Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2001-06-28
2004-09-14
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100, C536S023720, C536S024100
Reexamination Certificate
active
06790657
ABSTRACT:
FIELD
The invention relates to retroviral vectors, and their use in gene transfer.
BACKGROUND
The human immunodeficiency virus (HIV) is the etiological agent of the acquired immunodeficiency syndrome (AIDS) and related disorders. The expression of the virus in infected persons is regulated to enable the virus to evade the host's immune response. The HIV viruses (e.g. HIV-1 and HIV-2), as well as the simian immunodeficiency virus (SIV), share many structural and regulatory genes such as gag, pol, env, tat, rev and nef. See Guyader et al.,
Nature
328:662-669, 1987, which is incorporated by reference. HIV has been classified as a lentivirus because it causes slow infection, and has structural properties in common with such viruses (Haase.
Nature
322:130-136, 1986).
All known retroviruses share features of the replicative cycle, including packaging of viral RNA into virions, entry into target cells, reverse transcription of viral RNA to form the DNA provirus, and stable integration of the provirus into the target cell genome. Replication competent proviruses contain, at a minimum, regulatory long terminal repeats (LTRs) and the gag, pro, pol and env genes which encode core proteins, a protease, reverse transcriptase/RNAse H/integrase and envelope glycoproteins, respectively.
HIV shares the gag, pro, pol and env genes with other retroviruses. HIV-1 also possesses additional genes modulating viral replication, such as the vif, vpr, tat, rev, vpu and nef genes. HIV-2 contains a vpx gene which is not present in HIV-1, but lacks the HIV-1 vpu gene. Additionally the long terminal repeats (LTR) of both HIV-1 and HIV-2 contain cis-acting sequences that are important for integration, transcription and polyadenylation.
HIV, like other retroviruses, are RNA viruses that replicate through a DNA proviral intermediate which is integrated into the genome of the infected host cell. The virion particle contains a dimer of positive strand genomic RNA molecules, which is transcribed from the proviral DNA by the host RNA polymerase II. A portion of these full length RNAs which encode the gag and pol genes of the virus are translated by the host cell ribosomes to produce the structural and enzymatic proteins required for production of virion particles. The provirus also gives rise to a variety of smaller singly and multiply spliced mRNAs coding for the envelope proteins and regulatory proteins.
Wild type retroviruses have been modified to become vehicles for the delivery, stable integration, and expression of cloned genes into a wide variety of cells for experimental and therapeutic purposes. To achieve the aims of transfer and expression of nonviral genes, the vector behaves as a retroviral genome and passes as a virus from a producer cell line. Hence its DNA contains the regions of the wild-type retroviral genome required in cis for incorporation into a retroviral particle. In addition the vector also contains regulatory signals that lead to the optimization of the expression of the cloned gene once the vector is integrated in the target cell as a provirus.
All viral structural genes can be discarded and replaced by heterologous coding sequences, but certain essential sequence elements are retained within the vector. These sequence elements include the packaging sequence, a tRNA binding site, sequences in the LTR that permit “jumping” of the reverse transcriptase between RNA strands during DNA synthesis, sequences near the ends of the LTRs that are necessary for the integration of the vector DNA into the host cell chromosome, and sequences adjoining the 3′ LTR that serve as the priming site for synthesis of the plus strand DNA molecules. See Rapley and Walker, Molecular Biomethods Handbook, 1998, chapter 18 for a discussion of principles of retroviral vector construction, and Lewin, Genes V, 1995, chapter 35, for a discussion of the function of retroviral genes. Since vector genomes do not require that the viral structural genes gag, pol and env be retained, nonviral genes can be cloned into the space vacated by their removal.
A significant advance in the use of retroviral vectors has been the use of packaging cells that stably or constitutively express the viral gag, pol and env genes (for example from plasmids) that cannot themselves be packaged by their own encoded proteins, because they lack the essential packaging sequences. However, when a retroviral transfer vector genome is transfected into such a packaging cell, the viral proteins recognize and package the vector RNA genome into viral particles that are released into the culture supernatant. In such a vector system, the transfer vector (which includes the packaging sequence) shuttles the transgene with the potential for regulation and high titer encapsidation, while the packaging cell line encapsidates the transfer vector RNA but not the viral RNA, so that the packaging cell line does not act as a helper virus. The viral particles produced in this manner can be used to deliver the encapsidated retroviral vector to a target cell with high efficiency.
For HIV-2, it has previously been reported that the leader sequence of this lentivirus contains a packaging signal downstream of the splice donor site (Garzino-Demo et al.,
Hum. Gene Ther
. 6:177-184, 1995). Another report suggested that the downstream sequence elements made only a minor or no contribution to RNA encapsidation, and that the major element was located upstream of the splice donor site (McCann and Lever,
J. Virol
. 71:4133-4137, 1997). Since a knowledge of the packaging signals of HIV-2 is important to the optimal construction of packaging deficient vectors, this uncertainty about the location of the packaging signals has impeded the use of HIV-2 retroviral vector systems.
Moreover, it would be advantageous to express a transgene using an HIV-2 retroviral vector, in such a manner that packaging of the vector RNA is maximized, without an increase in the packaging of viral RNA.
SUMMARY
The invention derives from the discoveries that:
1) deletion of sequences both upstream and downstream of the 5′ splice donor (SD) region of the HIV-2 provirus (packaging vector) results in suppressed encapsidation of packaging vector genomes without critical loss of gene expression, thus the production of “helper virus” is suppressed while making adequate structural viral protein available for encapsidation of foreign nucleotides; and
2) that functional deletion of the SD site of the HIV-2 provirus (transfer vector) results in enhanced encapsidation of the transfer vector's own genome, especially when the host cell has been co-transfected with the packaging vector as described under (1) above; and
3) that the HIV-2, but not HIV-1, packaging vector specifically and faithfully packages its own optimally constructed transfer vector as described under (2) above; and
4) that HIV-2 packaging vector gives both better quality and titer of vector.
Transfer and packaging vectors incorporating one or a combination of these features are useful as gene delivery agents, for example gene therapeutic agents, and provide an improved HIV-2 viral vector system that allows transfer of a transgene into the genome of non-dividing cells. The vectors of the invention also may be used to create a high-efficiency packaging cell line that provides greatly enhanced packaging of foreign DNA, especially when such DNA is carried within the SD deleted transfer vector of the invention. Additionally, it has been discovered that, for the transfer vector of the invention, deletion of the 3′ LTR and its replacement with a puromycin-poly(A) cassette results in still further suppression of encapsidation of packaging virus genomes, without substantial loss of viral particle expression.
The invention includes the transfer vector derived from an HIV lentivirus, such as HIV-2/ST, wherein the vector is functionally deleted for the splice donor site (SD), and contains a functional packaging signal and a transgene operably linked to a promoter. When susceptible cells (such as 293 cells) are co-transfected with t
Hill Myron G.
Housel James
Klarquist & Sparkman, LLP
The United States of America as represented by the Department of
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