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Biomedical subjects

K W Peng

Publications and source records attributed to K W Peng.

13 recordsLinked to original sources
PubMed2003

Electrophoretic deposition of porous hydroxyapatite scaffold.

Bioactive porous hydroxyapatite (HA) scaffold was fabricated using electrophoretic deposition (EPD) technique in the present work. Bulk HA scaffold was achieved by repeated deposition. The green scaffold was sintered at 1200 degrees C to 82% of the theoretical density. Scanning electron microscopy examination and mercury porosimetry measurement have shown that the porosity remains interconnected and a range of pore size from several microns to hundreds of microns was obtained. X-ray diffraction analysis was performed and confirmed that there is no HA decomposition during the sintering process. Mechanical characterization has also shown that the EPD scaffold possesses excellent properties. Cell culturing experiment was carried out and the result shows that the scaffold bioactivity is not only dependent on the interconnectivity of the pores, but also the pore size.

Biocompatible Materials↗
PubMed2001

Systemic therapy of myeloma xenografts by an attenuated measles virus.

Conditionally replicating viruses are promising agents for the treatment of malignancy. Here it is shown that the live attenuated Edmonston-B vaccine strain of measles virus (MV-Edm) replicates selectively in human myeloma cells and has potent antitumor activity. In vitro, replication of MV-Edm was restricted in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBLs) but proceeded efficiently in a panel of 6 myeloma cell lines-ARH-77, RPMI 8226, JJN-3, MM1, KAS-6/1, and KMS-11-and in primary myeloma cells isolated by CD138 sorting from the bone marrow aspirates of 6 patients. MV-Edm infection induced potent cytopathic effects in these myeloma cells, resulting in the formation of multinucleated syncytia that eventually became nonviable. In contrast, syncytial formation in PHA-stimulated PBLs was minimal after MV-Edm infection. In vivo, MV-Edm was antitumorigenic and inhibited the establishment of myeloma cells as xenografts in immunocompromised mice. When injected directly into ARH-77 myeloma xenografts in the mice, MV-Edm caused complete regression of these xenografts. MV-Edm administered intravenously into the tail veins of mice also showed significant antineoplastic activity against established RPMI 8226 and ARH-77 xenografts. In particular, the ARH-77 myeloma xenografts were exquisitely sensitive to MV-Edm therapy, and tumors in all mice regressed completely. In light of its selectivity for myeloma cells and its potent antineoplastic activity against myeloma xenografts in vivo, MV-Edm merits further development for the treatment of multiple myeloma.

Animals↗
PubMed2001

Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors.

Lentiviral vectors represent an attractive technology platform from which to develop a targetable injectable gene delivery system for transduction of specific cell populations in vivo, irrespective of their cell cycle status. Targeted HIV-1-based lentiviral vectors were generated by pseudotyping them with chimeric murine leukemia virus (MLV) envelope glycoproteins displaying N-terminal targeting polypeptides. Vectors displaying an EGF polypeptide were fully infectious on EGF receptor-negative cells, but were inactive on cells with abundant EGF receptors (inverse targeting). Receptor-mediated inactivation of gene transfer was overcome by competing the EGF receptors on the target cells with soluble EGF or by removing the displayed EGF domain from the surface of the vector particles by factor Xa cleavage of a specific protease substrate engineered into its tethering linker (protease targeting). Intravenous infusion of nontargeted HIV-1 vectors led to maximal luciferase activity in liver and spleen with moderate or minimal activity in heart, skeletal muscle, lung, brain, kidney, ovaries and bone marrow. In contrast, intravenous EGF-displaying vectors were expressed maximally in spleen with very low level luciferase expression detectable in liver (EGF-receptor rich). Liver transduction by the EGF-displaying vector was restored by pretreating the animals with soluble EGF suggesting that these vectors are inversely targeted to spleen.

Animals↗
PubMed1999

Viral vector targeting.

The field of viral vector targeting is advancing rapidly. Recent advances include the successful use of bifunctional crosslinkers to target adenoviral and retroviral vectors, elucidation of the crystal structures of an adenoviral and a retroviral receptor-binding domain, and definition of strategies for inserting short targeting peptides and larger polypeptide-binding domains into the coat proteins of a number of different viral vectors. Novel targeting strategies based on host range restriction and protease activation have been developed, targeted replication-competent vectors have shown promise as anti-cancer agents and the possibility of selecting targeted vectors from vector display libraries has been established.

Animals↗
PubMed1999

Strategies for targeting therapeutic gene delivery.

A major goal for gene therapy is to obtain targeted vectors that transfer genes efficiently to specific cell types. In theory, this can be achieved by targeting entry of the vector or by building gene expression cassettes that restrict gene expression to certain cell types. This review summarizes recent strategies to alter vector tropism for targeted gene delivery.

Animals↗
PubMed1999

Selective transduction of protease-rich tumors by matrix-metalloproteinase-targeted retroviral vectors.

We recently showed that retroviral vectors can be targeted through protease substrate interactions. Infectivity is blocked by a polypeptide fused to the viral envelope glycoprotein (SU) and is restored when a protease cleaves the connecting linker, releasing the inhibitory polypeptide from the viral surface. Protease specificity is achieved by engineering the sequence of the linker. Here, using two different matrix-metalloproteinase (MMP)-activatable vectors, we demonstrated highly efficient and selective transduction of MMP-rich target cells in a heterogeneous cell population. In vivo, the MMP-targeted vectors showed strong selectivity for MMP-rich tumor xenografts. Protease-activatable vectors offer new possibilities for in vivo targeting of gene delivery.

Endopeptidases↗
PubMed1999

The mudskipper, Periophthalmodon schlosseri, actively transports NH4+ against a concentration gradient.

Periophthalmodon schlosseri can maintain ammonia excretion rates and low levels of ammonia in its tissues when exposed to 8 and 30 mM NH4Cl, but tissue ammonia levels rise when the fish is exposed to 100 mM NH4Cl in 50% seawater. Because the transepithelial potential is not high enough to maintain the NH4+ concentration gradient between blood and water, ammonia excretion under such a condition would appear to be active. Branchial Na+-K+-ATPase activity is very high and can be activated by physiological levels of NH4+ instead of K+. Ammonia excretion by the fish against a concentration gradient is inhibited by the addition of ouabain and amiloride to the external medium. It is concluded that Na+-K+-ATPase and an Na+/H+ exchanger may be involved in the active excretion of ammonia across the gills. This unique ability of P. schlosseri to actively excrete ammonia is related to the special structure of its gills and allows the fish to continue to excrete ammonia while air exposed or in its burrow.

Ammonia↗
PubMed1998

In vivo selection of protease cleavage sites from retrovirus display libraries.

Phage display libraries are widely used for selection and optimization of polypeptide ligands or protease substrates. Because they are expressed and amplified in bacterial hosts, phage are not ideal for displaying eukaryotic polypeptides or for probing mammalian cells. As retroviruses do not suffer from these limitations we constructed plasmids encoding replication-competent murine leukemia viruses displaying a virally encoded epidermal growth factor (EGF) domain at the N-terminus of the envelope glycoprotein. The EGF-displaying viruses replicated freely on EGF receptor-poor cells without deleting the displayed EGF domain but did not propagate on EGF receptor-rich cells because they were sequestered by the EGF receptors. A retrovirus display library was then generated by diversifying the seven-residue linker between the envelope glycoprotein and the displayed EGF domain. Selective pressure for loss of EGF receptor-binding activity was applied to the library by serial passage on EGF receptor-rich HT1080 cells. The selected viruses propagated on these cells with wild-type efficiencies, a phenotype that was conferred by intracellular cleavage of their displayed linker sequences. The selected linker sequences invariably presented arginine-rich motifs matching the consensus cleavage signal for furin-like proteases. Retrovirus display libraries can be used for the selection of polypeptides interacting with components of living mammalian cells.

3T3 Cells↗
PubMed1997

Masking of retroviral envelope functions by oligomerizing polypeptide adaptors.

We have constructed chimeric retroviral envelopes displaying N-terminal polypeptides that are known to form homotrimeric associations. The amphotropic receptor (RAM-1) binding domain from the trimeric surface (SU) glycoprotein of 4070A murine leukemia virus (MLV)-inhibited ecotropic receptor (Rec-1) mediated infection by the SU glycoprotein of Moloney MLV when grafted to its N-terminus. The block to Rec-1-mediated infection was reversed when the RAM-1 binding domain was cleaved from the vector particles using an engineered factor Xa protease-sensitive cleavage signal between the envelope glycoprotein and its N-terminal extension. Trimeric leucine zipper peptides and the trimeric C-terminal domain of CD40 ligand were shown to inhibit RAM-1-mediated infection of NIH3T3 cells by the 4070A envelope when fused to its N-terminus, whereas monomeric helical peptides and the monomeric epidermal growth factor domain did not. The block to RAM-1-mediated infection was reversed when the trimeric polypeptides were cleaved from the vector particles by addition of factor Xa protease. Envelope binding assays using cleaved and uncleaved chimeric 4070A envelopes revealed that binding to RAM-1 receptors on mammalian cells was hindered by trimeric, but not by monomeric, N-terminal polypeptides. These results have important implications for the design of protease-activatable vectors for targeted gene delivery.

3T3 Cells↗
PubMed1997

A gene delivery system activatable by disease-associated matrix metalloproteinases.

We are developing protease-activatable gene delivery vehicles for selective gene delivery to protease-expressing cells. Angiogenesis, inflammation, and cancer invasion are linked to the overexpression of matrix metalloproteinases (MMPs), which destroy the extracellular matrix. Therefore, the MMPs are promising targets for therapy. We have displayed epidermal growth factor (EGF) on retroviral vector particles as an MMP-cleavable amino-terminal extension of the 4070A murine leukemia virus (MLV) envelope glycoprotein. This was achieved by engineering an MMP-cleavage signal (PLGLWA) into the linker between the EGF domain and the 4070A SU. The chimeric envelope was expressed and incorporated into viral particles, and the EGF domain could be cleaved from the surface of the viral particles by gelatinase A (MMP-2). The MMP-sensitive vector and control MMP-insensitive vectors could bind, via their displayed EGF domains, to EGF receptors on A431 cells but were unable to infect them because the EGF receptor (EGFR) does not support postbinding steps required for retroviral entry. In the presence of exogenous MMPs, the infectivity of the MMP-sensitive vector, but not of the MMP-insensitive vectors, was restored on A431 cells, and this cleavage activation could be partially blocked by MMP inhibitors. Endogenous MMPs produced by EGFR-positive HT 1080 cells could selectively activate the MMP-sensitive vector giving rise to a titer that was 1,000-fold higher on HT 1080 cells than on MMP-negative A431 cells. Inhibitor studies and gelatin zymograms indicated that the membrane-associated MT-MMP expressed on the HT 1080 cells played an important role in cleavage activation of the vector. When presented simultaneously with both EGFR-positive cell lines A431 and HT 1080, the vector could efficiently discriminate between the two different cell types, infecting the MMP-positive HT 1080 cells in preference over the A431 cells.

Binding Sites↗
PubMed1996

Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity.

Five different single-chain antibody fragments (scFv) against human cell-surface antigens were displayed on murine ecotropic retroviral vectors by fusing them to the Moloney SU envelope glycoprotein. The spacing between the scFv and the SU glycoprotein was varied by fusing the scFv to residue +7 or to residue +1 of Moloney SU and by inserting linker sequences of different lengths between the domains. All of the chimeric envelopes were efficiently incorporated into vector particles and could bind to human cells through their displayed antibody fragments, but did not infect them. The spacing between the scFvs and the SU glycoproteins had no significant effect on the efficiency of envelope expression or viral incorporation and did not affect the binding properties of the chimeric envelopes, nor did it influence the efficiency of targeted gene delivery to human cells by scFv-displaying vectors. However, on murine fibroblasts the infectivity of vectors incorporating the chimeric envelopes was strongly influenced by the length of the interdomain spacer. The titers were very low when the single-chain antibodies were fused through a tripeptide linker to SU residue +7 and were greatly enhanced (up to 10(5)-fold) when they were fused to SU residue +1 through a heptapeptide linker. These results point to the importance of steric interactions between the domains of chimeric envelope glycoproteins and may have implications for retroviral vector design for human gene therapy.

Amino Acid Sequence↗
PubMedDate not supplied

Concentration of viral vectors by co-precipitation with calcium phosphate.

BACKGROUND: The envelope glycoproteins, surface unit (SU) and transmembrane (TM) of the murine leukemia virus (MLV) are not covalently linked and tend to dissociate upon high-speed centrifugation, leading to loss of vector infectivity. This study describes a gentle and simple method to concentrate MLV vectors or HIV vectors pseudotyped with MLV envelopes. Having a fast and inexpensive method to concentrate large volumes of vector supernatant will facilitate in vivo experiments and clinical trials that require high titer vector stocks. METHODS: The methods employed in the study were co-precipitation of viral supernatant with calcium phosphate, low-speed centrifugation, dialysis, and infection assays with Lac-Z transducing vectors. RESULTS: Murine leukemia virus vectors and HIV vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV.G) or MLV envelopes were concentrated successfully using the calcium phosphate co-precipitation method. Parameters that influence virus yield and the reproducibility of the method were investigated. The optimized protocol involves virus harvest in serum-free media, co-precipitation using 60mM calcium chloride, pelleting at 2,000 g, resuspending the pellet in a small volume of 0.1M EDTA-saline, and dialysis against saline to remove EDTA. Volumes were decreased from 300 ml to 10 ml, with 50-100% recovery, and titers can be concentrated up to 1,000-fold. CONCLUSIONS: The calcium phosphate co-precipitation method to concentrate virus is applicable to retrovirus and lentivirus preparations. It uses simple techniques and does not require expensive equipment. Multiple rounds of co-precipitation can be carried out if required.

Animals↗