WO2004044157A2 - Gp64-pseudotyped vectors and uses thereof - Google Patents

Abstract

The present invention relates to vectors pseudo typed with GP-64 that can be used for gene transfer as well as for large-scale production of high titer virus to be utilized in clinical and commercial applications.

Description

GP64-PSEUDOTYPED VECTORS AND USES THEROF

This application claims priority to U.S. provisional application Serial No. 60/425,853, filed November 12, 2002, which is herein incorporated by this reference in its entirety.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates to pseudotyped lentiviral vectors that can be used for gene transfer as well as for large-scale production of high titer vims to be utilized in clinical and commercial applications.

BACKGROUND ART

Lentiviral vectors can insert large genes and can target both dividing and non- dividing cells. Therefore, they hold unique promise as gene transfer agents, the past, the native lentiviral envelope glycoprotein has been replaced with that of vesicular stomatitis vims G (VSV-G), and the genes of interest were packaged in non-replicating vectors using transient transfection with three plasmids. However, due to the cytotoxic effects of VSV-G expression in producer cells (293T cells) it has been difficult to generate a packaging cell line, required for even modest scale-up of vector production. The present invention provides a pseudotyped lentiviras vector using the baculovims gp64 envelope glycoprotein. When compared to VSV-G, gp64 vectors exhibit similar broad tropism and ~30% higher native titers. Gp64-pseudotyped vectors are also highly concentrated without losing titer. Since, unlike VSV-G, gp64 expression does not kill cells, 293T-based cell lines constitutively expressing gp64 were generated. The present invention demonstrates that the baculovims gp64 protein is an alternative to VSV-G for viral vectors for the large-scale production of high titer vims required for clinical and commercial applications. SUMMARY OF THE INVENTION

The present invention provides a recombinant retroviral vector expression system comprising: a) a first vector comprising a retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising a retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein.

Further provided by the present invention is a method of producing a recombinant retroviral particle,comprising transfecting a cell with: a) a first vector comprising a retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein, wherein the cell produces a recombinant retroviral particle.

Also provided by the present invention is a composition comprising: a) a first vector comprising an retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein.

The present invention further provides a method of producing a recombinant retroviral particle,comprising: a) transfecting a cell with a first vector comprising a viral nucleic acid sequence, wherein the first vector expresses the GP64 envelope protein; and b)transfecting the GP64 expressing cells of a) with a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest and a third vector comprising an retroviral nucleic acid sequence encoding retroviral gag and retroviral pol wherein the cell produces a recombinant retroviral particle. Also provided by the present invention is a retroviral particle comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env.

Further provided by the present invention is a population of retroviral particles comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 A shows the production of baculovims GP64-pseudotyped recombinant HΓV-1. The arrow indicates the GP64 band. Left two lanes: 293T cells were either transfected or not with plasmid pBsd-gp64, then grown for 48 hrs. Samples of total cellular proteins were separated on SDS-PAGE, transferred to nitrocellulose membrane for immunob lotting using the anti-GP64 monoclonal antibodies ACV5. Right two lanes: Cells were transfected or not with three plasmids for 60 hrs. Cell media then were spun at 1000 x g for 10 minutes, and then 15 μl of the supernatant was solubilized in Laemmli buffer and separated by 10% SDS-PAGE, followed by immunoblot analysis using the anti-GP64 monoclonal antibodies ACN5. Figure IB shows the production of baculovims GP64-pseudotyped recombinant

HIN-1. Purified recombinant vims was applied to formavar-coated copper grids, followed by negative staining and visualized by transmission electron microscopy.

Figure 1C shows the production of baculovims GP64-pseudotyped recombinant HIN-1. 200 μl of purified GP64-pseudotyped vims suspension containing 10 mM sodium azide was overlaid over a monolayer of HOS cells in a 10 cm dish on ice for 30 min. The cells were then washed 4-5 times in excess chilled PBS containing 10 mM sodium azide to remove unbound vims. Cells were then incubated in blocking solution (PBS containing 5% BSA) followed by incubation with 1:20 diluted anti-GP64 monoclonal antibody (ACN1), next washed to remove excess antibody, and finally incubated with Cy3 -labeled goat anti-mouse IgG (Amersham Pharmacia Biotech, ΝJ). Each step (blocking, primary and secondary antibody incubation) was carried out on ice for 1 hr. Labeled cells were visualized using a fluorescence microscope. Cells incubated with mock inoculum or cells transfected with GP64 plasmid were used as controls.

Fig. 2 A shows transduction of a broad range of target cells by GP64- pseudotyped HIN- 1. Pseudotyped vims was created by three-plasmid transfection methods in 293T cells. The same amount of unconcentrated vims-containing culture supernatant (lOOμl) was added to the medium on monolayers of HOS, MDCK, 293T and HeLa cells. Cells were observed for GFP (trans gene cloned into the transfer vector) expression using fluorescence microscopy 60h post-infection. Figure 2B shows that the number of GFP-positive cells increases linearly with the amount of vims used. HOS cells were infected with indicated amounts of GP64- pseudotyped HJV-GFP and imaged 60h post-infection.

Figure 2C shows that HOS cell monolayers growing in 6-well plates were infected with indicated amounts of GP64-pseudotyped vims. At 60h post-infection cells were analyzed by FACS for percent GFP positive cells as described in methods. (D) Genomic DΝA was prepared from transduced cell lines and tested by Alu PCR for integration of the transfer vector, as described (Butler et al., 2001). The PCR products were separated on a 1% agarose gel and visualized by ethidium bromide staining. U, uninfected cells; I, Infected cells. Figure 3A shows a permanent human cell line expressing GP64. The level of expression of GP64 in various passages of the permanent cell line (KZ64) was measured using FACS analysis. Cells from each passage were labeled with anti-GP64 monoclonal antibodies followed by cy5-labeled goat anti-mouse IgG as secondary and analyzed by FACS as described in the methods. Figure 3B shows that the presence of gp64 gene was detected in each passage of

KZ64 by PCR of the genomic DΝA followed by separation of the PCR product on agarose gel. Arrow indicates the gp64 gene (~1.5kb in size). Genomic DΝA from untransfected 293T cells was used as negative control, designated as "-ve". (C) Relative comparison of GP64 mRΝA in different passages of KZ64 cells was carried out by qRT-PCR as described in methods. Small "p" represents passage number and the number following it denotes the passage number from which RΝA was made. 293T cells transfected with pBsdGP64 and 48 hr later the transfected cells were plated into complete DMEM containing lOμg/ml Blasticidin. Resistant cells were harvested 8 days post-selection and used for total RNA purification. This sample was designated passage zero (pO).

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Example included herein. Before the present compounds and methods are disclosed and described, it is to be understood that this invention is not limited to specific proteins, specific vectors, specific methods, or specific nucleic acids, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes multiple copies of the nucleic acid and can also include more than one particular species of nucleic acid molecule. The present invention provides a recombinant retroviral vector expression system comprising: a) a first vector comprising a retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising a retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein.

As used herein "retroviral nucleic acid" means that the sequence comprises at least part of a retroviral genome. In the expression system described above, the retroviral nucleic acid can be a nucleic acid from an HJN-1 genome or from an HIN-2 genome. The entire human HIN-1 genome can be accessed via GenBank Accession

No. NC_001802 and is incorporated herein in its entirety by this reference. The human HIN-1 genome accessible via Accession No. NC_001802 includes nucleic acids encoding retroviral gag and pol, as well as nucleic acids encoding tat, vif, vpu, vpr, nef and env all of which are incorporated herein by this reference. The entire human HIN- 2 genome can be accessed via GenBank Accession No. NC_001722 and is incorporated herein in its entirety by this reference. The human HIV-2 genome accessible via Accession No. NC_001722 includes nucleic acids encoding retroviral gag and pol, as well as nucleic acids encoding tat, vif, vpu, vpr, nef and env all of which are incorporated herein by this reference. One of skill in the art will know how to obtain and modify these nucleic acid sequences, according to the methods described herein, in order to make the vectors of the present invention. The vectors can also comprise a retroviral nucleic acid sequence from a self inactivating HIV sequence. The retroviral nucleic acid sequences can also be sequences derived from bovine immunodeficiency vims, Jembrana disease vims, Equine infectious anemia vims, feline immunodeficiency vims, caprine arthritis-encephalitis vims, ovine lentivirus, Visna vims, simian immunodeficiency vims, simian-human immunodeficiency vims. As mentioned above, the first vector is a gag/pol retroviral expression vector which expresses proteins required for assembly and release of viral particles from cells, and includes the nucleic acids encoding retroviral gag and pol. This vector can also comprise nucleic acids encoding vpr and vpu as well as a promoter sequence. The promoter sequence may comprise a promoter of eukaryotic or prokaryotic origin . Suitable promoters include, but are not limited to, the cytomegalovims promoter, the Rous Sarcoma vims promoter, promoters derived from immunoglobulin genes, SV40, Adenovims and Bovine Papilloma Vims. All of the vectors described herein can comprise a promoter, hi addition to the guidance provided in the Examples provided herein, standard techniques for the constraction of the vectors of the present invention are well-known to one of skill in the art and can be found in references such as Sambrook et al., Molecular Cloning: A Laboratory Manual 2^nd Ed. (Cold Spring Harbor, N.Y., 1989). One of skill in the art can select from a variety of techniques available for the ligation of nucleic acids to construct the vectors of the present invention. The first vector can also comprise a defective env gene. As used herein,

"defective" means that a nucleic acid sequence is not functional with regard to either encoding its gene product or serving as a signaling sequence. For example, a defective env nucleic acid sequence will not encode the env protein; in another example, a defective packaging signal will not facilitate the packaging of the nucleic acid molecule the defective packaging signal is located on. Nucleic acid sequences may be rendered defective by means known in the art, including the deletion of some or all of the sequence, by mutating the nucleic acid sequence, by placing the sequence out of frame or by otherwise interfering with the sequence.

The second vector comprises a retroviral nucleic acid sequence comprising cis- acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted. The cis-acting sequences of the present invention include rev, tat and the long terminal repeats (LTRs) containing the packaging signals. This second vector can also comprise a defective env gene. The present invention also provides an expression system in which a defective env nucleic acid or gene is present in the first vector as well as in the second vector.

The second vector can also be deficient or defective for expression of at least one retroviral structural protein selected from the group consisting of: gag, pol, vif, vpu, vpr, nef and env. The vectors of the present invention include such defective nucleic acids in order to exclude the possibility of generating replication-competent retrovims. In fact, the second vector need only include the LTRs and the gene of interest. Another possibility is that the second vector contains a CMN promoter and one of the LTRs in order to minimize gene silencing. In another example, the second vector can have active gag, pol, vif, vpu and vpr genes and inactive nef and env genes. The present invention also contemplates the use of the self-inactivating (SLΝ) construct of the HJN vims, hi this construct, a 400 base pair fragment from the 3' LTR is deleted such that the HIN genome can integrate only once and the chances of forming replication competent vims by random recombination is much lower, h these vectors, gag, pol, vif, vpr, vpu, nef and env are usually deleted.

The second vector of the expression system of the present invention is designed to serve as the vector for gene transfer and contains all of the cis-acting sequence elements required to support reverse transcription of the vector genome, as well as a multiple cloning site for insertion of a nucleic acid encoding a heterologous gene of interest that will be delivered to cells. In the present invention, the vector encoding the gene of interest is a recombinant retroviral vector, for example, an HIN-1 or HIN-2 vector, that comprises a gene of interest to be transduced into a cell as well as cis-acting sequences necessary for the packaging and integration of the viral genome.

By " nucleic acid sequence encoding the heterologous gene of interest" or "heterologous nucleic acid" is meant that any heterologous or exogenous nucleic acid, i.e. not normally found in the wildtype retroviral genome, can be inserted into the vector for transfer into a cell, tissue or organism. Once cells are transfected with the plasmids or vectors of the expression systems described herein, particles comprising a heterologous nucleic acid encoding a gene of interest can be isolated for delivery to cells. The heterologous nucleic acid can be functionally linked to a promoter. By

"functionally linked" is meant that the promoter can promote expression of the heterologous nucleic acid, as is known in the art, and can include the appropriate orientation of the promoter relative to the heterologous nucleic acid. Furthermore, the heterologous nucleic acid preferably has all appropriate sequences for expression of the nucleic acid. The nucleic acid can include, for example, expression control sequences, such as an enhancer, a silencer and necessary information processing sites, such as ribosome binding sites, RΝA splice sites, polyadenylation sites, and transcriptional terminator sequences.

The heterologous nucleic acid can encode beneficial proteins or polypeptides that replace missing or defective proteins required by the cell or subject into which the vector is transferred or can encode a cytotoxic polypeptide that can be directed, e.g., to cancer cells or other cells whose death would be beneficial to the subject. The heterologous nucleic acid can also encode antisense RΝAs that can bind to, and thereby inactivate, mRΝAs made by the subject that encode harmful proteins. The heterologous nucleic acid can also encode ribozymes that can effect the sequence-specific inhibition of gene expression by the cleavage of mRΝAs. In one embodiment, antisense polynucleotides can be produced from a heterologous expression cassette in a retroviral vector construct where the expression cassette contains a sequence that promotes cell-type specific expression (Wirak et al, EMBO 10:289 (1991)). For general methods relating to antisense polynucleotides, see Antisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988).

Examples of heterologous nucleic acids that can be utilized in the expression systems and methods of the present invention, include but are not limited to the following: nucleic acids encoding secretory and nonsecretory proteins, nucleic acids encoding therapeutic agents, such as tumor necrosis factors (TNF) and TNF-α; interferons, such as interferon-α, interferon-β, and interferon-γ; interleukins, such as LL-1, IL-lβ, and ILs -2 through -14; GM-CSF; adenosine deaminase; cellular growth factors, such as lymphokines; soluble CD4; Factor NIII; Factor IX; T-cell receptors; LDL receptor; ApoE; ApoC; alpha- 1 antitrypsin; ornithine transcarbamylase (OTC); cystic fibrosis fransmembrane receptor (CFTR); insulin; anti-apoptotic gene products; proteins promoting neuronal survival, such as growth factors and glutamate receptors; Fc receptors for antigen binding domains of antibodies, such as immunoglobulins; anti- HIN decoy tar elements; and antisense sequences which inhibit viral replication, such as antisense sequences which inhibit replication of hepatitis B or hepatitis non- A, non- B vims.

Using the expression system described herein, particles comprising a heterologous nucleic acid encoding a gene of interest can be made and used to target various cells. The nucleic acid is chosen considering several factors, including the cell to be transfected. Where the target cell is a blood cell, for example, particularly useful nucleic acids to use are those which allow the blood cells to exert a therapeutic effect, such as a gene encoding a clotting factor for use in treatment of hemophilia. Another target cell is the lung airway cell, which can be used to administer nucleic acids, such as those coding for the cystic fibrosis fransmembrane receptor, which could provide a gene therapeutic treatment for cystic fibrosis.. Other target cells include muscle cells where useful nucleic acids, such as those encoding cytokines and growth factors, can be transduced and the protein the nucleic acid encodes can be expressed and secreted to exert its effects on other cells, tissues and organs, such as the liver. Neurons and retinal cells can also serve as targets for delivery of heterologous nucleic acids. Furthermore, the nucleic acid can encode more than one gene product, limited only, if the nucleic acid is to be packaged, by the size of nucleic acid that can be packaged. Furthermore, suitable nucleic acids can include those that, when transferred into a primary cell, such as a blood cell, cause the transferred cell to target a site in the body where that cell's presence would be beneficial. For example, blood cells such as TIL cells can be modified, such as by transfer into the cell of a Fab portion of a monoclonal antibody, to recognize a selected antigen. Another example would be to introduce a nucleic acid that would target a therapeutic blood cell to tumor cells. Nucleic acids useful in treating cancer cells include those encoding chemotactic factors which cause an inflammatory response at a specific site, thereby having a therapeutic effect.

Cells, particularly blood cells, muscle cells, airway epithelial cells, brain cells and endothelial cells having such nucleic acids transferred into them can be useful in a variety of diseases, syndromes and conditions. For example, suitable nucleic acids include nucleic acids encoding soluble CD4, used in the treatment of AIDS and α- antitrypsin, used in the treatment of emphysema caused by α-antitrypsin deficiency. Other diseases, syndromes and conditions in which such cells can be useful include, for example, adenosine deaminase deficiency, sickle cell deficiency, brain disorders such as Alzheimer's disease, Huntington's disease, lysosomal storage diseases, Gaucher's disease, Hurler's disease, Rrabbe's disease, motor neuron diseases such as amylotrophic lateral sclerosis and dominant spinal cerebellar ataxias (examples include SCA1, SCA2, and SCA3), thalassemia, hemophilia, diabetes, phenylketonuria, growth disorders and heart diseases, such as those caused by alterations in cholesterol metabolism, and defects of the immune system.

The heterologous nucleic acid can also encode a reporter gene sequence or a selectable marker gene sequence. A reporter gene sequence is any gene sequence which, when expressed, results in the production of a protein whose presence or activity can be monitored. Thus, the reporter protein can be specifically detected when expressed. Many reporter proteins are known to one of skill in the art. These include, but are not limited to, beta-galactosidase, chloramphenicol acetyltransferase, beta- lactamase, luciferase, and alkaline phosphatase that produce specific detectable products. Fluorescent reporter proteins can also be used, such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP) and yellow fluorescent protein (YFP). A selectable marker sequence is any gene sequence capable of expressing a protein whose presence permits one to selectively propagate a cell which contains it. Examples of selectable marker genes include gene sequences capable of conferring host resistance to antibiotics, for example, arnpicillin, tetracycline, kanamycin etc., or of conferring host resistance to amino acid analogues, or of permitting the growth of bacteria on additional carbon sources.

Expression of the heterologous nucleic acid may also provide an immunogenic or antigenic protein or polypeptide that can be used to achieve an antibody response. These antibodies can then be collected from an animal in a body fluid such as blood, serum or ascites. In the expression system of the present invention, the third vector comprises a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein. Therefore, this vector comprises a nucleic acid sequence encoding GP64. GP64 is a class I membrane glycoprotein, wliich constitutes the major envelope protein of the AcMNPN budded baculovims virion (Nolkman and Goldsmith, 1984; Nolkman et al, 1984). In essence, the third vector is pseudotyped, which means that the viral particles made utilizing the expression system described herein contains nucleic acid from one vims and the envelope protein of another, different vims. Therefore, based on the teachings of the present invention, any recombinant vector system can utilize a psuedotyped GP64 vector for the production of recombinant viral particles. Nucleic acids encoding GP-64 may be identical in sequence to the sequences which are naturally occurring for the GP-64 protein or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. These nucleic acids can also be modified from their typical structure. Such modifications include, but are not limited to, methylated nucleic acids, the substitution of a non-bridging oxygen on the phosphate residue with either a sulfur (yielding phosphorothioate deoxynucleotides), selenium (yielding phosphorselenoate deoxynucleotides), or methyl groups (yielding methylphosphonate deoxynucleotides). Any of the nucleic acid sequences described herein, such as the GP-64 nucleic acid sequences and the heterologous nucleic acid sequences provided for by the present invention may be obtained in any number of ways. One example of a method of obtaining a DNA molecule encoding a specific GP64 protein is to synthesize a recombinant DNA molecule which encodes the GP64 protein. For example, oligonucleotide synthesis procedures are routine in the art and oligonucleotides coding for a particular protein region are readily obtainable through automated DNA synthesis. A nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand. One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5' or 3' overhangs at the termini for cloning into an appropriate vector. Double-stranded molecules coding for relatively large proteins can readily be synthesized by first constmcting several different double-stranded molecules that code for particular regions of the protein, followed by ligating these DNA molecules together. For example, Cunningham, et al, "Receptor and Antibody Epitopes in Human Growth Hormone Identified by Homolog-Scanning Mutagenesis," Science, 243:1330-1336 (1989), have constructed a synthetic gene encoding the human growth hormone gene by first constructing overlapping and complementary synthetic oligonucleotides and ligating these fragments together. See also, Ferretti, et al, Proc. Nat. Acad. Sci. 82:599-603 (1986), wherein synthesis of a 1057 base pair synthetic bovine rhodopsin gene from synthetic oligonucleotides is disclosed. See also, U.S. Patent No. 5,503,995 which describes an enzyme template reaction method of making synthetic genes. Techniques such as this are routine in the art and are well documented. By constmcting a GP64 nucleic acid in this manner, one skilled in the art can readily obtain any particular GP64 protein with desired amino acids at any particular position or positions within the GP64 protein. These nucleic acids or fragments of a nucleic acid encoding a GP64 protein can then be expressed in vivo or in vitro as discussed below.

Once the nucleic acid sequence of the desired GP64 protein is obtained, the sequence encoding specific amino acids can be modified or changed at any particular amino acid position by techniques well known in the art. For example, PCR primers can be designed which span the amino acid position or positions and which can substitute any amino acid for another amino acid. Then a nucleic acid can be amplified and inserted into the wild-type GP64 protein coding sequence in order to obtain any of a number of possible combinations of amino acids at any position of the GP64 protein.

Alternatively, one skilled in the art can introduce specific mutations at any point in a particular nucleic acid sequence through techniques for point mutagenesis. General methods are set forth in Smith, M. "In vitro mutagenesis" Ann. Rev. Gen., 19:423-462 (1985) and Zoller, M.J. "New molecular biology methods for protein engineering" Curr. Opin. Struct. Biol., 1:605-610 (1991). These techniques can be used to alter the coding sequence without altering the amino acid sequence that is encoded. These techniques can be utilized to obtain any nucleic acid encoding GP64, any nucleic acid encoding a heterologous nucleic acid of the present invention or any nucleic acid encoding retroviral nucleic acid sequences.

The present invention also provides an expression system in wliich the first vector is pHIN-PV, the second vector is pHIV-IRES-G^"PΕT^"V^" and the third vector is pcDΝA-gp64. Also provided by this invention is an expression system in which the first vector is pHFN-PN, the second vector is pHIN- G^'P^'ETN^" IRES-GFP and the third vector is pcDΝA-gp64. These vectors and the methods of constructing these vectors are described in the Examples herein. Examples of other vectors that can be utilized as a third vector in this system include plasmids containing other envelope genes, e.g. pME-VSVG, pMD-G (also contains VSVG), pCB5HA (contains influenza HA), pLTRMVG (contains Makola Vims envelope), pHIT456 (contains murine leukemia virus amphotrophic envelope), pCG6-Ebo-GP (contains ebola virus glycoprotein) and others.

The present invention also provides a method of producing a recombinant retroviral particle,comprising transfecting a cell with: a) a first vector comprising a retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein, wherein the cell produces a recombinant retroviral particle.

The present invention further provides a method of producing a recombinant retroviral particle,comprising transfecting a cell with: a) a first vector comprising a retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest; c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein, wherein the cell produces a recombinant retroviral particle; d) growing the cell to allow production of recombinant retroviral particles in the cell; and e) obtaining the recombinant retroviral particles from the cell.

The cells that can be utilized to make the particles of the present invention include, but are not limited to, 293 cells, COS7 cells, COS cells and CEM cells. The methods of the present invention allow the preparation of infectious, replication-defective retroviral particles. These methods can be utilized with those available in the art to prepare viral particles. Viral particles can be made by contacting lentivims-permissive cells or producer cells with the vector expression system or the compositions comprising the vectors of the present invention, producing the retro viral- derived particles in the transfected cells and collecting the vims particles from the cell. Transfection of cells can be accomplished utilized any standard method in the art such as electorporation, LEPTOFECTAMiNE mediated transfection, DEAE-dextran transfection etc. Production of the infectious viral particles in the cells can be carried out using conventional techniques, such as standard cell culture techniques. With regard to collecting the particles, collection can be carried by techniques known in the art. For example, infectious particles can be collected by cell lysis, or collection of the supernatant of the cell culture, as described in the Examples and as is known in the art. Once the vims particles are collected, the vims particles can be further purified. Such purification techniques are known in the art. The GP64-pseudotyped viral particles of the present invention can be concentrated without significant loss of titer.

The present invention also provides recombinant retroviral particles produced by the methods of the present invention as well as cells comprising the recombinant particles produced by the methods of the present invention. Further provided by the present invention is a composition comprising: a) a first vector comprising an retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein. All of the compositions described herein can further comprise a carrier.

Also provided by the present invention are cells comprising the compositions of the present invention.

The present invention also provides a method of producing a recombinant retroviral particle,comprising: a) transfecting a cell with a first vector comprising a viral nucleic acid sequence, wherein the first vector expresses the GP64 envelope protein; and b) transfecting the GP64 expressing cells of a) with a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest and a third vector comprising an retroviral nucleic acid sequence encoding retroviral gag and retroviral pol wherein the cell produces a recombinant retroviral particle.

Also provided by the present invention is a method of producing a recombinant retroviral particle, comprising: a) transfecting a cell with a first vector comprising a viral nucleic acid sequence, wherein the first vector expresses the GP64 envelope protein; b) transfecting the GP64 expressing cells of a) with a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest and a third vector comprising an retroviral nucleic acid sequence encoding retroviral gag and retroviral pol wherein the cell produces a recombinant retroviral particle; c) growing the cell to allow production of recombinant retroviral particles in the cell; and d) obtaining the recombinant retroviral particles from the cell.

The present invention also provides a retroviral particle comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env. Thus, the genome can lack any one of these structural proteins or any combination thereof. Therefore, the genome can lack all of the structural proteins (gag, pol, vif, vpu, vpr, nef and env). The retroviral particle of the present invention can further comprise rev and/or tat. The retroviral particle of the present invention can be a particle wherein the genome is an HIN-1 genome and vpu, vpr, vif, tat and nef are absent or are dismpted. Any of the above-described particles of the present invention can further comprise a nucleic acid sequence encoding a gene of interest. The present invention also provides an isolated cell or cells comprising the particles of the present invention.

The present invention also provides a method of expressing a gene of interest comprising contacting a cell with a retroviral particle of the present invention comprising a nucleic acid encoding a gene of interest. The cells in which a gene of interest can be expressed include, but are not limited to, fibroblasts, neurons, retinal cells, kidney cells, lung cells, bone marrow stem cells, hematopoietic stem cells, retinal cells and neurons. The cells in which the gene of interest can be expressed can be dividing cells such as MDCK cells, BHK cells, HeLa cells, 3T3 cells, CN1 cells, COS7 cells, HOS cells and 293 cells. The cells can also be embryonic stem cells of mouse, rhesus, human, bovine or sheep origin, as well as stem cells of neural, hematopoietic, muscle, cardiac, immune or other origin. The embryonic stem cells of the present invention, such as stem cells of neural, hematopoietic, muscle, cardiac, immune or other origin can be dividing or nondividing embryonic stem cells.

Νondividing cells can also be contacted with a particle of the present invention to express a gene of interest. Such cells include, but are not limited to hematopoietic stem cells and embryonic stem cells that have been rendered non-dividing by specific media. Stem cells can be rendered non-dividing by growing them in media lacking growth factors required by stem cells, namely interleukins, LL-3, IL-6, GM-CSF and stem cell factor. In the present invention cells can be contacted ex vivo, in vitro or in vivo with the particles of the present invention. Administration can be an ex vivo administration directly to a cell removed from a subject, such as any of the cells listed above, followed by replacement of the cell back into the subject, or administration can be in vivo administration to a cell in the subject. For ex vivo administration, cells are isolated from a subject by standard means according to the cell type and placed in appropriate culture medium, again according to cell type (see, e.g., ATCC catalog). Viral particles are then contacted with the cells as described above, and the vims is allowed to transfect the cells. Cells can then be transplanted back into the subject's body, again by means standard for the cell type and tissue (e. g., for neural cells, Dunnett, S.B. and Bjδrklund, A., eds., Transplantation: Neural Transplantation-A Practical Approach,

Oxford University Press, Oxford (1992)). If desired, prior to transplantation, the cells can be studied for degree of transfection by the vims, by known detection means and as described herein. For example, prior to undertaking ex vivo or in vivo uses, one of skill in the art could contact a particular cell or tissue in vitro with a particle of the present invention comprising a reporter protein. By measuring the expression of the reporter protein in the cell or tissue, one of skill in the art can determine whether the cell is amenable for transduction with the particles of the present invention. The skilled artisan can also determine dosages utilizing such in vitro studies.

In vivo administration to a human subject or an animal model can be by any of many standard means for administering vimses, depending upon the target organ, tissue or cell. Vims particles can be administered orally, parenterally (e.g., intravenously), by intramuscular inj ection, by direct tissue or organ inj ection, by intraperitoneal inj ection, topically, transdermally, via aerosol delivery, via the mucosa or the like. The present compositions can include various amounts of the selected viral particle in combination with a pharmaceutically acceptable carrier and, in addition, if desired, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. Parental administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Dosages will depend upon the mode of administration, the disease or condition to be treated, and the individual subject's condition, but will be that dosage typical for and used in administration of viral vectors. For example, a typical dosage can be from about 10 to about 10⁷ viral particles, depending on the age and the species of the subject. Often a single dose can be sufficient; however, the dose can be repeated if desirable.

Administration methods can be used to treat brain disorders such as Parkinson's disease, Alzheimer's disease, and demyelination disease. Other diseases that can be treated by these methods include metabolic disorders such as , muscoloskeletal diseases, cardiovascular disease, cancer, and autoimmune disorders.

Administration of recombinant particles to the cell can be accomplished by any means, including simply contacting the particle, optionally contained in a desired liquid such as tissue culture medium, or a buffered saline solution, with the cells. The particle can be allowed to remain in contact with the cells for any desired length of time, and typically the particle is administered and allowed to remain indefinitely. For such in vitro methods, the particle can be administered to the cell by standard viral fransduction methods, as known in the art and as exemplified herein. Titers of vims to administer can vary, particularly depending upon the cell type, but will be typical of that used for retroviral fransduction in general which is well known in the art. Additionally the titers used to transduce the particular cells in the present examples can be utilized. The cells that can be transduced by the present particles of the present invention particle can include any desired cell, such as the following cells and cells derived from the < following tissues, human as well as other mammalian tissues, such as primate, horse, sheep, goat, pig, dog, rat, and mouse: Adipocytes, Adenocyte, Adrenal cortex, Airway epithelial cells, Alveolar cells, Amnion, Aorta, Ascites, Astrocyte, Bladder, Bone, Bone marrow, Brain, Breast, Bronchus, Cardiac muscle, Cecum, Cerebellar, Cervix, Chorion, Colon, Conjunctiva, Connective tissue, Cornea, Dennis, Duodenum, Endometrium, Endothelium, Endothelial cells, Ependymal cells, Epithelial tissue, Epithelial cells, Epidermis, Esophagus, Eye, Fascia, Fibroblasts, Foreskin, Gastric, Glial cells, Glioblast, Gonad, Hepatic cells, Histocyte, Ileum, Intestine, small Intestine, Jejunum, Keratinocytes, Kidney, Larynx, Leukocytes, Lipocyte, Liver, Lung, Lymph node, Lymphoblast, Lymphocytes, Macrophages, Mammary alveolar nodule, Mammary gland, Mastocyte, Maxilla, Melanocytes, Mesenchymal, Monocytes, Mouth, Myelin, Myoblasts Nervous tissue, Neuroblast, Neurons, Neuroglia, Osteoblasts, Osteogenic cells, Ovary, Palate, Pancreas, Papilloma, Peritoneum, Pituicytes, Pharynx, Placenta, Plasma cells, Pleura, Prostate, Rectum, Retinal, Salivary gland, Skeletal muscle, Skin, Smooth muscle, Somatic, Spinal cord, Spleen, Squamous, Stomach, Submandibular gland, Submaxillary gland, Synoviocytes, Testis, Thymus, Thyroid, Trabeculae, Trachea, Turbinate, Umbilical cord, Ureter, and Uterus. Thus, the particles of the present invention can be used to deliver a nucleic acid to these cells. Also provided by the present invention are cells comprising the particles of the present invention such as, but not limited to, fibroblasts, kidney cells, neurons, retinal cells, lung cells, bone manow stem cells, hematopoietic stem cells, MDCK cells, BHK cells, HeLa cells, 3T3 cells, CV1 cells, COS7 cells, HOS cells and 293 cells. Other cells that can comprise the particles of the present invention include embryonic stem cells of mouse, rhesus, human, bovine or sheep origin, as well as stem cells of neural, hematopoietic, muscle, cardiac, immune or other origin. Nondividing cells can also comprise the particles of the present invention. These cells include, but are not limited to hematopoietic stem cells and embryonic stem cells that have been rendered non- dividing by specific media.

The present invention also provides a population of retroviral particles, e.g., more than one particle, comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env. Also provided by the present invention is a composition comprising a population of retroviral particles of the present invention.

Further provided by the present invention is a method of expressing a gene of interest comprising contacting a population of cells with a population of retroviral particles of the present invention. The cells can be, but are not limited to, fibroblasts, neurons, retinal cells, kidney cells, lung cells, bone marrow stem cells and hematopoietic stem cells. The population of cells can be a population of dividing cells. The population of cells can also be a population of non-dividing cells. Also provided by the present invention are populations of cells comprising a population of retroviral particles comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env. Also provided by the present invention is a composition comprising a population of retroviral particles of the present invention. The cells can be, but are not limited to, fibroblasts, neurons, retinal cells, kidney cells, lung cells, bone marrow stem cells and hematopoietic stem cells. The population of cells can be a population of dividing cells. The population of cells can also be a population of non-dividing cells.

The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLES

Since many native envelope glycoproteins have restricted target cell ranges, investigators have developed novel vectors by replacing the native envelope glycoprotein with ones from other vimses, a process called pseudotyping¹. The most prevalent pseudotyped lentiviral vector is HJN-1 with the vesicular stomatitis viras G (NSN-G) glycoprotein substituting for the env glycoprotein^2"8. These recombinant gene transfer vectors are produced by transient co-transfection of human embryonic kidney 293T cells using three different plasmids encoding the helper (packaging), gene transfer and envelope functions²'^9"11. They can transduce a wide range of cells and can be concentrated more than 1000 fold by ulfracentrifugation without losing infectivity, in contrast to oncoretroviral vectors¹²'¹³. To upscale vector production for in vivo experiments in larger animals and human patients, however, development of a stable lentivirus vector packaging cell lines is highly desirable¹⁴'¹⁵. NSNG, the cunent pseudotype of choice, is cytotoxic to the cells used to produce the recombinant HIN- based gene therapy vectors¹ . This prevents isolation and propagation of stable producer clones in which it is constitutively expressed, an essential step in the production of large quantities of standardized HIN-based vectors repetitively. Recently, it has also been reported that NSN-G pseudotyped vims is inactivated by the human complement system in the blood¹⁷. To circumvent this problem, several attempts have been made to construct packaging cell lines with inducible expression of NSN-G ' ^" . Although such packaging cell lines have been shown to initially produce reasonable titers of lentiviral vectors, they do not retain their expression properties long-term, presumably due to negative selection of NSN-G expressing cells. An alternative method is to use other non-toxic heterologous envelope gene. Several envelope genes have been tried for pseudotyping human immunodeficiency vims type-1 (HιN-l)-based vectors¹³'²¹'²². Although different envelope proteins do pseudotype with HIN-based vectors to different extents, none are considered better than NSNG either due to the lower titers of the pseudotyped vectors (RSN, influenza, Moloney, amphotrophic Moloney, MLN envelopes), reduced tropism (Ebola Z, Ebola R) or difficulties in concentrating the vector (HA, RSN), making NSN-G the best option so far.

Gp64 is the major baculoviral envelope protein. It is a bonafide fusion protein as the expression of gp64 in insect cells is necessary and sufficient for a cell-cell fusion phenotype²³'²⁴. Gp64 has been extensively studied to understand the mechanism of membrane fusion^23"28. The present invention provides the use of gp64 for pseudotyping

HIN-1 based vectors. By transient transfection of three plasmids (one containing gp64) into 293T cells, retroviral vectors that efficiently transduced various cell types were produced. More notably, gp64 expression was not cytotoxic to host cells. Thus, derivation of a permanent cell line constitutively expressing gp64 at high levels over many passages was successful. This novel cell line produced gp64 pseudotyped lentiviral vectors when transfected with helper and vector plasmids. The present invention demonstrates that baculovims gp64 meets the requirements of an envelope glycoprotein for pseudotyping lentiviruses without the associated cytotoxic effects of NSN-G. Packaging cell lines based on gp64-pseudotyping can thus be easily created, making the production of large amounts of standardized vector necessary for efficient gene transfer in in vivo and ex vivo studies practical.

Generation ofbaculovirus GP64-pseudotyped recombinant HIV-1

The plasmid for GP64 expression (pBsd-gp64) was transfected into 293T cells and expression of GP64 confirmed by Western blotting using anti-GP64 monoclonal antibodies (ACV5) (Fig. 1 A). GP64-pseudotyped vims containing the gene for green fluorescence protein (GFP) was created using three-plasrnid transient transfection followed by purification. Electron microscopic analysis of the purified viral particles showed uniform virions ~100 nm in diameter (Fig. IB). Western blot analysis of the purified GP64-pseudotyped recombinant vims using anti-GP64 monoclonal antibodies showed the presence of GP64 in the viral particles (Fig. 1A). Membrane fusion catalyzed by baculovims GP64 requires the protein to be exposed to the acidic endosomal environment (Liekina et al., 1992, Plonsky and Zimmerberg, 1996). Hence blocking endocytosis specifically blocks GP64-mediated fusion. To test for baculovims gp64-mediated binding of the GP64-pseudotyped vims, purified vims was overlaid on target cells in the presence of 10 mM sodium azide (to block endocytosis) followed by labeling of cells using anti-GP64 monoclonal antibodies as described in methods. Immunofluroscence analysis showed a GP64-mediated binding of pseudotyped vims to target cells (Fig. 1C). Cells exposed to mock inoculum did not exhibit any labeling. HIV-1 pseudotyped with GP64 is a vector with wide tropism

GP64-pseudotyped virus could transduce a variety of cell types (293T, HOS, HeLa, MDCK, BHK, NTH3T3) indicating a wide cell tropism (Fig 2A). Using the same amount of vims different numbers of GFP-positive cells were obtained in different cell types (Fig. 2A). Titers of the two vector preparations were assayed on several cell lines in parallel, using the expression of GFP in the target cell to detect successful fransduction. GP64- and VSVG-pseudotyped vimses were prepared in parallel as described in below and titers for GP64-pseudotyped vimses on different cell lines were compared to that for VSVG pseudotyped vimses by two methods (Table 1). Titers obtained from GP64-pseudotyped vims are 15-50% higher than the VSVG- pseudotyped vims, depending on the cell type used and this difference is statistically significant, as determined by two-way ANOVA (with replication) (Table IB). The titers depend on the type of cells used with interaction between the cell type and the vector used (Table IB). Variation in titer determination as a function of the method used has been described earlier (Cashion et al., 1999, Sastry et al., 2002). We also observed some variation in the titers obtained by different methods for the same viras preparation apparently due to the difference in the sensitivity of the method used to quantify the reporter gene (GFP). However, when subjected to the statistical tests, both titer methods gave similar results. Not surprisingly, the titers were lowest with murine cells (NIH3T3) while human, canine or monkey cells gave similar titers (Table 2). Furthermore, the pseudotyped viras could be concentrated to about 100 fold in most cases (293T, HOS, CV1 and HeLa cells, Table 2). However, the increase in titer was not linear to the concentration increase of the vector for COS 7 and NTH3T3 cells although the GP64- and VSVG-pseudotyped vimses did yield similar increase in titer in all cases. Preliminary data indicates that the GP64-pseudotyped viras could also efficiently transduce CD34⁺ cells and rhesus monkey embryonic stem cells, R366.4.. These experiments demonstrate that the GP64-pseudotyped virus gives broad tropism and high titers comparable to VSVG pseudotyped vims.

Heat-inactivating the viras by incubating the supernatant at 56°C for 30 min. before infection, or elimination of the plasmid encoding the envelope gene (GP64 or VSVG) from the triple transfections of 293T cells to produce the viras, led to undetectable titer, thus serving as negative controls to confirm pseudotyped virus- mediated delivery of the marker gene GFP. The number of GFP-positive foci increased linearly with the amount of GP64-pseudotyped viras used without increase in the average fluorescent intensity of GFP-positive cells (Fig. 2B and 2C), and the GFP- expression pattern was distinct and not diffused, ruling out pseudotransduction. Integration of the vector DNA into the genome of the transduced cells was confirmed by Alu PCR (Fig. 2D), which amplifies DNA sequences that have conjoint elements of HIV and host genome. In addition to HIV- 1 -based vectors, GP64 could also efficiently pseudotype HIV-2-based gene transfer vectors.

Baculovims GP64 expression does not induce cytotoxic effects in 293T

One of the major problems for large-scale production of VSVG-pseudotyped HIN-1 gene delivery vectors has been the fact that VSVG expression is cytotoxic to 293T cells (Yang et al. 1995, Ory et al, 1996). To test the effect of GP64 expression on 293T cell-viability, cells were transfected with a GP64 expression plasmid containing the Blasticidin resistance gene. Two days after transfection, the cells were split into selection medium and Blasticidin-resistant clones of cells were isolated. The Blasticidin-resistant cell clones were tested for GP64 expression by immunofluorescence and Western blotting, and one clone (called KZ64) was selected based on a high level constitutive expression of GP64. KZ64 cells exhibit a steady high-level constitutive expression of GP64 up to 12 passages as tested by FACS analysis (Fig. 3A). The GP64 gene could also be detected by genomic PCR (Fig. 3B). Relative comparison of GP64 mRNA in different passages of KZ64 cells by qRT-PCR exhibited a stable expression of GP64 gene (Fig. 3C). Transfection of all passages of KZ64 cells with the HIN-1 packaging and transfer plasmids alone lead to the production of high-titer infectious GP64-pseudo typed HIN-1 vectors (Table 3). The present invention shows that the major envelope protein of insect Baculovims, GP64, is an efficient and non-toxic substitute for env in an HIN-based vector system. GP64-pseudotyped vectors efficiently transduced various mammalian cell types. Vector particles were usually concentrated without apparent loss of titer. For any given target cell type, GP64-pseudotyped vector titers were modestly higher than

VSVG pseudotyped HIN vectors. Lack of toxicity was shown by the generation of a cell line from 293T cells constitutively expressing high levels of GP64 up to 16 passages. High-titers of GP64-pseudotyped recombinant virus were simply and efficiently produced from this new cell line by transient transfection of the packaging and gene transfer plasmids alone. Incorporation of GP64 did not influence the size, shape or maturation of the HIN vector as far as we could detect. Thus the marked reduction of cytopathic effects of cells expressing GP64, compared to cells expressing NSNG, is a distinct advantage for the large-scale production of vectors.

Baculovims GP64 protein has been studied extensively as the protein essential for the entry of the baculovims nucleocapsid into insect cells. It is known that native baculovims produced in insect cells, and insect cells expressing GP64 fuse with mammalian cells (Hofmann et al., 1995, Boyce and Bucher, 1996, Plonsky et al.,

1999). Indeed, both native and pseudotyped baculovims, produced in insect cells, have been used as vectors for the transfection of mammalian cells (Hofmann et al., 1995, Barsoum et al., 1997, Huser et al., 2001). But the insect virus has components of insect origin that activate the classical complement pathway in humans, hampering in vivo gene transfer using baculoviras-based vectors (Hofmann and Strauss, 1998). It is likely that differences in surface protein glycosylation contribute to this rejection of baculovims because glycoproteins synthesized in insect cells lack the sialic acid residues found on glycoproteins of human cells (Marchal et al., 2000). Since GP64 expressed in mammalian cells is glycosylated like other mammalian proteins (Jarvis and Finn, 1995), the vectors of the present invention should not have the foreign patterns of glycosylation that activate complement.

Although GP64 may pseudotype a variety of enveloped viral vectors, lentiviral vectors have several advantages over conventional oncoretro viruses: 1) Transduction of cells without requiring passage through the M phase of the cell cycle to traverse the nuclear membrane (Νaldini et al., 1996, Miyoshi et al., 1999). 2) A higher efficiency of transduction (Burns et al., 1993, Kafri et al., 1997, Kafri et al., 1999, Kumar et al., 2001) now enhanced further by using a central DΝA flap, (Zennou et al., 2001). And 3) lentiviral vectors can accommodate large, strong and complex genetic regulatory- elements conferring high-level expression and lineage-specificity (e.g. Kumar et al, 2001, Cui et al., 2002), critical to the success of many experimental and therapeutic gene-transfer strategies. Recent modifications to the HIN sequences in the helper and vector genomes, like the creation of self-inactivating (SLN) transfer vectors (Zufferey et al., 1998) and the removal of most of the HIV genes from the vector plasmid (Dull et al., 1998), are thought to get rid of the possibility of recombination and productive HIN infection (Dunbar, 2002).

When GP64 was expressed in human cell lines, it trafficked to the plasma membrane to incorporate into the budding envelope of a recombinant mammalian viras (HIN-1). This budded vims was able to fuse to native target cells of human origin, and integrate its engineered gene into that target cell for subsequent expression. Earlier, Moloney murine leukemia virus-based retroviral vector has been successfully pseudotyped by an insect retroviral envelope to infect insect cells (Teysset et al, 1998). The present invention providse the first study where a protein from an insect viras has been used to pseudotype a HINl-based vector for infecting mammalian cells. GP64 confers all the advantages of a heterologous envelope for pseudotyping gene therapy vectors without any associated cytotoxicity. In contrast, attempts to construct packaging cell lines constitutively expressing NSNG were of limited use (Oil et al., 1996, Kafri et al, 1999). The production of a permanent cell line for GP64 demonstrates the ease of constructing packaging cell lines for HIN-based vectors, something that until now has been a major hurdle in their use for clinical gene therapy. Towards that end, it now should be straightforward to repetitively create large amounts of reasonably homogeneous, GP64-pseudotyped, minimal lentiviral vectors incorporating both SIN LTRs and a central DNA flap.

Cells

293T, HOS, HeLa, MDCK, BHK, COS7, CV1 and NTH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%o fetal bovine serum and antibiotics.

Plasmid vector construction pHIN-rRES eGFP GTΕ V^" (Agag, Apol, Aenv, Avi Avpr) (IRES, internal ribosome entry site from encephalomyocarditis viras), pHJN-PN (PN, packaging vector) and pME NSNG was provided by Dr. Richard E. Sutton, Baylor College of Medicine, Houston, TX (Sutton et al., 1998, Kumar et al., 2001). The fransfer vector is based upon T-tropic isolate NL4-3, has large deletions in Gag, Pol, Env, Vif and Vpr; and carries LRES followed by the enhanced green fluorescence protein (GFP) gene (Clontech) in place of nef. Baculovims AcMNPV gp64 gene was cut out from the insect expression vector ATH-11 (Kingsley et al., 1999) and sub-cloned into mammalian expression vector pCDNA 3.1 (Invitrogen, CA). The blasticidin resistance gene from pCMV-Bsd (Invitrogen, CA) was subsequently sub-cloned into the above plasmid. The resulting plasmid, pBsd-gp64, had both the gp64 and Blasticidin resistance genes with separate CMV promoters.

Preparation and titering of vector supernatant

Pseudotyped HIV supematants were prepared as described earlier, without the addition of pcRev or butyrate (Sutton and Littman, 1996). In brief, 293T cells, maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin (complete DMEM), were passaged into 25 cm² culture flasks and then transiently transfected at 50-60% confluency with equal mass amounts of pHIN-PV, vector plasmid and either pME-VSVG or pBsd-gp64 using TransIT-LTl transfection reagent (Minis, WI). Vims-containing supematants were collected approximately 60h later, centrifuged at 3000 x g for 10 min. at 25°C to remove cell debris, and used for infections. Both the GP64- and VSV G-pseudotyped vimses were titered on various target cells (HOS, 293T, HeLa, COS7, CV1 , ΝTH3T3, MDCK or BHK) in two different ways. Target cells grown in large flasks were lifted and seeded into 6-well plates (~5 x 10⁵ cells per well). After 24 hrs, wells were infected with increasing dilutions of both viras preparations in complete DMEM. The first method for titer determination (counting GFP -foci method) was based upon the counting of GFP-positive foci using fluorescence microscopy as described earlier (Cashion et al, 1999), with modification. After 48-60h, foci of GFP-positive cells (defined as small clusters (2-8 cells) of GFP-positive cells apparently due to division of a transduced cell occurring between the times of infection and counting) were counted using a calibrated field of a digital cooled CCD camera (ORCA-ER, Hamamatsu) on a fluorescent microscope. The total number of positive foci per well was estimated by counting green foci in 9 distinct calibrated areas of the well randomly selected and knowing the total surface area of a well. The viral titer was determined by multiplying the number of positive foci per well by the viral dilution. The biological end-point titer was estimated from the three highest dilution wells that contained GFP positive foci. In the second method, viral titers were calculated from the percent GFP-positive cells in a well, obtained by FACS analysis, multiplied by the mean number of cells present at the time of infection in a parallel set of wells, to give the number of cells infected followed by calculation of infectious titer per ml. For FACS analysis, cells were lifted with trypsin-EDTA, made into single cell suspensions by pipetting, and fixed with 4% parafonnaldehyde. GFP expressing cells were analyzed with FACScan (Beckton Dickinson). Cells were counted for scatter characteristics, and the data was analyzed with WinList software (Verity Software House, ME) using uninfected cells as negative controls. As an alternative to FACS analysis, a qualitative method for titer estimation, paired percentage method, was used to compare the titers of different vims preparations. For this method, cells plated in 6-well plates were infected with different dilution of the respective unconcentrated or concentrated viras preparations so as to give 20-80% GFP positive cells. The percent GFP-positive cells were estimated visually using fluorescence microscopy and multiplied by the mean number of cells present at the time of infection for each dilution followed by calculation of infectious titer per ml of the viras preparation. To concentrate the viras, the culture supernatant was centrifuged at 100, 000 X g for 1 hr at 4°C to pellet the viras and the viras pellet was resuspended in 1/100 of the volume in DMEM and used for infection.

For infection of CD34+ HPCs, the virus-containing culture supernatant was ultracentrifuged as described above and the pellet suspended in either 1/100 of the volume or the same volume of LMDM (to correspond for unconcentrated supernatant). CD34+ cells were typically transduced overnight with the GFP-containing viras, using 3 X 10⁵ cells in 500 μl volume then infected with 100 μl unconcentrated or concentrated vims suspension in the presence of 4μg of polybrene per ml, either in the presence or absence of the cytokine cocktail as described above. Initial marker analysis was carried out 48 to 96 hr later. GFP expressing cells were analyzed with FACScan (Beckton Dickinson). Cells were counted for scatter characteristics, and the data was analyzed with CELLQUEST software (Beckton Dickinson). Confrols were uninfected cells.

Confirming the presence ofGP64 on the pseudotyped virus

Recombinant viras was concentrated from the culture supernatant by ulfracentrifugation as described above and further purified by another cycle of ulfracentrifugation on a 50% sucrose bed. Such purified viras was tested for integrity and presence of GP64 by electron microscopy (EM), Western blotting and immunofluorescence. For EM purified virus was applied to formavar coated copper grids, negatively stained in 2% phosphotungstic acid (PTA) solution for 1 min, and then visualized using transmission electron microscopy (100CX, JEOL, Japan). For immunofluorescence, HOS cells were infected with GP64 pseudotyped virus in cold and in the presence of lOmM sodium azide (a potent inhibitor of endocytosis, Schwartz, et al., 1982), followed by labeling with anti-GP64 monoclonal (ACV1, that binds to native conformation of GP64) as primary and Cy3 labeled goat anti-mouse IgG polyclonal antibodies (Amersham Pharmacia, NJ) as secondary. The labeled cells were visualized using an inverted fluorescence microscope (Zeiss Axiovert 25) and ^■ imaged with a 16-bit cooled CCD camera (ORCA-ER, Hamamatsu, Japan) using Metamorph imaging software (Universal Imaging, PA). Western blot analysis was carried out by separating purified viras on SDS-PAGE, followed by Western blot analysis using anti-GP64 monoclonal antibodies.

Testing for stable integration of the vector in the genomic DNA of transduced cells

Genomic DNA was prepared from transduced cells using Wizard genomic DNA purification kit (Promega, WI). The genomic DNA was subjected to Alu PCR as described earlier (Butler et al., 2001) to test for integration of the vector DNA.

Generation of 293T-derived cell lines expressing gp64 constitutively

293T cells were transfected with pBsd-gp64 plasmid using TransIT LT1 transfection reagent. 48 hr post-transfection the cells were lifted and plated into selection medium (complete DMEM containing 1 Oμg per ml Blasticidin (Life Tech., NY)). The resistant colonies were propagated till 10-14 days. Individual cell colonies were grown and tested for GP64 expression by immunofluorescence, FACS analysis using anti-GP64 monoclonal antibodies, Western blotting and PCR. Cell lines with high expression level were selected and passaged in complete medium containing Blasticidin. Each passage of the selected GP64-expressing cell line (called KZ64) was tested for the level of GP64 expression by FACS analysis, immunofluorescence and quantitative RT-PCR (qRT-PCR) for comparing level of GP64 rnRNA in different passages of KZ64 cells. For qRT-PCR, total RNA from ~2 X 10⁶ cells was extracted using Trizol reagent (Life Tech., MD) and 5μg of total RNA from each preparation was used for each test using QuantiTech S YBR green RT-PCR kit (Qiagen, CA) and primers 5'-ATGCGGCCGCA-TGGTAAGCGCTATTGTT-3' and 5'-

GATCCTCGAGTTAATATTGTCTATTAC-GG-3'. The qRT-PCR were carried on the ABI 7700 system (Applied Biosystems, CA) with conditions of 94°C, 58°C and 72°C each for 45 sec. To produce GP64-pseudotyped viras, packaging and gene transfer plasmids were transiently transfected in the KZ64 cells and the virus- containing culture supernatant harvested 60h post-transfection. This supernatant was tested for viras titers as described above.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Although the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.

References

1. Landau, N. R., Page, K. A. & Liftman, D. R. Pseudotyping with Human T-cell leukemia viras type I broadens the human immunodeficiency vims host range. J Virol 65, 162-169 (1991).

2. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 272, 263-267 (1996). 3. Naldini, L. et al Efficient transfer, integration and sustained long-term expression of the transgene in adult rat brains injected with lentiviral vectors. Proc. Nail. Acad. Sci. USA 93, 11382-11388 (1996).

4. Aiken, C. Pseudotyping human immunodeficiency vims type 1 (HIN-1) by the glycoprotein of vehicular stomatitis viras targets HIV-l entry to an endocytic pathway and suppresses both the requirement for Νef and the sensitivity to cyclosporin A. J. Virol. 71, 5871-5877 (1997)

5. Reiser, J. et al. Development of multigene and regulated lentiviral vectors. J Virol. 74, 10589-10599 (2000).

6. Buchschacher, G. L. & Wong-Staal, F. Development of lentiviral vectors for gene therapy for human diseases. Blood, 95, 2499-2504 (2000).

7. Klages, Ν., Zufferey, R. & Trono, D. A stable system for the high-titer production of multiply attenuated lentiviral vectors. Mol Ther. 2, 170-176 (2000).

8. Trono, D. Lentiviral vectors: turning a deadly foe into a therapeutic agent. Gene Ther. 7, 20-23 (2000).

9. Dull, T. et al. A third generation lentiviral vector with a conditional packaging system. J Virol. 72, 8463-8471 (1998).

10. Kafri, T. et al. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat. Genet. 17, 314-317 (1997). ll. Miyoshi, H., Takahashi, M., Gage, F.H. & Nerma, I.M. Stable and efficient gene transfer into the retina using an HIN-based lentiviral vector. Proc. Natl Aca. Sci. USA 94, 10319-10323 (1997).

12. Bums, J.C. et al. Vesicular stomatitis viras G protein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Aca. Sci. USA 90, 8033-8037 (1993).

13. Reiser, J. Production and concentration of pseudotyped HIV-1 -based gene transfer vectors. Gene Titer. 7, 910-913 (2000).

14. Pawliuk, R. et al. Conection of sickle cell disease in transgenic mouse models by gene therapy. Science 294, 2368-2371 (2001).

15. Dunbar, C. Lentivirases get specific. Blood99, 397 (2002). 16. Yang, Y. et al. friducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis vims G envelope protein. Hum. Gene Tfier. 6, 1203-1213 (1995).

17. Depolo, N. J., et al. VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum. Mol Ther. 2, 218-222 (2000).

18. Ory, D.S., Neugeboren, B.A. & Mulligan, R.C. A stable human-derived packaging cell line for production of high titer retroviras/vesicular stomatitis viras G pseudotypes. Proc. Natl. Aca. Sci. USA 93, 11400-11406 (1996).

19. Yoshida, Y., Emi, N. & Hamada, H. VSV-G-pseudotyped retroviral packaging through Adenoviras-mediated inducible gene expression. Biochem. Biophysic. Res. Com. 232, 379-382 (1997).

20. Kafri, T. et al A packaging cell line for lentiviras vectors. J. Virol. 73, 576-584 (1999).

21. Stitz, J. et al. Lentiviral vectors pseudotyped with envelope glycoproteins derived from Gibbon ape leukemia viras and murine leukemia viras 10A1. Virology 273, 16-20 (2000).

22. Kobinger, G. P. et al. Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo. Nature Biotech. 19, 225-230 (2001).

23. Blissard, G. W. & Wenz, J. R. Baculovims gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion. J. Virol. 66, 6829-6835 (1992).

24. Leikina, E, Onaran, H.O. & Zimmerberg, J. Acidic pH induces fusion of cells infected with baculovims to form syncytia. FEBSLett. 304, 221-224 (1992).

25. Plonsky, I. & Zimmerberg, J. Initial fusion pore induced by baculoviras gp64 is large and forms quickly. J. CellBiol. 135, 1831-1839 (1996).

26. Chemomordik, L.V. et al. Control of baculovims gp64-induced syncytium formation by membrane lipid composition. J. Virol. 69, 3049-3058 (1995).

27. Plonsky, I. et al. An analysis of the role of target membrane on the gp-64-induced fusion pore. Virology 253, 65-76 (1999).

28. Kingsley D. H. et al. A discrete stage of baculovims GP64-mediated membrane fusion. Mol. Biol Cell 10, 4191-200 (1999).

29. Sutton, R.E. et al. Human Immunodeficiency viras type I vectors efficiently transduce human hematopoietic stem cells. J. Virol. 72, 5781-5788 (1998). 30. Miyoshi, H. et al. Transduction of Human CD34+ cells that mediate long-term engraftment of NOD/SCLD mice by HIN vectors. Science 283, 682-686 (1999).

31. Hofmann, C. et al. Efficient gene transfer into human hepatocytes by baculoviras vectors. Proc. Natl. Aca. Sci. USA 92, 10099-11103 (1995).

32. Boyce, F.M. & Buchner, Ν.L. Baculoviras-mediated gene transfer into mammalian cells. Proc. Natl. Aca. Sci. USA 93, 2348-2352 (1996).

33. Barsoum, J., Brown, R., Mckee, M. & Boyce, F.M. Efficient transduction of mammalian cells by a recombinant baculovims having the vesicular stomatitis vims G glycoprotein. Hum. Gene Ther. 8, 2011-2018 (1997).

34. Huser, A., Rudolph, M. & Hofmann, C. Incorporation of decay-accelerating factor into the baculovims envelope generates complement-resistant gene transfer vectors. Nature Biotech. 19, 451-455 (2001)

35. Hofmann, C. & Strauss, M. Baculoviras-mediated gene transfer in the presence of human serum of blood facilitated by inhibition of the complement system. Gene Ther. 5, 531-536 (1998).

36. Marchal, I. et al. Glycoproteins of insect cells: Sialylated or not? Biol Chem. 382, 152-159 (2000).

37. Jarvis, D.L. & Finn, E.E. Biochemical analysis of the Ν-glycosylation pathway in baculovirus-infected lepidopteran insect cells. Virology 212, 500-511 (1995).

38. Kumar, M. et al. Systematic determination of the packaging limit of lentiviral vectors. Hum. Gene Ther. 12, 1893-1905 (2001).

39. Zennou, N. et al. The HIN-1 DΝA flap stimulates HJN vector-mediated cell transduction in the brain. Nature Biotech. 19, 446-450 (2001).

40. Cui, Y. et al. Targeting transgene expression to antigen-presenting cells derived from lentivirus-induced engrafting human-hematopoietic stem/progenitor cells. Blood 99, 399-408 (2002).

41. Zufferey, R. et al. Self-inactivating lentiviras vector for safe and efficient in vivo gene delivery. J. Virol. 72, 9873-9880 (1998).

42. Sutton, R.E. et al. Transduction of human progenitor hematopoietic stem cells by human immunodeficiency viras type 1 -based vectors is cell cycle dependent. J. Virol. 73, 3649-3660 (1999). 43. Sutton, R.E. & Liftman, D.R. Broad host range of human T-cell leukemia viras type 1 demonstrated with an improved psuedotyping system. J Virol. 70, 7322-7326 (1996).

44. Schwartz, A.L., Fridovich, S.E. & Lodish H.F. Kinetics of intemalization and recycling of the asialoglycoprotein receptor in a hepatoma cell line. J. Biol Chem. 257, 4230-4237 (1982).

45. Butler, S. L., Hansen, M. S. T. & Bushman, F. D. A quantitative assay for HIN DΝA integration in vivo. Nat. Med. 7, 631-634 (2001).

2-way ANOVA with replication (triplicate experiment)

* Cell types are CV1, BHK, HOS TK^_ and 293T.

* GP64- or VSVG-pseudotyped virus.

* between cell type and the pseudotyped virus used to infect.

Table 1: A, Transduction of several cell types by GP64-pseudotyped HIN-1. Paired dishes of the appropriate cells were grown at the same time and then freated with equal volumes of either GP-64 vector or NSV-G vector at differing dilutions. After 48-60 hrs, titers were determined by three methods as discussed in the text. In each case, dilutions were tested in triplicate and the mean and SD is given. B, Statistical analysis of variance between infections of cell lines infected with either GP64- or VSVG- pseudotyped virus. Titers obtained by each method were analyzed by two-way ANOVA with replication and the results of the analysis for each tifration type are given.

Titers measured by Paired percentage method as described in Methods Table 2: Transduction of several cell types by GP64-pseudotyped HIV-1. Results are given from two independent experiments.

*Titers measured by Paired percentage method as described in Methods

Table 3: High titer GP64-pseudotyped viras could be created from KZ64 cells. Each passage of KZ64 cells was fransfected with transfer and packaging vectors alone, and supernatant harvested 60hr post-transfection. Unconcentrated viras from each passage indicated was used to fransduce MDCK cells, and titers per ml were calculated by the paired percentage method as described in the text. Each observation is average of two independent experiments. o

Claims

What is claimed is:

1. A recombinant retroviral vector expression system comprising: a) a first vector comprising a retroviral nucleic acid sequence encoding refroviral gag and refroviral pol; b) a second vector comprising a retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein.

2. The vector system of claim 1, wherein the first vector comprises a defective env gene.

3. The vector system of claim 2, wherein the defect in the env gene is a deletion mutation.

4. The vector system of claim 1, wherein the first vector and the second vector each comprise a defect in the env gene.

5. The vector system of claim 1, wherein the second vector is deficient for expression of at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env.

6. The vector system of claim 1, wherein the second vector comprises a gene of interest.

7. The vector system of claim 1 , wherein the first vector is pHJN-PV.

8. The vector system of claim 1 , wherein the second vector is pH-N-IRES G -P- E- F- N.

9. The vector system of claim 1 , wherein the third vector is pcDNA-gp64.

10. A method of producing a recombinant refroviral particle,comprising transfecting a cell with: a) a first vector comprising a retroviral nucleic acid sequence encoding refroviral gag and retroviral pol; b) a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein, wherein the cell produces a recombinant retroviral particle.

11. The method of claim 10, further comprising: d) growing the cell to allow production of recombinant retroviral particles in the cell; and e) obtaining the recombinant retroviral particles from the cell.

12. The method of claim 11, wherein the cell is a human 293 cell.

13. A recombinant retroviral particle produced by the method of claim 10.

14. A cell comprising a recombinant retroviral particle produced by the method of claim 10.

15. A composition comprising: a) a first vector comprising an refroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising an refroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein.

16. The composition of claim 15, further comprising a carrier.

17. A cell comprising the composition of claim 15.

18. A method of producing a recombinant retroviral particle,comprising: a) transfecting a cell with a first vector comprising a viral nucleic acid sequence, wherein the first vector expresses the GP64 envelope protein; and b) transfecting the GP64 expressing cells of a) with a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest and a third vector comprising an refroviral nucleic acid sequence encoding retroviral gag and retroviral pol wherein the cell produces a recombinant retroviral particle.

19. The method of claim 18, further comprising: d) growing the cell to allow production of recombinant refroviral particles in the cell; and e) obtaining the recombinant retroviral particles from the cell.

20. The method of claim 18, wherein the cell is a human 293 cell.

21. A retroviral particle comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env.

22. The particle of claim 21, further comprising rev.

23. The particle of claim 21 , further comprising tat.

24. The retroviral particle of claim 21, wherein the genome is an HJN-1 genome and vpu, vpr, vif, tat and nef are absent or are disrupted.

25. The refroviral particle of claim 21, wherein the genome is an HIN-1 genome and vpu, vpr, vif, tat, rev and nef are absent or are dismpted.

26. The particle of claim 21, further comprising a nucleic acid sequence encoding a gene of interest.

27. An isolated cell comprising the refroviral particle of claim 21.

28. An isolated cell comprising the retroviral particle of claim 26.

29. A composition comprising the retroviral particle of claim 21 and a carrier.

30. A composition comprising the retroviral particle of claim 26 and a carrier.

I

31. A method of expressing a gene of interest comprising contacting a cell with the retroviral vector particle of claim 26.

32. The method of claim 31 , wherein the cell is a fibroblast.

33. The method of claim 31 , wherein the cell is a kidney cell.

34. The method of claim 31 , wherein the cell is a lung cell.

35. The method of claim 31 , wherein the cell is a bone manow stem cell.

36. The method of claim 31 , wherein the cell is a hematopoietic stem cell.

37. The method of claim 31 , wherein the cell is an embryonic stem cell.

38. The method of claim 31 , wherein the cell is a dividing cell.

39. The method of claim 31 , wherein the cell is a non-dividing cell.

40. A fibroblast comprising the particle of claim 21.

41. A kidney cell comprising the particle of claim 21.

42. A lung cell comprising the particle of claim 21.

43. A bone marrow stem cell comprising the particle of claim 21.

44. A hemapoietic stem cell comprising the particle of claim 21.

45. An embryonic stem cell comprising the particle of claim 21.

46. A population of retroviral particles comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral stractural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env.

47. A composition comprising the population of claim 46.

48. A population of cells comprising the population of claim 46.

49. A method of expressing a gene of interest comprising contacting a population of cells with the population of claim 46.

50. The method of claim 49, wherein the cells are fibroblasts.

51. The method of claim 49, wherein the cells are kidney cells.

52. The method of claim 49, wherein the cells are lung cells.

53. The method of claim 49, wherein the cells are bone manow stem cells.

54. The method of claim 49, wherein the cells are hematopoietic stem cells.

55. The method of claim 49, wherein the cells are embryonic stem cells.

56. The method of claim 49, wherein these cells are dividing cells.

57. The method of claim 49, wherein the cells are non-dividing cells.

58. A population of fibroblasts comprising the population of claim 46.

59. A population of kidney cells comprising the population of claim 46.

60. A population of lung cells comprising the population of claim 46.

61. A population of bone marrow stem cells comprising the population of claim 46.

62. A population of hemapoietic stem cells comprising the population of claim 46.

63. A population of embryonic stem cells comprising the population of claim 46.