EP1358342A2

EP1358342A2 - Retroviral vectors for transduction into quiescent cells and packaging systems for them

Info

Publication number: EP1358342A2
Application number: EP02714708A
Authority: EP
Inventors: Candace Summerford; John T. Gray; Jeng-Shin Lee; Richard C. Mulligan
Original assignee: Harvard College; Childrens Medical Center Corp
Current assignee: Harvard College; Childrens Medical Center Corp
Priority date: 2001-01-06
Filing date: 2002-01-07
Publication date: 2003-11-05
Also published as: WO2002059338A3; WO2002059338A2; CA2433867A1; AU2002246958B2

Abstract

Methods of producing retroviral vector particles capable of transducing therapeutic genes into quiescent cells and methods of gene transfer to quiescent cells comprising infection with a retroviral particle described herein.

Description

VECTORS AND PACKAGING SYSTEMS FOR TRANSDUCTION INTO QUIESCENT CELLS

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/260,199, filed on January 6, 2001. The entire teachings of the above application are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant H P50HL59316 from the National Institutes of Health. The United States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Retroviruses are enveloped RNA viruses that, after infection of a host cell, reverse transcribe their RNA genomes into a DNA intermediate, or provirus. Viruses containing an RNA genome and producing an RNA-dependent DNA polymerase are contained in the retroviral family. Retroviruses contain at least three types of proteins encoded by the viral genome, i.e., gag proteins (the group antigen internal structural proteins), pol proteins (the RNA-dependent DNA polymerase and the protease and integrase proteins) and env proteins (the viral envelope protein or proteins). Retroviral vectors have been the vehicle of choice for clinical gene transfer because of their efficacy, safety and stable long-term gene expression. Murine leukemia virus (MLV)-based retroviral vectors are the most widely used gene delivery vehicles in gene therapy clinical trials (Ali, M. et al., Gene Ther., 1 :367-384 (1994); and Marshall, E., Science, 269:1050-1055 (1995)). ' A drawback of retroviral vectors is their inability to infect and transduce genes into non-proliferating (quiescent, non-dividing, resting) cells, such as neurons, macrophages, hematopoietic stem cells, myofibers.and hepatocytes. These cells are important targets for gene therapy. SUMMARY OF THE INVENTION

The present invention is related to Applicants' surprising and unexpected discovery that retroviral vector particles which encode the wild-type spleen necrosis virus (SNV) gag-pol gene products can infect and transduce a DNA sequence of interest into quiescent (non-dividing, resting, non-proliferating) cells. The present invention is also related to Applicants' unexpected discovery that retroviral vector particles encoding wild-type SNV gag-pol gene products do not require a nuclear localization sequence in the matrix protein to transduce into quiescent cells. Quiescent cells can be naturally quiescent or produced by artificial means. As a result, Applicants' invention relates to novel packaging cell lines useful for generating retroviral vector particles capable of infecting and transducing a DNA sequence of interest into quiescent cells, to construction of such cell lines and to methods of using the particles to introduce DNA of interest into quiescent cells (e.g., quiescent animal cells (particularly mammalian cells)). In a particular embodiment, the packaging cell lines are stable packaging cell lines. As described herein, integration of provirus is not required for gene transfer and expression of the DNA of interest in the quiescent cells.

Packaging cell lines for producing retroviral particles capable of infecting and transducing a DNA sequence of interest into quiescent cells comprise (a) a cell (e.g., mammalian cell); (b) a first retroviral nucleotide sequence in the cell which comprises a coding sequence for wild-type SNV gag-pol; (c) a second retroviral nucleotide sequence in the cell which comprises a coding sequence for a heterologous envelope protein; and (d) a third retroviral nucleotide sequence in the cell which comprises the DNA sequence of interest and sequences required for packaging and reverse transcription. Sequences required for packaging and reverse transcription can be derived from Moloney leukemia virus (MLV), SNV or other viruses.

In a particular embodiment, the heterologous envelope protein is the G glycoprotein of vesicular stomatitis virus (VSV G). In another embodiment, the heterologous envelope protein is the amphotropic envelope of the Moloney leukemia virus (MLV). Cell lines for producing retroviral vector particles capable of infecting and transducing a DNA sequence of interest into quiescent cells are produced by transfecting host cells (e.g., mammalian host cells) with (a) a plasmid comprising a DNA sequence which encodes wild-type SNV gagpol proteins; (b) a plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and (c) a plasmid comprising the DNA sequence of interest and sequences required for packaging and reverse transcription.

The present invention also relates to methods of producing retroviral vector particles capable of infecting and transducing a DNA sequence of interest into quiescent cells, comprising co-transfecting host cells (e.g., mammalian host cells) with (a) a first plasmid comprising a DNA sequence which encodes wild-type SNV gagpol proteins; (b) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and (c) a third plasmid comprising the DNA sequence of interest and sequences required for packaging and reverse transcription. In a particular embodiment, the present invention relates to novel packaging cell lines useful for generating retroviral vector particles that comprise a MLV- derived retroviral genome and are capable of infecting and transducing a DNA sequence of interest into quiescent cells, to construction of such cell lines and to methods of using the particles to introduce DNA of interest into quiescent cells (e.g., animal cells (particularly mammalian cells). By "retroviral vector particles that comprise a MLV-derived retroviral genome" is meant to refer to retroviral vector particles that include in their genome MLV-derived sequences required for packaging and reverse transcription.

Packaging cell lines for producing retroviral particles that comprise a MLV- derived retroviral genome and are capable of infecting and transducing a DNA sequence of interest into quiescent cells comprise (a) a cell (e.g., mammalian cell); (b) a first retroviral nucleotide sequence in the cell which comprises a coding sequence for wild-type SNV gag-pol; (c) a second retroviral nucleotide sequence in the cell which comprises a coding sequence for a heterologous envelope protein; and (d) a third retroviral nucleotide sequence in the cell which comprises the DNA sequence of interest and MLV-derived sequences required for packaging and reverse transcription.

Cell lines for producing retroviral vector particles that comprise a MLV- derived retroviral genome and are capable of infecting and transducing a DNA sequence of interest into quiescent cells are produced by transfecting host cells (e.g., mammalian host cells) with (a) a plasmid comprising a DNA sequence which encodes wild-type SNV gagpol proteins; (b) a plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and (c) a plasmid comprising the DNA sequence of interest and MLV-derived sequences required for packaging and reverse transcription.

The invention also relates to methods of producing retroviral vector particles that comprise a MLV-derived retroviral genome and are capable of infecting and transducing a DNA sequence of interest into quiescent cells, comprising co- transfecting host cells (e.g., mammalian host cells) with (a) a first plasmid comprising a DNA sequence which encodes wild-type SNV gagpol proteins; (b) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and (c) a third plasmid comprising the DNA sequence of interest and MLV-derived sequences required for packaging and reverse transcription.

In another embodiment, the present invention relates to novel packaging cell lines useful for generating retroviral vector particles that comprise a SNV-derived retroviral genome and are capable of infecting and transducing a DNA sequence of interest into quiescent cells, to construction of such cell lines and to methods of using the particles to introduce DNA of interest into quiescent cells (e.g., animal cells (particularly mammalian cells). By "retroviral vector particles that comprise a SNV-derived retroviral genome" is meant to refer to retroviral vector particles that include in their genome SNV-derived sequences required for packaging and reverse transcription.

Packaging cell lines for producing retroviral particles that comprise a SNV- derived retroviral genome and are capable of infecting and transducing a DNA sequence of interest into quiescent cells comprise (a) a cell (e.g., mammalian cell); (b) a first retroviral nucleotide sequence in the cell which comprises a coding sequence for wild-type SNV gag-pol; (c) a second retroviral nucleotide sequence in the cell which comprises a coding sequence for a heterologous envelope protein; and (d) a third retroviral nucleotide sequence in the cell which comprises the DNA sequence of interest and SNV-derived sequences required for packaging and reverse transcription.

Cell lines for producing retroviral vector particles that comprise a SNV- derived retroviral genome and are capable of infecting and transducing a DNA sequence of interest into quiescent cells are produced by transfecting host cells (e.g., mammalian host cells) with (a) a plasmid comprising a DNA sequence which encodes wild-type SNV gagpol proteins; (b) a plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and (c) a plasmid comprising the DNA sequence of interest and SNV-derived sequences required for packaging and reverse transcription.

The invention also relates to methods of producing retroviral vector particles that comprise a SNV-derived retroviral genome and are capable of infecting and transducing a DNA sequence of interest into quiescent cells, comprising co- transfecting host cells (e.g., mammalian host cells) with (a) a first plasmid comprising a DNA sequence which encodes wild-type SNV gagpol proteins; (b) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and (c) a third plasmid comprising the DNA sequence of interest and SNV-derived sequences required for packaging and reverse transcription.

The present invention also relates to a method of gene transfer to quiescent cells comprising infecting quiescent cells with a genetically engineered retroviral vector described herein encoding a wild-type SNV gag-pol gene product. The present invention further relates to isolated retroviral nucleotide sequences and plasmids used to produce the retroviral vector particles described herein. The retroviral nucleotide sequences and plasmids can comprise one or more of the nucleic acid products described herein, thereby reducing the number of retroviral nucleotide sequences or plasmids transfected into cells. The invention also relates to the plasmids illustrated in the figures. The packaging cell lines and viral particles of the present invention can be used in gene therapy or gene replacement to introduce genes into a variety of quiescent cells. The packaging cell lines and viral particles of the present invention can also be used in development and production of vaccines, and in production of biochemical reagents. Gene therapy vectors produced with the cell lines of the present invention are expected to be valuable medical therapeutics and have many commercial, industrial and medical research applications.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a circular map of pHDM-SNVgp (a helper vector), which encodes wild-type SNV gag-pol gene products. The amino acid sequence of the matrix protein (MA), amino acid positions 24 through 29, is phe-lys-lys-arg-ala-gly.

Figure 2 is a circular map of pMMP-IC-nLacZ-lN (a transfer vector), which encodes the β-galactosidase (LacZ) gene product.

Figures 3A through 3B depict the nucleotide sequence (SEQ ID NO:l) of pHDM-tatlb, a high copy expression plasmid encoding HIV tat. Figure 3C is a circular map of pHDM-tatlb.

Figures 4A through 4C depict the nucleotide sequence (SEQ ID NO:2) of pRC/CMV-revlb, an expression plasmid encoding HIV rev. Figure 4D is a circular map of pRC/CMV-revlb. Figures 5A through 51 depict the nucleotide sequence (SEQ ID NO:3) of pHR'CMVLacZSINTrip, a pHR'CMVLacZ derivative comprising a self inactivating element (SIN) and a central ppt fragment (Trip).

Figure 5 is a circular map of pHR'CMVLacZSINTrip. Figures 6 A through 6D depict the nucleotide sequence (SEQ ID NO:4) of pHDM-SNVgpM7, which comprises a nucleic acid sequence encoding a mutant SNV gag-pol containing a mutant matrix protein whose amino acid sequence at positions 24 through 29 is phe-lys-lys-arg-tyr-lys, cloned into pHDM. Figure 6E is a circular map of pHDM-SNVgpM7. Figures 7 A through 7D depict the nucleotide sequence (SEQ ID NO: 5) of pHDM-SNVgpM8, which comprises a nucleic acid sequence encoding a mutant SNV gag-pol containing mutant matrix protein whose amino acid sequence at positions 24 through 29 is gly-lys-lys-lys-tyr-lys (HIV nuclear localization sequence), cloned into pHDM.

Figure 7E is a circular map of pHDM-SNVgpM8. Figures 8A through 8N depict the annotated nucleotide sequence (SEQ ID

NO: 7) and the deduced amino acid sequence (SEQ ID NO: 8) for SNV gag-pol.

Figure 9 is a table which depict the results of the experiment described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Applicants' surprising discovery that retroviral vector particles encoding wild-type spleen necrosis virus (SNV) gag-pol gene products can infect quiescent cells. By "quiescent cells" is meant non-dividing cells, resting cells or non-proliferating cells. The present invention also relates to Applicants' unexpected discovery that retroviral vector particles encoding wild-type SNV gag-pol gene products do not require a nuclear localization sequence in the matrix protein to transduce into quiescent cells.

As a result, Applicants' invention relates to novel packaging cell lines useful for generating retroviral vector particles capable of infecting and transducing a DNA sequence of interest into quiescent cells, to construction of such cell lines and to methods of using the particles to introduce DNA of interest into quiescent cells (e.g., animal cells (particularly mammalian cells). The retroviral vector particles of the invention comprise retroviral sequences encoding wild-type SNV gag-pol proteins. As described herein, integration of provirus is not required for gene transfer and expression of the DNA of interest in the quiescent cells. In a particular embodiment, the invention relates to novel packaging cell lines useful for generating retroviral vector particles that comprise a MLV-derived retroviral genome and are capable of infecting quiescent cells, to construction of such cell lines and to methods of using the retroviral vector particles comprising a MLV-derived retroviral genome to introduce DNA of interest into quiescent cells (e.g., animal cells (particularly mammalian cells). In another embodiment, the invention relates to novel packaging cell lines useful for generating retroviral vector particles that comprise a SNV-derived retroviral genome and are capable of infecting quiescent cells, to construction of such cell lines and to methods of using the retroviral vector particles comprising a SNV-derived retroviral genome to introduce DNA of interest into quiescent cells. In a particular embodiment, the packaging cell lines of the present invention are stable packaging cell lines.

By "capable of infecting quiescent cells" is meant to include retroviral particles which can infect quiescent cells.

The viral particles and vector particles of the invention, as described herein, are capable of transducing a DNA sequence of interest (e.g., gene) into quiescent cells. By "capable of transducing a DNA sequence of interest into quiescent cells" is meant to include particles which can transduce a DNA sequence of interest (e.g., a gene) into quiescent cells. Thus, the invention also relates to methods for transducing a DNA sequence of interest (e.g., gene) into quiescent cells using a retroviral vector particle encoding wild type SNV gag-pol proteins, as described herein.

Packaging cell lines for producing retroviral particles capable of infecting quiescent cells comprise (a) a cell (e.g., mammalian cell); (b) a first retroviral nucleotide sequence in the cell which comprises a coding sequence for wild-type SNV gag-pol; (c) a second retroviral nucleotide sequence in the cell which comprises the coding sequence for a heterologous envelope protein; and (d) a third retroviral nucleotide sequence in the cell which comprises a DNA sequence of interest. Sequences required for packaging and reverse transcription are generally included in the third retroviral nucleotide sequence. Optionally, sequences required for integration can be included in the third retroviral nucleotide sequence.

Sequences required for packaging and reverse transcription can be derived from MLV, SNV or other viruses. Sequences required for integration can be derived from MLV, SNV or other viruses. The retroviral sequences can be contained in one or more plasmids. A plasmid including retroviral sequences encoding gag-pol proteins is also referred to herein as a helper vector. A plasmid including retroviral sequences encoding sequences required for packaging and reverse transcription is also referred to herein as a transfer vector.

A retroviral nucleotide sequence or plasmid comprising a DNA sequence which encodes wild-type SNV gag-pol proteins includes a promoter which drives the expression of the gag-pol proteins. An example of such a promoter is the human cytomegalovirus (hCMV) immediate early promoter. Other suitable promoters are well known in the art. A plasmid comprising DNA sequence which encodes wild- type SNV gag-pol proteins is depicted in Figure 1 (plasmid pHDM-SNVgp).

The retroviral envelope protein interacts with a specific cellular protein to determine host cell range. The retroviral envelope protein can be altered to enable the transduction of target cells of interest. Methods of generating retroviral vectors capable of specifically infecting target cell types are well known in the art (see, e.g., Engelstadter et al., Human Gene Therapy 11:293-303 (2000), and references cited therein). A heterologous envelope protein can be any appropriate envelope protein, including the G glycoprotein of vesicular stomatitis virus (VSV G) and the amphotropic envelope of the Moloney leukemia virus (MLV). Other heterologous envelope proteins are generally known in the art. A heterologous envelope protein permits pseudotyping of particles generated by the packaging construct. Retroviral vectors of the invention can be engineered to include an envelope protein that allows the vectors to infect human cells (Engelstadter et al., Human Gene Therapy 11 :293- 303 (2000)).

As used herein, the term "animal" includes mammals, as well as other animals, vertebrate and invertebrate (e.g., birds, fish, reptiles, insects (e.g., Drosophila species)). The terms "mammal" and "mammalian", as used herein, refer to any vertebrate animal, including monotremes, marsupials and placental, that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals). Examples of mammalian species include humans and other primates (e.g., monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs) and ruminents (e.g., cows, pigs, horses). Examples of mammalian cells include human (such as U2OS cells, HeLa cells, 293T cells, NTH 3T3 cells), bovine, ovine, porcine, murine (such as embryonic stem cells), rabbit and monkey (such as COS1 cells) cells. The cell may be an embryonic cell, bone marrow stem cell or other progenitor cell. Where the cell is a somatic cell, the cell can be, for example, an epithelial cell, fibroblast, smooth muscle cell, blood cell (including a hematopoietic cell, red blood cell, T-cell, B-cell, etc.), hepatocytes, myofϊbers, tumor cell, cardiac muscle cell, macrophage, dendritic cell, neuronal cell (e.g., a neuron, a glial cell or astrocyte), or pathogen-infected cell (e.g., those infected by bacteria, viruses, virusoids, parasites, or prions). Quiescent cells are non-dividing, non-proliferating or resting cells.

Quiescent cells may be naturally non-dividing (including hepatocytes, myofϊbers, hematopoietic stem cells, neurons, macrophages) or can be artificially induced to be non-dividing with reagents generally known in the art. For example, U2OS cells can be induced to become non-dividing cells upon treatment with amphidicolin. The cells can be obtained commercially or from a depository or obtained directly from an animal, such as by biopsy. Alternatively, the cell need not be isolated at all from the animal where, for example, it is desirable to deliver the virus to the animal in gene therapy.

To produce the cell lines of the present invention for producing retroviral vector particles capable of infecting quiescent cells, mammalian host cells are co- transfected with (a) a first plasmid comprising a DNA sequence which encodes wild- type SNV gag-pol proteins; (2) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and (3) a third plasmid comprising a DNA sequence of interest, under conditions appropriate for transfection of the cells. Conditions appropriate for transfection of cells are well known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1998)). Sequences required for packaging and reverse transcription are generally included in the third plasmid. Optionally, sequences required for integration can be included in the third plasmid. Sequences required for packaging and reverse transcription can be derived from MLV, SNV or other viruses.

Sequences required for integration can be derived from MLV, SNV or other viruses. Virus stocks consisting of retroviral vector particles capable of infecting quiescent cells of the present invention are produced by maintaining the transfected cells under conditions suitable for virus production (e.g., in an appropriate growth media and for an appropriate period of time). Such conditions, which are not critical to the invention, are generally known in the art. See, e.g., Sambrook et al,

Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor University Press, New York (1989); Ausubel et ah, Current Protocols in Molecular Biology, John Wiley & Sons, New York (1998); U.S. Patent No. 5,449,614; and U.S. Patent No. 5,460,959, the teachings of which are incorporated herein by reference.

To generate retroviral vector particles capable of infecting quiescent cells, mammalian host cells can be co-transfected with (a) a first plasmid comprising a DNA sequence which encodes wild-type SNV gag-pol proteins; (b) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and (c) a third plasmid comprising a DNA sequence of interest. Sequences required for packaging and reverse transcription are generally included in the third plasmid. Optionally, sequences required for integration can be included in the third plasmid.

DNA sequence of interest, as used herein, includes all or a portion of a gene or genes encoding a nucleic acid product whose expression in a cell or a mammal is desired. In a particular embodiment, the nucleic acid product is a heterologous therapeutic protein. A heterologous therapeutic protein, as used herein, refers to a protein which alleviates or attenuates a clinical condition, malady, pain or symptom of a patient. The duration of the therapeutic benefit to the patient can be temporary or more preferably, long term.

Examples of therapeutic proteins include antigens or immunogens, such as a polyvalent vaccine, cytokines, tumor necrosis factor, interferons, interleukins, adenosine deaminase, insulin, T-cell receptors, soluble CD4, growth factors, such as epidermal growth factor, human growth factor, insulin-like growth factors, fibroblast growth factors), blood factors, such as Factor VHL Factor LX, cytochrome b, glucocerebrosidase, ApoE, ApoC, ApoAL the LDL receptor, negative selection markers or "suicide proteins", such as thymidine kinase (including the HSV, CMV, VZV TK), anti-angiogenic factors, Fc receptors, plasminogen activators, such as t- PA, u-PA and streptokinase, dopamine, MHC, tumor suppressor genes such as p53 and Rb, monoclonal antibodies or antigen binding fragments thereof, drug resistance genes, ion channels, such as a calcium channel or a potassium channel, adrenergic receptors, hormones (including growth hormones) and anti-cancer agents.

In another embodiment, the nucleic acid product is a gene product to be expressed in a cell or a mammal and which product is otherwise defective or absent in the cell or mammal. For example, the nucleic acid product can be a functional gene(s) which is defective or absent in the cell or mammal. Alternatively, the nucleic acid product can regulate a defective, hyperactive, hypoactive or overexpressed gene or gene product in the cell or mammal.

DNA sequence of interest includes DNA sequences (control sequences) which are necessary to drive the expression of the gene or genes. The control sequences are operably linked to the gene. The term "operably linked", as used herein, is defined to mean that the gene is linked to control sequences in a manner which allows expression of the gene (or the nucleic acid sequence). Generally, operably linked means contiguous.

Control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites and sequences which control termination of transcription and translation. In a particular embodiment, a recombinant gene encoding a desired nucleic acid product can be placed under the regulatory control of a promoter which can be induced or repressed, thereby offering a greater degree of control with respect to the level of the product produced.

As used herein, the term "promoter" refers to a sequence of DNA, usually upstream (5') of the coding region of a structural gene, which controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors which may be required for initiation of transcription. Suitable promoters are well known in the art. Exemplary promoters include the SV40, CMV and human elongation factor (EFI) promoters. Other suitable promoters are readily available in the art (see, e.g., Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1998); Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor University Press, New York (1989); and U.S. Patent No. 5,681,735). A DNA sequence of interest can be isolated from nature, modified from native sequences or manufactured de novo, as described in, for example, Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York (1998); and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor University Press, New York. (1989). DNA sequences can be isolated and fused together by methods known in the art, such as exploiting and manufacturing compatible cloning or restriction sites.

The packaging cell lines and viral particles of the present invention can be used, in vitro, in vivo and ex vivo, to introduce DNA of interest into a eukaryotic cell (e.g., a mammalian cell) or a mammal (e.g., a human or other mammal or vertebrate). The cells can be obtained commercially or from a depository or obtained directly from a mammal, such as by biopsy. The cells can be obtained from a mammal to whom they will be returned or from another/different mammal of the same or different species. For example, using the packaging cell lines or viral particles of the present invention, DNA of interest can be introduced into nonhuman cells, such as pig cells, which are then introduced into a human. Alternatively, the cell need not be isolated from the mammal where, for example, it is desirable to deliver vial particles of the present invention to the mammal in gene therapy.

Ex vivo therapy has been described, for example, in Kasid et al, Proc. Natl Acad. Sci. USA, 87:473 (1990); Rosenberg et al, N. Engl J. Med., 323:570 (1990); Williams et al, Nature, 310:476 (1984); Dick et al, Cell, 42:71 (1985); Keller et al, Nature, 3 5:149 (1985); and Anderson et al, United States Patent No. 5,399,346.

Methods for administering (introducing) viral particles directly to a mammal are generally known to those practiced in the art. For example, modes of administration include parenteral, injection, mucosal, systemic, implant, intraperitoneal, oral, intradermal, transdermal (e.g., in slow release polymers), intramuscular, intravenous including infusion and/or bolus injection, subcutaneous, topical, epidural, etc. Viral particles of the present invention can, preferably, be administered in a pharmaceutically acceptable carrier, such as saline, sterile water, Ringer's solution, and isotonic sodium chloride solution. The dosage of a viral particle of the present invention administered to a mammal, including frequency of administration, will vary depending upon a variety of factors, including mode and route of administration; size, age, sex, health, body weight and diet of the recipient mammal; nature and extent of symptoms of the disease or disorder being treated; kind of concurrent treatment, frequency of treatment, and the effect desired.

The present invention will now be illustrated by the following Example, which is not intended to be limiting in any way.

EXAMPLES

EXAMPLE 1 Plasmids. pHDM-SNVgp comprises a nucleic acid sequence encoding wild-type SNV gag-pol cloned into pHDM (Figure 1). pMMP-IC-nLacZ-lN is a transfer vector encoding β-galactosidase (Figure 2). pHDM-tatlb is a high copy expression plasmid encoding HIV tat (Figures 3A-3C). pRC/CMV-revlb is an expression plasmid encoding HIV rev (Figures 4A-4D). pHR'CMVLacZSINTrip is a pHR'CMVLacZ derivative comprising a self inactivating element (SIN) and a central ppt fragment (Trip) (Figures 5A-5J). pHDM-SNVgpM7 comprises a nucleic acid sequence encoding a mutant

SNV gag-pol containing a mutant matrix protein whose amino acid sequence at positions 24 through 29 is phe-lys-lys-arg-tyr-lys, cloned into pHDM (Figures 6A-6E). pHDM-SNVgpM8 comprises a nucleic acid sequence encoding a mutant SNV gag-pol containing mutant matrix protein whose amino acid sequence at positions 24 through 29 is gly-lys-lys-lys-tyr-lys (HJV nuclear localization sequence), cloned into pHDM (Figures 7A-7E). pHDM is a high copy plasmid comprising multiple cloning sites, a CMV IE gene promoter, the 2^nd and 3^rd exon and 2^nd intron of the human b-globin and a poly A signal (International Publication No. WO 00/15819). pHDM-G comprises a nucleic acid sequence encoding a VSV G envelope protein cloned into pHDM (International Publication No. WO 00/15819; and Naldini et al., Science 272:263-267 (1996)). pHDM-Hgpm2 is a high copy expression plasmid that encodes codon optimized HIV gag-pol (International Publication No. WO 00/15819). pRC/CMV is a high copy plasmid comprising a CMV promoter (Invitrogen, Inc.). pCMVdR8.2 comprises most of the HTV genome, a CMV IE gene promoter and an inactivated Env gene (International Publication No. WO 00/15819). pHR'CMVLacZ is a high copy plasmid comprising the HIV LTR, a packaging signal, the rev response element (RRE) and an internal CMV promoter (Naldini et al, Science 272:263-267 (1996)).

EXAMPLE 2 Production of recombinant retroviruses by calcium phosphate transfection.

1. Split 293T cells 20-24 hours before transfection.

Plate 2 x 10⁶ cells in 4 ml of 293 media in 6 cm plates. or 4.0 x 10⁶ cells in 10 ml of 293 media in 10 cm plates. (The optimal number of cells to be plated depends on how fast the cells grow. The goal is to generate a cell density (e.g. 30%) at the time of transfection which yields many cells capable of producing viruses.) 2. Calcium phosphate transfection:

- Change medium to LMDM (GibcoBRL cat#12440-053, containing L- glutamine and 25 M Hepes buffer) + 10% heat-activated fetal calf serum + Pen Strep about three hours before transfection. For a 6 cm plate, a typical transfection mix contains:

- DNA/Calcium mix: 6 μg pMD.MLV gag.pol (helper)

8 μg pMMP-nLacZ (transfer) 2 μg pMD.G (VSVG pseudotype) 31 μl 2M CaCl₂ add dH₂O to 250 μl

- Add 250 μl 2x HBS to the DNA/Calcium mix slowly. Flick the tube several times to mix well. Let the tubes sit at room temperature for about 20 minutes. Slowly add the solution to the cells. Do not change the media of the cells before adding the transfection solution. * for 10 cm plates, increase the amount of the plasmids by 2.5 fold and use

62 μl 2M CaCl₂ and 500 μl 2x HBS pH 7.12.

-12-16 hours after transfection, replace the media with 293 media with HEPES.

- 40-48 hours after transfection, harvest the supernatant. Pass the supernatant through 0.45 μm membrane. The supernatant can be directly frozen and stored at -80° C for an extended time.

3. Titering by infection on NIH 3 T3 :

1) Split NIH 3T3 cells the day before infection. Plate 8 x 10⁴ cells/2ml 3T3 media/well in 12- well plates, 10⁵ cells/3 ml 3T3 media/well in 6-well plates, 3 x 10⁵ cells/4 ml 3T3 media/6 cm plate or 10⁶ cells/10 ml 3T3 media/10 cm plate.

2) The virus titer generated is normally in the range of 10⁷/ml. Do proper dilution to obtain an appropriate level of infection. The percentage of infection is preferably between 5 to 40%. Too few infection events may create more variation for the result as one samples a number of high power fields to count lacZ staining cells. Too many infection events will result in multiple copies per cells. The supernatants are usually diluted in 293 media with HEPES and supplemented with polybrene at the final concentration of 8 μg/ml. To cover the cells during the time of infection, use: 0.5 ml supernatant/well in 12-well plate

1 ml supernatant/well in 6-well plate

2 ml supernatant/6 cm plate 4 ml supernatant/10 cm plate

Incubate the cells for 4-6 hours and replace the supernatant with 3T3 media. Culture the cells for another 2 days before X-Gal staining.

Notes:

(1) 293 cells are human embryonic kidney epithelial cells transformed with adenovirus El A/EIB oncoproteins. 293T cells are 293 cells further transfected with SV40 large T antigen and a G418 selection plasmid. 293T cells are known to be highly transfectable.

(2) 293 media: DME + 10% fetal calf serum (heat-activated) + 4 mM L-Glutamine + Pen/Strep (penicillin/streptomycin).

(3) 2 x HBS (HEPES buffered saline):

281 mM NaCl 100 mM HEPES

1.5 mM Na₂HPO₄ Adjust the pH to 7.12 with 0.5 N NaOH. Sterilize the solution by passing through a 0.22 μm filter.

Results Retroviral vectors were evaluated for the ability to produce retroviral particles capable of infecting quiescent cells. A series of experiments were conducted to examine whether mutations in the matrix (MA) protein coding sequences of the spleen necrosis virus (SNV) are required to generate retroviral vectors capable of transducing quiescent cells. A three plasmid system was used to generate virus. Briefly, 293T cells were transfected with helper, transfer and heterologous envelope retroviral vectors. Viral particles were harvested, titered oh quiescent cells (e.g. cell-cycle arrested human U2OS cells, hepatocytes, primary macrophages or primary rat neurons) and infection of quiescent cells was measured by assaying for β-galactosidase activity via staining with 5-bromo-4-chloro- 3-indolyl-β-D-galactopyranoside (X-gal) by standard protocol.

Retroviral helper constructs tested encoded wild type SNV gag-pol (pHDM-SNVgp (Figure 1)) and two MA mutants (ρHDM-SNVgρM7 (Figures 6A-6D) and pHDM-SNVgpM8 (Figures 7A-7E)). ρHDM-SNVgpM7 encodes a mutant MA whose amino acid sequence at positions 24 through 29 is phe-lys-lys- arg-tyr-lys. pHDM-SNVgpM8 encodes a mutant MA whose amino acid sequence at positions 24 through 29 is gly-lys-lys-lys-tyr-lys. Both MA mutants of SNV gag-pol provide nuclear localization signal sequences (NLS) similar to that found in the HLV MA.

Results obtained with various retroviral vectors are provided in Figure 9. The results show that retroviral vector particles encoding wild type (see plate 7) or the M7 MA mutant (see plate 8) infected non-dividing U2OS cells. Surprisingly, virus encoding the wild type SNV gag-pol was found to transduce nearly 100% of the resting cells, while the negative control MLV virus typically showed transduction efficiency of no more than 3%. The MA mutants failed to show any appreciable advantage over the wild type SNV gag-pol in the ability to transduce resting cells, h fact, one of the mutants, M8, appeared to be non-viable. Similar results were obtained in hepatocytes, primary macrophages and primary rat neurons. That is, virus packaged by the wild type SNV gag-pol was found to transduce hepatocytes, primary macrophages and primary rat neurons. Thus, wild type SNV gag-pol derived virus can transduce diverse types of quiescent cells.

Lentiviral vector particles were used as positive controls since they can infect both proliferating and non-proliferating cells (see Figure 9, plate 20 and 22).

To investigate the extent of viral integration the SNV integrase was mutated (inactivated). X-gal staining on SNV vector infected primary macrophages was very intense. Unexpectedly, there was no significant difference in the amount of X-gal staining between cells transduced with a retroviral particle encoding the wild type SNV gag-pol and its integrase mutant. Since the level of gene expression observed in aphidicolin treated human U2OS cells transduced with wild type SNV gag-pol and with vectors packaged by a wild type SNV gag-pol harboring a specific integrase mutation were comparable, gene expression may not be directed from integrated provirus. This surprising finding suggests that SNV gag-pol may possess unique nuclear transport capability to move the preintegration complex into the nucleus of the infected resting cells, allowing gene expression to occur. The preliminary results of these experiments suggest that further experiments may be necessary to optimize the process of integration, hi addition, experiments in other systems, such as rat neuronal cultures, can be conducted with a longer time frame to examine the physical conformation of pro viral DNA products at various time points after infection in both proliferating and resting cells. Experiments directed at utilizing different viral integrase genes to increase viral integration and expression a gene of interest can also be performed.

EXAMPLE 3 In vivo retroviral rat brain injection - Infection of neurons in vivo. The viruses used were pMMP-IC-GFP-Ws (pHDM-SNVgp) (8.4xl0⁷pfu ml), pMMP-IC-GFP-Ws (pHDM-MLVgp) (5.1xl0⁷pfu/ml) and HRST- cGFP (a lentiyirus control derived from pHR'CMV-lacZ) (1.7xl0⁹pfu/ml). The retroviral vector particles were generated as described above. To facilitate the identification of cell transduction, the retroviruses also encode a GFP reporter. Two Male Sprague-Dawly rats (225-250 grams) were used for each virus per time point of sacrifice (at injection, 2 weeks post-injection and 8 weeks post- injection). Animals were anesthetized by injection intraperitoneally with 14 mg/kg

Xylazene and 80 mg/kg Keratimine in PBS.

Animals were prepared for direct injection by fixing the skull in Kopf sterotactic frame and drilling bilateral burr holes into the left hemisphere (striatal injection) and right hemisphere (hippocampal injection) of the skull. Four μl of viral suspension (5 xl0⁷pfu/ml) was injected into the dura mater of the brain at coordinates indicated below with a SGE Gastight syringe (10 μl). As an additional control, two animals were injected with the lentivirus control at 1.7xl0⁹pfu/ml (~50x).

Striatal injection coordinates used were +0.2 mm antero-posterior, 3.5 mm medio-lateral and -4.5 dorso-ventral. Hippocampal injection coordinates used were -3.5 antero-posterior, 3.0 mm mediolateral and -4.0 mm dorso-ventral.

Following injections, burr holes were sealed with dental cement. Animals were sacrificed 2 weeks and 8 weeks post-injection after Xylazene/Ketamine anesthesia (intraperitoneal injection with 14 mg/kg Xylazene and 80 mg/kg Keratimine in PBS), followed by perfusion with PBS and then injection with 4% paraformaldehyde. Subsequently, brains were removed, post-fixed in 4% paraformaldehyde for 24 hours at 4°C and transferred into 20% sucrose overnight for cryoprotection. Brains were embedded in TissueTek O.C.T and kept at -20°C until sectioning. Histological sections were prepared and GFP activity visualized under ultraviolet illumination by standard protocol. After immunofluorescent staining, red color indicates neurons, stained with antibody against the neuronal-specific nuclear protein (NeuN). Green color indicates green fluorescent protein (GFP) expression in the cytoplasm of one of the neurons in this picture. This co-localization of GFP and NeuN, two weeks and eight weeks after in vivo SNV infection, shows that the resting cells can be transduced in vivo with SNV vectors. The results indicated that direct injection of retroviruses encoding wild type SNV gag-pol proteins into the rat brain can transduce rat brain cells in vivo significantly better than the MLV derived control viruses.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of producing a retroviral vector particle capable of infecting quiescent cells comprising co-transfecting mammalian host cells with: a) a first plasmid comprising a DNA sequence which encodes wild-type

SNV gag-pol; b) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and c) a third plasmid comprising a DNA sequence of interest and sequences required for packaging and reverse transcription.

2. The method of Claim 1 wherein the heterologous envelope protein is the G glycoprotein of vesicular stomatitis virus (VSV G) or the amphotropic envelope of the Moloney leukemia virus.

3. The method of Claim 1 wherein the sequences required for packaging and reverse transcription are derived from MLV or SNV.

4. The method of Claim 1 wherein the DNA sequence of interest is a heterologous therapeutic protein.

5. The method of Claim 1 wherein the first plasmid is pHDM-SNVgp.

6. A packaging cell line for producing a retroviral vector particle capable of infecting quiescent cells comprising: a) a mammalian cell; b) a first retroviral nucleotide sequence in the cell which comprises a coding sequence for wild-type SNV gag-pol; c) a second retroviral nucleotide sequence in the cell which comprises the coding sequence for a heterologous envelope protein; and d) a third retroviral nucleotide sequence in the cell which comprises a DNA sequence of interest and sequences required for packaging and reverse transcription.

7. The packaging cell line of Claim 6 wherein the heterologous envelope protein is the G glycoprotein of vesicular stomatitis virus (VSV G) or the amphotropic envelope of the Moloney leukemia virus.

8. The method of Claim 6 wherein the sequences required for packaging and reverse transcription are derived from MLV or SNV.

9. The packaging cell line of Claim 6 wherein the DNA sequence encodes a heterologous therapeutic protein.

10. A method of producing a packaging cell line which produces a retroviral vector particle capable of infecting quiescent cells comprising co-transfecting mammalian host cells with: a) a first plasmid comprising a DNA sequence which encodes wild-type SNV gag-pol; b) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and c) a third plasmid comprising a DNA sequence of interest and sequences required for packaging and reverse transcription.

11. The method of Claim 10 wherein the heterologous envelope protein is the G glycoprotein of vesicular stomatitis virus (VSV G) or the amphotropic envelope of the Moloney leukemia virus.

12. The method of Claim 10 wherein the sequences required for packaging and reverse transcription are derived from MLV or SNV.

13. The method of Claim 10 wherein the DNA sequence encodes a heterologous therapeutic protein.

14. A method of producing a retroviral vector particle capable of infecting quiescent cells comprising the steps of: a) co-transfecting mammalian host cells with: i) a first plasmid comprising a DNA sequence which encodes wild-type SNV gag-pol; ii) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and iii) a third plasmid comprising a DNA sequence of interest and . sequences required for packaging and reverse transcription; b) maintaining the transfected cells under conditions suitable for virus particle production; and c) recovering virus particles produced in step b).

15. The method of Claim 14 wherein the heterologous envelope protein is the G glycoprotein of vesicular stomatitis virus (VSV G) or the amphotropic envelope of the Moloney leukemia virus.

16. The method of Claim 14 wherein the sequences required for packaging and reverse transcription are derived from MLV or SNV.

17. The method of Claim 14 wherein the DNA sequence encodes a heterologous therapeutic protein.

18. A method of producing a retroviral vector particle capable of infecting quiescent cells comprising co-transfecting mammalian host cells with: a) pHDM-SNVgp; b) pMMP-IC-nLacZ-lN; and c) pHDM-G.

19. A method of gene transfer to quiescent cells comprising infecting said quiescent cells with a retroviral vector particle encoding a wild-type SNV gag-pol gene product.

20. A method of producing a retroviral vector particle that comprises a MLV- derived retroviral genome and is capable of infecting quiescent cells, comprising the steps of: a) co-transfecting mammalian host cells with: i) a first plasmid comprising a DNA sequence which encodes wild-type SNV gag-pol; ii) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and iii) a third plasmid comprising a DNA sequence of interest and

MLV-derived sequences required for packaging and reverse transcription; b) maintaining the transfected cells under conditions suitable for virus particle production; and c) recovering virus particles produced in step b).

21. A packaging cell line for producing a retroviral vector particle that comprises a MLV-derived retroviral genome and is capable of infecting quiescent cells, comprising: a) a mammalian cell; b) a first retroviral nucleotide sequence in the cell which comprises a coding sequence for wild-type SNV gag-pol; c) a second retroviral nucleotide sequence in the cell which comprises the coding sequence for a heterologous envelope protein; and d) a third retroviral nucleotide sequence in the cell which comprises a DNA sequence of interest and MLV-derived sequences required for packaging and reverse transcription.

22. A method of producing a packaging cell line which produces a retroviral vector particle that comprises a MLV-derived retroviral genome and is capable of infecting quiescent cells, comprising co-transfecting mammalian host cells with: a) a first plasmid comprising a DNA sequence which encodes wild-type SNV gag-pol; b) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and c) a third plasmid comprising a DNA sequence of interest and MLV- derived sequences required for packaging and reverse transcription.

23. A method of producing a retroviral vector particle that comprises a SNV- derived retroviral genome and is capable of infecting quiescent cells, comprising the steps of: a) co-transfecting mammalian host cells with: i) a first plasmid comprising a DNA sequence which encodes wild-type SNV gag-pol; ii) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and iii) a third plasmid comprising a DNA sequence of interest and SNV-derived sequences required for packaging and reverse transcription; b) maintaining the transfected cells under conditions suitable for virus particle production; and c) recovering virus particles produced in step b).

24. A packaging cell line for producing a retroviral vector particle that comprises a SNV-derived retroviral genome and is capable of infecting quiescent cells, comprising: a) a mammalian cell; b) a first retroviral nucleotide sequence in the cell which comprises a coding sequence for wild-type SNV gag-pol; c) a second retroviral nucleotide sequence in the cell which comprises the coding sequence for a heterologous envelope protein; and d) a third retroviral nucleotide sequence in the cell which comprises a DNA sequence of interest and SNV-derived sequences required for packaging and reverse transcription.

25. A method of producing a packaging cell line which produces a retroviral vector particle that comprises a SNV-derived retroviral genome and is capable of infecting quiescent cells, comprising co-transfecting mammalian host cells with: a) a first plasmid comprising a DNA sequence which encodes wild-type SNV gag-pol; b) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and c) a third plasmid comprising a DNA sequence of interest and SNV- derived sequences required for packaging and reverse transcription.