Note: Descriptions are shown in the official language in which they were submitted.
W093/2~698~ PCT/~S93/04706
~137361
VE~TOR PARTICLES RESISTANT TO INACTIVATION
BY ~nMAN 5ERUM
' ~
This invention relates to "injectable" vector
particles. More particularly, this invention relates to
vector particles, such as retroviral vector particles,
wherein such vector particles are resistant to i~activation
by human serum.
Vector particles are u~eful agents for introducing
gene(~) or DNA (RNA) into a cell, such as a eukaryotic cell.
The gene(~) is controlled by an appropriate promoter.
Examples of vectors which may be employed to generate vector
particles includ~ prokaryotic vectors, such as bacterial
vectors; eukaryotic vectors; including fungal vectors such
as yeast vectors; and viral vectors such as DNA virus
vectors, RNA virus vectors, and retroviral vectors.
Retroviruses which have been employed for generating vector
particles for introducing genes or DNA (RNA) into a cell
include Moloney Murine Leukemia Virus, Spleen Necrosis
Virus, Rous Sarcoma Virus and Harvey Sarcoma Virus. The
term "introducing" as u~ed herein encompasses a variety of
methods of transfering genes or DNA tRNA) into a cell. Such
methods include transformation, transduction, transfection,
and infection.
Vector particles have been used for introducing DNA
(RNA) into cells for gene therapy purposes. In general,
such a procedure involves obtaining cells from a patient and
using a vector particle to introduce desired DNA (RNA) into
the cells and then providing the patient with the engineered
cells for a therapeutic purpose. It would be desirable to
provide alternative procedures for gene therapy. Such an
alternative procedure would involve genetically engineering
SUBSTITUTE SH~ET (RVLE 26)
:
W093/2569~ 2 1 3 7 3 ~ 1 PC~/US93/047~6
r~
cells in vivo. In such a procedure, a vector particle which
includes the desired DNA (RNA) would be administered to a
patient for in v vo delivery to the cells of a patient.
It is therefore an object of the present invention to
provide gene therapy by introduction of a vector particle,
uch as, for example, a retroviral vector particle, into a
patient, wherein the vector particle is resistant to
inactivation ~y human serum.
In accordance with an aspect of the present invention,
there is provided a vector particle which is resistant to
inactivation by human serum. Preferably, the vector
particle is a viral vector particle, and more preferably the
viral vector particle is a retroviral vector particle.
The envelope portion of retroviruses include a protein
known as pl5E, and Applicants have found that retroviruses
are susceptible to inactivation by human serum as a result
of the action of complement protein(s) present in serum on
the pl5E protein portion of the retrovirus. Applicants have
further found that such retroviruses can be made resistant
to inactivation by human serum by mutating such pl5E
protein.
In one embodiment, there is provided a retroviral
vector wherein a portion of the DNA encoding pl5E protein
(shown in the accompanying sequence listing), has been
mutated to render the vector particle resistant to
inactivation by human serum. The terms "mutated" and
"mutation" as used herein mean that the DNA encoding pl5E
protein has been changed such that at least one but not àll
of the amino acids of pl5E protein have been changed (such
changes can include point mutations, deletions, and/or
insertions).
pl5E protein is a viral protein having 196 amino acid
residues. ln viruses, sometimes all 196 amino acid residues
SUBSlITUTE SHEET (RULE 26)
W093/~5698 ~ ~ 3 7 ~ 6 1 PCT/US93/047~6
f~
are present, and other times, amino acid residues 181 to 196
(known as the "r" peptide), are not present, and the
resulting protein is the "mature" form of pl5E known as
pl2E. Thus, viruses can contain both the pl5E and pl2E
proteins. pl5E protein is anchored in the viral membrane
such that amino acid residues 1 to 134 are present on the
outside of the virus. Although the present invention is not
to be limited to any of the following reasoning, Applicants
believe complement proteins may bind to this region whereby
such binding leads to inactivation and/or lysis of the
retrovirus. In particular, the pl5E protein includes two
regions, amino acid residues 39 to 61 (sometimes hereinafter
referred to as region 1), and amino acid residues 101 to 123
(sometimes hereinafter referred to as region 2), which
Applicants believe have 'an external location in the
three-dimensional structure of the pl5E protein; i.e., such
regions are directly exposed to human serum. Region 2 is a
highly conserved region in many retroviruses, even though
the amino acid ~eguences of this region are not identical in
all retroviruses. Such regions are complement binding
regions. Examples of complement proteins which may bind to
the complement binding regions are ClS and ClQ, which bind
to regions 1 and 2.
In order to inactivate the retrovirus, complement proteins
bind to both region 1 and region 2. Thus, in a preferred
embodiment, at least one portion of DNA encoding a
complement binding region of pl5E protein has been mutated.
Such a m~ltation results in a change of at least one amino
acid residue of a complement binding region of pl5E protein.
The change in at least one amino acid residue of a
complement binding region of pl5E protein prevents binding
of a complement protein to the complement binding region,
thereby preventing complement inactivation of the
SUBST~TIJTE SHEET (RULE 26~
W093/~5698 21373~1 PCT/US93/04706
retrovirus. In one embodiment, at least one amino acid
residue in both complement binding region~ of pl5E protein
is changed, whereas in another embodiment, at least one
amino acid residue in one of the complement binding regions
i 9 cha~ged.
It is to be understood, howaver, that the entire DNA
se-guence encoding pl5E protein cannot be mutated because
such a change renders the vectors unsuitable for in vivo
use.
In one embodiment, the at least one portion of DNA
encoding pl5E protein is mutated such that at least one
positively charged amino acid residue or negatively charged
amino acid residue is changed to an amino acild residue
having the opposite charge.
The positively charged amino acids are His, Lys, and
Arg.
The negatively charged amino acids are Asp and Glu.
In another~embodiment, the at least one portion of DNA
encoding pl~E protein is mutated such that at least one
positively charged amino acid or negatively charged amino
acid is changed to a noncharged amino acid.
In one embodiment, the at least one portion of DNA
encoding a complement binding region of pl5~ protein, which
is mutatad, encodes one or more of amino acid residues 101
to 123 of pl5E protein. In one em~odiment, the at least one
portion of DNA encoding pl5E protein is mutated such that
amino acid residue 122 is changed.
In one embodiment, the at least one portion of DNA
encoding pl5E protein is mutated such that at least one of
amino acid residues 117~ and 122 are changed. Preferably,
amino acid residue 117 is changed from Arg to Glu, and amino
acid residue 122 i8 changed from Glu to Gln.
SUBSTITUTE SHEET (RULE 26)
W093/25698 ~ PCT/US93/04706
2137361
In another embodiment, the at least one portion of DNA
encoding pl5~ protein is mutated ~uch that amino acid
residues 104, 105, 109, and 111 are changed. Preferably,
amino acid residue 104 is changed from Arg to His, amino
acid residue 105 is ch~nged from Asp to Asn, amino acid
residue lO9 is changed from Lys to Gln, and amino acid
residue 111 is changed from Arg to Gln.
In another embodiment, the at least one portion of DNA
encoding pl~E protein is mutated such that amino acid
residues 104, 105, 109, 111, 117, and 122 are changed.
Preferably, the at lea~t one portion of DNA is mutated such
that amino acid resid~e 104 is changed from Arg to His,
amino acid residue 105 is changed from Asp to Asn, amino
acid residue 109 is changed from Lys to Gln, amino acid
residue 111 is changed from Arg to Gln, amino acid residue
117 is changed from Arg to Glu, and amino acid residue 122
is changed from Glu to Gln.
In yet another alternative embodiment, the mutation of
DNA encoding pl5E protein may be effected by deleting a
portion of the pl5E gene, and replacing the deleted portion
of the pl5E gene, with fragment(s) or portion(s) of a gene
encoding another viral protein. In one embodiment, one
portion of DNA encoding the pl5E protein is replaced with a
fragment of the gene encoding the p21 protein, which is an
HTLV-I transmembrane protein. HTLV-I virus has been found
to be resistant to binding by complement proteins and thus
HTLV-I is resistant to inactivation by human serum (Hoshino,
et al., Nature, Vol. 310, pgs. 324-325 ~1984)). Thus, in
one embodiment, there is also provided a retroviral vector
particle wherein a portion of the plSE protein has been
deleted and replaced with a portion of another viral
protein, such as a portion of the p21 protein.
SU~STITUTE SHEET (RULE 26)
wn 93/25698 213 7 ~ 61 PCT/US93/04706
p21 protein (as shown in the ac~ompanying sequence
listing) is a protein having 176 amino acid residues, and
which, in relation to pl5E, has significant amino acid
sequence homology. In one embodiment, at least amino acid
residues 39 to 61, and lOl to 123 are deleted from pl5E
protein, and replaced with amino acid residues 34 to 56 and
96 to 118 of p21 protein. In one alternative, at least
amino acid residues 39 to 123 of plSE protein are deleted
and repla~ed with amino acid residues 34 to 118 of p21
protein.
In another embodiment, amino acid residues 39 to 69 of
pl5E protein are deleted and replaced with amino acid
residues 34 to 64 of p21 protein, and amino acid residues 96
to 123 of pl5E protein are deleted and replaced with amino
acid residues 91 to 118 of p21 protein.
Vector particles thu~ generated, and which are
resistant to inactivation by human serum, may be engineered
surh that the vector particles may, when introduced into a
patient, travel directly to a target cell or tissue. Thus,
in a preferred embodiment, the vector particle further
includes a protein which contains a receptor binding region
that binds to a receptor of a human target cell, such as,
for example, but not limited to, the amphotropic cell
receptor.
The retroviral vectors hereinabove described, may be
constructed by genetic engineering techniques known to those
skilled in the art.
In one embodiment, the retroviral vector may be of the
LN series of vectors, as described in Bender, et al.,
J.Virol., Vol. 61, pgs. 1639-1649 (1987) and Miller, et al.,
Biotechniques, Vol. 7, pgs. 980-99O (1989).
In another embodiment, the retroviral vector includes a
multipLe r-~triction enzyme site, or multiple cloning site.
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i
SUBSTiTUTE SHEET (RULE 26)
W093/2~698 ; PCT/~S93/04706
i-` 2137361
The multiple cloning site includes at least four cloning, or
restriction enzyme sites, wherein at least two of the sites
have an average freguency of appearance in eukaryotic genes
of less than once in lO,OO0 ba3e pairs; i.e., the
restriction product has an average ~ize of at least 10,000
base pairs.
In general, such restriction sites, also sometimes
hereinafter referred to as "rare" sites, which have an
average frequency of appearance in eukaryotic genes of less
than once in lO,OOO base pairs, contain a CG doublet within
thsir recognition ~eguence, such doublet appearing
particularly infrequently in the mammalian genome. Another
measure of rarity or scarcity of a restriction enæyme site
in mammals is its representation in mammalian ~iruses, such
as SV40. In general, an en~yme whose recognition sequence
is absent in SV40 may be a candidate for being a "rare"
mammalian cutter.
Examples of restriction enzyme sites having an average
frequency of appearance in eukaryotic genes of less than
once in 10,000 base pairs include, but are not limited to
the NotI, SnaBI, SalI, XhoI, ClaI, SacI, EagI, and SmaI
sites. Preferred cloning sites are selected from the group
consisting of NotI, SnaBI, SalI, and XhoI.
Preferably, the multiple cloning site has a length no
greater than about 70 base pairs, and preferably no greater
than about 60 base pairs. In general, the multiple
restriction enzyme site, or multiple cloning site is located
between the 5'LTR and 3'LTR of the retroviral vector. The
5' end of the multiple cloning site is no greater than about
895 base pairs from the 3'~end of the 5' LTR, and preferably
at least about 375 base pairs from the 3' end of the 5' LTR.
The 3' end of the multiple cloning ~ite is no greater than
about 40 base pairs from ~he 5' end of the 3' LTR, and
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W093~25698 ~ 1 3 ~ 3 6 1 PCT~US~3/04706
f
preferably at least 11 baYe pairs from the 5' end of the 3'
LTR.
Such vectors may be engineered from existing retroviral
vectors through genetic engineering techniques known in the
art such that the resulting retroviral vector includes at
least four cloning sites wherein at least two of the cloning
sites are ~elected from the group consisting of the NotI,
SnaBI, SalI, and XhoI cloning sites. In a preferred
embodiment, the retroviral vector includes each of the NotI,
SnaBI, SalI, and XhoI cloning sites.
Such a retroviral vector may serve as part of a cloning
system for the transfer of genes to eukaryotic cells. Thus,
there may be provided a cloning system for the manipulation
of genes in a retroviral vector which încludes a retroviral
vector including a multiple cloning site of the type
hereinabove described, and a shuttle cloning vector which
includes at least two cloning sites which are compatible
with at least two cloning sites selected from the group
consisting of NotI, SnaBI, SalI, and XhoI located on the
retroviral vector. The shuttle cloning vector also includes
at least one desired gene which is capable of being
transferred from said shuttle cloning vector to said
retroviral vector.
The shuttle cloning vector may be constructed from a
basic "backbone" vector or fragment to which are ligated one
or more linkers which include cloning or restriction enzyme
recognition sites. Incl~ded in the cloning sites are the
compatible, or complementary cloning sites hereinabove
described. Genes and/or promoters having ends corresponding
to the restriction sites of the shuttle vector may be
ligated into the shuttle vector through techniques known in
the art.
Il
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2137~61
The shuttle cloning vector c~n be employed to amplify
DNA sequences in prokaryotic systems. The shuttle cloning
vector may be prepared from plasmids generally used in
prokaryotic systems and in particular in bacteria. Thus,
for example, the shuttle cloning vactor may be derived from
plasmids such as pBR322; pUC18; etc.
It is also contemplated that within the scope of the
present invention that the DNA encoding pl5E protein which
has been mutated to render a vector particle resistant to
inactivation by human serum, may be contained in an
expression vehicle other than a retroviral vector. Such
expression vehicles include, for example, viral vectors
other ~han retroviral vectors, or any expression plasmid
which is capable of being transferred into a cell l:ine which
is capable of producing vector particles which include the
mutated pl5E protein.
Such vectors or expression vehicles which contain DNA
encoding a mutated env protein such as the mutated pl5E
protein hereinabove described, are transferred into
pre-packaging cell line to generate vector particles. In
general, the pre-packaging cell line contains the gag and
pol proteins of the virus, plus a retroviral vector lacking
the structural gag, pol, and env proteins. An example of
such a pre-packaging cell line is the GPL pre-packaging cell
line which consists of an NIH 3T3 mouse fibroblast cell line
, which contains an expression plasmid for MoMuLV gag-pol
i protein as well as the retroviral vector LNL6 (Miller, et
al., Biotechniques, Vol. 7, pgs. 980-990 (1989)). It is to
be understood, however, that the scope of the present
invention is not to be limited to any particular
I pre-packaging cell line.
Upon transfection of the pre-packaging cell line with
an expression vehicle containing DNA encoding a mutated env
r
Sl.~STITUTE SHEET (RULE 26)
W093/25698 2 1 3 7 3 6 1 PCT/US93/04706
protein, the pre-packaging cell line will generate vector
particles. The vector particles are then tested for
complement res~stance. The vector particles which are shown
to be complement resi stant (i.e., not inactivated by human
serum)j therefore, contain complement resistant envelope
proteins encoded by a specific envelope expression vehicle.
Such an expr~ssion vehicle can then be u~ed, by techniques
known to those skilled in the art, to produce a packaging
cell line which contains an expression vehicle encoding the
retroviral gag and pol proteins, and an expression vehicle
rontaining a gene encoding the mutated env protein ~such as,
for example, an expression vehicle or expression plasmid
containing a mutated pl5E protein such as hereinabove
described), whereby such packaging cell line may be employed
to generate vector particles which are _esistant to
inactivation by human serum. In particular, a retroviral
vector which lacks the structural gag, pol, and env genes,
but includes a desired gene of interest, may be transferred
into such a packaging cell line. Thus, the packaging cell
line may generate vector particles which contain a desired
gene(s) of interest, and which are resistant to inactivation
by human serum.
The vector particles generated from the packaging cell
line will not be inactivated when contacted with human
serum; and in addition, such vector particles, when
engineered with protein containing a receptor binding region
for a human receptor, are targetable, whereby the receptor
binding region for a human receptor enables the vector
particlec to bind to a target cell. Thus, such retroviral
vector particles may be directly introduced into the body
(e.g., by intravenous, intramuscular, or subcutaneous
injection, intranasally, orally, rectally or vaginally), and
travel to a desired target cell. Such vector particles,
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!
therefore, are useful for ~he introduction of desired
heterologous genes into target cells ~ as a gene
therapy procedure.
Thus, preferably, the vectors of the present invention
~urther include at least one heterologous gene.
Heterologous or foreign genes which may be placed into the
vector or vector particles include, but are not limited to,
genes which encode cytokines or cellular growth factors,
such as ly~phokines, which are growth factors for
lymphocytes. Other examples of foreign gen~s include, but
are not limited to, genes encoding Factor VIII, Factor IX,
tumor nerosis factors (TNF's), ADA, ApoE, ApoC, and Protein
C.
The vectors of the present invention include one or
more promoters. Suitable promoters which may be employed
include, but are not limited to, the retroviral LTR; the
SV40 promoter; and the human cytomegalovirus (CMV) promoter
described in Miller, et al., Biotechniques, Vol. 7, No~ 9,
pgs. 980-990 (1989), or any other promoter (eg., cellular
promoters such as eukaryotic cellular promoters including,
but not limited to, the histone, pol III, and B-actin
promoters). Other viral promoters which may be employed
include, but are not limited to adenovirus promoters, TK
promoters, and Bl9 parvovirus promoters. The selection of a
suitable promoter will be apparent to those skilled in the
art from the teachings contained herein.
The vectors of the present invention may contain
regulatory elements, where necessary, to ensure tissue
specific expression of the desired heterologous gene(s),
and/or to regulate expression of the heterologous gene(s) in
response to cellular or metabolic signals.
- Although the invention has been d~scribed with respect
to retroviral vector particles, other viral vector particles
' -11-
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~ ~r~ i . ' ' ' ' ' ' '
W093/25698 2 1 3 7 3 6 1 PCT/US93/04706
~,
(such as, for example, adenovirus and adeno-associated virus
particles), or synthetic particles may be constructed
wherein a region of the envelope protein in the vector
particle may be mutated such that the vector particle
becomes resistant to inactivation by human serum, thereby
making such vector particles suitable for in vivo
administration.
The invention will now be described with respect to the
following example; however, the scope of the present
invention is not intended to be limited thereby.
ExamPle
Plasmid pCE2 was constructed from pBR322 such that the
resulting plasmid pCE2 includes genes encoding the envelope
proteins gp70 and pl5E. pBR322 (Figure 1) was cut with
EcoRI and filled in to dèstroy the EcoRI site to give
pBR3Z2 RI. pBR322 Rl was then cut with NdeI and filled in
to destroy the NdeI site to give pBR322 R N.
pBR322 R N was digested with HindIII and EcoRV, and
cloned into the HindIII/EcoRV fragment was a HindIII/FspI
cassette containing the gp70 and pl5E genes under the
control of a cytomegalovirus (CMV) intermediate aarly
promoter with a polyA (adenine) tail from SV40 (Figure 2)
from plasmid pCEE. (Fi~ure 3). The HindIII/FspI cassette
obtained from plasmid pCEE contains a CMV intermediate early
promoter in which the BalI/SacII (bp 21 to bp 766) was
converted to an HindIII~SalI fragment by linker addition;
the ecotropic envelope BglII/NheI fragment (bp 5408 to bp
7847 of MoMuLV, encoding gp70 and pl5E) was filled and EcoRI
linkers were added; and the SV40 poly A signal from BclI to
BamHI (bp 2770 to bp 2533) was cloned into a BamHI site
(thereby destroying the BclI site). A BglII site was added
at the 3' end of the gp70 gene. (Thi~ addition does not
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2137~61. -
, .
alter any amino acids). The resulting plasmid is pCE2~Figure 4.~
To create the mutations in ~he pl5E gene, subcloning is
carried out in a different plasmid called pUC-E2.
(Figure 6.) This plasmid is pUC18 (Figure 5) with the PvuII
fragment removed and replaced with EcoRI linker~. Such was
accomplished by digesting pUC18 with PvuII to remove a 322
bp P~II fragment, and EcoRI linkers were then added. Into
the EcoRI site was cloned the MoMuLV ecotropic envelope gene
(i.e., the gp70 and pl5E genes from pCE2) from the BglII
site (5408) to the NheI site (7847), which ha~e been blunted
and had EcoRI linkers added. The resulting pUC-E2 plasmid
(Figure 6) therefore has unique BglII, SpeI, ClaI, and PvuII
sites in and around the pl5E gene.
PCR primers are then synthesized to encode the
following mutations i~ the pl5E protein (using pCE2 as a
template):
1. Amino acid residue 117 is changed from Arg to Glu,
and amino acid residue 122 i5 changed from Glu to Gln;
2. Amino acid residue 104 is changed from Arg to His,
amino acid residue 105 is changed from Asp to Asn, amino
acid residue 109 is changed from Lys to Gln, and amino acid
residue 111 is changed from Arg to Gln; and
3. Amino acid residue 104 is changed from Arg to His,
amino acid residue 105 is changed from Asp to Asn, amino
acid residue 109 is changed from Lys to Gln, amino acid
residue 111 is changed from Arg to Gln, amino acid residue
117 is changed from Arg to Glu, and amino acid residue 122
is changed from Glu to Gln.
Each PCR prod~ct is digested with SpeI and PvuII, and
cloned into pUC-E2 at the unique SpeI and PvuII sites. The
resulting plasmids are then seguenced to confirm the point
mutations.
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f ~
_,
DNA fra~ments bearing these mutations are then
subcloned into the expression plasmid pCE2. pCEZ is
digested with EcoRI and the envelope DNA fragment is removed
and replaced with the EcoRI envelope fragment ~rom the
pUC-E2 plasmids. The resulting pCE2 plasmids are then
checked for orientation of the EcoRI fragment and seguenced
again (only at the cloning site junctions and at the regions
bearing the point mutations) to confirm the presence of the
newly created mutated pl5E genes. The resulting expression
plasmids are identified as follows:
pCR68 - includes mutations in which amino acid residue
117 is changed from Arg to Glu, and amino acid residue
122 is changed from Glu to Gln;
pCR69 - includes mutations in which amino acid residue
104 is changad from Arg to His, amino acid residue 105
is changed from Asp to Asn, amino acid residue lO9 is
changed from Lys to Gln, and amino acid residue 111 is
changed from Arg to Gln; and
pCR70 - includes mutations in which amino acid residue
104 is changed from Arg to His, amino acid residue 105
is changed from Asp to Asn, amino acid residue lO9 is
changed from Lys to Gln, amino acid residue 111 is
changed from Arg to Gln, amino acid residue 117 is
changed from Arg to Glu, and amino acid residue 122 is
changed from &lu to Gln.
Plasmids pCR68, pCR69, and pCR70 are transfected
separately into the GPL pre-packaging cell line. The GPL
pre-packaging cell line consists of an NIH 3T3 mouse
fibroblast cell line which contains an expression plasmid
for MoMuLV gag-pol protein as well as the retroviral vector
LNL6 (Miller, et al., 1989). Upon transfection with pCR68,
pCR69, or pCR70, the GPL packaging cell line produces vector
paxticles.
r.~S~ ITE SHE~ (RULE ~
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; 2~3736~
Transiently expressed vector particles are collected
with cell supernatant at 48-72 hrs. post-transfection.
The vector particles generated as hereinabove described
may then be assayed for vector titer by techniques known to
those ~killed in the art. The vector particles may also be
collected in viral supernatant and concentrated, if
necéssary, according to procedures Xnown to those skilled in
the art in order to employ such vector particles in assays
or in therapeutic procedures.
Advantages of the present invention include the ability
to introduce vector particles directly into a human patient
whereby the vector particle is not lysed or inactivated by
human serum upon such introduction. Thus, the vector
particles of the present invention enable one to deliver
desired genes to a patient in vivo. Such vector particles
may also be engineered such that they are "targetable", as
well as injectable, thereby enabling the vector particles to
travel directly to a target cell or tissue without being
lysed or inactivated by human serum.
It is to be understood, however, that the scope of the
present invention is not to be limited to the specific
embodiments described above. The invention may be practiced
other than as particularly described and still be within the
scope of the accompanying claims.
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~ . '
SEQUENCE LISTING
51~ GE~ERAL INFORMATION:
(i) APPLICANT: Anderson, W. French
Mason, Jam~s M.
(ii) TITLE OF INVENTION: Vector Particles
Resistant to Inactivation
- by Human Serum
(iii) NUMBER QF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ~DDRESSEE: Carella, Byrne, Bain,
Gilfillan, Cecchi & St:ewart
(B) STREET: 6 Becker Farm Road
(C) CITY: Ro~eland
(~) STATE: N~w Jersey
(E) COUNTRY: ' USA
(F) ZIP: 07068
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch diskette
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: DW4.V2
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILIN~ DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATI~N:
: (A) NAME: Olstein, Elliot M.
(B) REGISTRATION NUMBER: 24,025
(C) REFERENCE/DOCKET NUMBER: 271010-73
(ix) TELECOMMUNICATION INFORMATION:
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IT~TE SHE~ ~RU~ _ ?6'
WO 93/25698 ~ 1 3 7 3 6 1 PCr/US93/0
(A) TELEP~IONE: 201-994-1700
(B~ TELEFA~C: 201-994-1744
( 2 ) INFORMATION FOR SEQ ID NO ~
t ~: ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 196 amino acids
B ) TYP~5 ~ amino acid
~ C ) STRANDEDNESS:
( D ) TOPOLOG~Y: linear
( ii ) MOLECULE TYPE: protein
( ix ) FEATURE:
(A) NAME/KEY: pl5E protein.
(xi ) SEQUENCE; DESCRIPTION: SEQ ID NO: 1:
Giu Pro Val Ser Leu Thr Leu Al~ Leu Leu
` 5 10
Leu C;ly Gly Leu Thr Met Gly Gly I le Ala
Ala Gly I le Gly Thr Gly Thr Thr Ala Leu
Met Ala Thr Gln Gln Phe Gln Gln Leu Gln
Ala Ala Val Gln Asp Aæp Leu Arg Glu Val
Glu Lys Ser I le Ser Asn Leu Glu Lys Ser
~5 60
Leu Thr Ser Leu Ser Glu Val Val Leu Gln
Asn Ar~ Arg Gly Leu Asp Leu Leu Phe Leu
Lys Glu Gly Gly Leu Cys Ala Ala Leu Lys
Glu Glu Cys Cys Phe Tyr Ala Asp His Thr
100
q
~ -17-
,: . . ~ , .
UTE SHEET ~RU!F ~
W093t2569$ 2 1 ~ 7 3 ~ i P~/USg3/04706
Gly Leu Val Arg Asp Ser Met Ala Lys Leu
105 110
Arg Glu Arg Leu Asn Gln Arg Gln Lys Leu
115 120
Phe Glu Ser Thr Gln Gly Trp Phe Glu Gly
125 130
Leu Phe Asn Arg Ser Pro Trp Phe Thr Thr
135 14
Leu Ile Ser Thr Ile Met Gly Pro Leu Ile
145 150
Val Leu Leu Met Ile Leu Leu Phe Gly Pro
155 l~iO
Cys Ile Leu Asn Arg Leu Val Gln Phe Val
165 1'70
Lys Asp Arg Ile Ser Val Val Gln Ala Leu
175 1~0
Val Leu Thr Gln Gln Tyr His Gln Leu Lys
1~5 190
Pro Ile Glu Tyr Glu Pro
195
(2) INFO~MATION EOR SEQ ID NO:2:
~ (i) SEQUENCE CH~RACTERISTICS:
i (A~ LENGTH: 176 amino acids
(B) TYPE: amino acid
(C) 5TRANDEDNESS:
(D) TOPOLOGY: linear
,
l (ii) MOLECULE TYPE: protein
: .
. (ix) FEATURE
. (A) NAME/KEY: p21 protein
: ; ~
~ -18-
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TUT~ SHEFr !R~IL~ r.
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WO 93/25~98 ~ ' PCT/US93/04706
2~37361 ` -
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:2:
Ala Val Pro Val Ala Val Trp Leu Val Ser
Ala Leu Ala Met Gly Ala Gly Val Ala Gly
15 20 ;
Arg Ile Thr Gly Ser Met Ser Leu Ala Ser
~5 30
Gly Lys Ser Leu Leu His Glu Val Asp Lys
Asp Ile Ser Gln Leu Thr Gln Ala Ile Val
Lys Asn His Lys A~n Leu Leu 1ys Ile Ala
Gln Tyr Ala Ala Gln Asn Arg Arg Gly Leu
' 65 70
Asp Leu Leu Phe Trp Glu Gln Gly Gly Leu
Cys Lys ~la Leu Gln Glu Gln Cys Cys Phe
Leu Asn Ile Thr Asn Ser Hi s Val Ser I le
100
~ Leu Gln Glu Arg Pro Pro Leu Glu Asn Arg
i 105 llO
Val Leu Thr Gly Trp Gly Leu Rsn Trp Asp
~- 115 120
Leu Gly Leu Ser Gln Trp Ala Arg Glu Ala
` 125 130
Leu Gln Thr Gly Ile Thr Leu Val Ala Leu
135 140
Leu Leu Leu Val Ile Leu Ala Gly Pro Cys
~ 145 150
;. Ile Leu Arg Gln Leu Arg His Leu Pro Ser
: 155 160
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SUBS~ITUTE SHEET ~RVLE 26)
r~
WO 93~25698 2 ~ ~ 7 3 6 i P~/US93/04706
Arg Val Arg Tyr Pro Hi 5 Tyr Ser Leu I le
165 170
Asn Pro Glu Ser Ser Leu
175
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