Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-03-13
2003-09-02
Chen, Shin-Lin (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100, C536S023500, C424S130100, C424S147100, C530S350000
Reexamination Certificate
active
06613563
ABSTRACT:
TECHNICAL FIELD
The present invention relates to gene therapy. In particular, therapeutic agents and methods useful in targeting and delivering genes more efficiently to particular cells are disclosed, wherein re-targeted, tropism-modified viral vectors presenting ligand on the surface and including a nucleotide sequence encoding a therapeutic gene product are used to facilitate targeting and delivery.
BACKGROUND OF THE INVENTION
The primary impediment to the transfer of non-native or foreign DNA into mammalian cells is that the genetic material must be transported across multiple cellular barriers before it enters the host cell nucleus and initiates gene expression. Previously established methods have utilized artificial means to introduce DNA into the cell although these methods are associated with significant cell toxicity (Graham et al.,
Virology
52:456-467 (1973); Felgner et al,
PNAS USA
84:7413-7417 (1987)).
More recently, enhanced transfer of DNA conjugates into cells has been achieved with adenovirus, a human DNA virus which readily infects various cell types (Horwitz, Adenoviridae and their replication, in
Virology
, Fields and Knipe, eds., Raven Press, NY (1990) pp. 1679-1740). Since adenovirus efficiently disrupts the membranes of endocytic vesicles, co-internalization of the virus with the DNA conjugate allows rapid transfer of the conjugate into the cell cytoplasm before it can be subjected to lysosomal degradation. The fact that adenovirus exhibits selective tropism has also been exploited to reconstitute these cells in vivo with the human cystic fibrosis transmembrane conductance regulator (CFTR) (Rosenfeld et at.,
Cell
68:143-155 (1992)) and the alpha 1-antitrypsin genes (Rosenfeld et al.,
Science
252:431-434 (1991)).
A number of features make adenoviruses very attractive for gene delivery applications. Knowledge of the adenovirus genetic system is fairly extensive, including the viral life cycle, DNA replication, transcription and RNA processing, and regulation of virus gene expression. In addition, the size of the adenovirus (Ad) genome allows relatively easy manipulation of the viral DNA while still having the capacity for insertion of most cDNAs into the viral genome. Additional advantages of adenovirus vectors include their ability to infect both dividing and nondividing cells efficiently, to induce high-level foreign protein expression without replication or integration of the viral genome, and to grow to high yields when propagated in an appropriate complementing cell line.
If a target tissue lacks sufficient levels of adenovirus attachment receptors to mediate viral adsorption, however, this may also be a barrier to efficient gene transfer. Infection by most viruses requires viral attachment to its host cell receptor. Adenovirus attaches to its host cell receptor via its fiber protein (see, e.g., Wickham et al.,
Cell
73:309-319 (1993)).
The Ad fiber protein is a long, trimeric protein that protrudes from the surface of the virion. At the distal end of the fiber protein is a knob-like C-terminus that interacts with an unidentified cellular receptor present on HeLa and other epithelial-derived carcinoma cell lines (see, e.g., Defer et al.,
J. Virol
. 64:3661-3673 (1990)). The receptor, generally identified as FibR, is assumed to be expressed by cells that are the normal targets for adenovirus infection.
Thus, reduced gene delivery to certain tissues may well result from a low expression of the adenovirus receptor (FibR). A lack of functional receptors is thus likely to be directly correlated with dramatic reductions in gene transfer efficiency.
In general, adenoviral vectors possess the capacity for in vivo gene transfer that are critical to effective gene therapy. Following administration of the adenovirus vector, three distinct, sequential steps are required for expression of the therapeutic gene in target cells: (1) attachment of the adenovirus vector to specific receptors on the surface of the target cell; (2) internalization of the virus; and (3) transfer of the gene to the nucleus where it can be expressed. Thus, any attempt to modify the tropism of an adenovirus vector—that is, its native ability to target its cognate receptor must reserve its ability to perform these three functions efficiently.
Investigators have met with greater or lesser success in this regard. For example, the methodology proposed by Krasnykh et al. (
J. Virol
. 70: 6839-46 (1996) generates recombinant adenovirus with chimeric fibers having a fiber shaft from one Ad serotype and a knob from another, thereby altering the adenovirus' receptor recognition profile. (Also see Gall et al.,
J. Virol
. 70:2116-2123 (1996), which describes an Ad 5/7 capsid chimera.) However, such constructs would appear to have limited utility, as they still rely on the less-than-ubiquitous (and less-than-efficient) Ad receptors for targeting. Moreover, Ad vectors that rely upon Ad receptors for targeting (and putative internalization) are not able to target as wide a variety of cells as one might wish, and depending on the nature of the chimeric fiber, any alterations in its conformation may have a negative impact on targeting and/or delivery.
Further, the modifications described in the aforementioned articles do not alter viral tropism in a manner that enhances viral targeting or increases trafficking to the nucleus, contrary to what is disclosed herein. In addition, the art fails to disclose targeting and delivery constructs and systems that achieve the unexpectedly high level of “infectivity” and expression shown herein. Finally, the constructs and methods of the present invention successfully achieved delivery of therapeutic agents to cells that are normally resistant to viral-mediated delivery.
In view of the aforementioned problems, the design and construction of the within-disclosed vectors and conjugates provides a novel and elegant solution, as described further herein. The use of the recombinant sequences and vectors of this invention to mediate the transfer of foreign genes into recipient cells both in vitro and in vivo overcomes the limitations of the above-described gene transfer systems. This invention utilizes recombinant constructs which confer the advantages of targeting via the fibroblast growth factor receptor upon adenovirus—in place of the adenovirus usual targeting via fiber protein—and thus represents an improved method for gene therapy as well as for therapeutic applications involving delivery of a gene.
BRIEF SUMMARY OF THE INVENTION
In contrast to the disadvantages of using intact adenovirus, modified adenovirus vectors requiring a helper plasmid or virus, or so-called replication-deficient adenovirus, the use of recombinant adenovirus-derived vectors according to the present invention provides certain advantages for gene delivery. In particular, adenoviral vectors having their native tropism modified or ablated, which are then re-targeted via a targeting ligand, are disclosed herein as advantageous for a variety of gene therapy applications.
The Ad-derived vectors of the present invention possesses all of the functional properties required for gene therapy including binding to specific cell receptors and penetration of endocytic vesicles. They further include those in which all or part of the fiber head or tail is replaced with—or conjugated to—a ligand-encoding gene. Use of the vectors and conjugates disclosed herein allows one to target a wide variety of cells and to deliver therapeutic agents—irrespective of whether those agents are proteins, polypeptides, nucleotide sequences, or some other molecular species—directly into specific target cells.
The presently-disclosed constructs, systems and methods afford a level of flexibility in therapeutic approaches not seen with other systems and methods. Therefore, the vectors, systems and methods of the present invention are ideal for use in a wide variety of therapeutic applications.
Therefore, in one embodiment, the present invention provides a tropism-modified adenoviral vector system that specifically targe
Baird Andrew
Curiel David T.
Douglas Joanne T.
Pierce Glenn F.
Rogers Buck E.
Chen Shin-Lin
Seed Intellectual Property Law Group PLLC
Selective Gentics, Inc.
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