Pcj

receptor collagen

Fig. 3 Efforts to improve transduction of distinct cell populations have involved modification of viral envelope proteins. A. Retroviral infection begins with interaction of the native retroviral Env with a susceptible cellular receptor. B. Replacement of the native Env receptor binding domain with a peptide ligand may direct retroviral binding to a specific receptor. C. Adaptor proteins contain ligand binding sites from natural Env receptors linked to alternative ligands in an effort to direct retroviral binding to specific receptors. D. Trans-complementation of binding-defective influenza hemagglutinin proteins and fusion-defective retroviral Env proteins expands the retroviral host range while circumventing fusion defects often seen with retroviral envelopes possessing an alternative ligand at the receptor binding site. E. Attachment of a peptide ligand to the retroviral envelope protein outside of the receptor binding domain allows concentration of retroviral particles in the vicinity of target cells by interaction with receptors on nearby structures, e.g., collagen.

receptor collagen

Fig. 3 Efforts to improve transduction of distinct cell populations have involved modification of viral envelope proteins. A. Retroviral infection begins with interaction of the native retroviral Env with a susceptible cellular receptor. B. Replacement of the native Env receptor binding domain with a peptide ligand may direct retroviral binding to a specific receptor. C. Adaptor proteins contain ligand binding sites from natural Env receptors linked to alternative ligands in an effort to direct retroviral binding to specific receptors. D. Trans-complementation of binding-defective influenza hemagglutinin proteins and fusion-defective retroviral Env proteins expands the retroviral host range while circumventing fusion defects often seen with retroviral envelopes possessing an alternative ligand at the receptor binding site. E. Attachment of a peptide ligand to the retroviral envelope protein outside of the receptor binding domain allows concentration of retroviral particles in the vicinity of target cells by interaction with receptors on nearby structures, e.g., collagen.

targeting of ALV-pseudotyped MLV vectors to cells expressing the tumor-specific EGF receptor.[9]

Finally, trans-complementation represents a strategy for circumventing the fusion defect. Orthomyxoviruses and paramyxoviruses possess two distinct envelope proteins which mediate binding and fusion functions separately, suggesting that segregation of the two functions may be possible for retroviral vectors as well. Lin et al. have demonstrated improved transduction efficiency into cells expressing Flt-3 by coexpression of a binding-defective influenza hemagglutinin protein and a fusion-defective MLV Env protein possessing the Flt-3 ligand at the receptor binding domain on an MLV vector.[10]

As an alternative method to improve transduction of tumors in vivo, the use of replication-competent retroviral (RCR) vectors has recently become of interest. As the majority of cells in the body are quiescent, transduction by MLV-based retroviral vectors should be selective for tumor cells. Following the initial transduction event, RCR vectors would replicate, essentially turning each transduced cell into a virus producer cell, and perpetuating gene delivery throughout the tumor. Importantly, RCR vectors typically contain a suicide gene which is toxic to the transduced cell following prodrug administration, thus eliminating tumor cells and minimizing the spread of virus to noncancerous cells. Injection of human glioma cell tumors in rats with MLV-based RCR vectors delivering the cytosine deaminase suicide gene showed 70-90% transduction rates, compared to 1% transduction with replication-defective vectors, and 100% survival of the rats for at least 60 days.[11] The RCR vectors were not detected in other tissues.

Improving Gene Expression

Variable levels of gene expression are observed following integration of current retroviral vectors into target cells. These can be attributed to chromosomal position effects at the site of integration, as well as limitations of vector function. Depending on the site of integration, vector genes may be silenced by a closed chromatin environment or subject to inappropriate regulation by host elements. Expression of retroviral vectors is restricted in several cell types, including embryonic stem cells and murine fibroblasts, as a result of the association of repressive factors with sequences in or near the 5'LTR and methylation of viral promoter elements. Additionally, host cells have a tendency to recognize foreign promoters, especially strong viral promoters such as SV40 and CMV typically used to drive expression of the gene of interest, and inactivate them by various mechanisms, including methylation. Even if gene expression remains active, transduced cells often lose viability over time because of an immune response elicited by the product of the delivered gene.

Replacement of repressive viral sequences with similar sequences from heterologous retroviruses and the minimization of CpG dinucleotides within viral promoters has lessened, but not eliminated, silencing of transgene expression.[12] Silencing of viral promoters has sparked an interest in the use of gene-specific regulatory sequences to direct transgene expression. Logg et al. have reported prostate-specific gene expression which persists over time by incorporation of the prostate-specific probasin promoter into RCR vectors, demonstrating the benefits of improved gene expression and tissue-specificity achieved with an authentic human promoter.[13] Incorporation of gene-specific locus control regions (LCR) into retroviral vectors confers integration site-independent and tissue-specific vector gene expression.[14] Additionally, the use of authentic genomic elements such as scaffold or matrix attachment regions and insulator elements has been reported to improve transgene expression when included in a retroviral vector.[15]

Improving Vector Safety

As retroviral transduction efficiencies and gene expression levels improve, so will the potential for serious adverse effects including retroviremia, generation of replication-competent revertants, germline transmission, and insertional mutagenesis. The recent demonstration that retroviral vectors favor integration sites within transcriptionally active loci has increased the probability of insertional mutagenesis caused by vector integra-tion.[16] Additionally, the development of leukemia-like disease in two French SCID-X1 gene therapy patients as a result of retroviral vector integration in the vicinity of a known oncogene, LMO2, has underscored the necessity for improved safety mechanisms within gene therapy

Many of the vector modifications described above to improve transduction efficiency and gene expression may also improve vector safety. Elimination of dispensable viral sequences from the vector, separation of necessary viral genes (gag, pol, env) onto individual packaging vectors, and replacement of viral sequences with heterol-ogous retroviral components or exogenous c/s-acting regulatory elements all combine to reduce the risk of homologous recombination between vectors and endogenous viruses and the development of chimeric replication-competent vectors. Additionally, transcriptional targeting, or the use of gene-specific regulatory elements to limit gene expression to desired cell types, will help prevent expression in inappropriate cell types. Targeting of vectors to specific cell types or tissues by reengineering of envelope proteins will also limit gene expression to appropriate tissues. The use of insulator elements and matrix attachment regions within vectors may prevent inappropriate activation of cellular oncogenes by confining the action of vector regulatory elements to vector transgenes. Finally, attempts to target integration to specific genomic locations by modification of the integrase enzyme may eventually direct vector integration to benign sites within the genome. Tan et al. have shown that fusion of the synthetic polydactyl zinc finger protein, E2C, to the HIV-1 integrase results in preferential vector integration near the E2C binding site in vitro.[18] The E2C binding sequence is a unique site within the human genome; however, preferential integration has not yet been demonstrated in vivo.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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