Genetic Modification Of The Fiber Knob

Genetic manipulation may be advantageous over adapters in that all virions harbor an identical modification and lack of stability is less likely. Although conceptually elegant, genetic targeting efforts must work within narrow structural constraints. Key to this approach is the successful modulation of complex protein structure instead of function relationships that result in Ad tropism modification without disrupting native molecular interactions indispensable for proper function. Specifically, genetic modification can affect capsid protein production, fiber trimerization, and packaging of virions. Based on an increasing understanding of native Ad entry biology, development of genetically targeted vectors has rationally focused on the fiber knob domain, the primary determinant of Ad tropism (Fig. 2).

To circumvent variable expression of CAR, targeting lig-ands have been incorporated into the Ad knob domain without ablating native CAR binding. This results in Ad vectors with expanded, rather than restricted, cell recognition. These efforts are based on rigorous structural analysis of the knob domain and have exploited two separate locations within the knob that tolerate genetic manipulation without loss of fiber function, the C-terminus and the HI loop. Because the C-terminus of the Ad knob is exposed, extension of the knob peptide to include a targeting peptide moiety is conceptually simple.

Figure 2 Genetic targeting of adenovirus. (A) Trimeric wildtype adenovirus fiber; tail, shaft, and knob regions are shown. (B) Knob containing targeting ligand in the flexible HI loop. (C) Knob containing a C-terminal targeting ligand. (D) Pseudotyped chimeric fiber bearing an alternate human serotype knob domain. (E) Knobless fiber bearing a heterologous trimerization sequence and a C-terminal targeting moiety.

Figure 2 Genetic targeting of adenovirus. (A) Trimeric wildtype adenovirus fiber; tail, shaft, and knob regions are shown. (B) Knob containing targeting ligand in the flexible HI loop. (C) Knob containing a C-terminal targeting ligand. (D) Pseudotyped chimeric fiber bearing an alternate human serotype knob domain. (E) Knobless fiber bearing a heterologous trimerization sequence and a C-terminal targeting moiety.

Ads with C-terminal RGD and polylysine ligands have yielded some promising results in vitro and in vivo, but other peptide ligands were rendered ineffective in the C-terminus context (57). To circumvent this problem, Dmitriev and coworkers introduced an RGD peptide sequence (RGD-4C) into an exposed loop structure that connects beta-sheets H and I (Hi-loop) within the knob (31). The expanded tropism of this vector has been useful for several purposes, including carcinomas of the ovary, pancreas, colon, and head and neck, all of which exhibit frequent CAR deficiency (33,35,58-60). Incorporation of various other ligands into the HI-loop has been attempted, but based on the limited number of publications available, the loop may not be a suitable locale for all targeting moieties. The combination of C-terminus and HI-loop targeting has also been performed and was reported to increase gene transfer over either approach alone (61).

Was this article helpful?

0 0

Post a comment