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Vector Titers (vector + titer)
Selected AbstractsChimeric HIV-1 and HIV-2 lentiviral vectors with added safety insurance,JOURNAL OF MEDICAL VIROLOGY, Issue 2 2007Geetanjali Sachdeva Abstract Lentiviruses are unique in their ability to infect both dividing and non-dividing cells. This makes the vectors derived from them particularly useful for gene transfer into non-dividing cells, including stem cells. Lentiviral vectors are becoming the vectors of choice for si/shRNA delivery. The utility of the lentiviral vectors will be enhanced if additional elements of safety are built into their design. One safety concern is the generation of replication competent virus by recombination. We reasoned that HIV-1 and HIV-2 hybrid or chimeric lentiviral vectors will have added safety insurance in this regard. This is based on the premise that HIV-1 and HIV-2 are dissimilar enough in sequence to curtail recombination, yet similar enough to complement functionally. For hybrid vectors, we found that both HIV-1 and HIV-2 transfer vector RNAs could be packaged to equivalent titer by the HIV-1 packaging machinery. However, HIV-2 packaging machinery was unable to package HIV-1 transfer vector as well as it did HIV-2 transfer vector. This non-reciprocacity suggested that the requirement for HIV-2 vectors was more stringent and that for HIV-1 vectors more promiscuous. When the HIV-1 transfer vector was packaged with the chimeric packaging construct where the leader-gag region of HIV-2 was replaced with that of HIV-1 packaging construct, the titer of the vector went up. This suggests that at least some of the determinants of specificity for vector assembly reside in the leader-gag region. Incorporation of central polypurine tract (cPPT) and woodchuck post-transcriptional enhance element (WPRE) into the HIV-2 vectors had only modest effect on vector titer. Thus, chimeric lentiviral vectors with added safety features can be designed without compromising transduction efficiency. J. Med. Virol. 79:118,126, 2007. © 2006 Wiley-Liss, Inc. [source] Retrograde adenoviral vector targeting of nociresponsive pontospinal noradrenergic neurons in the rat in vivoTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2009Patrick W. Howorth Abstract The spinal dorsal horn receives a dense innervation of noradrenaline-containing fibers that originate from pontine neurons in the A5, locus coeruleus (LC), and A7 cell groups. These pontospinal neurons are believed to constitute a component of the endogenous analgesic system. We used an adenoviral vector with a catecholaminergic-selective promoter (AVV-PRS) to retrogradely label the noradrenergic neurons projecting to the lumbar (L4,L5) dorsal horn with enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein (mRFP). Retrogradely labeled neurons (145 ± 12, n = 14) were found in A5-12%, LC-80% and A7-8% after injection of AVV-PRS-EGFP to the dorsal horn of L4,L5. These neurons were immunopositive for dopamine ,-hydroxylase, indicating that they were catecholaminergic. Retrograde labeling was optimal 7 days after injection, persisted for over 4 weeks, and was dependent on viral vector titer. The spinal topography of the noradrenergic projection was examined using EGFP- and mRFP-expressing adenoviral vectors. Pontospinal neurons provide bilateral innervation of the cord and there was little overlap in the distribution of neurons projecting to the cervical and lumbar regions. The axonal arbor of the pontospinal neurons was visualized with GFP immunocytochemistry to show projections to the inferior olive, cerebellum, thalamus, and cortex but not to the hippocampus or caudate putamen. Formalin testing evoked c-fos expression in these pontospinal neurons, suggesting that they were nociresponsive (A5-21%, LC-16%, and A7-26%, n = 8). Thus, we have developed a viral vector-based strategy to selectively, retrogradely target the pontospinal noradrenergic neurons that are likely to be involved in the descending control of nociception. J. Comp. Neurol. 512:141,157, 2009. © 2008 Wiley-Liss, Inc. [source] Lentiviral vectors that carry anti-HIV shRNAs: problems and solutionsTHE JOURNAL OF GENE MEDICINE, Issue 9 2007Olivier ter Brake Abstract Background HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. Methods Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. Results RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. Conclusions Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production. Copyright © 2007 John Wiley & Sons, Ltd. [source] Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus-based lentiviral vectorsTHE JOURNAL OF GENE MEDICINE, Issue 4 2002Seraphin Kuate Abstract Background Lentiviral vectors allow gene transfer into non-dividing cells. Further development of these vector systems requires stable packaging cell lines that enable adequate safety testing. Methods To generate a packaging cell line for vectors based on simian immunodeficiency virus (SIV), expression plasmids were constructed that contain the codon-optimized gag-pol gene of SIV and the gene for the G protein of vesicular stomatitis virus (VSV-G) under the control of an ponasterone-inducible promoter. Stable cell lines expressing these packaging constructs were established and characterized. Results The RT activity and vector titers of cell clones stably transfected with the inducible gag-pol expession plasmid could be induced by ponasterone by more than a factor of 1000. One of these clones was subsequently transfected with the ponasterone-inducible VSV-G expression plasmid to generate packaging cells. Clones of the packaging cells were screened for vector production by infection with an SIV vector and subsequent induction by ponasterone. In the supernatant of selected ponasterone-induced producer clones vector titers of more than 1×105 transducing units/ml were obtained. Producer cell clones were stable for at least five months, as tested by vector production. Conclusions The packaging cells described should be suitable for most preclinical applications of SIV-based vectors. By avoiding regions of high homology between the vector and the packaging constructs, the design of the SIV packaging cell line should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the SIV vector constructs that can be packaged. Copyright © 2002 John Wiley & Sons, Ltd. [source] | ||||||||||