1. Field of the Invention
The present invention is in the field of targeted gene delivery. Specifically, the invention relates to recombinant viral vectors for targeted delivery to selected cells, wherein the recombinant virus is a small, encapsidated virus, such as adenovirus or adeno-associated virus.
2. Background Art
Recent attempts to target gene transfer to human cells have focused on the use of retroviral and adenoviral vectors. However, the most promising current vectors have resulted in limited success, due in part to the inability to target specific cell types such as hematopoietic stem cells, and the need to culture target cells in vitro to promote cell cycling which can result in the loss of stem cell function. The ability to target gene transfer to specific cell types in situ would greatly enhance current approaches to gene therapy.
Several approaches have been taken to target viral and non viral vectors that include using ligands, antibodies or peptides in the vector construction thereby redirecting virus infection through antigens or receptors expressed on specific cell types. Early experiments designed to redirect the host range of retroviruses by molecular modification of ecotropic envelopes to recognize cellular receptors through antigen binding, ligand or peptide sequences were unsuccessful. It has subsequently been shown that efficient uncoating of the virus requires a conformational change in a subunit of the envelope protein which is only induced upon interaction of the retrovirus particle with its cognate receptor; thus retroviruses directed to alternate receptors do not yield high frequencies of infection.
Similarly, the use of bifunctional antibodies which recognize both viral epitopes and target cell antigens to redirect the tropism of amphotropic retroviruses have not resulted in targeted gene transfer. Successful targeting of retroviruses has required construction of chimeric amphotropic envelopes which contain a protease cleavage site and a targeting moiety. Following recognition of the target cell by the targeting moiety, proteases cleave the chimeric sequences to leave the intact amphotropic envelope. Because the virus is now in close proximity to the cell surface, the viral envelope can interact with its authentic receptor and viral infection will proceed.
The present invention utilizes the biology of adenovirus infections to confer several advantages to the use of recombinant adenoviruses for targeted gene transfer. Infection of a cell by adenovirus requires two distinct receptors, one for recognition and one to mediate internalization of the virus. First, the knob portion of the capsid fiber interacts with a recently identified receptor on the cell surface, then internalization of the adenovirus is mediated by interaction of the penton base protein with integrins (fibronectin and vitronectin) on the cell surface. The adenovirus capsid uncoats in the acidic endosome (i.e., does not require interaction with a receptor to mediate a conformational switch) and the DNA is delivered to the nuclear pore.
It has been demonstrated that targeting of adenoviruses can be accomplished by redirection of host range by linkage to bispecific antibodies. Several groups have also reported that molecular modifications to introduce chimeric viral proteins have met with success. However, bispecific antibodies limit the usefulness of targeted vectors for in situ gene transfer due to the effects of serum on antigen-antibody interactions, and introduction of sequences encoding chimeric viral proteins into the viral genome can adversely affect virus function. Therefore, there is a need for more efficient, effective, specifically targeted adenoviral vectors that retain their infectivity characteristics for gene delivery for a variety of purposes.
The present invention fills this need by providing a vector, and particularly an adenoviral vector, that can be linked to any selected targeting moiety for targeting to any selected cell type. The vector is highly efficient, achieving high levels of expression of the transferred gene specifically in the targeted cells, The present invention provides a vector wherein biotin is covalently linked to recombinant adenovirus particles such that through an avidin bridge we could redirect the virus using biotinylated growth factors and antibodies.
Targeted gene transfer to specific populations of hematopoietic progenitor cells represents an advance towards therapeutic use of gene transfer. The c-kit receptor has been shown to be expressed on the surface of primitive hematopoietic cells with long term reconstitution activity, and represents an attractive receptor for targeted gene transfer. Therefore, a molecular conjugate vector consisting of plasmid DNA encoding the luciferase reporter gene complexed with polylysine coupled to avidin (for addition of the biotinylated ligand) and defective adenovirus particles was used to target hematopoietic progenitors (Blood 87:472-478, 1996). Upon the addition of biotinylated steel factor (SLF) we demonstrated that this vector specifically targets c-kit+hematopoietic cell lines and results in up to a ten fold increase in luciferase activity as compared to a control vector. Furthermore, transient gene expression is observed with maximum expression at 30 hours, and greater than 90% of target cells are transfected. However, the potential for use of this vector for in vivo gene transfer is limited by the inclusion of polylysine in the molecular conjugate for two reasons: 1) polylysine yields a high background due to non-specific electrostatic interactions with the cell surface, 2) the electrostatic charges required to hold the molecular conjugate vector together may be rapidly neutralized in the presence of human serum. Other laboratories have approached targeted gene transfer by modifying the tropism of recombinant adenoviral vectors through a neutralizing anti-fiber antibody chemically conjugated to cell-specific ligands (Nature Biotechnology 14:1574-1578, 1996). These vectors represent an improvement over the molecular conjugate vector in that the targeting moiety is no longer attached to the vector through electrostatic charges, resulting in a decrease in the non-specific background, but the affinity of the antibody-antigen interaction required for targeting of these vectors is variable and is not stable in the presence of serum. A third approach to targeted adenoviral vectors has been to molecularly clone sequences encoding the targeting moiety into the fiber or penton base structural genes of the adenoviral vector. This approach is expected to avoid the disadvantages of targeting through antibody-antigen interactions, but necessitates extensive manipulation of the adenoviral genome to introduce each targeting moiety.
The present invention provides more efficient vectors for gene transfer. The vectors were generated by direct high affinity linkage of the targeting moiety to a recombinant virus particle. To achieve these inventive vectors, the invention includes a protocol for the covalent addition of biotin to the capsid of recombinant encapsidated small virus particles. The present invention demonstrates that biotin can be linked to the capsid of recombinant adenovirus while maintaining wild-type infectivity. Following incubation with streptavidin, any biotinylated ligand or antibody could be added to target this vector to any cell type.
The present invention provides a composition for targeted delivery of a nucleic acid to a cell comprising a biotinylated recombinant encapsidated virus, wherein the recombinant virus comprises the nucleic acid to be delivered, and wherein the biotinylated recombinant virus is linked via streptavidin to a biotinylated targeting moiety.
The present invention specifically provides a composition for targeted delivery of a nucleic acid to a cell comprising a biotinylated recombinant adenovirus, wherein the recombinant adenovirus comprises the nucleic acid to be delivered, and wherein the biotinylated recombinant adenovirus is linked via streptavidin to a biotinylated targeting moiety.
The present invention further provides a method for targeted delivery of a nucleic acid to a selected cell in a subject comprising administering to the subject a composition comprising a biotinylated recombinant encapsidated virus comprising the nucleic acid to be delivered, wherein the virus is linked via streptavidin to a biotinylated targeting moiety that specifically targets the selected cell.
The present invention further provides a method for targeted delivery of a nucleic acid to a selected cell in a subject comprising administering to the subject a composition comprising a biotinylated recombinant adenovirus comprising the nucleic acid to be delivered, wherein the virus is linked via streptavidin to a biotinylated targeting moiety that specifically targets the selected cell.