CA2235347A1 - In vitro construction of sv40 viruses and pseudoviruses - Google Patents

Abstract

The invention relates to constructs capable of infecting mammalian cells comprising at least one semi-purified or pure SV40 capsid protein and a constituent selected from the group consisting of an exogenous DNA, a vector comprising an exogenous DNA, an exogenous RNA, a vector comprising 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 said mammalian cell and optionally further comprising operatively linked regulatory elements sufficient for the expression and/or replication of said exogenous protein in a mammalian cell. The protein product is preferably a therapeutic protein or peptide product which is not made or contained in mammalian cells, or is made or contained in such cells in abnormally low amount, or is made or contained in such cells in defective form, or is made or contained in mammalian cells in physiologically abnormal or normal amount and can be an enzyme, a receptor, a structural protein, a regulatory protein or a hormone. The invention further relates to a method for the in vitro construction of SV40 viruses or pseudoviruses constructs according to the invention. In a further aspect, the invention relates to mammalian, preferably human cells infected with the constructs of the invention or with constructs obtained by any of the methods of the invention.
Still further, the invention relates to a method of providing a therapeutic DNA, RNA, protein or peptide product or antisense RNA to a patient in need of such product by administering to the patient a therapeutically effective amount of the SV40 viruses or pseudoviruses of the invention or a therapeutically effective amount of infected cells according to the invention.
Pharmaceutical compositions comprising as active ingredient a therapeutically effective amount of the SV40 viruses or pseudoviruses according to the invention or a therapeutically effective amount of infected cells according to the invention are also within scope of this application.

Description

CA 0223~347 l998-04-30 W O 97/17456 PCTnL96/00143 PSEUDOVIRUSES

FIELD OF THE INVENTION

s The invention relates to methods of in vitro construction of SV40 viruses orpseudoviruses conl~lisi-lg exogenous nucleic acid or exogenous protein or peptide which are particularly suitable for use in gene therapy.

BACKGROUND OF T~IE INVENTION

lo Previous studies have shown that SV40 virions disrupted at pH 10.6 [Christensen, M. & Rachmeler, M. (1976) Virology 75:433-41] or by reducing disulfide bonds [Colomar, M.C., et al. (1993) J. Virol. 67:2779-2788] may be reassociated to form infectious SV40 aggregates. The early attempts to package in vitro foreign DNA in these aggregates [Christensen & Rachmeler (1976) ibid.] produced infectious S products which did not resemble SV40 virions. Furthermore, their resistance to DNase has not been tested. Later, in vitro p~çk~ging experiments [Colomar et al.(1993) ibid. ] did not yield particles with infectivity above the level of naked DNA.

Recently, pseudocapsids of the closely related murine polyoma virus, prepared from polyoma VP1, were used as carriers for heterologous DNA into m~mm~ n cells [Forstova, J., et al. (1995) Hum. Gene Therapy 6:297-306]. The pseudo-capsid protected 2-30% of the input DNA from DNase I digestion. When a plasmid carrying the cat gene was tested, most of the DNA which was protected from DNase I appeared as a ~2kb fragment, while the input plasmid was significantly larger (exact size was not reported), suggesting that each DNA molecule was onlypartially protected against DNases. Infectious units were not measured in those CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 experiments. The DNA transferred into recipient cells was functional in gene expression, albeit at a very low efficiency. With a 1.6kb DNA fragment which carries the polyoma middle T-antigen, <30 transformed foci were obtained per l,~Lg of input DNA. Similarly, a low level of CAT activity was observed with the plasmid carrying the cat gene.

SV40 is a simian papovirus, with a small double-skanded circular DNA genome of 5.2kb [reviewed in Tooze, J. (1981) DNA Tumor Viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York]. The viral capsid, surrounding the viral mini-chromosome, is composed of three viral-coded proteins, VPl, VP2, and VP3.
o Recent X-ray crystallographic studies on SV40 structure at 3.8A resolution [T i~l-lin~on, R., et al. (1991) Nature 354:278-282] revealed that the outer shell of the virion particle is composed of 72 pentamers of VPl, 60 hexavalent and 12 pentavalent. The VP2 and VP3 appear to bridge between the VP 1 outer shell and the chromatin core. The VPl 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 facilitates the use of identical building blocks in the formation of a structure that is sufficiently flexible as required for the variability in packing geometry [T icl-lin~on et al. (1991) ibid.].

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 LPl. This is ploteil, 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. Virol. 60:1098-1106; Ng, S.C., et al. (1985) J. Biol. Chem. 260:1127-1132; Carswell, S.& Alwine, J. C. (1986~ J. Virol. 60:1055-1061].

The major hindrance in be~innin~ to use the SV40 pseudovirions in prelimin~ry experiments in hllm~n~ is the present need for a viral helper for encapsidation. This results in pseudoviral stocks that contain also wild type SV40. Because of the CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 simil~rity in properties (shape, size and density) between the pseudovirions and the helper, they cannot be separated by physical means. An ideal way to ~lG~ale pseudovirions for therapeutic purposes for human use would be by in vitro pack~gin~ This would provide maximal safety, since all steps ofthe ~i~dlion can s be well controlled. Ex vivo ~(1mini.~tration would circumvent problems associated with immllne response.

Viral p~ck~ging in vivo occurs by gradual addition and or~ni7~tion of capsid proteins around the SV40 chromatin [Garber, E.A., et al. (1980) Virology 107: 389-401; Bina, M. (1986) Comments Mol. Cell Biophys. 4:55]. The three capsid 0 proteins VPl, VP2 and VP3 bind to DNA non-specifically [Soussi, T. (1986) J.
Virol. 59:740-742, Clever, J., et al. (1993) J. Biol. Chem. 268:20877-20883]. How the specific recognition between the viral capsid proteins and its DNA is achieved remains unclear. The p~ck~ging of SV40 using pseudovirions, in which most of theviral DNA is replaced by other sequences has been investigated [Oppenheim, A., et 15 al. (1986) Proc. Natl. Acad. Sci. USA 83:6925-6929]. The pseudoviral particles are pr~aled 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 model suggests that ses serves several functions in SV40 packaging: as a sensor for the level of the late viral proteins in the transition from replication and/or 20 transcription to pack~ing, in nucleosomal reorg~ni7~tion and the initiation of viral assembly [Oppenheim, A., et al. (1994). J. Mol. Biol. 238:501-513] and probably also as a nucleation center for viral assembly [Dalyot-Herman, N. et al. (1996) J.
Mol. Biol. 259:69-80].

The pseudovirions, carrying various genes of therapeutic interest, are very efficient 25 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; Dalyot, N. &
Oppenheim, A. (1989) Efficient transfer of the complete human beta-globin gene CA 0223~347 1998-04-30 W O 97/174~6 PCT~L96/00143 into hllm~n and mouse hemopoietic cells via SV40 pseudovirions. In: Gene Transfer and Gene Therapy (Beaudet, A.L., Mlllli~n R., I.M. Verrna, eds), pp. 47-56, AlanR. Liss, Inc., New York; Oppenheim, A., et al. (1992) Development of somatic gene therapy: A simian virus 40 pseudoviral vector for hemopoietic cells. In Genetic s Among Jews (Bonne-Tamir, B., A. Adams, eds), pp. 365-373, Oxford University Press, Oxford]. The ideal way to pr~ pseudovirions for therapeutic purposes for human use is by in vitro packaging. This would provide maximal safety, since allsteps of the preparation can be well controlled.

0 SUl\~MARY OF THE INVENTION

The present invention relates to construct capable of infecting a m~mm~ n cell comprising at least one semi-purified or pure SV40 capsid protein and a constituent selected from the group con~ ting of an exogenous DNA encoding an exogenous protein or peptide product, or encoding therapeutic RNA, or itself a therapeuticproduct, 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, and antisense RNA, ribozyme RNA or any RNA or DNA which inhibits or prevents the expression of undesired protein/s in said m~mm~ n cell, and optionally further comprising operatively linked regulatory elements sufficient for the expression and/or replication of said exogenous protein in a m~mmz li~n cell.

The construct of the invention may optionally further comprise additional SV40 protein or proteins, preferably SV40 agnoprotein.

=
CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 Constructs according to the invention may comprise as said con~t~ ent exogenous circular or linear DNA encoding an exogenous protein or peptide product, or is itself a therapeutic product, or a vector comprising exogenous DNA encoding a therapeutic RNA, or encoding an exogenous protein or peptide product.

s The said protein product is preferably a therapeutic protein or peptide product which is not made or contained in m~mm~ n cells, or is DNA which encodes a therapeutic protein or peptide product which is made or cont~ine-l in such cells in abnormally low amount, or is DNA which encodes a therapeutic protein or peptide product which is made or contained in such cells in defective form or is DNA which o encodes a therapeutic protein or peptide product which is made or contained inm~mm~ n cells in physiologically abnormal or norrnal amount and can be an enzyme, a receptor, a structural protein, a regulatory protein or a hormone.

The constructs of the invention may comprise SV40-derived ori DNA sequence as said replication regulatory element and may further comprise DNA sequences 5 encoding one or more regulatory eléments sufficient for the expression of saidexogenous RNA or exogenous protein or peptide in said m~mm~ n cell.

In other embodiments, in constructs ,a~ccording to the invention said constituent is exogenous RNA, preferably RNA which encodes a therapeutic protein or peptide product which is not made or contained in said cell, or is RNA which encodes a therapeutic ~,rotehl or peptide product which is made or contained in said cell in abnormally low amount, or is RNA which encodes a therapeutic protein or peptide product which is made contained in said cell in defective form, or is RNA which encodes a therapeutic protein or peptide product which is made or contained in said ~ cell in physiologically abnormal or norrnal arnount, said RNA having regulatory 2s elements, including translation signal/s sufficient for the translation of said protein or peptide product in said m~mm~ n ce ~, operatively linked thereto.

CA 0223~347 1998-04-30 W O 97/174S6 PCT~L96/00143 In other embo~liment~, the constructs according to the invention may comprise assaid constituent an exogenous protein or peptide product, which can be a therapeutic protein or peptide product which is not made or contained in m~mm~ n cells, or is a therapeutic protein or peptide product which is made or contained in such cells in 5 abnormally low amount, or is a therapeutic protein or peptide product which is made or c~nt~ined in such cells in defective form or is a therapeutic protein or peptide product which is made or contained in m~mm~ n cells in physiologically abnormal or normal arnount.

In further embodiments, the constructs according to the invention may comprise as 0 said constituent antisense RNA or DNA or ribozyme RNA, or any RNA or DNA
which inhibits or prevents the expression of undesired protein/s in m~mm~ n cells.

The m~mm~ n cells can be hemopoietic cells, such as bone marrow cells, peripheral blood cells and cord blood cells or liver cells., epithelial cells, endothelial cells, liver cells, epidermal cells, muscle cells, tumor cells, nerve cells and germ line 15 cells.

The invention further relates to a method for the in vitro construction of SV40 viruses or pseudoviruses comprising exogenous nucleic acid comprising the steps of bringing a semi-purified or pure SV40 capsid protein or a mixture of at least two such proteins into contact with the exogenous nucleic acid to give recombinant 20 SV40 viruses or with a vector comprising the exogenous nucleic acid to give pseudoviruses; and optionally subjecting the SV40 viruses or pseudo-viruses thusformed to digestion by nuclease to remove non-packaged DNA.

In the method of the invention, at least one other SV40 protein, preferably SV40agnoprotein, can be added to the mixture of the SV40 capsid protein/s and the 25 nucleic acid.

The DNA employed in the method of the invention can be DNA which encodes a therapeutic protein or peptide product which is not made or contained in m~mm~ n CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 cells, or is DNA which encodes a therapeutic protein or peptide product which ismade or contained in such cells in abnormally low amount, or is DNA which encodes a therapeutic protein or peptide product which is made or cont~ined in such cells in defective form or is DNA which encodes a therapeutic protein or peptide~ s product which is made or contained in such cells in physiologically abnormal or normal amount or is DNA which encodes a therapeutic RNA.

The therapeutic protein or peptide product can be an enzyme, a receptor, a structural protein, a regulatory protein or a hormone.

The nucleic acid employed in the method of the invention can alternatively be 0 exogenous RNA, wherein said RNA is RNA which encodes a therapeutic protein orpeptide product which is not made or contained in m71mm~ n cells, or is RNA
which encodes a therapeutic protein or peptide product which is made or cont~ine~l in such cells in abnormally low amount, or is RNA which encodes a therapeutic protein or peptide product which is made or contained in such cells in defectiveS form or is RNA which encodes a therapeutic protein or peptide product which is made or contained in such cells in physiologically abnormal or normal amount andwherein said RNA has regulatory elements, including translation signal, sufficient for the translation of said protein product in m~mm~ n cells, operatively linkedthereto.

The method of the invention can also be used for the in vitro construction of recombinant SV40 viruses or pseudoviruses comprising an exogenous protein or peptide comprising the steps of bringing a semi-purified or purified SV40 capsidprotein or a mixture of at least two such proteins into contact with the exogenous ~ protein to give recombinant SV40 viruses or pseudoviruses; and optionally 25 purifying the recombinant viruses or pseudoviruses thus obtained from any non-packaged protein.

CA 0223~347 l998-04-30 W O 97/17456 PCT~L96/00143 In addition, the method of the invention can be used for the in vitro construction of SV40 pseudoviruses Gomprising exogenous antisense RNA, or ribozyme RNA or RNA or DNA which inhibits or prevents the ~Les~ion of undesired protein/s in a m~mm~ n cell, comprising the steps of bringing a semi-purified or pure SV40 s capsid protein or a mixture of at least two such proteins into contact with the exogenous antisense RNA, or ribozyme RNA, or RNA or DNA which inhibits or prevents the ex~lession of undesired protein/s in a m~nnm~ n cell, to give recombinant SV40 pseudoviruses; and optionally subjecting the SV40 pseudoviruses thus formed to digestion by nuclease to remove non-packaged DNA.

o In a further aspect, the invention relates to m~mm~ n cells infected with the constructs of the invention or with constructs obtained by any of the methods of the invention.

Still further, the invention relates to a method of providing a therapeutic DNA,RNA, protein or peptide product or antisense RNA to a patient in need of such 5 product by ~lmini~tering to the patient a therapeutically effective amount of the SV40 viruses or pseudoviruses of the invention or a therapeutically effective amount of infected cells according to the invention.

Ph~rmslceutical compositions comprising as active ingredient a therapeutically effective amount of the SV40 viruses or pseudoviruses according to the invention or a therapeutically effective amount of infected cells according to the invention are also within scope of this application.

CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 BRIEF DESCRIPTION OF THE F IGURES

Figure 1(a) and 1(b): Self-Assembly of the SV40 capsid proteins Nuclear extracts [Schreiber, E., et al, (1989) Nucl. Acids Res. 15:6419-6436] were prepared from Sfg cells infected with the three recombinant baculoviruses expressing VP1, VP2 and VP3. Samples were adsorbed onto Follllv~-carbon-coated copper grids and stained with 1% phospho~mgct~e, pH 7.2. The sarnples were viewed in a Philips CM-12 electron microscope, used at a voltage of 100kV, and photographed at a m~nification of x60,000. The bar represents 50nm.

Fig. 1(a): Three fields of nuclear extracts of Sfg cells infected with three recombinant baculoviruses, expressing VPl, VP2 and VP3.

Fig. 1(b): Wild type SV40, shown for comparison.

Figure 2(a)-2(c): Infectivity of SV40 virions and pS03cat pseudovirions rq~ ed in vi~ro, Products of the in vitro p~ck~ging reaction were assayed for infectious units in situ hybridization, following infection of CMT4 monolayers.

Fig. 2(a): Autoradiograms showing infections centers produced by pS03cat DNA
packaged in vitro, using nuclear extracts of Sfg cells infected either with recombinant baculovirus expressing VP1 or with the three recombinant viruses, as designated.

Fig. 2(b): Quantification of the results shown in Fig. 2(a).

Fig. 2(c): In vitro p~ck~ging of SV40 DNA, using nuclear extracts of Sfg cells, uninfected or infected, as designated.

W O 97/17456 PCT~L96/00143 Figure 3(a)-3(d): Physical association of pSO3caf plasmid DNA with the capsid protein.

Fig. 3(a): The reaction products were placed on top of a 5-35% sucrose gradient in s 10mM TrisHCI pH 7.4/lSOrnM NaCl, with a 2M sucrose cushion, and centrifuged (SW 50.1 rotor) at 35000 rpm for 120 min., at 4~C. Fractions were collected from the bottom. ~ -SV40 capsid proteins, assayed by SDS-PAGE and western blotting with anti-VP1 antibody (Sandalon, Z., ~erm~n-Dalyot, N., Oppenheim, A. B. And Oppenheim, A. Submitted for publication). Cl- DNA was analyzed following treatment with 0.4M
NaOH in 25mM EGTA and 20rnM DTT at 37~C for 30 min., by electrophoresis on 1% agarose gels and Southern blotting, with pML2 as a probe. O -IU were assayed as described in Fig. 2.

Fig 3(b): Nuclear extracts of Sf9 cells infected with the three recombinant S baculoviruses. The fractions were analyzed by SDS-PAGE as in (a).

Fig. 3(c): SV40 virions, analyzed as described for (a). ~ -SV40 capsid proteins, O -SV40 DNA.

Fig. 3(d): pS03cat DNA (l~g). The fractions were analyzed by electrophoresis on 1% agarose gels and EtdBr st~ining-Figure 4(a) and 4(b): Expression of the transmitted DNA molecules.

Fig. 4(a): CMT4 cells, infected with in vitro packaged pS03cat DNA, were assayedfor CAT activity 3 days post infection. Essentially as previously described [Oppenheim. A., et al., (1986) Proc. Natl. Acad. U.S.A.
2s 83:6925-6929], for 60min. at 37~, using extracts of 106 cells/ assay.

CA 0223~347 1998-04-30 W O 97/174~6 PCT~L96/00143 1 - No extract negative control;

2 - Mock infected CMT4 control;

3 - Control cells "infected" with pS03cat DNA only;

4 - Cells infected with in vitro packaged pS03cat; moi of l-IU per 2x104 s cells;

5 - Cells infected at a moi of l-IU per lx104 cells.

Fig. 4(b): Lysis of CV1 cells infected with in vitro packaged virions.

A - Mock infected;

B -"Infected" with DNA only;

o C - Infected with in vitro packaged SV40.

DET~TT,T~'~ DESCRIPTION OF THE INVENTION

The present invention relates to constructs capable of infecting a m~mm~ n cell,comprising at least one semi-purified or pure SV40 capsid protein and a constituent 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, an exogenous RNA encoding an exogenous protein or peptide product or itself a 20 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, and antisense RNA, ribozyme RNA or any RNA or DNA which inhibits or prevents the expression of undesired protein/s in a m~mm~ n cell, and optionally further comprising operatively linked regulatory CA 0223~347 1998-04-30 6 PCT~L96/00143 elements sufficient for the ~,ession and/or replication of said exogenous therapeutic RNA or of an exogenous protein or peptide in a m~mm~ n cell.

The construct of the invention may optionally further comprise additional SV40 protein or proteins, preferably SV40 agnoprotein.

s In specific embodiments, the constructs of the invention comprise a mixture of at least two semi-purified or pure SV40 capsid proteins.

In a further specific embo-liment, the constructs of the invention comprise a mixture of three semi-purified or pure SV40 capsid proteins.

The SV40 capsid proteins of the invention can be semi-purified or pure VP 1 or VP2 o or VP3.

The constructs of the invention may comprise as said 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 S peptide product. Delivery into cells of linear DNA, by infecting the cells with constructs of the invention comprising such linear DNA, may be advantageous for recombination, i.e. integration into the cellular genome for stable expression.

Specifically, said 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 con~in~-l in said cell in abnormally low amount, or is DNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in defective forrn or is DNA which encodes a therapeutic protein or peptide which is made or contained in said cell in physiologically abnormal or normal amount or encodes a25 therapeutic RNA.

CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 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 mi~ing or defective in patients ~uLr~ g genetic disorders. A specific example may be ~-globin, missing in patients with ,~-- 5 ~h~ emia.

The constructs of the invention may optionally comprise SV40-derived ori DNA
sequence as said replication regulatory element. The exogenous DNA may optionally have, operatively linked thereto, additional DNA sequence/s encoding one or more regulatory elements sufficient for the expression of the exogenous o protein or peptide encoded thereby in m~mm~ n cells.

In an additional aspect, in constructs of the invention said constituent is exogenous RNA, particularly RNA which encodes a therapeutic protein or peptide product which is not made or contained in said cell, or is RNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in abnormally low amount, or is RNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in defective form, or is RNA which encodes a therapeutic protein or peptide product which 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 20 product in said m~mm~ n cell, opel~Li~ely linked thereto.

As in the embodiments cont~ining RNA, the therapeutic protein or peptide productencoded 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.
- 25 Packaging of RNA in SV40 pseudovirions, in~te~d of, or in addition to DNA, will allow delivery of mRNA into m~mm~ n cells. The rnRNA should include CA 0223~347 1998-04-30 W O 97/174S6 PCTnL96/00143 m~mm~ n translation signal, for example Kozak sequences. Such constructs will facilitate transient production of proteins, having high specific function, in vivo.

The constructs of the invention will also enable the delivery of ribozyme RNA, which can be used in any application where specific RNA cleavage is desired, as an s anti-AIDS agent or as an agent against other viral infections or for other therapeutic purposes.

A specific example may be chronic myelogenous leukemia (CML). CML is a clonal stem-cell disorder which accounts for about 25 percent of all leukemias, with anannual incidence of one per 100,000 population, affecting all age groups, with a0 peak incidence in the fifth and sixth decades of life. Clinically, CML is characterized by a triphasic course. The initial chronic phase, often develops insidiously and is marked by an increased pool of commit~e~l myeloid progenitor cells. After a few weeks to several years (median duration 42 months) the disease turns into a phase of"acceleration", which later progresses to the acute phase. The median survival, from diagnosis, in the acute phase is approximately 4 months.

CML is genetically characterized by the presence of the Philadelphia chromosome (Ph'), which is the result of reciprocal translocation between chromosomes 9 and22. At the molecular level, the proto-oncogene abl from chromosome 9 is translocated to the breakpoint cluster region (bcr) on chromosome 22, creating two 20 major types of junctions: L-6 and K-28, each resulting in the formation of bcr/abl hybrid gene, expressing a fusion protein of 210kd, which causes the disease.
Possible use of antisense (18-mer) oligonucleotides, or DNA encoding ~nti~n.ce RNA, directed against the expression of the bcr/abl gene as tumor specific agents which alter the transformed phenotype of leukemia cells cultured in vitro has 25 already been demonstrated [Szczylik, C. et al. (1991). Science, 2~i3:562-565,Garcia-Hernandaz, B. & Sanchez-Garcia, I. (1996) Mol. Medicine, 2:1076-1551].

CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 Therefore, the constructs according to the invention may comprise as said constituent antisense RNA or DNA encoding antisense or ribozyme RNA, or any RNA or DNA which inhibits or prevents the expression of lln~esired protein/s or peptide/s in m~mm~ n cells.

5 In this specific embodiment, said antisense RNA or DNA encoding ~nti~en~e RNA
can be directed against the expression of the bcr/abl transcript, or ~in~t an ~Vtranscript.

In additional embodiments, the constructs of the invention may comprise as said constituent an exogenous protein or peptide product.

o In preferred such constructs, said constituent is exogenous protein or peptideproduct 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, or is a therapeutic protein or peptide product which is made or contained in said cell in defective form or is a 15 therapeutic protein or peptide product which is made or contained in said cell in physiologically abnormal or normal amount.

The delivery of packaged proteins or peptides will also facilitate their transient function in vivo. This approach will be used when long term effects of the packaged protein are not required or may be dangerous. Thus, for example, the delivery of20 packaged proteins may be useful in cases where transient local production of a~ u~liate growth factors, for example, FGF (Fibroblast Growth Factor) is required, to accelerate internal wound healing or post-operative incision he~lin~?.
Local transient introduction of blood clotting factors may be desirable for prevention of hemorrhage and introduction of anti-coagulating factors may be - 25 desirable for dissolving unwanted blood clots. Application of infecting pseudo-virions on site may be by catheters or any other suitable physical means.

CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 Some proteins may have specific function on the fate of DNA delivery. The constructs of the invention will enable the delivery of mRNA encoding ffir a protein which promotes homologous recombination, or the delivery of such protein itself.Pseudovirions carrying a gene will be used in co-infection, together with constructs s comprising as said constituent mRNA coding for proteins which promote homologous recombination such as REC A, or construct comprising as a constituentsuch protein/s. This technique will enable gene replacement therapy.

The constructs of the invention are capable of infecting m~mm~ n, particularly human cells. Specific cells may be hemopoietic cells, such bone marrow cells, o peripheral blood cells and cord blood cells, or liver cells, epithelial cells, endothelial cells, liver cells, epidermal cells, muscle cells, tumor cells, nerve cells and germ line cells.

In another aspect, the invention relates to a method for the in vitro construction of SV40 viruses or pseudoviruses comprising exogenous nucleic acid, comprising the steps of bringing a semi-purified or pure SV40 capsid protein or a mixture of atleast two such proteins into contact with said exogenous nucleic acid to give recombinant SV40 viruses or with a vector comprising said exogenous nucleic acidto give pseudoviruses; and optionally subjecting the SV40 viruses or pseudoviruses thus formed to digestion by nuclease to remove non-packaged DNA.

The SV40 capsid protein are preferably semi-purified or pure SV40 VPl, VP2 or VP3.

The method of the invention may employ, in addition to said semi-purified or pure SV40 capsid protein/s and said nucleic acid, at least one other SV40 protein, preferably SV40 agnoprotein.

The exogenous nucleic acid is preferably circular or linear DNA or is RNA. The exogenous nucleic acid preferably encodes a therapeutic protein or peptide product or is itself therapeutic product.

CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 In specific embo-liment~, said DNA or RNA are DNA or RNA which encode a therapeutic protein or peptide product which is not made or cont~ined in said cell, or which encode a therapeutic protein or peptide product which is made or containedin said cell in abnormally low amount, or which encode a therapeutic protein or - s peptide product which is made or contained in said cell in defective form or which encode a therapeutic protein or peptide product which is made or contained in said cell in physiologically abnormal or normal amount or is a DNA which encodes a ~erapeutic RNA.

Said exogenous DNA or RNA preferably encode a therapeutic protein or peptide o product which is an enzyme, a receptor, a structural protein, a regulatory ~lotehl or a hormone.

In the method of the invention, SV40-derived ori DNA sequence may be added and said exogenous nucleic acid optionally has DNA sequence encoding one or more regulatory element~, sufficient for the expression of said exogenous protein or 5 peptide in said m~mm~ n cell, operatively linked thereto.

When said nucleic acid is exogenous RNA, it has to have the necessary regulatorysignals, including a translation signal, sufficient for the translation of said protein or peptide product in a m~mm~ n cell, operatively linked thereto.

In a further embo-liment, the invention relates to a method for the in vitro 20 construction of recombinant SV40 viruses or pseudoviruses comprising as said constituent an exogenous protein or peptide comprising the steps of bringing a semi-purified or pure SV40 capsid protein or a mixture of at least two such proteins into contact with an exogenous protein or peptide, to give recombinant SV40 viruses or pseudoviruses, and optionally purifying the recombinant viruses or pseudoviruses25 thus obtained from any non-packaged protein.

CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 In this embo-liment, the exogenous protein or peptide can be, respectively, a naturally occurring or recombinant protein or peptide, a chemically modified protein or peptide, or a synthetic protein or peptide.

The exogenous protein or peptide product are, respectively, a therapeutic protein or peptide product not made or contained in a m~mm~ n cell, or are a therapeutic protein or peptide product made or contained in said cell in abnormally low amount or are a therapeutic protein or peptide product made or cont~ine~l in said cell in defective form or are a therapeutic protein or peptide product made or contained in said cell in physiologically abnormal or normal amount.

o In addition, the method of the invention can be used for the in vitro construction of SV40 pseudoviruses comprising antisense RNA, or exogenous DNA encoding antisense RNA, or ribozyme RNA, or RNA or DNA which inhibits or prevents the expression of undesired protein/s or peptide/s in a m~mm~ n cell, comprising thesteps of bringing a semi-purified or pure SV40 capsid protein or a mixture of al15 least two such proteins into contact with the exogenous antisense RNA or exogenous DNA encoding antisense RNA, or ribozyme RNA, or RNA or DNA
which inhibits or prevents the expression of undesired protein/s or peptide/s in a m~rnm~ n cell, to give recombinant SV40 pseudoviruses, and optionally subjecting the SV40 pseudoviruses thus formed to digestion by nuclease to remove20 non-packaged DNA.

In this embodiment, the said antisense RNA or DNA encoding ~n~ieçnee RNA can be directed ~inet the expression of bcr/abl transcripts, or against an HIV
transcripts.

The method of the invention is suitable for the ~lcpa d~ion of constructs which are 25 capable of infecting any suitable m~mm~ n cell. Specific cells are hemopoietic cells, such as bone marrow cell, peripheral blood cells and cord blood cells, or liver CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 cells, epithelial cells, endothelial cells, liver cells, epi-l~rm~l cells, muscle cells, tumor cells, nerve cells and germ line cells.

In yet a further aspect, the invention relates to a m~mm~ n, preferably human cell infected with any of the constructs of the invention, or constructs obtained by any of s the methods of the invention.

Still further, the invention concerns a method of providing a therapeutic DNA, RNA, protein or peptide product or antisense RNA or DNA encoding ~nti~çn~e to a patient in need of such product by ~-lminictering to said patient a therapeutically effective amount of any of the said SV40 viruses or pseudoviruses or a o therapeutically effective amount of said infected cells.

The invention also relates to pharm~(~ell~ical compositions comprising as activeingredient a therapeutically effective amount of the SV40 virus or pseudoviruses of the invention or a therapeutically effective amount of the infected cells of theinvention.

The constructs of the invention are very efficient in gene transfer into a variety of cells, including human hemopoietic cells and probably also stem cells. Thus, they may be suitable for treating a wide spectrum of diseases. Plasmids carrying the desired gene and the SV40 ori and, optionally ses, are encapsidated in COS cells, optionally with helpers, as SV40 pseudovirions, and tr~n~mitte~ into the target cells by viral infection. The prokaryotic DNA is removed after propagation in bacteriaand before encapsidation. The constructs include only 200bp of SV40 DNA, with cloning capacity of over 5kb. Thus plasmids carrying over 95% human DNA are efficiently transferred into human hemopoietic cells.

The invention provides for safer and cheaper products for medical use, which maybe prepared under aseptic conditions. A major advantage is that proteins are readily made in insect cells. While semi-purified proteins (nuclear extracts) are exemplified, purified proteins can be employed. Further, DNA is pl~aled in CA 0223~347 l998-04-30 W O 97/17456 PCT~L96/00143 bacteria and can be purified before it is packaged. This ensures high purity and high quality DNA minimi7:ing the chance for picking spontaneous mutations and/or led,~ gements. In addition, in vivo pseudovirions are present in a solution which also cont~in~ cons~ çnt~ of the cells in which they were grown. In contrast, 5 infection by retroviral vectors plc~al~d in vivo is done by co-culturing of the patients cells with the producer cell-lines (usually murine). Although in vivo ~)l~dlc;d viral sectors (such as adenoviruses or adeno-associated virus) can be purified, this may sub~t~nti~lly increase production cost, as purification is also associated with loss of virion particles. In addition, in helper-free p~çk~ging cell-l0 lines (of any virus) there is always a risk of cont~min~tion by recombinants. Thesecould be either the wild-type virus or unknown recombination products, carrying potentially harmful (cellular) genes. The risk is completely abolished when packaging is done in vitro. Moreover, in vitro packaging can accommodate larger plasmids than in vivo packaged SV40 pseudovirions: The in vivo packaged pseudovirions accommodate only up to about 5.4kb of DNA [Oppenheim, A. &
Peleg, A. (1989) Gene 77:79-86]. In the present in vitro method about 7.5kb havebeen packaged successfully, and larger plasmids can be packaged. Regulatory elements (e.g. ,~-globin~ LCR), which interfere with packaging in vivo, are not expected to interfere with packaging in vitro. An additional important advantage is 20 that the ses element is not required for in vitro packaging (Tables 2 and 3), reducing the size of the required SV40 sequences to about 100bp, comprising the ori, in the exemplified experiments. Embo~liment~ without even this element are also contemplated. In the present examples, the ori element was required for the assay of infectious units. The high flexibility afforded by the method and constructs of the 25 invention may allow the development of gene targeting (or gene replacement therapy).

CA 0223~347 1998-04-30 W ~ 97/17456 PCT~L96/00143 EXAl~PLES

Cloning ~*e genes of ~he SV40 capsid proteins for e~cpression in bacferia First, plasmids designed to express the complete VP1, VP2 and VP3 polypeptides as fusion proteins to glutathion-S-kansferase (GST) in E. coli [Smith, D.B. & Johnson, s K.S. (1988) Gene 67:31-40]. The respective SV40 fragments were cloned into the vector with the aid of PCR. Expression level was high, leading to the production of insoluble inclusion bodies. Similarly, it was recently reported [Clever et al., ibid.]
that a trllnc~te~l VP2 fused to GST also yielded an insoluble product.

Preparation of antibodies o The three GST-fusion capsid proteins were used to raise polyclonal antibodies in rabbits. Antibodies against GST-VP1 did not cross-react with VP2 and VP3. As expected, antibodies against GST-VP2 reacted both with VP2 and VP3, and did not cross-react with VP1.

Cloning the SV40 late genes for expression in insect cells 15 SV40 DNA fr~ nt~ were cloned into the plasmid vectors pVL1393 and pVL1392 (comrnercially available from PharMingen, San Diego, California), derived from Autographa californica nuclear polyhedrosis virus (AcMNPV) [Luckow, V.A. &
Summers, M.D. (1988) 6:47-55, Luckow, V.A. & Summers, M.D. (1989) Virology 170:31-39] In these vectors expression of the foreign gene is driven by the strong promoter for the viral occlusion protein, polyhedrin. The genes for the capsid proteins were cloned into pVL1393 as follows: VPl was cloned by introducing a StuI-Bcll DNA fragment (SV40 coordinates 1463-2770) into the plasmid cleaved by restriction endonucleases SmaI and BglII. The VP2 gene was cloned by ligating a HincII-EcoR~ fr~nent (522-1782) between the SmaI and EcoRI sites. The VP3 2s gene, which is nested in the VP2 gene (it is tr~n~l~te~l from an internal AUG signal), was cloned by using a Sau3AI-EcoRI fr~ nt (874-1782) and the BamHI and CA 0223~347 1998-04-30 W O 97/17456 PCTnL96/00143 EcoRI sites of pVL1393. A fourth late polypeptide, the agnoprotein (or LPl), encoded by the leader region of the late 16S mRNA, appears to play a role in expediting virion assembly [Carswell, S. & Alwine, J.C (1986) J: Yirol. 60:1055-1061; Resnick, J. & Shenk, T. (1986) J. Virol. 60:1098-1106] The agnogene, a s PvuII-MboI fragment (273-873), was cloned between the SmaI and BamE~ sites of plasmid pVL1392.

The structures of the four recombinant plasmids were confirmed by restriction analysis and after propagation in F~. coli. Sequence analysis was pc.rol,l-ed for The recombinant plasmids carrying VP2 and VP3. Recombinant baculovirus carrying o the four respective genes were produced using the BaculoGold kit (kindly provided by PharMingen, California). The technique relies on homologous recombination between the plasmid and a modified type of baculovirus with a lethal deletion. Each of the recombinant plasmids was co-transfected, together with linearized DNA of the defective baculovirus, into Spodoptera frugiperda (Sf9) cells. Virus was 15 harvested 4 days later, according to the protocol supplied by the m~nllf~ctllrer. To obtain high titer stocks each of the recombinant virus was amplified by 3 cycles of infection (5 cycles for the VP2 recombinant virus) of freshly seeded Sf9 cells [Summers, M.D. & Smith, G.E. (1988) A m~n~l~l of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station, 20 College Station, Texas] The final titers of the 4 recombinant baculoviruses stocks were 2-4x108 pfu/ml.

The SV40 proteins were produced in Sf9 cells, infected at a multiplicity of 10 pfu/cell and grown at 27~C. Cells were harvested and the soluble ~Lo~eins were analyzed by SDS-P~GE [Laemrnli, E.K. (1970) Nat~re 277:80-685] and Western 2s blotting [Harlow, E. & Lane, D. (1988) Antibodies, a laboratory manual. Cold Spring Harbor Laboratory, N.Y., Cold Spring Harbor] using antibodies raised against the corresponding GST-fusion proteins. Kinetic studies showed increasinglevels of the SV40 proteins from 3 to 6 days postinfection.

CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 T*e three capsid pro~eins ~emble spontaneously ~o form SV40-like parficles The SV40 capsid proteins were produced, separately, in Spodoptera frugiperda (SF9) cells from recombinant baculovirus each carrying the genes coding for VPl,VP2 or VP3. The cells were harvested and nuclear and cytoplasmic fractions were analyzed by SDS-PAGE and western blotting. The results demonstrated that VPl and VP3 were preferentially present in the nuclear fraction, whereas VP2 was preferentially cytoplasmic (not shown). When the three proteins were co-expressed in the same cells (following infection with the three recombinant baculoviruses together), all three proteins were present in the nuclear fraction. The capsid proteins o self-assembled to form SV40-like structures of various sizes. Electron microscopy of nuclear extracts of the infected cells showedl abundance of "empty" SV40-likecapsids and heterogeneous aggregates of variable size, mostly 20-45 nm (Fig. l(a)).
Under the same staining conditions wild type SV40 virions are 45 nm with well defined boundaries (Fig. l(b)). Occasionally smaller, presumable empty or defective particles, are also seen in wild type SV40 stocks.

To purify VPl, nuclear extracts of infected cells were placed on 15-35% glycerolgradient with a 2M sucrose cushion. Western blot analysis and EM studies demonstrated that the majority of VPl was present as high MW structures in 2 peaks, one at the cushion and the other at the bottom of the tube. About a third of the protein was present as pentamers (lOS units; MW ~210kd). In most of the experiments, monomers were not seen. The results indicate that VPl molecules readily form pentamers and high MW structures at this high concentration. VPl pentamers and higher MW structures are stabilized by S-S bonds [Ghar~kh~ni~n, E.
et al. (1995) Virology 207:251-254] and by Ca [Liddington et al., ibid.]. It appears that only at very low concentrations VPl molecules may remain monomeric [Ghar~kh~ni~n et al., ibid.].

Similar experiments, performed with nuclear extracts of cells con~ining the 3 capsid proteins, demonstrated that the three proteins co-precipitated together in the W O 97/17456 PCT~L96/00143 glycerol gradients in two high MW fractions, at the very bottom of the tube (fraction I) and at the cushion (fraction II). In addition, peaks corresponding to VPl pentarners and to VP2 monomers were also seen.

In SV40, the three capsid proteins are expressed from a single promoter with complex regulatory controls, including several transcription start sites, ~lt~rn~tive splicing to two major species, 16S RNA, producing the agnoprotein (which is not part of the capsid) and VPl and l9S, producing VP2 and VP3. The two bicistronic mes~ges contain int~rn~l translation initiation signals. This org~ni7~tion is thought to facilitate coor 1in~te-1 expression at the correct ratio for p~k~ging (Se~lm~n, S.A., 10 et al. (1989) J: Virol. 63:3884-3893; Ser1m~n~ S.A. et al. (1990) J: Virol. 64:453-457]. Co-production of the capsid proteins VPl, VP2 and VP3 in Sf9 cells was pclro~llled by infecting with the 3 baculovirus species at equal multiplicities.Nevertheless, the ratio of the 3 ploteills in fraction II was similar to the ratio obtained in monkey CMT4 cells tGerard, R.D. & Gl--7m~n, Y. (1985) Mol. Cell.
Biol. ~i:3231-3240] infected with wild type SV40. This was not true for fraction I.
Furthermore, EM studies revealed that fraction II was relatively homogeneous, cont~ining particles of approximately SV40 size, which appeared "empty", as theyallow penetration of the stain. On the other hand the particles in fraction I were highly heterogeneous in size.

20 DNA is packaged in fhe SV40 capsids in vifro, fo. ~,.i,.~ infectious parficles A. Pack~ging experiments were performed with SV40 DNA and with heterologous plasmid DNA. Nuclear extracts of Sf9 cells were prepared according to Schreiber [Schreiber, (1989) ibidl. 2~Ll of nuclear extracts (protein concentration 1-2~Lg/,ul) were mixed by vortex with l~Lg DNA in a total volume of 4 ~Ll and placed at 37~C25 for 6hr. Prelimin~ry experiments showed that shorter incubation periods (1-4hr) gave lower yields of infectious particles. CaC12 and MgC12 were added to final concentrations of 100,uM and 8mM respectively, to a total volume of 6,ul, and the reactions were incubated for an additional lhr on ice. DNase I digestion was CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 performed using 0.5 unit of enzyme for lOmin on ice, and stopped by the addition of EDTA to a final concentration of SmM.

- The DNase I tre~trn~nt was used to remove DNA which was not stably packaged.
The reaction products were assayed for infectious units (IU) on CM~4 monolayers,s 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. & Gln7m~n, Y. (1985) Mol. Cell. Biol.
5:3231-3240]. Sub-confluent monolayers were incubated with the packaging o mixture for 120 min at 37 C, with occasional agitation, followed by the addition of fresh medium cont~ining O.lmM ZnC12 and l~LM CdS04 for the induction of T-~ntigçn expression. Infective centers were scored by in situ hybridization. The number of infective centers obtained for a 6~L1 reaction mixture was used in computing the titer of IU/ml (Fig. 2(a)).

5 The procedure yielded infectious units both with SV40 DNA (Fig 2(c)) and with pS03cat DNA (Figs. 2(a) and 2(b)). A typical ~c.illlent, demonstrating infectivity of in vitro packaged pS03cat, is shown in Fig. 2(a). Under these conditions naked DNA sometimes also entered the cells. However, the DNase I treatment completely removed the naked DNA background from the assay. The experiments showed that using the nuclear extracts cont~inin~ VPl+VP2+VP3 directly, without purificationon glycerol gradients, yielded 90 infectious centers, per 6~L1 p~ck~ging reaction, equivalent to 1.5x104 infective units IU per ml. Pre-treatment before the infection with DNase I re~ ce~ the number to 77 (1.2x104 IU/ml). Incubation with nuclear extracts cont~ining VP1 alone produced 75 infectious centers (1.2x104 IU/ml), almost the same as with the three capsid proteins. However, The pre-tre~tm~nt with DNase reduced the number almost 2 fold, to 40 (a titer of 6.6x103 IU/ml). This was seen both for pS03cat DNA (Fig. 2(b)) and for SV40 DNA (Fig. 2(c)), suggesting that the DNA in those particles was not as effectively protected from DNase I, under CA 0223~347 l998-04-30 W O 97/17456 PCT~L96/00143 the conditions described below. These results suggest that VP2 and VP3 contribute to the stability of the DNA-capsid complexes. In the absence of DNase I tre~tment naked DNA was also sometimes infectious. However, DNase I digestion completely removed the naked background from the assay. Therefore, all subsequent s experiments included DNase I digestion. Fig. 2(c) also demonstrates that presence of the SV40 capsid proteins is required for the production of infectious particles, as "packaging" with nuclear extracts of uninfected Sf9 gave negative results. Few "infectious units" were seen in cells treated with SV40 DNA only, demonstrating the ability of naked DNA to penetrate cells. DNase I treatment completely removed o this background.

Insect cells were infected with three recombinant baculoviruses encoding for thethree capsid proteins, VP1, VP2 and VP3, at moi 10 each. After 4 days nuclear extracts were ~ d essentially as described by Schreiber [Schreiber, E., et al.
(1989) Nucleic Acids Res. 15:6419-6436] by ~h~king the nuclei, isolated with 10%NP-40, in a buffer cont~ining 20mM HEPES pH 7.9, 0.4M NaCl, lmM EDTA, PMSF and leupeptin were added just before use. The nuclei from each 75x cm2 culture bottle were extracted with 1 S0~L1 buffer.

The nuclear extract was used in p~k~ging experiments performed exactly as described above. For each packaging reaction 2,u1 nuclear extracts were mixed with 20 l,ug DNA (either SV40 or pS03cat) in a total volume of 4,~L1, for several hours at 37~C. The reaction mixture was transferred to ice, 2~11 of 0.4mM CaCl2 were added and incubated for 60 min on ice. This was followed by the addition of 2~1 cont~inin~ 0.5 unit of DNase I, and the incubation continlle~ for 10 min. on ice. The reaction was stopped by the addition of 2~L1 of 25mM EDTA (final concentration 25 SmM). Serum-free medium was added (300~L1) and the ll~i~lul~ was applied to asub-confluent CMT4 culture in a 6cm diameter culture plate. The cultures were incubated at 37~C with gentle agitation every 20 min. Two hours later the infection mixture was sucked off and 5ml fresh medium co~ g 5% FBS was added. After CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 about 40hr, to allow replication of the DNA tr~n~mitte~l by the infectious particles, the monolayers were transferred to nitrocellulose layers and processed for hybridization.

B. In another study, the inventors investigated the properties of the self-assembled s empty capsids shown in Fig. 1 and Fig. 3(b). It was found that DTT and EGTA
induced dissociation of the self-assembled empty capsids. Therefore, an alternative protocol was devised, in which the proteins are pre-treated with DTT and EGTA
prior to their incubation with the DNA. The DTT and EGTA are dialyzed out and replaced by Ca~ ions. Nuclear extracts of Sf9 were plepal~d as described above o [Shreiber et al. (1989), ibid.] from cells infected with the three recombinantbaculoviruses expressing VPl, VP2 and VP3 at multiplicity of infection (moi) 10 for each recombinant baculovirus. For each packaging reaction, 10~1 of nuclear extracts, cont~ining 10-15~Lg protein, are incubated with DTT at a final concentration of lOmM for 5 min at 33~C, with gentle ~h~kin~ to allow the capsid5 proteins to dissociate. The reactions are than cooled on ice, l~Lg of DNA is added to each reaction and the ingredients are thoroughly mixed by vortexing and incubated on ice for 30min. Total volume for each reaction is 20111. The reaction mixtures are then dialyzed against a buffer cont~ining lOmM CaCl2, 150mM NaCl, lOmM Tris-HCl pH 7.2 for 24hr in the cold, with two changes.

This protocol yielded, in 4 different experiments, a titer of l-2.5xl04 IU/ml, which is similar to the titer obtained in the above described protocol A.

Physical associa~ion befween capsids and DNA

To obtain physical evidence for the association of DNA with capsid particles, the reaction products were sedimented in a sucrose gradient. To prevent the action of - 2s endonucleases present in the nuclear extract, EDTA was added to a final concentration of 4mM. Under these conditions, non-packed DNA, which sedimented at the top of the gradient (as does free plasmid DNA, Fig.3(d)), CA 0223~347 1998-04-30 W O 97/17456 PCTnL96/00143 rPn-~ined intact and supercoiled, as was evident from agarose gel electrophoresis The majority of the capsid proteins to the sucrose cushion, similarly to ~llthentic SV40 (Figs. 3(a) and 3(c)). A small portion of the DNA (pS03cat) co-se-limentel with the capsid proteins to the sucrose cushion. That DNA was visible on agaroses gel electrophoresis and Southern blotting only after the particles were dissociated by tre~tment with EGTA and DTT at ~Ik~line pH, suggests that it had been cont~ined within particles. The DNA in those fractions migrated on the gel as did supercoiled pS03cat DNA (not shown), indicating the presence of intact plasmid molecules.
Analyses of the fractions that contained both capsid proteins and DNA (Fig. 3(a)o fractions 2-4) demonstrated the presence of infectious units. The results taken together indicate the formation of capsid particles which contain supercoiled plasmid DNA and which are infective.

Proteins presenf in nuclear extrac~s assisf in packaging in vitro SV40 assembly requires precise insertion of the carboxy-termin~l arms of the VPlmolecules into the neighboring p~ ..ers. However, an arm can easily insert, instead, into its own pentamer, thus in~e,r~ g with assembly. To explain how such mistakes are avoided, the participation of chaperones has been invoked [Li<1tlington, R.D, et al. (1991) ibid.]. The inventors ration~li7~?~1 that such chaperones may also be present in nuclear extracts of the Sfg cells, and that they may facilitate capsid 20 formation and entry of DNA into pre-formed aggregates. Indeed, experiments with crude nuclear extracts consistently yielded appr~ xim~tely 10 fold more infectious units than those perforrned with capsid aggregates which had been partially purified by glycerol gradients (Table 2). These results suggest that additional proteins present in the nuclear extract of Sf~ cells, presumably, chaperons, enhance DNA
packaging in vitro. As chaperones require ATP for their activity we investigatedwhether the addition of ATP to the reaction improves pack~ing Packaging was pelro~l"ed as above, in the presence of SmM ATP. The results shown in Table 1 W O 97/174S6 PCT~L96/00143 suggest that ATP may indeed improve in vitro p~Ck~ging, although the titers obtained in these e~l~clhllents were lower than the usual.

- The inventors have recently started to investigate whether the agnoprotein can also enhance DNA packaging in vitro. Nuclear extracts of Slg were prepared as s described above from cells infected with the four recombinant baculoviruses expressing VP1, VP2 and VP3 and the agnoprotein, at moi 10 for each recombinant baculovirus. Packaging of DNA was pl,.rollned after treatment of the nuclear extracts with DTT, as described above for the nuclear extract which contained the three capsid proteins. The results presented in Table 1, although inconclusive, 0 suggest that the agnoprotein may improve p~ck~ing The effect of the agnoprotein on packaging may be more critical when purified capsid l,roteil1s are used in the p~ck~ging reaction.

Table 1.

The effect of ATP on in vitro pac~ of pS03cat Infectious units/ml*

Capsid ~lotei,ls No ATP added + ATP

Nuclear extracts containing 2.7xlO~ 8xlO~
VP1+VP2+VP3 Nuclear extracts co,.l~i"i~-g 3.8x103 1.4x104 VP 1 +VP2+VP3+agnoprotein *titers after DNase I tre:ltment CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 The infectious particles Ir~~ il comp~ete DNA Ir.olec~ s which are biologically functional The results shown in Fig. 2(c) may ~le~ bly reflect DNA which did not penetrate the cells but remained adsorbed on the cell surface, in structures which are DNase I
5 resi~t~nt To prove that the in vitro packaged DNA entered the cells, and that the DNA is biologically active in gene expression, the inventors asked whether the cat gene tr~n~mi1te~1 by pS03cat expressed the CAT enzyme. CMT4 cells were harvested 3 days postinfection with in vitro packaged pS03cat. CAT assays [Oppenheim et al. (1986) ibidl demonstrated enzyme activity at a significant level o (Fig. 4(a)), although the moi in this experiment was very low (less than 1 IUx104 cells). These results indicate that the infectious units tr~ncmitt~ biologically active DNA into the target cells.

It was then to be verified whether complete DNA molecules became packaged in this experimental protocol. For production of SV40 virions, the complete SV40 5 molecule is required, including the regulatory region, and the early and the late genes. SV40 DNA was packaged as described above and used to infect CV-1 cells at a low moi (~lIU/104 cells). After 2 weeks extensive cell lysis was visible, indicating that productive SV40 infection was going on (Fig. 4(b)). Only complete SV40 DNA, which can produce functional T-antigen as well as the late proteins, can 20 produce virions on CV- 1 cells. It was therefore concluded that the in vitro packaging system described herein produces particles which contain the complete circular DNA molecules.

In vitro packaging is not dependent on ses A small DNA element, ses, is required for SV40 assembly in vivo. ses appears to 2s play multiple roles in pack~ing It is probably a recognition site for the capsid proteins, serving as a nucleation center in the initiations of viral assembly [Dalyot-Herman et al., ibidl. In addition, ses functions in regulating the late stages of the CA 02235347 l998-04-30 W O 97/17456 PCT~L96/00143 SV40 life cycle, in tllrnin~ off viral gene activity and by allowing the capsid proteins to induce nucleosomal reor~ ;on and chromatin conclen~tion in the transition from replication and late transcription to p~ck~ging [Oppenheim et al.
(1992) ibid.]. In vitro, the use of non-transcribing, naked plasmid DNA may be - 5 predicted to ch~;ulllvent part of the requirements for ses. As shown in Tables 1 and 2, ses+ (pSO~y-S) and ses~ (pSOy-N) plasmids are indeed packed in vitro at similar efficiencies, indicating that ses is dispensable for in vitro p~çk~ging Possibly, in vitro packaging of SV40 pseudovirions may allow efficient transfer of human genes without any accessory viral DNA.

0 Table 2 P~cl~gin~ with purified capsid proteins in comparison with total nuclear extracts of Sf9 cells infected with recombinant baculovirus.

Infectious units/ml Capsid protein SV40 pS03cat pSOyS pSO~N

Nuclear extract cont~ining l.5xl0~ 4x104 7.5xlO~ 9.5xlO~
VPl+VP2+VP3 Purifieda VPl+VP2+VP3 1.7x103 2.6x103 <1.7x103 1.6x103 Nuclear extract cont~inin~ 7.5x103 5.3x103 N.D. N.D.
VPl Purifieda VPl 6.7x103 <1.7x1()2 <1.7 X102 5 X102 Nuclear extract of 0 0 N.D. N.D.
uninfected SF9 cells apartially purified by glycerol gradient W O 97/17456 PCT~L96/00143 Plasmid signif cantly larger t*an SV40 can be packaged in vifro The genome size of SV40 plasmid is 5,243bp, and the upper limit of plasmid packaging is ~5.4-5.7 kb [Chang, X.B. & Wilson, J.H. (1986) J. Virol. ~8:393-401;
Dalyot, N. (1994) Regulation of human globin genes and the development of a 5 model for gene therapy of ,B-thalassemia, Ph.D. Thesis, The Hebrew University,Jerusalem]. SV40 DNA is packed in vivo as a minichromosome, complexed in nucleosomes, occupying large space within the virion particle [Martin, R.G. (1977) Virology 83:433-437]. Further experiments were pc~r~ -ed under the same conditions as before, except that instead of SV40 or pS03cat DNA other plasmids,lO significantly larger than SV40 size, were used. In each experimPnt~ Lg of plasmid DNA was incubated with 2~11 of nuclear extracts of Sf9 cells infected with VP1+VP2+VP3. The experiments were repeated at least twice for each pl~mi~l Typical results are shown in Table 3.

W O 97/17456 PCT~L96/00143 Table 3 In Vitro P~clr~in~ of Various Plasmid Plasmid units Properties Size(kb) Infectious units/ml SV~0 5.2 4.2x104 pS03cat 4.1 2.4x104 pSOl~-S ses+ 4.3 7.5x103 pSO~-N ses~ 4.2 9.5x103 pS06,B-lb carries ,B-globin 7.3 3.3x104 pS06,B-5 carries ~-globin+LCR element 7.4 1.2x104 pS06,B 9 b carries ,B-globin+LCR element 7.0 1.2x104 pSMl C carries MDRl 7.1 7.3x103 described in Oppenheim, A., ibid.
b described in Dalyot, N., Ph.D Thesis (1996) The Hebrew University, Jerusalem s c described in ,e.g., WO95/30762.

Impo~ ly, the experiments demonstrated that various pl~mid~, carrying useful genes, can be packaged. Furthermore, packaging in vitro is not limite-1 to 5.4-5.7kb of plasmid DNA, and plasmids over 7kb can be almost as efficiently packaged. This is ple~...-.~hly because under in vitro conditions naked DNA is packaged, ratherl0 than a minichromosome (which includes the DNA complexes in nucleosomes). The minichromosome occupies much more space, as coln~aled to DNA ofthe same size, within the pseudoviral particle. It is also possible that in the absence of histones, the internal capsid proteins VP2 and VP3 assist in plasmid DNA con(l~n~tion. The CA 0223~347 1998-04-30 W O 97/17456 PCT~L96/00143 titers obtained in these experiments, around 1-5x104 IU/ml, are co~ alable to titers obtained by in vitro packaging of a number of c~lenlly used vectors.

In vitro pslr.kz~ging is probably accomplished by a mech~ni~m which is differentfrom the packaging in vivo. In the latter process, the viral capsid proteins arethought to assemble around the viral minichromosome, while in vitro, empty capsid-like structure (Fig. 1) serve as starting material. Furthermore, in vitro packaging I~tili7~s naked DNA, prepared in E. coli. This result, combined with many of theinventors' previous studies on in vivo p~ck~ging, leads to the prediction that potent regulatory sequences, such as ,B-globin LCR, which interfere with viral packaging in o vivo [Chang et al. (1992) ibid, Dalyot et al. (1994) ibid.], will not interfere with in vitro pack~ging It is hypothesized that the LCR elements interfere with in vivo packaging of SV40 pseudovirions by the formation of higher order nucleoprotein structures which is not compatible with chromatin condensation [Dalyot, N. (1994) ibid.]. The use of supercoiled plasmid DNA, in the absence of regulatory proteins which bind to these regulatory elements, is predicted to relieve this problem in vitro.
Indeed, two LCR cont~ining plasmids, pS06~-5 and pS06,~-9, which are poorly packed in vivo (producing titers of 103 and ~104 IU/ml, respectively [Dalyot, N.(1994) ibid.], yield here titers similar to pS03cQt (Table 3). The present results suggest that in vitro packaging will allow to combine in the constructs for gene20 therapy the optimal regulatory signal, which will lead to important improvement in expression of the delivered genes.

Claims (48)

CLAIMS:

1. A construct capable of infecting a mammalian cell comprising at least one semi-purified or pure SV40 capsid protein; and a constituent selected from the group consisting of - an exogenous DNA encoding a[n] therapeutic exogenous protein or peptide product, or encoding therapeutic RNA, or itself a therapeutic product, - a vector comprising an exogenous DNA encoding a[n] therapeutic exogenous protein or peptide product, or encoding therapeutic RNA, or itself a therapeutic product, - an exogenous RNA encoding a[n] therapeutic exogenous protein or peptide product or itself a therapeutic product, - a vector comprising an exogenous RNA encoding a[n] therapeutic exogenous protein or peptide product or itself a therapeutic product, - a[n] therapeutic 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 said mammalian cell; and [optionally] further comprising operatively linked regulatory elements sufficient for the expression and/or replication of said exogenous protein in a mammalian cell.

2. A construct according to claim 1 [optionally] further comprising additional SV40 protein or proteins, preferably SV40 agnoprotein.

3. A construct according to claim 1 or claim 2 comprising a mixture of at least two semi-purified or pure SV40 capsid proteins.

4. A construct according to any one of claims 1 to 3 comprising a mixture of three semi-purified or pure SV40 capsid proteins.

5. A construct according to claim 1 to 4 wherein said SV40 capsid protein is semi-purified or pure VP1 or VP2 or VP3.

6. A construct according to any one of claims 1 to 5 wherein said constituent is exogenous circular or linear DNA encoding a[n] therapeutic 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 a[n] therapeutic exogenous protein or peptide product.

7. A construct according to claim 6 wherein said DNA is DNA which encodes a therapeutic protein or peptide 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, or is DNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in defective form or is DNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in physiologically abnormal or normal amount, or encodes a therapeutic RNA.

8. A construct according to claim 6 or claim 7 wherein said therapeutic protein or peptide product is an enzyme, a receptor, a structural protein, a regulatory protein or a hormone.

9. A construct according to any one of claims 6 to 8 comprising SV40-derived ori DNA sequence as a replication regulatory element and further comprising a DNA sequence encoding one or more regulatory elements sufficient for the expression of said exogenous RNA or exogenous protein or peptide in said mammalian cell.

10. A construct according to any one of claims 1 to 5 wherein said constituent is exogenous RNA, wherein said RNA is RNA which encodes a therapeutic protein or peptide product which is not made or contained in said cell, or is RNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in abnormally low amount, or is RNA which encodes a therapeutic protein or peptide product which is made contained in said cell in defective form, or is RNA which encodes a therapeutic protein or peptide product which 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.

11. A construct according to claim 10 wherein said therapeutic protein or peptide product is an enzyme, a receptor, a structural protein, a regulatory protein or a hormone.
[12. A construct according to any one of claims 1 to 5 wherein said constituent is an exogenous protein or peptide product.]

12. A construct according to any one of claims 1 to 5 [12] wherein said constituent is a therapeutic exogenous protein or peptide product which 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, or is a therapeutic protein or peptide product which is made or contained in said cell in defective form or is a therapeutic protein or peptide product which is made or contained in said cell in physiologically abnormal or normal amount.

13. A construct according to any one of claims 1 to 5 wherein said constituent is antisense RNA or DNA or ribozyme RNA, or any RNA or DNA which inhibits or prevents the expression of undesired protein/s in said mammalian cell.

14. A construct according to claim 13 wherein said antisense RNA is antisense RNA directed against the bcr/abl transcript.

15. A construct according to claim 13 wherein said antisense RNA is antisense RNA directed against a HIV transcript.

16. A construct according to any one of the preceding claims wherein said cell is a human cell selected from the group consisting of hemopoietic cells, epithelial cells, endothelial cells, liver cells, epidermal cells, muscle cells, tumor cells, nerve cells and germ line cells.

17. A construct according to claim l6 wherein said hemopoietic cells are bone marrow cells, peripheral blood cells and cord blood cells, or liver cells.

18. A method for the in vitro construction of SV40 viruses or pseudoviruses comprising exogenous nucleic acid comprising the following steps:
a. [bringing] allowing a semi-purified or pure SV40 capsid protein or a mixture of at least two such proteins to self-assemble into SV40-like particles; and b. bringing the SV40-like particles assembled in step (a) into contact with said exogenous nucleic acid to give recombinant SV40 viruses or with a vector comprising said exogenous nucleic acid to give pseudoviruses. [and b. optionally subjecting the SV40 viruses or pseudoviruses formed in step (a) to digestion by nuclease to remove non-packaged DNA.]

19. The method of claim 18 wherein said recombinant SV40 viruses or pseudoviruses are subjected to digestion by nuclease to remove non-packaged DNA.

20. A method according to claim 18 or 19 wherein in step (a) at least one other SV40 protein, preferably SV40 agnoprotein, is added to the mixture of said SV40 capsid protein/s and said nucleic acid.

21. A method according to any one of claims 18 to 20 wherein said SV40 capsid protein is semi-purified or pure SV40 VPI, VP2, or VP3.

22. A method according to any one of claims 18 to 21 wherein said exogenous nucleic acid is circular or linear DNA.

23. A method according to any one of claims 18 to 21 wherein said exogenous nucleic acid is RNA.

24. A method according to any one of claims 18 to 23 wherein said exogenous nucleic acid encodes a therapeutic protein or peptide product or itself a therapeutic product.

25. A method according to claim 22 wherein said DNA is DNA which encodes a therapeutic protein or peptide 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, or is DNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in defective form or is DNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in physiologically abnormal or normal amount or is DNA which encodes a therapeutic RNA.

26. A method according to claim 25 wherein said exogenous DNA encodes a therapeutic protein or peptide product which is an enzyme, a receptor, a structural protein, a regulatory protein or a hormone.

27. A method according to any one of claims 18 to 20 wherein in step (b) SV40-derived ori DNA sequence is added and said exogenous nucleic acid [optionally] has DNA sequence encoding one or more regulatory elements sufficient for the expression of said exogenous protein in said mammalian cell operatively linked thereto.

28. A method according to any one of claims 18 to 20 wherein said nucleic acid is exogenous RNA, wherein said RNA is RNA which encodes a therapeutic protein or peptide product which is not made or contained in said cell, or is RNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in abnormally low amount, or is RNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in defective form or is RNA which encodes a therapeutic protein or peptide product which is made or contained in said cell in physiologically abnormal or normal amount and wherein said RNA has regulatory elements, including translation signal, sufficient for the translation of said protein product in said mammalian cell, operatively linked thereto.

29. A method for the in vitro construction of recombinant SV40 viruses or pseudoviruses comprising an exogenous therapeutic protein or peptide comprising the following steps:
a. [bringing] allowing a semi-purified or purified SV40 capsid protein or a mixture of at least two such proteins to self-assemble into SV40-like particles; and b. bringing the SV40-like particles assembled in step (a) into contact with said exogenous protein to give recombinant SV40 viruses or pseudoviruses. [and b. optionally purifying the recombinant viruses or pseudoviruses obtained in step (a) from any non-packaged protein.]

30. A method according to claim 29 wherein said recombinant SV40 viruses or pseudoviruses are purified from any non-packaged protein.

31. A method according to claim 29 or claim 30 wherein said exogenous protein or peptide are, respectively, a naturally occurring or recombinant protein or peptide, a chemically modified protein or peptide, or a synthetic protein or peptide.

32. A method according to claim 31 wherein said exogenous protein or peptide product are, respectively, a therapeutic protein or peptide product not made or contained in said cell, or are a therapeutic protein or peptide product made or contained in said cell in abnormally low amount, or are a therapeutic protein orpeptide product made or contained in said cell in defective form or are a therapeutic protein or peptide product made or contained in said cell in physiologically abnormal or normal amount.

33. A method according to any one of claims 18 to 32 wherein said cell is a human cell selected from the group consisting of hemopoietic cells, muscle cells, tumor cells, nerve cells and germ line cells.

34. A method according to claim 33 wherein said hemopoietic cells are bone marrow cells, peripheral blood cells and cord blood cells, or liver cells.

35. A method for the in vitro construction of SV40 pseudoviruses comprising exogenous antisense RNA, or ribozyme RNA or RNA or DNA which inhibits or prevents the expression of undesired protein/s in a mammalian cell, comprising the following steps:
a. [bringing] allowing a semi-purified or pure SV40 capsid protein or a mixture of at least two such proteins to self assemble into SV40-lilke particles: and b. bringing said SV40-like particles obtained in step (a) into contact with said exogenous antisense RNA, or ribozyme RNA, or RNA or DNA
which inhibits or prevents the expression of undesired protein/s in a mammalian cell, to give recombinant SV40 pseudoviruses. [and b. optionally subjecting the SV40 pseudoviruses formed in step (a) to digestion by nuclease to remove non-packaged DNA.]

36. The method of claim 35 wherein said pseudoviruses are subjected to digestion by nuclease to remove non-packaged DNA.

37. A method according to claim 35 or 36 wherein in step (a) at least one other SV40 protein, preferably SV40 agnoprotein, is added to the mixture of SV40 capsid protein/s and the exogenous nucleic acid or antisense nucleic acid.

38. A method according to any one of claims 35 to 37 wherein said SV40 capsid protein is semi-purified or pure SV40 VP1, VP2, or VP3.

39. A method according to any one of claims 35 to 38 wherein said antisense RNA
is antisense RNA directed against the bcr/abl transcript.

40. A method according to any one of claims 35 to 38 wherein said antisense RNA
is antisense RNA directed against a HIV transcript.

41. A mammalian cell infected with a construct of any one of claims 1 to 17.

42. An infected human cell according to claim 41 selected from the group consisting of hemopoietic cells, muscle cells, tumor cells, nerve cells and germline cells.

43. A mammalian cell infected with a construct obtained by the method of any one of claim 18 to 40.

44. An infected human cell according to claim 43 selected from the group consisting of hemopoietic cells, muscle cells, tumor cells, nerve cells and germline cells.

45. A method of providing a therapeutic DNA, RNA, antisense RNA, ribozyme RNA, protein or peptide product to a patient in need of such product by administering to said patient a therapeutically effective amount of the SV40 viruses or pseudoviruses according to any one of claims 1 to 17.

46. A method of providing a therapeutic DNA, RNA, antisense RNA, ribozyme RNA, 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 41 to 44.

47. Pharmaceutical compositions comprising as active ingredient a therapeutically effective amount of the SV40 viruses or pseudoviruses according to any one of claims 1 to 17.

48. Pharmaceutically compositions comprising as active ingredient a therapeutically effective amount of infected cells according to any one of claims 41 to claim 44.