Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome

Lehmberg, Elisabeth; Traina, Joseph A.; Chakel, John A.; Chang, Ray-Jen; Parkman, Maria; McCaman, Michael T.; Murakami, Peter K.; Lahidji, Vafa; Nelson, Jim; Hancock, William S.; Nestaas, Eirik; Püngor, E.

doi:10.1016/s0378-4347(99)00316-3

Cited by 79 publications

(93 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In particular, the latest observation argues against the hypothesis that the inflammatory or immune response of individual mice may be determined solely by stochastic factors, but rather suggests that something in the HD-mEPO preparation increases the likelihood that individual mice mount an immune response. The nature and identity of the putative contaminant may be investigated by means of HPLC and SDS͞PAGE (30).…”

Section: Discussionmentioning

confidence: 99%

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus

Maione

Rocca

Giannetti

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance-i.e., 100% of treated animals showing sustained expression after 4 months-was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible. R eplication-deficient adenoviral (Ad) vectors deleted of one or more early genes (first-and second-generation Ad vectors) are among the most efficient vehicles for in vivo gene delivery, but their utilization for therapeutic purposes is limited by the transient nature of transgene expression and the systemic toxicity, which are both due to inflammatory and immune responses triggered by the residual expression of viral proteins (1-5). The development of Ad vectors deleted of all viral coding sequences offers the prospect of a safer and more efficient way to deliver genes (6). Production of fully deleted Ad vectors, also called helper-dependent (HD) Ad vectors, is possible thanks to the supply in trans of the viral proteins required for replication and packaging by a helper first-generation virus (7).Previous studies by several laboratories have shown improved performances of HD vs. first-generation vectors after intravenous (i.v.) injection as to efficacy of transduction, longevity of transgene expression, and systemic toxicity (8-11). These studies strongly suggested that latest generation adenoviruses may be useful for human applications. However, delivery of Ad vectors (including HD vectors) to human recipients may be rendered particularly difficult when a preexisting immunity against an adenovirus serotype identical to, or cross-reactive with, the one used for gene transfer has previously developed as a consequence of a natural infection. This interfering effect is expected to be particularly pronounced in the case of i.v. delivery, because transducing vector particles will be exposed to circulating neutralizing antibodies before reaching the main target organ, the liver. It is therefore important to evaluate performance, therapeutic efficacy, an...

show abstract

Section: Discussionmentioning

confidence: 99%

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus

Maione

Rocca

Giannetti

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…72 In this assay, the virus is irreversibly denatured by an organic solvent acetonitrile. Hydrophobic interaction of the exposed regions of the denatured viral proteins with the hydrophobic surface of the resin mediates binding to the reverse-phase column.…”

Section: Analytical Chromatographic Methods For Process Development Amentioning

confidence: 99%

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

Burova

Ioffe

2005

Gene Ther

View full text Add to dashboard Cite

“…No extra band indicative of contaminating protein could be detected. In addition, an analysis of the protein composition by reverse-phase HPLC on a C-4 Jupiter column 5 (250 × 2mm; 5 m; Phenomenex) showed no quantitative Table 2. Aliquots from amplification II were titered by the standard biological assays (pfu and tdu) as described in Materials and methods.…”

Section: Purity Of Purified Preparationsmentioning

confidence: 99%

“…It leads to the generation of AV 1.0 CMV.lacZ adenovirus. Plasmid pXL3497 derives from pXL3185 and has a sequence inserted at position 32784 (Ad5 coordinates) which corresponds to the addition of a (Gly-Ser) 5 -(Lys) 7 peptide at the C-terminus of the fiber protein. 20 The corresponding adenovirus, AV 1.k CMV.lacZ, was described as AdZ.F(pK7)␤gal by Wickham et al 20 Plasmid pXL3615 is an RK2-derived vector containing an Ad5 genome carrying deletions in the E1 region (from position 386 to position 3512), in the E3 region (from position 28 592 to position 30 470) and in the E4 region (from position 33 423 to position 35 356).…”

Section: Adenoviral Genomementioning

confidence: 99%

An improved anion-exchange HPLC method for the detection and purification of adenoviral particles

Blanche¹,

Cameron²,

Barbot³

et al. 2000

Gene Ther

View full text Add to dashboard Cite

We have developed an anion-exchange high-performance liquid chromatography (HPLC) method using Q Sepharose XL (Amersham Pharmacia Biotech) as adsorbent to analyze samples containing adenovirus. This method has several major advantages over the HPLC method previously described for quantitating particles, namely (1) a Ͼ10-fold improvement in the detection limit of adenovirus in crude preparations; (2) absence of interferences originating from nucleic acids and proteins which usually contaminate crude samples; (3) unprecedented sharpness and symmetry of adenovirus peak, rendering the identification of the viral peak unambiguous, even in extremely crude and dilute prep-

show abstract

Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome

Cited by 79 publications

References 19 publications

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

An improved anion-exchange HPLC method for the detection and purification of adenoviral particles

Contact Info

Product

Resources

About