Characterization of capsid-modified adenovirus vectors containing heterologous peptides in the fiber knob, protein IX, or hexon

Abstract

Adenovirus (Ad) vectors are widely used in gene therapy and in vitro/in vivo gene transfer because of their high transduction efficiency. However, Ad vector application in the gene therapy field is limited by poor transduction into cells not expressing the primary receptor, coxsackievirus and adenovirus receptor. To overcome this problem, several types of capsid-modified Ad vectors have been developed. The HI loop or C-terminus of the fiber knob, the C-terminus of the protein IX (pIX) and the hypervariable region 5 of the hexon are promising candidate locations for displaying foreign peptide sequences. In the present study, we constructed Ad vectors in which each of the above region was modified by a simple in vitro ligation-based method, and examined the characterization of each Ad vector containing the FLAG tag (DYKDDDDK) or RGD (CDCRGDCFC) peptide. Enzymelinked immunosorbent assay examining the surface expression of foreign peptides on the virus suggested that foreign peptides are exposed on virion surfaces in all types vectors and that the hexon was the most efficiently reacted, reflecting the copy number of the modification. However, in the case of the transduction efficiency of Ad vectors containing the RGD peptides, the modification of pIX and the hexon showed no effect. The modification of the HI loop of the fiber knob was the most efficient, followed by the modification of the C-terminus region of the fiber knob. These comparative analyses, together with a simple construction method for each modified Ad vector, could provide basic information for the generation of capsid-modified Ad vectors.

Fiber-modified adenovirus vectors containing the TAT peptide derived from HIV-1 in the fiber knob have efficient gene transfer activity

“…Several groups, including us, have generated various types of capsid-modified hAd5 vectors, such as hAd5 vectors containing an RGD (Arg-Gly-Asp) peptide or a poly-lysine (KKKKKKK;K7) peptide in the fiber knob, pIX, or hexon. [6][7][8][9][10][11][12][13][14] These capsid-modified hAd5 vectors efficiently infect CAR-negative cells via interaction between cellular av integrin/heparan sulfate proteoglycans and RGD/poly-lysine peptides on the capsid. However, in blood cells, which are known to resist efficient transduction, the enhancement of transduction by the use of fiber-modified hAd5 vectors was limited compared with that in adherent cells.…”

Development of fiber-substituted adenovirus vectors containing foreign peptides in the adenovirus serotype 35 fiber knob

“…Various sites for genetic incorporation of target ligands in Ad capsids, including the fiber knob, penton base and hexon, have been reported for Ad5 vectors. 25,26 Among these sites, the HI loop and C-terminal region in the fiber knob are considered to be the most promising sites for the insertion of target ligands for Ad5 vectors. 26,27 However, regions suitable for the insertion of target ligands have not been identified in the Ad35 fiber knob.…”

“…25,26 Among these sites, the HI loop and C-terminal region in the fiber knob are considered to be the most promising sites for the insertion of target ligands for Ad5 vectors. 26,27 However, regions suitable for the insertion of target ligands have not been identified in the Ad35 fiber knob. In this study, to achieve both ablation of CD46 binding and addition of targeting ligands to the Ad35 fiber knob, we selected the regions crucial for binding to CD46 as an insertion site for foreign peptides and developed fiber-mutant AdF35 vectors with foreign peptides incorporated into the fiber knob.…”

“…31 A unique XbaI restriction site was introduced into the proximity of regions encoding these amino-acid residues, between Gln243 and Thr244 in the FG loop, Met280 and Ile281 in the HI loop, or Glu302 and Ser303 in the IJ loop, in the vector plasmid for AdF35 vectors to allow easy insertion of the oligonucleotides corresponding to foreign peptides by means of in vitro ligation. 26,34,35 The vector plasmids pAdHM75-XbaI(FG), -XbaI(HI) and -XbaI(IJ) were constructed as described below. First, the fragments encoding the FG, HI or IJ loop of the Ad35 fiber knob were amplified by PCR with the following primers: the AdF35 primer (5 0 -CTTAAGTGT TTAACCCCACTAACAACCACAGGC-3 0 ) and FG loop-1R (5 0 -GATATCTCTAGAGTTGAAGGGATAAGCTGTA GTACTTGGC-3 0 ) for the FG loop (Ad35 genome, 30 974-31 555 bp), the AdF35 primer and HI loop a-1R (5 0 -GATATCTCTAGACATACGGCTGTTTAGCATTATAG AAATGTTCAAGGG-3 0 ) for the HI loop (Ad35 genome, 30 974-31 675 bp), or the AdF35 primer and IJ loop-R (5 0 -ATCGATTTCGAATCTAGATTCTGGAGATT CACTTGCATTTAGATTCCATTCAAATTGTATGGC-3 0 ) for the IJ loop (Ad35 genome, 30 974-31 732 bp) (the XbaI sites are underlined).…”

“…The ClaI/SrfI fragment of pAdHM41 35 (Ad5 genome, 27 526-32 787 bp) was ligated with the ClaI/SrfI fragments of pEco-35FG-XbaI, -35HI-XbaI and -35IJ-XbaI, resulting in pAdHM75-XbaI(FG), -XbaI(HI) and -XbaI(IJ), respectively ( Figure 1). pAdHM75-XbaI(FG) was digested by I-CeuI/PI-SceI and ligated with I-CeuI/PI-SceIdigested pCMVL1a, 26 which contains a cytomegalovirus promoter-driven firefly luciferase expression cassette, resulting in pAdHM75-XbaI(FG)-L2. To insert a foreign oligonucleotide corresponding to the RGD-4C peptide, Chimeric Ad vectors with foreign peptides in the Ad35 fiber knob H Matsui et al CDCGRDCFC, which binds with high affinities to integrins (a v b 3 and a v b 5 ) on the cell surface, 36,37 pAdHM75-XbaI(FG)-L2 was digested with XbaI and ligated with oligonucleotides Ad35RGD-1 (5 0 -CTAGGTGTGACTGCCGCGGAGACTGTTTCTGCC-3 0 ) and Ad35RGD-2 (5 0 -CTAGGGCAGAAACAGTCTCCGC GGCAGTCACAC-3 0 ), which contain binding sites with an XbaI site, resulting in pAdHM75-RGD(FG)-L2.…”