Note: Descriptions are shown in the official language in which they were submitted.
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IN-VITRO CONSTRUCTION OF SV40 VIRUSES AND
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
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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
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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
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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.
=
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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.
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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
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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.
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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.
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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.
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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.
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11
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
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12
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.
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13
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
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14
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].
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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.
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16
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.
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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.
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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
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19
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
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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).
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21
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
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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.
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23
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
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24
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
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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
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26
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
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27
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)),
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28
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
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29
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
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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
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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
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32
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.
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33
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
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34
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.
