Lentiviral vector system for high quantity screening

Details Lentiviral vector system for high quantity screening Lentiviral vector system for high quantity screening

2001-09-14

2004-12-14

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S320100, C435S455000, C435S456000, C435S457000, C435S005000, C435S006120, C435S069100, C435S462000, C435S463000, C435S325000, C435S366000

Reexamination Certificate

active

06830892

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to a vector system wherein multiple lentiviral vectors are used to transfer a large number (library) of nucleic acid segments to host cells. Preferably the system uses an inducible expression system to express the nucleic acid segments, and the lentiviral vector are pseudotyped lentiviral vectors. Still more preferably, the system uses nucleic acid segments encoding antibodies that are expressed intracellularly and bind to their target antigens intracellularly (intrabodies).
BACKGROUND OF THE INVENTION
In recent years considerable effort has been directed at applying gene delivery techniques. That term describes a wide variety of methods using recombinant biotechnology techniques to deliver a variety of different materials to a cell. These methods include, for example, vectors such as viral vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. The different techniques used depend in part upon the gene being transferred and the purpose therefore. Thus, for example, there are situations where only a short-term expression of the gene is desired in contrast to situations where a longer term, even permanent expression of the gene is desired.
Vectors that have been looked at include both DNA viral vectors and RNA viral vectors. For example, DNA vectors include pox vectors such as orthopox or avipox vectors (see, e.g., U.S. Pat. No. 5,656,465), herpes virus vectors, such as herpes simplex I Virus (HSV) vector [Geller, A. I. et al.,
J. Neurochem.
64:487 (1995); Lim, F., et al.,
DNA Cloning: Mammalian Systems,
D. Glover, Ed., Oxford Univ. Press, Oxford, England (1995); Geller, A. I. et al.,
Proc. Natl. Acad. Sci.,
U.S.A. 90:7603 (1993)]; Adenovirus vectors [Legal Lasalle et al.,
Sci.
259-988 (1993); Davidson et al.,
Nat. Genet.
3:219 (1993); Yang et al.,
J. Virol.,
69:2004 (1995)]; and Adeno Associated Virus Vectors [Kaplitt, M. G., et al.,
Nat. Genet.
8;148 (1994)]. Retroviral vectors include Moloney murine leukemia viruses (MMLV) and human immunodeficiency viruses (HIV) [See, U.S. Pat. No. 5,665,577].
While much attention has been focused on the use of viral vectors, particularly for in vivo therapy, for example, in somatic cell therapy or direct in vivo applications, other applications exist.
For example, a retroviral vector can be used to infect a host cell and have the genetic material integrated into that host cell with high efficiency. One example of such a vector is a modified Moloney murine leukemia virus (MMLV), which has had its packaging sequences deleted to prevent packaging of the entire retroviral genome. However, that retrovirus does not transduce resting cells. Additionally, since many retroviruses typically enter cells via specific receptors, if the specific receptors are not present on a cell or are not present in large enough numbers, the infection is either not possible or is inefficient. Concerns have also been expressed as a result of outbreaks of wild-type viruses from the recombinant MMLV producing cell lines, i.e., reversions.
Recently, attention has focused on lentiviral vectors such as those based upon the primate lentiviruses, e.g., human immunodeficiency viruses (HIV) and simian immunodeficiency virus (SIV). HIV vectors can infect quiescent cells in addition to dividing cells. Moreover, by using a pseudotyped vector (i.e., one where an envelope protein from a different species is used), problems encountered with infecting a wide range of cell types can be overcome by selecting a particular envelope protein based upon the cell you want to infect. Moreover, in view of the complex gene splicing patterns seen in a lentiviruses such as HIV, multivalent vectors (i.e., those expressing multiple genes) having a lentiviral core, such as an HIV core, are expected to be more efficient. Despite the advantages that HIV based vectors offer, there is still a concern with the use of HIV vectors in view of the severity of HIV infection. Thus, means for providing additional attenuated forms that are less likely to revert to a wild type virus are desirable.
Variations can be made where multiple modifications are made, such as deleting nef, rev, vif and vpr genes. One can also have the 3′ and 5′ U3 deleted LTRs.
However, in such instances the vectors are intended to deliver a single heterologous gene or small group of genes.
In recent years, advances such as the use of expression sequence tags (ESTs) have led to the identification of numerous genes, putative genes and their expression products. While comparisons between nucleotide and amino acid sequence may lead to classifications of these genes, putative genes, and expression products, frequently the specific function of the genes product remains unknown. It would be desirable to have a rapid means for identifying the function of such genes and gene products.
Marasco et al. discovered a method by which one could express antibodies within a cell and have them bind to a target within that cell. [See U.S. Pat. No. 5,851,829 to Marasco and Haseltine]. These intracellularly expressed antibodies (intrabodies) can be used in a method of functional genomics. In this manner, one can take a specific unknown gene express its gene product, use that gene product to generate an antibody thereto and use the antibody intracellularly to “knock-out” the putative protein in the cell. Thereafter one can compare that cell to a control to determine the effect the loss of its gene product has on the cell in both in vitro and in vivo systems. This method requires generation of a specific antigen and antibody thereto. It would be desirable to have a method to take advantage of the efficiencies of this approach with large numbers of members of a particular group.
In recent years, attention has been directed to developing large libraries consisting of multiple members of related groups. For example, libraries of antibodies, typically monoclonal antibodies. For example, antigen binding antibody fragments have been expressed on the surface of filamentous phage [G. P. Smith,
Science
228: 1315 (1985)], and used to create large libraries of such antibodies—e.g., 10
7
members or more, referred to as phage display libraries.
In phage display libraries the carboxyl-terminal end of the Fd or Fv region is tethered to a fragment of a phage coat protein, which anchors, for example, Fab fragment to the surface of the phage. The antigen binding site is formed from the combination of the V
H
and V
L
-domain. Phage display libraries can be selected for binding to specific antigens by affinity chromatography [R. P. Hawkins et al.,
J. Mol. Biol.,
226: 889 (1992)] or by panning phage on antigen coated surfaces [C. F. Barbas et al.,
Proc. Natl. Acad. Sci. USA
88: 4363 (1991)]. Antibodies are selected by affinity binding to specific proteins. However, if the antigen has an unknown function, this methodology does not permit you to determine the function of that protein.
It would be highly desirable to have a method where one could look for any molecule resulting in a particular function and rapidly determine that molecule, e.g. protein. It would be very desirable to be able to do this in an automated manner permitting rapid identification of the desired molecule.
SUMMARY OF THE INVENTION
We have now discovered a method to identify and obtain a molecule resulting in a desired function from a large pool of molecules. This method involves using a plurality of vectors, wherein the group of vectors contain a plurality of different target molecules. The target molecules can be any molecules having diversity, e.g. genetic diversity. The molecules can be proteins such as antibodies, growth factors, receptors, cytokines, peptides, ribozymes and antisense molecules. Preferably the target molecules are genes encoding proteins such as antibodies. More preferably the nucleic acid sequences are operably linked to an inducible promoter. The vectors can be used to transduce a plurality of cell

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