WO2001094560A2

WO2001094560A2 - In vitro assembly of sv40 viruses and pseudoviruses

Info

Publication number: WO2001094560A2
Application number: PCT/IL2001/000520
Authority: WO
Inventors: Ariella Oppenheim; Ariela Gordon-Shaag
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem; Hadasit Medical Research Services & Development Ltd.
Priority date: 2000-06-06
Filing date: 2001-06-06
Publication date: 2001-12-13
Also published as: AU2001274444A1; WO2001094560A3; IL136610A0

Abstract

The present invention relates to a method for the improved in vitro assembly of pseudoviruses. These viruses or pseudoviruses comprise at least one pure or semi-purified SV40 capsid protein and at least one exogenous constituent selected from nucleic acids, proteins and peptides. In the method of the invention at least one semi-purified or pure SV40 capsid protein is brought into contact with at least one exogenous constituent in the presence of semi-purified or pure PARP [Poly (ADP-ribose) polymerase]. The invention further relates to use of pseudoviruses assembled by the method of the invention, in the preparation of a pharmaceutical composition for the treatment of an individual suffering from pathological disorder.

Description

O 01/94560

In Vitro Assembly of SV40 Viruses and Pseudoviruses

Field of the Invention

The invention relates to an improved method for in vitro assembly of pseudoviruses by using SV40 capsid proteins and exogenous nucleic acid or exogenous protein or peptide in the presence of PARP [Poly (ADP-ribose) polymerase]. More particularly, such method is used for in vitro construction of pseudoviruses comprising exogenous nucleic acid or exogenous protein or peptide which have improved assembly and therefore improved infectivity and which are particularly suitable for use in gene therapy.

Background of the Invention

SN40 is a simian papovirus, with a small double-stranded circular DΝA genome of 5.2kb [reviewed in Tooze, J. (1981) DΝA Tumor Viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York].

The icosahedral capsid of SN40, surrounding the viral mini-chromosome, is composed of three viral-coded proteins, VP1, VP2, and VP3. Recent X-ra crystallographic studies on SV40 structure at 3.8A resolution [Liddington, R., et al. (1991) Nature 354:278-282] revealed that the outer shell of the virion particle is composed exclusively of VP1 pentamers. The VP2 and VP3 appear to bridge between the VP1 outer shell and the chromatin core. The VP1 pentamers have identical conformations, except for the carboxy-terminal arms, which tie them together. Five arms extend from each pentamer and insert into the neighboring pentamers in three distinct kinds of interactions. It appears that this construction facihtates the use of identical building blocks in the formation of a structure that is sufficiently flexible as required for the variability in packing geometry [Liddington et al. (1991) ibid.}. 01/94560

Another protein encoded by the late regions of SV40 (which also encoded the three capsid proteins VPl, VP2 and VP3) is the agnoprotein, also called LP1. This is protein a small, 61 amino acid protein. Although the agnoprotein was not found in the viral capsid, it is thought to expedite viral assembly in vivo [Resnick, J & Shenk, T. (1986) J. Nirol. 60:1098-1106; Νg, S.C., et al. (1985) J. Biol. Chem. 260:1127-1132; Carswell, S. & Alwine, J.C. (1986) J. Virol. 60:1055-1061].

Assembly occurs in the nucleus, probably by the gradual addition and organization of the capsid protein around the viral minichromosome. [Garber, E.A., et al. (1980) Virology 107:389-401; Bina, M. (1986) Comments Mol. Cell Biophys. 4:55]. However, the three capsid proteins VPl, VP2 and VP3 bind to DΝA non-specifically [Soussi, T. (1986) J. Virol. 59:740-742; Clever, J., et al. (1993) J. Biol. Chem. 268:20877-20883]. This presents the dilemma of how they find the SV40 minichromosome in the nucleus in a large excess of cellular DΝA. The packaging of SV40 using pseudovirions, in which most of the viral DΝA is replaced by other sequences has been investigated by the inventors - [Oppenheim, A., et al. (1986) Proc. Νatl. Acad. Sci. USA 83:6925-6929]. The pseudoviral particles are .prepared by encapsidating plasmids that ' carry- the SV40 origin of replication (ori) and the packaging signal (ses) [Oppenheim, A., et al. (1992) J. Virol. 66:5320-5328].

The inventors have recently demonstrated that the cellular transcription factor Spl recruits the cap.sid proteins to the SV40 packaging signal, ses. Thus, the packaging signal acts as a recognition signal and a nucleation center for capsid formation [Gordon-Shaag et al. (1998) J. Mol Bio. 275(2): 187-195].

SV40 capsids have a unique packing geometry in which the C-terminal arm of each VPl molecule is intercalated into a neighboring pentamer in a very precise and specific manner [Liddington, et al. (1991) ibid.]. As 01/94560

suggested by Liddington, et. al., the formation of such complex and delicate structures most likely requires the participation of chaperones. The possible interaction of VPl with such chaperones has been investigated by using the major capsid protein VPl, immobilized on resin, as "bait" and testing cellular extracts for such chaperones.

The present invention is based on the surprising finding that instead of chaperones, the nuclear enzyme PARP was specifically "fished" out of cellular extracts by the immobilized VPl. Furthermore, the present invention demonstrates that PARP is involved in SV40 capsid assembly, and improves the efficiency of packaging in vitro.

PARP is an abundant nuclear protein that catalyzes the formation of extensive branched polymers of poly(ADP-ribose) attached to a protein acceptor using NAD+ as a substrate [Jeggo et al. (1998) Curr. Biol. 8(2):49-51]. PARP has several targets which are primarily DNA-binding proteins such as h stones and transcription factors, but is most active, in auto-modification of itself. Because of the high negative charge on ADP-ribose polymers, poly(ADP-ribosylated) proteins lose their affinity for DNA [Oliver et al. ,(1999) Am. J. Hum. Genet. 64(5): 1282-8]. PARP 'has been implicated in functioning in several key cellular processes: repair of DNA damage, recombination, telomer length and chromosome stability, apoptosis, transcription regulation and histone shuttling [Scovassi et al. (1999) Mol. Cell. Biochem. 199(l-2):125-37; Soldatenkov et al. (2000) Int. J. Cancer 90(2):59-67; Ziegler (2000) Eur. J. Biochem. 267(6): 1550-64]. PARP has been reported as participating in retroviral infection by facilitating integration of the provirus into the host genome [Gaken et al. (1996) J. Virol. 70(6):3992-4000]. The role of PARP in retroviral integration is fully consistent with its known properties: binding to DNA strand breaks followed by the activation of its catalytic property, resulting in its auto-ADP-ribosylation and release from DNA strand breaks. PARP was also shown to modify core proteins (V and VII) of adenovirus, both of which are intimately associated with the viral genome acting in its organization inside the virion particle [Dery et al. (1986) Virus Res. 4(4):313- 29]. Participation of PARP in SV40 assembly/infectivity through interaction with VPl, a protein of the virus outer shell, has not been hitherto implicated.

Several functional domains of PARP have been defined and well studied. The DNA-binding domain located at the amino-terminus is composed of two zinc-fingers and is essential for enzymatic activity, as PARP activity is stimulated >500 fold on binding to DNA strand breaks. Adjacent to the DNA-binding domain is the auto-modification domain, which is rich in glutamate residues. The C-terminal end has the poly(ADP-ribosyl) catalytic domain [De Murcia et al. (1994) Mol. Cell. Biochem. 138(l-2):15-24].

Pseudovirions carrying various genes of therapeutic interest are very efficient in DNA transfer into a wide range of cells, including human bone marrow cells, and are therefore potential vectors for gene therapy [Oppenheim et al. (1986) ibid.; Oppenheim A., et al. (1987) Ann. New York Acad. Sci. 511: 418-427; Daliot, N. & Oppenheim,- ^'A- (1989)]. Daliot et al. have shown efficient expression of the human^' β-^'globin gene in human primary erythroid progenitors of β-thalassemia patients using SV40 pseudovirions as a delivery system [Daliot et al. (1999), J. Hematother. and Stem Cell Res. 8:593-599], and efficient expression of the human MORI gene was demonstrated in murine MEL cells as well as in primary human BM cells, using SV40/MDR1 pseudovirions [Rund et al. (1998), Human Gene Therapy 9:649-657].

The major hindrance in beginning to use the SV40 pseudovirions in preliminar}^ experiments in humans is the present need for a viral helper for encapsidation. This results in pseudoviral stocks that contain also wild type SV40. Because of the similarity in properties (shape, size and density) between the pseudovirions and the helper, they cannot be separated by physical means. An ideal way to prepare pseudovirions for therapeutic purposes for human use would be by in vitro packaging. This would provide maximal safety, since all steps of the preparation can be well controlled.

SV40 vectors were recently shown not to induce immune response, permitting repeated administration and in vivo delivery [Srayer, (1999) Seminars in Liver Disease 19:71-81].

One solution by the present inventors is offered in WO 97/17456, which describes constructs capable of infecting mammalian cells, comprising semi-purified or pure SV40 capsid proteins and heterologous constituent such as exogenous DNA, an exogenous RNA, an exogenous protein or peptide product, and antisense RNA, ribozyme RNA or any RNA or DNA which inhibits or prevents the expression of undesired protein/s in a mammalian cell. .Such construct was shown to have the advantageous ability of packaging large DNA fragments, of about 7.5Kb, compared to the in vivo packaged pseudovirions which could accommodate only up to about 5.4Kb of DNA. ^{• ■}

The present invention relates to a method for the assembly of constructs having improved capability of infecting mammalian cells. This improvement is based on the novel finding that PARP increases the infectivity of SV40 pseudovirions comprising capsid proteins and a heterologous constituent.

Summary of the Invention

The present invention relates to an improved method for in vitro assembly of pseudoviruses having increased infectivity. These viruses or pseudoviruses comprise at least one pure or semi-purified SN40 capsid protein and at least one exogenous constituent selected from nucleic acids, proteins and peptides. In the method of the invention at least one semi-purified or pure SN40 capsid protein is brought into contact with at least one said exogenous constituent in the presence of semi-purified or pure PARP [Poly (ADP-ribose) polymerase].

In a preferred embodiment the method of the invention may further comprise the step of purifying the assembled recombinant viruses or pseudoviruses from any non-packaged exogenous constituent.

In a specifically preferred embodiment the method of the invention may further comprise the addition of at least one other SN40 protein, preferably SN40 agnoprotein, to the mixture of the SN40 capsid protein/s and the exogenous constituent.

The exogenous constituent is selected from the group consisting of an

. .exogenous DΝA encoding an exogenous protein or- peptide product, or encoding therapeutic RΝA, or itself a therapeutic product, a vector

-comprising an exogenous DΝA encoding an exogenous protein or peptide

• product, or encoding therapeutic RΝA, or itself a therapeutic product, an exogenous RΝA encoding an exogenous protein or peptide product or itself

. a therapeutic product, a vector comprising an exogenous RΝA encoding an exogenous protein or peptide product or itself a therapeutic product, an exogenous protein or peptide product, antisense RΝA, ribozyme RΝA or any RΝA or DΝA which inhibits or prevents the expression of undesired protein/s in said mammahan cell; and, chimeric RΝA-DΝA oligonucleotide for targeted corrections of mutated genes or for targeted gene modifications.

In a specifically preferred embodiment the method of the invention employs SV40 capsid protein that are semi-purified or pure SV40 VPl, VP2. or VP3, preferably, the VPl. Specifically, the pseudoviruses prepared by the method of the present invention may comprise a mixture of at least two of semi-purified or pure SV40 VPl, VP2, and VP3.

The exogenous nucleic acid may be any one of circular DNA, linear DNA and RNA.

Further, the therapeutic protein or peptide product is at least one of a therapeutic protein or peptide which is not expressed, expressed in abnormally low amount, expressed in defective form or expressed in physiologically abnormal amount in a target cell.

More specifically the therapeutic protein or peptide product is at least one of an enzyme, a receptor, a structural protein, a regulatory protein and a hormone.

In another embodiment, where exogenous nucleic acids are used as the exogenous constituent,- they may be operably attached to SV40-derivee_ ori DNA sequence and optionally operably linked additional DNA sequences encoding one or more regulatory elements sufficient for the expression of the protein they encode in said target cell.

Alternatively, the method of the present invention may make use of an exogenous nucleic acid that is selected from the group consisting of antisense RNA, ribozyme RNA, chimeric RNA-DNA oligonucleotide and RNA and DNA which inhibit or prevent the expression of undesired protein/s in a target cell.

In an alternative embodiment the exogenous constituent is a protein or peptide which is a naturally occurring or recombinant protein or peptide, a chemically modified or peptide, or a synthetic protein and peptide. More specifically, this exogenous protein or peptide product is at least one of therapeutic protein or peptide which is not expressed, expressed in abnormally low amount, expressed in defective form or expressed in physiologically abnormal amount in a target cell.

Particular examples for such therapeutic proteins are β-globin, MDRl and glucocerebrosidase.

In a second aspect, the invention relates to an SV40 virus or pseudovirus prepared by the method of the invention.

Another aspect of the present invention relates to a mammalian cell infected with pseudovirus prepared by the method of the invention. More specifically, an infected human cell of the invention is selected from the group consisting of hemopoietic cells, muscle cells, epithelial cells, endothelial cells, fibroblasts, spleen cells, pancreatic cells, liver cells, tumor cells, cells of the central or peripheral nervous system and germ line ^; cells. The hemopoietic cells are bone marrow cells, peripheral blood cells and cord blood cells.

As a further aspect the invention relates to a method of providing a therapeutic DNA, RNA, antisense RNA, ribozyme RNA, chimeric RNA-DNA oligonucleotide, protein or peptide product to a patient in need of such product by administering to the patient a therapeutically effective amount of the pseudoviruses comprising these therapeutic constituents, prepared by the method of the invention.

A preferred embodiment of this aspect relates to a method of providing a therapeutic DNA, RNA, antisense RNA, ribozyme RNA, chimeric RNA-DNA oligonucleotide protein or peptide product to a patient in need of such product by administering to the patient a therapeutically effective amount of infected cells of the invention. The invention further relates to pharmaceutical compositions comprising as active ingredient a therapeutically effective amount of the pseudoviruses prepared by the method of the invention. Alternatively, such pharmaceutically compositions may comprise as active ingredient a therapeutically effective amount of infected cells of the present invention.

Brief Description of the Figures

Fig. 1 - Binding of cellular proteins to VPl

GST-VP1 was immobilized on agarose-GSH resin and washed several times with PBS. The bound (B) GST- VPl material (lane 3) was incubated with K562 cells nuclear extracts (NE, lane 2). The mixture was centrifuged separating the resin with the immobilized GST- VPl and putative bound proteins (=prot) from pellet (P lane 4) and from the supernatant (S, lane 5).

The fractions were analyzed by 8% SDS-PAGE following TCA precipitation as required and visualized by silver-staining. Molecular weight, marker —

(M) in lane 1. - •

Fig. 2 - The PARP inhibitor 3-AB inhibits SV40 production . ^■ -

Subconfluent CV-1 cells were infected with SV40 (m.o.i. (multiplicity of infection) of 5) and grown in the presence of increasing concentrations (cone.) of 3-AB. After 5 days the virus was harvested. The histogram shows SV40 titer at three 3-AB concentrations.

Fig. 3 - PARP inhibitor does not affect viral DNA replication Subconfluent CV-1 cells were infected with SV40 (m.o.i. of 5) and grown for 3 days in the presence of increasing concentrations of 3-AB. The cells were harvested and SV40 DNA was isolated. Ahquots were analyzed by electrophoresis on 1% agarose and Southern blotting. The left and right sides of the gel show 1/10 and 1/100 ahquots of the total Hirt supernatant, respectively. fo-I and fo-II designate form I or II, respectively. Abbreviations: o (=mock).

Fig. 4 - PARP assists assembly of SV40 pseudovirions in vitro In vitro packaging reaction was performed in the presence (175 ng/re action) or in the absence of PARP. The histogram shows the infectious units (Infec U) counts of four packaging experiments (#l-#4). Control (cont) without PARP.

Fig. 5 — Addition of PARP enhances packaging reaction with VPl only In vitro packaging reaction was performed using the plasmid EGFP-Cl DNA and nuclear extracts containing VPl in the presence (9 ng/reaction) or in the absence of PARP. The histogram shows the infectious units (Infec U) counts of the packaging experiment. As negative control, similar reaction was performed in the presence of DNA only (DNA).

Detailed Description of the Invention

A number of methods of the art of molecular biology are not detailed .herein, as they are well known to the person of skill in the art. Such methods may include site-directed mutagenesis, expression of cDNAs, analysis of recombinant proteins or peptides, transformation of insect cells, transfection of bacterial or mammalian cells, and the like. Textbooks describing such methods are e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory; ISBN: 0879693096, 1989, Current Protocols in Molecular Biology ,by F. M. Ausubel, ISBN: 047150338X, John Wiley & Sons, Inc. 1988, and Short Protocols in Molecular Biology, by F. M. Ausubel et al. (eds.) 3^rd ed. John Wiley & Sons; ISBN: 0471137812, 1995. These publications are incorporated herein in their entiretjf by reference. Furthermore, a number of immunological techniques are not in each instance described herein in detail, as they are well known to the person of skill in the art. See e.g., Current Protocols in Immunology, Coligan et al. (eds), John Wiley & Sons. Inc., New York, NY.

The invention relates to an improved method for the preparation of SV40 pseudovirions with increased infectivity, involving the use of PARP.

The finding that PARP interacts with SV40 capsid proteins, for example VPl, was very surprising. PARP is an extremely abundant nuclear protein, in fact cells have 10° PARP molecules in the nucleus at a given time [Lindahl et al. (1995) Trends. Biochem. Sci. 20(10):405-11]. Intuitively it would be of great disadvantage for the major external viral capsid protein to have high affinity for such an abundant protein, since all the VPl would then be sequestered by PARP and not be available to form capsids. Unexpectedly, the results in the Examples of the present application demonstrate that PARP has an active role in SV40 assembly, substantially increasing the infectivity of the assembled constructs.

Thus, in a first aspect the present invention relates to a method for improved in vitro assembly of SV40 pseudoviruses having increased infectivity. Such viruses' or pseudoviruses comprise at least one pure or semi-purified SV40 capsid protein and at least one exogenous constituent selected from nucleic . acids, protein and peptides. These viruses • or pseudoviruses are prepared by contacting at least one semi-purified or pure SV40 capsid protein with at least one exogenous constituent in the presence of semi-purified or pure PARP.

The method of the invention may further comprise the step of purifying the assembled recombinant viruses or pseudoviruses from any non-packaged exogenous constituent. In case the exogenous constituent is a nucleic acid, the recombinant viruses or pseudoviruses may be subjected to digestion by nuclease to remove non-packaged DNA, and when the exogenous constituent is protein or peptide, the recombinant viruses or pseudoviruses may be purified from any non-packaged protein by protein purification techniques.

The method of the invention may optionally further comprise the addition of at least one other SV40 protein or proteins, preferably SV40 agnoprotein to the mixture of the SV40 capsid protein/s and the exogenous constituent.

In a preferred embodiment, the method of the invention relates to the assembly of recombinant pseudoviruses having improved infectivity, by bringing a mixture of at least one semi-purified or pure SV40 capsid protein and an exogenous constituent into a contact with semi-purified or pure PARP protein. The exogenous constituent is selected from the group consisting of an exogenous DNA encoding an exogenous protein or peptide product, or encoding a therapeutic RNA, or itself a therapeutic product, a vector comprising exogenous DNA encoding an exogenous protein or peptide product, or encoding a therapeutic RNA, or itself a therapeutic product, ail exogenous RNA encoding an . exogenous protein or peptide product or itself a therapeutic product, a vector comprising an exogenous RNA encoding an exogenous protein or peptide product, or itself a therapeutic product, an exogenous protein or peptide product, antisense RNA, ribozyme RNA or any RNA or DNA which inhibits or prevents the expression of undesired protein/s in a mammalian. target cell and chimeric RNA-DNA oligonucleotide for targeted corrections of mutated genes. Particular examples for such exogenous constituent, are genes coding for the human β-globin, human MDRl or human glucocerebrosidase gene products, as presented in Example 5.

By the term " target cell' as used herein is meant particularly a mammalian cell, preferably a human cell which can be infected with the recombinant pseudoviruses assembled by the method of the present invention. The expression or the function of undesired protein/s in such a mammalian target cell, can be altered by the infection with the recombinant SV40 pseudoviruses that were assembled by the method of the present invention.

The term DNA used herein also encompasses cDNA, i.e. complementary or copy DNA produced from an RNA template by the action of reverse transcriptase (RNA-dependent DNA polymerase).

The term vector as used herein means an expression vector derived, e.g., from a plasmid, bacteriophage, mammalian or insect virus, or any other suitable source, into which fragments of DNA may be inserted or cloned. A vector will contain one or more unique restriction sites and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. A DNA sequence may be recombined with vector DNA in accordance with conventional techniques. Conventional techniques include techniques such as: blunt-ended or staggered-ended ligation, restriction enzyme digestion to provide appropriate .termini, filling, of cohesive ends as appropriate, /alkaline . phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases, or PCR of the vector and the insert. Techniques for such cloning manipulations are well known in the art. A DNA molecule, can •be capable of being expressed if it contains transcriptional. and translational regulatory elements. Such regulatory elements should be operably linked to the encoding nucleotide sequences. An operable linkage is a linkage in which the regulatory elements and the DNA sequence of the invention are connected in such a way as to permit gene expression. The precise nature of the regulatory elements may vary from organism to organism, but in general will include a promoter region comprising non-coding sequences as TATA box, capping sequence, CAAT sequence, initiation codon (for initiation of transcription and translation) at the 5 end, and the non coding region at the 3 end including termination sequences as well as polyadenylation signal. In specific embodiments, the SV40 capsid proteins used in the method of the invention may be semi-purified or pure VPl, VP2 or NP3. In a further specific embodiment, the method of the invention employs a mixture of at least two semi-purified or pure SN40 VPl, VP2 or VP3 capsid proteins. In a particular embodiment, the method of the invention may use the semi-purified or pure SV40 VPl alone.

The following Examples demonstrate association between PARP and the SV40 capsid protein VPl, and possibly VP1/VP2/VP3 complex (Example 1). PARP used in the process of the invention may be semi-purified or pure and improves the efficiency of pseudoviruses assembly as described in Example 4. Furthermore, extremely efficient assembly of three therapeutic genes in the presence of PARP, by the method of the present invention, is described in Example 5.

Without being bound to theory, it is possible that PARP participates in assembly in a similar manner to. Spl, by recruiting the external capsid protein to the minichromosome. Although PARP binds DΝA strand breaks non- specifically, it also recognized cruciform structures and loops in the DΝA [Oei et al. (1997) Rev. Physiol. Biochem. Pharmacol. 131:127-73]. It may recognize such secondary structure in the SV40 regulatory region and thus recruit VPl to this region.

It is possible that Spl and PARP cooperate in the recruitment of VPl to the viral minichromosome. Conversely, they could provide two alternative routes in which the virus utilizes host proteins to recruit the major capsid protein to SV40 DΝA.

Thus, the participation of PARP in the SV40 life-cycle, as demonstrated in the present application, was unexpected and remote from its role in other cellular processes, including viral recombination and integration. The method of the present invention improves the efficiency of the in vitro packaging and the infectivity of the pseudovirions. The high flexibility afforded by the method of the invention may allow the development of gene targeting and/or gene replacement therapies.

In a preferred embodiment, the method of the invention may use as said exogenous constituent an exogenous circular or linear DNA encoding an exogenous protein or peptide product, or itself a therapeutic product, or encoding therapeutic RNA, or a vector comprising exogenous DNA encoding therapeutic RNA or encoding an exogenous protein or peptide product. Delivery into cells of linear DNA, by infecting the cells with recombinant pseudovirions that were prepared by the method of the invention, may be advantageous for recombination, i.e. integration into the cellular genome for stable expression.

Specifically, the DNA is DNA which encodes a therapeutic protein product or is itself, a therapeutic, product which is not made or contained in said cell, or is DNA which encodes a therapeutic protein or peptide product which is made or. contained in said cell in abnormally low amount, in defective form, in physiologically abnormal or normal amount or may encode a therapeutic RNA.

The therapeutic protein or peptide product can be any protein of interest, such as an enzyme, a receptor, a structural protein, a regulatory protein or a hormone. Of particular interest are proteins which are missing or defective in patients suffering genetic disorders.

As a non-limiting examples of such additional therapeutic products are enzymes, e.g. adenosine deaminase (ADA), glucocerebrosidase and hexoaminidase; receptors, e.g. low density lipoprotein receptor (LDL receptor) and IL-6 receptor; transporters, e.g. cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance (MDR); structural proteins, e.g. APO A-I; regulatory proteins, e.g. p53 and retinoblastoma (Rb); hormones, e.g. insuhn, growth hormone; and growth factors, e.g. IL-2 and IL-6.

In a preferred embodiment, the method of the invention may use as said exogenous constituent a chimeric RNA-DNA oligonucleotide for targeted corrections of mutated genes or any type of directed modification of genomic sequences. Correction of the mutation responsible for Sickle Cell Anemia in the hemoglobin β^s gene, through a gene conversion mechanism by use of a chimeric oligonucleotide composed of RNA and DNA residues has been described [Cole-Strauss et al. (1996) Science 273:1386-1388]. This strategy for targeted mutation correction or gene modification is based on the observation that RNA-DNA hybrids are highly active in homologous pairing reactions. Cole-Strauss et al. (1996) showed that single base pair alterations in the β-globin genes can be introduced to the mammalian genome upon transfection with a chimeric RNA-DNA hybrid.

In a particular embodiment, the therapeutic protein is one which is lacking or is defective in hemopoietic disorders such as β-thalassemia, α-thalassemia, anemias due to deficiencies* in red blood cell cytoskeletal or membrane proteins or enzymes, deficiencies in heme synthesis enzymes and deficiencies in erythroid transcription factors. Thus, in this particular embodiment the therapeutic protein is any one of α-globin, β-globin or γ-globin, preferably β-globin, which is lacking or defective in β-thalassemia and related diseases.

In a further preferred embodiment, the invention relates to therapeutic exogenous nucleic acid sequence, which is the human MDRl gene. Enhancement of drug resistance is particularly important in connection with autografting hemopoietic stem cells, conferring resistance of the bone marrow cells to high dose chemotherapy. This mammalian gene confers resistance against variety of drugs such as colchicine, vinblastine, adriamycin and others.

In yet another specific embodiment, the therapeutic protein according to the invention may be glucocerebrosidase. This enzyme is defective in Gaucher disease, and therefore overexpression of this enzyme by the method of the invention has a potential application for the treatment of Gaucher patients.

The exogenous nucleic acids used in the method of the invention are preferably operably attached to SV40-derived ori DNA sequence as replication regulatory element. The exogenous DNA may optionally contain additional DNA sequence/s encoding one or more regulatory elements sufficient for the expression of the exogenous protein or peptide encoded thereby in mammalian cells. It is to be appreciated that the exogenous DNA may operably further contains elements conferring sustained expression. . . . .

Using the pseudoviruses that were prepared by the method of the present application as , an ; .efficient vehicle for delivery of chimeric -RNA-DNA oligonucleotide introducing site specific mutation correction ^' . of gene modification, may be promising as a therapeutic method for the treatment of genetic diseases.

In an additional embodiment, the method of the invention may use as exogenous constituent an exogenous RNA, particularly RNA which encodes a therapeutic protein or peptide product which is not made or contained in a mammalian cell, is made or contained in said cell in abnormally low amount, is made or contained in said cell in defective form, and is made or contained in said cell in physiologically abnormal or normal amount, said RNA having regulatory elements, including translation signal/s sufficient for the translation of said protein or peptide product in said mammalian cell, operatively linked thereto.

As in the embodiments containing DNA, the therapeutic protein or peptide product encoded by the exogenous RNA may be any protein of interest, such as an enzyme, a receptor, a structural protein, a regulatory protein or a hormone.

Packaging of RNA may be advantageous for " short term" , transient gene activity. Packaging of RNA in SV40 pseudovirions, instead of, or in addition to DNA, will allow delivery of mRNA into mammalian cells. The niRNA should include mammalian translation signal, for example Kozak sequences. Such constructs will facihtate transient production of proteins, having high specific function, in vivo.

■ Alternatively, the method of the invention may use as the exogenous constituent an exogenous antisense RNA or DNA encoding antisense or ribozyme RNA, or any RNA or DNA which inhibits or prevents the expression of undesired protein/s or peptide/s in mammalian cells.

In an additional embodiment the method of the invention employ use as .the exogenous constituent an exogenous protein or peptide product. Such protein or peptide product is, respectively, a therapeutic protein or peptide product which is not made or contained in said cell, or is a therapeutic protein or peptide product which is made or contained in said cell in abnormally low amount, in defective form or is made or contained in said cell in physiologically abnormal or normal amount. The exogenous protein or peptide may be a naturally occurring or recombinant protein or peptide, a chemically modified or peptide, or a synthetic protein and peptide.

The delivery of packaged proteins or peptides will also facihtate their transient function in vivo. This approach may be used when long term effects of the packaged protein are not required or may be dangerous. Thus, for example, the delivery of packaged proteins may be useful in cases where transient local production of appropriate growth factors, is required, to accelerate internal wound healing or post-operative incision healing. Local transient introduction of blood clotting factors may be desirable for prevention of hemorrhage and introduction of anti-coagulating factors may be desirable for dissolving unwanted blood clots.

Some proteins may have specific function in the fate of DNA delivery. The constructs of the invention will enable the dehvery of mRNA encoding for a protein which promotes homologous recombination, or the delivery of such protein itself. Pseudovirions carrying a potentially therapeutic gene may be used in co-infection, together with constructs comprising as said constituent mRNA coding for proteins which promote homologous recombination such as RECA, or construct comprising as a constituent such protein/s. This technique will enable gene replacement therapy.

The method of the invention is suitable for the assembly of recombinant SV40 pseudovirions, which are capable of infecting in improved efficiency any suitable mammalian cell. Specific cells are hemopoietic cells, such as bone marrow cell, peripheral blood cells and cord blood cells, or liver cells, epithelial cells, endothelial cells, epidermal cells, spleen cells, fibroblasts, pancreatic cells, muscle cells, tumor cells, cells of the peripheral or central nervous system and germ fine cells.

In a second aspect, the invention relates to an SV40 pseudovirus prepared by the method of the invention. This SV40 virus or pseudovirus has an improved infectivity due to its assembly, in the presence of PARP.

Particular examples for such ^'Pseudovirus assembled in the presence of PARP according to the method of the invention, are described in Example 5 and include pseudovirus comprising the β-globin gene, the MDRl gene or the glucocerebrosidase gene.

In a third aspect, the invention relates to a mammalian, preferably human cell infected with any of the SV40 pseudoviruses that were assembled by the method of the invention.

In a preferred embodiment, these infected human cells are selected from the group consisting of hemopoietic cells, liver cells, pancreatic cells, muscle cells, epithelial cells, endothelial cells, fibroblasts, spleen cells, tumor cells, cells of the peripheral or central nervous system and germ line cells.

As a specifically preferred embodiment the infected human hemopoietic cells of the invention are bone marrow cells, peripheral blood cells and cord blood cells, or liver cells.

Still further, the invention concerns a method of providing a therapeutic DNA, RNA, protein or peptide product or antisense RNA or DNA encoding antisense, chimeric RNA-DNA or ribozyme RNA to a patient in need of such product by administering to said patient a therapeutically effective amount of any of the said pseudoviruses or a therapeutically effective amount of said infected cells prepared by the method of the invention.

In yet another aspect, the invention relates to a method for ex vivo treating an individual suffering an acquired or hereditary pathological disorder in which a product is not made by said individual, or is made in abnormal amounts or in defective form.

It is to be appreciated that the therapeutic product may be any element that can modulate expression of undesired product produced in any pathological disorder including immune -related disorders, viral or bacterial infections and mahgnant situations. Such modulating element may be any one of DNA, RNA, protein or peptide product or antisense RNA or DNA encoding antisense, chimeric RNA-DNA or ribozyme RNA.

The ex vivo treatment according to the method of the invention comprising obtaining cells from an individual suffering said disorder and optionally culturing said cells under suitable conditions, infecting the cells using the pseudoviruses that were assembled in the presence of PARP according to the method of the invention, under suitable conditions, selecting and testing the cells for sufficient expression; and reintroducing the selected cells to the suffering individual.

The therapeutic composition according to the method of the invention may be further used for vaccination and immune-modulating treatment such as in viral or bacterial in mahgnant processes.

The present invention further relates to the use of pseudoviruses prepared by the method of the invention or cells infected by said pseudoviruses, in the preparation of a pharmaceutical composition for the treatment of an individual suffering from pathological disorder.

A particular embodiment relates to preparation of pharmaceutical compositions for the treatment of an individual suffering from pathological disorder requiring expression of a product which is normally not made by said individual, or modulation of a product that is made in abnormally low or high amounts or in a defective or abnormal form. Products expressed in defective or abnormal form may be, by non-limiting example, fusion proteins created by translocations and rearrangements occurring in cancer.

The invention also relates to pharmaceutical compositions comprising as active ingredient a therapeutically effective amount of the pseudoviruses of the invention or a therapeutically effective amount of the infected cells of the invention.

The invention provides for improved products for medical use, which may be prepared under aseptic conditions. A major advantage is that the SV40 capsid proteins are readily made in insect cells. While semi-purified proteins (nuclear extracts) are exemplified, purified proteins can be employed, PARP protein as well can be commercially available or may be semi-purified or purified f om expressing cells.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be -limiting since the scope of the present invention will be limited only by the _: appended claims and equivalents thereof. . . -. -■.. ^■ - ^'. .

Throughout this specification and the claims which follow, unless the context requires otherwise, the word " comprise" , and variations such as " comprises" and " comprising' , will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in this specification and the appended claims, the singular forms " ά' , " an' and " the" include plural referents unless the content clearly dictates otherwise.

The following examples are representative of techniques employed by the inventors in cardin out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

Examples

Materials and methods

Constructs pEGFP-Cl - (Green Fluorescent Protein - GeneBank ACCESSION No.

U55763), was purchased from Clontech (cat # 6084-1).

pSO6β-9 - (human β-globin) - ATCC Accession No. 75596 (ATCC 75596).

pSM2 - (human multi-drug resistance) - Identical to the pSMl plasmid [that was previously cloned by the inventors and was deposited under ATCC Accession No. 97125 (ATCC 97125)], except that it carries the cDNA for the wild type, human MDR1 gene, whereas the pSMl has a -single mutation in amino acid 185 (glycine to valine), [Cardarelli CO. et al., (1995) Cancer Research 55:1086-9]. The pSM2 was cloned by the inventors as well.

pSLE-GC - (human glucocerebrosidase) — was constructed by the inventors by cloning of glucocerebrosidase, that was excised from plasmid pLSVglu2 [obtained from Horowitz. M., Tel Aviv University - DNA 6:101-108, (1987)], into filled-in Bglll sites of the pSLE plasmid (5562bp).

Cell lines

K562- Human erythroleukemia cell - ATCC # CCL-243.

CV-1 — Fibroblast cell line derived from African Green Monkey Kidney — ATCC # CCL-70. CMT4 cells — Fibroblast cell-line derived from CV-1 cells. CV-1 cells were transfected with a plasmid carrying the SV40 genome with a deletion at the origin of replication and the early promoter, and with insertion of the mouse metallothionein promoter upstream to the T-antigen coding region [Gerard, R.D. and Gluzmen, Y., Mol. Cell. Biol. 5:3231-3240 (1985].

Example 1

Binding of cellular proteins to VPl

SV40 capsids have a unique packing geometry in which the C-terminal arm of each VPl molecule is intercalated into a neighboring pentamer in a very precise and specific manner [Liddington, et al. (1991) ibid.]. As suggested by Liddington et al., the formation of such complex and delicate structures most likely requires the participation of chaperones.

To. identify host proteins that interact with Pl, a. "fishing" , experiment was performed. GST- VPl was immobilized on an agarose-GSH resin, washed several times with PBS and was used as "bait" to specifically "fish" proteins with affinity to VPl. • •^• ..^'

The bound (B) GST- VPl material (Fig. 1, lane 3) was . incubated with samples of K562 cells nuclear extracts (Fig. 1, lane 2) containing approximately 20 μg of total protein, for 20 minutes at 4°C. The mixture was centrifuged separating the resin with the immobihzed GST- VPl and putative bound proteins (Fig. 1, lane 4) from the supernatant (Fig. 1, lane 5). The resin fraction was then washed three times with PBS. The fractions were analyzed by 8% SDS-PAGE following TCA precipitation as required and visualized by silver- staining.

The results showed a clear major protein of about 100 KD that specifically bound to VPl (Fig. 1). This strong distinct band was present in the pellet (Fig. 1, lane 4, marked by dots), but not in the bound GST-VP1 (Fig. 1, lane 3). The band was excised and was identified by mass spectrometry amino-terminus sequencing to be poly (ADP-ribose) polymerase (PARP).

To determine whether PARP also binds to the minor capsid protein VP3, a similar experiment using GST-VP3 was carried out. Western blotting using specific anti-PARP antibody showed that PARP also binds to VP3 (not shown). Thus, PARP probably binds to VP1-VP2/3 complex.

Example 2

The involvement of PARP in the SV40 life-cycle

To investigate whether the observed interaction between PARP and the SV40 VP1-VP2/3 complex indicates that PARP has a role in the SV40 life-cycle, the effect of the PARP inhibitor 3-AB on SV40 titer was examined.

The PARP inhibitor 3-AB is an analog of NAD⁺ that cannot be enzymatically modified by PARP. Thus PARP binds to DNA but is' enzymatically inactive. Subconfluent CV-1 cells were infected with SV40 (m.o.i. of 5) and grown in the presence of increasing concentrations of 3-AB. Affcer 5 days the virus was harvested.

As shown in Fig. 2 the PARP inhibitor 3-AB reduces SV40 titer in a dose dependent manner. 3-AB also delays and reduces the magnitude of cytopathic effects which are the hallmark of SV40 infection.

Example 3

PARP is not involved in SV4G DNA replication

Inhibition of PARP dramatically reduced the SV40 titer and cytopathic effects, indicating that PARP could participate in one or more of several steps in the SV40 life-cycle, such as infection, DNA replication or capsid assembly.

To determine whether PARP has a role in DNA replication, experiments testing viral replication in the presence of the PARP inhibitor 3-AB were performed.

Subconfluent CV-1 cells were infected with SV40 (m.o.i. of 5) and grown for 3 days in the presence of increasing concentrations of 3-AB. The cells were then harvested and SV40 DNA was isolated by the Hirt method. Ahquots ^' were analyzed by electrophoresis on 1% agarose followed by Southern blotting.

Phosphorimage analysis showed that 3-AB did not significantly affect the SV40 DNA level present in the Hirt supernatants (Fig 3). Thus, SV40 DNA replication was not affected by the PARP inhibitor (Fig. 3). This suggests ^■ that PARP does not have a role in replication. ^{■ ■} -.!- .>

Example 4 ^" ::

PARP assists assembly of SV40 pseudovirions in vitro

The results support the idea that PARP has a role in SV40 assembly, since the PARP inhibitor reduces viral titer and cytopathic effects. The fact that PARP specifically binds to the major capsid protein VPl suggests that it has a role in capsid assembly. This is further supported by the findings that viral replication is not inhibited by 3-AB. Therefore, the involvement of PARP in capsid assembly was examined by performing in vitro packaging reaction.

The in vitro Tiac gin^p" reaction was carried out essentially as previouslv described [Sandalon et al, (1997) Hum. Gene. Ther. 8:843-849]. Briefly, nuclear extracts of Sf9 cells containing VPl, VP2, VP3 and the agnoprotein were prepared according to Schreiber [Schreiber, et al. (1989) ibid.]. 2.5μl of nuclear extracts (total protein Iμg) were mixed by vortex with lμg of pEGFP-Cl plasmid DNA in a total volume of 8 μl and placed at 37°C for 6hr. The buffer (HEPES pH 7.9) contained 160 mM NaCl, 12.5 mM KC1, 2.5 mM MgC12, 2.5 micromolar ZnSO4, 5% glycerol. Tubes #1-4 contained, in addition, 175 ng PARP each. Tubes #2,3,4 also contained 0.5, 1.5 and 5.0 mM NAD+, respectively, Tube #1 and the control tube (without PARP) did not contain any NAD+. CaCl2 and MgCl2 were added to final concentrations of lOOμM and 8mM respectively, to a total volume of lOμl, and the reactions were incubated for an additional lhr on ice. DNAse l digestion was performed using 0.5 unit of enzyme for lO in on ice, and stopped by the addition of EDTA to a final concentration of 5mM.

DNAse I treatment was used to remove DNA which was not stably packaged. The reaction products were assayed for infectious units (IU) on CMT4 monolayers, grown in Dulbecco's modified Eagle's medium with 10% FBS, using a standard SV40 infection protocol. CMT4 are permissive' African green monkey kidney cells that harbor the gene for SV40 T-antigen expressed from the inducible metallothionein promoter [Gerard, R. D. et al. (1985) Mol. Cell. Biol. - 5:3231-3240]. Sub-confluent monolayers were ^" incubated with the packaging mixture for 120 min at 37 C, with occasional agitation, followed by the addition of fresh medium containing O.lmM ZnCl and lμM CdSO for the induction of T-antigen expression. Infective centers were scored by in situ hybridization.

When PARP was added to the in vitro packaging reaction at 175 ng/reaction (ICN Biomedicals), the efficiency of the reaction was increased approximately by 5 fold. The titer obtained was increased from 2xl0⁴ to 9-10X10⁴, in four different experiments (Fig. 4). It may be noted that addition of NAD⁺ did not affect the results. Presumably, since the nuclear extracts contained sufficient levels of NAD⁺ for PARP enzymatic activity. Addition of PARP enhances the packaging reaction with VPl only In order to find out whether the observed PARP enhancement of SV40 pseudovirions assembly may occur in the presence of VPl alone, a similar packaging experiment was performed, using nuclear extracts containing VPl only. Nuclear extract (one microliter) of Sf9 cells which were infected with recombinant baculovirus expressing VPl at moi ~10 (1.5 microgram total protein) was mixed with 1 microgram of pEGFP-Cl DNA, 9 nano grams of PARP and 5 mM ATP in a total volume of 10 microliters. The nuclear extracts was prepared according to Schreiber [Schreiber, et al., Nucleic Acids Research, 15: 6419-6436 (1989)]. The buffer (HEPES pH 7.9) contained 100 mM NaCl, 100 M KC1, 3 mM MgC12, 2.5 micromolar ZnS04, 5% glycerol, 0.025% NP-40. After 6 hrs incubation at 37°c, CaCl₂ and MgCl₂ were added to final concentrations of 1 mM and 8 mM respectively, in a total volume of 11 μl and the reactions were incubated for an additional lhr on ice. DNAse I digestion was performed using 0.5 unit of enzyme for 10 min on ice, and stopped by the addition of EDTA to a final -concentration of 5 mM. Infectious units- ere scored in. CMT4 monolayers as described hereinabove.

Fig. 5. clearly indicates that addition of 9 ng PARP in the presence of VPl only, significantly increased the titer of infectious units (by 4 fold), from - 1.6xl04/ml to 7xl04/ml. Plasmid DNA incubated in parallel and treated with DNase I under the same conditions did not lead to any ineffective centers on CMT4 cells (Fig. 5). Thus, PARP may enhance the packaging reaction in the presence of VPl only.

Example 5

PARP assists in vitro assembly of SV40 pseudovirions containing differenΛ therapeutic genes

The possibility of using the enhanced packaging properties of PARP-containing reactions of the invention for creation of therapeutic gene containing pseudovirions, was next validated. For that purpose, three different therapeutic genes, the human multi-drug resistance gene, the human β-globin gene or the human glucocerebrosidase gene were packaged in the presence of PARP.

Nuclear extract (one microliter) of Sf9 cells which were infected with recombinant baculovirus expressing VPl at moi ~10 (1.5 microgram total protein) was mixed with 1 microgram of pSO6β-9 (human β-globin), pSM2 (human multi-drug resistance) or pSLE-GC (human glucocerebrosidase) plasmid DNA, 175 nanograms of PARP and 5 mM ATP in a total volume of 10 microliters. The buffer (HEPES pH 7.9) contained 100 mM NaCl, 100 mM KC1, 3 M MgC12, 2.5 micromolar ZnSO4, 5% glycerol, 0.025% NP-40. After 6 hrs incubation at 37°c, CaCl₂ and MgC were added to final concentrations of 1 mM and 8 mM respectively, in a total volume of 11 μl and the reactions were incubated for an additional lhr on ice. DNAse I digestion was performed using 0.5 unit of enzyme for 10 min on ice, and stopped by. -the. addition of EDTA to a final concentration- of 5 - mM. Infectious units were scored in CMT4 monolayers as described herein-above.

As shown in Table 1; addition of PARP to packagin reaction (even in the presence of VPl only-table 1) containing vectors carrying different ■ therapeutic genes, efficiently enhances pseudovirions assembly. Similar enhancement of packaging in the presence of PARP, was also resulted in packaging experiments of the same therapeutic genes, using nuclear extracts containing VPl, VP2, VP3 and agnoprotein (not shown). Table 1

Therapeutic genes packaged in the presence of PARP using with nuclear extracts containing VPl only

Plasmid Transgene Infectious Units/ml pSO6β-9 Human β-globin 4.4 x lO⁴ pSM Human MDR1 4.0 x lO⁴ pSLE-GC Human glucocerebrosidase 5.7 x lO*

Claims

Claims:

1. A method for an improved in vitro assembly of SV40 pseudoviruses comprising at least one pure or semi-purified SV40 capsid protein and at least one exogenous constituent selected from nucleic acids, protein and peptides, wherein at least one semi-purified or pure SV40 capsid protein is brought into contact with at least one said exogenous constituent in the presence of semi-purified or pure PARP [Poly (ADP-ribose) polymerase].

2. A method according to claim 1, further comprising the step of purifying the assembled recombinant viruses or pseudoviruses from any non-packaged exogenous constituent.

3. A method according to any one of claims 1 and 2, further comprising adding at least one other SV40 protein, preferably SV40 agnoprotein, to the mixture of said SV40 capsid protein/s and said exogenous constituent.

4. . A method according to any one of claims 1 to .-3, ■wherein- said exogenous constituent selected from the group consisting of : an exogenous DNA encoding an exogenous protein or peptide product, or encoding therapeutic RNA, or itself a therapeutic product; -- • a vector comprising an exogenous DNA encoding an exogenous protein or peptide product, or encoding therapeutic RNA, or itself .a therapeutic product; an exogenous RNA encoding an exogenous protein or peptide product or itself a therapeutic product; a vector comprising an exogenous RNA encoding an exogenous protein or peptide product or itself a therapeutic product; an exogenous protein or peptide product; antisense RNA, ribozyme RNA or any RNA or DNA which inhibits or prevents the expression of undesired protein's in said mammalian cell; and chimeric RNA-DNA oligonucleotide for targeted corrections of mutations or gene modifications.

5. A method according to any one claims 1 to 4 wherein said SV40 capsid protein is any one of the semi-purified or pure SV40 VPl, VP2, or VP3.

6. A method according to claim 5 wherein said SV40 capsid protein is the semi-purified or pure SV40 VPl.

7. A method according to claim 5 wherein said SV40 pseudoviruses comprise a mixture of at least two of semi-purified or pure SV40 VPl, VP2, and VP3.

8. A method according to any one of claims 6 to 7, wherein said exogenous constituent is a nucleic acid encoding a therapeutic protein or

, peptide product or is itself a therapeutic product..

9. A method according to claim 8, herein said exogenous nucleic acid is, any one of circular DNA, linear DNA and RNA...

10. A method according to claim 9 wherein said therapeutic protein or peptide product is at least one of a therapeutic protein or peptide which is not expressed, is expressed in abnormally low amount, is expressed in defective form or is expressed in physiologically abnormal amount, in a target cell.

11. A method according to claim 10, wherein said therapeutic protein or peptide product is at least one of an enzyme, a receptor, a structural protein, a regulatory protein and a hormone.

12. A method according to claim 11, wherein said therapeutic protein or peptide product encoded by the exogenous nucleic acid is human β-globin.

13. A method according to claim 11, wherein said therapeutic protein or peptide product encoded by the exogenous nucleic acid is the human MDR1.

14. A method according to claim 11, wherein said therapeutic protein or peptide product encoded by the exogenous nucleic acid is the human glucocerebrosidase.

15. A method according to any one of claims 11 to 14 wherein said exogenous nucleic acid is operably attached to SV40-derived ori DNA sequence and optionally to operably hnked additional DNA sequence encoding one or more regulatory elements sufficient for the expression of said exogenous protein in said target cell and/or elements conferring sustained, expression. •—. • -« -..-.•----.

16. A method according to any one of claims 6 or 7, wherein said exogenous nucleic acid is selected from the group consisting of-antisense RNA, ribozyme RNA, chimeric RNA-DNA oligonucleotide and RNA and DNA which inhibits, or prevents or corrects the expression of undesired protein/s in a target cell.

17. A method according to any one of claims 6 or 7, wherein said exogenous constituent is a protein or peptide, which protein or peptide is a naturally occurring or recombinant protein or peptide, a chemically modified or peptide, or a synthetic protein and peptide.

18. A method according to claim 17, wherein said exogenous protein or peptide product is at least one of therapeutic protein or peptide which is not expressed, is expressed in abnormally low amount, is expressed in defective form or is expressed in physiologically abnormal amount in a target cell.

19. An SV40 virus or pseudovirus prepared by the method of any one of claims 1 to 11.

20. An SV40 pseudovirus prepared by the method of any one of claims 12 to 14.

21. A mammalian cell infected with pseudovirus prepared by the method of any one of claim 1 to 11.

22. A mammalian cell infected with SV40 virus or pseudovirus prepared by the method of any one of claim 12 to 14.

23. An infected human cell according . to claim 21, selected from the group consisting of hemopoietic cells, liver.. cells, pancreatic cells, epithelial cells, endothelial cells, spleen cells, fibroblasts, muscle cells, tumor cells, cells of the peripheral or central nervous system and germ line cells.

24. An infected human cell according to claim 22, selected from "the group consisting of hemopoietic cells, liver cells, pancreatic cells, epithelial cells, endothelial cells, spleen cells, fibroblasts, muscle cells, tumor cells, cells of the peripheral or central nervous system and germ hne cells.

25. An infected human cell according to any one of claims 23 and 24, wherein said hemopoietic cells are bone marrow cells, peripheral blood cells and cord blood cells, or liver cells.

26. A method of providing a therapeutic DNA, RNA, antisense RNA, ribozyme RNA, chimeric RNA-DNA, protein or peptide product to a patient in need of such product by administering to said patient a therapeutically effective amount of the pseudoviruses of claims 19 or 20, prepared by the method according to any one of claims 1 to 11 and 12 to 14 respectively.

27. A method of providing a therapeutic DNA, RNA, antisense RNA, ribozyme RNA, chimeric RNA-DNA, protein or peptide product to a patient in need of such product by administering to said patient a therapeutically effective amount of infected cells according to any one of claims 21 or 22.

28. Use of pseudoviruses according to any one of claims 19 and 20 or infected cells according to claims 21 or 22, in the preparation of a pharmaceutical composition for the treatment of an individual suffering from pathological disorder.

29. Use according to claim 28, in the preparation of a pharmaceutical composition for the treatment of an individual suffering from pathological' disorder requiring expression of a product normally not made by said individual, or modulation of a product that is made in -abnormal amounts or in a defective or abnormal form.

30. Use according to claim 29, wherein said composition is intended for increasing the expression of the human MDRl gene.

31. Use according to claim 29, wherein said composition is intended for increasing the expression of the human β-globin gene.

32. Use according to claim 29, wherein said composition is intended for increasing the expression of the human glucocerebrosidase gene.

33. Use according to any one of claims 28 to 32, wherein said pharmaceutical composition is intended for the treatment of hereditary diseases, mahgnant diseases, bacterial and viral infection diseases or autoimmune diseases.

34. Use according to any one of claims 31 and 33, wherein said hereditary disease is β-thalassemia and said pseudovirions comprise the human β-globin gene.

35. Use according to any one of claims 32 and 33, wherein said hereditary diseases is Gaucher disease and said pseudovirions comprise the human glucocerebrosidase gene.

36. Pharmaceutical compositions for the treatment of hereditary diseases, malignant diseases, bacterial and viral infection diseases or autoimmune diseases, comprising as active ingredient a therapeutically effective amount of the pseudoviruses prepared by the method according to any one of claims 1 to 14.

37. Pharmaceutical compositions for the treatment of hereditary diseases, malignant diseases,-, --bacterial and viral infection diseases or autoimmune diseases, comprising as active ingredient a therapeutically effective amount of infected cells according to any one of claims 21 to claim

24. ^• : . . .. ^{• ■} . .. .