells using recombinant adenovirus vectors /Francis, Murray A. January 2000 (has links)
Thesis (Ph.D.) -- McMaster University, 2000. / Includes bibliographical references (leaves 244-294). Also available via World Wide Web.
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A lentiviral gene transfer vector for the treatment of cystic fibrosis airway disease /Limberis, Maria. January 2002 (has links) (PDF)
Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003. / "16th September 2002." Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). Bibliography: leaves xxix-li.
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