Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-07-10
2004-11-09
Priebe, Scott D. (Department: 1635)
Chemistry: molecular biology and microbiology
Vector, per se
C435S235100, C435S455000, C435S456000, C424S093200
Reexamination Certificate
active
06815200
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of vector biology and gene therapy. More specifically, the present invention relates to the production of recombinant adenoviral vectors with replacement of fibers for cell-specific targeting with concomitant elimination of endogenous tropism.
2. Description of the Related Art
Approaches to target adenoviral vectors to specific cell types should be based on an understanding of the mechanism of cell entry exploited by the majority of human adenoviruses and on the identification of the components of the adenoviral virion which are involved in the early steps of the virus-cell interaction. Adenoviruses are non-enveloped viruses containing a double stranded DNA genome packaged into an icosahedral capsid. Whereas the most abundant capsid protein, the hexon, performs structural functions and is not involved in the active cell entry process, the other two major protein components of the capsid, the fiber and the penton base, have been shown to play key roles in the early steps of virus-cell interaction. The fiber and penton base together form penton capsomers consisting of five penton base subunits embedded in the virus capsid tightly associated with a homotrimer of fiber proteins protruding from the virion.
Each of the five subunits of the penton base contains a flexible loop structure, which corresponds to a hypervariable domain of the otherwise highly conserved protein. Amino acid sequence analysis of penton base proteins of different adenoviral serotypes showed that each loop consists of two stretches of alpha helices flanking an arginine-glycine-aspartic acid (RGD) tripeptide positioned in the middle of the loop. Cryo-electron micrography (cryo-EM) studies of Ad2 virions revealed that these loops form 22 Å protrusions on the surface of penton base, thereby facilitating interaction of the RGD motif, localized at the apex of the protrusion with cellular integrins.
The fiber has a well-defined structural organization with each of its three domains, the tail, the shaft, and the knob, performing a number of functions vital for the virus. The short amino terminal tail domain (46 amino acid residues in Ad2 and Ad5 fibers) of the fiber protein is highly conserved among most adenoviral serotypes. In addition to being involved in the association with the penton base protein through an FNPVYD (SEQ ID NO: 15) motif at residues 11-16, which results in anchoring the fiber to the adenoviral capsid, the tail domain also contains near its amino terminus the nuclear localization signal KR&lgr;R (where indicates a small amino acid residue), which directs the intracellular trafficking of newly synthesized fibers to the cell nucleus, where the assembly of the adenoviral particle takes place.
The central domain of the fiber is the shaft, which extends the carboxy terminal knob domain away from the virion, thereby providing optimal conditions for receptor binding. The shaft is organized as a sequence of pseudorepeats, each 15 amino acids in length, with a characteristic consensus sequence containing hydrophobic residues at highly conserved positions. This sequence, X-X&phgr;-X-&phgr;-X-&phgr;-G-X-G-&phgr;-X-&phgr;-X-X or X-X-&phgr;-X-&phgr;-X-&phgr;X-X-P-&phgr;-X-&phgr;-X-X, contains hydrophobic amino acids at “&phgr;”-positions, with either the eighth and tenth positions being occupied with two glycines or with a proline in the tenth position. The models for the secondary structure corresponding to these repeats describe the shaft as a triple &bgr;-spiral in which the &bgr;-strands are oriented more along the fiber axis and the hydrophobic residues at the 7
th
and 13
th
position are located at greater radius. The trimer is stabilized with extensive intra- and inter-chain hydrogen bonding. Due to its rod-like shape, the shaft domain basically determines the length of the entire molecule, which depends on the number of pseudorepeats contained within the shaft. The fibers of various human adenoviral serotypes contain different number of repeats, resulting in a significant variation in the fiber length: from 160 Å (Ad3) to 373 Å (Ad2 and Ad5).
The carboxy terminal knob domain (180-225 amino acid residues) carries out two distinct functions, i.e., initiation of fiber and trimerization and binding of the virus to its primary cellular receptor. X-ray crystallography studies on
E. coli
-expressed Ad5 fiber knob protein have shown that the trimeric knob is arranged around a three-fold crystallographic symmetry axis and resembles a three bladed propeller when viewed along this axis. Each monomer of the knob is a &bgr;-sandwich structure, formed by two antiparallel &bgr;-sheets R and V. The surface of the V-sheet, which consists of the strands A, B, C, and J, points towards the virion, while the R-sheet, formed, by strands D, I, H, and G, points outside the virion and towards the surface of the target cell. These findings have been then corroborated with X-ray crystallography data obtained with recombinant Ad2 fiber knob protein.
A number of studies employing recombinant knobs have shown that these proteins are capable of self-trimerization, which does not require any cellular chaperons. The exact trimerization motif within the fiber knob is largely unknown, which makes mutagenesis or modification of this protein quite difficult: indeed, any new mutation or modification of the fiber may affect amino acid(s) involved in the fiber trimerization and may therefore destabilize the entire molecule, thereby rendering it non-functional. The mutant knobs revealed that deletions in the knob sequence, even as short as two amino acid residues, may result in monomeric fibers, which cannot associate with penton base and, therefore, cannot be incorporated into mature adenoviral particles.
The second function performed by the knob is binding to a cellular receptor and, therefore, mediating the very first step of the virus-cell interaction. This receptor-binding ability of the knob has been demonstrated by utilization of recombinant knob proteins as specific inhibitors of adenoviral binding to cells. Based on the &bgr;-sandwich structure of the knob, it was originally hypothesized by Xia et al. that the strands constituting the R-sheet form a receptor binding structure. Recently, however, analysis of fiber knob mutants has revealed that segments outside the R-sheet constitute the receptor-binding site. The Ad5 binding site is located at the side of the knob monomer and specifically involves sequences within the AB and DE loops and B, E, and F &bgr;-strands. The binding site of Ad37 that binds to a different receptor involves a critical residue in the CD loop at the apex of the trimer.
The two penton proteins, the penton base and fiber, work in a well-orchestrated manner to provide the early steps of the cell infection mechanism developed by adenoviruses. Importantly, each of these early events is mediated by either fiber or penton base; therefore, both proteins play distinct and well defined roles in this process.
The fiber knob provides the initial high-affinity binding of the virus to its cognate cell surface receptor, coxsackievirus and adenovirus receptor (CAR), which does not possess any internalization functions and merely works as a docking site for Ad attachment.
Human adenoviruses (Ad) of serotype 2 and 5 have been extensively used for a variety of gene therapy applications. This is largely due to the ability of these vectors to efficiently deliver therapeutic genes to a wide range of different cell types. However, the promiscuous tropism of adenovirus resulting from the widespread distribution of coxsackie virus and adenovirus receptor (CAR) (1, 2), limits the utility of adenoviral vectors in those clinical contexts where selective delivery of therapeutic transgene to a diseased tissue is required. Uncontrolled transduction of normal tissues with adenoviral vectors expressing potentially toxic gene products may lead to a series of side effects, thereby undermining the efficacy of the therap
Curiel David T.
Krasnykh Victor N.
Frommer Lawrence & Haug
Kowalski Thomas J.
Lu, Ph. D. Deborah L.
Priebe Scott D.
The UAB Research Foundation
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