Identification of HI-like loop in CELO adenovirus fiber for incorporation of receptor binding motifs.
Vectors based on the chicken embryo lethal orphan (CELO) avian adenovirus (Ad) have two attractive properties for gene transfer applications: resistance to preformed immune responses to human Ads and the ability to grow in chicken embryos, allowing low-cost production of recombinant viruses. However, a major limitation of this technology is that CELO vectors demonstrate decreased efficiency of gene transfer into cells expressing low levels of the coxsackie-Ad receptor (CAR). In order to improve the efficacy of gene transfer into CAR-deficient cells, we modified viral tropism via genetic alteration of the CELO fiber 1 protein. The alphav integrin-binding motif (RGD) was incorporated at two different sites of the fiber 1 knob domain, within an HI-like loop that we identified and at the C terminus. Recombinant fiber-modified CELO viruses were constructed containing secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein genes as reporter genes. Our data show that insertion of the RGD motif within the HI-like loop of the fiber resulted in significant enhancement of gene transfer into CAR-negative and CAR-deficient cells. In contrast, CELO vectors containing the RGD motif at the fiber 1 C terminus showed reduced transduction of all cell lines. CELO viruses modified with RGD at the HI-like loop transduced the SEAP reporter gene into rabbit mammary gland cells in vivo with an efficiency significantly greater than that of unmodified CELO vector and similar to that of Ad type 5 vector. These results illustrate the potential for efficient CELO-mediated gene transfer into a broad range of cell types through modification of the identified HI-like loop of the fiber 1 protein.
Logunov
Denis Y
DY
Gamaleya Research Institute for Epidemiology and Microbiology (GIEM), 123098, Gamaleya Street 18, Moscow, Russia.
Zubkova
Olga V
OV
Karyagina-Zhulina
Anna S
AS
Shuvalova
Eugenia A
EA
Karpov
Andrei P
AP
Shmarov
Maxim M
MM
Tutykhina
Irina L
IL
Alyapkina
Yulia S
YS
Grezina
Natalia M
NM
Zinovieva
Natalia A
NA
Ernst
Lev K
LK
Gintsburg
Alexsandr L
AL
Naroditsky
Boris S
BS
D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D002642:Chick Embryo; D002645:Chickens; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D006224:Cricetinae; D003412:Cricetulus; D025681:Fowl adenovirus A / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy; D006801:Humans; D017434:Protein Structure, Tertiary / Q000235:genetics; D011817:Rabbits; D011991:Receptors, Virus / Q000172:deficiency / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D011991:Receptors, Virus; D011994:Recombinant Proteins; D014764:Viral Proteins