Episomally replicating lentiviral vectors

Details Episomally replicating lentiviral vectors Episomally replicating lentiviral vectors

2001-11-21

2004-10-26

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Vector, per se

C435S069100, C435S455000, C435S456000, C435S457000, C435S325000, C536S023100, C536S023720, C536S024100, C536S024500, C536S023200, C536S023500, C536S023510, C536S023520, C536S023530

Reexamination Certificate

active

06808923

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to a viral vector that can introduce a desired nucleic acid sequence into a targeted host cell by retroviral infection where the nucleic acid sequence replicates episomally. Preferably, the viral vector is a lentiviral vector that also contains a heterlogous viral origin of replication (ori) and a second gene that functions as a replication transactivator.
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].
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 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. These vectors like MMLV also result in having the genetic material integrated into the host cell with high efficiency.
Variations in the lentiviral vectors 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.
While those vectors provide many advantages, one of their prime advantages—the ability to stably integrate into a host cell's chromosomes—can also be a major safety concern. This ability to integrate into a chromosome can cause insertional mutagenesis [Shiramiza, B., et al.,
Cancer Res
., 54:2069-2072 (1994); Verma, I. M. and Somia, N.,
Nature
, 389:239-242 (1997)]. One method of dealing with this problem has been to fuse a specific DNA binding domain to the integrase (IN) polypeptide to direct integration into specific DNA sequences [Bushman, F.,
Science
, 267:1443-1444 (1995); Bushman, F. and Miller, M. D.,
J. Virol
., 71:458-464 (1997); Katz, R. A., et al.,
Virology
, 217:178-190 (1996)]. However, additional improvements are still useful.
Further, there are many instances where one does not want to have a gene stably integrated, but only expressed for a limited time period. For example, such as approach is useful with “suicide therapy” where the gene product is designed to negatively impact the integrity of the host cell. It is also useful with angiogenesis proteins. These proteins can promote wound healing, growth of blood vessels, etc. Thus, they can be useful in dealing with individuals having circulatory problems, heart problems etc. However, these proteins can also cause the growth of blood vessel regulated tumors. Accordingly, while some expression of the protein can be beneficial, its unlimited expression can ultimately cause more harm than benefit.
One type of expression where a gene is not integrated into a chromosome is episomal replication. Adenovirus, which replicates episomally, is the most widely used nonintegrating viral vector. It produces very high titers and has a broad target range. However, that broad target range is a disadvantage in using “suicide therapy”. It would be desirable to have a method of gene therapy where one can target specific cells. In addition, adenoviruses, have immunogenicity problems [Haddada, et al.,
Curr. Top. Microbiol. Immunol
., 199:297-306 (1995); Verma and Somia,
Nature
, 389, supra.]. Thus, instances where repeated use of the vector is necessary are problematic. SV40-based vectors also replicate episomally, and have been looked at for use in suicide therapy [Cooper, M. J., et al.,
Proc. Natl. Acad. Sci. USA
, 94:6450-6455 (1997)]. However, these vectors are currently introduced into cells by transfection, typically ex vivo. It would be desirable to have an alternative episomal replicating vector, that can readily be introduced into a cell.
SUMMARY OF THE INVENTION
We have now discovered a viral vector system that takes advantage of retroviral infection to bring a desired nucleic acid sequence to a targeted host cell without resulting in stable integration.
In one preferred embodiment the plurality of vectors used, include lentiviral vectors. These lentiviral vectors preferably contain a selectable marker.
The lentivirus vectors include, for example, primate lentiviruses such as human immunodeficiency virus (HIV) (e.g. HIV-1 and HIV-2) and simian immunodeficiency virus (SIV); feline immunodeficiency virus (FIV); or visna virus. The primate lentiviruses show a remarkable ability to utilize a range of heterologous envelopes resulting in pseudotyped virions.
The lentiviral virion (particle) is expressed by a vector system encoding the necessary viral proteins to produce a virion (viral particle). Preferably, there is at least one vector containing a nucleic acid sequence encoding the lentiviral pol proteins necessary for reverse transcription, operably linked to a promoter. Although the vector encoding pol sequences should not encode an IN capable of integration, it preferably does encode an IN.
For example, this can be accomplished by the substitution of sites that bind to DNA, negating the peptides ability to bind to DNA and integrate. For instance, substituting Gln for the third IN wild type active site, such as in HIV-1 (E152Q). This can be done by PCR introducting the Q into N/N yielding N/N/Q. Other changes can also be carried out. For example, if a triple leucine mutant is made it will require 9 nucleotide changes to revert all three amino acids to wild type (WT

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